WO2022185319A1 - Ubiquitin ligase kpc1-peptide based proteolysis targeting chimeras (protac) and uses thereof - Google Patents

Ubiquitin ligase kpc1-peptide based proteolysis targeting chimeras (protac) and uses thereof Download PDF

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WO2022185319A1
WO2022185319A1 PCT/IL2022/050243 IL2022050243W WO2022185319A1 WO 2022185319 A1 WO2022185319 A1 WO 2022185319A1 IL 2022050243 W IL2022050243 W IL 2022050243W WO 2022185319 A1 WO2022185319 A1 WO 2022185319A1
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amino acid
molecule
conjugate
xaa
peptide
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Aaron Ciechanover
Ashraf Brik
Yelena KRAVTSOVA
Gilad GOLDHIRSH
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Technion Research & Development Foundation Limited
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present disclosure relates to NF- ⁇ B modulators. More specifically, the present disclosure provides bifunctional hybrid-molecules, conjugates, complexes, and compositions enhancing ubiquitination and proteasomal processing of NF- ⁇ B 1 p105 to p50 and their uses for treating pathological disorders, e.g., cancer.
  • Nuclear factor- kB/r65 (Rel A) is constitutively activated in human prostate adenocarcinoma and correlates with disease progression. Neoplasia 6, 390-400 (2004).
  • NF- ⁇ B is a key transcriptional regulator involved in inflammation and cell proliferation, survival, and transformation. Upregulation of NF- ⁇ B is frequently observed in a variety of tumors, and probably play a role in malignant transformation. The mechanism(s) that underlie the pro-tumorigenic activity of NF- ⁇ B involve upregulation of expression of pro-proliferative and anti-apoptotic genes, as well as stimulation of the inflammatory process [1, 2, 3].
  • the NF- ⁇ B family consists of five different proteins - three Rel proteins (RelA, RelB, and cRel), and two proteins - p50 and p52 - which are derived from limited, UPS-mediated - processing of longer precursors, p105 (NF- ⁇ B 1) and p100 (NF- ⁇ B2), respectively.
  • RelA, RelB, and cRel RelA, RelB, and cRel
  • p50 and p52 - proteins derived from limited, UPS-mediated - processing of longer precursors, p105 (NF- ⁇ B 1) and p100 (NF- ⁇ B2), respectively.
  • NF- ⁇ B 1 p105
  • NF- ⁇ B2 p100
  • the tumorigenicity is related to the p50 ⁇ p65 dimer [6, 7, 8].
  • Ub ubiquitin
  • KPC1 was previously identified as the Ub ligase (E3) that binds to the ankyrin repeats domain of p105, ubiquitinates it, and mediates its processing both under basal conditions and following signalling [9].
  • E3 Ub ligase
  • the inventors previously showed that KPC1 interacts with p105 via a short, seven amino acids - WILVRLW - sequence (residues 968-974, also denoted by SEQ ID NO: 4) in KPC1 [10].
  • Overexpression of p50 resulted in a strong tumor- suppressive effect which was due to up-regulation of various tumor suppressors [9], and modulation of the tumor microenvironment by recruiting and activating the immune system [11]: excess of KPC1 or p50 up-regulates expression of CCL3, CCL4, and CCL5, which are pro- inflammatory chemokines, which in turn recruit to the tumor NK cells and macrophages that contribute to the tumor-suppressive effect. Also, p50 down-regulates the expression of the immune checkpoint programmed cell death-ligand 1 (PD-L1) [11]. Furthermore, the inventors previously showed that a truncated KPC1 containing the 7 amino acids and the minimal domains required for its enzymatic activity, is sufficient to stimulate ubiquitination of p105 and to suppress growth of model human tumors in mice.
  • PD-L1 programmed cell death-ligand 1
  • sites of Ub ligases that bind their cognate substrates are rather long.
  • the interaction site of KPC1 which regulates the degradation of p27 at the G1 phase of the cell cycle spans 766 amino acids at the N-terminal part of the molecule [12].
  • Another example is the interaction site of MDM2 with p53. It was shown that deletion of the N-terminal 61 am aicniods of the ligase abrogated its interaction with the tumor suppressor [13, 14].
  • WO 2020/110114 [10] by the preset inventors, discloses a peptide comprising the seven amino acids - WILVRLW - sequence (residues 968-974) of KPC1, conjugates or chimeras of this peptide and uses thereof in treating cancer. These conjugates or chimeras comprise the KPC1 -derived peptide linked to a RING domain of KPC1, or to molecules or peptides that recruits E3 ligases. There is need in the art to develop effective drugs that enhance processing of the NF- ⁇ B 1 precursor p105 to the p50 active subunit, for the treatment of NF- ⁇ B 1 associated disorders.
  • a first aspect of the present disclosure relates bifunctional hybrid-molecule, conjugate or complex comprising the at least two following components: component (a), comprises at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue.
  • the peptide that may be also referred to herein as a bio-scaffold, comprises the amino acid sequence of Xaa (n) -Zaa-Xaa (m) -Zaa-Xaa (n) , as denoted by SEQ ID NO: 1. More specifically, Zaa, as used herein is any aromatic amino acid residue and Xaa is any amino acid residue.
  • m is an integer between 1 to 7
  • n as used herein is zero or an integer between 1 to 7.
  • Component (b), of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least one proteasome degradation mediating moiety.
  • a further aspect of the invention relates to a composition
  • a composition comprising at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the bioactive molecule of the present disclosure.
  • the composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
  • the bifunctional hybrid-molecule, conjugate or complex comprises: (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amino acid sequence of Xaa (n) -Zaa-Xaa (m) - Zaa-Xaa (n) , as denoted by SEQ ID NO: 1. It should be noted that Zaa is any aromatic amino acid residue. Still further, in some embodiments, Xaa is any amino acid residue. In some specific embodiments, n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7, as discussed above in connection with the previous aspect of the present disclosure; and (b), at least one proteasome degradation mediating moiety.
  • a further aspect of the present invention relates to a method for inducing ubiquitination and proteasomal processing of NF- ⁇ B1 p105, thereby generating the NF- ⁇ B p50 in a cell or in a cell- free system comprising said NF- ⁇ B 1 p105. More specifically, the method comprising the step of contacting said cell or said cell-free system with an effective amount of at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the same, or any composition thereof.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise: (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue.
  • the peptide comprises the amino acid sequence of Xaa (n) -Zaa-Xaa (m) -Zaa-Xaa (n) , as denoted by SEQ ID NO: 1.
  • Zaa is any aromatic amino acid residue.
  • Xaa is any amino acid residue.
  • n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7.
  • the bifunctional hybrid-molecule, conjugate or complex of the methods disclosed herein further comprises a second component (b), at least one proteasome degradation mediating moiety.
  • a further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder or condition in a subject in need thereof. More specifically, the method comprising the step of administering to said subject a therapeutically effective amount of at least one bifunctional hybrid- molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the same, or any composition thereof.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise: (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amin aocid sequence of Xaa (n) -Zaa-Xaa (m) -Zaa-Xaa (n) , as denoted by SEQ ID NO: 1.
  • Zaa is any aromatic amino acid residue.
  • Xaa is any amino acid residue.
  • n is zero or an integer between 1 to 7
  • m is an integer between 1 to 7.
  • the bifunctional hybrid- molecule, conjugate or complex of the compositions disclosed herein further comprises a second component (b), at least one proteasome degradation mediating moiety.
  • the present disclosure further provides peptides that recruit the NF- ⁇ B1 p105.
  • Still further aspect of the present disclosure relates to therapeutically effective amount of at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro- particle comprising the same, or any composition thereof for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder or condition in a subject in need thereof, and/or in a method for inducing ubiquitination and proteasomal processing of NF- ⁇ B1 p105, thereby generating the NF- ⁇ B p50 in a cell or in a cell-free system comprising said NF- ⁇ B 1 p105.
  • FIG. 1A-1B Ubiquitination of p105 by KPC1 is dependent on amino acid sequence WILVRLW in KPC1
  • Fig. 1Ai-1Aii In vitro translated 35 S-labeled p105 was ubiquitinated by the indicated purified species of KPC1 in a reconstituted cell-free system as described under Experimental procedures.
  • Fig. Ai Ubiquitination of p105 by KPC1 ⁇ 1-967-Hisx6 (lanes 2-4) andKPC1 ⁇ 1-973-Hisx6 (lanes 5-7).
  • the numbers indicate the amount of the added ligase in ⁇ g. It was calculated by dividing the amount of p105 and its monoubiquitinated species (generated by the E2 present in all reaction mixtures) remained under each condition by the amount of p105 (and its monoubiquitinated species) in lane 1.
  • Fig. 1Ai-1Aii In vitro translated 35 S-labeled p105 was ubiquitinated by the indicated purified species of KPC1 in a reconstituted cell-free system as described under Experimental procedures.
  • Fig. Ai Ub
  • 1Aii Ubiquitination of p105 using the following species of KPC1: KPC1 ⁇ 1-967 (lane 2); KPC1 ⁇ 1-974 (lane 3); WIFVRFW-KPC1 ⁇ 1-1039 (lane 4); and WIFVRFW-KPC1 ⁇ 1-1061 (lane 5).
  • KPC1 ⁇ 1-967 Lane 2
  • KPC1 ⁇ 1-974 Lane 3
  • WIFVRFW-KPC1 ⁇ 1-1039 lane 4
  • WIFVRFW-KPC1 ⁇ 1-1061 lane 5
  • Each enzyme was added at 0.5 ⁇ g. Also shown is the fraction of the free p105 remained for each condition (compared to a system to which KPC1 was not added; lane 1; arbitrarily was designated as 1).
  • Fig. IB Addition of either WILVRLW or RIWVWLL peptide inhibits ubiquitination of p105 by KPC1 ⁇ 1-967 in a dose-dependent manner.
  • KPC1 ⁇ 1-967 (lanes 2-12, SEQ ID NO: 68, in a cell-free system in the presence of the indicated peptides derived from KPC1: ILVRLW (lanes 3-5, SEQ ID NO: 5); WILVRLW (lanes 6-8, SEQ ID NO: 4); and RIWVWLL (lanes 9-11, SEQ ID NO: 2).
  • the numbers indicate the peptide concentration in mM.
  • the fraction of remained free unconjugated p105 for each condition is as described under Ai. Visualization of p105 and its conjugates was carried out using Phosphor Imaging. Also shown is the fraction of the free p105 remained for each condition (compared to a system to which a peptide was not added; lane 2; arbitrarily was designated as 1).
  • FIG. 2A-2B Overexpression of truncated species of KPC1 that contain the amino acid sequence WILVRLW or RIWVWLL attract leukocytes into a xenograft tumor model and inhibits tumor growth
  • Fig. 2Ai-2Aiv. Fig. 2Ai Growth rates (* represents P-value ⁇ 0.05) and Fig. 2Aii weights (at the end of the experiment, 25 days after inoculation) of U87-MG cells-derived xenografts grown in SCID mice.
  • the tumors express either KPC1-FLAG, WILVRLW-KPC 1 A 1 - 1039-FLAG, RIWVWLL- KPC 1 Al - 1039-FLAG, or KPC1 ⁇ 1-1039-FLAG proteins (SEQ ID NOs: 66, 62, 65, and 40, respectively). Control cells were transfected with an empty vector.
  • Fig 2Aiii Growth rates (* represents P-value ⁇ 0.05) and Fig. 2Aii weights (at the end of the experiment, 25 days after inoculation) of U87-MG cells-derived xenografts grown in SCID mice.
  • the tumors express either KPC1-FLAG, WILV
  • Fig. 2B Tumors’ sections expressing the indicated KPC1 species attract leucocytes as is evident from immunohistochemical staining with anti-CD45.
  • FIG. 3A-3B Proteome analysis of glioblastoma xenografts expressing different species of KPC1
  • Fig. 3A Euclidean Heat map diagram presenting an unsupervised hierarchical clustering of the cellular proteome under expression of the different KPC1 species. The clustering was based on the intensities of the identified proteins (low intensity - green, high intensity - red), and was generated by the Perseus software.
  • Fig. 3B Functional analysis of proteins that are up- (red) or down- (green) regulated (compared to tumors transfected with an empty vector with a p-value ⁇ 0.05). Shown are selected annotation clusters with false discovery rate (FDR) ⁇ 0.05 (the numbers on the flanks of each column represent the FDR of the indicated cluster). Functional analysis was carried out using the string software [Szklarczyk D, et ak, Nucleic Acids Res. Jan 8;49(D1):D605-12 (2021)]. Proteome analysis was carried out on the xenografts described under Figure 2. The raw proteomic data are disclosed by the present inventors, in Goldhirsh G.
  • PROTACs containing the peptides WILVRLW or RIWVWLL induce ubiquitination of p105 by the E3 ligase pVHL in a reconstituted cell-free system
  • Fig. 4Ai— 4Avi Schematic structure of the components of different PROTACs.
  • Fig. 4Ai. shows PROTAC 1 of Formula V, that comprises the peptide of SEQ ID NO: 28.
  • Fig. 4Aii. shows PROTAC 2 of Formula XII, that comprises the peptide of SEQ ID NO: 42.
  • Fig. 4Aiii. shows PROTAC 6 of Formula XIV, that comprises the peptide of SEQ ID NO: 43.
  • Fig. 4Aiv. shows PROTAC 5 of Formula VI, that comprises the peptide of SEQ ID NO: 39.
  • Fig. 4Av. shows PROTAC 4 of Formula XIII, that comprises the peptide of SEQ ID NO: 8, with acetylated Arg.
  • Fig. 4Avi shows PROTAC 3 of Formula IV, that comprises the peptide of SEQ ID NO: 8.
  • Fig. 4Bi-4BiiL In vitro translated 35 S-labeIed p105 was ubiquitinated by purified VHL complex mediated by PROTAC molecules.
  • Fig. 4Bi. dose dependent ubiquitination of in vitro translated 35 S-labeIed p105 by WILVRLW-SG-PEG-VHL PROTAC 1 molecule (Fig. 4Bii) and (Fig.
  • Fig. 4C In vitro processing of p105 to p50 by Fr2 increases in the presence of different concentrations of WILVRLW-SG-PEG-VHL PROTAC 1 (lanes 1-3). Visualization was carried out using Phosphorimaging.
  • FIG. 5A-5B Schematic representation of the PROTACs’ synthesis.
  • FIG. 5A Schematic representation of the peptide-based PROTACs’ synthesis.
  • Fig. 5B Attachment of the fluorophore FITC to the PROTAC RIWVWLLCG-PEG-pVHL ligand.
  • FIG. 6A-6G HPLC-MS analysis of the purified PROTACs
  • Fig. 6A HPLC-MS analysis of the purified WILVRLWSG-PEG-pVHL ligand PROTAC1 with an observed mass of 1686.4 ⁇ 0.1 Da (calculated 1687.4 Da, average isotopes).
  • Fig. 6B HPLC-MS analysis of the purified ILVRLSG-PEG-pVHL ligand PROTAC2 with an observed mass of 1313.9 ⁇ 0.2 Da (calculated 1314.9 Da, average isotopes).
  • Fig. 6C HPLC-MS analysis of the purified RIWVWLLSG-PEG-pVHL ligand PROTAC3 with an observed mass of 1686.3 ⁇ 0.5 Da (calculated 1687.4 Da, average isotopes).
  • Fig. 6D HPLC-MS analysis of the purified Acetyl-RIWVWLLSG-PEG-pVHL ligand PROTAC 4, with an observed mass of 1727.5 ⁇ 0.1 Da (calculated 1728.4 Da, average isotopes).
  • Fig. 6E HPLC-MS analysis of the purified IWVWLLSG-PEG-pVHL ligand PROTAC5 with an observed mass of 1529.9 ⁇ 0.1 Da (calculated 1530.2 Da, average isotopes).
  • Fig. 6F HPLC-MS analysis of the purified IWVWLLS-PEG-pVHL ligand PROTAC6 with an observed mass of 1473.4 ⁇ 0.4 Da (calculated 1473.1 Da, average isotopes).
  • FIG. 6G HPLC-MS analysis of the purified RIWVWLLC(-FITC)G-PEG-pVHL ligand PROTAC8 with an observed mass of 2129.4 ⁇ 0.1 Da (calculated 2129.8 Da, average isotopes).
  • Figure 7A-7D PROTACs that contain the WILVRLW or RIWVWLL peptides stimulate interaction between p105 and pVHL and subsequent processing of p105 to p50, and restrict cell growth
  • Fig. 7Ai-Aii The PROTAC RIWVWLL-C(-FITC)G-PEG-pVHL(L) is cell permeable.
  • Fig. 7Ai Schematic representation of the PROTAC RIWVWLL-C(-FITC)G-PEG-pVHL(L).
  • Fig. 7Aii Confocal microscopy imaging of HEK293 cells 24 h after addition of the RIWVWLL-C(FITC)G- PEG-pVHL ligand PROTAC to the growth medium (25 mM).
  • Fig. 7Bi-Biv The interaction between p105 and pVHL in cells is stimulated by WILVRLW or RIWVWLL-based PROTACs (25 pM).
  • HEK293 cells that stably express pVHL-FLAG were transfected with p105-HA (lanes 1-9).
  • pVHL-FLAG was immunoprecipitated from the cell lysate using immobilized anti-FLAG.
  • p105-HA (Fig. 7Bi) and pVHL-FLAG (Fig. 7Bii) were visualized using the appropriate antibodies as indicated.
  • Fig. 7Biii) and (Fig. 7Biv) display the two proteins in TCL.
  • Fig. 7Ci-Cii WILVRLW and RIWVWLL-based PROTACs enhance cellular processing of p105 to p50.
  • HEK293 cells that stably express pVHL-FLAG were transfected with cDNAs coding for FLAG-p105 along with Myc-Ub.
  • WILVRLW or RIWVWLL-based PROTACs 25 pM were added to the growth medium as indicated.
  • FLAG-p105 and FLAG-p50 were visualized using anti- FLAG antibody (Fig. 7Ci). Actin was used as a loading control (Fig. 7Cii).
  • Fig. 7Di-Dii WILVRLW or RIWVWLL-based PROTACs restrict growth of U87-MG cells.
  • Fig. 7Di Growth curve of U87-MG cells in the presence of WILVRLWSG-PEG-pVHL(L) PROTAC (25 pM).
  • Fig. 7Dii Similar to 7Di, but in the presence of RIWVWLLSG-PEG-pVHL(L) PROTAC. (L) denotes - ligand. * represents P- value ⁇ 0.05.
  • FIG. 8A-8B PROTAC that contains RIWVWLL peptide, infiltrates the tumor xenografts Lig. 8A.
  • Xenografts derived from U87-MG cells were grown in NSG mice. After tumors were established, DMSO or RIWVWLL-C(-FITC)-G-PEG-VHL PROTAC were injected to the tumor surroundings. 24 hr later mice were visualized using IVIS device, following by sacrificing, and dissection of tumors.
  • Fig. 8B The slides were prepared using cryotechnology and visualized under confocal microscope.
  • Figs. 9A-9C In vitro translated 35 S-labeled p105 was ubiquitinated by purified KPC1 ⁇ 1-967 (SEQ ID NO: 67) in a reconstituted cell-free system with the addition of permutated peptides derived from the peptide RIWVWLL (as denoted by SEQ ID NO: 4). In vitro translated and 35 S- labeled p105 was ubiquitinated by purified KR01D1-967 in a cell-free system in the presence of the indicated peptides. The numbers indicate the peptide concentration in mM.
  • Fig. 9A shows peptides KVS-4, KVS-1, KVS-5, KVS-3.
  • Fig. 9B. shows peptides KVS-4, KVS-2A, KVS-2B, KVS-2C.
  • Fig. 9C. shows peptides KVS-4, KVS- 2D, KVS-6, KVS-7.
  • Fig. 9D Schematic representation of the peptides used in the experiment shown in Figs 9A-9C.
  • the peptides comprise the ami ancoid sequence as denoted by SEQ ID NO: 8 (KVS-4), SEQ ID NO: 44 (KVS-1), SEQ ID NO: 45 (KVS-5), SEQ ID NO: 46 (KVS-3), SEQ ID NO: 47 (KVS-2A), SEQ ID NO: 48 (KVS-2B), SEQ ID NO: 49 (KVS-2C), SEQ ID NO: 50 (KVS-2D), SEQ ID NO: 51 (KVS-6), SEQ ID NO: 17 (KVS-7, is a cyclic form of the peptide of SEQ ID NO: 17).
  • p105 is not the only substrate of the ligase, and other substrates such as p27 [12] are recognized by different domains.
  • the inventors synthesized a PROTAC that contains the binding sequence, with the hope that it will generate excess of p50 and serve as a prototype for a tumor- suppressive modality. Indeed, the PROTAC simulated conjugation of Ub to p105 in a reconstituted cell-free system ( Figure 4B) and processing of p105 to p50 in cells. Further, it restricted cell growth when added to the growth medium.
  • the first component (a) comprises at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue.
  • the peptide that may be also referred to herein as a bio- scaffold, comprises the amino acid sequence of Xaa (n) -Zaa-Xaa (m) -Zaa-Xaa (n) , as denoted by SEQ ID NO: 1. More specifically, Zaa, as used herein is any aromatic amino acid residue and Xaa is any amino acid residue (that may be any of the 20 amino acids, including aromatic amino acids, or any derivatives or mimetics thereof).
  • Zaa as used herein is also denoted in the sequence listing with respect to SEQ ID NO: 1, as residue “Xaa 2 " and as “Xaa 4 " , and refer to any aromatic amino acid residue as disclosed herein.
  • sequence Xaa (n) -Zaa-Xaa (m) -Zaa-Xaa (n) is also equivalent to Xaa (n) -Xaa 2 -Xaa (m) -Xaa 4 -Xaa (n), both are denoted by SEQ ID NO: 1.
  • the at least two aromatic amino acid residues, or any derivatives or mimetics thereof, Zaa, of the peptide disclosed herein may be either identical, or different aromatic amino acids.
  • the interspacing amino acid residue/s, Xaa (m) may be any amino acid residue, or any derivatives or mimetics thereof. Since in some embodiments, "m" is an integer between 1 to 7, the peptide may comprise 1, 2, 3, 4, 5, 6, or 7 interspacing amino acid residue/s, located between the at least two aromatic am aicniod residues.
  • These interspacing, gaping separating, amin aocid residues may be any identical or different amino acid residues, or any derivative, analogs, or mimetics thereof.
  • the peptide of the bifunctional hybrid-molecule further comprises Xaa (n) am aincoid residues, that may be located at any possible position of the peptide, and/or at any of the N' or C termini thereof. Since "n” as used herein is zero or an integer between 1 to 7, the peptide may comprise none (0), or 1, 2, 3, 4, 5, 6 or 7, amino acid residues at the N' terminus of the peptide, optionally, between one or both of the at least two aromatic amino acid residues and the interspacing residue/s, and/or at the at the C terminus of the peptide. These amino acid residues may be any identical or different amino acid residue/s, or any derivative, analogs, or mimetics thereof.
  • the second component of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least one proteasome degradation mediating moiety.
  • the peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure comprises at least two aromatic amino acid residues.
  • An aromatic amino acid (AAA) is an amino acid that includes a hydrophobic side chain, specifically, an aromatic ring. More specifically, a cyclic (ring-shaped), planar (flat) structures with a ring of resonance bonds that gives increased stability compared to other geometric or connective arrangements with the same set of atoms.
  • An aromatic functional group or other substituent is called an aryl group.
  • Aromatic amino acids absorb ultraviolet light at a wavelength above 250 nm and produce fluorescence.
  • Aromatic amino acid includes natural as well as unnatural amino acids.
  • Unnatural, aromatic amino acids include those that include an indole moiety in their amino acid side chain, wherein the indole ring structure can be substituted with one or more aryl group substituents.
  • Additional examples of aromatic amino acids include but are not limited to 1-naphthylalanine, biphenylalanine, 2- napthylalananine, pentafluorophenylalanine, and 4-pyridylalanine.
  • aromatic refers to a mono-, bi-, or other multi-carbocyclic, aromatic ring system.
  • the aromatic group may optionally be fused to one or more rings chosen from aromatics, cycloalkyls, and heterocyclyls.
  • Aromatics can have from 5-14 ring members, such as, e.g., from 5-10 ring members.
  • One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aromatic, aryloxy, azido, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, cycloalkyl, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, and ureido.
  • aromatic groups include phenyl, naphthyl, indolyl, biphenyl, and anthracenyl
  • the at least two aromatic amino acid residues of the peptide component of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure may be at least one of Tryptophan, Tyrosine, and Phenylalanine, or any combinations thereof.
  • at least one of the at least two aromatic amino acid residues comprised in the peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure is Tryptophan, or any derivative, analogs, or mimetics thereof.
  • Tryptophan symbol Trp or W
  • Tryptophan is also a precursor to the neurotransmitter serotonin, the hormone melatonin and vitamin B3.
  • at least one of the at least two aromatic amino acid residues of the peptide comprised in the bifunctional hybrid-molecule, conjugate or complex of the present disclosure is Tyrosine, or any derivative, analogs, or mimetics thereof.
  • Tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is a non-essential amino acid with a polar side group, having the formula C 9 H 11 NO 3 .
  • L-Tyrosine has the following chemical structure, as denoted by Formula X:
  • tyrosine While tyrosine is generally classified as a hydrophobic amino acid, it is more hydrophilic than phenylalanine. It is encoded by the codons UAC and UAU in messenger RNA (mRNA). Mammals synthesize tyrosine from the essential amino acid phenylalanine. The conversion of phe to tyr is catalyzed by the enzyme phenylalanine hydroxylase. In dopaminergic cells in the brain, tyrosine is converted to L-DOPA by the enzyme tyrosine hydroxylase (TH). TH is the rate-limiting enzyme involved in the synthesis of the neurotransmitter dopamine.
  • TH tyrosine hydroxylase
  • Dopamine can then be converted into other catecholamines, such as norepinephrine (noradrenaline) and epinephrine (adrenaline).
  • catecholamines such as norepinephrine (noradrenaline) and epinephrine (adrenaline).
  • the thyroid hormones triiodothyronine (T 3 ) and thyroxine (T 4 ) in the colloid of the thyroid are also derived from tyrosine.
  • At least one of the at least two aromatic amino acids of the peptide comprised in the bifunctional hybrid-molecule, conjugate or complex of the present disclosure is Phenylalanine, or any derivative, analogs, or mimetics thereof.
  • Phenylalanine symbol Phe or F
  • Phenylalanine is an essential ⁇ -amino acid with the formula C 9 H 11 NO 2 . It can be viewed as a benzyl group substituted for the methyl group of alanine, or a phenyl group in place of a terminal hydrogen of alanine.
  • L-Phenylalanine has the following chemical structure, as denoted by Formula XI: Formula XI
  • Phenylalanine is a precursor for tyrosine, the monoamine neurotransmitters dopamine, norepinephrine (noradrenaline), and epinephrine (adrenaline), and the skin pigment melanin. It is encoded by the codons UUU and UUC.
  • the peptide comprised in the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least two tryptophan residues, or any derivative, analogs, or mimetics thereof.
  • the peptide comprised in the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least one tryptophan and at least one tyrosine, or any derivative, analogs, or mimetics thereof.
  • such peptide may comprise the amino acid sequence of Xaa (n) -Trp-Xaa (m) -Tyr-Xaa (n), as denoted by SEQ ID NO: 30, Xaa (n) -Tyr-Xaa (m) -Trp-Xaa (n), as denoted by SEQ ID NO: 31, or any peptide derived therefrom, wherein Xaa is any amin aocid, n is zero or an integer between 1 to 7 and m is an integer between 1 to 7.
  • the peptide comprised in the bifunctional hybrid- molecule, conjugate or complex of the present disclosure may comprise at least one tryptophan and at least one phenyl alanine, or any derivative, analogs, or mimetics thereof.
  • such peptide may comprise the amino acid sequence of Xaa (n) -Trp-Xaa (m) -Phe- Xaa (n), as denoted by SEQ ID NO: 32, Xaa (n) -Phe-Xaa (m) -Trp-Xaa (n), as denoted by SEQ ID NO: 33, or any peptide derived therefrom, wherein Xaa is any amino acid, n is zero or an integer between 1 to 7 and m is an integer between 1 to 7.
  • the peptide comprised in the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least two tyrosine residues.
  • such peptide may comprise theamino acid sequence of Xaa (n) -Tyr-Xaa (m) -Tyr-Xaa (n), as denoted by SEQ ID NO: 34, or any peptide derived therefrom, wherein Xaa is any amino acid, n is zero or an integer between 1 to 7 and m is an integer between 1 to 7.
  • the peptide comprised in the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least one tyrosine and at least one phenylalanine, or any derivative, analogs, or mimetics thereof.
  • such peptide may comprise the amino acid sequence of Xaa (n) -Phe-Xaa (m) - Tyr-Xaa (n), as denoted by SEQ ID NO: 35, Xaa (n) -Tyr-Xaa (m) - Phe-Xaa (n), as denoted by SEQ ID NO: 36, or any peptide derived therefrom, wherein Xaa is any amino acid, n is zero or an integer between 1 to 7 and m is an integer between 1 to 7. Still further, in some embodiments, the peptide comprised in the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least two phenylalanine residues.
  • such peptide may comprise the amino acid sequence of Xaa (n) -Phe-Xaa (m) -Phe-Xaa (n), as denoted by SEQ ID NO: 37, or any peptide derived therefrom, wherein Xaa is any amino acid, n is zero or an integer between 1 to 7 and m is an integer between 1 to 7. Still further, the disclosure contemplates the use of at least two aromatic amino acid residues or mimetics thereof, in the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure.
  • amino acid mimetics refers to chemical compounds having a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • tryptophan mimetic and “W mimetic”, “tyrosine mimetic” and “Y mimetic” and “phenylalanine mimetic” and “F mimetic”, are used interchangeably to refer to any agent that either emulates the biological effects of tryptophan, tyrosine, and/or phenylalanine in the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure, on recruitment of NF- ⁇ B1 p105.
  • the W, Y and/or F mimetic in the peptide of bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure can be any kind of agent.
  • Exemplary W, Y and/or F mimetics include, but are not limited to, small organic or inorganic molecules; L-tyrosine, L-tryptophan and/or L- phenylalanine, D-tyrosine, D-tryptophan and/or D-phenylalanine, an NF- ⁇ B1 p105 recruiting tyrosine, tryptophan and/or phenylalanine mimetic, saccharides, oligosaccharides, polysaccharides, a biological macromolecule that may be any one of peptides, non-standard peptides, polypeptides, non-standard polypeptides, proteins, non-standard proteins, peptide analogs and derivatives enriched for L- tyrosine , L- tyrosine tryptophan and/or L-phenylalanine.
  • the at least two aromatic amino acid residues W, Y and/or F of the peptide, or any mimetics thereof comprise at least one of the native amino acid tyrosine, tryptophan and/or phenylalanine.
  • native amino acid refers to the L-form of the amino acid which naturally occurs in proteins; thus, the term “native amino acid tryptophan, tyrosine and/or phenylalanine” refers to L- tryptophan, L- tyrosine and/or L-phenylalanine.
  • the amino acid residues in the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure can be in D-configuration or L-configuration (referred to herein as D- or L- enantiomers).
  • the at least two of W, Y and/or F in the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure comprises the native amino acid tryptophan, tyrosine and/or phenylalanine (W, Y and/or F).
  • the native amino acid tyrosine, tryptophan and/or phenylalanine is isolated and/or purified.
  • the at least two of W, Y and/or F, and/or mimetics thereof, of the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure may comprise a derivative, variant or analog of the native amino acid tryptophan, tyrosine, and/or phenylalanine.
  • the at least two of W, Y and/or F, and/or mimetics thereof, in the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure may comprise a combination of the native amino acid tryptophan, tyrosine, and/or phenylalanine, a derivative of the native amino acid tryptophan, tyrosine, and/or phenylalanine and/or an analog of the native amino acid tryptophan, tyrosine, and/or phenylalanine.
  • derivative of a peptide comprising at least two of W, Y and/or F may comprise a C'-terminus modification to at least one of W, Y and/or F.
  • C-terminus modification refers to the addition of a moiety or substituent group to the amino acid via a linkage between the carboxylic acid group of the amino acid and the moiety or substituent group to be added to the amino acid.
  • the disclosure contemplates any C-terminus modification to a peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure, that may occur in at least one of the at least two of W, Y and/or F, while the peptide still retains the ability to recruit NF- ⁇ B1 p105.
  • the C-terminus modification to a peptide comprising at least two of W, Y and/or F may comprise a carboxy alkyl ester.
  • alkyl refers to saturated non-aromatic hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms (these include without limitation methyl, ethyl, propyl, allyl, or propargyl), which may be optionally inserted with N, O, S, SS, SO2, C(0), C(0)0, OC(O), C(0)N or NC(O).
  • C i-Ce indicates that the group may have from 1 to 6 (inclusive) carbon atoms in it.
  • the C- terminus modification to L comprises a carboxy alkenyl ester.
  • alkenyl refers to an alkyl that comprises at least one double bond.
  • exemplary alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl and the like.
  • the C-terminus modification to at least one of W, Y, F comprises a carboxy alkynyl ester.
  • alkynyl refers to an alkyl that comprises at least one triple bond.
  • the carboxy ester comprises tyrosine, tryptophan and/or phenylalanine carboxy methyl ester.
  • the carboxy ester comprises tyrosine, tryptophan and/or phenylalanine carboxy ethyl ester.
  • derivative of a peptide comprising at least two of W, Y and/or F may comprise an N-terminus modification to at least one of W, Y and/or F.
  • N-terminus modification refers to the addition of a moiety or substituent group to the amino acid via a linkage between the alpha amino group of the amino acid and the moiety or substituent group to be added to the amino acid.
  • the disclosure contemplates any N'-terminus modification to at least one of Y, W and/or F in which the peptide comprising N- terminus modified W, Y and/or F, still retains the ability to recruit NF- ⁇ B1 p105.
  • the derivative of a peptide comprising at least two of W, Y, and/or F may comprise at least two of W, Y, and/or F modified by an amino bulky substituent group.
  • amino bulky substituent group refers to a bulky substituent group which is linked to the amino acid via the alpha amino group.
  • An exemplary amino bulky substituent group is a carboxybenzyl (Cbz) protecting group.
  • the derivative of a peptide comprising at least two of W, Y, and/or F comprises Y, W, and/or F modified by an amino carboxybenzyl (Cbz) protecting group.
  • Cbz amino carboxybenzyl
  • the derivative of a peptide comprising at least two of W, Y, and/or F may comprise a side-chain modification to Y, W and/or F.
  • side-chain modification refers to the addition of a moiety or substituent group to the side-chain of the amino acid via a linkage (e.g., covalent bond) between the side-chain and the moiety or chemical group to be added.
  • linkage e.g., covalent bond
  • the present disclosure further encompasses any Deuterated, Fluorinated, Acetylated or Methylated forms of any one of the amino acid residues of the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure, being either an aromatic amino acid residue or any other amino acid residue of the discussed peptide.
  • deuterium-substituted amino acids applicable as analogs of the present invention may include but are not limited to L-Tyrosine- (phenyl-3, 5-d 2 ), L-4-Hydroxyphenyl-2,3,5,6-d4-alanin and L-Tryptophan-(indole-d 5 ).
  • Methylated aromatic amino acids residues include but are not limited to any one of L-Tyrosine methyl ester, O-Methyl-L-tyrosine, ⁇ -Methyl-L-tyrosine, ⁇ -Methyl-DL-tyrosine methyl ester hydrochloride, ⁇ -Methyl-L-tyrosine, ⁇ -Methyl-DL-tyrosine, ⁇ -Methyl-DL-tryptophan, O-Methyl- L-tyrosine, N-Methylphenethylamine, ⁇ -Methylphenethylamine, N, N-Dimethylphenethylamine, 3-Methylphenethylamine, (R)-(+)- ⁇ -Methylphenethylamine, N-Methyl-N-(l-phenylethyl)amine, 2-methylphenethylamine, 4- Bromo-N-methylphen
  • Fluorinated tyrosine, phenylalanine or tryptophan include but are not limited to any one of 5-Fluoro-L-tryptophan, 5-Fluoro-DL-tryptophan, 4-Fluoro-DL- tryptophan, 6-Fluoro-L-Tryptophan, 5-Methyl-DL-tryptophan, 5-Bromo-DL-tryptophan, 7- Azatryptophan, m-Fluoro-DL-tyrosine, p-Fluoro-L-phenylalanine, o-Fluoro-DL-phenylalanine, p- Fluoro-DL -phenylalanine, 4-Chloro-DL-phenylalanine, m-Fluoro-L-phenylalanine, 3-Nitro-L- tyrosine.
  • Acetylated aromaticamino acids residues include but are not limited to any one of N-acetyl-L-tyrosine, N- Acetyl -L-phenylalanine, L-Phenylalanine methyl ester hydrochloride, N-Acetyl-D-phenylalanine, N-Acetyl-L-tryptophan.
  • Exemplary analogs of tyrosine and/or phenylalanine that may be applicable in accordance with the peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure include but are not limited to any one of (2R, 3S)/(2S, 3R) - Racemic Fmoc - b - hydroxyphenylalanine, Boc - 2 - cyano - L - phenylalanine, Boc - L - thyroxine, Boc - O - methyl
  • exemplary analogs of tryptophan that may be applicable in accordance with the present disclosure include but are not limited to any one of Boc - 4 - methyl - DL - tryptophan, Boc - 4 - methyl - DL - tryptophan, Boc - 6 - fluoro - DL - tryptophan, Boc - 6 - methyl - DL - tryptophan, Boc - DL - 7 azatryptophan, Fmoc - (R) - 7 - Azatryptophan, Fmoc - 5 - benzyloxy
  • the present disclosure provides the peptide of the bifunctional hybrid-molecule, conjugate or complex of the invention that comprises at least two aromatic amino acid residues, specifically, tryptophan, tyrosine and/or phenylalanine and/or any serogates thereof, any salt, base, ester or amide thereof, any enantiomer, stereoisomer or disterioisomer thereof, or any combination or mixture thereof.
  • Pharmaceutically acceptable salts include salts of acidic or basic groups present in compounds, specifically, the at least two aromatic amino acid residues of the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure.
  • Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., 1,1'- methylene-bis-(2-hydroxy-3-naphthoate)) salts.
  • hydrochloride hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphat
  • Certain aromatic amino acid residues of the present disclosure can form pharmaceutically acceptable salts.
  • Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.
  • at least one of the at least two aromatic amino aromatic amino acid residues of the peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may be tryptophan.
  • one of the aromatic amino acid residues in the peptide of the bifunctional hybrid-molecule of the present disclosure may be tryptophan, and the other aromatic amino acid residue may be tyrosine.
  • the peptide of the bifunctional hybrid-molecule of the present disclosure may comprise at least one tryptophan and at least one phenylalanine. In yet some further embodiments, the bifunctional hybrid-molecule of the invention may comprise at least two tryptophan residues. It should be understood that the peptide of the bifunctional hybrid-molecule of the present disclosure may comprise any further aromatic amino acid residues as one or more of the Xaa residues located in various positions of the peptide as discussed above.
  • At least one of the "interspacing", “gapping”, “separating” amino acid residue/s located between the two aromatic amino acid residues may be one or more amino acid residue/s having a non-polar side chain, also referred to herein as a non-polar amino acid.
  • Such residues may be any one of Valine (V, Val), Glycine (G, Gly), Leucine (L, Leu), Isoleucine (I, IIe), Methionine (M, Met), Phenylalanine (F, Phe), Tryptophane (W, Trp) and/or Proline (P, Pro).
  • the interspacing amino acid residue may be any hydrophobic amino acid residue, for example, any one of Valine (V, Val), Leucine (L, Leu) and/or Isoleucine (I, IIe), or any derivative, analogs, or mimetics thereof, as disclosed herein above.
  • the peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least two aromatic amino acid residues that are interspaced by at least one of Valine (V), Glycine (G) and/or Alanine (A).
  • V Valine
  • G Glycine
  • A Alanine
  • the at least two aromatic amino acid residues of the peptide of the bifunctional hybrid-molecule disclosed herein are interspaced by at least one Valine residue.
  • the peptide of the bifunctional hybrid- molecule, conjugate or complex in accordance with the invention may comprise the amino acid sequence of Xaa (n) -Trp (W)-Xaa (n) -Val (V)-Xaa (n) -Trp (W)-Xaa (n), as denoted by SEQ ID NO: 7, or any mimetics thereof.
  • Xaa is any amino acid residue
  • n is zero or an integer between 1 to 7.
  • These amino acid residues may be any identical or different amino acid residues.
  • the at least one peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise three to ten amino acid residues, specifically, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. As indicated above, at least two of these residues are aromatic amino acids, specifically, W, Y and/or F, or any derivative, analogs, or mimetics thereof.
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise any amino acid residue, Xaa that is any one of Arg (R), IIe (I) and Leu (L), or any derivative, analogs, or mimetics thereof, as disclosed herein above.
  • the peptides of the bi-functional hybrid-molecule of the present disclosure or any conjugate or complex thereof may comprise at least one Arg residue, at any position of the peptide, for example, at the N-termini thereof, at the C-termini thereof, and/or the interspacing residue/s.
  • Non-limiting embodiments for such peptides are provided by the peptides that comprises the amino acid sequence as denoted by SEQ ID NOs: 27 and 59 (KVS-18); 24 and 58 (KVS-14); 25 (KVS-15) and 26 (KVS-16), or any derivatives thereof, that clearly retain the ability of requiring the NF- ⁇ B1 p105.
  • the peptide of the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise the amino acid sequence of at least one of any one of: (a) RIWVWLL, as dented by SEQ ID NO: 2, or any variants and derivatives thereof; (b), IWVWLL, as dented by SEQ ID NO: 3, or any variants and derivatives thereof; (c) WILVRLW, as dented by SEQ ID NO: 4, or any variants and derivatives thereof; (d) RIFVFLL, as dented by SEQ ID NO: 9; (e) RIYVFLL, as dented by SEQ ID NO: 10; (f) Citrulline-IWVWLL, as dented by SEQ ID NO: 15; (g) GRIWVWLL, as dented by SEQ ID NO: 16; (h) RRRIWVWLL, as dented by SEQ ID NO: 27; and/or (i) WVW, or any variants and derivatives thereof.
  • the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise any of the peptides disclosed herein, for example, any of the peptides discussed in Example 8 and Figure 9, specifically, any of the peptides of SEQ ID NOs: 8, 11 to 28, and 52 to 59.
  • the peptide of the bifunctional hybrid-molecule, conjugate or complex of the invention recognizes, binds and therefore recruits the NF- ⁇ B1 p105. It should be understood that any variant or derivative of any of the above-mentioned peptides may be comprised within the bifunctional hybrid-molecule, conjugate or complex of the invention. In some embodiments, the invention contemplates any variant or derivative of each one of the above indicated peptides, provided that the variant and/or derivative retains the ability of the peptide (specifically, any one of SEQ ID NO: 1 to 5, 7 to 39, and 42 to 59), to recruit and bind the NF- ⁇ B1 p105.
  • the bifunctional hybrid-molecule of the present disclosure comprises a polypeptide component that recruits and binds the NF- ⁇ B1 p105. It is therefore understood that in some embodiments, the polypeptide or peptide may be considered as an isolated polypeptide or peptide.
  • a "polypeptide” refers to a polymer of amino acids linked by peptide bonds.
  • a protein is a molecule comprising one or more polypeptides.
  • a peptide is a relatively short polypeptide, typically between about 3 and 100 amino acids (aa) in length, e.g., between 4 and 60 aa; between 8 and 40 aa; between 10 and 30 aa.
  • the terms “protein”, “polypeptide”, and “peptide” may be used interchangeably.
  • a polypeptide may contain only standard amino acids or may comprise one or more non-standard amino acids (which may be naturally occurring or non-naturally occurring amino acids) and/or amino acid analogs in various embodiments.
  • a “standard amino acid” is any of the 20 L- ami no acids that are commonly utilized in the synthesis of proteins by mammals and are encoded by the genetic code.
  • a “non-standard amino acid” is an amino acid that is not commonly utilized in the synthesis of proteins by mammals. Non-standard amino acids include naturally occurring amino acids (other than the 20 standard amino acids) and non-naturally occurring amino acids. In some embodiments, a non-standard, naturally occurring amino acid is found in mammals.
  • nonstandard amino acids include, e.g., singly or multiply halogenated (e.g., tluorinated) amino acids, D-amino acids, homo-ammo acids, N-alkyl amino acids (other than proline), dehydroamino acids, aromatic amino acids (other than histidine, phenylalanine, tyrosine and tryptophan), and a, a disubstituted amino acids,
  • halogenated e.g., tluorinated amino acids
  • D-amino acids homo-ammo acids
  • N-alkyl amino acids other than proline
  • dehydroamino acids aromatic amino acids (other than histidine, phenylalanine, tyrosine and tryptophan)
  • a disubstituted amino acids e.g., an amino acid, e.g., one or more of the amino acids in a polypeptide, may be modified, for example, by addition, e
  • Modifications may occur anywhere in a polypeptide, e.g., the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • a given polypeptide may contain many types of modifications.
  • Polypeptides may be branched or they may be cyclic, with or without branching.
  • Polypeptides may be conjugated with, encapsulated by, or embedded within a polymer or polymeric matrix, dendrimer, nanoparticle, microparticle, liposome, or the like. Modification may occur prior to or after an amino acid is incorporated into a polypeptide in various embodiments.
  • Polypeptides may, for example, be purified from natural sources, produced in vitro or in vivo in suitable expression systems using recombinant DNA technology (e.g., by recombinant host cells or in transgenic animals or plants), synthesized through chemical means such as conventional solid phase peptide synthesis, and/or methods involving chemical ligation of synthesized peptides.
  • recombinant DNA technology e.g., by recombinant host cells or in transgenic animals or plants
  • chemical means such as conventional solid phase peptide synthesis, and/or methods involving chemical ligation of synthesized peptides.
  • a protein may be composed of a single amino acid chain or multiple chains associated covalently or noncovalently.
  • an 'isolated polypeptide' or "isolated peptide” is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature.
  • a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
  • isolated polypeptide does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • isolated peptides are also non-naturally occurring, synthetic peptides. Methods for isolating or synthesizing peptides of interest with known amino acid sequences are well known in the art. The polypeptides of the invention are therefore considered as proteinaceous material.
  • a “proteinaceous material” is any protein, or fragment thereof, or complex containing one or more proteins formed by any means, such as covalent peptide bonds, disulfide bonds, chemical crosslinks, etc., or non-covalent associations, such as hydrogen bonding, van der Waal's contacts, electrostatic salt bridges, etc.
  • the peptides of the bifunctional hybrid-molecule, conjugate or complex of the invention are composed of an amino acid sequence.
  • An "amino acid/s” or an “amino acid residue/s” can be a natural or non-natural amino acid residue/s linked by peptide bonds or bonds different from peptide bonds.
  • the amino acid residues can be in D-configuration or L-configuration (referred to herein as D- or L- enantiomers).
  • An amino acid residue comprises an amino terminal part (N3 ⁇ 4) and a carboxy terminal part (COOH) separated by a central part (R group) comprising a carbon atom, or a chain of carbon atoms, at least one of which comprises at least one side chain or functional group.
  • NH 2 refers to the amino group present at the amino terminal end of an amino acid or peptide
  • COOH refers to the carboxy group present at the carboxy terminal end of an amino acid or peptide.
  • the generic term amino acid comprises both natural and non-natural amino acids. Natural amino acids of standard nomenclature are listed in 37 C.F.R. 1.822(b)(2). Examples of non-natural amino acids are also listed in 37 C.F.R.
  • non-natural amino acid residues include, but are not limited to, modified amino acid residues, L, -amino acid residues, and stereoisomers of D-amino acid residues.
  • Naturally occurring amino acids may be further modified, e.g., hydroxyproline, g-carboxy glutamate, and O- phosphoserine.
  • the peptides of the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise natural or non-natural amino acid residues, or any combination thereof.
  • amino acids may be amino acid analogs or amino acid mimetics.
  • Amino acid analogs refer to compounds that have the same fundamental chemical structure as naturally occurring amino acids, but modified R groups or modified peptide backbones, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • the peptides of the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise "equivalent amino acid residues'.
  • This term refers to an amino acid residue capable of replacing another amino acid residue in a polypeptide without substantially altering the structure and/or functionality of the polypeptide.
  • Equivalent amino acids thus have similar properties such as bulkiness of the side- chain, side chain polarity (polar or non-polar), hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral or basic) and side chain organization of carbon molecules (aromatic/aliphatic).
  • equivalent amino acid residues can be regarded as conservative amino acid substitutions.
  • equivalent amino acid substitutioiT is meant that in certain embodiments one amino acid may be substituted for another within the groups of amino acids indicated herein below:
  • Amino acids having polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys); (ii) Amino acids having non-polar side chains (Gly, Ala, Val, Leu, IIe, Phe, Trp, Pro, and Met); (iii) Amino acids having aliphatic side chains (Gly, Ala Val, Leu, IIe); (iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro); (v) Amino acids having aromatic side chains (Phe, Tyr, Trp); (vi) Amino acids having acidic side chains (Asp, Glu); (vii) Amino acids having basic side chains (Lys, Arg, His); (viii) Amino acids having amide side chains (Asn, Gin); (ix) Amino acids having hydroxy side chains (Ser, Thr); (x) Amino acids having sulphur-containing
  • peptides of the bifunctional hybrid-molecule, conjugate or complex of the invention may have secondary modifications, such as phosphorylation, acetylation, glycosylation, sulfhydryl bond formation, cleavage and the likes, as long as said modifications retain the functional properties of the original protein, specifically, the ability to interact with, bind and recruit NF- ⁇ B 1 p105.
  • Secondary modifications are often referred to in terms of relative position to certain amino acid residues. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • the invention further encompasses any derivatives, enantiomers, analogues, variants or homologues of any of the peptides of the bifunctional hybrid-molecule, conjugate or complex disclosed herein, specifically, any of the peptides that comprise the amin aocid sequence of any one of SEQ ID NO: 1 to 5, 7 to 39, and 42 to 59.
  • derivative is used to define amino acid sequences (polypeptide), with any insertions, deletions, substitutions and modifications to the amino acid sequences (polypeptide) that do not alter the activity of the original polypeptides (e.g., recruiting NF- ⁇ B 1 p105).
  • derivative it is also referred to homologues, variants and analogues thereof, as well as covalent modifications of a polypeptides made according to the present invention.
  • the peptides of the bifunctional hybrid-molecule, conjugate or complex according to the invention can be produced either synthetically, or by recombinant DNA technology. Methods for producing polypeptides peptides are well known in the art.
  • derivatives include, but are not limited to, polypeptides that differ in one or more amino acids in their overall sequence from the polypeptides defined herein, polypeptides that have deletions, substitutions, inversions or additions.
  • derivatives refer to polypeptides, which differ from the polypeptides specifically defined in the present invention by insertions of amino acid residues.
  • insertions or “deletions”, as used herein it is meant any addition or deletion, respectively, of amino acid residues to the polypeptides used by the invention, of between 1 to 50 amino acid residues, between 20 to 1 amino acid residues, and specifically, between 1 to 10 amino acid residues. More particularly, insertions or deletions may be of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. It should be noted that the insertions or deletions encompassed by the invention may occur in any position of the modified peptide, as well as in any of the N' or C termini thereof.
  • peptides of the bifunctional hybrid-molecule, conjugate or complex of the invention of the invention may all be positively charged, negatively charged or neutral.
  • they may be in the form of a dimer, a multimer or in a constrained conformation, which can be attained by internal bridges, short-range cyclization, extension or other chemical modifications.
  • a non-limiting example for a cyclized peptide may be the KVS-7 peptide, that comprises the amino acid sequence of SEQ ID NO: 17, wherein as shown by Fig.
  • cyclization is performed by linking the amine of the N-terminal Arg residue with the Cys residue of the linker.
  • the polypeptides of the bifunctional hybrid-molecule, conjugate or complex of the invention can be coupled (conjugated) through any of their residues to another peptide or agent.
  • the peptides of the bifunctional hybrid-molecule, conjugate or complex of the invention can be coupled through their N-terminus to a lauryl-cysteine (FC) residue and/or through their C-terminus to a cysteine (C) residue.
  • FC lauryl-cysteine
  • C cysteine
  • the peptides of the bifunctional hybrid-molecule, conjugate or complex of the invention may be extended at the N- terminus and/or C-terminus thereof with various identical or different amino acid residues.
  • the peptide may be extended at the N-terminus and/or C-terminus thereof with identical or different amino acid residue/s, which may be naturally occurring or synthetic amino acid residue/s.
  • An additional example for such an extension may be provided by peptides extended both at the N-terminus and/or C-terminus thereof with a cysteine residue.
  • cysteine residue a residue that is a cysteine residue.
  • Another example may be the incorporation of an N-terminal lysyl-palmitoyl tail, the lysine serving as linker and the palmitic acid as a hydrophobic anchor.
  • the peptides may be extended by aromaticamino acid residue/s, which may be naturally occurring or syntheticamino acid residue/s.
  • the peptides may be extended at the N- terminus and/or C-terminus thereof with various identical or different organic moieties, which are not naturally occurring or synthetic amino acids.
  • the peptides of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may be extended at the N-terminus and/or C- terminus thereof with an N-acetyl group.
  • Figure 4 shows the effectivity of a bifunctional hybrid-molecule, conjugate or complex of the present disclosure that comprises an N-acetylated peptide (Formula XIII, also indicated herein as PROTAC 4).
  • this invention includes the corresponding retro-inverse sequence wherein the direction of the peptide chain has been inverted and wherein all or part of the amino acids belong to the D-series. It should be understood that the present invention includes embodiments wherein one or more of the L- amino acids is replaced with its D isomer, thus providing peptides comprising 1 , -amino acids, D-amino acids, and any combination of 1 , -amino acids and 1 , -amino acids.
  • a non-limiting example for a peptide that comprises at least one residue in the D-form may comprise SEQ IS NO: 14, 49.
  • the peptides of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least one amino acid residue in the D-form. It should be noted that every amino acid (except glycine) can occur in two isomeric forms, because of the possibility of forming two different enantiomers (stereoisomers) around the central carbon atom. By convention, these are called L- and D- forms, analogous to left-handed and right-handed configurations. It should be appreciated that in some embodiments, the enantiomer or any derivatives of the peptides of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may exhibit at least one of enhanced activity, and superiority.
  • such derivatives and enantiomers may exhibit increased affinity to the NF- ⁇ B1 p105, increased specificity, enhanced stability, and increased resistance to proteolytic degradation.
  • increased stability, specificity, affinity and the like as disclosed herein with respect to any of the peptides of the bi-functional hybrid-molecule of the present disclosure or any conjugate or complex thereof relate to the increase, elevation, enhancement, escalation, of at least one of the stability, specificity, affinity, binding and recruitment of the NF- ⁇ B p105 by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 9
  • the invention also encompasses any homologues of the peptides of the bifunctional hybrid- molecule, conjugate or complex of the present disclosure, particularly of those specifically defined by their amino acid sequence according to the invention.
  • homologues is used to define amino acid sequences (polypeptide) which maintain a minimal homology to the amino acid sequences defined by the invention, e.g. specifically have at least about 65%, more specifically, at least about 70%, at least about 75%, even more preferably at least about 80%, at least about 85%, most preferably at least about 90%, at least about 95% overall sequence homology with the entire amino acid sequence of any of the polypeptide as structurally defined above, e.g.
  • a native polypeptide and its functional derivative is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- nor C- terminal extensions nor insertions or deletions shall be construed as reducing identity or homology.
  • the present invention also encompasses peptides of the bifunctional hybrid- molecule, conjugate or complex of the present disclosure which are variants of, or analogues to, the polypeptides specifically defined in the invention by their amino acid sequence.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to peptide, polypeptide, or protein sequence thereby altering, adding or deleting a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant”, where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art and disclosed herein before.
  • amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amin aocid replacements.
  • Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • the peptides of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may be modified by omitting their N-terminal sequence.
  • the invention further encompasses the omission of about 1, 2, 3, 4, 5, 6, 7, 8 and more amino acid residues from both, the N' and/or the C termini of the peptides of the invention.
  • Certain commonly encountered amino acids which also provide useful substitutions include, but are not limited to, ⁇ -alanine ( ⁇ -Ala) and other omega-amino acids such as 3- aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4- aminobutyric acid and so forth; ⁇ - aminoisobutyric acid (Aib); e-aminohexanoic acid (Aha); d- aminovaleric acid (Ava); N- methylglycine or sarcosine (MeGIy); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine
  • a non- limiting embodiments for a peptide of the bi-functional hybrid-molecule of the present disclosure or any conjugate or complex thereof that comprises citrulline (Cit), is provided by the peptides of SEQ ID NO: 15, and SEQ ID NO: 50 (with linker).
  • Covalent modifications of the peptide are included and may be introduced by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Cysteinyl residues most commonly are reacted with ⁇ -haloacetates (and corresponding amines) to give carboxymethyl or carboxyamidomethyl derivatives.
  • Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, ⁇ -bromo- ⁇ -(5- imidozoyl)propionic acid, chloroacetyl phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4- nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3- diazole. Histidyl residues are derivatized by reaction with diethylprocarbonate. Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides.
  • Suitable reagents for derivatizing a- amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, including phenylglyoxal, 2,3- butanedione, 1,2-cyclohexanedione, and ninhydrin.
  • Such derivatization requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine e-amino group. Modification of tyrosyl residues has permits introduction of spectral labels into a peptide. This is accomplished by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to create O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Carboxyl side groups are selectively modified by reaction with carbodiimides (R'-N-C-N-R') such as l-cyclohexyl-3-(2-morpholinyl- (4-ethyl) carbodiimide or 1- ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.
  • Aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues.
  • Deamidation can be performed under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • Derivatization with bifunctional agents is useful for cross-linking the peptide to a water-insoluble support matrix or other macromolecular carrier.
  • cross- linking agents include 1,1- bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, esters with 4- azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8- octane.
  • Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • the present disclosure encompasses any derivative or variant of the peptide of the present disclosure as disclosed throughout the specification, or any orientation, isomer or form thereof, provided that the peptide/s retain/s the biologic activity indicated herein. More specifically, in some embodiments the biological activity of the peptide is requiting, targeting, and/or binding the NF ⁇ B p105 such that it will be in close proximity to the proteasome degradation mediating moiety (e.g., the ligand disclosed herein), in a manner that enables the proteasomal degradation of NF ⁇ B p105 to form the NF ⁇ B p50 product.
  • the proteasome degradation mediating moiety e.g., the ligand disclosed herein
  • any derivative or variant or form of the disclosed peptides, and or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein are those that at least retain the ability to requite, target, and/or bind the NF ⁇ B p105, and/or mediate the degradation of NF ⁇ B p105 to form the NF ⁇ B p50 product.
  • the derivative or variant or form of the disclosed peptides, and or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein are able to mediate, increase, and enhance the production of the NF ⁇ B p50 product in a cell, in a subject and/or in a cell free system, in about any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more, as compared to a cell, in a subject and/or a cell free system in the absence of the peptides
  • the activity of the peptides of the invention, the derivative or variant or form of the disclosed peptides, and/or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein is meant herein, the induction of the production of the NF ⁇ B p50 product that leads and mediates either directly or indirectly a tumor suppressive effect.
  • any derivative or variant or form of the disclosed peptides or any conjugates thereof act to increase the tumor suppressive effect by increasing p50 levels.
  • the peptides of the invention and constructs thereof change the expression pattern of various groups of proteins.
  • the peptides of the invention, the derivative or variant or form of the disclosed peptides, and/or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein lead to an increase, elevation, upregulation, enhancement and or enlargement of the expression of proteins connected to immune system processes and/or proteins connected with regulation of cell migration, and/or proteins that relate to ECM organization, and/or proteins that relate to cell adhesion, and/or proteins that relate to regulation of cell adhesion.
  • the peptides of the invention, the derivative or variant or form of the disclosed peptides, and/or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein lead to a decrease, reduction, attenuation, inhibition and downregulation of the expression of proteins connected to metabolic processes, and/or proteins that relate to cell cycle, and/or proteins that relate to DNA metabolic processes.
  • the invention further encompasses any of the peptides of the invention referred herein, any serogates thereof, any salt, base, ester or amide thereof, any enantiomer, stereoisomer or disterioisomer thereof, or any combination or mixture thereof.
  • Pharmaceutically acceptable salts include salts of acidic or basic groups present in compounds of the invention.
  • Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., l,T-methylene-bis-(2-hydroxy-3- naphthoate)) salts.
  • Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.
  • the peptides of the bi-functional hybrid-molecule of the present disclosure or any conjugate or complex thereof may be any of the peptides encompassed by SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 29, with the proviso that the peptide is not the peptide of SEQ ID NO: 4.
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure comprises two components. One component comprises at least one peptide as discussed above that recruits NF- ⁇ B1 p105.
  • the second component is at least one proteasome degradation mediating moiety.
  • proteasomes are protein complexes which degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds, mediated by proteases. Proteasomes are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins. Proteins are tagged for degradation with a small protein called ubiquitin. The tagging reaction is catalyzed by enzymes called ubiquitin ligases. The degradation process yields peptides of about seven to eight amino acids long, which can then be further degraded into shorter amino acid sequences and used in synthesizing new proteins.
  • proteasomes are found inside all eukaryotes and archaea, and in some bacteria.
  • the proteasome is a cylindrical complex containing a "core" of four stacked rings forming a central pore. Each ring is composed of seven individual proteins.
  • the inner two rings are made of seven b subunits that contain three to seven protease active sites. These sites are located on the interior surface of the rings, so that the target protein must enter the central pore before it is degraded.
  • the outer two rings each contain seven a subunits whose function is to maintain a "gate” through which proteins enter the barrel. These a subunits are controlled by binding to "cap” structures or regulatory particles that recognize polyubiquitin tags attached to protein substrates and initiate the degradation process.
  • the overall system of ubiquitination and proteasomal degradation is known as the ubiquitin-proteasome system (UPS).
  • the proteasome subcomponents are often referred to by their Svedberg sedimentation coefficient (denoted S).
  • the proteasome most exclusively used in mammals is the cytosolic 26S proteasome, which is about 2000 kilodaltons (kDa) containing one 20S protein subunit (also referred to herein as the core proteasome, or CP) and two 19S regulatory cap subunits (also referred to herein as the regulatory proteasome or RP).
  • the core is hollow and provides an enclosed cavity in which proteins are degraded. Openings at the two ends of the core allow the target protein to enter.
  • Each end of the core particle associates with a 19S regulatory subunit that contains multiple ATPase active sites and ubiquitin binding sites. This structure recognizes polyubiquitinated proteins and transfers them to the catalytic core.
  • An alternative form of regulatory subunit called the 11 S particle may play a role in degradation of foreign peptides and can associate with the core in essentially the same manner as the 19S particle.
  • the proteasomal degradation pathway is essential for many cellular processes, including the cell cycle, the regulation of gene expression, and responses to oxidative stress.
  • the second component of the bifunctional hybrid-molecule, conjugate or complex of the preset disclosure is at least one proteasome degradation mediating moiety.
  • proteasome degradation mediating moiety refers to any element, component or moiety that may mediate either directly or indirectly proteasome degradation and processing of a target molecule.
  • the degradation mediating moiety as disclosed herein may be any natural, synthetic, organic or inorganic molecule or moiety, that is involved in recruiting, translocating or modifying any target molecule in a manner that increases proteasomal degradation thereof.
  • the degradation mediating moiety of the bifunctional hybrid-molecule of the present disclosure mediates the proteasomal degradation and processing of the NF- ⁇ B1 p105, recruited by the peptide component of the bifunctional hybrid- molecule, conjugate or complex of the present disclosure.
  • the degradation mediating moiety may be any compound that participates and/or mediates degradation of a target protein by the proteasome.
  • the degradation mediating moiety may be a hydrophobic tag mimicking protein misfolding.
  • the hydrophobic tagging (HyT) technology extends the concept of inducing protein instability to a broader range of protein targets by mimicking protein misfolding
  • the HyT consists of a hydrophobic fragment and a ligand fragment of the protein of interest (POI), e.g., any of the peptides disclosed herein, which is capable of causing degradation of the POI, specifically, NF- ⁇ B 1 p105.
  • POI protein of interest
  • HyT destabilizes the POI, thereby recruiting an endogenous chaperone protein to the misfolded protein and then degrading the protein by the proteasome.
  • Another mechanism is the direct recognition of the HyT by chaperones, mediating the proteasomal degradation of the tagged protein. The hydrophobic marker then is released and the POI can be degraded and procesed in successive rounds. More specifically, protein ubiquitination and degradation can be achieved by recruiting chaperones using lipophilic small molecule tags. For example, HSP70 family members recognize the exposed hydrophobic cores of misfolded proteins to hijack misfolded protein reactions.
  • HSP70 is highly conserved and ubiquitous in microorganisms, plants and animals, and is involved in many cellular processes, including protein folding, transmembrane protein translocation and protein degradation regulation. Proteins with mild or partial misfolding are ubiquitinated by HSP40 and HSP70 and then degraded by HSP70 and 26S proteasomes.
  • the at least one proteasome degradation mediating moiety of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may be a hydrophobic tag (HyT), that recruits chaperones and mediates misfolded protein reactions.
  • HyT hydrophobic tag
  • the at least one proteasome degradation mediating moiety of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may be a HaloTag.
  • a HaloTag is a modified bacterial dehalogenase enzyme that covalently binds with a hexyl chloride label. HaloTag forms stable covalent bonds with compounds containing alkyl chlorides via a very simple binding moiety with low molecular weight and reasonable cell permeability.
  • the HaloTag-based bifunctional molecule in accordance with some embodiments of the present disclosure, contain an alkyl chain HaloTag and a peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, that binds the target protein (NF- ⁇ B 1 p105), in accordance with the preset invention.
  • This bifunctional molecule transfers the fusion domain onto the POI, binds the bacterial HaloTag protein and generates a hydrophobic group on its surface, which is mediated by a chaperone, e.g., HSP70.
  • the proteasome degradation mediating moiety of the present disclosure may comprise at least one E3 ubiquitin ligase recruiting moiety, or at least one E3 ubiquitin ligase active moiety.
  • E3 ubiquitin ligase active moiety may be any domain or fragment of an E3 ligase, that is capable of forming of an isopeptide bond between the carboxy terminus of ubiquitin and a lysine residue of a target protein.
  • the E3 ubiquitin ligase active moiety may comprise at least one of RING finger and U-box E3s, the HECT E3s, and the RING/HECT-hybrid type E3s of an E3 ligase.
  • Non-limiting embodiment for such bifunctional hybrid molecules that comprises target (p105) recruitment component (SEQ ID NO: 2) is disclosed herein by SEQ ID NO: 65.
  • the proteasome degradation mediating moiety of the bifunctional hybrid-molecule, conjugate or complex of the invention may be any moiety that recruits at least one E3 ligase.
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure acts in some embodiments as an adaptor compound that recruits the NF- ⁇ B 1 p105 via the peptide component, and recruits E3 ligase using the proteasome degradation mediating component.
  • the invention further encompasses bifunctional hybrid-molecule, conjugate or complexes, that induce targeted proteasome degradation of the NF- ⁇ B 1 p105 and processing thereof to form the p50. As indicated above, degradation of the NF- ⁇ B 1 p105 is performed by the UPS.
  • Ubiquitin Proteasome Pathway System relates to the ubiquitin proteasome pathway, conserved from yeast to mammals, and is required for the targeted degradation of most short-lived proteins in the eukaryotic cell. Targets include cell cycle regulatory proteins, whose timely destruction is vital for controlled cell division, as well as proteins unable to fold properly within the endoplasmic reticulum. Ubiquitin modification is an ATP - dependent process carried out by three classes of enzymes. An “ubiquitin activating enzyme” (El) forms athio-ester bond with ubiquitin.
  • El ubiquitin activating enzyme
  • E3 ubiquitin conjugating enzyme
  • E3 ligases can be single- or multi-subunit enzymes. More specifically, an active site Cys on the E2 subsequently facilitates the transfer of the covalently linked ubiquitin from the E 1 to a Cys residue on the E2 through a trans-thio-esterification reaction.
  • an E3 ligase recruits a specific downstream target protein and mediates the transfer of the ubiquitin from the E2 enzyme to the terminal substrate through either a covalent or non - covalent mechanism.
  • Each ubiquitin is ligated to a protein through either a peptide bond with the N-terminal amino group or an isopeptide bond formed between a side chain e -amino group of a select Lys residue on the target protein and the ubiquitin.
  • Ubiquitin a small protein with 76 amino acids, covalently conjugates to lysine (K) residues of substrate proteins. Ubiquitin conjugation is mediated by a three-step enzymatic process.
  • the carboxyl group at the end of U glycine is linked to the thiol group of the U-activating enzyme El to form a thioester bond between U and El.
  • El transfers the activated U to E2 through a lactide process.
  • E3 binds E2 U to the target protein and releases E2 to leave a specific ubiquitinated protein.
  • ubiquitinated proteins are recognized by specific proteasomes and degraded into short peptides or amino acids by proteases.
  • proteasome degradation refers to the degradation of the ubiquitinated proteins, specifically the NF- ⁇ B1 p105, by the proteasome, as discussed herein above, into shorter peptides, for example, the p50 peptide, and optionally to other shorter peptides and amino acid residues.
  • proteasome degradation of the NF- ⁇ B1 p105 mediated by the bifunctional hybrid molecules of the present disclosure leads to an increase in the production, and/or the levels of the NF- ⁇ B 1 p50 peptide, an increase of about 5% to 100%, specifically, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, as compared to the levels of NF- ⁇ B 1 p50 peptide in the absence of the bifunctional hybrid molecules of the present disclosure.
  • E3s Fluman genome encodes over 600 E3s, which serve as catalytic intermediates in ubiquitination (HECT domain E3s) or directly catalyze the transfer of ubiquitin from E2 to substrates (RING or RING-like domain E3s).
  • HECT domain E3s ubiquitination
  • RING or RING-like domain E3s There are eight different linkage types of ubiquitination, including K6, K11, K27, K29, K33, K48, K63, and Ml, in which the C terminus of ubiquitin is attached to lysine residues or the N-terminal methionine (Ml) of another ubiquitin.
  • Ml N-terminal methionine
  • E3 ligases provide specificity in that each can modify only a subset of substrate proteins. Further specificity is achieved by post-translational modification of substrate proteins, including, but not limited to, phosphorylation. However, multiple ubiquitination cycles resulting in a polyubiquitin chain are required for targeting a protein to the proteasome for degradation.
  • the multisubunit 26S proteasome recognizes, unfolds, and degrades polyubiquitinated substrates into small peptides. The reaction occurs within the cylindrical core of the proteasome complex, and peptide bond hydrolysis employs a core threonine residue as the catalytic nucleophile.
  • lipids may lie between the polyubiquitination and degradation steps. These receptors react with a subset of polyubiquitinated substrates, aiding in their recognition by the 26S proteasome, and thereby promoting their degradation.
  • “Ligase” as used herein, is an enzyme that can catalyze the joining of two or more compounds or biomolecules by bonding them together with a new chemical bond.
  • Ubiquitin-protein (E3) ligases are a large family of highly diverse enzymes selecting proteins for ubiquitination. “Ub Ligases” are involved in disease pathogenesis for oncology, inflammation and infectious disease. The E3 ligases originate in three major classes - the RING finger and U-box E3s, the HECT E3s, and the RING/HECT-hybrid type E3s.
  • the E3 ubiquitin ligase recruiting moiety of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may be suitable for recruiting any E3 ubiquitin ligase.
  • the E3 ubiquitin ligase recruiting moiety of the bifunctional hybrid-molecule is suitable for at least one of von- Hippel-Lindau (VHL), Cereblon (CRBN), Mouse double minute 2 homolog (MDM2), cellular inhibitor of apoptosis protein- 1 (cIAPl) and X-linked inhibitor of apoptosis protein (XIAP) and DDB1 and CUL4 associated factor 15 (DCAF15).
  • any moiety capable of recruiting E3 ubiquitin ligase may be used in the bifunctional hybrid- molecule, conjugate or complex of the invention.
  • the moiety is a ligand for an E3 ubiquitin ligase.
  • any ligand may be used in the bifunctional hybrid-molecule, conjugate or complexes of the present disclosure, for recruiting any E3 ligase.
  • MDM2 E3 can be recruited by using a known MDM2-p53 PPI inhibitor, nutlin, as the E3 ligand [Nutlins, a group of cis-diphcnyl substituted imidazoline- containing compounds (nutlin- 1, -2, and-3)], as well as Nutlin carboxylic acid (MDM2 ligand 1; E3 ligase Ligand 16),(4R,5S)-Nutlin carboxylic acid (MDM2 ligand 2; E3 ligase Ligand 15).
  • MDM2 ligand 1 E3 ligase Ligand 16
  • E3 ligase Ligand 16 Nutlin carboxylic acid
  • ligands such as bestatin methyl esters can be used. More specifically, any one of cIAPl ligand 1 (E3 ligase Ligand 12), Bestatin- amido-Me (PROTAC IAP binding moiety 1) and ⁇ -Naphthoflavone-CH2-OH ( ⁇ -NH-CH2-OH), may be applicable. Still further, for recruiting Cereblon (CRBN), immunomodulatory drugs (IMiDs), may be useful.
  • E3 ligase Ligand 12 E3 ligase Ligand 12
  • Bestatin- amido-Me PROTAC IAP binding moiety 1
  • ⁇ -Naphthoflavone-CH2-OH ⁇ -NH-CH2-OH
  • IiDs immunomodulatory drugs
  • suitable CRBN ligands applicable in the bifunctional hybrid-molecule, conjugate or complexes of the present disclosure may include, but are not limited to Lenalidomide (CC-5013), Pomalidomide (CC-4047), ThaIidomide-NH-CH2- COOH, Thalidomide-O-COOH (Cereblon ligand 3; E3 ligase Ligand 3), Cereblon modulator 1 , Lenalidomide hemihy dr ate (CC-5013 hemihydrate), Thalidomide-5-OH, Thalidomide 4- fluoride (Cereblon ligand 4; E3 ligase Ligand 4), ThaIidomide-4-OH (Cereblon ligand 2; E3 ligase Ligand 2), ThaIidomide-O-C8-COOH, Thalidomide-propargyl, ThaIidomide-O-C8-Boc, CRBN modulator- 1 and Thalidomide 5 -
  • the bifunctional hybrid- molecule, conjugate or complex of the present disclosure comprises at least one recruiting moiety for VHL E3 ubiquitin ligase.
  • the recruiting moiety comprises at least one small molecule or peptide, or any combinations thereof.
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise VHL ligands that comprise small molecules.
  • Suitable ligands for VHL may include, but are not limited to any one of (S,R,S)-AHPC hydrochloride (VH032-NH2 hydrochloride; VHL ligand 1 hydrochloride), (S,R,S)-AHPC-Me hydrochloride (VHL ligand 2 hydrochloride; E3 ligase Ligand 1), (S,R,S)-AHPC-Me dihydrochloride (VHL ligand 2 dihydrochloride; E3 ligase Ligand 1 dihydrochloride), VL285, (S,S,S)-AHPC hydrochloride ((S,S,S)-VH032-NH2 hydrochloride), VH032-cyclopropane-F (VHL ligand 3; E3 ligase Ligand 19), VHL Ligand 8, VH032 thiol (VHL ligand 6), (S,R,S)-AHPC (VH032-NH2; VHL ligand 1)
  • the at least one recruiting moiety for VHL E3 ubiquitin ligase, of the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise the VHL ligand of Formula I, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof: Formula I
  • the bifunctional hybrid-molecule, conjugate or complex of the invention comprises at least two components, one component, is at least one peptide, that in some embodiments recruits NF- ⁇ B1 p105.
  • the second component is an E3 ligase recruiting component (also referred to herein as a ligand).
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may further comprise at least one linker.
  • At least one of the linkers of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure is a bridging linker that bridges between said at least one peptide component or moiety and the at least one proteasome degradation mediating component or moiety.
  • the linker may take any form, and any length appropriate to bring together the target protein NF- ⁇ B1 p105 and ubiquitinating machinery and thereby elicit the ubiquitination of NF- ⁇ BI p105 and its subsequent degradation in the proteasome.
  • the linker may take any form, provided that it does not significantly interfere with binding of the ligand to the target, specifically, the NF- ⁇ B1 p105.
  • the linker is constituted by those parts of the ligand which are exposed to solvent when the ligand is bound to the target.
  • the linker may be a series of stable covalent bonds incorporating one or more (e.g., 1-500) non-hydrogen atoms selected from the group consisting of C, N, O, S and P.
  • Exemplary linkers therefore include moieties comprising — C(0)NH-- , -- C(0)0— , -- NH— , S-- and --O-- groups.
  • linkers may also be comprised of the atoms or groups including (but not limited to), carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl and imine.
  • the linker chain may also comprise part of a saturated, unsaturated or aromatic ring, including polycyclic and heteroaromatic rings wherein the heteroaromatic ring is an aryl group containing from one to four heteroatoms, N, O or S.
  • Specific examples include, but are not limited to, saturated alkanes, unsaturated alkanes, polyethylene glycols and dextran polymers.
  • the linker or spacer is a substituted or unsubstituted polyglycol, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkyl ene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene. Still further, the linker is of a length appropriate to bring together target protein and ubiquitinating machinery and thereby elicit the ubiquitination of the protein of interest and its subsequent degradation in the proteasome.
  • the linker of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the invention serves as a spacer, physically separating the target and ligase ligands to a degree sufficient to ensure that binding with their respective targets is not rendered mutually exclusive as a result of steric inhibition.
  • the interaction between the target specifically, the NF- ⁇ B1 p105 recruiting peptide and ligase ligands of the bifunctional hybrid-molecule, conjugate or complex/es disclosed herein, and their respective NF- ⁇ B1 p105 target and E3 ligase, could be disrupted.
  • the linker should also not be too long, since in such cases the bound E3 ligase might not be in sufficiently close spatial proximity to the NF- ⁇ B1 p105 target protein to trigger its ubiquitination and processing to produce the p50.
  • the length of the linker is optimized by reference inter alia to target and E3 ligase binding efficiency as well as target protein ubiquitination.
  • the linker may be 1-21 bonds in length, for example 1-20, 1-19, 1-18, 1- 17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 bonds in length.
  • the linker may be 2-21 bonds in length, for example 3-21, 4-21, 5-21, 6-21, 7-21, 8-21, 9-21, 10-21, 11-21, 12-21, 13-21, 14-21, 15-21, 16-21, 17-21, 18-21, 18-21, 19-21 or
  • linker is also referred to herein as a "linker unit”).
  • the linker may be a bond.
  • the linker may be an amide bond.
  • the linker may be an amide bond between the peptide and the ligand.
  • the peptide forms an amide bond with the ligand.
  • the ligand forms an amide bond with the peptide.
  • the ligand forms an amide bond with the linker.
  • the linker forms an amide bond with the peptide.
  • the NH of the amide bond is originated from the linker unit.
  • the linker may comprise one or more ethylene glycol units.
  • the linker comprises one ethylene glycol unit.
  • the linker is an ethylene glycol derivative that forms an amide bond with the ligand and/or the peptide, and/or the optional peptide linker.
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise any of the linkers of Formulas II or XVI to XXVII, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof.
  • linkers may be used by the bifunctional hybrid- molecule of the present disclosure:
  • m is an integer between 1 to 10.
  • m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise the linker of Formula II, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof, Formula II wherein m is an integer between 1 to 10.
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprises the linker of Formula III, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof: Formula III
  • the linker of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may further comprise at least one ami ancoid residue. It should be noted that such amin-acid linker may be of any length and may comprise any amino acid residues, either identical or different.
  • the linker of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may further comprise an additional amino acid linker.
  • such linker in the context of the invention concerns an amino acid sequence of from about 1 to about 10 or more amino acid residues flanking the peptide component of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure.
  • the linker may be positioned in some embodiments, in at least one of its termini, namely at the C-terminus and/or at the N-terminus of the polypeptide of the bifunctional hybrid-molecule of the invention.
  • the linker is covalently linked or joined to the amino acid residues in its vicinity.
  • an additional amino acid linker in accordance with the invention may be of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acid residues long.
  • the linker/s used by the invention may be a combinatorial linker comprising all possible linkers composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues.
  • the peptide linker may be composed of various amino acid residues that may be either different or identical amino acid residues.
  • the amino-acid linker further comprised in the linker of the bifunctional hybrid-molecule, conjugate or complex of the invention may comprises at least one of: at least one Serine (S), at least one cysteine (C) and at least one Glycine (G) amino acid residues, attached therewith.
  • the peptides of the present disclosure comprise peptide linkers, such peptide include for example any of the peptides of SEQ ID NOs: 8, 28, 39, 42 to 59.
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure that comprises an NF- ⁇ B1 p105 recruiting peptide, an E3 ligase recruiting ligand and at least one linker may be the conjugate or complex of any of the compounds of Formula IV, V, VI and VII, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof.
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise the compound of Formula IV(also indicated herein as PROTAC 3, also shown in Fig. 4Avi), being: Formula IV.
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise the compound of Formula V (also indicated herein as PROTAC 1, also shown in Fig. 4Ai), being: Formula V.
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise the compound of Formula VI (also indicated herein as PROTAC 5, also shown in Fig. 4Aiv), being: Formula VI.
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise the compound of Formula VII , being:
  • the present disclosure relates to bifunctional hybrid-molecule, conjugate or complex, that induce targeted proteasomal degradation and processing of NF- ⁇ B 1 p105.
  • Bifunctional hybrid-molecules that target UPS mediated degradation of a target protein by bridging between the target and the E3 ligase, are also known as Proteolysis Targeting Chimeric ligands (PROTAC compounds) that induce ubiquitination by the use of a ligase, such as E3 ligase and degrade a protein of interest.
  • PROTAC compounds Proteolysis Targeting Chimeric ligands
  • the present disclosure provides PROTAC compounds, and more specifically, the bifunctional hybrid-molecule, conjugate or complex of the present invention are in some embodiments, PROTAC molecules.
  • PROTACs as used herein are typically designed with three parts: (1) a ligand/molecule that binds to and/or modulates ubiquitin ligases; (2) a binding moiety that targets and recruits the protein of interest for proteolysis, e.g., the peptide of the invention that recruits NF- ⁇ B1 p105; and (3) a linker that links the two molecules together.
  • PROTACs thus function by allowing the ligand/molecule to bind to the ubiquitin ligases, thereby recruiting the target of protein of interest to the ligase for ubiquitination and ultimately proteolysis and degradation.
  • PROTACs hijack the catalytic activity of ubiquitin E3 ligases to mediate proteasome dependent degradation of selected protein of interest (POI), by bringing the ligase and POI into close spatial proximity and initiating the poly-ubiquitination process.
  • POI protein of interest
  • the bifunctional hybrid-molecule, conjugate or complex disclosed by the present disclosure are according to some embodiments PROTAC molecules.
  • the present disclosure further encompasses also similar or corresponding CLIPTAC molecules.
  • CLIPTAC defines a proteolysis targeting chimeric molecule (PROTAC) formed from the intracellular self-assembly of precursors via bioorthogonal click chemistry (CLlckable Proteolysis TArgeting Chimera chimeric molecule), that refers to any chemical reaction that can occur inside of living systems.
  • the present disclosure encompasses any PROTAC, CLIPTAC, or any bifunctional hybrid- molecule, conjugate or complexes that comprise any of the E3 ligands, any of the NF- ⁇ B1 p105 recruiting peptides and any of the linkers disclosed herein and any combinations thereof. Moreover, the molecules disclosed by the present disclosure are effective in targeted degradation and processing of the NF- ⁇ B1 p105 to produce NF- ⁇ B1 p50.
  • the bifunctional hybrid-molecules, conjugates or complexes, PROTACs of the present disclosure may comprise any combination of the discussed components (E3 ligase, recruiting peptide and linker), with the proviso that such molecule is not the PROTAC molecule of Formula VIII, being:
  • the bifunctional hybrid-molecule, conjugate or complex of the invention e.g., PROTACs
  • the at least one peptide component thereof, that comprises the at least two aromatic amino acid residues or any mimetics thereof may be in certain embodiments, associated with, combined with or conjugated with at least one "enhancing" moiety.
  • Such moiety may be any moiety that facilitating cell penetration, targeting to specific cell target and/or by increasing stability and reducing clearance thereof.
  • association with means that such moiety may be linked non- covalently, or covalently bound to, conjugated to, cross-linked to, incorporated within (e.g., such as an amino acid sequence within the NF- ⁇ B1 p105 recruiting peptide that comprise at least one of the aromatic amino acid residues or any mimetics thereof), or present in the same composition as the bifunctional hybrid-molecule, conjugate or complex of the present invention or the NF- ⁇ B 1 p105 recruiting peptide thereof, in such a way as to allow such moiety to carry out its function.
  • cell penetration moiety means a moiety that enhances the ability of the bifunctional hybrid-molecule, conjugate or complex of the present invention or the NF- ⁇ B 1 p105 recruiting peptide thereof, with which it is associated to penetrate the cell membrane.
  • the "cell penetration moiety” may be an amino acid sequence within or connected to the bifunctional hybrid-molecule, conjugate or complex of the present invention or the NF- ⁇ B 1 p105 recruiting peptide thereof.
  • Examples of cell penetration sequences include, but are not limited to, Arg-Gly-Asp (RGD), Tat peptide, oligoarginine, MPG peptides, Pep- land the like.
  • specific organ directing moiety means a moiety that enhances the ability of the bifunctional hybrid-molecule, conjugate or complex of the invention or its peptide component that comprises at least two aromatic amino acid residue/s or any mimetics thereof, non- standard peptide, polypeptide, non-standard polypeptide, protein or non-standard protein thereof, with which it is associated to be targeted to a specific organ.
  • the "specific organ directing moiety” is an amino acid sequence, small molecule or antibody that binds to a cell type present in the specific organ.
  • the "specific organ directing moiety” is anamino acid sequence, small molecule or antibody that binds to a receptor or other protein characteristically present in the specific organ.
  • specific cell-type directing moiety means a moiety that enhances the ability of the bifunctional hybrid-molecule, conjugate or complex of the invention, with which it is associated to be targeted to a specific cell type.
  • the "specific cell-type directing moiety” is an ami ancoid sequence, small molecule or antibody that binds to a specific receptor or other protein characteristically present in or on the surface of the specific target cell type.
  • the bifunctional hybrid-molecule, conjugate or complex of the invention e.g., PROTACs
  • the at least one peptide component thereof, that comprises the at least two aromatic amino acid residues or any mimetics thereof may be in certain embodiments, associated with, combined with or conjugated with at least one "labeling" moiety.
  • labeling moiety or label may include any tag (e.g., His Tag), or any dye, for example, FITS molecule as shown in Figures 5, 6 (synthesis and characterization) and 7 and 8 (function).
  • the present disclosure provides bifunctional hybrid molecules and compositions thereof, that on one hand recruit the target NF- ⁇ B1 p105, and in the other hand recruit an E3 ligase.
  • the invention further encompasses any multiple forms of the bifunctional hybrid molecules.
  • the present disclosure further provides plurality of NF- ⁇ B1 p105 recruiting peptide/s as disclosed herein (that may potentially target the same or different locations of the target NF- ⁇ B1 p105 protein), and a plurality of E3 ubiquitin ligase recruiting moieties that may recruit the same or various E3 ubiquitin ligases (e.g., VHL, CRBN, MDM2, clAPl, XIAP and DCAF15).
  • E3 ubiquitin ligases e.g., VHL, CRBN, MDM2, clAPl, XIAP and DCAF15.
  • the NF- ⁇ B1 p105 recruiting peptide/s, E3 ligase recruiting moieties (E3 ligand), and optionally, other moieties that bind specifically to another E3 ubiquitin ligase can be coupled directly or via one or more chemical linkers or a combination thereof.
  • the moieties can be for the same E3 ubiquitin ligase or each respective moiety can bind specifically to a different E3 ubiquitin ligase.
  • a compound has multiple NF- ⁇ B1 p105 recruiting moieties (e.g., any of the various peptides disclosed herein), such moieties may be the same or, optionally, different.
  • the plurality of E3 ligase recruiting moieties are each connected to a NF- ⁇ B1 p105 recruiting peptide/a via a chemical linker.
  • the bifunctional hybrid-molecule, conjugate or complex may comprise a plurality of E3 ligase recruiting moieties that may be either identical of different, and moreover, may recruit the same or various E3 ligases, and further comprises multiple NF- ⁇ B 1 p105 recruiting moieties, that may be either identical or different (e.g., the same peptide, or any combination of the peptides disclosed herein).
  • the compounds of the invention specifically, bifunctional hybrid- molecule, conjugate or complex or their pharmaceutically acceptable salts can contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as ( R )- or (.S')- or, as (D)- or (L)- for amino acids.
  • the present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms whether or not they are specifically depicted herein.
  • Optically active (+) and (-), ( R )- and (.S' )- or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization.
  • Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).
  • HPLC high pressure liquid chromatography
  • a “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable.
  • the present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers”, which refers to two stereoisomers whose molecules are nonsuperimposable mirror images of one another.
  • a “tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule.
  • the present disclosure includes tautomers of any said bifunctional hybrid-molecule, conjugate or complex. It should be appreciated that each and every bifunctional hybrid-molecule/s, conjugate/s or complex/s of the present disclosure as disclosed herein above, are applicable for any of the aspects disclosed herein after.
  • a further aspect of the invention relates to a composition
  • a composition comprising at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the bioactive molecule of the present disclosure.
  • the composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
  • the bifunctional hybrid-molecule, conjugate or complex comprises: First (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amino acid sequence of Xaa (n) -Zaa-Xaa (m) -Zaa-Xaa (n) , as denoted by SEQ ID NO: 1.
  • Zaa is any aromatic amino acid residue.
  • Zaa as used herein is also denoted in the sequence listing with respect to SEQ ID NO: 1, as residue “Xaa 2 - and as residue "Xaa 4 ”, both refer to any aromatic amino acid residue as disclosed herein.
  • the sequence Xaa (n) -Zaa-Xaa (m) -Zaa-Xaa (n) is also equivalent to Xaa (n) -Xaa 2 -Xaa (m) - Xaa 4 -Xaa (n), as also denoted by SEQ ID NO: 1.
  • Xaa is any amino acid residue.
  • n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7, as discussed above in connection with the previous aspect of the present disclosure.
  • the bifunctional hybrid-molecule, conjugate or complex of the compositions disclosed herein further comprises a second component (b), at least one proteasome degradation mediating moiety.
  • the composition disclosed herein may comprise at least one bifunctional hybrid-molecule, conjugate or complex that is any of the bifunctional hybrid-molecule/s, conjugate/s or complex/es as defined by the present disclosure, in connection with other aspects of the invention.
  • the peptides of the bifunctional hybrid-molecule/s, conjugate/s or complex/es comprise at least two aromatic am aicnido residues. Specifically, at least two of at least one W, at least one Y, at least one F, and any mimetics thereof.
  • the peptide of the bifunctional hybrid-molecule, conjugate or complex of the compositions of the invention may comprise the amino acid sequence of at least one of any one of: (a) RIWVWLL, as dented by SEQ ID NO: 2, or any variants and derivatives thereof; (b), IWVWLL, as dented by SEQ ID NO: 3, or any variants and derivatives thereof; (c) WILVRLW, as dented by SEQ ID NO: 4, or any variants and derivatives thereof; (d) RIFVFLL, as dented by SEQ ID NO: 9; (e) RIYVFLL, as dented by SEQ ID NO: 10; (f) Citrulline-IWVWLL, as dented by SEQ ID NO: 15; (g) GRIWVWLL, as dented by SEQ ID NO: 16; (h) RRRIWVWLL, as dented by SEQ ID NO: 27; and/or (i) WVW, or any variants and derivatives thereof.
  • the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise any of the peptides disclosed herein, specifically, any of the peptides of SEQ ID NOs: 8, 11 to 28, and 52 to 59.
  • the at least one recruiting moiety for VHL E3 ubiquitin ligase, of the bifunctional hybrid-molecule, conjugate or complex of the compositions of the invention may comprise the VHL ligand of Formula I, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof.
  • the bifunctional hybrid-molecule, conjugate or complex of the compositions of the invention comprises at least two components.
  • One component is at least one peptide, that in some embodiments, recruits NF ⁇ B p105.
  • the second component is an E3 ligase recruiting component (also referred to herein as a ligand).
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may further comprise at least one linker.
  • the bifunctional hybrid- molecule, conjugate or complex of the compositions of the present disclosure may comprise the linker of Formula II, or of Formula III, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof.
  • the linker of the bifunctional hybrid-molecule, conjugate or complex of the compositions of the present disclosure may further comprises at least one amino acid residue. It should be noted that such amin-acid linker may be of any length and may comprise any amino acid residues, either identical or different.
  • the amino-acid linker further comprised in the linker of the bifunctional hybrid-molecule, conjugate or complex of the compositions of the invention may comprises at least one of: at least one Ser (S), at least one cysteine (C) and at least one Gly (G) amino acid residues, attached therewith.
  • the bifunctional hybrid-molecule, conjugate or complex of the composition of the present disclosure that comprises an NF- ⁇ B1 p105 recruiting peptide, an E3 ligase recruiting ligand and at least one linker may be the conjugate or complex of any of the compounds of Formula IV, V, VI and VII, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof. More specifically, in some embodiments, the bifunctional hybrid-molecule, conjugate or complex of the composition of the present disclosure may comprise the compound of Formula IV, being: Formula IV.
  • the bifunctional hybrid-molecule, conjugate or complex of the composition of the present disclosure may comprise the compound of Formula V, being: Formula V.
  • the bifunctional hybrid-molecule, conjugate or complex of the composition of the present disclosure may comprise the compound of Formula VI, being: Formula VI.
  • the bifunctional hybrid-molecule, conjugate or complex of the composition of the present disclosure may comprise the compound of Formula VII, being: Formula VII.
  • compositions provided by the present disclosure may comprise any of the bifunctional hybrid-molecule/s, conjugate/s or complex/es, that in some embodiments may be PROTACs molecules of the invention and any combinations thereof.
  • present disclosure further encompasses any compositions comprising any of the compounds of the invention, specifically, the bifunctional hybrid-molecule/s, conjugate/s or complex/es and PROTACs, and any combinations thereof with any additional therapeutic compounds.
  • any of the compositions of the present disclosure may be formulated as a pharmaceutical composition for delivery to a specific organ or cell type (e.g., brain, muscle, fibroblasts, bone, cartilage, liver, lung, breast, skin, bladder, kidney, heart, smooth muscle, adrenal, pituitary, pancreas, melanocytes, blood, adipose, and intestine).
  • a specific organ or cell type e.g., brain, muscle, fibroblasts, bone, cartilage, liver, lung, breast, skin, bladder, kidney, heart, smooth muscle, adrenal, pituitary, pancreas, melanocytes, blood, adipose, and intestine.
  • compositions of bifunctional hybrid-molecule, conjugate or complex of the invention that may be available as (but not limited to) a solution (e.g., tea), powder, tablet, capsule, elixir, topical, or injection.
  • a solution e.g., tea
  • the at least one bifunctional hybrid-molecule, conjugate or complex, any dosage form or composition thereof may be an add-on to any type of drugs or therapeutic compounds administered orally, intravenously, intradermaly, by inhalation or intrarectaly.
  • the compositions of the invention may comprise an effective amount of at least one bifunctional hybrid-molecule, conjugate or complex of the invention as disclosed herein and/or any vehicle, matrix, nano- or micro-particle thereof.
  • the term "effective amount” relates to the amount of an active agent present in a composition, specifically, the bifunctional hybrid-molecule, conjugate or complex of the invention as described herein that is needed to provide a desired level of active agent in the bloodstream or at the site of action in an individual (e.g., the specific site of the tumor) to be treated to give an anticipated physiological response when such composition is administered.
  • the precise amount will depend upon numerous factors, e.g., the active agent, the activity of the composition, the delivery device employed, the physical characteristics of the composition, intended patient use (i.e., the number of doses administered per day), patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein.
  • compositions used to treat subjects in need thereof according to the invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s).
  • formulations are prepared by uniformly and intimately bringing into association the active ingredients, specifically, the bifunctional hybrid-molecule, conjugate or complex of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the compositions may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas.
  • compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions of the present invention also include, but are not limited to, emulsions and liposome-containing formulations. It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question.
  • pharmaceutical preparations are compositions that include one or more bifunctional hybrid- molecule, conjugate or complex present in a pharmaceutically acceptable vehicle.
  • “Pharmaceutically acceptable vehicles” may be vehicles approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in any organism, specifically any vertebrate organism, for example, any mammal such as human.
  • vehicle refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is formulated for administration to a mammal.
  • Such pharmaceutical vehicles can be lipids, e.g., liposomes, e.g., liposome dendrimers; liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline; gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
  • compositions may be formulated into preparations in solid, semisolid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • administration of the bifunctional hybrid-molecule, conjugate or complex /s of the invention can be achieved in any of the various ways disclosed by the invention.
  • a further aspect of the present invention elates to a method for inducing ubiquitination and proteasomal processing of NF- ⁇ B1 p105, thereby generating the NF- ⁇ B p50 in a cell or in a cell- free system that comprise the NF- ⁇ B 1 p105. More specifically, the method comprising the step of contacting the cell or the cell-free system with an effective amount of at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the same, or any composition thereof.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise: First component (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue.
  • the peptide comprises the amino acid sequence of Xaa (n) -Zaa-Xaa (m) -Zaa- Xaa (n) , as denoted by SEQ ID NO: 1.
  • Zaa is any aromatic amino acid residue.
  • “Zaa” as used herein is also denoted in the sequence listing with respect to SEQ ID NO: 1, as residue "Xaa 2 - and as residue "Xaa 4 ⁇ .
  • Xaa is any amino acid residue.
  • n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7, as discussed above in connection with the other aspect of the present disclosure.
  • the bifunctional hybrid-molecule, conjugate or complex of the methods disclosed herein further comprises a second component (b), at least one proteasome degradation mediating moiety.
  • the peptide comprised in the bifunctional hybrid molecules of the preset disclosure recruit the target NF- ⁇ B1 p105, thereby enhancing ubiquitination and proteasomal processing of NF- ⁇ B1 p105 to p50.
  • NF- ⁇ B forms a family of transcription factors that play essential roles in multiple physiological and pathological processes.
  • NF- ⁇ B is typically a heterodimer that can be made of either p50 or p52 and RelA, RelB, or cRel.
  • p50 and p52 homodimers are also described as well.
  • the p50 and p52 are the products of ubiquitination and proteasome-dependent limited processing of the long precursors- p105 and plOO, respectively.
  • p50 and p52 NF ⁇ B subunits lack a transactivation domain, which is present in the Rel proteins.
  • homodimers based on p50 or p52 promote transcription only in case of complex formation with additional transcriptional activators, such as Bcl3, F1DAC3, or I ⁇ BZ.
  • additional transcriptional activators such as Bcl3, F1DAC3, or I ⁇ BZ.
  • the dimers are trapped in the cytoplasm by specific inhibitors known as I ⁇ BS (e.g., IkB ⁇ , plOO, p105, and Bcl3).
  • I ⁇ BS In response to a broad array of signals (e.g., oxidative stress, viral and bacterial infections, proinflammatory cytokines, and DNA damage), certain I ⁇ BS are phosphorylated on specific serine residues by the I ⁇ B kinase (IKK) complex and, consequently, are ubiquitinated and degraded by the proteasome. This releases the dimers that are translocated to the nucleus to initiate the specific transcriptional program.
  • IKK I ⁇ B kinase
  • the crucial step of activation of the canonical NF- ⁇ B is phosphorylation-dependent activation of the IKKs (I ⁇ B kinases) complex. Consequently, the inhibitory I ⁇ B proteins are phosphorylated and subjected to the ubiquitination-dependent degradation by proteasome, liberating the ⁇ B transcription factor to translocate to nucleus and activate the target genes.
  • the activation is quick but transient, since NF- ⁇ B also induces expression of the negative regulators like IkB ⁇ , A20, and p105, forming a negative feedback mechanism.
  • the non-canonical NF- ⁇ B is activated only through a handful of TNF superfamily receptors, indicating that the biological functions of this branch of pathway are more specific.
  • NIK NF- ⁇ B-inducing kinase
  • TRAF3 TNFR-associated factor 3
  • E3 ubiquitin ligase cIAP cellular inhibitor of apoptosis
  • RelA and p50 heterodimers are responsible for transcription of target genes when the canonical NF- ⁇ B pathway is activated, while RelB and p52 form a heterodimer in non-canonical NF- ⁇ B pathway.
  • RelA and p50 are sequestered in the cytoplasm by the I ⁇ B (inhibitor of NF- ⁇ B) proteins, which consist of three groups: the typical I ⁇ B proteins (IkB ⁇ , IkBb, and IkBe), the precursor proteins (plOO and p105), and the atypical I ⁇ B proteins (IkBz, BCL-3 and I ⁇ BNS).
  • IKK The central event in canonical NF- ⁇ B activation is the signal-induced phosphorylation of I ⁇ B molecules by IKKs.
  • IKK consists of two homologous catalytic subunits IKK ⁇ (also known as IKK1) and IKKb (also known as IKK2), and a regulatory subunit IKKg (also known as NF- ⁇ B essential modulator, NEMO).
  • the NF- ⁇ B 1 p105 as used herein refers to the human NF- ⁇ B 1 p105. Still further, in some embodiments, such protein is denoted by UniProtKB - P19838. In yet some further embodiments, p105 as used herein comprises the amino acid sequence as denoted by SEQ ID NO: 60. In yet some further embodiments, the Nuclear factor NF-kappa-B p50 product of UniProtKB P19838, comprises the amino acid sequence as denoted by SEQ ID NO: 61. Thus, in some embodiments, generation the NF- ⁇ B p50, leads to various effects.
  • the generation of p50 by the methods of the invention may be involved in modulation of NF- ⁇ B canonical pathway. In some embodiments, such modulation may involve inhibition of the NF- ⁇ B canonical pathway. Still further, in some embodiments, the generation of p50 by the methods of the invention may lead to activation of transcription of at least one tumor suppressor.
  • the generation of p50 by the methods of the invention may lead to suppression of programmed cell death-ligand l(PD-Ll) expression. In yet some further embodiments, the generation of p50 by the methods of the invention may result in increased secretion of at least one proinflammatory cytokine by the cell. In some embodiments, the peptides of the invention and constructs thereof change the expression pattern of various groups of proteins.
  • the peptides of the invention, the derivative or variant or form of the disclosed peptides, and/or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein lead to an increase, elevation, upregulation, enhancement and or enlargement of the expression of proteins connected to immune system processes and/or proteins connected with regulation of cell migration, and/or proteins that relate to ECM organization, and/or proteins that relate to cell adhesion, and/or proteins that relate to regulation of cell adhesion.
  • the peptides of the invention, the derivative or variant or form of the disclosed peptides, and/or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein lead to a decrease, reduction, attenuation, inhibition and downregulation of the expression of proteins connected to metabolic processes, and/or proteins that relate to cell cycle, and/or proteins that relate to DNA metabolic processes.
  • the at least one bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may be any of the bifunctional hybrid-molecule, conjugate or complex disclosed by the present disclosure, in connection with other aspects of the present disclosure.
  • the peptides of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the methods of the invention comprise at least two aromatic amino acid residues. Specifically, at least two of at least one W, at least one Y, at least one F, and any mimetics thereof. In some embodiments, at least one of the at least two aromatic amino aromatic amino acid residues of the peptides of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure, may be tryptophan. In some embodiments, one of the aromatic amino acid residues in the peptides of the bifunctional hybrid-molecule of the present disclosure may be tryptophan, and the other may be tyrosine.
  • the peptides of the bifunctional hybrid-molecule of the present disclosure may comprise at least one tryptophan and at least one phenylalanine. In yet some further embodiments, the peptides of the bifunctional hybrid-molecule of the invention may comprise at least two tryptophan residues. It should be understood that the peptides of the bifunctional hybrid-molecule of the methods of the present disclosure may comprise any further aromatic amino acid residues as one or more of the Xaa residues located in various positions of the peptide as discussed above.
  • At least one of the "interspacing" amino acid residue/s located between the two aromatic amino acid residues, peptides of the bifunctional hybrid-molecule/s used by the methods of the invention may be one or more amino acid residue/s having a non-polar side chain, also referred to herein as a non-polar amino acid.
  • Such residues may be any one of Valine (V, Val), Glycine (G, Gly), Leucine (L, Leu), Isoleucine (I, IIe), Methionine (M, Met), Phenylalanine (F, Phe), Tryptophane (W, Trp) and/or Proline (P, Pro).
  • the interspacing amino acid residue may be any hydrophobic amino acid residue, for example, Valine (V, Val), Leucine (L, Leu) and/or Isoleucine (I, IIe).
  • the peptides of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise at least two aromatic amino acid residues that are interspaced by at least one of Valine (V), Glycine (G) and/or Alanine (A).
  • the at least two aromatic amino acid residues of the peptides of the bifunctional hybrid-molecule disclosed herein are interspaced by at least one Valine residue.
  • the peptides of the bifunctional hybrid-molecule, conjugate or complex used by the methods in accordance with the invention may comprise the amino acid sequence of Xaa (n) -Trp (W)-Xaa (n) -Val (V)-Xaa (n) -Trp (W)-Xaa (n), as denoted by SEQ ID NO: 7, or any mimetics thereof.
  • Xaa is any amino acid residue
  • n is zero or an integer between 1 to 7.
  • These amino acid residues may be any identical or different am aincoid residues.
  • the at least one peptide of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise three to ten amino acid residues, specifically, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues.
  • the peptides of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise any amino acid residue, more specifically, in some embodiments Xaa that may be any one of Arg (R), IIe (I) and Leu (L), amino acid residues.
  • the peptide of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise the amino acid sequence of at least one of any one of: (a) RIWVWLL, as dented by SEQ ID NO: 2, or any variants and derivatives thereof; (b), IWVWLL, as dented by SEQ ID NO: 3, or any variants and derivatives thereof; (c) WILVRLW, as dented by SEQ ID NO: 4, or any variants and derivatives thereof; (d) RIFVFLL, as dented by SEQ ID NO: 9; (e) RIYVFLL, as dented by SEQ ID NO: 10; (f) Citrulline-IWVWLL, as dented by SEQ ID NO: 15; (g) GRIWVWLL, as dented by SEQ ID NO: 16; (h) RRRIWVWLL, as dented by SEQ ID NO: 27; and/or (i) WVW, or any variants and derivatives thereof.
  • the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise any of the peptides disclosed herein, for example, any of the peptides of SEQ ID NO: 8, 11 to 28, and 52 to 59.
  • the peptide of the bifunctional hybrid-molecule, conjugate or complex of the invention binds and therefore recruits the NF- ⁇ B1 p105. It should be understood that any variant or derivative of the above-mentioned peptides may be comprised within the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention.
  • any variant or derivative of each one of the above indicated peptides may be applicable in any of the methods of the invention, provided that the variant and/or derivative retains the ability of the peptide to recruit and bind the NF- ⁇ B1 p105.
  • the proteasome degradation mediating moiety of the bifunctional hybrid- molecule/s, conjugate/s or complex/es of the present disclosure may comprises at least one E3 ubiquitin ligase recruiting moiety, or at least one E3 ubiquitin ligase active moiety.
  • such E3 ubiquitin ligase active moiety may be any domain or fragment of an E3 ligase, that is capable of forming of an isopeptide bond between the carboxy terminus of ubiquitin and a lysine residue of a target protein.
  • the E3 ubiquitin ligase active moiety may comprise at least one of RING finger and U-box E3s, the HECT E3s, and the RING/HECT-hybrid type E3s of an E3 ligase.
  • the proteasome degradation mediating moiety of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may be any moiety that recruits at least one E3 ligase.
  • the E3 ubiquitin ligase recruiting moiety of the bifunctional hybrid- molecule, conjugate or complex of the methods of the present disclosure may be suitable for recruiting any E3 ubiquitin ligase.
  • the E3 ubiquitin ligase recruiting moiety of the bifunctional hybrid-molecule is suitable for at least one of von-Hippel-Lindau (VHL), Cereblon (CRBN), Mouse double minute 2 homolog (MDM2), cellular inhibitor of apoptosis protein- 1 (clAPl) and X-linked inhibitor of apoptosis protein (XI AP) and DDB1 and CUL4 associated factor 15 (DCAF15).
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure comprises at least one recruiting moiety for VHL E3 ubiquitin ligase.
  • the recruiting moiety comprises at least one small molecule or peptide, or any combinations thereof. It should be understood that any of the L3 ligase ligands, and specifically, an of the VHL ligands disclosed by the invention are applicable for this aspect as well.
  • the at least one recruiting moiety for VHL E3 ubiquitin ligase, of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise the VHL ligand of Formula I, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof:
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention comprises two components, one component, is at least one peptide, that in some embodiments recruits NF- ⁇ B p105.
  • the second component is an E3 ligase recruiting component.
  • the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may further comprise at least one linker.
  • At least one of said linkers is a bridging linker that bridges between said at least one peptide and the at least one proteasome degradation mediating moiety.
  • the linker may take any form, and any length appropriate to bring together the target protein NF- ⁇ BI p105 and ubiquitinating machinery and thereby elicit the ubiquitination of NF- ⁇ B 1 p105 and its subsequent degradation in the proteasome.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the linker of Formula II as disclosed herein, or the linker of Formula III as disclosed herein, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof.
  • the linker of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may further comprises at least one amino acid residue.
  • amin-acid linker may be of any length and may comprise anyamino acid residues, either identical or different.
  • the amino- acid linker further comprised in the linker of he the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprises at least one of: at least one Serine (S), at least one cysteine (C) and at least one Glycine (G) amino acid residues, attached therewith.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise at least one of the compounds of Formula IV, V, VI and VII, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula IV, being: Formula IV.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula V, being: Formula V.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula VI, being: Formula VI.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula VII, being: Formula VII.
  • the methods of the present disclosure may be applicable for inducing ubiquitination and proteasomal processing of NF- ⁇ B 1 p105 to generate the NF- ⁇ B p50 in a subject.
  • the step of contacting the cell that comprise the NF- ⁇ B p105 with the bi-functional hybrid-molecule of the present disclosure may comprise administering to the subject an effective amount of the bi-functional hybrid-molecule of the present disclosure.
  • the effective amount as used herein, refers to the amount required for the induction of the processing of the NF- ⁇ B p105, to generate the NF- ⁇ B p50 in the subject.
  • generation the NF- ⁇ B p50 leads to various effects in the subject.
  • the generation of p50 by the methods of the invention may be involved in modulation of NF- ⁇ B canonical pathway in a subject. In some embodiments, such modulation may involve inhibition of the NF- ⁇ B canonical pathway in the subject. Still further, in some embodiments, the generation of p50 by the methods of the invention may lead to activation of transcription of at least one tumor suppressor in the subject.
  • the generation of p50 by the methods of the invention may lead to suppression of programmed cell death-ligand l(PD-Ll) expression in the subject. In yet some further embodiments, the generation of p50 by the methods of the invention may result in increased secretion of at least one proinflammatory cytokine in the subject. In some further embodiments, the generation of p50 by the methods of the invention may result in recruitment of immune cells to a diseased tissue or organ in the subject. As indicated above, in some embodiments, the peptides of the invention and constructs thereof change the expression pattern of various groups of proteins.
  • the peptides of the invention, the derivative or variant or form of the disclosed peptides, and/or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein lead to an increase, elevation, upregulation, enhancement and or enlargement of the expression of proteins connected to immune system processes and/or proteins connected with regulation of cell migration, and/or proteins that relate to ECM organization, and/or proteins that relate to cell adhesion, and/or proteins that relate to regulation of cell adhesion.
  • the peptides of the invention, the derivative or variant or form of the disclosed peptides, and/or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein lead to a decrease, reduction, attenuation, inhibition and downregulation of the expression of proteins connected to metabolic processes, and/or proteins that relate to cell cycle, and/or proteins that relate to DNA metabolic processes.
  • a decrease or alternatively, an increase in the level of the discussed proteins is meant any increase or alternatively, decrease of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more, as compared to a cell, in a subject and/or a cell free system in the absence of the peptides of the invention or any derivative or variant or form of the disclosed peptides, and or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein.
  • NF- ⁇ B is a major transcriptional regulator for the cell response to external signals. It orchestrates a broad range of cellular processes, among them cell division and differentiation and cell death and survival. Importantly, NF- ⁇ B controls the immune and inflammatory response. Dysregulated activity of NF- Kb has been reported to be involved in a broad array of immune system-related disorders and malignant transformation. Canonical NF- ⁇ B is activated rapidly, inducing numerous proinflammatory mediators and molecules that lead to inflammatory response as well as activation and differentiation of immune cells. However, aberrant activation of NF- ⁇ B cause chronic inflammation, oncogenesis and autoimmune disease.
  • Activation of canonical NF- ⁇ B is often linked to the inflammation response to infection and injury, which is a part of host defense.
  • Well-regulated inflammation response is essential for host homeostasis. Tumorigenic pathogens cause chronic infections and inflammation, leading to malignancy. It has been shown that chronic infections and inflammation contribute to certain cancers.
  • HBV human hepatitis B virus
  • HCC hepatocellular carcinoma
  • Chronic Helicobacter pylori infection is linked to MALT (mucosa- associated lymphoid tissue) lymphoma and gastric cancer.
  • RA rheumatoid arthritis
  • IBD inflammatory bowel disease
  • psoriasis chronic inflammatory diseases
  • IBD is tightly correlated with colorectal cancer
  • RA and psoriasis do not show significant tumor-promoting effect.
  • RA rheumatoid arthritis
  • psoriasis do not show significant tumor-promoting effect.
  • tobacco smoke, silica particles and obesity are other factors also contributing to chronic inflammation-related cancer, such as tobacco smoke, silica particles and obesity.
  • the invention provides powerful means for modulating NF- ⁇ B 1 canonical pathway, and moreover, for extending the tumor suppressive effect of NF- ⁇ B 1 p50.
  • a further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder or condition in a subject in need thereof. More specifically, the method comprising the step of administering to said subject a therapeutically effective amount of at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the same, or any composition thereof.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise: First (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amino acid sequence of Xaa (n) -Zaa-Xaa (m) -Zaa-Xaa (n) , as denoted by SEQ ID NO: 1. It should be noted that Zaa is any aromatic amino acid residue. Still further, in some embodiments, Xaa is any amino acid residue.
  • Zaa as used herein is also denoted in the sequence listing with respect to SEQ ID NO: 1, as residue “Xaa 2 " and as residue “Xaa 4 " , both refer to any aromatic amino acid residue as disclosed herein.
  • sequence Xaa (n) -Zaa- Xaa (m) -Zaa-Xaa (n) is also equivalent to Xaa (n) -Xaa 2 -Xaa (m) -Xaa 4 -Xaa (n), as also denoted by SEQ ID NO: 1.
  • n is zero or an integer between 1 to 7
  • m is an integer between 1 to 7, as discussed above in connection with the previous aspect of the present disclosure.
  • the bifunctional hybrid-molecule, conjugate or complex of the compositions disclosed herein further comprises a second component (b), at least one proteasome degradation mediating moiety.
  • the peptide of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the methods of the invention may comprise at least two aromatic amino acid residues. Specifically, at least two of at least one W, at least one Y, at least one F, and any mimetics thereof.
  • the peptide of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the methods of the invention may comprise two tryptophan residues (WW).
  • the peptide of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the methods of the invention comprise at least one tryptophan and at least one tyrosine (WY).
  • WY tryptophan residues
  • the peptide of the bifunctional hybrid- molecule/s, conjugate/s or complex/es of the methods of the invention may comprise at least one tryptophane and at least one phenylalanine (WF).
  • the peptide of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the methods of the invention may comprise at least two tyrosine resides (YY).
  • the peptide of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the methods of the invention comprise may comprise at least one tyrosine and at least one phenylalanine residues (YF).
  • the peptide of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the methods of the invention comprise at least two phenylalanine residues (FF).
  • At least one of the at least two aromatic amino aromatic amino acid residues of the peptide of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may be tryptophan.
  • one of the aromatic amino acid residues in the peptide of the bifunctional hybrid-molecule of the present disclosure may be tryptophan, and the other may be tyrosine.
  • the peptide of the bifunctional hybrid-molecule of the present disclosure may comprise at least one tryptophan and at least one phenylalanine.
  • the peptide of the bifunctional hybrid-molecule of the invention may comprise at least two tryptophan residues.
  • the peptide of the bifunctional hybrid-molecule of the present disclosure may comprise any further aromatic amino acid residues as one or more of the Xaa residues located in various positions of the peptide as discussed above. Still further, in some embodiments, at least one of the "interspacing" amino acid residue/s located between the two aromatic amino acid residues peptide of the bifunctional hybrid-molecule, conjugate or complex of the invention, may be one or more amino acid residue/s having a non-polar side chain, also referred to herein as a non-polar amino acid.
  • Such residues may be any one of Valine (V, Val), Glycine (G, Gly), Leucine (L, Leu), Isoleucine (I, lie), Methionine (M, Met), Phenylalanine (F, Phe), Tryptophane (W, Trp) and/or Proline (P, Pro).
  • the interspacing amino acid residue may be any hydrophobic amino acid residue, for example, Valine (V, Val), Leucine (L, Leu) and/or Isoleucine (I, IIe).
  • the peptide of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise at least two aromatic amino acid residues that are interspaced by at least one of Valine (V), Glycine (G) and/or Alanine (A).
  • V Valine
  • G Glycine
  • A Alanine
  • the at least two aromatic amino acid residues of the peptide of the bifunctional hybrid-molecule disclosed herein are interspaced by at least one Valine residue.
  • the peptide of the bifunctional hybrid- molecule, conjugate or complex used by the methods in accordance with the invention may comprise the amino acid sequence of Xaa (n) -Trp (W)-Xaa (n) -Val (V)-Xaa (n) -Trp (W)-Xaa (n), as denoted by SEQ ID NO: 7, or any mimetics thereof.
  • Xaa is any amino acid residue
  • n is zero or an integer between 1 to 7.
  • These am aicniod residues may be any identical or different amino acid residues.
  • the at least one peptide of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise three to ten amino acid residues, specifically, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues.
  • the peptide of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise any amino acid residue, Xaa that is any one of Leu (L), Arg (R) and IIe (I) amino acid residues.
  • the peptide of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise the amino acid sequence of at least one of any one of: (a) RIWVWLL, as dented by SEQ ID NO: 2, or any variants and derivatives thereof; (b), IWVWLL, as dented by SEQ ID NO: 3, or any variants and derivatives thereof; (c) WILVRLW, as dented by SEQ ID NO: 4, or any variants and derivatives thereof; (d) RIFVFLL, as dented by SEQ ID NO: 9; (e) RIYVFLL, as dented by SEQ ID NO: 10; (f) Citrulline-IWVWLL, as dented by SEQ ID NO: 15; (g) GRIWVWLL, as dented by SEQ ID NO: 16; (h) RRRIWVWLL, as dented by SEQ ID NO: 27; and/or (i) WVW, or any variants and derivatives thereof.
  • the peptide of the bifunctional hybrid-molecule, conjugate or complex of the methods of the invention binds and therefore recruits the and bind the NF- ⁇ B1 p105. It should be understood that any variant or derivative of the above-mentioned peptides may be comprised within the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention. In some embodiments, any variant or derivative of each one of the above indicated peptides is encompassed by the invention, provided that the variant or derivative retains the ability of the peptide to recruit and bind the NF- ⁇ B1 p105.
  • the proteasome degradation mediating moiety of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the present disclosure may comprises at least one E3 ubiquitin ligase recruiting moiety, or at least one E3 ubiquitin ligase active moiety.
  • E3 ubiquitin ligase active moiety may be any domain or fragment of an E3 ligase, that is capable of forming of an isopeptide bond between the carboxy terminus of ubiquitin and a lysine residue of a target protein.
  • the E3 ubiquitin ligase active moiety may comprise at least one of RING finger and U-box E3s, the HECT E3s, and the RING/HECT-hybrid type E3s of an E3 ligase.
  • the proteasome degradation mediating moiety of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may be any moiety that recruits at least one E3 ligase.
  • the E3 ubiquitin ligase recruiting moiety of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may be suitable for recruiting any E3 ubiquitin ligase.
  • the E3 ubiquitin ligase recruiting moiety of the bifunctional hybrid-molecule is suitable for at least one of von- Hippel-Lindau (VHL), Cereblon (CRBN), Mouse double minute 2 homolog (MDM2), cellular inhibitor of apoptosis protein- 1 (clAP1) and X-linked inhibitor of apoptosis protein (XI AP) and DDB1 and CUL4 associated factor 15 (DCAF15).
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure comprises at least one recruiting moiety for VHL E3 ubiquitin ligase.
  • the recruiting moiety comprises at least one small molecule or peptide, or any combinations thereof.
  • the at least one recruiting moiety for VHL E3 ubiquitin ligase, of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise the VHL ligand of Formula I, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof, being:
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention comprises two components, one component, is at least one peptide, that in some embodiments recruits NFKB p105.
  • the second component is an E3 ligase recruiting component (also referred to herein as a ligand).
  • the bifunctional hybrid- molecule, conjugate or complex of the present disclosure may further comprise at least one linker.
  • at least one of the linkers may be a bridging linker that bridges between the at least one peptide and the at least one proteasome degradation mediating moiety.
  • the linker may take any form, and any length appropriate to bring together the target protein NF- ⁇ B1 p105 and ubiquitinating machinery and thereby elicit the ubiquitination of NF- ⁇ B1 p105 and its subsequent degradation in the proteasome.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the linker of Formula II (as disclosed herein), or the linker of Formula III (as disclosed herein), or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof.
  • the linker of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may further comprises at least one amino acid residue.
  • amino-acid linker may be of any length and may comprise anyamino acid residues, either identical or different.
  • the amino- acid linker further comprised in the linker of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise at least one of: at least one Serine (S), at least one cysteine (C) and at least one Glycine (G) amino acid residues, attached therewith.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise at least one of the compounds of Formula IV, V, VI and VII, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof, or any compositions thereof.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula IV, being: Formula IV.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula V, being: Formula V.
  • the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula VI, being: Formula VI. Still further, in some embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula VII, being: Formula VII.
  • the present disclosure encompasses methods using any PROTAC, CLIPTAC, or any bifunctional hybrid-molecule, conjugate or complexes that comprise any of the E3 ligands, any of the NF- ⁇ B1 p105 recruiting peptides and any of the linkers disclosed herein and any combinations thereof, and any compositions thereof.
  • the molecules used by the methods of the present disclosure are effective in targeted degradation and processing of the NF- ⁇ BI p105 to produce NF- ⁇ B1 p50.
  • the bifunctional hybrid- molecules, conjugates or complexes, PROTACs use by the methods of the present disclosure may comprise any combination of the discussed components (E3 ligase, recruiting peptide and linker), with the proviso that such molecule is not the PROTAC molecule of Formula VIII, being: Formula VIII.
  • NF- ⁇ B The crucial role of NF- ⁇ B in tumorigenesis is well established. In many solid malignances, the activity of NF- ⁇ B is upregulated, modulating tumor initiation, promotion, and metastases. In most cases, the tumor-promoting activity of NF- ⁇ B is related to the p65p50 heterodimeric complex. Thus, in ovarian cancer, strong expression of both subunits was found compared to borderline and benign ovarian tumors. Enhanced p65 staining was found in human prostate adenocarcinoma, correlating with increased tumor grade. The study revealed that the DNA-binding complex is mainly made of NF- ⁇ B p50p65 heterodimers. Although in most cases, NF- ⁇ B appears to be oncogenic, in some studies it has been demonstrated as a tumor suppressor, which is due in particular to its p50 subunit.
  • SKP2 is the ubiquitin ligase that negatively regulates the abundance of the cyclin- dependent kinase inhibitor p27KIP, thus promoting malignant transformation by increased transition to S phase.
  • one of the well-defined p50- mediated tumor- suppressive mechanisms was described in human glioblastoma and breast cancer cells and xenografts, and in tumors derived from patients: it was shown that excessive generation of p50 by the ubiquitin ligase KPC1 triggers up-regulation of transcripts of numerous tumor suppressors, suggesting that p50p50 homodimers control their transcription (rather than the “canonical” tumorigenic p50p65).
  • NF-xB-dependent modulation of the tumor microenvironment is another essential mechanism that regulates cancer dynamics-progression or suppression.
  • a tumor suppression mechanism based on modulation of the tumor microenvironment is the induction of mitochondrial outer-membrane permeabilization that causes activation of NF- ⁇ B in tumor cells with subsequent anticancer immune response, including macrophage activation and T cell infiltration.
  • activation of NF- ⁇ B up-regulates the T cell-recruiting chemokines CCL2 and CCL5, resulting in tumor rejection.
  • NF- ⁇ B activity was linked to high infiltration of T cells to the tumor.
  • Chemokines play an important role in cancer progression not only through the direct autocrine effect on the tumor cells, but also through recruitment of specific immune cells.
  • Genomic expression signature of twelve chemokines (CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11, and CXCL13) was identified in genomic arrays of colorectal carcinoma and of aboutl5,000 distinct solid tumors. It was correlated with the presence of tertiary lymph node-like structures and was associated with better survival of a subset of melanoma patients.
  • PD-L1 programmed cell death-ligand 1
  • NK natural killer
  • p65- containing NF- ⁇ B dimers were shown to up-regulate CSN5-a deubiquitinating enzyme-which resulted in stabilization of PDL1, thus bypassing immune suppression of the cancer cells [3].
  • the present inventors recently showed that the ubiquitin ligase KPC1 acts as a tumor suppressor via its excessive activity on the p105 NF- ⁇ B precursor, generating excess of the p50 subunit.
  • Recent initial results of the present inventors [3] demonstrated that p50 stimulates transcription of a broad array of tumor suppressors. The inventors showed that excess of p50 down-regulates the surface expression of PD-L1.
  • KPC1 and its downstream product p50 act on several layers to suppress tumor growth. They 1) activate transcription of tumor suppressors; 2) suppress expression of PD-L1; and 3) recruit immune cells via stimulation of secretion of an array of cytokines. In some embodiments, generation the NF- ⁇ B p50, leads to various effects in the treated subject.
  • the generation of p50 by the methods of the invention may be involved in modulation of NF- ⁇ B canonical pathway in a treated subject. In some embodiments, such modulation may involve inhibition of the NF- ⁇ B canonical pathway in the treated subject. Still further, in some embodiments, the generation of p50 by the methods of the invention may lead to activation of transcription of at least one tumor suppressor in the treated subject. In yet some further embodiments, the generation of p50 by the methods of the invention may lead to suppression of programmed cell death-ligand l(PD-Ll) expression in the treated subject. In yet some further embodiments, the generation of p50 by the methods of the invention may result in increased secretion of at least one proinflammatory cytokine in the treated subject.
  • the generation of p50 by the methods of the invention may result in recruitment of immune cells to a diseased tissue, specifically, tumor tissue or organ in the treated subject.
  • the methods disclosed herein may be applicable for any pathologic disorder, for example, a proliferative disorder.
  • such proliferative disorder or cancer may be at least one solid and non-solid tumor and any related conditions.
  • the pathologic disorder or condition treated by the methods of the present disclosure is cancer. It should be understood that the present invention is further apphcable to any metastatic tissue, organ or cavity of any proliferative disorder/s.
  • cancer As used herein to describe the present invention, “cancer”, “proliferative disorder”, “tumor” and “malignancy” all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non-solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. In general, the methods, compositions and bifunctional hybrid-molecule, conjugate or complex of the present invention may be applicable for a patient suffering from any one of non-solid and solid tumors. Malignancy, as contemplated in the present invention may be any one of carcinomas, melanomas, lymphomas, leukemia, myeloma and sarcomas.
  • carcinoma refers to an invasive malignant tumor consisting of transformed epithelial cells.
  • a malignant tumor composed of transformed cells of unknown histogenesis, but which possess specific molecular or histological characteristics that are associated with epithelial cells, such as the production of cytokeratins or intercellular bridges.
  • Melanoma is a malignant tumor of melanocytes. Melanocytes are cells that produce the dark pigment, melanin, which is responsible for the color of skin.
  • Leukemia refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood-leukemic or aleukemic (subleukemic).
  • Sarcoma is a cancer that arises from transformed connective tissue cells. These cells originate from embryonic mesoderm, or middle layer, which forms the bone, cartilage, and fat tissues. This is in contrast to carcinomas, which originate in the epithelium. The epithelium lines the surface of structures throughout the body, and is the origin of cancers in the breast, colon, and pancreas. Myeloma as mentioned herein is a cancer of plasma cells, a type of white blood cell normally responsible for the production of antibodies. Collections of abnormal cells accumulate in bones, where they cause bone lesions, and in the bone marrow where they interfere with the production of normal blood cells.
  • lymphoma is a cancer in the lymphatic cells of the immune system. Typically, lymphomas present as a solid tumor of lymphoid cells. These malignant cells often originate in lymph nodes, presenting as an enlargement of the node (a tumor). It can also affect other organs in which case it is referred to as extranodal lymphoma.
  • lymphoma include Hodgkin's disease, non-Hodgkin's lymphomas and Burkitt's lymphoma.
  • the methods of the present disclosure may be applicable for any solid tumor.
  • the methods disclosed herein may be applicable for any malignancy that may affect any organ or tissue in any body cavity, for example, the peritoneal cavity (e.g., liposarcoma), the pleural cavity (e.g., mesothelioma, invading lung), any tumor in distinct organs, for example, the urinary bladder, ovary carcinomas, and tumors of the brain meninges.
  • the peritoneal cavity e.g., liposarcoma
  • the pleural cavity e.g., mesothelioma, invading lung
  • any tumor in distinct organs for example, the urinary bladder, ovary carcinomas, and tumors of the brain meninges.
  • tumors applicable in the methods, compositions and bifunctional hybrid-molecule, conjugate or complex of the present disclosure may include but are not limited to at least one of glioblastoma, breast cancer, ovarian cancer, liver carcinoma, colorectal carcinoma, pancreatic cancer, brain tumors and any related conditions, as well as any metastatic condition, tissue or organ thereof.
  • the methods, the bifunctional hybrid-molecule, conjugate or complex and compositions of the present disclosure may be applicable for treating breast cancer.
  • the methods, the bifunctional hybrid-molecule, conjugate or complex and compositions of the present disclosure may be applicable for treating glioblastoma.
  • GBM Glioblastoma
  • GBM Glioblastoma
  • GBMs can arise in the brain de novo or evolve from lower-grade astrocytoma. In adults, GBM occurs most often in the cerebral hemispheres, especially in the frontal and temporal lobes of the brain.
  • the methods, compositions and bifunctional hybrid-molecule, conjugate or complex of the invention are relevant to colorectal carcinoma, or any malignancy that may affect all organs in the peritoneal cavity, such as liposarcoma for example.
  • the method of the invention may be relevant to tumors present in the pleural cavity (mesothelioma, invading lung) the urinary bladder, and tumors of the brain meninges.
  • the methods, compositions and bifunctional hybrid-molecule, conjugate or complex of the invention may be applicable for ovarian cancer.
  • the invention further encompasses any tissue, organ or cavity barring ovarian metastasis, as well as any cancerous condition involving metastasis in ovarian tissue.
  • the methods, the bifunctional hybrid-molecule, conjugate or complex and compositions of the present disclosure may be suitable for liver cancer.
  • the invention further encompasses any tissue, organ or cavity barring liver originated metastasis, as well as any cancerous condition having metastasis of any origin in liver tissue.
  • Liver cancer also known as hepatic cancer and primary hepatic cancer, is cancer that starts in the liver. Cancer which has spread from elsewhere to the liver, known as liver metastasis, is more common than that which starts in the liver.
  • Symptoms of liver cancer may include a lump or pain in the right side below the rib cage, swelling of the abdomen, yellowish skin, easy bruising, weight loss and weakness.
  • the most common types are hepatocellular carcinoma (HCC), which makes up 80% of cases, and cholangiocarcinoma. Less common types include mucinous cystic neoplasm and intraductal papillary biliary neoplasm.
  • HCC hepatocellular carcinoma
  • cholangiocarcinoma cholangiocarcinoma
  • mucinous cystic neoplasm and intraductal papillary biliary neoplasm.
  • the methods, the bifunctional hybrid-molecule, conjugate or complex and compositions of the present disclosure may be applicable for pancreatic cancer.
  • the invention further encompasses any tissue, organ or cavity barring pancreatic metastasis, as well as any cancerous condition having metastasis of any origin in the pancreas.
  • Pancreatic cancer arises when cells in the pancreas, a glandular organ behind the stomach, begin to multiply out of control and form a mass.
  • the methods, compositions and the bifunctional hybrid-molecule, conjugate or complex of the present disclosure are applicable for any type and/or stage and/or grade of any of the malignant disorders discussed herein or any metastasis thereof. Still further, it must be appreciated that the methods, compositions and bifunctional hybrid-molecule, conjugate or complex of the invention may be applicable for invasive as well as non-invasive cancers.
  • non-invasive cancer when referring to “non-invasive” cancer it should be noted as a cancer that do not grow into or invade normal tissues within or beyond the primary location.
  • invasive cancers it should be noted as cancer that invades and grows in normal, healthy adjacent tissues.
  • the methods, compositions and bifunctional hybrid-molecule, conjugate or complex of the present disclosure are applicable for any type and/or stage and/or grade of any metastasis, metastatic cancer or status of any of the cancerous conditions disclosed herein.
  • metastatic cancer or “metastatic status” refers to a cancer that has spread from the place where it first started (primary cancer) to another place in the body.
  • Further malignancies that may find utility in the present invention can comprise but are not limited to hematological malignancies (including lymphoma, leukemia, myeloproliferative disorders, Acute lymphoblastic leukemia; Acute myeloid leukemia), hypoplastic and aplastic anemia (both virally induced and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune mediated and idiopathic) and solid tumors (including GI tract, colon, lung, liver, breast, prostate, pancreas and Kaposi's sarcoma.
  • the invention may be applicable as well for the treatment or inhibition of solid tumors such as tumors in lip and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbladder, extraliepatic bile ducts, ampulla of vater, exocrine pancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma, carcinoma and malignant melanoma of the skin, breast, vulva, vagina, cervix uteri, corpus uteri, ovary, fallopian tube, gestational trophoblastic tumors, penis, prostate, testis, kidney, renal pelvis, ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma of the conjunctiva, malignant melanoma of the conjunctiva, malignant
  • the methods disclosed herein involve in some embodiments thereof the administration of an effective amount of the bifunctional hybrid-molecule/s, conjugate/s or complex/es or compositions of the invention.
  • An “effective amount” of the bifunctional hybrid-molecule, conjugate or complex/s of the invention, or any compositions thereof can be administered in one administration, or through multiple administrations of an amount that total an effective amount, preferably within a 24-hour period, to achieve the therapeutic effect as discussed herein, specifically, to induce in the treated subject the degradation of the NF- ⁇ B1 p105, to generate the NF- ⁇ B1 p50 product that exhibits a tumor suppressive effect.
  • the "effective amount" as discussed herein is the amount sufficient for a therapeutic effect in the treated subject.
  • the "effective amount" can be the result of empirical and/or individualized (case-by-case) determination on the part of the treating health care professional and/or individual. Still further, local administration to the area in need of treatment may be achieved by, for example, by local infusion during surgery, or using any permanent or temporary infusion device, topical application, direct injection into the specific organ, etc.
  • the bifunctional hybrid-molecule, conjugate or complex/s and compositions disclosed herein may be adapted for administration by parenteral, intraperitoneal, transdermal, oral (including buccal or sublingual), rectal, topical (including buccal or sublingual), vaginal, intranasal and any other appropriate routes.
  • Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).
  • the composition of the invention may optionally further comprise at least one of pharmaceutically acceptable carrier/s, excipient/s, additive/s diluent/s and adjuvant/s.
  • treat means preventing, ameliorating or delaying the onset of one or more clinical indications of disease activity in a subject having a pathologic disorder.
  • Treatment refers to therapeutic treatment. Those in need of treatment are subjects suffering from a pathologic disorder. Specifically, providing a "preventive treatment” (to prevent) or a “prophylactic treatment” is acting in a protective manner, to defend against or prevent something, especially a condition or disease.
  • treatment or prevention refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, pathologic disorder involved with at least one short term cellular stress condition/process and any associated condition, illness, symptoms, undesired side effects or related disorders. More specifically, treatment or prevention of relapse or recurrence of the disease, includes the prevention or postponement of development of the disease, prevention or postponement of development of symptoms and/or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing- additional symptoms and ameliorating or preventing the underlying metabolic causes of symptoms.
  • the terms “inhibition”, “moderation”, “reduction”, “decrease” or “attenuation” as referred to herein, relate to the retardation, restraining or reduction of a process by any one of about 1 % to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more.
  • inhibitor and all variations of this term is intended to encompass the restriction or prohibition of the progress and exacerbation of pathologic symptoms or a pathologic process progress, said pathologic process symptoms or process are associated with.
  • laminate relates to the substantial eradication or removal of the pathologic symptoms and possibly pathologic etiology, optionally, according to the methods of the invention described herein.
  • delay and delaying the onset
  • retard and all variations thereof are intended to encompass the slowing of the progress and/or exacerbation of a disorder associated with the at least one short term cellular stress condition/process and their symptoms, slowing their progress, further exacerbation or development, so as to appear later than in the absence of the treatment according to the invention.
  • the methods and compositions provided by the present invention may be used for the treatment of a “pathological disorder”, i.e., pathologic disorder or condition involved with at least one short term cellular stress condition/process, which refers to a condition, in which there is a disturbance of normal functioning, any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with that person.
  • pathological disorder i.e., pathologic disorder or condition involved with at least one short term cellular stress condition/process, which refers to a condition, in which there is a disturbance of normal functioning, any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with that person.
  • pathological disorder i.e., pathologic disorder or condition involved with at least one short term cellular stress condition/process, which refers to a condition, in which there is a disturbance of normal functioning, any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with that person.
  • the methods, bifunctional hybrid-molecule, conjugate or complexes and compositions of the present disclosure may be suitable for any subject that may be any multicellular organism, specifically, any vertebrate subject, and more specifically, a mammalian subject, avian subject, fish or insect.
  • the therapeutic and non- therapeutic methods presented by the enclosed disclosure may be applicable to mammalian subjects, specifically, human subjects.
  • patient or “subject” it is meant any mammal that may be affected by the above-mentioned conditions, and to whom the treatment methods herein described is desired, including human, bovine, equine, canine, murine and feline subjects.
  • the subject is a human.
  • the present disclosure provides in an additional aspect thereof a therapeutically effective amount of at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the same, or any composition thereof for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder or condition in a subject in need thereof.
  • the bifunctional hybrid-molecule, conjugate or complex used as described herein may comprise: First (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amino acid sequence of Xaa (n) -Zaa-Xaa (m) -Zaa-Xaa (n) , as denoted by SEQ ID NO: 1.
  • Zaa is any aromatic amino acid residue.
  • “Zaa” as used herein is also denoted in the sequence listing with respect to SEQ ID NO: 1, as residue "Xaa 2 ” and as residue "Xaa 4 - .
  • Xaa is any amino acid residue.
  • n is zero or an integer between 1 to 7
  • m is an integer between 1 to 7, as discussed above in connection with the previous aspect of the present disclosure.
  • the bifunctional hybrid-molecule, conjugate or complex of the compositions disclosed herein further comprises a second component (b), at least one proteasome degradation mediating moiety.
  • the indicated use may be applicable for treating cancer. Specifically, an of the cancers disclosed by the present invention.
  • the present disclosure provides an effective amount of at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro- particle comprising the same, or any composition thereof for use in a method for inducing ubiquitination and proteasomal processing of NF- ⁇ B1 p105, thereby generating the NF- ⁇ B p50 in a cell or in a cell-free system comprising said NF- ⁇ B 1 p105.
  • the bifunctional hybrid-molecule, conjugate or complex used as described herein may comprise: First (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amino acid sequence of Xaa (n) -Zaa-Xaa (m) -Zaa- Xaa (n) , as denoted by SEQ ID NO: 1.
  • Zaa is any aromatic amino acid residue.
  • “Zaa” as used herein is also denoted in the sequence listing with respect to SEQ ID NO: 1, as residue “Xaa 2 ” and as residue “Xaa 4 ”, both refer to any aromatic amino acid residue as disclosed herein.
  • the sequence Xaa (n) -Zaa-Xaa (m) -Zaa- Xaa (n) is also equivalent toXaa (n) -Xaa 2 -Xaa (m) -Xaa 4 -Xaa (n), as also denoted by SEQ ID NO: 1.
  • Xaa is any amino acid residue.
  • n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7, as discussed above in connection with the previous aspect of the present disclosure.
  • the bifunctional hybrid-molecule, conjugate or complex of the compositions disclosed herein further comprises a second component (b), at least one proteasome degradation mediating moiety.
  • bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro- particle comprising the same, or any composition thereof used herein in the disclosed method are any of the bifunctional hybrid-molecule, conjugate or complex, disclosed by the resent invention in connection with other aspects.
  • a further aspect of the present disclosure elates to a peptide comprising the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1, wherein Zaa is any aromatic amino acid residue, wherein Xaa is any amino acid residue.
  • SEQ ID NO: 1 Zaa is any aromatic amino acid residue
  • Xaa is any amino acid residue.
  • n is zero or an integer between 1 to 7
  • m is an integer between 1 to 7, with the proviso that said peptide is not the peptide of SEQ ID NO: 4.
  • At least one of the interspacing amino acid residue/s Xaa (m) located between the two aromatic amino acid residues Zaa may be one or more amino acid residue/s having a non-polar side chain, also referred to herein as a non-polar amino acid.
  • Such residues may be any one of Valine (V, Val), Glycine (G, Gly), Leucine (L, Leu), Isoleucine (I, IIe), Methionine (M, Met), Phenylalanine (F, Phe), Tryptophane (W, Trp) and/or Proline (P, Pro).
  • the interspacing amino acid residue may be any hydrophobic amino acid residue, for example, Valine (V, Val), Leucine (L, Leu) and/or Isoleucine (I, IIe).
  • the peptide of the present disclosure may comprise at least two aromatic amino acid residues that are interspaced by at least one of Valine (V), Glycine (G) and/or Alanine (A).
  • the at least two aromatic amino acid residues of the peptide disclosed herein are interspaced by at least one Valine residue.
  • the peptide in accordance with the invention may comprise the amino acid sequence of Xaa (n) -Trp (W)-Xaa (n) -Val (V)-Xaa (n) -Trp (W)-Xaa (n), as denoted by SEQ ID NO: 7, or any mimetics thereof.
  • Xaa is any amino acid residue
  • n is zero or an integer between 1 to 7.
  • these amino acid residues may be any identical or different amino acid residues.
  • the at least one peptide of the present disclosure may comprise three to ten amino acid residues, specifically, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues.
  • the peptide of the present disclosure may comprise any amino acid residue, Xaa that is any one of Leu (L), Arg (R) and IIe (I) amino acid residues.
  • a variant of the heptapeptide of SEQ ID NO: 2 may be a peptide comprising the amino acid sequence IWVWLL, as dented by SEQ ID NO: 3, or any variants and derivatives thereof, or any conjugate, complex, chimera and composition thereof.
  • the peptide disclosed herein comprises the amino acid sequence RIFVFLL, as dented by SEQ ID NO: 9. Still further, in some embodiments, the peptide disclosed herein comprises the amino acid sequence RIYVFLL, as dented by SEQ ID NO: 10. In certain embodiments, the peptide disclosed herein comprises the amino acid sequence Citrulline-IWVWLL, as dented by SEQ ID NO: 15. In yet some further embodiments, the peptide disclosed herein comprises the amino acid sequence GRIWVWLL, as dented by SEQ ID NO: 16. In some embodiments, the peptide disclosed herein comprises the amino acid sequence RRRIWVWLL, as dented by SEQ ID NO: 27.
  • the peptide disclosed herein comprises the amino acid sequence WVW, or any variants and derivatives thereof.
  • the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise any of the peptides disclosed herein, for example, any of the peptides of SEQ ID NOs: 8, 11 to 28, and 52 to 59. It should be understood that all definitions that relate to polypeptides and derivatives disclosed in connection with other aspects of the invention are also applicable for the present aspect as well.
  • the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of’ “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
  • the phrase “at least one,” in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • ranging/ranges between" a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number "to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment.
  • Ub aldehyde (UbAl) was from BIOMOL. TNT ® T7 Quick Coupled Transcription/Translation System was from Promega. Synthesis of KPC1 -derived peptides was carried out by Bio Basic Inc. Recombinant Human VHL/ELOB/ELOC/CUL2/RBX1 complex was from R&D Systems. Restriction and modifying enzymes were from New England Biolabs. Oligonucleotides were from Syntezza Bioscience. C.B-17/IcrHsd-Prkdc scid mice were from Envigo. Analytical grade N,N-dimethyIformamide (DMF) was from Biotech. Resins were from Creosalus.
  • DMF N,N-dimethyIformamide
  • HCTU 1,3,3- tetramethyluronium hexafluorophosphate
  • HATU 1-[Bis(dimethylamino)methylene]-1H- 1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate]
  • DIPEA N.N-Diisopiopylethylamine
  • TIPS Triisopropylsilane
  • KPC1 ⁇ 1041-1061 and KPC1A968-974 amplified using primers containing the restriction sites EcoRI and Notl
  • KPC1A969-1314 and KPC1 ⁇ 1-973 amplified using primers containing the restriction sites BamHI and Xhol
  • KPC1 ⁇ 1-967 and WILVRLW-KPC1 ⁇ 1-1039 amplified using primers containing the restriction sites Ndel and Xhol or Bam HI and Notl, respectively
  • cDNA coding for p105-HA was cloned into the CMV-5-B expression vector (amplified using primers containing the restriction sites Notl and Kpnl).
  • the following cDNAs, coding for truncated species of KPC1 were cloned into the pT7b-6xHIS expression vector: KPC1 ⁇ 1-967; KPC1 ⁇ 1-974; WILVRLW-KPC1 ⁇ 1-1039; and WILVRLW-KPC1 ⁇ 1-1061 (were all amplified using primers containing the restriction sites Ndel and HindIII).
  • the following cDNAs were cloned into the NSPI-CMV MCS lentiviral expression vector: WILVRLW-KPC1 ⁇ 1- 1039; KPC1 ⁇ 1-1039; and RIWVWLL-KPC1 ⁇ 1-1039 (were amplified using primers containing the restriction site Xhol).
  • U87-MG and HEK293 were grown at 37°C and 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with penicillin-streptomycin and 10% fetal calf serum. Stable expression
  • HEK293 cells were transfected with various cDNAs coding for WT and the truncated species of KPC 1 -FLAG, or with an empty vector along with p105-HA. The transfection was carried out using the jetOPTIMUS transfection reagent. After 48 h, cells were lysed with RIPA buffer [150 mM NaCl, 50 mM Tris-FiCl (pFi 8.0), 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, and freshly added protease inhibitors cocktail]. Lysates were incubated with immobilized anti-FLAG at 4°C for 2 h.
  • the pLysS bacteria were grown at 37°C and brought to an O.D. of 0.7. Protein expression was induced by IPTG (500 mM). 4 h after induction, cells were lysed by sonication in a buffer containing 0.1 M NaCl, 20 mM Tris-HCl (pH 7.6), 10 mM ⁇ -mercaptoethanol, and EDTA-free protease inhibitor cocktail.
  • Exponentially growing U87-MG cells that stably express V0, KPC1-FLAG, WILVRLW- KPC 1 D 1 - 1039-FLAG, KPC1 ⁇ 1- 1039-FLAG, or RIWVWLL-KPC1 ⁇ 1 - 1039-FLAG were dissociated with trypsin and diluted to a concentration of 60x10 6 cells/ml in PBS. 6-10 weeks old C.B-17/IcrHsd-Prkdc scid , or NOD.Cg-Prkdc sc id Il2rg tmlWjl l SzJ (NSG) mice were injected subcutaneously with 6x10 6 cells /0.1 ml at both flanks.
  • Formalin-fixed xenografts tumors were embedded in paraffin. 5 pm thick sections were stained by rabbit anti-CD45 (1 :3,000) using the Ventana BenchMark ULTRA IHC/ISH system. Visualization was performed by the iVIEW DAB detection kit according to the manufacturer’s protocols.
  • HEK293 cells that stably express VHL-FLAG were transiently transfected with cDNA coding for HA-p105 using jetOPTIMUS transfection reagent, according to the manufacturer’s instructions. 24 hr after transfection, the growth medium was replaced with the fresh one, supplemented with the PROTAC molecules.
  • VHL-FLAG was immunoprecipitated with immobilized anti-FLAG at 4°C for 2 hr, followed by extensive washing in RIPA buffer. Proteins were resolved by SDS-PAGE and blotted onto nitrocellulose membrane. The HA-p105 was visualized with anti-NF-kB1, and VHL with anti-VHL antibodies. 10% of total cell lysates (TCL) were analyzed for the expression of p105 and VHL.
  • HEK293 cells that stably express VHL were transfected with cDNAs coding for HA-p105 and Myc-Ub. 6 hr after transfection, fresh medium that contained PROTACs molecules was added, and cells were moved to the hypoxic conditions (1.5% O2). After 16 hr, cells were lysed with RIPA buffer, proteins were resolved by SDS-PAGE, and blotted onto nitrocellulose membrane. FLAG- p105/FLAG-p50 were visualized using anti-FLAG, and VHL was visualized with anti-VHL antibodies.
  • HEK293 cells were seeded on a glass-bottomed 35mm dishes and were incubated in the presence of either Fluorescein isothiocyanate (FITC) or FITC-PROTAC for 16 hr. Cells were washed with PBS and stained with 4’-6’-diamidino-2-phenylindole (DAPI) in mounting medium. Imaging of the cells was carried out using the Zeiss LSM-700 confocal microscope.
  • FITC Fluorescein isothiocyanate
  • DAPI 4’-6’-diamidino-2-phenylindole
  • U87-MG VHL xenografts were grown in NSG mice for 3 weeks, as described under the "Tumorigenicity", followed by subcutaneous injection of DMSO or RIWVWLL-C(-FITC)-G- PEG-VHL PROTAC into the tumor surroundings. After 20 hr mice were visualized using in vivo imaging device (IVIS; Xenogen Corp., Waltham, MA). Further, mice were sacrificed and tumors were dissected and frozen in iso-pentane. Cryo-sections were stained with DAPI in mounting medium, and imaging was carried out using the Zeiss LSM-700 confocal microscope.
  • IVIS in vivo imaging device
  • the proteins were then digested overnight at 37°C by modified trypsin (Promega; dissolved in a solution of 1 M urea and 50 mM ammonium bicarbonate) at a ratio of 1:50 enzyme-to-substrate. A second similar amount of trypsin was added for additional 4 h. Proteomic analysis of the peptides was carried out as described elsewhere [9], [Hakim-Eshed, etal., Proc. Natl. Acad. Sci. U. S. A. 117, 18661-18669 (2020)].
  • Peptides were prepared by SPPS either manually in Teflon filter fitted syringes (purchased from Torviq) or by using an automated peptide synthesizer (CS336X, CSBIO). Analytical grade N,N- dimethylformamide (DMF) was purchased from Biotech.
  • Resins were purchased from Creosalus, protected amino acids were purchased from GL Biochem and activating reagents, O-(1H-6- Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), 1- [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidhexafluorophosphate (HATU)] were purchased from Luxembourg Bio Technologies. Chemicals were purchased from Aldrich, Strem Chemicals and Alfa Aesar.
  • Analytical HPLC was performed on a Thermo instrument (Dionex Ultimate 3000) using analytical column X Select (Waters, CSH C18, 3.5 pm, 4.6 x 150 mm) at a flow rate of 1.2 mL/min and semi-preparative HPLC was performed on a Thermo instrument (Dionex Ultimate 3000) using Phenomenex Jupiter C18 10 pm, 300 A, 250 x 10 mm column, at a flow rate of 4 mL/min. All synthetic peptides were purified by HPLC and characterized by mass spectrometry using LCQ Fleet Ion Trap (Thermo Scientific). All calculated masses have been reported as an average isotope composition. Buffer A: 0.1% TFA in water; buffer B: 0.1% TFA in acetonitrile.
  • New Ubiquitin ligase KPC1- derived peptides comprising the W-X-W motif A heptapeptide containing amino acid residues WILVRLW, also denoted by SEQ ID NO:4, derived from KPC1 ubiquitin ligase positions 968-974 (of the Wild type KPC1 amino acid sequence as denoted by SEQ ID NO: 6), was previously identified by the inventors as the binding site of the ligase and its substrate p105. The inventors further demonstrated that truncated species of KPC1 that contain the 7 amino acid sequence WILVRLW-KPC1 ⁇ 1-1039, still display a full ligase activity in cell free system and in cells as described in WO 2020/110114 [10].
  • the 7 amino acids peptide WILVRLW inhibited the ubiquitination of p105 by KPC L ⁇ 1 -967 (SEQ ID NO: 68) to a larger extent compared to 6 amino acids ILVRLW (SEQ ID NO: 64) peptide that lacks the first tryptophan (lanes 3-5).
  • RIWVWLL also denoted by SEQ ID NO: 2
  • SEQ ID NO: 2 displayed a dramatically increased inhibition of the ubiquitination of p105, even stronger than the WT peptide (lanes 9-11, as compared to lanes 6-8).
  • WILVRLW or RIWVWLL attract leukocytes into a xenograft tumor model and inhibit tumor growth
  • a tumor xenograft model was next used. More specifically, using a SCID mice model, various U87 (glioblastoma)-based xenografts were generated.
  • KPC1-LLAG also denoted by SEQ ID NO: 66
  • RIWVWLL-KPC1 ⁇ 1 - 1039- FLAG also denoted by SEQ ID NO: 65
  • WILVRLW-KPC1 ⁇ 1 - 1039-FLAG also denoted by SEQ ID NO: 62
  • KPC1 ⁇ 1 - 1039-FLAG SEQ ID NO: 40
  • WT KPC1 and its truncated species that lack most of the sequence of the enzyme - but contain either the WT binding domain (WILVRLW, SEQ ID NO: 4) or the scrambled one (RIWVWLL, SEQ ID NO: 2) - elicit similar alterations in the protein profile of the cells in which they are expressed
  • the proteome of the tumors shown in Figure 2 were analyzed by mass spectrometry. The reason was that the deleted part of the enzyme might affect cellular processes that are not affected by the deleted species.
  • proteolysis targeting chimera (PROTAC) molecules were designed based on the 7 amino acid sequences mentioned in Examples 1 and 2, and a VHL-binding head ( Figure 4A).
  • the various peptides were conjugated to the VHL ligand of Formula I, via a PEG liker, optionally with a further linking peptide of at least one of: at least one glycine (G), at least one serine (S), at least one Cysteine (C) or any combinations thereof.
  • Figures 5A and 5B illustrate the synthesis of the peptide based PROTACs.
  • CTC 2-Chlorotritylchloride
  • PROTAC 1 The synthesis of PROTAC 1 was carried out following the general procedure described herein, with ⁇ 56% isolated yield.
  • Figure 6A shows HPLC-MS analysis of the purified PROTAC 1 with the observed mass 1686.4 ⁇ 0.1 Da (calcd 1687.4 Da, average isotopes).
  • PROTAC 3 was carried out following the general procedure with ⁇ 54 isolated yield.
  • Figure 6C shows HPLC-MS analysis of the purified PROTAC 3 with the observed mass 1686.3 ⁇ 0.5 Da (calcd 1687.4 Da, average isotopes).
  • PROTAC 4 (Formula XIII, also shown in Fig.4Av), Acetyl-RIWVWLLSG-PEG- Formula XIII
  • PROTAC 4 was carried out following the general procedure described herein with ⁇ 46 isolated yield.
  • Figure 6D shows HPLC-MS analysis of the purified PROTAC 4, with the observed mass 1727.5 ⁇ 0.1 Da (calcd 1728.4 Da, average isotopes).
  • PROTAC 5 was carried out following the general procedure described herein with - 51 isolated yield.
  • Figure 6E shows HPLC-MS analysis of the purified PROTAC 5, IWVWLLSG-PEG-VHL ligand with the observed mass 1529.9 ⁇ 0.1 Da (calcd 1530.2 Da, average isotopes).
  • Peptide 7 (SEQ ID NO: 17) was carried out following the general procedure with ⁇ 52 isolated yield. To a solution of peptide 7 (5.0 mg, 1.0 equiv.) in 6M Gn.HCl/200 mM phosphate buffer (pH 7.3) was added FITC-maleimide (1.5 mg, 1.2 equiv.) at 0 °C and kept for 1 hour. After 1 hour, the reaction mixture was diluted with 6M Gn.HCl/200 mM phosphate buffer and purified using semi preparative to obtain PROTAC 8 in 76% yield. The synthesis of PROTAC 8 is also illustrated by Figure 5B.
  • Figure 6G shows HPLC-MS analysis of the purified PROTAC 8, RIWVWLLC(FITC)G-PEG- VHL ligand with the observed mass 2129.4 ⁇ 0.1 Da (calcd 2129.8 Da, average isotopes).
  • WILVRLW- and RIWVWLL-pVHL ligand-based PROTACs induce ubiquitination of p105 by the E3 Ub ligase pVHL in a reconstituted cell-free system
  • the PROTAC1 WILVRLW-SG-PEG-VHL stimulates the ubiquitination of p105 in a dose- dependent manner (Figure 4Bi).
  • a series of substitutions in the peptide, as well as in the linker part of the PROTAC was generated. Shortening the distance between two tryptophan residues in the peptide part of the PROTAC 3 (RIWVWLL- SG-PEG-VHL), enhanced ubiquitination of p105 compared to ubiquitination by WILVRLW-SG- PEG-VHL PROTAC 1 that has native sequence (Figure 4Bii, compare lanes 8 and 9).
  • PROTACs that contain the SerGly linker stimulate the ubiquitination of p105 more efficient than the PROTAC that contains the G linker ( Figure 4BB, compare lanes 5 and 6) which highlights the importance of the linker in the conjugation-stimulating activity. Also, removal of the two Trp (W) residues weakened the activity significantly, even in the presence of the GlySer linker ( Figure 4BB, lane 4).
  • PROTAC RIWVWLL-SG-PEG-VHL mediates the strongest ubiquitination ( Figure 4BB and 4Biii).
  • the seven amino acid-based PROTACs stimulate the interaction between p105 and pVHL and enhance the generation ofp50 in cells , and consequently restrict cell growth After confirming the activity of the PRPTACs in a cell-free system, it was important to test their effects in cells.
  • the RIWVWLLC(-FITC)G- PEG-pVHL-Ligand PROTAC 8 which contains the fluorophore FITC ( Figures 7Ai, 5B and 6G), was synthesized as described in Example 4 and Figure 5B.
  • PROTACs ability to restrict growth of U87-MG cells.
  • the RIWVWLL- and WILVRLW-based PROTACs were added to the growth medium of the cells (as described under Experimental procedures ) and tracked their growth for three days. In all measuring points, the PROTACs inhibited growth rate of the cells (in a statistically significant manner) compared to DMSO-treated cells ( Figure 7D), thereby demonstrating the feasibility of using the PROTACs of the present disclosure in cancer therapy.
  • PROTAC molecule infiltrates the xenografts derived from U87-MG cell in NSG mice
  • U87-MG glioblastoma cells were inoculated to NSG mice.
  • 25 mg/kg/tumor PROTAC labeled with FITC were injected to the tumor area. 24 hr later the tumors were visualized using in vivo imaging system (IVIS).
  • IVIS in vivo imaging system
  • Peptide KVS-4 that comprises the original RIWVWLL sequence (SEQ ID NO: 4), with the SG linker (as denoted by SEQ ID NO: 8), peptide SKV-17 having the sequence of the original peptide of SEQ ID NO: 4, with no linker, and peptide KVS-19, comprising the wildtype sequence as denoted by SEQ ID NO: 4, with the SG linker (SEQ ID NO: 28) were used as controls.
  • Figure 9 discloses an example for p105 ubiquitination by the various peptides.
  • an in vitro translated and 35 S-labeled p105 was ubiquitinated by purified KPC1 ⁇ 1-967 (SEQ ID NO: 67) in a cell-free system in the presence of the indicated peptides.
  • the numbers indicate the peptide concentration in mM.
  • the fraction of remained free unconjugated p105 for each condition is compared to a system where a peptide was not added (arbitrarily designated as 1), as shown in Figures 9A, 9B and 9C.
  • Figure 9D provides a schematic representation of the various peptides sequences and activity.
  • Trp amino acid residues as in the original peptide of SEQ ID NO: 7 and 4 can be replaced by any aromatic amino acid residue (e.g., F, and) Y. Still further, it appears that the initial Arg residue is required and addition of two or more Arg resides may approve the effectivity of the peptide.
  • the effect of the PROTAC molecules on xenografts derived from human tumors The prototypic PROTAC molecules of Formulas IV, V, VI and VII are tested in several human tumor models in mice.
  • An inactive PROTAC is used as a control. More specifically, an amount of approximately 100 microliters of various concentrations of the different PROTACs ranging between 10 to 500 micromolar, is injected into the tumor bed about 1 to 4 times every day and every other day for two weeks after the tumors solidify. The following parameters are monitored in the treated animals: tumors size, tumors volume, cytokines secretion by the tumors, infiltration of lymphocytes to the tumors, expression of PD-L1 by the tumor and expression of tumor suppressors by the tumors.

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Abstract

The present disclosure relates to bifunctional hybrid-molecules comprising (a) at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, the peptide comprises the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1, wherein Zaa is any aromatic amino acid residue, and Xaa is any amino acid residue, n is zero or an integer between 1 to 7, and m is an integer between 1 to 7; and (b) at least one proteasome degradation mediating moiety, specifically, E3 ligase requiting moiety. The present disclosure further provides any conjugates, complexes, and compositions of the bifunctional hybrid-molecules and uses thereof in enhancing ubiquitination and proteasomal processing of NF-kB p105 to p50, specifically for treating pathological disorders, e.g., cancer.

Description

UBIQUITIN LIGASE KPC1-PEPTIDE BASED PROTEOLYSIS TARGETING CHIMERAS (PROTAC) AND USES THEREOF
TECHNOLOGICAL FIELD
The present disclosure relates to NF-κB modulators. More specifically, the present disclosure provides bifunctional hybrid-molecules, conjugates, complexes, and compositions enhancing ubiquitination and proteasomal processing of NF-κB 1 p105 to p50 and their uses for treating pathological disorders, e.g., cancer.
BACKGROUND ART
References considered to be relevant as background to the presently disclosed subject matter are listed below:
[1] J. A. Didonato, F. Mercurio, M. Karin, NF-κB and the link between inflammation and cancer. Immunol. Rev. 246, 379-400 (2012).
[2] M. S. Hayden, S. Ghosh, NF-κB, the first quarter-century: Remarkable progress and outstanding questions. Genes Dev. 26, 203-234 (2012).
[3] K. Taniguchi, M. Karin, NF-B, inflammation, immunity and cancer: Coming of age. Nat. Rev. Immunol. 18, 309-324 (2018).
[4] J. C. Betts, G. J. Nabel, Differential regulation of NF-kappaB2(p100) processing and control by amino-terminal sequences. Mol. Cell. Biol. 16, 6363-6371 (1996).
[5] V. J. Palombella, O. J. Rando, A. L. Goldberg, T. Maniatis, The ubiquitinproteasome pathway is required for processing the NF-κB 1 precursor protein and the activation of NF-κB . Cell 78, 773-785 (1994).
[6] Y. Xia, S. Shen, I. M. Verma, NF-κB, an active player in human cancers. Cancer Immunol. Res. 2, 823-830 (2014).
[7] W. Weichert, et al, High expression of RelA/p65 is associated with activation of nuclear factor- κB -dependent signaling in pancreatic cancer and marks a patient population with poor prognosis. Br. J. Cancer 97, 523-530 (2007).
[8] S. Shukla, et al, Nuclear factor- kB/r65 (Rel A) is constitutively activated in human prostate adenocarcinoma and correlates with disease progression. Neoplasia 6, 390-400 (2004).
[9] Y. Kravtsova-Ivantsiv, et al, KPC1 -mediated ubiquitination and proteasomal processing of nf-Kbl p105 to p50 restricts tumor growth. Cell 161, 333-347 (2015). [11] Kravtsova-Ivantsiv, Y. et al. PNAS 117 (47), 29823-29831 (2020).
[12] S. Kotoshibai, T. Kamura, T. Hara, N. Ishida, K. I. Nakayama, Molecular dissection of the interaction between p27 and Kipl ubiquitylation-promoting complex, the ubiquitin ligase that regulates proteolysis of p27 in G1 phase. J. Biol. Chem. 280, 17694-17700 (2005).
[13] Y. Haupt, Y. Barak, M. Oren, Cell type-specific inhibition of p53-mediated apoptosis by mdm2. EMBO J. 15, 1596-1606 (1996).
[14] Y. Haupt, R. Mayat, A. Kazazt, M. Orent, letters to nature Mdm2 promotes the rapid degradation of p53. Nature 387, 296-299 (1997).
Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.
Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.
BACKGROUND OF THE INVENTION
NF-κB is a key transcriptional regulator involved in inflammation and cell proliferation, survival, and transformation. Upregulation of NF-κB is frequently observed in a variety of tumors, and probably play a role in malignant transformation. The mechanism(s) that underlie the pro-tumorigenic activity of NF-κB involve upregulation of expression of pro-proliferative and anti-apoptotic genes, as well as stimulation of the inflammatory process [1, 2, 3].
The NF-κB family consists of five different proteins - three Rel proteins (RelA, RelB, and cRel), and two proteins - p50 and p52 - which are derived from limited, UPS-mediated - processing of longer precursors, p105 (NF-κB 1) and p100 (NF-κB2), respectively. Members of the two groups generate different heterodimers which trigger unique transcriptional programs [2, 4, 5]. In most cases, the tumorigenicity is related to the p50●p65 dimer [6, 7, 8]. Several key steps in its activation are mediated by the ubiquitin (Ub) system. One crucial step is limited proteasomal processing of the NF-κB 1 precursor p105 to the p50 active subunit of the ‘canonical’ heterodimeric transcription factor p50●p65. KPC1 was previously identified as the Ub ligase (E3) that binds to the ankyrin repeats domain of p105, ubiquitinates it, and mediates its processing both under basal conditions and following signalling [9]. In addition, the inventors previously showed that KPC1 interacts with p105 via a short, seven amino acids - WILVRLW - sequence (residues 968-974, also denoted by SEQ ID NO: 4) in KPC1 [10]. Overexpression of p50 resulted in a strong tumor- suppressive effect which was due to up-regulation of various tumor suppressors [9], and modulation of the tumor microenvironment by recruiting and activating the immune system [11]: excess of KPC1 or p50 up-regulates expression of CCL3, CCL4, and CCL5, which are pro- inflammatory chemokines, which in turn recruit to the tumor NK cells and macrophages that contribute to the tumor-suppressive effect. Also, p50 down-regulates the expression of the immune checkpoint programmed cell death-ligand 1 (PD-L1) [11]. Furthermore, the inventors previously showed that a truncated KPC1 containing the 7 amino acids and the minimal domains required for its enzymatic activity, is sufficient to stimulate ubiquitination of p105 and to suppress growth of model human tumors in mice.
Interestingly, sites of Ub ligases that bind their cognate substrates are rather long. For example, the interaction site of KPC1 which regulates the degradation of p27 at the G1 phase of the cell cycle, spans 766 amino acids at the N-terminal part of the molecule [12]. Another example is the interaction site of MDM2 with p53. It was shown that deletion of the N-terminal 61 am aicniods of the ligase abrogated its interaction with the tumor suppressor [13, 14].
WO 2020/110114 [10] by the preset inventors, discloses a peptide comprising the seven amino acids - WILVRLW - sequence (residues 968-974) of KPC1, conjugates or chimeras of this peptide and uses thereof in treating cancer. These conjugates or chimeras comprise the KPC1 -derived peptide linked to a RING domain of KPC1, or to molecules or peptides that recruits E3 ligases. There is need in the art to develop effective drugs that enhance processing of the NF-κB 1 precursor p105 to the p50 active subunit, for the treatment of NF-κB 1 associated disorders.
SUMMARY OF THE INVENTION
A first aspect of the present disclosure relates bifunctional hybrid-molecule, conjugate or complex comprising the at least two following components: component (a), comprises at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue. In some embodiments, the peptide, that may be also referred to herein as a bio-scaffold, comprises the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1. More specifically, Zaa, as used herein is any aromatic amino acid residue and Xaa is any amino acid residue. In some embodiments, "m" is an integer between 1 to 7, and "n" as used herein is zero or an integer between 1 to 7. Component (b), of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least one proteasome degradation mediating moiety.
A further aspect of the invention relates to a composition comprising at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the bioactive molecule of the present disclosure. In some embodiments, the composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
In some embodiments, the bifunctional hybrid-molecule, conjugate or complex comprises: (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amino acid sequence of Xaa(n)-Zaa-Xaa(m)- Zaa-Xaa(n), as denoted by SEQ ID NO: 1. It should be noted that Zaa is any aromatic amino acid residue. Still further, in some embodiments, Xaa is any amino acid residue. In some specific embodiments, n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7, as discussed above in connection with the previous aspect of the present disclosure; and (b), at least one proteasome degradation mediating moiety.
A further aspect of the present invention relates to a method for inducing ubiquitination and proteasomal processing of NF-κB1 p105, thereby generating the NF-κB p50 in a cell or in a cell- free system comprising said NF-κB 1 p105. More specifically, the method comprising the step of contacting said cell or said cell-free system with an effective amount of at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the same, or any composition thereof. In some embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise: (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue. The peptide comprises the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1. It should be noted that Zaa is any aromatic amino acid residue. Still further, in some embodiments, Xaa is any amino acid residue. In some specific embodiments, n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7. The bifunctional hybrid-molecule, conjugate or complex of the methods disclosed herein further comprises a second component (b), at least one proteasome degradation mediating moiety.
A further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder or condition in a subject in need thereof. More specifically, the method comprising the step of administering to said subject a therapeutically effective amount of at least one bifunctional hybrid- molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the same, or any composition thereof. In some embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise: (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amin aocid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1. It should be noted that Zaa is any aromatic amino acid residue. Still further, in some embodiments, Xaa is any amino acid residue. In some specific embodiments, n is zero or an integer between 1 to 7, and m is an integer between 1 to 7. The bifunctional hybrid- molecule, conjugate or complex of the compositions disclosed herein further comprises a second component (b), at least one proteasome degradation mediating moiety. The present disclosure further provides peptides that recruit the NF-κB1 p105.
Still further aspect of the present disclosure relates to therapeutically effective amount of at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro- particle comprising the same, or any composition thereof for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder or condition in a subject in need thereof, and/or in a method for inducing ubiquitination and proteasomal processing of NF-κB1 p105, thereby generating the NF-κB p50 in a cell or in a cell-free system comprising said NF-κB 1 p105. These and other aspects of the present disclosure will become apparent by the hand of the following description.
BRIEF DESCRIPTION OF THE DRAWINGS
In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
Figure 1A-1B. Ubiquitination of p105 by KPC1 is dependent on amino acid sequence WILVRLW in KPC1
Fig. 1Ai-1Aii. In vitro translated 35S-labeled p105 was ubiquitinated by the indicated purified species of KPC1 in a reconstituted cell-free system as described under Experimental procedures. Fig. Ai. Ubiquitination of p105 by KPC1Δ1-967-Hisx6 (lanes 2-4) andKPC1Δ1-973-Hisx6 (lanes 5-7). The numbers indicate the amount of the added ligase in μg. It was calculated by dividing the amount of p105 and its monoubiquitinated species (generated by the E2 present in all reaction mixtures) remained under each condition by the amount of p105 (and its monoubiquitinated species) in lane 1. Fig. 1Aii. Ubiquitination of p105 using the following species of KPC1: KPC1Δ1-967 (lane 2); KPC1Δ1-974 (lane 3); WIFVRFW-KPC1Δ1-1039 (lane 4); and WIFVRFW-KPC1Δ1-1061 (lane 5). Each enzyme was added at 0.5 μg. Also shown is the fraction of the free p105 remained for each condition (compared to a system to which KPC1 was not added; lane 1; arbitrarily was designated as 1). Fig. IB. Addition of either WILVRLW or RIWVWLL peptide inhibits ubiquitination of p105 by KPC1Δ1-967 in a dose-dependent manner. In vitro translated and 35S-labeled p105 was ubiquitinated by purified KPC1Δ1-967 (lanes 2-12, SEQ ID NO: 68, in a cell-free system in the presence of the indicated peptides derived from KPC1: ILVRLW (lanes 3-5, SEQ ID NO: 5); WILVRLW (lanes 6-8, SEQ ID NO: 4); and RIWVWLL (lanes 9-11, SEQ ID NO: 2). The numbers indicate the peptide concentration in mM. The fraction of remained free unconjugated p105 for each condition is as described under Ai. Visualization of p105 and its conjugates was carried out using Phosphor Imaging. Also shown is the fraction of the free p105 remained for each condition (compared to a system to which a peptide was not added; lane 2; arbitrarily was designated as 1).
Figure 2A-2B. Overexpression of truncated species of KPC1 that contain the amino acid sequence WILVRLW or RIWVWLL attract leukocytes into a xenograft tumor model and inhibits tumor growth
Fig. 2Ai-2Aiv. Fig. 2Ai. Growth rates (* represents P-value <0.05) and Fig. 2Aii weights (at the end of the experiment, 25 days after inoculation) of U87-MG cells-derived xenografts grown in SCID mice. The tumors express either KPC1-FLAG, WILVRLW-KPC 1 A 1 - 1039-FLAG, RIWVWLL- KPC 1 Al - 1039-FLAG, or KPC1Δ1-1039-FLAG proteins (SEQ ID NOs: 66, 62, 65, and 40, respectively). Control cells were transfected with an empty vector. Fig 2Aiii. Tumors derived from U87-MG at the end of the experiment. Fig. 2Aiv.The different species of KPC1 stably expressed in the U87-MG cells used in the experiment. Western blotting of extracts derived from the different cells was carried out using an anti-FLAG antibody.
Fig. 2B. Tumors’ sections expressing the indicated KPC1 species attract leucocytes as is evident from immunohistochemical staining with anti-CD45.
Figure 3A-3B. Proteome analysis of glioblastoma xenografts expressing different species of KPC1
Fig. 3A. Euclidean Heat map diagram presenting an unsupervised hierarchical clustering of the cellular proteome under expression of the different KPC1 species. The clustering was based on the intensities of the identified proteins (low intensity - green, high intensity - red), and was generated by the Perseus software.
Fig. 3B. Functional analysis of proteins that are up- (red) or down- (green) regulated (compared to tumors transfected with an empty vector with a p-value <0.05). Shown are selected annotation clusters with false discovery rate (FDR) <0.05 (the numbers on the flanks of each column represent the FDR of the indicated cluster). Functional analysis was carried out using the string software [Szklarczyk D, et ak, Nucleic Acids Res. Jan 8;49(D1):D605-12 (2021)]. Proteome analysis was carried out on the xenografts described under Figure 2. The raw proteomic data are disclosed by the present inventors, in Goldhirsh G. et al., Proc Natl Acad Sci U S A.118(49)(Dec 7, 2021). Figure 4A-4C. PROTACs containing the peptides WILVRLW or RIWVWLL induce ubiquitination of p105 by the E3 ligase pVHL in a reconstituted cell-free system Fig. 4Ai— 4Avi. Schematic structure of the components of different PROTACs.
Fig. 4Ai. shows PROTAC 1 of Formula V, that comprises the peptide of SEQ ID NO: 28. Fig. 4Aii. shows PROTAC 2 of Formula XII, that comprises the peptide of SEQ ID NO: 42. Fig. 4Aiii. shows PROTAC 6 of Formula XIV, that comprises the peptide of SEQ ID NO: 43. Fig. 4Aiv. shows PROTAC 5 of Formula VI, that comprises the peptide of SEQ ID NO: 39. Fig. 4Av. shows PROTAC 4 of Formula XIII, that comprises the peptide of SEQ ID NO: 8, with acetylated Arg. Fig. 4Avi shows PROTAC 3 of Formula IV, that comprises the peptide of SEQ ID NO: 8.
Fig. 4Bi-4BiiL In vitro translated 35S-labeIed p105 was ubiquitinated by purified VHL complex mediated by PROTAC molecules. Fig. 4Bi. dose dependent ubiquitination of in vitro translated 35S-labeIed p105 by WILVRLW-SG-PEG-VHL PROTAC 1 molecule (Fig. 4Bii) and (Fig. 4Biii) ubiquitination of in vitro translated and 35S-labeIed p105 by VHL complex (4Bii, lanes 2-9, and 4Biii, lanes 2-6) in the presence of different PROTAC molecules (PROTAC 2, 6, 5, 4, 3, 1, respectively), as indicated (4Bii, lanes 3-9, and 4Biii, lanes 3-6).
Fig. 4C. In vitro processing of p105 to p50 by Fr2 increases in the presence of different concentrations of WILVRLW-SG-PEG-VHL PROTAC 1 (lanes 1-3). Visualization was carried out using Phosphorimaging.
Figure 5A-5B. Schematic representation of the PROTACs’ synthesis Fig. 5A. Schematic representation of the peptide-based PROTACs’ synthesis.
Fig. 5B. Attachment of the fluorophore FITC to the PROTAC RIWVWLLCG-PEG-pVHL ligand.
Figure 6A-6G. HPLC-MS analysis of the purified PROTACs
Fig. 6A. HPLC-MS analysis of the purified WILVRLWSG-PEG-pVHL ligand PROTAC1 with an observed mass of 1686.4 ± 0.1 Da (calculated 1687.4 Da, average isotopes).
Fig. 6B. HPLC-MS analysis of the purified ILVRLSG-PEG-pVHL ligand PROTAC2 with an observed mass of 1313.9 ± 0.2 Da (calculated 1314.9 Da, average isotopes).
Fig. 6C. HPLC-MS analysis of the purified RIWVWLLSG-PEG-pVHL ligand PROTAC3 with an observed mass of 1686.3 ± 0.5 Da (calculated 1687.4 Da, average isotopes).
Fig. 6D. HPLC-MS analysis of the purified Acetyl-RIWVWLLSG-PEG-pVHL ligand PROTAC 4, with an observed mass of 1727.5 ± 0.1 Da (calculated 1728.4 Da, average isotopes). Fig. 6E. HPLC-MS analysis of the purified IWVWLLSG-PEG-pVHL ligand PROTAC5 with an observed mass of 1529.9 ± 0.1 Da (calculated 1530.2 Da, average isotopes).
Fig. 6F. HPLC-MS analysis of the purified IWVWLLS-PEG-pVHL ligand PROTAC6 with an observed mass of 1473.4 ± 0.4 Da (calculated 1473.1 Da, average isotopes).
Fig. 6G. HPLC-MS analysis of the purified RIWVWLLC(-FITC)G-PEG-pVHL ligand PROTAC8 with an observed mass of 2129.4 ± 0.1 Da (calculated 2129.8 Da, average isotopes). Figure 7A-7D. PROTACs that contain the WILVRLW or RIWVWLL peptides stimulate interaction between p105 and pVHL and subsequent processing of p105 to p50, and restrict cell growth
Fig. 7Ai-Aii. The PROTAC RIWVWLL-C(-FITC)G-PEG-pVHL(L) is cell permeable. Fig. 7Ai. Schematic representation of the PROTAC RIWVWLL-C(-FITC)G-PEG-pVHL(L). Fig. 7Aii. Confocal microscopy imaging of HEK293 cells 24 h after addition of the RIWVWLL-C(FITC)G- PEG-pVHL ligand PROTAC to the growth medium (25 mM).
Fig. 7Bi-Biv. The interaction between p105 and pVHL in cells is stimulated by WILVRLW or RIWVWLL-based PROTACs (25 pM). HEK293 cells that stably express pVHL-FLAG were transfected with p105-HA (lanes 1-9). pVHL-FLAG was immunoprecipitated from the cell lysate using immobilized anti-FLAG. p105-HA (Fig. 7Bi) and pVHL-FLAG (Fig. 7Bii) were visualized using the appropriate antibodies as indicated. (Fig. 7Biii) and (Fig. 7Biv) display the two proteins in TCL.
Fig. 7Ci-Cii. WILVRLW and RIWVWLL-based PROTACs enhance cellular processing of p105 to p50. HEK293 cells that stably express pVHL-FLAG were transfected with cDNAs coding for FLAG-p105 along with Myc-Ub. WILVRLW or RIWVWLL-based PROTACs (25 pM) were added to the growth medium as indicated. FLAG-p105 and FLAG-p50 were visualized using anti- FLAG antibody (Fig. 7Ci). Actin was used as a loading control (Fig. 7Cii).
Fig. 7Di-Dii. WILVRLW or RIWVWLL-based PROTACs restrict growth of U87-MG cells. Fig. 7Di. Growth curve of U87-MG cells in the presence of WILVRLWSG-PEG-pVHL(L) PROTAC (25 pM). Fig. 7Dii. Similar to 7Di, but in the presence of RIWVWLLSG-PEG-pVHL(L) PROTAC. (L) denotes - ligand. * represents P- value <0.05.
Figure 8A-8B. PROTAC that contains RIWVWLL peptide, infiltrates the tumor xenografts Lig. 8A. Xenografts derived from U87-MG cells were grown in NSG mice. After tumors were established, DMSO or RIWVWLL-C(-FITC)-G-PEG-VHL PROTAC were injected to the tumor surroundings. 24 hr later mice were visualized using IVIS device, following by sacrificing, and dissection of tumors. Fig. 8B. The slides were prepared using cryotechnology and visualized under confocal microscope.
Figure 9A-9D. Peptide optimization
Figs. 9A-9C. In vitro translated 35S-labeled p105 was ubiquitinated by purified KPC1Δ1-967 (SEQ ID NO: 67) in a reconstituted cell-free system with the addition of permutated peptides derived from the peptide RIWVWLL (as denoted by SEQ ID NO: 4). In vitro translated and 35S- labeled p105 was ubiquitinated by purified KR01D1-967 in a cell-free system in the presence of the indicated peptides. The numbers indicate the peptide concentration in mM. The fraction of remained free unconjugated p105 for each condition is compared to a system where a peptide was not added; lanes 2; arbitrarily was designated as 1. Visualization of p105 and its conjugates was carried out using Phosphorimaging. Fig. 9A. shows peptides KVS-4, KVS-1, KVS-5, KVS-3. Fig. 9B. shows peptides KVS-4, KVS-2A, KVS-2B, KVS-2C. Fig. 9C. shows peptides KVS-4, KVS- 2D, KVS-6, KVS-7. Fig. 9D. Schematic representation of the peptides used in the experiment shown in Figs 9A-9C. The peptides comprise the ami ancoid sequence as denoted by SEQ ID NO: 8 (KVS-4), SEQ ID NO: 44 (KVS-1), SEQ ID NO: 45 (KVS-5), SEQ ID NO: 46 (KVS-3), SEQ ID NO: 47 (KVS-2A), SEQ ID NO: 48 (KVS-2B), SEQ ID NO: 49 (KVS-2C), SEQ ID NO: 50 (KVS-2D), SEQ ID NO: 51 (KVS-6), SEQ ID NO: 17 (KVS-7, is a cyclic form of the peptide of SEQ ID NO: 17).
DETAILED DESCRIPTION OF THE INVENTION
The inventors have previously shown that the Ub ligase KPC1 conjugates Ub to p105, the NF-κB long precursor, resulting in its limited proteasomal processing to the p50 active subunit of the mature transcriptional regulator [9]. Furthermore, we have shown that excess of p50 which results from overexpression of KPC1, exerts a strong tumor-suppressive effect mediated by several mechanisms, mostly related to recruitment of the immune system [11]. In the current study we identified the domain in KPC1 to which p105 binds, and found that it is an exceptionally short, seven amino acids stretch that comprises residues 968-974 in the KPC1 sequence (WILVRLW). A synthetic peptide that spans this sequence, inhibited KPC1 -mediated conjugation of Ub to p105 (Figure IB). A truncated species of the enzyme that lack the major part of the protein (amino acids 1039 out of 1314 residues), but contains the RING finger domain necessary for binding the E2 component of the conjugation machinery and the seven amino acids binding domain, can conjugate Ub to p105 (Figure lAii), suppress tumor growth (Figure 2A), and attract leucocytes to the tumor (Figure 3B). Proteomic analysis revealed that the seven amino acids binding domain-containing short KPC1 species, altered the expression of several sets of proteins related to tumorigenesis in a similar way induced by WT KPC1 (Figure 3). An interesting question related to the ability of the truncated enzyme to catalyze conjugation of p105 is why such a long protein is necessary at all. One answer is that p105 is not the only substrate of the ligase, and other substrates such as p27 [12] are recognized by different domains.
Based on this information, the inventors synthesized a PROTAC that contains the binding sequence, with the hope that it will generate excess of p50 and serve as a prototype for a tumor- suppressive modality. Indeed, the PROTAC simulated conjugation of Ub to p105 in a reconstituted cell-free system (Figure 4B) and processing of p105 to p50 in cells. Further, it restricted cell growth when added to the growth medium.
When attempting to optimize the binding sequence and identify critical residues within it, we noted a discrepancy between the activity of a scrambled peptide (RIWVWLL, SEQ ID NO: 2) in cell- free system and in cells/tumors. Thus, the peptide was a slightly better competitor than its WT counterpart (WILVRLW, SEQ ID NO: 4) in a cell-free reconstituted conjugation system (Figure IB). Also, when it was part of a PROTAC, it stimulated conjugation of Ub to p105 more efficiently than the WT peptide-based PROTAC (Figure 4Bii), although in suppressing tumors (Figure 2) and altering the cellular proteome to display a “tumor-suppressive“ landscape (Figure 3), it was a bit less potent than the wildtype peptide. This may require further optimization, but clearly demonstrates the feasibility of using PROTACs based on the peptide of SEQ ID NOs: 1 and 2. Thus, a first aspect of the present disclosure relates bifunctional hybrid-molecule, conjugate or complex comprising the following components: The first component (a), comprises at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue. In some embodiments, the peptide, that may be also referred to herein as a bio- scaffold, comprises the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1. More specifically, Zaa, as used herein is any aromatic amino acid residue and Xaa is any amino acid residue (that may be any of the 20 amino acids, including aromatic amino acids, or any derivatives or mimetics thereof). It should be understood that "Zaa" as used herein is also denoted in the sequence listing with respect to SEQ ID NO: 1, as residue "Xaa2" and as "Xaa4", and refer to any aromatic amino acid residue as disclosed herein. Thus, in some embodiments the sequence Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n) is also equivalent to Xaa(n)-Xaa2-Xaa(m)-Xaa4-Xaa(n), both are denoted by SEQ ID NO: 1. It should be understood that the at least two aromatic amino acid residues, or any derivatives or mimetics thereof, Zaa, of the peptide disclosed herein, may be either identical, or different aromatic amino acids. Similarly, the interspacing amino acid residue/s, Xaa(m), may be any amino acid residue, or any derivatives or mimetics thereof. Since in some embodiments, "m" is an integer between 1 to 7, the peptide may comprise 1, 2, 3, 4, 5, 6, or 7 interspacing amino acid residue/s, located between the at least two aromatic am aicniod residues. These interspacing, gaping separating, amin aocid residues may be any identical or different amino acid residues, or any derivative, analogs, or mimetics thereof. Still further, the peptide of the bifunctional hybrid-molecule further comprises Xaa(n) am aincoid residues, that may be located at any possible position of the peptide, and/or at any of the N' or C termini thereof. Since "n" as used herein is zero or an integer between 1 to 7, the peptide may comprise none (0), or 1, 2, 3, 4, 5, 6 or 7, amino acid residues at the N' terminus of the peptide, optionally, between one or both of the at least two aromatic amino acid residues and the interspacing residue/s, and/or at the at the C terminus of the peptide. These amino acid residues may be any identical or different amino acid residue/s, or any derivative, analogs, or mimetics thereof. The second component of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure (b), may comprise at least one proteasome degradation mediating moiety. As discussed herein, the peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure comprises at least two aromatic amino acid residues. An aromatic amino acid (AAA) is an amino acid that includes a hydrophobic side chain, specifically, an aromatic ring. More specifically, a cyclic (ring-shaped), planar (flat) structures with a ring of resonance bonds that gives increased stability compared to other geometric or connective arrangements with the same set of atoms. An aromatic functional group or other substituent is called an aryl group. Aromatic amino acids absorb ultraviolet light at a wavelength above 250 nm and produce fluorescence. Among the 20 standard amino acids, the following are aromatic: tryptophan, phenylalanine and tyrosine. "Aromatic amino acid" as used herein, includes natural as well as unnatural amino acids. Unnatural, aromatic amino acids include those that include an indole moiety in their amino acid side chain, wherein the indole ring structure can be substituted with one or more aryl group substituents. Additional examples of aromatic amino acids include but are not limited to 1-naphthylalanine, biphenylalanine, 2- napthylalananine, pentafluorophenylalanine, and 4-pyridylalanine. More specifically, the term "aromatic" as used herein, refers to a mono-, bi-, or other multi-carbocyclic, aromatic ring system. The aromatic group may optionally be fused to one or more rings chosen from aromatics, cycloalkyls, and heterocyclyls. Aromatics can have from 5-14 ring members, such as, e.g., from 5-10 ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aromatic, aryloxy, azido, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, cycloalkyl, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, and ureido. Nonlimiting examples of aromatic groups include phenyl, naphthyl, indolyl, biphenyl, and anthracenyl.
As indicated above, in some particular embodiments, the at least two aromatic amino acid residues of the peptide component of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure, may be at least one of Tryptophan, Tyrosine, and Phenylalanine, or any combinations thereof. In some specific embodiments, at least one of the at least two aromatic amino acid residues comprised in the peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure, is Tryptophan, or any derivative, analogs, or mimetics thereof. Tryptophan (symbol Trp or W) is an α-amino acid that is used in the biosynthesis of proteins, having the formula C11H12N2O2. L-Tryptophan has the following chemical structure, as denoted by Formula IX:
Figure imgf000014_0001
making it a non-polar aromatic amino acid. It is encoded by the codon UGG. Like other amino acids, tryptophan is a zwitterion at physiological pH where the am girnooup is protonated (-NH3+; pKa = 9.39) and the carboxylic acid is deprotonated (-COO-; pKa = 2.38).Tryptophan functions as a biochemical precursor for the following compounds: Serotonin (a neurotransmitter), synthesized by tryptophan hydroxylase; Melatonin (a neurohormone) is in turn synthesized from serotonin, via N-acetyltransferase and 5-hydroxyindole-O-methyltransferase enzymes; Niacin, also known as vitamin B3, is synthesized from tryptophan via kynurenine and quinolinic acids; Auxins (a class of phytohormones) are synthesized from tryptophan. Tryptophan is also a precursor to the neurotransmitter serotonin, the hormone melatonin and vitamin B3. In yet some further specific embodiments, at least one of the at least two aromatic amino acid residues of the peptide comprised in the bifunctional hybrid-molecule, conjugate or complex of the present disclosure, is Tyrosine, or any derivative, analogs, or mimetics thereof. Tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is a non-essential amino acid with a polar side group, having the formula C9H11NO3. L-Tyrosine has the following chemical structure, as denoted by Formula X:
Figure imgf000015_0001
While tyrosine is generally classified as a hydrophobic amino acid, it is more hydrophilic than phenylalanine. It is encoded by the codons UAC and UAU in messenger RNA (mRNA). Mammals synthesize tyrosine from the essential amino acid phenylalanine. The conversion of phe to tyr is catalyzed by the enzyme phenylalanine hydroxylase. In dopaminergic cells in the brain, tyrosine is converted to L-DOPA by the enzyme tyrosine hydroxylase (TH). TH is the rate-limiting enzyme involved in the synthesis of the neurotransmitter dopamine. Dopamine can then be converted into other catecholamines, such as norepinephrine (noradrenaline) and epinephrine (adrenaline). The thyroid hormones triiodothyronine (T3) and thyroxine (T4) in the colloid of the thyroid are also derived from tyrosine.
Still further, in some specific embodiments, at least one of the at least two aromatic amino acids of the peptide comprised in the bifunctional hybrid-molecule, conjugate or complex of the present disclosure is Phenylalanine, or any derivative, analogs, or mimetics thereof. Phenylalanine (symbol Phe or F) is an essential α-amino acid with the formula C9H 11NO2. It can be viewed as a benzyl group substituted for the methyl group of alanine, or a phenyl group in place of a terminal hydrogen of alanine. L-Phenylalanine has the following chemical structure, as denoted by Formula XI:
Figure imgf000015_0002
Formula XI
This essential amino acid is classified as neutral, and nonpolar because of the inert and hydrophobic nature of the benzyl side chain. The L-isomer is used to biochemically form proteins, coded for by DNA. Phenylalanine is a precursor for tyrosine, the monoamine neurotransmitters dopamine, norepinephrine (noradrenaline), and epinephrine (adrenaline), and the skin pigment melanin. It is encoded by the codons UUU and UUC. As shown by Example 8, replacement of the two Trp residues with other aromatic amino acid residues, as shown for example in the KVS-1, and KVS-5 peptides of SEQ ID NOs: 9, 44, 10 and 45, clearly retains the function of inducing ubiquitination of the p105. However, replacement of the Trp residues with the non- aromatic amino acid reside His, could not retain recognition and ubiquitination of the p105. In some embodiments, the peptide comprised in the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least two tryptophan residues, or any derivative, analogs, or mimetics thereof. For example, any peptide comprising the amino acid sequence Xaa(n)-Trp-Xaa(m)-Trp-Xaa(n), as denoted by SEQ ID NO: 29, wherein Xaa is any amino acid, n is zero or an integer between 1 to 7 and m is an integer between 1 to 7, or any peptide derived therefrom, for example, any of the peptides of SEQ ID NOs: 2, 4, 7, 12 to 18, 20, 22 to 28. In some alternative embodiments, the peptide comprised in the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least one tryptophan and at least one tyrosine, or any derivative, analogs, or mimetics thereof. In some embodiments, such peptide may comprise the amino acid sequence of Xaa(n)-Trp-Xaa(m)-Tyr-Xaa(n), as denoted by SEQ ID NO: 30, Xaa(n)-Tyr-Xaa(m)-Trp-Xaa(n), as denoted by SEQ ID NO: 31, or any peptide derived therefrom, wherein Xaa is any amin aocid, n is zero or an integer between 1 to 7 and m is an integer between 1 to 7. In yet some further embodiments, the peptide comprised in the bifunctional hybrid- molecule, conjugate or complex of the present disclosure may comprise at least one tryptophan and at least one phenyl alanine, or any derivative, analogs, or mimetics thereof. In some embodiments, such peptide may comprise the amino acid sequence of Xaa(n)-Trp-Xaa(m)-Phe- Xaa(n), as denoted by SEQ ID NO: 32, Xaa(n)-Phe-Xaa(m)-Trp-Xaa(n), as denoted by SEQ ID NO: 33, or any peptide derived therefrom, wherein Xaa is any amino acid, n is zero or an integer between 1 to 7 and m is an integer between 1 to 7. In some further embodiments, the peptide comprised in the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least two tyrosine residues. In some embodiments, such peptide may comprise theamino acid sequence of Xaa(n)-Tyr-Xaa(m)-Tyr-Xaa(n), as denoted by SEQ ID NO: 34, or any peptide derived therefrom, wherein Xaa is any amino acid, n is zero or an integer between 1 to 7 and m is an integer between 1 to 7. In some further embodiments, the peptide comprised in the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least one tyrosine and at least one phenylalanine, or any derivative, analogs, or mimetics thereof. In some embodiments, such peptide may comprise the amino acid sequence of Xaa(n)-Phe-Xaa(m)- Tyr-Xaa(n), as denoted by SEQ ID NO: 35, Xaa(n)-Tyr-Xaa(m)- Phe-Xaa(n), as denoted by SEQ ID NO: 36, or any peptide derived therefrom, wherein Xaa is any amino acid, n is zero or an integer between 1 to 7 and m is an integer between 1 to 7. Still further, in some embodiments, the peptide comprised in the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least two phenylalanine residues. In some embodiments, such peptide may comprise the amino acid sequence of Xaa(n)-Phe-Xaa(m)-Phe-Xaa(n), as denoted by SEQ ID NO: 37, or any peptide derived therefrom, wherein Xaa is any amino acid, n is zero or an integer between 1 to 7 and m is an integer between 1 to 7. Still further, the disclosure contemplates the use of at least two aromatic amino acid residues or mimetics thereof, in the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure. More specifically, at least two of: at least one W mimetic, at least one Y mimetic, or at least one F mimetic in a peptide, such that the peptide retains the ability of recruiting NF-κB1 p105. "Amino acid mimetics", as used herein, refers to chemical compounds having a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. As used herein "tryptophan mimetic" and "W mimetic", "tyrosine mimetic" and "Y mimetic" and "phenylalanine mimetic" and "F mimetic", are used interchangeably to refer to any agent that either emulates the biological effects of tryptophan, tyrosine, and/or phenylalanine in the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure, on recruitment of NF-κB1 p105. The W, Y and/or F mimetic in the peptide of bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure, can be any kind of agent. Exemplary W, Y and/or F mimetics include, but are not limited to, small organic or inorganic molecules; L-tyrosine, L-tryptophan and/or L- phenylalanine, D-tyrosine, D-tryptophan and/or D-phenylalanine, an NF-κB1 p105 recruiting tyrosine, tryptophan and/or phenylalanine mimetic, saccharides, oligosaccharides, polysaccharides, a biological macromolecule that may be any one of peptides, non-standard peptides, polypeptides, non-standard polypeptides, proteins, non-standard proteins, peptide analogs and derivatives enriched for L- tyrosine , L- tyrosine tryptophan and/or L-phenylalanine. In some embodiments, the at least two aromatic amino acid residues W, Y and/or F of the peptide, or any mimetics thereof comprise at least one of the native amino acid tyrosine, tryptophan and/or phenylalanine. As used herein, "native amino acid" refers to the L-form of the amino acid which naturally occurs in proteins; thus, the term "native amino acid tryptophan, tyrosine and/or phenylalanine" refers to L- tryptophan, L- tyrosine and/or L-phenylalanine. In some embodiments, the amino acid residues in the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure, can be in D-configuration or L-configuration (referred to herein as D- or L- enantiomers). In some embodiments, the at least two of W, Y and/or F in the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure, comprises the native amino acid tryptophan, tyrosine and/or phenylalanine (W, Y and/or F). In some embodiments, the native amino acid tyrosine, tryptophan and/or phenylalanine is isolated and/or purified. In some embodiments, the at least two of W, Y and/or F, and/or mimetics thereof, of the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure, may comprise a derivative, variant or analog of the native amino acid tryptophan, tyrosine, and/or phenylalanine. In some embodiments, the at least two of W, Y and/or F, and/or mimetics thereof, in the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure, may comprise a combination of the native amino acid tryptophan, tyrosine, and/or phenylalanine, a derivative of the native amino acid tryptophan, tyrosine, and/or phenylalanine and/or an analog of the native amino acid tryptophan, tyrosine, and/or phenylalanine. In some embodiments, derivative of a peptide comprising at least two of W, Y and/or F may comprise a C'-terminus modification to at least one of W, Y and/or F. As used herein, a "C-terminus modification" refers to the addition of a moiety or substituent group to the amino acid via a linkage between the carboxylic acid group of the amino acid and the moiety or substituent group to be added to the amino acid. The disclosure contemplates any C-terminus modification to a peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure, that may occur in at least one of the at least two of W, Y and/or F, while the peptide still retains the ability to recruit NF-κB1 p105. In some embodiments, the C-terminus modification to a peptide comprising at least two of W, Y and/or F, may comprise a carboxy alkyl ester. As used herein, the term "alkyl" refers to saturated non-aromatic hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms (these include without limitation methyl, ethyl, propyl, allyl, or propargyl), which may be optionally inserted with N, O, S, SS, SO2, C(0), C(0)0, OC(O), C(0)N or NC(O). For example, C i-Ce indicates that the group may have from 1 to 6 (inclusive) carbon atoms in it. In some embodiments, the C- terminus modification to L comprises a carboxy alkenyl ester. As used herein, the term "alkenyl" refers to an alkyl that comprises at least one double bond. Exemplary alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl and the like. In some embodiments, the C-terminus modification to at least one of W, Y, F comprises a carboxy alkynyl ester. As used herein, the term "alkynyl" refers to an alkyl that comprises at least one triple bond. In some embodiments, the carboxy ester comprises tyrosine, tryptophan and/or phenylalanine carboxy methyl ester. In some embodiments, the carboxy ester comprises tyrosine, tryptophan and/or phenylalanine carboxy ethyl ester. In some embodiments, derivative of a peptide comprising at least two of W, Y and/or F may comprise an N-terminus modification to at least one of W, Y and/or F. As used herein, "N-terminus modification" refers to the addition of a moiety or substituent group to the amino acid via a linkage between the alpha amino group of the amino acid and the moiety or substituent group to be added to the amino acid. The disclosure contemplates any N'-terminus modification to at least one of Y, W and/or F in which the peptide comprising N- terminus modified W, Y and/or F, still retains the ability to recruit NF-κB1 p105. In some embodiments, the derivative of a peptide comprising at least two of W, Y, and/or F may comprise at least two of W, Y, and/or F modified by an amino bulky substituent group. As used herein "amino bulky substituent group" refers to a bulky substituent group which is linked to the amino acid via the alpha amino group. An exemplary amino bulky substituent group is a carboxybenzyl (Cbz) protecting group. Accordingly, in some embodiments, the derivative of a peptide comprising at least two of W, Y, and/or F, comprises Y, W, and/or F modified by an amino carboxybenzyl (Cbz) protecting group. Other suitable amino bulky substituent groups are apparent to those skilled in the art. In some embodiments, the derivative of a peptide comprising at least two of W, Y, and/or F may comprise a side-chain modification to Y, W and/or F. As used herein "side-chain modification" refers to the addition of a moiety or substituent group to the side-chain of the amino acid via a linkage (e.g., covalent bond) between the side-chain and the moiety or chemical group to be added. The disclosure contemplates the use of any side-chain modification that permits the side-chain modified amino acid to retain the ability of the peptide of the bifunctional hybrid- molecule, conjugate or complex provided by the present disclosure, to recruit NF-κB1 p105.
It should be understood that the present disclosure further encompasses any Deuterated, Fluorinated, Acetylated or Methylated forms of any one of the amino acid residues of the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure, being either an aromatic amino acid residue or any other amino acid residue of the discussed peptide. For example, in any one of: at least two of L- or D-tyrosine, the L- or D- phenylalanine or L- or D- tryptophan, or L- or D- Xaa where the Xaa is any amino acid residue (for example, where Xaa is the Arginine residue that is present in most peptides, e.g., of SEQ ID NOs: 2, 4, 5, 8 to 11, 14, 16 to 28), of the peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure. More specifically, deuterium-substituted amino acids (deuterated amino acids) applicable as analogs of the present invention may include but are not limited to L-Tyrosine- (phenyl-3, 5-d2), L-4-Hydroxyphenyl-2,3,5,6-d4-alanin and L-Tryptophan-(indole-d5). Methylated aromatic amino acids residues include but are not limited to any one of L-Tyrosine methyl ester, O-Methyl-L-tyrosine, α-Methyl-L-tyrosine, α-Methyl-DL-tyrosine methyl ester hydrochloride, α-Methyl-L-tyrosine, α-Methyl-DL-tyrosine, α-Methyl-DL-tryptophan, O-Methyl- L-tyrosine, N-Methylphenethylamine, β-Methylphenethylamine, N, N-Dimethylphenethylamine, 3-Methylphenethylamine, (R)-(+)-β-Methylphenethylamine, N-Methyl-N-(l-phenylethyl)amine, 2-methylphenethylamine, 4- Bromo-N-methylphenethylam,in 3e-Bromo-N-methylbenzylamine,(S)- β-Methylphenethylamine, p-Chloro-β-methylphenethylamine hydrochl, α-Methyl-DL- tryptophan, L-Tryptophan methyl ester hydrochloride, D-Tryptophan methyl ester hydrochloride, L-Tryptophan ethyl ester hydrochloride, L-Tryptophan benzyl ester, L-Tyrosine methyl ester hydrochloride, L-Phenylalanine methyl ester hydrochlori, DL-tryptophan methyl ester, N-acetyl- 1-tryptophan methyl ester. Still further, Fluorinated tyrosine, phenylalanine or tryptophan include but are not limited to any one of 5-Fluoro-L-tryptophan, 5-Fluoro-DL-tryptophan, 4-Fluoro-DL- tryptophan, 6-Fluoro-L-Tryptophan, 5-Methyl-DL-tryptophan, 5-Bromo-DL-tryptophan, 7- Azatryptophan, m-Fluoro-DL-tyrosine, p-Fluoro-L-phenylalanine, o-Fluoro-DL-phenylalanine, p- Fluoro-DL -phenylalanine, 4-Chloro-DL-phenylalanine, m-Fluoro-L-phenylalanine, 3-Nitro-L- tyrosine. In some further embodiments of the present disclosure Acetylated aromaticamino acids residues include but are not limited to any one of N-acetyl-L-tyrosine, N- Acetyl -L-phenylalanine, L-Phenylalanine methyl ester hydrochloride, N-Acetyl-D-phenylalanine, N-Acetyl-L-tryptophan. Exemplary analogs of tyrosine and/or phenylalanine that may be applicable in accordance with the peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure include but are not limited to any one of (2R, 3S)/(2S, 3R) - Racemic Fmoc - b - hydroxyphenylalanine, Boc - 2 - cyano - L - phenylalanine, Boc - L - thyroxine, Boc - O - methyl
- L - tyrosine, Fmoc - b - methyl - DL - phenylalanine, Fmoc - 2 - cyano - L - phenylalanine, Fmoc
- 3,4 - dichloro - L - phenylalanine, Fmoc - 3,4 - difluoro - L - phenylalanine, Fmoc - 3,4 - dihydroxy - L - phenylalanine, Fmoc - 3,4 - dihydroxy - phenylalanine, acetonide protected, Fmoc
- 3 - amino - L - tyrosine, Fmoc - 3 - chloro - L - tyrosine, Fmoc - 3 - fluoro - DL - tyrosine, Fmoc
- 3 - nitro - L - tyrosine, Fmoc - 4 - (Boc - amino) - L - phenylalanine, Fmoc - 4 - (Boc - aminomethyl) - L - phenylalanine, Fmoc - 4 - (phosphonomethyl) - phenylalanine, Fmoc - 4 - (phosphonomethyl) - phenylalanine, Fmoc - 4 - benzoyl - D - phenylalanine. Still further, in some embodiments, exemplary analogs of tryptophan that may be applicable in accordance with the present disclosure include but are not limited to any one of Boc - 4 - methyl - DL - tryptophan, Boc - 4 - methyl - DL - tryptophan, Boc - 6 - fluoro - DL - tryptophan, Boc - 6 - methyl - DL - tryptophan, Boc - DL - 7 - azatryptophan, Fmoc - (R) - 7 - Azatryptophan, Fmoc - 5 - benzyloxy
- DL - tryptophan, Fmoc - 5 - bromo - DL - tryptophan, Fmoc - 5 - chloro - DL - tryptophan, Fmoc
- 5 - fluoro - DL - tryptophan, Fmoc - 5 - fluoro - DL - tryptophan, Fmoc - 5 - hydroxy - L - tryptophan, Fmoc - 5 - hydroxy - L - tryptophan, Fmoc - 5 - methoxy - L - tryptophan, Fmoc - 5 - methoxy - L - tryptophan, Fmoc - 6 - chloro - L - tryptophan, Fmoc - 6 - methyl - DL - tryptophan, Fmoc - 7 - methyl - DL - tryptophan, Fmoc - DL - 7 - azatryptophan. It should be appreciated that the present disclosure provides the peptide of the bifunctional hybrid-molecule, conjugate or complex of the invention that comprises at least two aromatic amino acid residues, specifically, tryptophan, tyrosine and/or phenylalanine and/or any serogates thereof, any salt, base, ester or amide thereof, any enantiomer, stereoisomer or disterioisomer thereof, or any combination or mixture thereof. Pharmaceutically acceptable salts include salts of acidic or basic groups present in compounds, specifically, the at least two aromatic amino acid residues of the peptide of the bifunctional hybrid-molecule, conjugate or complex provided by the present disclosure. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., 1,1'- methylene-bis-(2-hydroxy-3-naphthoate)) salts. Certain aromatic amino acid residues of the present disclosure can form pharmaceutically acceptable salts. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts. In some embodiments, at least one of the at least two aromatic amino aromatic amino acid residues of the peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure, may be tryptophan. In some embodiments, one of the aromatic amino acid residues in the peptide of the bifunctional hybrid-molecule of the present disclosure may be tryptophan, and the other aromatic amino acid residue may be tyrosine. In some alternative embodiments, the peptide of the bifunctional hybrid-molecule of the present disclosure may comprise at least one tryptophan and at least one phenylalanine. In yet some further embodiments, the bifunctional hybrid-molecule of the invention may comprise at least two tryptophan residues. It should be understood that the peptide of the bifunctional hybrid-molecule of the present disclosure may comprise any further aromatic amino acid residues as one or more of the Xaa residues located in various positions of the peptide as discussed above.
Still further, in some embodiments, at least one of the "interspacing", "gapping", "separating" amino acid residue/s located between the two aromatic amino acid residues, may be one or more amino acid residue/s having a non-polar side chain, also referred to herein as a non-polar amino acid. Such residues may be any one of Valine (V, Val), Glycine (G, Gly), Leucine (L, Leu), Isoleucine (I, IIe), Methionine (M, Met), Phenylalanine (F, Phe), Tryptophane (W, Trp) and/or Proline (P, Pro). In yet some further embodiments, the interspacing amino acid residue may be any hydrophobic amino acid residue, for example, any one of Valine (V, Val), Leucine (L, Leu) and/or Isoleucine (I, IIe), or any derivative, analogs, or mimetics thereof, as disclosed herein above.
In some specific embodiments, the peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least two aromatic amino acid residues that are interspaced by at least one of Valine (V), Glycine (G) and/or Alanine (A). In some particular and non-limiting embodiments, the at least two aromatic amino acid residues of the peptide of the bifunctional hybrid-molecule disclosed herein, are interspaced by at least one Valine residue. Thus, in some particular and non-limiting embodiments, the peptide of the bifunctional hybrid- molecule, conjugate or complex in accordance with the invention may comprise the amino acid sequence of Xaa(n)-Trp (W)-Xaa(n)-Val (V)-Xaa(n)-Trp (W)-Xaa(n), as denoted by SEQ ID NO: 7, or any mimetics thereof. It should be noted that Xaa is any amino acid residue, and n is zero or an integer between 1 to 7. These amino acid residues may be any identical or different amino acid residues. In some embodiments, the at least one peptide of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure, may comprise three to ten amino acid residues, specifically, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. As indicated above, at least two of these residues are aromatic amino acids, specifically, W, Y and/or F, or any derivative, analogs, or mimetics thereof. In yet some further embodiments, the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise any amino acid residue, Xaa that is any one of Arg (R), IIe (I) and Leu (L), or any derivative, analogs, or mimetics thereof, as disclosed herein above. Still further, in some embodiments, the peptides of the bi-functional hybrid-molecule of the present disclosure or any conjugate or complex thereof, may comprise at least one Arg residue, at any position of the peptide, for example, at the N-termini thereof, at the C-termini thereof, and/or the interspacing residue/s. Specifically, at least one, at least two at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more arginine residues or any derivative, analogs, or mimetics thereof, as disclosed herein above, either at the N-termini thereof, at the C-termini thereof, and/or the interspacing residue/s. Non-limiting embodiments for such peptides are provided by the peptides that comprises the amino acid sequence as denoted by SEQ ID NOs: 27 and 59 (KVS-18); 24 and 58 (KVS-14); 25 (KVS-15) and 26 (KVS-16), or any derivatives thereof, that clearly retain the ability of requiring the NF-κB1 p105. Thus, in some specific embodiments, the peptide of the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise the amino acid sequence of at least one of any one of: (a) RIWVWLL, as dented by SEQ ID NO: 2, or any variants and derivatives thereof; (b), IWVWLL, as dented by SEQ ID NO: 3, or any variants and derivatives thereof; (c) WILVRLW, as dented by SEQ ID NO: 4, or any variants and derivatives thereof; (d) RIFVFLL, as dented by SEQ ID NO: 9; (e) RIYVFLL, as dented by SEQ ID NO: 10; (f) Citrulline-IWVWLL, as dented by SEQ ID NO: 15; (g) GRIWVWLL, as dented by SEQ ID NO: 16; (h) RRRIWVWLL, as dented by SEQ ID NO: 27; and/or (i) WVW, or any variants and derivatives thereof. In yet some further embodiments, the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise any of the peptides disclosed herein, for example, any of the peptides discussed in Example 8 and Figure 9, specifically, any of the peptides of SEQ ID NOs: 8, 11 to 28, and 52 to 59.
In some embodiments, the peptide of the bifunctional hybrid-molecule, conjugate or complex of the invention recognizes, binds and therefore recruits the NF-κB1 p105. It should be understood that any variant or derivative of any of the above-mentioned peptides may be comprised within the bifunctional hybrid-molecule, conjugate or complex of the invention. In some embodiments, the invention contemplates any variant or derivative of each one of the above indicated peptides, provided that the variant and/or derivative retains the ability of the peptide (specifically, any one of SEQ ID NO: 1 to 5, 7 to 39, and 42 to 59), to recruit and bind the NF-κB1 p105.
As indicated above, the bifunctional hybrid-molecule of the present disclosure comprises a polypeptide component that recruits and binds the NF-κB1 p105. It is therefore understood that in some embodiments, the polypeptide or peptide may be considered as an isolated polypeptide or peptide. A "polypeptide" refers to a polymer of amino acids linked by peptide bonds. A protein is a molecule comprising one or more polypeptides. A peptide is a relatively short polypeptide, typically between about 3 and 100 amino acids (aa) in length, e.g., between 4 and 60 aa; between 8 and 40 aa; between 10 and 30 aa. The terms "protein", "polypeptide", and "peptide" may be used interchangeably. In general, a polypeptide may contain only standard amino acids or may comprise one or more non-standard amino acids (which may be naturally occurring or non-naturally occurring amino acids) and/or amino acid analogs in various embodiments. A "standard amino acid" is any of the 20 L- ami no acids that are commonly utilized in the synthesis of proteins by mammals and are encoded by the genetic code. A "non-standard amino acid" is an amino acid that is not commonly utilized in the synthesis of proteins by mammals. Non-standard amino acids include naturally occurring amino acids (other than the 20 standard amino acids) and non-naturally occurring amino acids. In some embodiments, a non-standard, naturally occurring amino acid is found in mammals. For example, ornithine, citrulline, and homocysteine are naturally occurring non-standard amino acids that have important roles in mammalian metabolism. Exemplary nonstandard amino acids include, e.g., singly or multiply halogenated (e.g., tluorinated) amino acids, D-amino acids, homo-ammo acids, N-alkyl amino acids (other than proline), dehydroamino acids, aromatic amino acids (other than histidine, phenylalanine, tyrosine and tryptophan), and a, a disubstituted amino acids, An amino acid, e.g., one or more of the amino acids in a polypeptide, may be modified, for example, by addition, e.g., covalent linkage, of a moiety such as an alkyl group, an alkanoyl group, a carbohydrate group, a phosphate group, a lipid, a polysaccharide, a halogen, a linker for conjugation, a protecting group, etc. Modifications may occur anywhere in a polypeptide, e.g., the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. A given polypeptide may contain many types of modifications. Polypeptides may be branched or they may be cyclic, with or without branching. Polypeptides may be conjugated with, encapsulated by, or embedded within a polymer or polymeric matrix, dendrimer, nanoparticle, microparticle, liposome, or the like. Modification may occur prior to or after an amino acid is incorporated into a polypeptide in various embodiments. Polypeptides may, for example, be purified from natural sources, produced in vitro or in vivo in suitable expression systems using recombinant DNA technology (e.g., by recombinant host cells or in transgenic animals or plants), synthesized through chemical means such as conventional solid phase peptide synthesis, and/or methods involving chemical ligation of synthesized peptides. One of ordinary skill in the art will understand that a protein may be composed of a single amino acid chain or multiple chains associated covalently or noncovalently. An 'isolated polypeptide' or "isolated peptide", is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature. Typically, a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. One way to show that a particular protein preparation contains an isolated polypeptide is by the appearance of a single band following sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining of the gel. However, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms. By definition, isolated peptides are also non-naturally occurring, synthetic peptides. Methods for isolating or synthesizing peptides of interest with known amino acid sequences are well known in the art. The polypeptides of the invention are therefore considered as proteinaceous material. A "proteinaceous material" is any protein, or fragment thereof, or complex containing one or more proteins formed by any means, such as covalent peptide bonds, disulfide bonds, chemical crosslinks, etc., or non-covalent associations, such as hydrogen bonding, van der Waal's contacts, electrostatic salt bridges, etc. The peptides of the bifunctional hybrid-molecule, conjugate or complex of the invention are composed of an amino acid sequence. An "amino acid/s" or an "amino acid residue/s" can be a natural or non-natural amino acid residue/s linked by peptide bonds or bonds different from peptide bonds. The amino acid residues can be in D-configuration or L-configuration (referred to herein as D- or L- enantiomers). An amino acid residue comprises an amino terminal part (N¾) and a carboxy terminal part (COOH) separated by a central part (R group) comprising a carbon atom, or a chain of carbon atoms, at least one of which comprises at least one side chain or functional group. NH2 refers to the amino group present at the amino terminal end of an amino acid or peptide, and COOH refers to the carboxy group present at the carboxy terminal end of an amino acid or peptide. The generic term amino acid comprises both natural and non-natural amino acids. Natural amino acids of standard nomenclature are listed in 37 C.F.R. 1.822(b)(2). Examples of non-natural amino acids are also listed in 37 C.F.R. 1.822(b)(4), other non-natural amino acid residues include, but are not limited to, modified amino acid residues, L, -amino acid residues, and stereoisomers of D-amino acid residues. Naturally occurring amino acids may be further modified, e.g., hydroxyproline, g-carboxy glutamate, and O- phosphoserine. Thus, the peptides of the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise natural or non-natural amino acid residues, or any combination thereof. Further, amino acids may be amino acid analogs or amino acid mimetics. Amino acid analogs refer to compounds that have the same fundamental chemical structure as naturally occurring amino acids, but modified R groups or modified peptide backbones, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Further, the peptides of the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise "equivalent amino acid residues'. This term refers to an amino acid residue capable of replacing another amino acid residue in a polypeptide without substantially altering the structure and/or functionality of the polypeptide. Equivalent amino acids thus have similar properties such as bulkiness of the side- chain, side chain polarity (polar or non-polar), hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral or basic) and side chain organization of carbon molecules (aromatic/aliphatic). As such, equivalent amino acid residues can be regarded as conservative amino acid substitutions. In the context of the present invention, within the meaning of the term "equivalent amino acid substitutioiT as applied herein, is meant that in certain embodiments one amino acid may be substituted for another within the groups of amino acids indicated herein below:
(i) Amino acids having polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys); (ii) Amino acids having non-polar side chains (Gly, Ala, Val, Leu, IIe, Phe, Trp, Pro, and Met); (iii) Amino acids having aliphatic side chains (Gly, Ala Val, Leu, IIe); (iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro); (v) Amino acids having aromatic side chains (Phe, Tyr, Trp); (vi) Amino acids having acidic side chains (Asp, Glu); (vii) Amino acids having basic side chains (Lys, Arg, His); (viii) Amino acids having amide side chains (Asn, Gin); (ix) Amino acids having hydroxy side chains (Ser, Thr); (x) Amino acids having sulphur-containing side chains (Cys, Met); (xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr); (xii) Hydrophilic, acidic amino acids (Gin, Asn, Glu, Asp), and (xiii) Hydrophobic amino acids (Leu, IIe, Val).
Still further, peptides of the bifunctional hybrid-molecule, conjugate or complex of the invention, may have secondary modifications, such as phosphorylation, acetylation, glycosylation, sulfhydryl bond formation, cleavage and the likes, as long as said modifications retain the functional properties of the original protein, specifically, the ability to interact with, bind and recruit NF-κB 1 p105. Secondary modifications are often referred to in terms of relative position to certain amino acid residues. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence but is not necessarily at the carboxyl terminus of the complete polypeptide.
The invention further encompasses any derivatives, enantiomers, analogues, variants or homologues of any of the peptides of the bifunctional hybrid-molecule, conjugate or complex disclosed herein, specifically, any of the peptides that comprise the amin aocid sequence of any one of SEQ ID NO: 1 to 5, 7 to 39, and 42 to 59. The term "derivative" is used to define amino acid sequences (polypeptide), with any insertions, deletions, substitutions and modifications to the amino acid sequences (polypeptide) that do not alter the activity of the original polypeptides (e.g., recruiting NF-κB 1 p105). By the term “derivative” it is also referred to homologues, variants and analogues thereof, as well as covalent modifications of a polypeptides made according to the present invention. It should be noted that the peptides of the bifunctional hybrid-molecule, conjugate or complex according to the invention can be produced either synthetically, or by recombinant DNA technology. Methods for producing polypeptides peptides are well known in the art. In some embodiments, derivatives include, but are not limited to, polypeptides that differ in one or more amino acids in their overall sequence from the polypeptides defined herein, polypeptides that have deletions, substitutions, inversions or additions. In some embodiments, derivatives refer to polypeptides, which differ from the polypeptides specifically defined in the present invention by insertions of amino acid residues. It should be appreciated that by the terms "insertions" or "deletions", as used herein it is meant any addition or deletion, respectively, of amino acid residues to the polypeptides used by the invention, of between 1 to 50 amino acid residues, between 20 to 1 amino acid residues, and specifically, between 1 to 10 amino acid residues. More particularly, insertions or deletions may be of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. It should be noted that the insertions or deletions encompassed by the invention may occur in any position of the modified peptide, as well as in any of the N' or C termini thereof. It should be appreciated that in cases the deletion/s or insertion/s are in the N or C- terminus of the peptide, such derivatives may be also referred to as fragments. The peptides of the bifunctional hybrid-molecule, conjugate or complex of the invention of the invention may all be positively charged, negatively charged or neutral. In addition, they may be in the form of a dimer, a multimer or in a constrained conformation, which can be attained by internal bridges, short-range cyclization, extension or other chemical modifications. A non-limiting example for a cyclized peptide may be the KVS-7 peptide, that comprises the amino acid sequence of SEQ ID NO: 17, wherein as shown by Fig. 9D, cyclization is performed by linking the amine of the N-terminal Arg residue with the Cys residue of the linker. The polypeptides of the bifunctional hybrid-molecule, conjugate or complex of the invention can be coupled (conjugated) through any of their residues to another peptide or agent. For example, the peptides of the bifunctional hybrid-molecule, conjugate or complex of the invention can be coupled through their N-terminus to a lauryl-cysteine (FC) residue and/or through their C-terminus to a cysteine (C) residue. Further, the peptides of the bifunctional hybrid-molecule, conjugate or complex of the invention may be extended at the N- terminus and/or C-terminus thereof with various identical or different amino acid residues. As an example for such extension, the peptide may be extended at the N-terminus and/or C-terminus thereof with identical or different amino acid residue/s, which may be naturally occurring or synthetic amino acid residue/s. An additional example for such an extension may be provided by peptides extended both at the N-terminus and/or C-terminus thereof with a cysteine residue. Naturally, such an extension may lead to a constrained conformation due to Cys-Cys cyclization resulting from the formation of a disulfide bond. Another example may be the incorporation of an N-terminal lysyl-palmitoyl tail, the lysine serving as linker and the palmitic acid as a hydrophobic anchor. In addition, the peptides may be extended by aromaticamino acid residue/s, which may be naturally occurring or syntheticamino acid residue/s. The peptides may be extended at the N- terminus and/or C-terminus thereof with various identical or different organic moieties, which are not naturally occurring or synthetic amino acids. As an example for such extension, the peptides of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may be extended at the N-terminus and/or C- terminus thereof with an N-acetyl group. Figure 4 shows the effectivity of a bifunctional hybrid-molecule, conjugate or complex of the present disclosure that comprises an N-acetylated peptide (Formula XIII, also indicated herein as PROTAC 4). For every single peptide sequence encompassed by any of the peptides of the bifunctional hybrid- molecule, conjugate or complex of the present disclosure as disclosed herein, this invention includes the corresponding retro-inverse sequence wherein the direction of the peptide chain has been inverted and wherein all or part of the amino acids belong to the D-series. It should be understood that the present invention includes embodiments wherein one or more of the L- amino acids is replaced with its D isomer, thus providing peptides comprising 1 , -amino acids, D-amino acids, and any combination of 1 , -amino acids and 1 , -amino acids. A non-limiting example for a peptide that comprises at least one residue in the D-form may comprise SEQ IS NO: 14, 49.
In yet some further embodiments, the peptides of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise at least one amino acid residue in the D-form. It should be noted that every amino acid (except glycine) can occur in two isomeric forms, because of the possibility of forming two different enantiomers (stereoisomers) around the central carbon atom. By convention, these are called L- and D- forms, analogous to left-handed and right-handed configurations. It should be appreciated that in some embodiments, the enantiomer or any derivatives of the peptides of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may exhibit at least one of enhanced activity, and superiority. In more specific embodiments, such derivatives and enantiomers may exhibit increased affinity to the NF-κB1 p105, increased specificity, enhanced stability, and increased resistance to proteolytic degradation. It should be understood that increased stability, specificity, affinity and the like as disclosed herein with respect to any of the peptides of the bi-functional hybrid-molecule of the present disclosure or any conjugate or complex thereof, relate to the increase, elevation, enhancement, escalation, of at least one of the stability, specificity, affinity, binding and recruitment of the NF-κB p105 by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more, as compared to the wild type sequence, for example, the wild type sequence of SEQ IN NO: 4.
The invention also encompasses any homologues of the peptides of the bifunctional hybrid- molecule, conjugate or complex of the present disclosure, particularly of those specifically defined by their amino acid sequence according to the invention. The term " homologues ” is used to define amino acid sequences (polypeptide) which maintain a minimal homology to the amino acid sequences defined by the invention, e.g. specifically have at least about 65%, more specifically, at least about 70%, at least about 75%, even more preferably at least about 80%, at least about 85%, most preferably at least about 90%, at least about 95% overall sequence homology with the entire amino acid sequence of any of the polypeptide as structurally defined above, e.g. of a specified sequence, more specifically, an amino acid sequence of the polypeptides as denoted by any one of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 8, 11 to 28, and 52 to 59, and any derivatives, enantiomers and fusion proteins thereof. More specifically, "Homology" with respect to a native polypeptide and its functional derivative is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- nor C- terminal extensions nor insertions or deletions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art. In some embodiments, the present invention also encompasses peptides of the bifunctional hybrid- molecule, conjugate or complex of the present disclosure which are variants of, or analogues to, the polypeptides specifically defined in the invention by their amino acid sequence. With respect to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to peptide, polypeptide, or protein sequence thereby altering, adding or deleting a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant”, where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art and disclosed herein before. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues, and alleles and analogous peptides of the invention. More specifically, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amin aocid replacements. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. As noted above, the peptides of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may be modified by omitting their N-terminal sequence. It should be appreciated that the invention further encompasses the omission of about 1, 2, 3, 4, 5, 6, 7, 8 and more amino acid residues from both, the N' and/or the C termini of the peptides of the invention. Certain commonly encountered amino acids which also provide useful substitutions include, but are not limited to, β-alanine (β-Ala) and other omega-amino acids such as 3- aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4- aminobutyric acid and so forth; α- aminoisobutyric acid (Aib); e-aminohexanoic acid (Aha); d- aminovaleric acid (Ava); N- methylglycine or sarcosine (MeGIy); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (NIe); naphthylalanine (Nal); 4-chlorophenylalanine (Phe(4- Cl)); 2- fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); β-2- thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,4-diaminobutyric acid (Dab); p- aminophenylalanine (Phe(pNH.sub.2)); N-methyl valine (MeVal); homocysteine (hCys), homophenylalanine (hPhe) and homoserine (hSer); hydroxyproline (Flyp), homoproline (hPro), N-methylated amino acids (e.g., N-substituted glycine). Covalent Modifications of Amino Acids and the Peptide. A non- limiting embodiments for a peptide of the bi-functional hybrid-molecule of the present disclosure or any conjugate or complex thereof that comprises citrulline (Cit), is provided by the peptides of SEQ ID NO: 15, and SEQ ID NO: 50 (with linker). Covalent modifications of the peptide are included and may be introduced by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines) to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5- imidozoyl)propionic acid, chloroacetyl phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4- nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3- diazole. Histidyl residues are derivatized by reaction with diethylprocarbonate. Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents reverses the charge of the lysinyl residues. Other suitable reagents for derivatizing a- amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate. Arginyl residues are modified by reaction with one or several conventional reagents, including phenylglyoxal, 2,3- butanedione, 1,2-cyclohexanedione, and ninhydrin. Such derivatization requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine e-amino group. Modification of tyrosyl residues has permits introduction of spectral labels into a peptide. This is accomplished by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to create O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N-C-N-R') such as l-cyclohexyl-3-(2-morpholinyl- (4-ethyl) carbodiimide or 1- ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Conversely, glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues. Deamidation can be performed under mildly acidic conditions. Either form of these residues falls within the scope of this invention. Derivatization with bifunctional agents is useful for cross-linking the peptide to a water-insoluble support matrix or other macromolecular carrier. Commonly used cross- linking agents include 1,1- bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, esters with 4- azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8- octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Other chemical modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the a- amino groups of lysine, arginine, and histidine side chains (Creighton, supra), acetylation of the N-terminal amine, and, in some instances, amidation of the C-terminal carboxyl. Such chemically modified and derivatized moieties may improve the peptide's solubility, absorption, biological half-life, cell penetration, stability, biological availability, and the like. It should be understood that the present disclosure encompasses any derivative or variant of the peptide of the present disclosure as disclosed throughout the specification, or any orientation, isomer or form thereof, provided that the peptide/s retain/s the biologic activity indicated herein. More specifically, in some embodiments the biological activity of the peptide is requiting, targeting, and/or binding the NFκB p105 such that it will be in close proximity to the proteasome degradation mediating moiety (e.g., the ligand disclosed herein), in a manner that enables the proteasomal degradation of NFκB p105 to form the NFκB p50 product. Still further, in some embodiments, any derivative or variant or form of the disclosed peptides, and or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein, are those that at least retain the ability to requite, target, and/or bind the NFκB p105, and/or mediate the degradation of NFκB p105 to form the NFκB p50 product. Still further, in some embodiments, the derivative or variant or form of the disclosed peptides, and or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein, are able to mediate, increase, and enhance the production of the NFκB p50 product in a cell, in a subject and/or in a cell free system, in about any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more, as compared to a cell, in a subject and/or a cell free system in the absence of the peptides of the invention or any derivative or variant or form of the disclosed peptides, and or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein. In yet some further embodiments, the activity of the peptides of the invention, the derivative or variant or form of the disclosed peptides, and/or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein, is meant herein, the induction of the production of the NFκB p50 product that leads and mediates either directly or indirectly a tumor suppressive effect. Thus, in some embodiments, any derivative or variant or form of the disclosed peptides or any conjugates thereof act to increase the tumor suppressive effect by increasing p50 levels. As shown by Figure 3, the peptides of the invention and constructs thereof change the expression pattern of various groups of proteins. In yet some further embodiments, the peptides of the invention, the derivative or variant or form of the disclosed peptides, and/or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein lead to an increase, elevation, upregulation, enhancement and or enlargement of the expression of proteins connected to immune system processes and/or proteins connected with regulation of cell migration, and/or proteins that relate to ECM organization, and/or proteins that relate to cell adhesion, and/or proteins that relate to regulation of cell adhesion. In yet some further additional or alternative embodiments, the peptides of the invention, the derivative or variant or form of the disclosed peptides, and/or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein lead to a decrease, reduction, attenuation, inhibition and downregulation of the expression of proteins connected to metabolic processes, and/or proteins that relate to cell cycle, and/or proteins that relate to DNA metabolic processes. It should be appreciated that the invention further encompasses any of the peptides of the invention referred herein, any serogates thereof, any salt, base, ester or amide thereof, any enantiomer, stereoisomer or disterioisomer thereof, or any combination or mixture thereof. Pharmaceutically acceptable salts include salts of acidic or basic groups present in compounds of the invention. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., l,T-methylene-bis-(2-hydroxy-3- naphthoate)) salts. Certain compounds of the invention can form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts. In some particular embodiments, the peptides of the bi-functional hybrid-molecule of the present disclosure or any conjugate or complex thereof, may be any of the peptides encompassed by SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 29, with the proviso that the peptide is not the peptide of SEQ ID NO: 4. As indicated above, the bifunctional hybrid-molecule, conjugate or complex of the present disclosure comprises two components. One component comprises at least one peptide as discussed above that recruits NF-κB1 p105. The second component is at least one proteasome degradation mediating moiety. Proteasomes are protein complexes which degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds, mediated by proteases. Proteasomes are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins. Proteins are tagged for degradation with a small protein called ubiquitin. The tagging reaction is catalyzed by enzymes called ubiquitin ligases. The degradation process yields peptides of about seven to eight amino acids long, which can then be further degraded into shorter amino acid sequences and used in synthesizing new proteins. Proteasomes are found inside all eukaryotes and archaea, and in some bacteria. In structure, the proteasome is a cylindrical complex containing a "core" of four stacked rings forming a central pore. Each ring is composed of seven individual proteins. The inner two rings are made of seven b subunits that contain three to seven protease active sites. These sites are located on the interior surface of the rings, so that the target protein must enter the central pore before it is degraded. The outer two rings each contain seven a subunits whose function is to maintain a "gate" through which proteins enter the barrel. These a subunits are controlled by binding to "cap" structures or regulatory particles that recognize polyubiquitin tags attached to protein substrates and initiate the degradation process. The overall system of ubiquitination and proteasomal degradation is known as the ubiquitin-proteasome system (UPS). The proteasome subcomponents are often referred to by their Svedberg sedimentation coefficient (denoted S). The proteasome most exclusively used in mammals is the cytosolic 26S proteasome, which is about 2000 kilodaltons (kDa) containing one 20S protein subunit (also referred to herein as the core proteasome, or CP) and two 19S regulatory cap subunits (also referred to herein as the regulatory proteasome or RP). The core is hollow and provides an enclosed cavity in which proteins are degraded. Openings at the two ends of the core allow the target protein to enter. Each end of the core particle associates with a 19S regulatory subunit that contains multiple ATPase active sites and ubiquitin binding sites. This structure recognizes polyubiquitinated proteins and transfers them to the catalytic core. An alternative form of regulatory subunit called the 11 S particle may play a role in degradation of foreign peptides and can associate with the core in essentially the same manner as the 19S particle. The proteasomal degradation pathway is essential for many cellular processes, including the cell cycle, the regulation of gene expression, and responses to oxidative stress. As noted above, the second component of the bifunctional hybrid-molecule, conjugate or complex of the preset disclosure, is at least one proteasome degradation mediating moiety. The phrase " proteasome degradation mediating moiety", as used herein refers to any element, component or moiety that may mediate either directly or indirectly proteasome degradation and processing of a target molecule. The degradation mediating moiety as disclosed herein may be any natural, synthetic, organic or inorganic molecule or moiety, that is involved in recruiting, translocating or modifying any target molecule in a manner that increases proteasomal degradation thereof. In more specific embodiments, the degradation mediating moiety of the bifunctional hybrid-molecule of the present disclosure mediates the proteasomal degradation and processing of the NF-κB1 p105, recruited by the peptide component of the bifunctional hybrid- molecule, conjugate or complex of the present disclosure. The degradation mediating moiety may be any compound that participates and/or mediates degradation of a target protein by the proteasome. In some embodiments, the degradation mediating moiety may be a hydrophobic tag mimicking protein misfolding. The hydrophobic tagging (HyT) technology extends the concept of inducing protein instability to a broader range of protein targets by mimicking protein misfolding The HyT consists of a hydrophobic fragment and a ligand fragment of the protein of interest (POI), e.g., any of the peptides disclosed herein, which is capable of causing degradation of the POI, specifically, NF-κB 1 p105. One mechanism is that the HyT destabilizes the POI, thereby recruiting an endogenous chaperone protein to the misfolded protein and then degrading the protein by the proteasome. Another mechanism is the direct recognition of the HyT by chaperones, mediating the proteasomal degradation of the tagged protein. The hydrophobic marker then is released and the POI can be degraded and procesed in successive rounds. More specifically, protein ubiquitination and degradation can be achieved by recruiting chaperones using lipophilic small molecule tags. For example, HSP70 family members recognize the exposed hydrophobic cores of misfolded proteins to hijack misfolded protein reactions. HSP70 is highly conserved and ubiquitous in microorganisms, plants and animals, and is involved in many cellular processes, including protein folding, transmembrane protein translocation and protein degradation regulation. Proteins with mild or partial misfolding are ubiquitinated by HSP40 and HSP70 and then degraded by HSP70 and 26S proteasomes. Thus, in some embodiments, the at least one proteasome degradation mediating moiety of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure, may be a hydrophobic tag (HyT), that recruits chaperones and mediates misfolded protein reactions. Still Further, in some alternative embodiments, the at least one proteasome degradation mediating moiety of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure, may be a HaloTag. More specifically, a HaloTag is a modified bacterial dehalogenase enzyme that covalently binds with a hexyl chloride label. HaloTag forms stable covalent bonds with compounds containing alkyl chlorides via a very simple binding moiety with low molecular weight and reasonable cell permeability. Thus, the HaloTag-based bifunctional molecule in accordance with some embodiments of the present disclosure, contain an alkyl chain HaloTag and a peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, that binds the target protein (NF-κB 1 p105), in accordance with the preset invention. This bifunctional molecule transfers the fusion domain onto the POI, binds the bacterial HaloTag protein and generates a hydrophobic group on its surface, which is mediated by a chaperone, e.g., HSP70.
In some embodiments, the proteasome degradation mediating moiety of the present disclosure may comprise at least one E3 ubiquitin ligase recruiting moiety, or at least one E3 ubiquitin ligase active moiety.
In some specific embodiments, E3 ubiquitin ligase active moiety may be any domain or fragment of an E3 ligase, that is capable of forming of an isopeptide bond between the carboxy terminus of ubiquitin and a lysine residue of a target protein. In some embodiments, the E3 ubiquitin ligase active moiety may comprise at least one of RING finger and U-box E3s, the HECT E3s, and the RING/HECT-hybrid type E3s of an E3 ligase. Non-limiting embodiment for such bifunctional hybrid molecules that comprises target (p105) recruitment component (SEQ ID NO: 2) is disclosed herein by SEQ ID NO: 65.
In yet some other embodiments, the proteasome degradation mediating moiety of the bifunctional hybrid-molecule, conjugate or complex of the invention may be any moiety that recruits at least one E3 ligase. The bifunctional hybrid-molecule, conjugate or complex of the present disclosure acts in some embodiments as an adaptor compound that recruits the NF-κB 1 p105 via the peptide component, and recruits E3 ligase using the proteasome degradation mediating component. Thus, the invention further encompasses bifunctional hybrid-molecule, conjugate or complexes, that induce targeted proteasome degradation of the NF-κB 1 p105 and processing thereof to form the p50. As indicated above, degradation of the NF-κB 1 p105 is performed by the UPS. “Ubiquitin Proteasome Pathway System (UPS)” as used herein relates to the ubiquitin proteasome pathway, conserved from yeast to mammals, and is required for the targeted degradation of most short-lived proteins in the eukaryotic cell. Targets include cell cycle regulatory proteins, whose timely destruction is vital for controlled cell division, as well as proteins unable to fold properly within the endoplasmic reticulum. Ubiquitin modification is an ATP - dependent process carried out by three classes of enzymes. An “ubiquitin activating enzyme” (El) forms athio-ester bond with ubiquitin. This reaction allows subsequent binding of ubiquitin to a “ubiquitin conjugating enzyme” (E2), followed by the formation of an isopeptide bond between the carboxy-terminus of ubiquitin and a lysine residue on the substrate protein. The latter reaction requires a “ubiquitin ligase” (E3). E3 ligases can be single- or multi-subunit enzymes. More specifically, an active site Cys on the E2 subsequently facilitates the transfer of the covalently linked ubiquitin from the E 1 to a Cys residue on the E2 through a trans-thio-esterification reaction. Concomitantly, an E3 ligase recruits a specific downstream target protein and mediates the transfer of the ubiquitin from the E2 enzyme to the terminal substrate through either a covalent or non - covalent mechanism. Each ubiquitin is ligated to a protein through either a peptide bond with the N-terminal amino group or an isopeptide bond formed between a side chain e -amino group of a select Lys residue on the target protein and the ubiquitin. Ubiquitin, a small protein with 76 amino acids, covalently conjugates to lysine (K) residues of substrate proteins. Ubiquitin conjugation is mediated by a three-step enzymatic process. First, with ATP as the energy source, the carboxyl group at the end of U glycine is linked to the thiol group of the U-activating enzyme El to form a thioester bond between U and El. Second, El transfers the activated U to E2 through a lactide process. Third, E3 binds E2 U to the target protein and releases E2 to leave a specific ubiquitinated protein. Finally, ubiquitinated proteins are recognized by specific proteasomes and degraded into short peptides or amino acids by proteases.
Thus, according to some embodiments, the phrase "proteasome degradation" as used herein, refers to the degradation of the ubiquitinated proteins, specifically the NF-κB1 p105, by the proteasome, as discussed herein above, into shorter peptides, for example, the p50 peptide, and optionally to other shorter peptides and amino acid residues. In yet some further embodiments, proteasome degradation of the NF-κB1 p105 mediated by the bifunctional hybrid molecules of the present disclosure leads to an increase in the production, and/or the levels of the NF-κB 1 p50 peptide, an increase of about 5% to 100%, specifically, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, as compared to the levels of NF-κB 1 p50 peptide in the absence of the bifunctional hybrid molecules of the present disclosure.
Most of the specificity of ubiquitination is mainly determined by E3s. Fluman genome encodes over 600 E3s, which serve as catalytic intermediates in ubiquitination (HECT domain E3s) or directly catalyze the transfer of ubiquitin from E2 to substrates (RING or RING-like domain E3s). There are eight different linkage types of ubiquitination, including K6, K11, K27, K29, K33, K48, K63, and Ml, in which the C terminus of ubiquitin is attached to lysine residues or the N-terminal methionine (Ml) of another ubiquitin. Ubiquitin forms monoubiquitination chain and polymeric ubiquitin chain, leading to distinct signaling outcomes for the substrate proteins.
Numerous E3 ligases provide specificity in that each can modify only a subset of substrate proteins. Further specificity is achieved by post-translational modification of substrate proteins, including, but not limited to, phosphorylation. However, multiple ubiquitination cycles resulting in a polyubiquitin chain are required for targeting a protein to the proteasome for degradation. The multisubunit 26S proteasome recognizes, unfolds, and degrades polyubiquitinated substrates into small peptides. The reaction occurs within the cylindrical core of the proteasome complex, and peptide bond hydrolysis employs a core threonine residue as the catalytic nucleophile. It has been shown that an additional layer of complexity, in the form of multiubiquitin chain receptors, may lie between the polyubiquitination and degradation steps. These receptors react with a subset of polyubiquitinated substrates, aiding in their recognition by the 26S proteasome, and thereby promoting their degradation. “Ligase” as used herein, is an enzyme that can catalyze the joining of two or more compounds or biomolecules by bonding them together with a new chemical bond. The “ligation” of the two usually with accompanying hydrolysis of a small chemical group dependent to one of the larger compounds or biomolecules, or the enzyme catalyzing the linking together of two compounds, e.g., enzymes that catalyze joining of groups C-O, C-S, C- N, etc. Ubiquitin-protein (E3) ligases are a large family of highly diverse enzymes selecting proteins for ubiquitination. “Ub Ligases” are involved in disease pathogenesis for oncology, inflammation and infectious disease. The E3 ligases originate in three major classes - the RING finger and U-box E3s, the HECT E3s, and the RING/HECT-hybrid type E3s. In some embodiments, the E3 ubiquitin ligase recruiting moiety of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may be suitable for recruiting any E3 ubiquitin ligase. To name but few, the E3 ubiquitin ligase recruiting moiety of the bifunctional hybrid-molecule is suitable for at least one of von- Hippel-Lindau (VHL), Cereblon (CRBN), Mouse double minute 2 homolog (MDM2), cellular inhibitor of apoptosis protein- 1 (cIAPl) and X-linked inhibitor of apoptosis protein (XIAP) and DDB1 and CUL4 associated factor 15 (DCAF15).
Any moiety capable of recruiting E3 ubiquitin ligase may be used in the bifunctional hybrid- molecule, conjugate or complex of the invention. For example, the moiety is a ligand for an E3 ubiquitin ligase. Thus, any ligand may be used in the bifunctional hybrid-molecule, conjugate or complexes of the present disclosure, for recruiting any E3 ligase. To name but few, the mouse double minute 2 homologue (MDM2) E3 can be recruited by using a known MDM2-p53 PPI inhibitor, nutlin, as the E3 ligand [Nutlins, a group of cis-diphcnyl substituted imidazoline- containing compounds (nutlin- 1, -2, and-3)], as well as Nutlin carboxylic acid (MDM2 ligand 1; E3 ligase Ligand 16),(4R,5S)-Nutlin carboxylic acid (MDM2 ligand 2; E3 ligase Ligand 15). For recruiting the cellular inhibitor of apoptosis protein 1 (cIAPl), ligands such as bestatin methyl esters can be used. More specifically, any one of cIAPl ligand 1 (E3 ligase Ligand 12), Bestatin- amido-Me (PROTAC IAP binding moiety 1) and β-Naphthoflavone-CH2-OH ( β-NH-CH2-OH), may be applicable. Still further, for recruiting Cereblon (CRBN), immunomodulatory drugs (IMiDs), may be useful. More specifically, suitable CRBN ligands applicable in the bifunctional hybrid-molecule, conjugate or complexes of the present disclosure, may include, but are not limited to Lenalidomide (CC-5013), Pomalidomide (CC-4047), ThaIidomide-NH-CH2- COOH, Thalidomide-O-COOH (Cereblon ligand 3; E3 ligase Ligand 3), Cereblon modulator 1 , Lenalidomide hemihy dr ate (CC-5013 hemihydrate), Thalidomide-5-OH, Thalidomide 4- fluoride (Cereblon ligand 4; E3 ligase Ligand 4), ThaIidomide-4-OH (Cereblon ligand 2; E3 ligase Ligand 2), ThaIidomide-O-C8-COOH, Thalidomide-propargyl, ThaIidomide-O-C8-Boc, CRBN modulator- 1 and Thalidomide 5 -fluoride. In some specific embodiments, the bifunctional hybrid- molecule, conjugate or complex of the present disclosure comprises at least one recruiting moiety for VHL E3 ubiquitin ligase. In more specific embodiments, the recruiting moiety comprises at least one small molecule or peptide, or any combinations thereof. In yet some further specific embodiments, the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise VHL ligands that comprise small molecules. Suitable ligands for VHL may include, but are not limited to any one of (S,R,S)-AHPC hydrochloride (VH032-NH2 hydrochloride; VHL ligand 1 hydrochloride), (S,R,S)-AHPC-Me hydrochloride (VHL ligand 2 hydrochloride; E3 ligase Ligand 1), (S,R,S)-AHPC-Me dihydrochloride (VHL ligand 2 dihydrochloride; E3 ligase Ligand 1 dihydrochloride), VL285, (S,S,S)-AHPC hydrochloride ((S,S,S)-VH032-NH2 hydrochloride), VH032-cyclopropane-F (VHL ligand 3; E3 ligase Ligand 19), VHL Ligand 8, VH032 thiol (VHL ligand 6), (S,R,S)-AHPC (VH032-NH2; VHL ligand 1), (S,R,S)-AHPC-Boc (VH032-Boc), and (S,R,S)-AHPC TFA (VH032-NH2 TFA; VHL ligand 1 TFA).
In some embodiments, the at least one recruiting moiety for VHL E3 ubiquitin ligase, of the bifunctional hybrid-molecule, conjugate or complex of the invention, may comprise the VHL ligand of Formula I, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof:
Figure imgf000039_0001
Formula I
As indicated above, the bifunctional hybrid-molecule, conjugate or complex of the invention comprises at least two components, one component, is at least one peptide, that in some embodiments recruits NF-κB1 p105. The second component is an E3 ligase recruiting component (also referred to herein as a ligand). Still further, in some the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may further comprise at least one linker.
In some embodiments, at least one of the linkers of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure is a bridging linker that bridges between said at least one peptide component or moiety and the at least one proteasome degradation mediating component or moiety. Still further, the linker may take any form, and any length appropriate to bring together the target protein NF-κB1 p105 and ubiquitinating machinery and thereby elicit the ubiquitination of NF- κBI p105 and its subsequent degradation in the proteasome. More specifically, the linker, that may be also referred to herein as a spacer, may take any form, provided that it does not significantly interfere with binding of the ligand to the target, specifically, the NF-κB1 p105. In some embodiments, the linker is constituted by those parts of the ligand which are exposed to solvent when the ligand is bound to the target. In other embodiments, the linker may be a series of stable covalent bonds incorporating one or more (e.g., 1-500) non-hydrogen atoms selected from the group consisting of C, N, O, S and P. Exemplary linkers therefore include moieties comprising — C(0)NH-- , -- C(0)0— , -- NH— , S-- and --O-- groups. Other suitable linkers may also be comprised of the atoms or groups including (but not limited to), carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl and imine. The linker chain may also comprise part of a saturated, unsaturated or aromatic ring, including polycyclic and heteroaromatic rings wherein the heteroaromatic ring is an aryl group containing from one to four heteroatoms, N, O or S. Specific examples include, but are not limited to, saturated alkanes, unsaturated alkanes, polyethylene glycols and dextran polymers. The linker or spacer is a substituted or unsubstituted polyglycol, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkyl ene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene. Still further, the linker is of a length appropriate to bring together target protein and ubiquitinating machinery and thereby elicit the ubiquitination of the protein of interest and its subsequent degradation in the proteasome. It is therefore to be understood that the linker of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the invention serves as a spacer, physically separating the target and ligase ligands to a degree sufficient to ensure that binding with their respective targets is not rendered mutually exclusive as a result of steric inhibition. In the absence of a linker, or if the linker is too short, the interaction between the target, specifically, the NF-κB1 p105 recruiting peptide and ligase ligands of the bifunctional hybrid-molecule, conjugate or complex/es disclosed herein, and their respective NF-κB1 p105 target and E3 ligase, could be disrupted. Fiowever, it will also be understood that the linker should also not be too long, since in such cases the bound E3 ligase might not be in sufficiently close spatial proximity to the NF-κB1 p105 target protein to trigger its ubiquitination and processing to produce the p50.Those skilled in the art will therefore appreciate that the length of the linker is optimized by reference inter alia to target and E3 ligase binding efficiency as well as target protein ubiquitination.
In some embodiments, the linker may be 1-21 bonds in length, for example 1-20, 1-19, 1-18, 1- 17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 bonds in length. In other embodiments, the linker may be 2-21 bonds in length, for example 3-21, 4-21, 5-21, 6-21, 7-21, 8-21, 9-21, 10-21, 11-21, 12-21, 13-21, 14-21, 15-21, 16-21, 17-21, 18-21, 18-21, 19-21 or
20-21 bonds in length. It should be appreciated that any appropriate likers may be used by the present disclosure ("linker" is also referred to herein as a "linker unit"). In some embodiments, the linker may be a bond. In some further embodiments the linker may be an amide bond. Still further, in some embodiments the linker may be an amide bond between the peptide and the ligand. In yet some further embodiments, the peptide forms an amide bond with the ligand. In some embodiments, the ligand forms an amide bond with the peptide. In yet some further embodiments, the ligand forms an amide bond with the linker. In some alternative embodiments, the linker forms an amide bond with the peptide. Still further, in some embodiments, the C=0 of the amide bond is originated from the linker unit. In yet some further additional or alternative embodiments, the NH of the amide bond is originated from the linker unit. In some embodiments, the linker may comprise one or more ethylene glycol units. In some embodiments where m=l, the linker comprises one ethylene glycol unit. Still further, in some embodiments, the linker is an ethylene glycol derivative that forms an amide bond with the ligand and/or the peptide, and/or the optional peptide linker.
In some embodiments, the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise any of the linkers of Formulas II or XVI to XXVII, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof.
In yet some further embodiments, the following linkers may be used by the bifunctional hybrid- molecule of the present disclosure:
Figure imgf000041_0001
Figure imgf000042_0001
ormu a . wherein m is an integer between 1 to 10. Thus, it should be understood that in any one of the linkers of Formulas II, XVI to XXVII, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
In some specific embodiments, the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise the linker of Formula II, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof,
Figure imgf000043_0001
Formula II wherein m is an integer between 1 to 10.
In more particular and non-limiting embodiments, the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprises the linker of Formula III, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof:
Figure imgf000043_0002
Formula III
In yet some further embodiments, the linker of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may further comprise at least one ami ancoid residue. It should be noted that such amin-acid linker may be of any length and may comprise any amino acid residues, either identical or different.
Thus, in some embodiments, the linker of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may further comprise an additional amino acid linker. In some embodiments, such linker in the context of the invention concerns an amino acid sequence of from about 1 to about 10 or more amino acid residues flanking the peptide component of the bifunctional hybrid-molecule, conjugate or complex of the present disclosure. The linker may be positioned in some embodiments, in at least one of its termini, namely at the C-terminus and/or at the N-terminus of the polypeptide of the bifunctional hybrid-molecule of the invention. The linker is covalently linked or joined to the amino acid residues in its vicinity.
For example, an additional amino acid linker in accordance with the invention may be of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acid residues long. In yet some further embodiments, the linker/s used by the invention may be a combinatorial linker comprising all possible linkers composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues. Still further, it should be understood that the peptide linker may be composed of various amino acid residues that may be either different or identical amino acid residues. In some specific embodiments, the amino-acid linker further comprised in the linker of the bifunctional hybrid-molecule, conjugate or complex of the invention, may comprises at least one of: at least one Serine (S), at least one cysteine (C) and at least one Glycine (G) amino acid residues, attached therewith. As indicated herein, in some embodiments, the peptides of the present disclosure comprise peptide linkers, such peptide include for example any of the peptides of SEQ ID NOs: 8, 28, 39, 42 to 59.
Thus, in some embodiments, the bifunctional hybrid-molecule, conjugate or complex of the present disclosure, that comprises an NF-κB1 p105 recruiting peptide, an E3 ligase recruiting ligand and at least one linker may be the conjugate or complex of any of the compounds of Formula IV, V, VI and VII, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof.
More specifically, in some embodiments, the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise the compound of Formula IV(also indicated herein as PROTAC 3, also shown in Fig. 4Avi), being:
Figure imgf000044_0001
Formula IV.
In yet some further embodiments, the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise the compound of Formula V (also indicated herein as PROTAC 1, also shown in Fig. 4Ai), being: Formula V.
In some further embodiments, the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise the compound of Formula VI (also indicated herein as PROTAC 5, also shown in Fig. 4Aiv), being:
Figure imgf000045_0001
Formula VI.
Still further, in some embodiments, the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may comprise the compound of Formula VII , being:
NH2-RIWVWLLCG'
Figure imgf000045_0002
o Formula VII.
The present disclosure relates to bifunctional hybrid-molecule, conjugate or complex, that induce targeted proteasomal degradation and processing of NF-κB 1 p105. Bifunctional hybrid-molecules that target UPS mediated degradation of a target protein by bridging between the target and the E3 ligase, are also known as Proteolysis Targeting Chimeric ligands (PROTAC compounds) that induce ubiquitination by the use of a ligase, such as E3 ligase and degrade a protein of interest. Thus, in some embodiments thereof, the present disclosure provides PROTAC compounds, and more specifically, the bifunctional hybrid-molecule, conjugate or complex of the present invention are in some embodiments, PROTAC molecules. PROTACs as used herein, are typically designed with three parts: (1) a ligand/molecule that binds to and/or modulates ubiquitin ligases; (2) a binding moiety that targets and recruits the protein of interest for proteolysis, e.g., the peptide of the invention that recruits NF-κB1 p105; and (3) a linker that links the two molecules together. PROTACs thus function by allowing the ligand/molecule to bind to the ubiquitin ligases, thereby recruiting the target of protein of interest to the ligase for ubiquitination and ultimately proteolysis and degradation.
Other names can however be found in the literature: e.g., specific and non-genetic IAP-dependent protein erasers (SNIPER); degrader; degronimids; PROteolysis TArgeting Peptide (PROTAP); Protein Degradation Probe (PDP). PROTACs hijack the catalytic activity of ubiquitin E3 ligases to mediate proteasome dependent degradation of selected protein of interest (POI), by bringing the ligase and POI into close spatial proximity and initiating the poly-ubiquitination process.
Still further, the bifunctional hybrid-molecule, conjugate or complex disclosed by the present disclosure are according to some embodiments PROTAC molecules. However, it should be appreciated that the present disclosure further encompasses also similar or corresponding CLIPTAC molecules. In some embodiments, the term "CLIPTAC" defines a proteolysis targeting chimeric molecule (PROTAC) formed from the intracellular self-assembly of precursors via bioorthogonal click chemistry (CLlckable Proteolysis TArgeting Chimera chimeric molecule), that refers to any chemical reaction that can occur inside of living systems. It should be understood that the present disclosure encompasses any PROTAC, CLIPTAC, or any bifunctional hybrid- molecule, conjugate or complexes that comprise any of the E3 ligands, any of the NF-κB1 p105 recruiting peptides and any of the linkers disclosed herein and any combinations thereof. Moreover, the molecules disclosed by the present disclosure are effective in targeted degradation and processing of the NF-κB1 p105 to produce NF-κB1 p50. However, in some embodiments, the bifunctional hybrid-molecules, conjugates or complexes, PROTACs of the present disclosure may comprise any combination of the discussed components (E3 ligase, recruiting peptide and linker), with the proviso that such molecule is not the PROTAC molecule of Formula VIII, being:
Figure imgf000046_0001
It should be noted that the bifunctional hybrid-molecule, conjugate or complex of the invention (e.g., PROTACs), or the at least one peptide component thereof, that comprises the at least two aromatic amino acid residues or any mimetics thereof, may be in certain embodiments, associated with, combined with or conjugated with at least one "enhancing" moiety. Such moiety may be any moiety that facilitating cell penetration, targeting to specific cell target and/or by increasing stability and reducing clearance thereof. The term "associated with" as used herein in reference to a half-life increasing moiety, a cell penetration moiety, a specific tissue or organ-directing moiety or a specific cell type directing moiety means that such moiety may be linked non- covalently, or covalently bound to, conjugated to, cross-linked to, incorporated within (e.g., such as an amino acid sequence within the NF-κB1 p105 recruiting peptide that comprise at least one of the aromatic amino acid residues or any mimetics thereof), or present in the same composition as the bifunctional hybrid-molecule, conjugate or complex of the present invention or the NF-κB 1 p105 recruiting peptide thereof, in such a way as to allow such moiety to carry out its function. The term "cell penetration moiety" as used herein means a moiety that enhances the ability of the bifunctional hybrid-molecule, conjugate or complex of the present invention or the NF-κB 1 p105 recruiting peptide thereof, with which it is associated to penetrate the cell membrane. In some embodiments, the "cell penetration moiety" may be an amino acid sequence within or connected to the bifunctional hybrid-molecule, conjugate or complex of the present invention or the NF-κB 1 p105 recruiting peptide thereof. Examples of cell penetration sequences include, but are not limited to, Arg-Gly-Asp (RGD), Tat peptide, oligoarginine, MPG peptides, Pep- land the like.
The term "specific organ directing moiety" as used herein means a moiety that enhances the ability of the bifunctional hybrid-molecule, conjugate or complex of the invention or its peptide component that comprises at least two aromatic amino acid residue/s or any mimetics thereof, non- standard peptide, polypeptide, non-standard polypeptide, protein or non-standard protein thereof, with which it is associated to be targeted to a specific organ. In some embodiments, the "specific organ directing moiety" is an amino acid sequence, small molecule or antibody that binds to a cell type present in the specific organ. In some embodiments, the "specific organ directing moiety" is anamino acid sequence, small molecule or antibody that binds to a receptor or other protein characteristically present in the specific organ. The term "specific cell-type directing moiety" as used herein means a moiety that enhances the ability of the bifunctional hybrid-molecule, conjugate or complex of the invention, with which it is associated to be targeted to a specific cell type. In some embodiments, the "specific cell-type directing moiety" is an ami ancoid sequence, small molecule or antibody that binds to a specific receptor or other protein characteristically present in or on the surface of the specific target cell type. Still further, it should be appreciated that in some embodiments, the bifunctional hybrid-molecule, conjugate or complex of the invention (e.g., PROTACs), or the at least one peptide component thereof, that comprises the at least two aromatic amino acid residues or any mimetics thereof, may be in certain embodiments, associated with, combined with or conjugated with at least one "labeling" moiety. In some embodiments such labeling moiety or "label" may include any tag (e.g., His Tag), or any dye, for example, FITS molecule as shown in Figures 5, 6 (synthesis and characterization) and 7 and 8 (function). Still further, as indicated above, the present disclosure provides bifunctional hybrid molecules and compositions thereof, that on one hand recruit the target NF-κB1 p105, and in the other hand recruit an E3 ligase. However, it should be understood that the invention further encompasses any multiple forms of the bifunctional hybrid molecules. More specifically, in some embodiments, the present disclosure further provides plurality of NF-κB1 p105 recruiting peptide/s as disclosed herein (that may potentially target the same or different locations of the target NF-κB1 p105 protein), and a plurality of E3 ubiquitin ligase recruiting moieties that may recruit the same or various E3 ubiquitin ligases (e.g., VHL, CRBN, MDM2, clAPl, XIAP and DCAF15). More specifically, in any of the aspects of embodiments described herein, the NF-κB1 p105 recruiting peptide/s, E3 ligase recruiting moieties (E3 ligand), and optionally, other moieties that bind specifically to another E3 ubiquitin ligase can be coupled directly or via one or more chemical linkers or a combination thereof. In additional embodiments, where a compound has multiple moieties that bind specifically to another E3 ubiquitin ligase, the moieties can be for the same E3 ubiquitin ligase or each respective moiety can bind specifically to a different E3 ubiquitin ligase. In those embodiments where a compound has multiple NF-κB1 p105 recruiting moieties (e.g., any of the various peptides disclosed herein), such moieties may be the same or, optionally, different. In additional embodiments, the plurality of E3 ligase recruiting moieties, are each connected to a NF-κB1 p105 recruiting peptide/a via a chemical linker. Thus, in some further additional embodiments, the bifunctional hybrid-molecule, conjugate or complex may comprise a plurality of E3 ligase recruiting moieties that may be either identical of different, and moreover, may recruit the same or various E3 ligases, and further comprises multiple NF-κB 1 p105 recruiting moieties, that may be either identical or different (e.g., the same peptide, or any combination of the peptides disclosed herein). The compounds of the invention, specifically, bifunctional hybrid- molecule, conjugate or complex or their pharmaceutically acceptable salts can contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as ( R )- or (.S')- or, as (D)- or (L)- for amino acids. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms whether or not they are specifically depicted herein. Optically active (+) and (-), ( R )- and (.S' )- or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefmic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included. A “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers”, which refers to two stereoisomers whose molecules are nonsuperimposable mirror images of one another. A “tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any said bifunctional hybrid-molecule, conjugate or complex. It should be appreciated that each and every bifunctional hybrid-molecule/s, conjugate/s or complex/s of the present disclosure as disclosed herein above, are applicable for any of the aspects disclosed herein after.
A further aspect of the invention relates to a composition comprising at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the bioactive molecule of the present disclosure. In some embodiments, the composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s. In some embodiments, the bifunctional hybrid-molecule, conjugate or complex comprises: First (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1. It should be noted that Zaa is any aromatic amino acid residue. It should be understood that "Zaa" as used herein is also denoted in the sequence listing with respect to SEQ ID NO: 1, as residue "Xaa2- and as residue "Xaa4 ”, both refer to any aromatic amino acid residue as disclosed herein. Thus, in some embodiments the sequence Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n) is also equivalent to Xaa(n)-Xaa2-Xaa(m)- Xaa4-Xaa(n), as also denoted by SEQ ID NO: 1. Still further, in some embodiments, Xaa is any amino acid residue. In some specific embodiments, n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7, as discussed above in connection with the previous aspect of the present disclosure. The bifunctional hybrid-molecule, conjugate or complex of the compositions disclosed herein further comprises a second component (b), at least one proteasome degradation mediating moiety. In some embodiments, the composition disclosed herein may comprise at least one bifunctional hybrid-molecule, conjugate or complex that is any of the bifunctional hybrid-molecule/s, conjugate/s or complex/es as defined by the present disclosure, in connection with other aspects of the invention. As indicated above, the peptides of the bifunctional hybrid-molecule/s, conjugate/s or complex/es comprise at least two aromatic am aicnido residues. Specifically, at least two of at least one W, at least one Y, at least one F, and any mimetics thereof. In some specific embodiments, the peptide of the bifunctional hybrid-molecule, conjugate or complex of the compositions of the invention may comprise the amino acid sequence of at least one of any one of: (a) RIWVWLL, as dented by SEQ ID NO: 2, or any variants and derivatives thereof; (b), IWVWLL, as dented by SEQ ID NO: 3, or any variants and derivatives thereof; (c) WILVRLW, as dented by SEQ ID NO: 4, or any variants and derivatives thereof; (d) RIFVFLL, as dented by SEQ ID NO: 9; (e) RIYVFLL, as dented by SEQ ID NO: 10; (f) Citrulline-IWVWLL, as dented by SEQ ID NO: 15; (g) GRIWVWLL, as dented by SEQ ID NO: 16; (h) RRRIWVWLL, as dented by SEQ ID NO: 27; and/or (i) WVW, or any variants and derivatives thereof. In yet some further embodiments, the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise any of the peptides disclosed herein, specifically, any of the peptides of SEQ ID NOs: 8, 11 to 28, and 52 to 59. In some embodiments, the at least one recruiting moiety for VHL E3 ubiquitin ligase, of the bifunctional hybrid-molecule, conjugate or complex of the compositions of the invention, may comprise the VHL ligand of Formula I, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof. As indicated above, the bifunctional hybrid-molecule, conjugate or complex of the compositions of the invention comprises at least two components. One component is at least one peptide, that in some embodiments, recruits NFκB p105. The second component is an E3 ligase recruiting component (also referred to herein as a ligand). Still further, in some embodiments, the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may further comprise at least one linker. In some specific embodiments, the bifunctional hybrid- molecule, conjugate or complex of the compositions of the present disclosure may comprise the linker of Formula II, or of Formula III, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof. In yet some further embodiments, the linker of the bifunctional hybrid-molecule, conjugate or complex of the compositions of the present disclosure may further comprises at least one amino acid residue. It should be noted that such amin-acid linker may be of any length and may comprise any amino acid residues, either identical or different. In some specific embodiments, the amino-acid linker further comprised in the linker of the bifunctional hybrid-molecule, conjugate or complex of the compositions of the invention, may comprises at least one of: at least one Ser (S), at least one cysteine (C) and at least one Gly (G) amino acid residues, attached therewith. In some embodiments, the bifunctional hybrid-molecule, conjugate or complex of the composition of the present disclosure, that comprises an NF-κB1 p105 recruiting peptide, an E3 ligase recruiting ligand and at least one linker may be the conjugate or complex of any of the compounds of Formula IV, V, VI and VII, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof. More specifically, in some embodiments, the bifunctional hybrid-molecule, conjugate or complex of the composition of the present disclosure may comprise the compound of Formula IV, being:
Figure imgf000051_0001
Formula IV.
In yet some further embodiments, the bifunctional hybrid-molecule, conjugate or complex of the composition of the present disclosure may comprise the compound of Formula V, being:
Figure imgf000051_0002
Formula V.
In some further embodiments, the bifunctional hybrid-molecule, conjugate or complex of the composition of the present disclosure may comprise the compound of Formula VI, being: Formula VI.
Still further, in some embodiments, the bifunctional hybrid-molecule, conjugate or complex of the composition of the present disclosure may comprise the compound of Formula VII, being:
Figure imgf000052_0001
Formula VII.
It should be understood that the compositions provided by the present disclosure may comprise any of the bifunctional hybrid-molecule/s, conjugate/s or complex/es, that in some embodiments may be PROTACs molecules of the invention and any combinations thereof. In yet some further embodiments, the present disclosure further encompasses any compositions comprising any of the compounds of the invention, specifically, the bifunctional hybrid-molecule/s, conjugate/s or complex/es and PROTACs, and any combinations thereof with any additional therapeutic compounds. In even more specific embodiments, any of the compositions of the present disclosure may be formulated as a pharmaceutical composition for delivery to a specific organ or cell type (e.g., brain, muscle, fibroblasts, bone, cartilage, liver, lung, breast, skin, bladder, kidney, heart, smooth muscle, adrenal, pituitary, pancreas, melanocytes, blood, adipose, and intestine). It will be understood that formulation for delivery to the brain requires the ability of the active components to cross the blood-brain barrier or to be directly administered to the brain or CNS.
Also pertinent to the present context are any type of compositions of bifunctional hybrid-molecule, conjugate or complex of the invention, that may be available as (but not limited to) a solution (e.g., tea), powder, tablet, capsule, elixir, topical, or injection. Thus, in further embodiments, the at least one bifunctional hybrid-molecule, conjugate or complex, any dosage form or composition thereof, may be an add-on to any type of drugs or therapeutic compounds administered orally, intravenously, intradermaly, by inhalation or intrarectaly. The compositions of the invention may comprise an effective amount of at least one bifunctional hybrid-molecule, conjugate or complex of the invention as disclosed herein and/or any vehicle, matrix, nano- or micro-particle thereof. The term "effective amount” relates to the amount of an active agent present in a composition, specifically, the bifunctional hybrid-molecule, conjugate or complex of the invention as described herein that is needed to provide a desired level of active agent in the bloodstream or at the site of action in an individual (e.g., the specific site of the tumor) to be treated to give an anticipated physiological response when such composition is administered. The precise amount will depend upon numerous factors, e.g., the active agent, the activity of the composition, the delivery device employed, the physical characteristics of the composition, intended patient use (i.e., the number of doses administered per day), patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein.
More specifically, pharmaceutical compositions used to treat subjects in need thereof according to the invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general formulations are prepared by uniformly and intimately bringing into association the active ingredients, specifically, the bifunctional hybrid-molecule, conjugate or complex of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The compositions may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. The pharmaceutical compositions of the present invention also include, but are not limited to, emulsions and liposome-containing formulations. It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question. As indicated above, pharmaceutical preparations are compositions that include one or more bifunctional hybrid- molecule, conjugate or complex present in a pharmaceutically acceptable vehicle. "Pharmaceutically acceptable vehicles" may be vehicles approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in any organism, specifically any vertebrate organism, for example, any mammal such as human. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is formulated for administration to a mammal. Such pharmaceutical vehicles can be lipids, e.g., liposomes, e.g., liposome dendrimers; liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline; gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. Pharmaceutical compositions may be formulated into preparations in solid, semisolid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. As such, administration of the bifunctional hybrid-molecule, conjugate or complex /s of the invention can be achieved in any of the various ways disclosed by the invention.
A further aspect of the present invention elates to a method for inducing ubiquitination and proteasomal processing of NF-κB1 p105, thereby generating the NF-κB p50 in a cell or in a cell- free system that comprise the NF-κB 1 p105. More specifically, the method comprising the step of contacting the cell or the cell-free system with an effective amount of at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the same, or any composition thereof. In some embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise: First component (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue. The peptide comprises the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa- Xaa(n), as denoted by SEQ ID NO: 1. It should be noted that Zaa is any aromatic amino acid residue. It should be understood that "Zaa" as used herein is also denoted in the sequence listing with respect to SEQ ID NO: 1, as residue "Xaa2- and as residue "Xaa4·. both refer to any aromatic amino acid residue as disclosed herein. Thus, in some embodiments the sequence Xaa(n)-Zaa-Xaa(m)-Zaa- Xaa(n) is also equivalent to Xaa(n)-Xaa2-Xaa(m)-Xaa4-Xaa(n), as also denoted by SEQ ID NO: 1. Still further, in some embodiments, Xaa is any amino acid residue. In some specific embodiments, n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7, as discussed above in connection with the other aspect of the present disclosure. The bifunctional hybrid-molecule, conjugate or complex of the methods disclosed herein further comprises a second component (b), at least one proteasome degradation mediating moiety. The peptide comprised in the bifunctional hybrid molecules of the preset disclosure, recruit the target NF-κB1 p105, thereby enhancing ubiquitination and proteasomal processing of NF-κB1 p105 to p50. NF-κB forms a family of transcription factors that play essential roles in multiple physiological and pathological processes. NF-κB is typically a heterodimer that can be made of either p50 or p52 and RelA, RelB, or cRel. p50 and p52 homodimers are also described as well. The p50 and p52 are the products of ubiquitination and proteasome-dependent limited processing of the long precursors- p105 and plOO, respectively. p50 and p52 NFκB subunits lack a transactivation domain, which is present in the Rel proteins. As a result, homodimers based on p50 or p52 promote transcription only in case of complex formation with additional transcriptional activators, such as Bcl3, F1DAC3, or IκBZ. In resting cells, the dimers are trapped in the cytoplasm by specific inhibitors known as IκBS (e.g., IkBα, plOO, p105, and Bcl3). In response to a broad array of signals (e.g., oxidative stress, viral and bacterial infections, proinflammatory cytokines, and DNA damage), certain IκBS are phosphorylated on specific serine residues by the IκB kinase (IKK) complex and, consequently, are ubiquitinated and degraded by the proteasome. This releases the dimers that are translocated to the nucleus to initiate the specific transcriptional program. There are two different NF-κB pathways, the canonical and non-canonical NF-κB pathways, with different activating mechanisms. It is well established that the canonical NF-κB is activated to respond to a diversity of external stimuli involved in inflammation, immune response, cell proliferation, differentiation, and survival. The crucial step of activation of the canonical NF- κB is phosphorylation-dependent activation of the IKKs (IκB kinases) complex. Consequently, the inhibitory IκB proteins are phosphorylated and subjected to the ubiquitination-dependent degradation by proteasome, liberating the κB transcription factor to translocate to nucleus and activate the target genes. The activation is quick but transient, since NF-κB also induces expression of the negative regulators like IkBα, A20, and p105, forming a negative feedback mechanism. On the other hand, the non-canonical NF-κB is activated only through a handful of TNF superfamily receptors, indicating that the biological functions of this branch of pathway are more specific. NIK (NF-κB-inducing kinase), the key kinase in this pathway, remains below the detectable level in the steady-state condition due to the TRAF3 (TNFR-associated factor 3)-dependent ubiquitination-mediated degradation. Upon stimulation, TRAF3 is degraded by E3 ubiquitin ligase cIAP (cellular inhibitor of apoptosis), leading to NIK accumulation. Consequently, NIK, together with IKKα, phosphorylates plOO, which is further processed to p52, releasing RelB/p52 dimer to translocate into nuclear for target gene activation. It is well established that RelA and p50 heterodimers are responsible for transcription of target genes when the canonical NF-κB pathway is activated, while RelB and p52 form a heterodimer in non-canonical NF-κB pathway. In the steady-state settings, RelA and p50 are sequestered in the cytoplasm by the IκB (inhibitor of NF- κB) proteins, which consist of three groups: the typical IκB proteins (IkBα, IkBb, and IkBe), the precursor proteins (plOO and p105), and the atypical IκB proteins (IkBz, BCL-3 and IκBNS). The central event in canonical NF-κB activation is the signal-induced phosphorylation of IκB molecules by IKKs. IKK consists of two homologous catalytic subunits IKKα (also known as IKK1) and IKKb (also known as IKK2), and a regulatory subunit IKKg (also known as NF-κB essential modulator, NEMO).
In some embodiments, the NF-κB 1 p105 as used herein refers to the human NF-κB 1 p105. Still further, in some embodiments, such protein is denoted by UniProtKB - P19838. In yet some further embodiments, p105 as used herein comprises the amino acid sequence as denoted by SEQ ID NO: 60. In yet some further embodiments, the Nuclear factor NF-kappa-B p50 product of UniProtKB P19838, comprises the amino acid sequence as denoted by SEQ ID NO: 61. Thus, in some embodiments, generation the NF-κB p50, leads to various effects. In some embodiments, the generation of p50 by the methods of the invention may be involved in modulation of NF-κB canonical pathway. In some embodiments, such modulation may involve inhibition of the NF-κB canonical pathway. Still further, in some embodiments, the generation of p50 by the methods of the invention may lead to activation of transcription of at least one tumor suppressor.
In yet some further embodiments, the generation of p50 by the methods of the invention may lead to suppression of programmed cell death-ligand l(PD-Ll) expression. In yet some further embodiments, the generation of p50 by the methods of the invention may result in increased secretion of at least one proinflammatory cytokine by the cell. In some embodiments, the peptides of the invention and constructs thereof change the expression pattern of various groups of proteins. In yet some further embodiments, the peptides of the invention, the derivative or variant or form of the disclosed peptides, and/or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein lead to an increase, elevation, upregulation, enhancement and or enlargement of the expression of proteins connected to immune system processes and/or proteins connected with regulation of cell migration, and/or proteins that relate to ECM organization, and/or proteins that relate to cell adhesion, and/or proteins that relate to regulation of cell adhesion. In yet some further additional or alternative embodiments, the peptides of the invention, the derivative or variant or form of the disclosed peptides, and/or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein lead to a decrease, reduction, attenuation, inhibition and downregulation of the expression of proteins connected to metabolic processes, and/or proteins that relate to cell cycle, and/or proteins that relate to DNA metabolic processes.
In some embodiment, the at least one bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may be any of the bifunctional hybrid-molecule, conjugate or complex disclosed by the present disclosure, in connection with other aspects of the present disclosure.
As indicated above, the peptides of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the methods of the invention comprise at least two aromatic amino acid residues. Specifically, at least two of at least one W, at least one Y, at least one F, and any mimetics thereof. In some embodiments, at least one of the at least two aromatic amino aromatic amino acid residues of the peptides of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure, may be tryptophan. In some embodiments, one of the aromatic amino acid residues in the peptides of the bifunctional hybrid-molecule of the present disclosure may be tryptophan, and the other may be tyrosine. In some alternative embodiments, the peptides of the bifunctional hybrid-molecule of the present disclosure may comprise at least one tryptophan and at least one phenylalanine. In yet some further embodiments, the peptides of the bifunctional hybrid-molecule of the invention may comprise at least two tryptophan residues. It should be understood that the peptides of the bifunctional hybrid-molecule of the methods of the present disclosure may comprise any further aromatic amino acid residues as one or more of the Xaa residues located in various positions of the peptide as discussed above. Still further, in some embodiments, at least one of the "interspacing" amino acid residue/s located between the two aromatic amino acid residues, peptides of the bifunctional hybrid-molecule/s used by the methods of the invention, may be one or more amino acid residue/s having a non-polar side chain, also referred to herein as a non-polar amino acid. Such residues may be any one of Valine (V, Val), Glycine (G, Gly), Leucine (L, Leu), Isoleucine (I, IIe), Methionine (M, Met), Phenylalanine (F, Phe), Tryptophane (W, Trp) and/or Proline (P, Pro). In yet some further embodiments, the interspacing amino acid residue may be any hydrophobic amino acid residue, for example, Valine (V, Val), Leucine (L, Leu) and/or Isoleucine (I, IIe). In some specific embodiments, the peptides of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise at least two aromatic amino acid residues that are interspaced by at least one of Valine (V), Glycine (G) and/or Alanine (A). In some particular and non-limiting embodiments, the at least two aromatic amino acid residues of the peptides of the bifunctional hybrid-molecule disclosed herein, are interspaced by at least one Valine residue. Thus, in some embodiments, the peptides of the bifunctional hybrid-molecule, conjugate or complex used by the methods in accordance with the invention may comprise the amino acid sequence of Xaa(n)-Trp (W)-Xaa(n)-Val (V)-Xaa(n)-Trp (W)-Xaa(n), as denoted by SEQ ID NO: 7, or any mimetics thereof. It should be noted that Xaa is any amino acid residue, and n is zero or an integer between 1 to 7. These amino acid residues may be any identical or different am aincoid residues.
In some embodiments, the at least one peptide of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure, may comprise three to ten amino acid residues, specifically, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. In yet some further embodiments, the peptides of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise any amino acid residue, more specifically, in some embodiments Xaa that may be any one of Arg (R), IIe (I) and Leu (L), amino acid residues. Thus, in some specific embodiments, the peptide of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise the amino acid sequence of at least one of any one of: (a) RIWVWLL, as dented by SEQ ID NO: 2, or any variants and derivatives thereof; (b), IWVWLL, as dented by SEQ ID NO: 3, or any variants and derivatives thereof; (c) WILVRLW, as dented by SEQ ID NO: 4, or any variants and derivatives thereof; (d) RIFVFLL, as dented by SEQ ID NO: 9; (e) RIYVFLL, as dented by SEQ ID NO: 10; (f) Citrulline-IWVWLL, as dented by SEQ ID NO: 15; (g) GRIWVWLL, as dented by SEQ ID NO: 16; (h) RRRIWVWLL, as dented by SEQ ID NO: 27; and/or (i) WVW, or any variants and derivatives thereof.. In yet some further embodiments, the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise any of the peptides disclosed herein, for example, any of the peptides of SEQ ID NO: 8, 11 to 28, and 52 to 59. In some embodiments, the peptide of the bifunctional hybrid-molecule, conjugate or complex of the invention binds and therefore recruits the NF-κB1 p105. It should be understood that any variant or derivative of the above-mentioned peptides may be comprised within the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention. In some embodiments, any variant or derivative of each one of the above indicated peptides may be applicable in any of the methods of the invention, provided that the variant and/or derivative retains the ability of the peptide to recruit and bind the NF-κB1 p105.
In some embodiments, the proteasome degradation mediating moiety of the bifunctional hybrid- molecule/s, conjugate/s or complex/es of the present disclosure, may comprises at least one E3 ubiquitin ligase recruiting moiety, or at least one E3 ubiquitin ligase active moiety.
In some specific embodiments, such E3 ubiquitin ligase active moiety may be any domain or fragment of an E3 ligase, that is capable of forming of an isopeptide bond between the carboxy terminus of ubiquitin and a lysine residue of a target protein. In some embodiments, the E3 ubiquitin ligase active moiety may comprise at least one of RING finger and U-box E3s, the HECT E3s, and the RING/HECT-hybrid type E3s of an E3 ligase.
In yet some other embodiments, the proteasome degradation mediating moiety of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may be any moiety that recruits at least one E3 ligase.
In some embodiments, the E3 ubiquitin ligase recruiting moiety of the bifunctional hybrid- molecule, conjugate or complex of the methods of the present disclosure may be suitable for recruiting any E3 ubiquitin ligase. To name but few, the E3 ubiquitin ligase recruiting moiety of the bifunctional hybrid-molecule is suitable for at least one of von-Hippel-Lindau (VHL), Cereblon (CRBN), Mouse double minute 2 homolog (MDM2), cellular inhibitor of apoptosis protein- 1 (clAPl) and X-linked inhibitor of apoptosis protein (XI AP) and DDB1 and CUL4 associated factor 15 (DCAF15).
In some specific embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure comprises at least one recruiting moiety for VHL E3 ubiquitin ligase. In more specific embodiments, the recruiting moiety comprises at least one small molecule or peptide, or any combinations thereof. It should be understood that any of the L3 ligase ligands, and specifically, an of the VHL ligands disclosed by the invention are applicable for this aspect as well.
In some embodiments, the at least one recruiting moiety for VHL E3 ubiquitin ligase, of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention, may comprise the VHL ligand of Formula I, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof:
Figure imgf000059_0001
As indicated above, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention comprises two components, one component, is at least one peptide, that in some embodiments recruits NF-κB p105. The second component is an E3 ligase recruiting component. Still further, in some the bifunctional hybrid-molecule, conjugate or complex of the present disclosure may further comprise at least one linker.
In some embodiments, at least one of said linkers is a bridging linker that bridges between said at least one peptide and the at least one proteasome degradation mediating moiety.
The linker may take any form, and any length appropriate to bring together the target protein NF- κBI p105 and ubiquitinating machinery and thereby elicit the ubiquitination of NF-κB 1 p105 and its subsequent degradation in the proteasome.
In some specific embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the linker of Formula II as disclosed herein, or the linker of Formula III as disclosed herein, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof.
In yet some further embodiments, the linker of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may further comprises at least one amino acid residue. It should be noted that such amin-acid linker may be of any length and may comprise anyamino acid residues, either identical or different. In some specific embodiments, the amino- acid linker further comprised in the linker of he the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention, may comprises at least one of: at least one Serine (S), at least one cysteine (C) and at least one Glycine (G) amino acid residues, attached therewith. In some embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise at least one of the compounds of Formula IV, V, VI and VII, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof. In more specific embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula IV, being:
Figure imgf000060_0001
Formula IV.
In yet some further embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula V, being: Formula V.
In some further embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula VI, being:
Figure imgf000061_0001
Formula VI.
Still further, in some embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula VII, being:
Figure imgf000061_0002
Formula VII.
In some embodiments, the methods of the present disclosure may be applicable for inducing ubiquitination and proteasomal processing of NF-κB 1 p105 to generate the NF-κB p50 in a subject. In yet some further embodiments, where the step of contacting the cell that comprise the NF-κB p105 with the bi-functional hybrid-molecule of the present disclosure is performed in a subject in need thereof, the step of contacting the cell may comprise administering to the subject an effective amount of the bi-functional hybrid-molecule of the present disclosure. The effective amount as used herein, refers to the amount required for the induction of the processing of the NF-κB p105, to generate the NF-κB p50 in the subject. In some embodiments, generation the NF-κB p50, leads to various effects in the subject. In some embodiments, the generation of p50 by the methods of the invention may be involved in modulation of NF-κB canonical pathway in a subject. In some embodiments, such modulation may involve inhibition of the NF-κB canonical pathway in the subject. Still further, in some embodiments, the generation of p50 by the methods of the invention may lead to activation of transcription of at least one tumor suppressor in the subject.
In yet some further embodiments, the generation of p50 by the methods of the invention may lead to suppression of programmed cell death-ligand l(PD-Ll) expression in the subject. In yet some further embodiments, the generation of p50 by the methods of the invention may result in increased secretion of at least one proinflammatory cytokine in the subject. In some further embodiments, the generation of p50 by the methods of the invention may result in recruitment of immune cells to a diseased tissue or organ in the subject. As indicated above, in some embodiments, the peptides of the invention and constructs thereof change the expression pattern of various groups of proteins. In yet some further embodiments, the peptides of the invention, the derivative or variant or form of the disclosed peptides, and/or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein lead to an increase, elevation, upregulation, enhancement and or enlargement of the expression of proteins connected to immune system processes and/or proteins connected with regulation of cell migration, and/or proteins that relate to ECM organization, and/or proteins that relate to cell adhesion, and/or proteins that relate to regulation of cell adhesion. In yet some further additional or alternative embodiments, the peptides of the invention, the derivative or variant or form of the disclosed peptides, and/or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein lead to a decrease, reduction, attenuation, inhibition and downregulation of the expression of proteins connected to metabolic processes, and/or proteins that relate to cell cycle, and/or proteins that relate to DNA metabolic processes. It should be understood that as disclosed herein, a decrease or alternatively, an increase in the level of the discussed proteins is meant any increase or alternatively, decrease of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more, as compared to a cell, in a subject and/or a cell free system in the absence of the peptides of the invention or any derivative or variant or form of the disclosed peptides, and or of any bifunctional hybrid-molecule, conjugate or complex as disclosed herein. As indicated above, NF-κB, is a major transcriptional regulator for the cell response to external signals. It orchestrates a broad range of cellular processes, among them cell division and differentiation and cell death and survival. Importantly, NF-κB controls the immune and inflammatory response. Dysregulated activity of NF- Kb has been reported to be involved in a broad array of immune system-related disorders and malignant transformation. Canonical NF-κB is activated rapidly, inducing numerous proinflammatory mediators and molecules that lead to inflammatory response as well as activation and differentiation of immune cells. However, aberrant activation of NF-κB cause chronic inflammation, oncogenesis and autoimmune disease.
Activation of canonical NF-κB is often linked to the inflammation response to infection and injury, which is a part of host defense. Well-regulated inflammation response is essential for host homeostasis. Tumorigenic pathogens cause chronic infections and inflammation, leading to malignancy. It has been shown that chronic infections and inflammation contribute to certain cancers. For example, the HBV (human hepatitis B virus) is the major risk factor for HCC (hepatocellular carcinoma). Chronic Helicobacter pylori infection is linked to MALT (mucosa- associated lymphoid tissue) lymphoma and gastric cancer. However, immune dysregulation also causes chronic inflammation, leading to chronic or systemic inflammatory diseases such as RA (rheumatoid arthritis), IBD (inflammatory bowel disease) and psoriasis. Among these chronic inflammatory diseases, IBD is tightly correlated with colorectal cancer, while RA and psoriasis do not show significant tumor-promoting effect. There are other factors also contributing to chronic inflammation-related cancer, such as tobacco smoke, silica particles and obesity. As providing effective methods for inducing targeted proteasomal degradation and processing of the NF-κB 1 p105 to p50, the invention provides powerful means for modulating NF-κB 1 canonical pathway, and moreover, for extending the tumor suppressive effect of NF-κB 1 p50.
It should be understood that any of the peptides disclosed by the present disclosure and any of the PROTAC molecules or any variants, derivatives and combinations thereof disclosed by the present disclosure in connection with other aspects of the invention are applicable in the disclosed therapeutic methods discussed herein. Thus, a further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder or condition in a subject in need thereof. More specifically, the method comprising the step of administering to said subject a therapeutically effective amount of at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the same, or any composition thereof. In some embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise: First (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1. It should be noted that Zaa is any aromatic amino acid residue. Still further, in some embodiments, Xaa is any amino acid residue. It should be understood that "Zaa" as used herein is also denoted in the sequence listing with respect to SEQ ID NO: 1, as residue "Xaa2" and as residue "Xaa4", both refer to any aromatic amino acid residue as disclosed herein. Thus, in some embodiments the sequence Xaa(n)-Zaa- Xaa(m)-Zaa-Xaa(n) is also equivalent to Xaa(n)-Xaa2-Xaa(m)-Xaa4-Xaa(n), as also denoted by SEQ ID NO: 1. In some specific embodiments, n is zero or an integer between 1 to 7, and m is an integer between 1 to 7, as discussed above in connection with the previous aspect of the present disclosure. The bifunctional hybrid-molecule, conjugate or complex of the compositions disclosed herein further comprises a second component (b), at least one proteasome degradation mediating moiety. As indicated above, the peptide of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the methods of the invention may comprise at least two aromatic amino acid residues. Specifically, at least two of at least one W, at least one Y, at least one F, and any mimetics thereof. In some embodiments, the peptide of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the methods of the invention may comprise two tryptophan residues (WW). In yet some further embodiments, the peptide of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the methods of the invention comprise at least one tryptophan and at least one tyrosine (WY). Still further, in some embodiments, the peptide of the bifunctional hybrid- molecule/s, conjugate/s or complex/es of the methods of the invention may comprise at least one tryptophane and at least one phenylalanine (WF). In other embodiments, the peptide of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the methods of the invention may comprise at least two tyrosine resides (YY). In some further embodiments, the peptide of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the methods of the invention comprise may comprise at least one tyrosine and at least one phenylalanine residues (YF). Still further, in some embodiments the peptide of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the methods of the invention comprise at least two phenylalanine residues (FF).
In some embodiments, at least one of the at least two aromatic amino aromatic amino acid residues of the peptide of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure, may be tryptophan. In some embodiments, one of the aromatic amino acid residues in the peptide of the bifunctional hybrid-molecule of the present disclosure may be tryptophan, and the other may be tyrosine. In some alternative embodiments, the peptide of the bifunctional hybrid-molecule of the present disclosure may comprise at least one tryptophan and at least one phenylalanine. In yet some further embodiments, the peptide of the bifunctional hybrid-molecule of the invention may comprise at least two tryptophan residues. It should be understood that the peptide of the bifunctional hybrid-molecule of the present disclosure may comprise any further aromatic amino acid residues as one or more of the Xaa residues located in various positions of the peptide as discussed above. Still further, in some embodiments, at least one of the "interspacing" amino acid residue/s located between the two aromatic amino acid residues peptide of the bifunctional hybrid-molecule, conjugate or complex of the invention, may be one or more amino acid residue/s having a non-polar side chain, also referred to herein as a non-polar amino acid. Such residues may be any one of Valine (V, Val), Glycine (G, Gly), Leucine (L, Leu), Isoleucine (I, lie), Methionine (M, Met), Phenylalanine (F, Phe), Tryptophane (W, Trp) and/or Proline (P, Pro). In yet some further embodiments, the interspacing amino acid residue may be any hydrophobic amino acid residue, for example, Valine (V, Val), Leucine (L, Leu) and/or Isoleucine (I, IIe). In some specific embodiments, the peptide of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise at least two aromatic amino acid residues that are interspaced by at least one of Valine (V), Glycine (G) and/or Alanine (A). In some particular and non-limiting embodiments, the at least two aromatic amino acid residues of the peptide of the bifunctional hybrid-molecule disclosed herein, are interspaced by at least one Valine residue. Thus, in some embodiments, the peptide of the bifunctional hybrid- molecule, conjugate or complex used by the methods in accordance with the invention may comprise the amino acid sequence of Xaa(n)-Trp (W)-Xaa(n)-Val (V)-Xaa(n)-Trp (W)-Xaa(n), as denoted by SEQ ID NO: 7, or any mimetics thereof. It should be noted that Xaa is any amino acid residue, and n is zero or an integer between 1 to 7. These am aicniod residues may be any identical or different amino acid residues. In some embodiments, the at least one peptide of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure, may comprise three to ten amino acid residues, specifically, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. In yet some further embodiments, the peptide of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise any amino acid residue, Xaa that is any one of Leu (L), Arg (R) and IIe (I) amino acid residues. Thus, in some specific embodiments, the peptide of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may comprise the amino acid sequence of at least one of any one of: (a) RIWVWLL, as dented by SEQ ID NO: 2, or any variants and derivatives thereof; (b), IWVWLL, as dented by SEQ ID NO: 3, or any variants and derivatives thereof; (c) WILVRLW, as dented by SEQ ID NO: 4, or any variants and derivatives thereof; (d) RIFVFLL, as dented by SEQ ID NO: 9; (e) RIYVFLL, as dented by SEQ ID NO: 10; (f) Citrulline-IWVWLL, as dented by SEQ ID NO: 15; (g) GRIWVWLL, as dented by SEQ ID NO: 16; (h) RRRIWVWLL, as dented by SEQ ID NO: 27; and/or (i) WVW, or any variants and derivatives thereof. In yet some further embodiments, the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise any of the peptides disclosed herein, for example, any of the peptides of 8, 11 to 28, and 52 to 59.
In some embodiments, the peptide of the bifunctional hybrid-molecule, conjugate or complex of the methods of the invention binds and therefore recruits the and bind the NF-κB1 p105. It should be understood that any variant or derivative of the above-mentioned peptides may be comprised within the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention. In some embodiments, any variant or derivative of each one of the above indicated peptides is encompassed by the invention, provided that the variant or derivative retains the ability of the peptide to recruit and bind the NF-κB1 p105. In some embodiments, the proteasome degradation mediating moiety of the bifunctional hybrid-molecule/s, conjugate/s or complex/es of the present disclosure may comprises at least one E3 ubiquitin ligase recruiting moiety, or at least one E3 ubiquitin ligase active moiety. In some specific embodiments, E3 ubiquitin ligase active moiety may be any domain or fragment of an E3 ligase, that is capable of forming of an isopeptide bond between the carboxy terminus of ubiquitin and a lysine residue of a target protein. In some embodiments, the E3 ubiquitin ligase active moiety may comprise at least one of RING finger and U-box E3s, the HECT E3s, and the RING/HECT-hybrid type E3s of an E3 ligase. In yet some other embodiments, the proteasome degradation mediating moiety of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention may be any moiety that recruits at least one E3 ligase. In some embodiments, the E3 ubiquitin ligase recruiting moiety of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may be suitable for recruiting any E3 ubiquitin ligase. To name but few, the E3 ubiquitin ligase recruiting moiety of the bifunctional hybrid-molecule is suitable for at least one of von- Hippel-Lindau (VHL), Cereblon (CRBN), Mouse double minute 2 homolog (MDM2), cellular inhibitor of apoptosis protein- 1 (clAP1) and X-linked inhibitor of apoptosis protein (XI AP) and DDB1 and CUL4 associated factor 15 (DCAF15).In some specific embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure comprises at least one recruiting moiety for VHL E3 ubiquitin ligase. In more specific embodiments, the recruiting moiety comprises at least one small molecule or peptide, or any combinations thereof. It should be understood, that any VHL ligand is applicable by the methods of the invention, specifically, any of the VHL ligands disclosed by the invention. In some embodiments, the at least one recruiting moiety for VHL E3 ubiquitin ligase, of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention, may comprise the VHL ligand of Formula I, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof, being:
Figure imgf000067_0001
As indicated above, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention comprises two components, one component, is at least one peptide, that in some embodiments recruits NFKB p105. The second component is an E3 ligase recruiting component (also referred to herein as a ligand). Still further, in some embodiments, the bifunctional hybrid- molecule, conjugate or complex of the present disclosure may further comprise at least one linker. In some embodiments, at least one of the linkers may be a bridging linker that bridges between the at least one peptide and the at least one proteasome degradation mediating moiety. Still further, in some embodiments, the linker may take any form, and any length appropriate to bring together the target protein NF-κB1 p105 and ubiquitinating machinery and thereby elicit the ubiquitination of NF-κB1 p105 and its subsequent degradation in the proteasome. In some specific embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the linker of Formula II (as disclosed herein), or the linker of Formula III (as disclosed herein), or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof.
In yet some further embodiments, the linker of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may further comprises at least one amino acid residue. It should be noted that such amino-acid linker may be of any length and may comprise anyamino acid residues, either identical or different. In some specific embodiments, the amino- acid linker further comprised in the linker of the bifunctional hybrid-molecule, conjugate or complex used by the methods of the invention, may comprise at least one of: at least one Serine (S), at least one cysteine (C) and at least one Glycine (G) amino acid residues, attached therewith. In some embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise at least one of the compounds of Formula IV, V, VI and VII, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof, or any compositions thereof. In more specific embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula IV, being:
Figure imgf000068_0001
Formula IV.
In yet some further embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula V, being:
Figure imgf000068_0002
Formula V.
In some further embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula VI, being:
Figure imgf000068_0003
Formula VI. Still further, in some embodiments, the bifunctional hybrid-molecule, conjugate or complex used by the methods of the present disclosure may comprise the compound of Formula VII, being:
Figure imgf000069_0001
Formula VII.
It should be understood that the present disclosure encompasses methods using any PROTAC, CLIPTAC, or any bifunctional hybrid-molecule, conjugate or complexes that comprise any of the E3 ligands, any of the NF-κB1 p105 recruiting peptides and any of the linkers disclosed herein and any combinations thereof, and any compositions thereof. Moreover, the molecules used by the methods of the present disclosure are effective in targeted degradation and processing of the NF- κBI p105 to produce NF-κB1 p50. However, in some embodiments, the bifunctional hybrid- molecules, conjugates or complexes, PROTACs use by the methods of the present disclosure may comprise any combination of the discussed components (E3 ligase, recruiting peptide and linker), with the proviso that such molecule is not the PROTAC molecule of Formula VIII, being:
Figure imgf000069_0002
Formula VIII.
The crucial role of NF-κB in tumorigenesis is well established. In many solid malignances, the activity of NF-κB is upregulated, modulating tumor initiation, promotion, and metastases. In most cases, the tumor-promoting activity of NF-κB is related to the p65p50 heterodimeric complex. Thus, in ovarian cancer, strong expression of both subunits was found compared to borderline and benign ovarian tumors. Enhanced p65 staining was found in human prostate adenocarcinoma, correlating with increased tumor grade. The study revealed that the DNA-binding complex is mainly made of NF-κB p50p65 heterodimers. Although in most cases, NF-κB appears to be oncogenic, in some studies it has been demonstrated as a tumor suppressor, which is due in particular to its p50 subunit.
It was shown that in pancreatic cancer cells, IKKα regulates G1 to S-phase transition by exchange of the tumor promoter p52ReIB with the tumor-suppressive p50ReIB NF-κB dimers on the promoter of the F-box protein S-phase kinase-associated protein 2 (SKP2), inhibiting its expression. SKP2 is the ubiquitin ligase that negatively regulates the abundance of the cyclin- dependent kinase inhibitor p27KIP, thus promoting malignant transformation by increased transition to S phase. In another example, one of the well-defined p50- mediated tumor- suppressive mechanisms was described in human glioblastoma and breast cancer cells and xenografts, and in tumors derived from patients: it was shown that excessive generation of p50 by the ubiquitin ligase KPC1 triggers up-regulation of transcripts of numerous tumor suppressors, suggesting that p50p50 homodimers control their transcription (rather than the “canonical” tumorigenic p50p65). NF-xB-dependent modulation of the tumor microenvironment is another essential mechanism that regulates cancer dynamics-progression or suppression. It can combine two layers of activity: 1) it can affect immune cells in the adjacent tumor surroundings, ensuring their reactivity; and/or 2) it affects tumor cells by stimulating secretion of chemokines or generation of membrane-bound immune checkpoint molecules. An example of a tumor suppression mechanism based on modulation of the tumor microenvironment is the induction of mitochondrial outer-membrane permeabilization that causes activation of NF-κB in tumor cells with subsequent anticancer immune response, including macrophage activation and T cell infiltration. In a mouse lung cancer model, activation of NF-κB up-regulates the T cell-recruiting chemokines CCL2 and CCL5, resulting in tumor rejection. In lung cancer patient specimen, NF- κB activity was linked to high infiltration of T cells to the tumor. Chemokines play an important role in cancer progression not only through the direct autocrine effect on the tumor cells, but also through recruitment of specific immune cells. Genomic expression signature of twelve chemokines (CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11, and CXCL13) was identified in genomic arrays of colorectal carcinoma and of aboutl5,000 distinct solid tumors. It was correlated with the presence of tertiary lymph node-like structures and was associated with better survival of a subset of melanoma patients. An additional significant feature of immune cells invasion of different cancers is the surface expression of inhibitory ligands, programmed cell death-ligand 1 (PD-L1), for example. It is highly accepted that the PD-1/PD-L1 axis is one of the main mechanisms that inhibits the anticancer activity of T cells and macrophages. Recently, it was shown that PD-1 and PD-L1 blockade elicited a strong natural killer (NK) cell antitumoral activity. NF-κB can be involved in the regulation of immune checkpoints on the surface of tumor cells. For instance, p65- containing NF-κB dimers were shown to up-regulate CSN5-a deubiquitinating enzyme-which resulted in stabilization of PDL1, thus bypassing immune suppression of the cancer cells [3]. The present inventors recently showed that the ubiquitin ligase KPC1 acts as a tumor suppressor via its excessive activity on the p105 NF-κB precursor, generating excess of the p50 subunit. Recent initial results of the present inventors [3] demonstrated that p50 stimulates transcription of a broad array of tumor suppressors. The inventors showed that excess of p50 down-regulates the surface expression of PD-L1. In addition, it also up- regulates expression of the proinflammatory cytokines CCL3, CCL4, and CCL5, that recruit NK cells and macrophages to the tumor. These findings of p50 reveal multilayered mechanisms that are mostly driven by a change in the cellular equilibrium between the different NF-κB subunits which determine the dual character of this transcription factor moving on the broad range between a tumor promoter and suppressor. Thus, it appears that KPC1 and its downstream product p50 act on several layers to suppress tumor growth. They 1) activate transcription of tumor suppressors; 2) suppress expression of PD-L1; and 3) recruit immune cells via stimulation of secretion of an array of cytokines. In some embodiments, generation the NF-κB p50, leads to various effects in the treated subject. In some embodiments, the generation of p50 by the methods of the invention may be involved in modulation of NF-κB canonical pathway in a treated subject. In some embodiments, such modulation may involve inhibition of the NF-κB canonical pathway in the treated subject. Still further, in some embodiments, the generation of p50 by the methods of the invention may lead to activation of transcription of at least one tumor suppressor in the treated subject. In yet some further embodiments, the generation of p50 by the methods of the invention may lead to suppression of programmed cell death-ligand l(PD-Ll) expression in the treated subject. In yet some further embodiments, the generation of p50 by the methods of the invention may result in increased secretion of at least one proinflammatory cytokine in the treated subject. In some further embodiments, the generation of p50 by the methods of the invention may result in recruitment of immune cells to a diseased tissue, specifically, tumor tissue or organ in the treated subject. In more specific embodiments, the methods disclosed herein, may be applicable for any pathologic disorder, for example, a proliferative disorder. In yet some further embodiments, such proliferative disorder or cancer may be at least one solid and non-solid tumor and any related conditions. Thus, in some embodiments, the pathologic disorder or condition treated by the methods of the present disclosure is cancer. It should be understood that the present invention is further apphcable to any metastatic tissue, organ or cavity of any proliferative disorder/s. As used herein to describe the present invention, “cancer”, “proliferative disorder”, “tumor” and “malignancy” all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non-solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. In general, the methods, compositions and bifunctional hybrid-molecule, conjugate or complex of the present invention may be applicable for a patient suffering from any one of non-solid and solid tumors. Malignancy, as contemplated in the present invention may be any one of carcinomas, melanomas, lymphomas, leukemia, myeloma and sarcomas. Therefore, in some embodiments any of the methods of the invention, bifunctional hybrid-molecule, conjugate or complex, and compositions disclosed herein, may be applicable for any of the malignancies disclosed by the present disclosure. More specifically, carcinoma as used herein, refers to an invasive malignant tumor consisting of transformed epithelial cells. Alternatively, it refers to a malignant tumor composed of transformed cells of unknown histogenesis, but which possess specific molecular or histological characteristics that are associated with epithelial cells, such as the production of cytokeratins or intercellular bridges. Melanoma as used herein, is a malignant tumor of melanocytes. Melanocytes are cells that produce the dark pigment, melanin, which is responsible for the color of skin. They predominantly occur in skin but are also found in other parts of the body, including the bowel and the eye. Melanoma can occur in any part of the body that contains melanocytes. Leukemia refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood-leukemic or aleukemic (subleukemic). Sarcoma is a cancer that arises from transformed connective tissue cells. These cells originate from embryonic mesoderm, or middle layer, which forms the bone, cartilage, and fat tissues. This is in contrast to carcinomas, which originate in the epithelium. The epithelium lines the surface of structures throughout the body, and is the origin of cancers in the breast, colon, and pancreas. Myeloma as mentioned herein is a cancer of plasma cells, a type of white blood cell normally responsible for the production of antibodies. Collections of abnormal cells accumulate in bones, where they cause bone lesions, and in the bone marrow where they interfere with the production of normal blood cells. Most cases of myeloma also feature the production of a paraprotein, an abnormal antibody that can cause kidney problems and interferes with the production of normal antibodies leading to immunodeficiency. Hypercalcemia (high calcium levels) is often encountered. Lymphoma is a cancer in the lymphatic cells of the immune system. Typically, lymphomas present as a solid tumor of lymphoid cells. These malignant cells often originate in lymph nodes, presenting as an enlargement of the node (a tumor). It can also affect other organs in which case it is referred to as extranodal lymphoma. Non limiting examples for lymphoma include Hodgkin's disease, non-Hodgkin's lymphomas and Burkitt's lymphoma. In some embodiments, the methods of the present disclosure may be applicable for any solid tumor. In more specific embodiments, the methods disclosed herein may be applicable for any malignancy that may affect any organ or tissue in any body cavity, for example, the peritoneal cavity (e.g., liposarcoma), the pleural cavity (e.g., mesothelioma, invading lung), any tumor in distinct organs, for example, the urinary bladder, ovary carcinomas, and tumors of the brain meninges. Particular and non-limiting embodiments of tumors applicable in the methods, compositions and bifunctional hybrid-molecule, conjugate or complex of the present disclosure may include but are not limited to at least one of glioblastoma, breast cancer, ovarian cancer, liver carcinoma, colorectal carcinoma, pancreatic cancer, brain tumors and any related conditions, as well as any metastatic condition, tissue or organ thereof. In some embodiments, the methods, the bifunctional hybrid-molecule, conjugate or complex and compositions of the present disclosure may be applicable for treating breast cancer. In other embodiments, the methods, the bifunctional hybrid-molecule, conjugate or complex and compositions of the present disclosure may be applicable for treating glioblastoma. Glioblastoma (GBM), also referred to as a grade IV astrocytoma, is a fast-growing and aggressive brain tumor. It invades the nearby brain tissue, but generally does not spread to distant organs. GBMs can arise in the brain de novo or evolve from lower-grade astrocytoma. In adults, GBM occurs most often in the cerebral hemispheres, especially in the frontal and temporal lobes of the brain. It should be understood that the disclosed methods are applicable for any type or grade of GBM In some other embodiments, the methods, compositions and bifunctional hybrid-molecule, conjugate or complex of the invention are relevant to colorectal carcinoma, or any malignancy that may affect all organs in the peritoneal cavity, such as liposarcoma for example. In some further embodiments, the method of the invention may be relevant to tumors present in the pleural cavity (mesothelioma, invading lung) the urinary bladder, and tumors of the brain meninges. In some particular embodiments, the methods, compositions and bifunctional hybrid-molecule, conjugate or complex of the invention may be applicable for ovarian cancer. It should be further understood that the invention further encompasses any tissue, organ or cavity barring ovarian metastasis, as well as any cancerous condition involving metastasis in ovarian tissue. In yet some other embodiments, the methods, the bifunctional hybrid-molecule, conjugate or complex and compositions of the present disclosure may be suitable for liver cancer. It should be further understood that the invention further encompasses any tissue, organ or cavity barring liver originated metastasis, as well as any cancerous condition having metastasis of any origin in liver tissue. Liver cancer, also known as hepatic cancer and primary hepatic cancer, is cancer that starts in the liver. Cancer which has spread from elsewhere to the liver, known as liver metastasis, is more common than that which starts in the liver. Symptoms of liver cancer may include a lump or pain in the right side below the rib cage, swelling of the abdomen, yellowish skin, easy bruising, weight loss and weakness. The most common types are hepatocellular carcinoma (HCC), which makes up 80% of cases, and cholangiocarcinoma. Less common types include mucinous cystic neoplasm and intraductal papillary biliary neoplasm. In other embodiments, the methods, the bifunctional hybrid-molecule, conjugate or complex and compositions of the present disclosure may be applicable for pancreatic cancer. It should be further understood that the invention further encompasses any tissue, organ or cavity barring pancreatic metastasis, as well as any cancerous condition having metastasis of any origin in the pancreas. Pancreatic cancer arises when cells in the pancreas, a glandular organ behind the stomach, begin to multiply out of control and form a mass. It should be understood that the methods, compositions and the bifunctional hybrid-molecule, conjugate or complex of the present disclosure are applicable for any type and/or stage and/or grade of any of the malignant disorders discussed herein or any metastasis thereof. Still further, it must be appreciated that the methods, compositions and bifunctional hybrid-molecule, conjugate or complex of the invention may be applicable for invasive as well as non-invasive cancers. When referring to "non-invasive" cancer it should be noted as a cancer that do not grow into or invade normal tissues within or beyond the primary location. When referring to "invasive cancers” it should be noted as cancer that invades and grows in normal, healthy adjacent tissues. Still further, in some embodiments, the methods, compositions and bifunctional hybrid-molecule, conjugate or complex of the present disclosure are applicable for any type and/or stage and/or grade of any metastasis, metastatic cancer or status of any of the cancerous conditions disclosed herein. As used herein the term "metastatic cancer" or "metastatic status" refers to a cancer that has spread from the place where it first started (primary cancer) to another place in the body. A tumor formed by metastatic cancer cells originated from primary tumors or other metastatic tumors, that spread using the blood and/or lymph systems, is referred to herein as a metastatic tumor or a metastasis. Further malignancies that may find utility in the present invention can comprise but are not limited to hematological malignancies (including lymphoma, leukemia, myeloproliferative disorders, Acute lymphoblastic leukemia; Acute myeloid leukemia), hypoplastic and aplastic anemia (both virally induced and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune mediated and idiopathic) and solid tumors (including GI tract, colon, lung, liver, breast, prostate, pancreas and Kaposi's sarcoma. The invention may be applicable as well for the treatment or inhibition of solid tumors such as tumors in lip and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbladder, extraliepatic bile ducts, ampulla of vater, exocrine pancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma, carcinoma and malignant melanoma of the skin, breast, vulva, vagina, cervix uteri, corpus uteri, ovary, fallopian tube, gestational trophoblastic tumors, penis, prostate, testis, kidney, renal pelvis, ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma of the conjunctiva, malignant melanoma of the conjunctiva, malignant melanoma of the uvea, retinoblastoma, carcinoma of the lacrimal gland, sarcoma of the orbit, brain, spinal cord, vascular system, hemangiosarcoma, Adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; Anal cancer; Appendix cancer; Astrocytoma, childhood cerebellar or cerebral; Basal cell carcinoma; Bile duct cancer, extrahepatic; Bladder cancer; Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma; Brainstem glioma; Brain tumor; Brain tumor, cerebellar astrocytoma; Brain tumor, cerebral astrocytoma/malignant glioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Brain tumor, supratentorial primitive neuroectodermal tumors; Brain tumor, visual pathway and hypothalamic glioma; Breast cancer; Bronchial adenomas/carcinoids; Burkitt lymphoma; Carcinoid tumor, childhood; Carcinoid tumor, gastrointestinal; Carcinoma of unknown primary; Central nervous system lymphoma, primary; Cerebellar astrocytoma, childhood; Cerebral astrocytoma/Malignant glioma, childhood; Cervical cancer; Childhood cancers; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic myeloproliferative disorders; Colon Cancer; Cutaneous T-cell lymphoma; Desmoplastic small round cell tumor; Endometrial cancer; Ependymoma; Esophageal cancer; Ewing's sarcoma in the Ewing family of tumors; Extracranial germ cell tumor, Childhood; Extragonadal Germ cell tumor; Extrahepatic bile duct cancer; Eye Cancer, Intraocular melanoma; Eye Cancer, Retinoblastoma; Gallbladder cancer; Gastric (Stomach) cancer; Gastrointestinal Carcinoid Tumor; Gastrointestinal stromal tumor (GIST); Germ cell tumor: extracranial, extragonadal, or ovarian; Gestational trophoblastic tumor; Glioma of the brain stem; Glioma, Childhood Cerebral Astrocytoma; Glioma, Glioblastoma, Childhood Visual Pathway and Hypothalamic; Gastric carcinoid; Hairy cell leukemia; Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin lymphoma; Hypopharyngeal cancer; Hypothalamic and visual pathway glioma, childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi sarcoma; Kidney cancer (renal cell cancer); Laryngeal Cancer; Leukemias; Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia); Leukemia, acute myeloid (also called acute myelogenous leukemia); Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia); Leukemia, chronic myelogenous (also called chronic myeloid leukemia); Leukemia, hairy cell; Lip and Oral Cavity Cancer; Liver Cancer (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphomas; Lymphoma, AIDS-related; Lymphoma, Burkitt; Lymphoma, cutaneous T-Cell; Lymphoma, Hodgkin; Lymphomas, Non- Hodgkin (an old classification of all lymphomas except Hodgkin's); Lymphoma, Primary Central Nervous System; Marcus Whittle, Deadly Disease; Macroglobulinemia, Waldenstrom; Malignant Librous Histiocytoma of Bone/Osteosarcoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular (Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant; Mesothelioma, Childhood; Metastatic Squamous Neck Cancer with Occult Primary; Mouth Cancer; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Lungoides; Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Diseases; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple (Cancer of the Bone-Marrow); Myeloproliferative Disorders, Chronic; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral Cancer; Oropharyngeal cancer; Osteosarcoma/malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer (Surface epithelial- stromal tumor); Ovarian germ cell tumor; Ovarian low malignant potential tumor; Pancreatic cancer; Pancreatic cancer, islet cell; Paranasal sinus and nasal cavity cancer; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal astrocytoma; Pineal germinoma; Pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood; Pituitary adenoma; Plasma cell neoplasia/Multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell cancer; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary gland cancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sezary syndrome; Skin cancer (nonmelanoma); Skin cancer (melanoma); Skin carcinoma, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma - see Skin cancer (nonmelanoma); Squamous neck cancer with occult primary, metastatic; Stomach cancer; Supratentorial primitive neuroectodermal tumor, childhood; T-Cell lymphoma, cutaneous (Mycosis Lungoides and Sezary syndrome); Testicular cancer; Throat cancer; Thymoma, childhood; Thymoma and Thymic carcinoma; Thyroid cancer; Thyroid cancer, childhood; Transitional cell cancer of the renal pelvis and ureter; Trophoblastic tumor, gestational; Unknown primary site, carcinoma of, adult; Unknown primary site, cancer of, childhood; Ureter and renal pelvis, transitional cell cancer; Urethral cancer; Uterine cancer, endometrial; Uterine sarcoma; Vaginal cancer; Visual pathway and hypothalamic glioma, childhood; Vulvar cancer; Waldenstrom macroglobulinemia and Wilms tumor (kidney cancer).
The methods disclosed herein involve in some embodiments thereof the administration of an effective amount of the bifunctional hybrid-molecule/s, conjugate/s or complex/es or compositions of the invention. An “effective amount" of the bifunctional hybrid-molecule, conjugate or complex/s of the invention, or any compositions thereof can be administered in one administration, or through multiple administrations of an amount that total an effective amount, preferably within a 24-hour period, to achieve the therapeutic effect as discussed herein, specifically, to induce in the treated subject the degradation of the NF-κB1 p105, to generate the NF-κB1 p50 product that exhibits a tumor suppressive effect. In yet some further embodiments, the "effective amount" as discussed herein is the amount sufficient for a therapeutic effect in the treated subject. It can be determined using standard clinical procedures for determining appropriate amounts and timing of administration. It is understood that the "effective amount" can be the result of empirical and/or individualized (case-by-case) determination on the part of the treating health care professional and/or individual. Still further, local administration to the area in need of treatment may be achieved by, for example, by local infusion during surgery, or using any permanent or temporary infusion device, topical application, direct injection into the specific organ, etc. More specifically, the bifunctional hybrid-molecule, conjugate or complex/s and compositions disclosed herein, that are also used in any of the methods of the invention, described in connection with other aspects of the present disclosure, may be adapted for administration by parenteral, intraperitoneal, transdermal, oral (including buccal or sublingual), rectal, topical (including buccal or sublingual), vaginal, intranasal and any other appropriate routes. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). In yet some further embodiments, the composition of the invention may optionally further comprise at least one of pharmaceutically acceptable carrier/s, excipient/s, additive/s diluent/s and adjuvant/s.
It is to be understood that the terms "treat”, “treating”, “treatment" or forms thereof, as used herein, mean preventing, ameliorating or delaying the onset of one or more clinical indications of disease activity in a subject having a pathologic disorder. Treatment refers to therapeutic treatment. Those in need of treatment are subjects suffering from a pathologic disorder. Specifically, providing a "preventive treatment" (to prevent) or a "prophylactic treatment" is acting in a protective manner, to defend against or prevent something, especially a condition or disease. The term “treatment or prevention” as used herein, refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, pathologic disorder involved with at least one short term cellular stress condition/process and any associated condition, illness, symptoms, undesired side effects or related disorders. More specifically, treatment or prevention of relapse or recurrence of the disease, includes the prevention or postponement of development of the disease, prevention or postponement of development of symptoms and/or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing- additional symptoms and ameliorating or preventing the underlying metabolic causes of symptoms. It should be appreciated that the terms "inhibition", "moderation", “reduction”, "decrease" or "attenuation" as referred to herein, relate to the retardation, restraining or reduction of a process by any one of about 1 % to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more. With regards to the above, it is to be understood that, where provided, percentage values such as, for example, 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively. The term "amelioration" as referred to herein, relates to a decrease in the symptoms, and improvement in a subject's condition brought about by the compositions and methods according to the invention, wherein said improvement may be manifested in the forms of inhibition of pathologic processes associated with the disorders described herein, a significant reduction in their magnitude, or an improvement in a diseased subject physiological state. The term "inhibit" and all variations of this term is intended to encompass the restriction or prohibition of the progress and exacerbation of pathologic symptoms or a pathologic process progress, said pathologic process symptoms or process are associated with. The term "eliminate" relates to the substantial eradication or removal of the pathologic symptoms and possibly pathologic etiology, optionally, according to the methods of the invention described herein. The terms "delay", "delaying the onset", "retard" and all variations thereof are intended to encompass the slowing of the progress and/or exacerbation of a disorder associated with the at least one short term cellular stress condition/process and their symptoms, slowing their progress, further exacerbation or development, so as to appear later than in the absence of the treatment according to the invention. As indicated above, the methods and compositions provided by the present invention may be used for the treatment of a “pathological disorder”, i.e., pathologic disorder or condition involved with at least one short term cellular stress condition/process, which refers to a condition, in which there is a disturbance of normal functioning, any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with that person. It should be noted that the terms "disease", "disorder", "condition" and "illness", are equally used herein. It should be appreciated that any of the methods, bifunctional hybrid-molecule, conjugate or complexes, and compositions described by the invention may be applicable for treating and/or ameliorating any of the disorders disclosed herein or any condition associated therewith. It is understood that the interchangeably used terms "associated", “linked” and "related", when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder condition or pathology causes the second disease, disorder, condition or pathology. More specifically, as used herein, “disease”, “disorder”, “condition”, “pathology” and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms. It should be appreciated that the methods, bifunctional hybrid-molecule, conjugate or complexes and compositions of the present disclosure may be suitable for any subject that may be any multicellular organism, specifically, any vertebrate subject, and more specifically, a mammalian subject, avian subject, fish or insect. In some specific embodiments, the therapeutic and non- therapeutic methods presented by the enclosed disclosure may be applicable to mammalian subjects, specifically, human subjects. By “patient” or “subject” it is meant any mammal that may be affected by the above-mentioned conditions, and to whom the treatment methods herein described is desired, including human, bovine, equine, canine, murine and feline subjects. Specifically, the subject is a human. Still further, the present disclosure provides in an additional aspect thereof a therapeutically effective amount of at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the same, or any composition thereof for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder or condition in a subject in need thereof. More specifically, the bifunctional hybrid-molecule, conjugate or complex used as described herein may comprise: First (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1. It should be noted that Zaa is any aromatic amino acid residue. It should be understood that "Zaa" as used herein is also denoted in the sequence listing with respect to SEQ ID NO: 1, as residue "Xaa2 ” and as residue "Xaa4-. both refer to any aromatic am aicniod residue as disclosed herein. Thus, in some embodiments the sequence Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n) is also equivalent to Xaa(n)-Xaa2-Xaa(m)- Xaa4-Xaa(n), as also denoted by SEQ ID NO: 1. Still further, in some embodiments, Xaa is any amino acid residue. In some specific embodiments, n is zero or an integer between 1 to 7, and m is an integer between 1 to 7, as discussed above in connection with the previous aspect of the present disclosure. The bifunctional hybrid-molecule, conjugate or complex of the compositions disclosed herein further comprises a second component (b), at least one proteasome degradation mediating moiety. In some embodiments, the indicated use may be applicable for treating cancer. Specifically, an of the cancers disclosed by the present invention.
In yet a further aspect, the present disclosure provides an effective amount of at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro- particle comprising the same, or any composition thereof for use in a method for inducing ubiquitination and proteasomal processing of NF-κB1 p105, thereby generating the NF-κB p50 in a cell or in a cell-free system comprising said NF-κB 1 p105. More specifically, the bifunctional hybrid-molecule, conjugate or complex used as described herein may comprise: First (a), at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa- Xaa(n), as denoted by SEQ ID NO: 1. It should be noted that Zaa is any aromatic amino acid residue. It should be understood that "Zaa" as used herein is also denoted in the sequence listing with respect to SEQ ID NO: 1, as residue "Xaa2 ” and as residue "Xaa4 ”, both refer to any aromatic amino acid residue as disclosed herein. Thus, in some embodiments the sequence Xaa(n)-Zaa-Xaa(m)-Zaa- Xaa(n)is also equivalent toXaa(n)-Xaa2-Xaa(m)-Xaa4-Xaa(n), as also denoted by SEQ ID NO: 1. Still further, in some embodiments, Xaa is any amino acid residue. In some specific embodiments, n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7, as discussed above in connection with the previous aspect of the present disclosure. The bifunctional hybrid-molecule, conjugate or complex of the compositions disclosed herein further comprises a second component (b), at least one proteasome degradation mediating moiety. It should be appreciated that the bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro- particle comprising the same, or any composition thereof used herein in the disclosed method are any of the bifunctional hybrid-molecule, conjugate or complex, disclosed by the resent invention in connection with other aspects.
A further aspect of the present disclosure elates to a peptide comprising the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1, wherein Zaa is any aromatic amino acid residue, wherein Xaa is any amino acid residue. It should be noted that n is zero or an integer between 1 to 7, and m is an integer between 1 to 7, with the proviso that said peptide is not the peptide of SEQ ID NO: 4. In some embodiments, at least one of the interspacing amino acid residue/s Xaa(m) located between the two aromatic amino acid residues Zaa, may be one or more amino acid residue/s having a non-polar side chain, also referred to herein as a non-polar amino acid. Such residues may be any one of Valine (V, Val), Glycine (G, Gly), Leucine (L, Leu), Isoleucine (I, IIe), Methionine (M, Met), Phenylalanine (F, Phe), Tryptophane (W, Trp) and/or Proline (P, Pro). In et some further embodiments, the interspacing amino acid residue may be any hydrophobic amino acid residue, for example, Valine (V, Val), Leucine (L, Leu) and/or Isoleucine (I, IIe). In some specific embodiments, the peptide of the present disclosure may comprise at least two aromatic amino acid residues that are interspaced by at least one of Valine (V), Glycine (G) and/or Alanine (A). In some particular and non-limiting embodiments, the at least two aromatic amino acid residues of the peptide disclosed herein, are interspaced by at least one Valine residue. Thus, in some embodiments, the peptide in accordance with the invention may comprise the amino acid sequence of Xaa(n)-Trp (W)-Xaa(n)-Val (V)-Xaa(n)-Trp (W)-Xaa(n), as denoted by SEQ ID NO: 7, or any mimetics thereof. It should be noted that Xaa is any amino acid residue, and n is zero or an integer between 1 to 7. These amino acid residues may be any identical or different amino acid residues. In some embodiments, the at least one peptide of the present disclosure, may comprise three to ten amino acid residues, specifically, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. In yet some further embodiments, the peptide of the present disclosure may comprise any amino acid residue, Xaa that is any one of Leu (L), Arg (R) and IIe (I) amino acid residues. In some particular embodiments, the peptide disclosed herein the amino acid sequence RIWVWLL, as dented by SEQ ID NO: 2, any mimetics thereof, any variants and derivatives thereof, or any conjugate, complex, chimera and composition thereof. In some embodiments, a variant of the heptapeptide of SEQ ID NO: 2, may be a peptide comprising the amino acid sequence IWVWLL, as dented by SEQ ID NO: 3, or any variants and derivatives thereof, or any conjugate, complex, chimera and composition thereof. In some particular embodiments, the peptide disclosed herein comprises the amino acid sequence RIFVFLL, as dented by SEQ ID NO: 9. Still further, in some embodiments, the peptide disclosed herein comprises the amino acid sequence RIYVFLL, as dented by SEQ ID NO: 10. In certain embodiments, the peptide disclosed herein comprises the amino acid sequence Citrulline-IWVWLL, as dented by SEQ ID NO: 15. In yet some further embodiments, the peptide disclosed herein comprises the amino acid sequence GRIWVWLL, as dented by SEQ ID NO: 16. In some embodiments, the peptide disclosed herein comprises the amino acid sequence RRRIWVWLL, as dented by SEQ ID NO: 27. Still further, in some embodiments, the peptide disclosed herein comprises the amino acid sequence WVW, or any variants and derivatives thereof. In yet some further embodiments, the bifunctional hybrid-molecule, conjugate or complex of the invention may comprise any of the peptides disclosed herein, for example, any of the peptides of SEQ ID NOs: 8, 11 to 28, and 52 to 59. It should be understood that all definitions that relate to polypeptides and derivatives disclosed in connection with other aspects of the invention are also applicable for the present aspect as well.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. In some embodiments, the term "about" refers to ± 10 %. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of’ “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. Throughout this specification and the Examples and claims which follow, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Specifically, it should be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures. More specifically, the terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to". The term “consisting of means “including and limited to”. The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure. It should be noted that various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. Various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below find experimental support in the following examples. Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof. The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.
EXAMPLES Experimental procedures Materials
Materials for SDS-PAGE, Bradford, and enhanced chemiluminescence (ECL), were from Bio- Rad. Molecular weight markers and L-[35S] methionine were from GE Healthcare. Tissue culture media, sera, and supplements for growing HEK293 (ATCC®CRL1573™), and U87- MG (ATCC® HTB14™) cells, were from Biological Industries, Bet HaEmek, Israel. Formalin solution for histological tissue fixation was from Sigma. Immobilized mouse anti-FLAG (M2), mouse anti-FLAG (M2), and rabbit anti-NF-kB1, were from Sigma. Rabbit anti-Von Hippel- Lindau/VHL antibody (ab83307) and rabbit anti-CD45 (at>10558), were from Abeam. Mouse anti- actin was from Millipore. Peroxidase-conjugated secondary antibody was from Jackson ImmunoResearch Laboratories. Immunohistochemistry staining was carried out using the iVIEW DAB Detection Kit from Ventana Medical Systems. jetOPTIMUS® DNA Transfection Reagent was from Polyplus-transfection S.A. HEPES and Tris buffers, Adenosine 5 '-[γ-thio] triphosphate dithiothreitol (DTT), Ub-activating enzyme (El), and Ub, were from Sigma. Protease
Figure imgf000085_0001
inhibitors cocktail was from Roche. Ub aldehyde (UbAl) was from BIOMOL. TNT® T7 Quick Coupled Transcription/Translation System was from Promega. Synthesis of KPC1 -derived peptides was carried out by Bio Basic Inc. Recombinant Human VHL/ELOB/ELOC/CUL2/RBX1 complex was from R&D Systems. Restriction and modifying enzymes were from New England Biolabs. Oligonucleotides were from Syntezza Bioscience. C.B-17/IcrHsd-Prkdcscid mice were from Envigo. Analytical grade N,N-dimethyIformamide (DMF) was from Biotech. Resins were from Creosalus. The activating reagents, 0-(1H-6-Chlorobenzotriazole-1-yl)-1, 1,3,3- tetramethyluronium hexafluorophosphate (HCTU) and 1-[Bis(dimethylamino)methylene]-1H- 1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate] (HATU) were from Luxembourg Bio Technologies. The protected amino acids Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OF[, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Boc-Trp(Boc)-OH, Boc-Ile-OH, Boc-Arg(Pbf)-OF[ for synthesis of the peptide-based PROTACs were from GL Biochem. Teflon filter fitted syringes were from Torviq. N.N-Diisopiopylethylamine (DIPEA) and Triisopropylsilane (TIPS) were from Aldrich. All other high-grade chemicals were from Aldrich, Strem Chemicals, and Alfa Aesar.
Plasmids cDNA coding for FLAG-p105 and 6xHIS-Ubc5c [9], and for Myc-Ub [Belogurov, et al, J. Biol. Chem. 289, 17758-17766 (2014)], were described in the indicated references. For transient transfection in cells, the following cDNAs coding for truncated species of KPC1-FLAG, were cloned into the pCAGGS 3.2 FLAG expression vector: KPC1Δ1041-1061 and KPC1A968-974 (amplified using primers containing the restriction sites EcoRI and Notl); KPC1A969-1314 and KPC1Δ1-973 (amplified using primers containing the restriction sites BamHI and Xhol); KPC1Δ1-967 and WILVRLW-KPC1Δ1-1039 (amplified using primers containing the restriction sites Ndel and Xhol or Bam HI and Notl, respectively); KPC1; KPC1Δ1-389;A696-1314; WILVRLW-KPC1Δ1-1253; ILVRLW-KPC1Δ1-1253; LVRLW-KPC1Δ1-1253; VRLW- KPC1Δ1-1253; RLW-KPC1Δ1-1253; KPC1Δ1-1253; WILVRL-KPC1Δ1-1253; WILVR- KPC1Δ1-1253; WILV-KPC1Δ1-1253; and WIL-KPC1Δ1-1253 (amplified using primers containing the restriction sites EcoRI and Xhol). cDNA coding for p105-HA was cloned into the CMV-5-B expression vector (amplified using primers containing the restriction sites Notl and Kpnl). For protein expression in bacteria, the following cDNAs, coding for truncated species of KPC1 were cloned into the pT7b-6xHIS expression vector: KPC1Δ1-967; KPC1Δ1-974; WILVRLW-KPC1Δ1-1039; and WILVRLW-KPC1Δ1-1061 (were all amplified using primers containing the restriction sites Ndel and HindIII).For stable expression in cells, the following cDNAs were cloned into the NSPI-CMV MCS lentiviral expression vector: WILVRLW-KPC1Δ1- 1039; KPC1Δ1-1039; and RIWVWLL-KPC1Δ1-1039 (were amplified using primers containing the restriction site Xhol). KPC1-FLAG; and pVHL-FLAG was amplified using primers containing the restriction sites Bam HI and Xhol.
Cell culture
U87-MG and HEK293 were grown at 37°C and 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with penicillin-streptomycin and 10% fetal calf serum. Stable expression
Stable expression of cDNAs coding for KPC1, WILVRLW-KPC1Δ1 - 1039, KPC1Δ1-1039, RIWVWLL-KPC1Δ1-1039, and pVHL-FLAG in U87-MG and HEK293 cell lines was carried out as described previously [9]. Cells stably expressing the above proteins were selected by Puromycin (U87-MG, 5 μg/ml and HEK293, 2 μg/ml).
Interaction between p105 and KPC1 species
For analysis of the interaction between p105 and KPC1 species, HEK293 cells were transfected with various cDNAs coding for WT and the truncated species of KPC 1 -FLAG, or with an empty vector along with p105-HA. The transfection was carried out using the jetOPTIMUS transfection reagent. After 48 h, cells were lysed with RIPA buffer [150 mM NaCl, 50 mM Tris-FiCl (pFi 8.0), 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, and freshly added protease inhibitors cocktail]. Lysates were incubated with immobilized anti-FLAG at 4°C for 2 h. At the end of the incubation, the beads were washed five times with RIPA buffer. Proteins were resolved by SDS-PAGE and visualization of the co-immunoprecipitated p105-HA was carried out using anti-NF-kB1 antibody. Expression of proteins in bacteria cDNAs coding for KPC1Δ1-967-HISx6, KPC1Δ1-974-HISx6, WILVRLW-KPC1Δ1- 1039- HISx6, and WILVRLW-KPC1Δ1-1061-HISx6 (in pT7b-6xHIS) were transfected to pLysS E. coli competent cells [Rosetta™(DE3)pLysS - Novagen]. The pLysS bacteria were grown at 37°C and brought to an O.D. of 0.7. Protein expression was induced by IPTG (500 mM). 4 h after induction, cells were lysed by sonication in a buffer containing 0.1 M NaCl, 20 mM Tris-HCl (pH 7.6), 10 mM β-mercaptoethanol, and EDTA-free protease inhibitor cocktail.
In vitro translation
In vitro translation of L-[35S]methionine-IabeIed p105 was carried out using the TnT® Quick Coupled Transcription/Translation System according to the manufacturer’s protocols.
Conjugation of p105 in cell free system
In vitro translated and 35S-labeIed p105 was ubiquitinated in a reaction mixture that contained in a volume of 12.5mI: Ub (0.5μg), El (0.25mg), bacterially expressed 6xHis-tagged UbcH5c (0.75μg), and KPC1Δ1-967-HISx6 (0.5μg) or VHL complex, ATPγS (2 mM), and UbAI (0.5μg). The reaction was carried out in 37°C for 30 min in the presence or absence of the KPCl-derived peptides (ILVRLW, WILVRLW, or RIWVWLL, also denoted by SEQ ID NOs. 5, 4, 2, respectively) or PROTACs, at the indicated concentrations. Reaction was terminated by the addition of 4-fold concentrated sample buffer. Proteins were resolved by SDS-PAGE (7.5%) and visualized via Phosphorimaging. Processing of p105 to p50 in cell free system
Processing of p105 to p50 in cell free system was generated in an assay similar to that described for ubiquitination with following modifications: 1) ATP (0.5 mM) and an ATP-regenerating system (phosphocreatine [10 mM] and creatine phosphokinase 10.5 μg ] ) were added instead of ATPγS, 2) reaction was carried out without addition of UbAl, 3) reaction was carried out in the presence of Fraction II (Fr2, 60 pig).
Tumorigenicity
Exponentially growing U87-MG cells that stably express V0, KPC1-FLAG, WILVRLW- KPC 1 D 1 - 1039-FLAG, KPC1Δ1- 1039-FLAG, or RIWVWLL-KPC1Δ1 - 1039-FLAG were dissociated with trypsin and diluted to a concentration of 60x106 cells/ml in PBS. 6-10 weeks old C.B-17/IcrHsd-Prkdcscid, or NOD.Cg-Prkdcsc id Il2rgtmlWjll SzJ (NSG) mice were injected subcutaneously with 6x106 cells /0.1 ml at both flanks. The size of xenografts was monitored by external measuring of two parameters of the tumor (length and width) using caliper. Tumor volume was calculated by the equation: volume = length x width2 x 0.5. At the end of the experiment mice were sacrificed, and tumors were dissected, weighed, and fixed using formalin solution. Animal experiments were approved by the Technion ethics committee for animal experiments (Haifa, Israel).
Immunohistochemistry
Formalin-fixed xenografts tumors were embedded in paraffin. 5 pm thick sections were stained by rabbit anti-CD45 (1 :3,000) using the Ventana BenchMark ULTRA IHC/ISH system. Visualization was performed by the iVIEW DAB detection kit according to the manufacturer’s protocols.
Co-immunoprecipitation
For analyses of interaction between 105 and VHL in the absence or presence of PROTAC molecules, HEK293 cells that stably express VHL-FLAG, were transiently transfected with cDNA coding for HA-p105 using jetOPTIMUS transfection reagent, according to the manufacturer’s instructions. 24 hr after transfection, the growth medium was replaced with the fresh one, supplemented with the PROTAC molecules. Cells were incubated in a hypoxic conditions (1.5% O2) for another 16hr, followed by lysis in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.5% sodium deoxycholate, 0.1% SDS, and 1% NP-40, supplemented with freshly added protease inhibitors cocktail). VHL-FLAG was immunoprecipitated with immobilized anti-FLAG at 4°C for 2 hr, followed by extensive washing in RIPA buffer. Proteins were resolved by SDS-PAGE and blotted onto nitrocellulose membrane. The HA-p105 was visualized with anti-NF-kB1, and VHL with anti-VHL antibodies. 10% of total cell lysates (TCL) were analyzed for the expression of p105 and VHL.
Processing of p105 mediated by PROTACs molecules in HEK293 Cells
HEK293 cells that stably express VHL, were transfected with cDNAs coding for HA-p105 and Myc-Ub. 6 hr after transfection, fresh medium that contained PROTACs molecules was added, and cells were moved to the hypoxic conditions (1.5% O2). After 16 hr, cells were lysed with RIPA buffer, proteins were resolved by SDS-PAGE, and blotted onto nitrocellulose membrane. FLAG- p105/FLAG-p50 were visualized using anti-FLAG, and VHL was visualized with anti-VHL antibodies.
Cell counting. 0.5x106 U87-MG cells were seeded on a 60 mm dish and incubated (for 1 to 3 days) with either the RIWVWLLSG-PEG-pVHL-Ligand or the WILVRLWSG-PEG-pVHL- Ligand PROTAC (25 mM). On each day, a triplicate of the PROTACs’-treated dishes and a triplicate of control dishes were removed, and the cells were dispersed by trypsin and counted using the Beckman Coulter Vi-CELL™ XR Cell Viability Analyzer.
PROTAC cell’s intake
HEK293 cells were seeded on a glass-bottomed 35mm dishes and were incubated in the presence of either Fluorescein isothiocyanate (FITC) or FITC-PROTAC for 16 hr. Cells were washed with PBS and stained with 4’-6’-diamidino-2-phenylindole (DAPI) in mounting medium. Imaging of the cells was carried out using the Zeiss LSM-700 confocal microscope.
PROTAC tumor’s intake
U87-MG VHL xenografts were grown in NSG mice for 3 weeks, as described under the "Tumorigenicity", followed by subcutaneous injection of DMSO or RIWVWLL-C(-FITC)-G- PEG-VHL PROTAC into the tumor surroundings. After 20 hr mice were visualized using in vivo imaging device (IVIS; Xenogen Corp., Waltham, MA). Further, mice were sacrificed and tumors were dissected and frozen in iso-pentane. Cryo-sections were stained with DAPI in mounting medium, and imaging was carried out using the Zeiss LSM-700 confocal microscope.
Mass spectrometry analysis of U87-MG derived tumors. After grinding (Omni TH Tissue Homogenizer) and two rounds of sonication, proteins from frozen xenografts were isolated using a solution containing urea (9 M), ammonium bicarbonate (0.4 M) and DTT (10 mM). Aliquots of 20 μg protein each from the different tumors were treated with DTT (3 mM at 60°C for 30 min), and the -SH groups were modified with a solution of iodoacetamide (9 mM) dissolved in ammonium bicarbonate (0.4 M; in the dark at room temperature for 30 min). The proteins were then digested overnight at 37°C by modified trypsin (Promega; dissolved in a solution of 1 M urea and 50 mM ammonium bicarbonate) at a ratio of 1:50 enzyme-to-substrate. A second similar amount of trypsin was added for additional 4 h. Proteomic analysis of the peptides was carried out as described elsewhere [9], [Hakim-Eshed, etal., Proc. Natl. Acad. Sci. U. S. A. 117, 18661-18669 (2020)].
PROTAC synthesis:
General reagents:
Peptides were prepared by SPPS either manually in Teflon filter fitted syringes (purchased from Torviq) or by using an automated peptide synthesizer (CS336X, CSBIO). Analytical grade N,N- dimethylformamide (DMF) was purchased from Biotech. Resins were purchased from Creosalus, protected amino acids were purchased from GL Biochem and activating reagents, O-(1H-6- Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), 1- [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidhexafluorophosphate (HATU)] were purchased from Luxembourg Bio Technologies. Chemicals were purchased from Aldrich, Strem Chemicals and Alfa Aesar.
Protected amino acids used in peptides synthesis:
Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Ser(/Bu)-OH, Fmoc-Try(Boc)-OH, Boc-Try(Boc)-OH, Boc-Ile-OH, Boc-Arg(Pbf)-OH.
HPLC for peptide analysis and purification:
Analytical HPLC was performed on a Thermo instrument (Dionex Ultimate 3000) using analytical column X Select (Waters, CSH C18, 3.5 pm, 4.6 x 150 mm) at a flow rate of 1.2 mL/min and semi-preparative HPLC was performed on a Thermo instrument (Dionex Ultimate 3000) using Phenomenex Jupiter C18 10 pm, 300 A, 250 x 10 mm column, at a flow rate of 4 mL/min. All synthetic peptides were purified by HPLC and characterized by mass spectrometry using LCQ Fleet Ion Trap (Thermo Scientific). All calculated masses have been reported as an average isotope composition. Buffer A: 0.1% TFA in water; buffer B: 0.1% TFA in acetonitrile.
Statistical analysis
Data in graphs represent the mean ± SD. Experiments were statistically analyzed by the two-tailed student’s /-test. A p-value<0.05 was considered statistically significant. Scheme for the Synthesis of VHL ligand, a to f:
Figure imgf000091_0001
The compounds a-f were synthesized as described previously [Galdeano, C. et al., J. Med. Chem. 2014, 57, 8657-8663],
Compound f: 1H NMR (400 MHz, DMSO): d 8.99 (s, 1H), 8.74 (s, 1H), 8.05 (broad, 3H), 7.40 (broad, 4H), 4.54 (t, J = 10.0 Hz, 1H), 4.47-4.40 (m, 2H), 4.26-4.21 (m, 1H), 3.96 (d, J = 4.0 Hz, 1H), 3.71 (d, J = 12.0 Hz, 1H), 3.57-3.54 (m, 1H), 2.44 (s, 3H), 2.14-2.09 (m, 1H), 1.94-1.91 (m, 1H), 1.02 (s, 9H).
ESI-MS: Calculated for C22H30N4O3S: 430.2 Da; found: 430.0 Da.
Spectral data of the compound f (also denoted by Formula I) agreed with the values reported in the literature.
EXAMPLE 1
New Ubiquitin ligase KPC1- derived peptides comprising the W-X-W motif A heptapeptide containing amino acid residues WILVRLW, also denoted by SEQ ID NO:4, derived from KPC1 ubiquitin ligase positions 968-974 (of the Wild type KPC1 amino acid sequence as denoted by SEQ ID NO: 6), was previously identified by the inventors as the binding site of the ligase and its substrate p105. The inventors further demonstrated that truncated species of KPC1 that contain the 7 amino acid sequence WILVRLW-KPC1Δ1-1039, still display a full ligase activity in cell free system and in cells as described in WO 2020/110114 [10].
As previously shown by the inventors, the binding of p105 to KPC1 via the WILVRLW sequence (SEQ ID NO: 4), results in the ubiquitination of p!05. As is clearly shown in Figure 1Ai, KPC1Δ1- 967 ubiquitinates p105 in a dose-dependent manner in a cell-free system. In contrast, the truncated species KPC1Δ1-974 which lacks WILVRLW is inactive. In addition the inventors checked whether the location of the seven amino acids sequence in KPC1 is crucial for the conjugating activity. Towards this end, two truncated species of KPC1, WILVRLW-KPC1Δ1 - 1039 (also denoted by SEQ ID NO: 62, and WILVRLW-KPC1Δ1- 1061 (also denoted by SEQ ID NO: 63, were generated. The two “chimeric” KPCs ubiquitinated p105 in a cell-free system (Figure lAii). To further confirm that 7 amino acids WILVRLW of KPC1 are involved in the interaction with p105, an experiment was designed in whichKPC1Δ1 -967, the truncated but still active species of KPC1 competed on the binding of p105 to the ligase with synthetic peptides derived from KPC1. As shown by Figure IB, the 7 amino acids peptide WILVRLW (SEQ ID NO: 4) inhibited the ubiquitination of p105 by KPC L \ 1 -967 (SEQ ID NO: 68) to a larger extent compared to 6 amino acids ILVRLW (SEQ ID NO: 64) peptide that lacks the first tryptophan (lanes 3-5). Surprisingly, a scrambled peptide that contains the same content of 7 amino acid residues arranged in a completely different sequence, RIWVWLL, also denoted by SEQ ID NO: 2, displayed a dramatically increased inhibition of the ubiquitination of p105, even stronger than the WT peptide (lanes 9-11, as compared to lanes 6-8).
These results further confirm that the 7 amino acid sequence in KPC1 provides the specificity of binding between the ligase and the substrate p105. In addition, these results show that the sequence RIWVWLL (SEQ ID NO: 2) unexpectedly is an even stronger potential binding sequence than the sequence WILVRLW (SEQ ID NO: 4), present in a wild type KPC1. These results therefore establish the requirement of at least two aromatic amino acid residues, for example, as presented by the W-V-W sequence, as the essential motif for recruitment of NL-κB1 p105, and moreover, define a novel and more effective hepta-peptide.
EXAMPLE 2
Overexpression ofKPCl truncated species that contain the amino acid sequence WILVRLW or RIWVWLL attract leukocytes into a xenograft tumor model and inhibit tumor growth To further establish the therapeutic application of this hepta-peptide, a tumor xenograft model was next used. More specifically, using a SCID mice model, various U87 (glioblastoma)-based xenografts were generated. These various U87 -based xenografts overexpressed V0 (control), KPC1 WT (KPC1-LLAG, also denoted by SEQ ID NO: 66, a truncated species of KPC1 that lacks the first 1039 amino acids but contains RIWVWLL in its N-terminus (RIWVWLL-KPC1Δ1 - 1039- FLAG, (also denoted by SEQ ID NO: 65, a truncated species of KPC1 that lacks the first 1039 amino acids but contains WILVRLW in its N-terminus (WILVRLW-KPC1Δ1 - 1039-FLAG, also denoted by SEQ ID NO: 62), or a truncated species of KPC1 that lacks the first 1039 amino acids without WILVRLW sequence (KPC1Δ1 - 1039-FLAG, SEQ ID NO: 40). As can be seen in Figure 2A, the two truncated species had a strong suppressive effect on growth of U87-MG-derived xenografts. A truncated KPC1 species that does not contain any derivative of the seven amino acids p105-binding site, did not display a tumor-suppressive effect (Figure 2A). Of note is that despite the strongest competitive ability of the scrambled peptide on p105 conjugation (Figure IB), its tumor-suppressive effect was weaker than that of the WT sequence-containing truncated KPC1 (Figure 2A; see also the strong effect of a similar peptide-based PROTAC - Figure 4Bii). In correlation with the tumor-suppressive activity of WILVRLW-KPC1Δ1-1039 and RIWVWLL- KPC1Δ1-1039 (SEQ ID NO: 62 and 65, respectively), and in contrast to the lack of effect of KPC1Δ1-1039 (SEQ ID NO: 40), the two species also attracted leukocytes to the tumors (Figure 2B) which explains, at least partially, their suppressive effect.
These results clearly establish the feasibility of using KPC 1 -derived peptides for cancer treatment.
EXAMPLE 3
Proteomic analysis of Glioblastoma xenografts overexpressing either WT or WILVRLW- KPC1A1-1039, revealed alterations in proteins involved in biological pathways that modulate malignancies
In order to examine whether WT KPC1 and its truncated species that lack most of the sequence of the enzyme - but contain either the WT binding domain (WILVRLW, SEQ ID NO: 4) or the scrambled one (RIWVWLL, SEQ ID NO: 2) - elicit similar alterations in the protein profile of the cells in which they are expressed, the proteome of the tumors shown in Figure 2, were analyzed by mass spectrometry. The reason was that the deleted part of the enzyme might affect cellular processes that are not affected by the deleted species. Surprisingly, a strong similarity was found between tumors overexpressing the WT KPC1 or the truncated species WILVRLW-KPC1Δ1- 1039 (SEQ ID NO: 62, Figure 3). Overexpression of the enzyme that contains RIWVWLL (SEQ ID NO: 65) displayed a weaker similarity, possibly explaining the weaker tumor-suppressive activity of this ligase (Figure 2A). Expression of KPC1 species that lacks altogether the binding site (KPC1Δ1-1039, SEQ ID NO: 40, displayed a proteome that was almost identical to that displayed by expression of an empty vector (Figure 3). Functional analysis of proteins that were up-regulated significantly in WT KPC1- and WILVRLW-KPC1Δ1-1039-expressing tumors (compared to an empty vector-expressing tumors; p-value <0.05) revealed significant enrichment in proteins involved in cell migration, extracellular matrix (ECM), cell adhesion, and immune system processes (Figure 3B). Down-regulated proteins highlighted metabolic processes, and cell cycle and DNA metabolic process. The mild reduction in cell cycle proteins in KPC1Δ1 - 1039 and RIWVWLL-KPC1Δ1 - 1039 (number of proteins changed is 15 and 21, respectively) was much smaller than those changed in WT KPC1- and WILVRLW-KPC1Δ1 - 1039-ex pressing tumors (80 and 120 respectively). The raw data of the up-and down-regulated proteins are disclosed in Goldhirsh G. et al„ Proc Natl Acad Sci U S A.118(49)( 2021).
EXAMPLE 4
Design and synthesis of new Ubiquitin ligase KPCl-peptide based PROTACs At the next step, several proteolysis targeting chimera (PROTAC) molecules were designed based on the 7 amino acid sequences mentioned in Examples 1 and 2, and a VHL-binding head (Figure 4A). The various peptides were conjugated to the VHL ligand of Formula I, via a PEG liker, optionally with a further linking peptide of at least one of: at least one glycine (G), at least one serine (S), at least one Cysteine (C) or any combinations thereof. Figures 5A and 5B illustrate the synthesis of the peptide based PROTACs. More specifically, 2-Chlorotritylchloride (CTC) resin (0.7 mmol/g) was loaded with 0.6 eq. of Fmoc-PEG-OH and 2 eq. of DIEA in dry DCM for 1 hr. Following resin esterification, the Fmoc-PEG-resin was washed with DCM, methanol, and DMF. The final loading was verified by Fmoc group absorbance after the DBU treatment to be 0.42 mmol/gr. After initial resin loading, Fmoc-SPPS was carried on automated peptide synthesizer (CS336X, CSBIO) in presence of 4 eq. of amino acid, 4 eq. of HCTU, and 8 eq. of DIEA to the initial loading of the resin for 50 min. Upon peptide elongation, the resin was incubated with 20% HFIP in DCM for 30 min (three times) and flushed with nitrogen gas. The peptide was then treated with PyBOP (5.0 equiv), VHL ligand (5.0 equiv) and DIEA (5.0 equiv) in DMF for overnight. After overnight, the reaction mixture was diluted with acetonitrile-water and lyophilized. Further, the lyophilized material was treated with a cocktail of TFA: triisopropylsilane (TIS): water (95:2.5:2.5) and shaken for 2 hr at RT. The cleavage mixture was added dropwise to a 10-fold volume of cold ether and centrifuged. The precipitate was dissolved in acetonitrile-water for freeze drying in the lyophilizer to give the crude peptide. Finally, the crude peptide was purified using a semi-preparative column (Phenomenex Jupiter C18 10 pm, 300 A, 250 x 10 mm column), at a flow rate of 4 mL/min to obtain the purified PROTACs. 4.1 Synthesis of PROTAC 1 (Formula V, also shown in Fig.4Ai), WILVRLWSG-PEG-VHL lieand:
Figure imgf000095_0001
Formula V
The synthesis of PROTAC 1 was carried out following the general procedure described herein, with ~ 56% isolated yield. Figure 6A shows HPLC-MS analysis of the purified PROTAC 1 with the observed mass 1686.4 ± 0.1 Da (calcd 1687.4 Da, average isotopes).
4.2. Synthesis of PROTAC 2 (Formula XII, also shown in Fig.4Aii, ILVRLSG-PEG-VHL ligand:
Figure imgf000095_0002
Formula XII
The synthesis of PROTAC 2 was carried following the general procedure described herein with ~ 62% isolated yield. Figure 6B shows HPLC-MS analysis of the purified PROTAC 2, with the observed mass 1313.9 ± 0.2 Da (calcd 1314.9 Da, average isotopes).
4.3. Synthesis of PROTAC 3 ( Formula IV, also shown in Fig.4Avi), RIWVWLLSG-PEG-VHL ligand:
Figure imgf000095_0003
Formula IV
The synthesis of PROTAC 3 was carried out following the general procedure with ~ 54 isolated yield. Figure 6C shows HPLC-MS analysis of the purified PROTAC 3 with the observed mass 1686.3 ± 0.5 Da (calcd 1687.4 Da, average isotopes). 4.4. Synthesis of PROTAC 4 (Formula XIII, also shown in Fig.4Av), Acetyl-RIWVWLLSG-PEG-
Figure imgf000096_0001
Formula XIII
The synthesis of PROTAC 4 was carried out following the general procedure described herein with ~ 46 isolated yield. Figure 6D shows HPLC-MS analysis of the purified PROTAC 4, with the observed mass 1727.5 ± 0.1 Da (calcd 1728.4 Da, average isotopes).
4.5. Synthesis of PROTAC 5 (Formula VI, also shown in Fig.4Aiv), IWVWLLS-PEG-VHL ligand:
Figure imgf000096_0002
Formula VI
The synthesis of PROTAC 5 was carried out following the general procedure described herein with - 51 isolated yield. Figure 6E shows HPLC-MS analysis of the purified PROTAC 5, IWVWLLSG-PEG-VHL ligand with the observed mass 1529.9 ± 0.1 Da (calcd 1530.2 Da, average isotopes).
4.6. Synthesis of PROTAC 6 (Formula XIV , also shown in Fig.4Aiii), IWVWLLS-PEG-VHL ligand:
Figure imgf000096_0003
Formula XIV
The synthesis of PROTAC 6 was carried out following the general procedure disclosed herein with - 52 isolated yield. Figure 6F shows HPLC-MS analysis of the purified PROTAC 6, with the observed mass 1473.4 ± 0.4 Da (calcd 1473.1 Da, average isotopes).
Figure imgf000097_0001
The synthesis of Peptide 7 (SEQ ID NO: 17) was carried out following the general procedure with ~ 52 isolated yield. To a solution of peptide 7 (5.0 mg, 1.0 equiv.) in 6M Gn.HCl/200 mM phosphate buffer (pH 7.3) was added FITC-maleimide (1.5 mg, 1.2 equiv.) at 0 °C and kept for 1 hour. After 1 hour, the reaction mixture was diluted with 6M Gn.HCl/200 mM phosphate buffer and purified using semi preparative to obtain PROTAC 8 in 76% yield. The synthesis of PROTAC 8 is also illustrated by Figure 5B.
Figure imgf000097_0002
Formula XV
Figure 6G shows HPLC-MS analysis of the purified PROTAC 8, RIWVWLLC(FITC)G-PEG- VHL ligand with the observed mass 2129.4 ± 0.1 Da (calcd 2129.8 Da, average isotopes). EXAMPLE 5
WILVRLW- and RIWVWLL-pVHL ligand-based PROTACs induce ubiquitination of p105 by the E3 Ub ligase pVHL in a reconstituted cell-free system
To check their ability to stimulate the ubiquitination of p105, a cell-free system in the presence of VHL ubiquitin ligase complex, was next established.
The PROTAC1 WILVRLW-SG-PEG-VHL stimulates the ubiquitination of p105 in a dose- dependent manner (Figure 4Bi). In order to optimize the PROTAC molecule a series of substitutions in the peptide, as well as in the linker part of the PROTAC was generated. Shortening the distance between two tryptophan residues in the peptide part of the PROTAC 3 (RIWVWLL- SG-PEG-VHL), enhanced ubiquitination of p105 compared to ubiquitination by WILVRLW-SG- PEG-VHL PROTAC 1 that has native sequence (Figure 4Bii, compare lanes 8 and 9).
It was found that PROTACs that contain the SerGly linker stimulate the ubiquitination of p105 more efficient than the PROTAC that contains the G linker (Figure 4BB, compare lanes 5 and 6) which highlights the importance of the linker in the conjugation-stimulating activity. Also, removal of the two Trp (W) residues weakened the activity significantly, even in the presence of the GlySer linker (Figure 4BB, lane 4). Though all the PROTACs that contain both tryptophan residues and connected to the PEG via the SG/CG linker stimulate the ubiquitination of p105 by the VHL complex, the PROTAC RIWVWLL-SG-PEG-VHL mediates the strongest ubiquitination (Figure 4BB and 4Biii).
In addition, the enhanced generation of p50 in the presence of different concentrations of WILVRLW-SG-PEG-VHL PROTAC in cell free assay was clearly demonstrated by Figure 4C. EXAMPLE 6
The seven amino acid-based PROTACs stimulate the interaction between p105 and pVHL and enhance the generation ofp50 in cells , and consequently restrict cell growth After confirming the activity of the PRPTACs in a cell-free system, it was important to test their effects in cells. To track the penetration of the PROTACs into cells, the RIWVWLLC(-FITC)G- PEG-pVHL-Ligand PROTAC 8 which contains the fluorophore FITC (Figures 7Ai, 5B and 6G), was synthesized as described in Example 4 and Figure 5B. Using a confocal microscope, we observed that the FITC-labeled PROTAC penetrates into HEK293 cells (Figure 7AB) and can stimulate p105 conjugation in a cell-free system (Figure 4Biii). Co-immunoprecipitation experiment in HEK293 cells, reveled that the interaction between p105 and pVHL was negligible in the absence of the PROTACs, but increased significantly following the addition of either WILVRLW- or RIWVWLL-based PROTACs (Figure 6Bi). Importantly, the two PROTACs stimulated in cells the formation of p50 from the expressed p105 (Figure 7C).
Last, we checked for the PROTACs’ ability to restrict growth of U87-MG cells. The RIWVWLL- and WILVRLW-based PROTACs were added to the growth medium of the cells (as described under Experimental procedures ) and tracked their growth for three days. In all measuring points, the PROTACs inhibited growth rate of the cells (in a statistically significant manner) compared to DMSO-treated cells (Figure 7D), thereby demonstrating the feasibility of using the PROTACs of the present disclosure in cancer therapy.
EXAMPLE 7
PROTAC molecule infiltrates the xenografts derived from U87-MG cell in NSG mice In order to verify that PROTAC molecule is able to infiltrate the xenografts in mice, U87-MG glioblastoma cells were inoculated to NSG mice. When tumors were established, 25 mg/kg/tumor PROTAC labeled with FITC, were injected to the tumor area. 24 hr later the tumors were visualized using in vivo imaging system (IVIS). As shown in Figure 8A, a high concentration of FITC-PROTAC molecule was found in tumor surrounding area. In order to corroborate that PROTAC is competent to diffuse the tumor tissue, the slides prepared from dissected tumors were visualized using confocal microscope device. The presence of FITC-PROTAC molecule was observed inside the large regions of the tumor cells (Figure 8B).
EXAMPLE 8
Optimization of the pl05 recognition peptide of the PROTACs
To further optimize the_peptide component of the PROTACs of the present disclosure various substitutions were made in the peptide of SEQ ID NO: 4, that was shown as the most effective PROTAC in inducing p105 ubiquitination (see Fig. 4B). Therefore, the following peptides were synthesized: in peptide KVS-1 (SEQ ID NOs: 9 and 44, disclosing the peptide sequence and the peptide sequence that includes the SG linker, respectively), the two Trp residues were replaced by two Phenylalanine residues (F), in peptide KVS-5 (SEQ ID NOs: 10 and 45, disclosing the peptide sequence and the peptide sequence that includes the SG linker, respectively), the two Trp residues were replaced by Tyr and Phe, in peptide KVS-3 (SEQ ID NOs: 11 and 46, disclosing the peptide sequence and the peptide sequence that includes the SG linker, respectively), the two Trp residues were replaced by His, in peptide KVS-2A (SEQ ID NOs: 12 and 47, disclosing the peptide sequence and the peptide sequence that includes the SG linker, respectively), the Arg was replaced by Ala, in peptide KVS-2B (SEQ ID NOs: 13 and 48, disclosing the peptide sequence and the peptide sequence that includes the SG linker, respectively), the Arg was replaced by Lys, in peptide KVS-2C (SEQ ID NOs: 14 and 49, disclosing the peptide sequence and the peptide sequence that includes the SG linker, respectively), the L-Arg, was replaced by D-Arg, in peptide KVS-2D (SEQ ID NOs: 15 and 50, disclosing the peptide sequence and the peptide sequence that includes the SG linker, respectively), the Arg, was replaced by Citrulline, in peptide KVS-6 (SEQ ID NOs: 16 and 51, disclosing the peptide sequence and the peptide sequence that includes the CG linker, respectively), a Gly was added at the N-terminal end of the peptide, in peptide KVS-7 (SEQ ID NOs: 17 disclosing the peptide sequence (SEQ ID NO: 4) that further includes the CG linker), the peptide was cyclized (linking the N-terminal Arg and the Cys residue of the added linker), in each of the peptides KVS-8, KVS-9, KVS-10, KVS-11, KVS-12 and KSV-13 (denoted by SEQ ID NOs: 18, 52; 19, 53; 20, 54; 21, 55; 22, 56; 23, 57, for each disclosing the peptide sequence and the peptide sequence that includes the SG linker respectively), an Alanine residue (A), has been introduced, replacing each of the amino acid residues from position 2 in the peptide of SEQ ID NO: 4). In peptides KRS-14, KRS-15, KRS-16, and KRS-18, one, two or three Arginine and Lysin resides residues were added to the N-terminal end of the original peptide of SEQ ID NO: 4, as denoted by SEQ ID NOs: 24, 58 (RR(R)KRIWVWLL, where two arginine residues appear at the N-terminus of the peptide and a third arginine is linked to the lysine residue through the epsilon amine group); 25; 26 (R(R)KRIWVWLL, where one arginine residues appear at the N-terminus of the peptide and a second arginine is linked to the lysine residue through the epsilon amine group), and 27, 59; for each disclosing the peptide sequence and the peptide sequence that includes the SG linker, respectively).
Peptide KVS-4 that comprises the original RIWVWLL sequence (SEQ ID NO: 4), with the SG linker (as denoted by SEQ ID NO: 8), peptide SKV-17 having the sequence of the original peptide of SEQ ID NO: 4, with no linker, and peptide KVS-19, comprising the wildtype sequence as denoted by SEQ ID NO: 4, with the SG linker (SEQ ID NO: 28) were used as controls. Figure 9 discloses an example for p105 ubiquitination by the various peptides. More specifically, an in vitro translated and 35S-labeled p105 was ubiquitinated by purified KPC1Δ1-967 (SEQ ID NO: 67) in a cell-free system in the presence of the indicated peptides. The numbers indicate the peptide concentration in mM. The fraction of remained free unconjugated p105 for each condition is compared to a system where a peptide was not added (arbitrarily designated as 1), as shown in Figures 9A, 9B and 9C. Figure 9D provides a schematic representation of the various peptides sequences and activity. As shown by Figs 9A and 9D, replacement of the Trp residues by other aromatic amino acid residues in peptides KVS-1 and KVS-5, clearly retained the ability of the peptide to induce p105 ubiquitination. Additional peptides with replacement of the Arg with Citrulline or adding a Gly to the original peptide of SEQ ID NO: 4, improved the ability of the peptide to enhance p105 ubiquitination. Interestingly, peptide KVS-18 (SEQ ID NO: 27, 59) that comprise three Arg residues, showed the most effective enhancement of p105 ubiquitination (not shown). Taken together, in appears that the two Trp amino acid residues as in the original peptide of SEQ ID NO: 7 and 4, can be replaced by any aromatic amino acid residue (e.g., F, and) Y. Still further, it appears that the initial Arg residue is required and addition of two or more Arg resides may approve the effectivity of the peptide.
EXAMPLE 9
The effect of the PROTAC molecules on xenografts derived from human tumors The prototypic PROTAC molecules of Formulas IV, V, VI and VII are tested in several human tumor models in mice. An inactive PROTAC is used as a control. More specifically, an amount of approximately 100 microliters of various concentrations of the different PROTACs ranging between 10 to 500 micromolar, is injected into the tumor bed about 1 to 4 times every day and every other day for two weeks after the tumors solidify. The following parameters are monitored in the treated animals: tumors size, tumors volume, cytokines secretion by the tumors, infiltration of lymphocytes to the tumors, expression of PD-L1 by the tumor and expression of tumor suppressors by the tumors.

Claims

CLAIMS:
1. A bifunctional hybrid-molecule, conjugate or complex comprising:
(a) at least one peptide comprising at least two aromatic ami ancoid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1, wherein Zaa is any aromatic amino acid residue, wherein Xaa is any ami ancoid residue, wherein n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7; and
(b) at least one proteasome degradation mediating moiety.
2. The bifunctional hybrid-molecule, conjugate or complex according to claim 1 , wherein said aromatic amino acid residue comprise at least one Tryptophan (Trp, W).
3. The bifunctional hybrid-molecule, conjugate or complex according to any one of claims 1 and 2, wherein said at least two aromatic amino acid residues are interspaced by at least one of Valine (V), Glycine (G) and Alanine (A).
4. The bifunctional hybrid-molecule, conjugate or complex according to any one of claims 1 and 3, comprising the amino acid sequence of Xaa(n)-Trp (W)-Xaa(n)-Val (V)-Xaa(n)-Trp (W)- Xaa(n), as denoted by SEQ ID NO: 7, or any mimetics thereof.
5. The bifunctional hybrid-molecule, conjugate or complex according to any one of claims 1 to 4, wherein said peptide comprises 3 to 10 amino acid residues.
6. The bifunctional hybrid-molecule, conjugate or complex according to any one of claims 1 to 5, wherein each of said Xaa of said peptide is any one of Arg (R), IIe (I), and Leu (L), amino acid residues.
7. The bifunctional hybrid-molecule, conjugate or complex according to any one of claims 1 to 6, wherein said peptide comprises the amin aocid sequence of at least one of:
(a) RIWVWLL, as dented by SEQ ID NO: 2, or any variants and derivatives thereof;
(b) IWVWLL, as dented by SEQ ID NO: 3, or any variants and derivatives thereof;
(c) WILVRLW, as dented by SEQ ID NO: 4, or any variants and derivatives thereof;
(d) RIFVFLL, as dented by SEQ ID NO: 9;
(e) RIYVFLL, as dented by SEQ ID NO: 10;
(f) Citrulline-IWVWLL, as dented by SEQ ID NO: as dented by SEQ ID NO: 15;
(g) GRIWVWLL, as dented by SEQ ID NO: 16;
(h) RRRIWVWLL, as dented by SEQ ID NO: 27; and
(i) WVW, or any variants and derivatives thereof.
8. The bifunctional hybrid-molecule, conjugate or complex according to any one of claims 1 to 7, wherein said at least one proteasome degradation mediating moiety comprises at least one E3 ubiquitin ligase recruiting moiety, or at least one E3 ubiquitin ligase active moiety.
9. The bifunctional hybrid-molecule, conjugate or complex according to claim 8, wherein said E3 ubiquitin ligase is at least one of von-Hippel-Lindau (VHL), Cereblon (CRBN), Mouse double minute 2 homolog (MDM2), cellular inhibitor of apoptosis protein- 1 (clAPl) and X-linked inhibitor of apoptosis protein (XIAP) and DDB1 and CUL4 associated factor 15 (DCAF15).
10. The bifunctional hybrid-molecule, conjugate or complex according to any one of claims 1 to 9, wherein said at least one proteasome degradation mediating moiety comprises at least one recruiting moiety for VHL E3 ubiquitin ligase, said recruiting moiety comprises at least one small molecule or peptide, or any combinations thereof.
11. The bifunctional hybrid-molecule, conjugate or complex according to claims 10, wherein said at least one recruiting moiety for VHL E3 ubiquitin ligase comprises the VHL ligand of Formula I, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof:
Figure imgf000103_0001
12. The bifunctional hybrid-molecule, conjugate or complex according to any one of claims 1 to 11 , further comprising at least one linker.
13. The bifunctional hybrid-molecule, conjugate or complex according to any one of claims 1 to 12, wherein at least one of said linker/s comprises the linker of Formula II, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof,
Figure imgf000103_0002
Formula II wherein m is an integer between 1 to 10.
14. The bifunctional hybrid-molecule, conjugate or complex according to claims 13, wherein said linker comprises the linker of Formula III, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof:
Figure imgf000104_0001
Formula III
15. The bifunctional hybrid-molecule, conjugate or complex according to any one of claims 1 to 14, wherein said linker further comprises at least one amino acid residue.
16. The bifunctional hybrid-molecule, conjugate or complex according to claim 15, wherein said at least one amino acid residue further comprised in said linker comprises at least one of: at least one Serine (Ser, S), at least one cysteine (Cys, C) and at least one Glycine (Gly, G) amino acid residues, attached therewith.
17. The bifunctional hybrid-molecule, conjugate or complex according to any one of claims 1 to 16, wherein said bifunctional hybrid-molecule, conjugate or complex comprises at least one of the compounds of Formula IV, V, VI and VII, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof, wherein:
(a) Formula IV is:
Figure imgf000104_0002
Formula IV;
(b) Formula V is:
Figure imgf000104_0003
Formula V;
(c) Formula VI is:
Figure imgf000105_0001
18. A composition comprising at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the same, wherein said bifunctional hybrid-molecule, conjugate or complex comprises:
(a) at least one peptide comprising at least two aromatic amino acid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1, wherein Zaa is any aromatic amino acid residue, wherein Xaa is any amino acid residue, wherein n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7; and
(b) at least one proteasome degradation mediating moiety; said composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
19. The composition according to claim 18, wherein said at least one bifunctional hybrid- molecule, conjugate or complex is as defined in any one of claims 2 to 17.
20. The composition according to any one of claims 18 to 19, wherein said bifunctional hybrid- molecule, conjugate or complex comprises at least one of the compounds of Formula IV, V, VI and VII, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof, wherein:
(a) Formula IV is:
Figure imgf000106_0001
21. A method for inducing ubiquitination and proteasomal processing of NF-κB1 p105, thereby generating the NF-κB p50 in a cell or in a cell-free system comprising said NF-κB1 p105, the method comprising the step of contacting said cell or said cell-free system with an effective amount of at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro-particle comprising the same, or any composition thereof, wherein said bifunctional hybrid-molecule, conjugate or complex comprises:
(a) at least one peptide comprising at least two aromatic ami ancoid residues interspaced by at least one amino acid residue, wherein said peptide comprises the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1, wherein Zaa is any aromatic amino acid residue, wherein Xaa is any amino acid residue, wherein n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7; and (b) at least one proteasome degradation mediating moiety.
22. The method according to claim 21, wherein said at least one bifunctional hybrid-molecule, conjugate or complex is as defined in any one of claims 2 to 17.
23. The method according to any one of claims 21 to 22, wherein said bifunctional hybrid- molecule, conjugate or complex comprises at least one of the compounds of Formula IV, V, VI and VII, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof, wherein:
(a) Formula IV is:
Figure imgf000107_0001
Figure imgf000108_0001
24. The method according to any one of claims 22 to 23, for inducing ubiquitination and proteasomal processing of NF-κB1 p105 to generate the NF-κB p50 in a subject.
25. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder or condition in a subject in need thereof, the method comprising the step of administering to said subject a therapeutically effective amount of at least one bifunctional hybrid-molecule, conjugate or complex, or any vehicle, matrix, nano- or micro- particle comprising the same, or any composition thereof, wherein said bifunctional hybrid- molecule, conjugate or complex comprises:
(a) at least one peptide comprising at least two aromatic amino acid residues interspaced by at least oneamino acid residue, wherein said peptide comprises the amino acid sequence of Xaa(n ) Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1, wherein Zaa is any aromatic amino acid residue, wherein Xaa is any amino acid residue, wherein n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7 ; and
(b) at least one proteasome degradation mediating moiety.
26. The method according to claim 25, wherein said peptide comprises the amino acid sequence of Xaa(n)-Trp (W)-Xaa(n)-Val (V)-Xaa(n)-Trp (W)-Xaa(n), as denoted by SEQ ID NO: 7, or any mimetics thereof.
27. The method according to any one of claims 25 to 26, wherein said peptide comprises 3 to 10 amino acid residues .
28. The method according to any one of claims 25 to 27, wherein each of said Xaa of said peptide is any one of Arg (R), IIe (I), and Leu (L) amino acid residues.
29. The method according to any one of claims 25 to 28, wherein said peptide comprises the amino acid sequence of at least one of:
(a) RIWVWLL, as dented by SEQ ID NO: 2, or any variants and derivatives thereof;
(b) IWVWLL, as dented by SEQ ID NO: 3, or any variants and derivatives thereof;
(c) WILVRLW, as dented by SEQ ID NO: 4, or any variants and derivatives thereof;
(d) RIFVFLL, as dented by SEQ ID NO: 9;
(e) RIYVFLL, as dented by SEQ ID NO: 10;
(f) Citrulline-IWVWLL, as dented by SEQ ID NO: as dented by SEQ ID NO: 15;
(g) GRIWVWLL, as dented by SEQ ID NO: 16;
(h) RRRIWVWLL, as dented by SEQ ID NO: 27; and
(i) WVW, or any variants and derivatives thereof.
30. The method according to any one of claims 25 to 29, wherein said at least one proteasome degradation mediating moiety comprises at least one E3 ubiquitin ligase recruiting moiety, or at least one E3 ubiquitin ligase active moiety.
31. The method according to claim 30, wherein said E3 ubiquitin ligase is at least one of von- Hippel-Lindau (VHL), Cereblon (CRBN), Mouse double minute 2 homolog (MDM2), cellular inhibitor of apoptosis protein- 1 (clAPl) and X-linked inhibitor of apoptosis protein (XIAP) and DDB1 and CUL4 associated factor 15 (DCAF15).
32. The method according to any one of claims 25 to 31 , wherein said at least one proteasome degradation mediating moiety comprises at least one recruiting moiety for VHL E3 ubiquitin ligase, said recruiting moiety comprises at least one small molecule or peptide, or any combinations thereof.
33. The method according to claims 32, wherein said at least one recruiting moiety for VHL E3 ubiquitin ligase comprises the VHL ligand of Formula I, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof: Formula I
34. The method according to any one of claims 25 to 33, wherein said bifunctional hybrid- molecule, conjugate or complex further comprises at least one linker.
35. The method according to any one of claims 25 to 34, wherein at least one of said linker/s comprises the linker of Formula II, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof:
Figure imgf000110_0001
Formula II wherein m is an integer between 1 to 10.
36. The method according to claims 35, wherein said linker comprises the linker of Formula III, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof:
Figure imgf000110_0002
37. The method according to any one of claims 345 to 36, wherein said linker further comprises at least one amino acid residue.
38. The method according to claim 37, wherein said at least one amino acid residue further comprised in said linker comprises at least one of: at least one Ser (S), at least one cysteine (C) and at least one Gly (G) amino acid residues, attached therewith.
39. The method according to any one of claims 25 to 38, wherein said bifunctional hybrid- molecule, conjugate or complex comprises at least one of the compounds of Formula IV, V, VI and VII, or any pharmaceutically acceptable salt, solvate, ester, hydrate, stereoisomer or physiologically functional derivative thereof, and wherein: (a) Formula IV is:
Figure imgf000111_0001
Formula IV;
(b) Formula V is:
Figure imgf000111_0003
(d) Formula VII is:
Figure imgf000111_0002
Formula VII.
40. The method according to any one of claims 25 to 39, wherein said pathologic disorder or condition is cancer.
41. A peptide comprising the amino acid sequence of Xaa(n)-Zaa-Xaa(m)-Zaa-Xaa(n), as denoted by SEQ ID NO: 1, wherein Zaa is any aromaticamino acid residue, wherein Xaa is any amino acid residue, wherein n is zero or an integer between 1 to 7, and wherein m is an integer between 1 to 7, with the proviso that said peptide is not the peptide of SEQ ID NO: 4.
42. The peptide according to claim 41, comprising the amino acid sequence RIWVWLL, as dented by SEQ ID NO: 2, any mimetics thereof, any variants and derivatives thereof, or any conjugate, complex, chimera and composition thereof.
PCT/IL2022/050243 2021-03-03 2022-03-03 Ubiquitin ligase kpc1-peptide based proteolysis targeting chimeras (protac) and uses thereof WO2022185319A1 (en)

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JUNLONG (JACK) CHI, LI HONGCHUN, ZHOU ZHUAN, IZQUIERDO-FERRER JAVIER, XUE YIFAN, WAVELET CINDY M., SCHILTZ GARY E., ZHANG BIN: "A novel strategy to block mitotic progression for targeted therapy", EBIOMEDICINE, ELSEVIER BV, NL, vol. 49, 1 November 2019 (2019-11-01), NL , pages 40 - 54, XP055709006, ISSN: 2352-3964, DOI: 10.1016/j.ebiom.2019.10.013 *
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