WO2023166507A1 - Protéine associée à l'apoptose dans la voie de signalisation tgf-beta (arts) et composés à petites molécules mimétiques de arts pour réguler à la hausse le niveau p53 - Google Patents

Protéine associée à l'apoptose dans la voie de signalisation tgf-beta (arts) et composés à petites molécules mimétiques de arts pour réguler à la hausse le niveau p53 Download PDF

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WO2023166507A1
WO2023166507A1 PCT/IL2023/050212 IL2023050212W WO2023166507A1 WO 2023166507 A1 WO2023166507 A1 WO 2023166507A1 IL 2023050212 W IL2023050212 W IL 2023050212W WO 2023166507 A1 WO2023166507 A1 WO 2023166507A1
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arts
independently
alkyl
halogen
optionally substituted
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Sarit Larisch
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Carmel-Haifa University Economic Corporation Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the invention relates to the field of therapy. More particularly, the invention relates to apoptosis related protein in the TGF-beta signaling pathway (ARTS) and ARTS mimetic compounds and uses thereof as modulators of tumor suppressor p53, specifically, for upregulating the level of p53.
  • ARTS TGF-beta signaling pathway
  • Apoptosis is a programmed cell death essential for embryonic development and tissue homeostasis.
  • Abnormal regulation of the apoptotic pathway leads to various human diseases, including neurodegeneration and cancer (Fuchs and Sachr 2011 Cell 147 742- 758; Kerr et al. 1972 Br J Cancer 26 239-257; Meier et al. 2000 Nature 407 796-801).
  • the apoptotic pathway activates caspases (cysteine-aspartic proteases) through cleavage from their inactive zymogens.
  • XIAP X-linked inhibitor of apoptosis
  • XIAP contains three baculoviral IAP repeats (BIR), which serve as protein-protein interaction domains (Deveraux et al. 1999 EMBO J 18 5242- 5251; Deveraux and Reed 1999 Genes Dev 13 239-252; Schimmer 2004 Cancer Res 64 7183-7190; Suzuki et al. 2001 J Biol Chem 27627058-27063).
  • BIR baculoviral IAP repeats
  • XIAP-BIR3 is responsible for inhibiting caspase-9, and the linker region between XIAP-BIR1 and BIR2 is responsible for inhibiting caspase-3 and caspase-7 (Deveraux et al. 1999 EMBO J 18 5242-5251; Deveraux and Reed 1999 Genes Dev 13 239-252; Schimmer 2004 Cancer Res 647183-7190; Suzuki et al. 2001 J Biol Chem 276 27058-27063).
  • XIAP has a ubiquitin-associated (UBA) domain, which enables the binding of poly-ubiquitin conjugates, and a RING domain responsible for E3-ligase activity (Gyrd-Hansen et al.
  • ARTS (sept4-i2) is a splice variant derived from the septin4 gene, the only splice variant that regulates the apoptotic pathway (Mandel-Gutfreund et al. 2011 Biol Chem 392 783- 790).
  • ARTS is a pro-apoptotic and tumor suppressor protein localized on the mitochondrial outer membrane (MOM) (Edison et al. 2012 Cell Death Differ 19 356- 368). Over-expression of ARTS alone is sufficient to induce apoptosis in a variety of cultured cancer cell lines, in addition to increasing the sensitivity of cells toward apoptotic inducers (Larisch et al.
  • ARTS functions as a potent tumor suppressor protein
  • ALL acute lymphoblastic leukemia
  • HCC hepatocellular carcinoma
  • ARTS rapidly translocates to the cytosol to bind and antagonize XIAP (Edison et al. 2012 Cell Death Differ 19 356-368; Gottfried et al. 2004 EMBO J 23 1627-1635).
  • ARTS lacks the canonical lAP-binding motif, instead ARTS binds XIAP- BIR3 via its unique c-terminus sequence (Bornstein et al. 2011 Apoptosis 16 869-881; Gottfried et al. 2004 EMBO J 23 1627-1635; Reingewertz et al. 2011 PLoS One 6 e24655).
  • ARTS binds a distinct and unique sequence within the XIAP- BIR3, different from other IAP antagonists (Bornstein et al. 2011 Apoptosis 16 869-881; Mamriev et al. 2020 Cell Death Dis 11 483).
  • ARTS is the only IAP antagonist to promote XIAP degradation through the ubiquitin-proteasome system (UPS) (Garrison et al. 2011 Mol Cell 41 107-116).
  • UPS ubiquitin-proteasome system
  • the direct binding of ARTS to XIAP enables the release of caspases leading to cell death (Adrain et al. 2001 EMBO J 20 6627-6636; Braun et al. 2003 Mol Cancer Res 1 186-194, Edison et al.
  • P53 is a tumor suppressor protein found to be mutated in approximately 50% of human tumors (Kaelin 1999 Oncogene 18 7701-7705, Labrecque et al. 1993 Cancer Research 53 3468, Oren 1999 J Biol Chem 274 36031-36034, Tokino and Nakamura 2000 Crit Rev Oncol Hematol 33 1-6). P53 functions primarily as a sequence-specific transcription factor (Aylon and Oren 2007 Cell 130 597-600, Levine and Oren 2009 Nat Rev Cancer 9 749-758). The p53 unique binding sequence within the genome is called p53 binding site or p53 response element (Hassin and Oren 2022 Nat Rev Drug Discov 1-18, Wang et al.
  • ARTS binds directly to both XIAP and Bcl-2, acting as a scaffold to bring these proteins together. This binding leads to a UPS mediated degradation of Bcl-2.
  • ARTS comprise a BH3-like domain.
  • the inventors have further demonstrated a novel small molecule compound A4 and uses thereof in inducing differentiation and apoptosis in cancer cells ([9] WO 2017/077535).
  • the present disclosure describes the identification of XIAP as a novel E3-ligase of p53 and that ARTS upregulates p53 by antagonizing XIAP, indicating that ARTS and p53 regulate each other in a positive feedback loop manner.
  • the present disclosure presents the small molecule ARTS mimetics, B3, that binds directly to the unique sequence of ARTS in XIAP-BIR3. B3, just like ARTS, promoted apoptosis by simultaneously downregulating XIAP levels and upregulating p53 protein levels.
  • a first aspect of the present disclosure relates to an effective amount of at least one of apoptosis related protein in the TGF-beta signaling pathway (ARTS), any fragments thereof, or at least one mimetic compound thereof or any vehicle, matrix, nano- or microparticle comprising the same, for use in a method for upregulating p53 levels in a cell.
  • ARTS TGF-beta signaling pathway
  • compositions comprising effective amount of at least one of ARTS, any fragments thereof, at least one mimetic compound thereof or any vehicle, matrix, nano- or micro-particle comprising the same, for use in a method for upregulating p53 levels in a cell.
  • the composition optionally further comprises at least one of pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
  • Another aspect of the present disclosure relates to methods for upregulating the levels of p53 in a cell. More specifically, the disclosed method comprises the step of contacting the cell with an effective amount of ARTS, any fragments thereof, or at least one mimetic compound thereof or any vehicle, matrix, nano- or micro-particle comprising the same, or any composition thereof.
  • Another aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a pathological disorder in a subject in need thereof, by upregulating p53 levels in at least one cell of the subject.
  • the method comprising administering to the subject a therapeutically effective amount of at least one of ARTS, any fragments thereof, or at least one mimetic compound thereof or any vehicle, matrix, nano- or micro-particle thereof or of a composition comprising the same.
  • kits comprising:
  • ARTS In one component (a), ARTS, or any fragments thereof, or at least one mimetic compound thereof, optionally, in first dosage form.
  • the ARTS mimetic compound of the disclosed kits may have the general formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof, wherein formula (I) is: wherein
  • R 1 , R2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is L 1 '-R 3 '-L 1 ”-R 3 ” and R2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R 2 is L 2 '-R 4 '-L 2 ”-R 4 ”; or
  • kits further comprise at least one therapeutic compound, optionally, in a second dosage form.
  • FIG. 1A-1D ARTS is a transcriptional target of p53
  • Fig. 1A Scheme of the putative p53 binding site in ARTS promoter sequences.
  • the sept4 gene encodes two main splice variants, Sept4_il (PNUTL2, H5) and Sept4_i2 (ARTS). Only the ARTS protein can promote apoptosis.
  • the scheme shows the location of a putative p53 binding site at the ARTS promoter located between nucleotides -391 to -351 upstream of the ARTS TSS.
  • p53 binds to promoter sequences of ARTS rapidly upon induction of apoptosis.
  • Chromatin IP (ChIP) assay was performed on WT HCT116 cells. The binding of p53 is shown after 5 minutes of UV treatment. These results suggest that the ARTS promoter contains specific binding sequences for p53. Collectively, these results suggest that p53 acts as a transcriptional factor inducing upregulation of ARTS upon induction of DNA damage and apoptosis.
  • Fig. 1C UV irradiation induces p53-dependent upregulation of ARTS RNA.
  • Real-Time PCR results WT HCT116 and p53 KO HCT116 cells were induced for apoptosis with UV for the indicated time points. Real-Time PCR was performed, and cDNAs were normalized to GPI (housekeeping gene). Error bars represent +/- SER of three biologically independent experiments. (* p-value ⁇ 0.05, ** p-value ⁇ 0.01, ***p- value ⁇ 0.001).
  • FIG. 2A-2D ARTS regulates p53 protein levels through proteasome-mediated degradation
  • Sept4/ARTS KO MEFs were treated with 200ng/ml Nocodazole (NOC) for 1 hour following 40 hours of ARTS overexpression (Fig. 2C(i)).
  • NOC Nocodazole
  • Fig. 2C(ii) Densitometry analysis of four biologically independent repeats (Fig. 2C(ii)).
  • One Way ANOVA statistical analysis was done using GraphPad Prism version 8., Mean +/- SD; **p- value ⁇ 0.001; * p-value ⁇ 0.05.
  • Fig. 2D Bimolecular fluorescence complementary assay (BiFC). Etoposide promotes the formation of p53-ARTS complex. Bimolecular fluorescence complementation (BiFC) assay was performed on WT MEFs that were treated with 200 ⁇ M Etoposide for the indicated time points. Cells were transfected with VN-p53 and VC-ARTS plasmids for 36 hours. Fluorescence- activated cell sorting (FACS) analyses revealed an increase in the formation of ARTS-p53 complex upon Etoposide treatment.
  • Figure 3A-3C ARTS is important for the localization of p53 to the nucleus
  • MEFs cells Fig. 3A(i)
  • Sept4/ARTS KO MEFs cells Fig. 3A(ii)
  • Nuclei and cytosol fractions were dissected from the cells and Western blots were performed using p53, ARTS, Lamin, Tubulin and Her2 antibodies.
  • MEFs cells and Sept4/ARTS KO MEFs cells were induced for apoptosis with UV for 10 minutes.
  • Cells were immobilized and fluorescently stained for p53, mitotracker (a mitochondrial marker) and DAPI (a nuclei marker) (Fig. 3B(i)).
  • the graphs represent the numbers of cells displaying cytoplasmic/nuclear staining as a percent of total counted cells (mean+S.E, n1 ⁇ 4 300) (Fig. 3B(ii)).
  • MEFs cells and Sept4/ARTS KO MEFs cells were induced for apoptosis with UV for 10 minutes.
  • Cells were immobilized and fluorescently stained for p53, mitotracker (a mitochondrial marker) and DAPI (a nuclei marker) (Fig. 3C(i)).
  • the graphs represent the numbers of cells displaying cytoplasmic/nuclear staining as a percent of total counted cells (mean+S.E, n1 ⁇ 4 300) (Fig. 3C(ii)).
  • XIAP acts as an E3 ligase for p53
  • Western blot analysis of WT MEFs and XIAP KO MEFs (Fig. 4A(i)).
  • Densitometry analysis represents three biologically independent repeats (Fig. 4A(ii)).
  • XIAP binds p53.
  • Immunoprecipitation assay of XIAP shows that XIAP and p53 bind each other under normal conditions and to a lesser extent under apoptotic induction. Apoptosis was induced using 200 ⁇ M Etoposide (ETP) for 3 hours.
  • XIAP serves as an E3 ligase of p53.
  • In vitro ubiquitylation assay was performed using recombinant XIAP and p53 proteins, and UbcH5b as E2.
  • Western blot (Fig. 4C(i)).
  • Densitometry analysis of the non-ubiquitinated p53 Fig. 4C(ii)).
  • Fig. 4D p53 half-life in HCT 116 WT and XIAP KO HCP 116 cells treated with Cycloheximide (CHX) 200 ⁇ M for the indicated time points.
  • ARTS inhibits p53 ubiquitylation by antagonizing XIAP.
  • In vitro ubiquitylation assay was performed by incubating 0.2 ⁇ g of recombinant GST-XIAP and 0.2 ⁇ g of recombinant p53 together for 1 hour at 37°C. Different doses of ARTS (0.2, 0.4, and 0.8 ⁇ g) were added to the mix (Fig. 4E(i)). Densitometry analysis represents three biologically independent repeats (Fig. 4E(ii)).
  • Figure 5A-5C Small-molecule ARTS mimetics can bind XIAP
  • Fig. 5A Illustration of the 100 small molecules derived from the in “Silico screen”. In silico screen was done by “BioSolveit” to look for ARTS mimetic small molecules that fit into the binding pocket between ARTS -unique C-terminus and its distinct binding sequence in BIR3/XIAR
  • Fig. 5B Interaction of the small molecule compound "B3" with XIAP-BIR3 in 2D. The hydrogen bonds between B3 and the backbone carbonyls are depicted as dashed lines.
  • Fig. 5C MST (microscale thermophoresis) analysis of B3 binding to fluorescently labelled recombinant XIAP revealed a direct binding to XIAP-BIR3 with Kd of 36, u M +/-l l ⁇ M.
  • FIG. 6A-6I Small-molecule ARTS mimetics can reduce XIAP and increase p53 levels to promote apoptosis
  • FIG. 6A Western Blot analysis of A375 melanoma cells that were treated with 20uM of each of the indicated ARTS mimetic compounds for 24hrs. Western blot analysis was performed with the indicated antibodies, showing the levels of the proapoptotic cCasp3 and cP ARP in the presence or absence of different ARTS mimetic compounds.
  • Fig. 6B Western Blot analysis of WT MEFs and Sept4/ARTS KO MEFs cells that were induced for apoptosis with 20 or 40 ⁇ M of either A4 or B3 ARTS mimetic compounds for 24 hours, showing that B3 can restore elevation of p53.
  • Fig. 6C Co-immuno-precipitation between ARTS and p53. Western Blot analysis of lysates and immunoprecipitation of myc-ARTS using Sept4/ARTS KO MEFs cells. Cells were treated with 20 ⁇ M of B3 for 24hrs.
  • Fig. 6E(i)-6E(v) Western Blot analysis of HCT116 WT cells that were induced for apoptosis with 10, 20, 30 ⁇ M B3 for 24 hours (Fig. 6E(i)). Densitometry analysis of three biologically independent repeats (Fig. 6E(ii)-6E(v)).
  • WT and XIAP KO cells Western Blot analysis of MEF cells (WT and XIAP KO cells) that were induced for apoptosis with 20 ⁇ M B3 for 2,4,6,16 hours (Fig. 6F(i)).
  • Densitometry analysis of three biologically independent repeats Fig. 6F(ii)-6F(viii)
  • Figs. 6F(ii), (iv),(vi) and (viii) show the WT
  • Figs.6F(iii), (v), and (vii) show the KO cells.
  • B3 inhibits p53 ubiquitylation by XIAP.
  • In vitro ubiquitylation assay was performed by incubating 0.2 ⁇ g of recombinant GST-XIAP and 0.2 ⁇ g of recombinant p53 with 20 and 40 ⁇ M of B3 for 1 hour at 37C (Fig. 6H(i)).
  • Densitometry analysis represents three biologically independent repeats (Fig. 6H(ii)).
  • Fig. 61 B3 disrupts the binding between XIAP and p53.
  • Bimolecular fluorescence complementation (BiFC) assay was performed on WT MEFs that were transfected with BiFC pairs of plasmids: VN-p53 and VC-XIAP or VN-p53 and VC-ARTS. 24h hours post-transfection cells were treated with 20 ⁇ M B3 for 18 hours. The fluorescent signal indicating the proximity of each pair of proteins was measured by flow cytometry. MFI, mean fluorescence intensity. Fluorescence- activated cell sorting (FACS) results were normalized to the readings of the transfection efficiency reporter (pdsRED).
  • the apoptotic pathway is an ordered process of programmed cell death that is often altered in various pathologic conditions associated with either increased or decreased apoptosis.
  • apoptosis by external means provides an important and promising approach that paves the way for a variety of therapeutically opportunities.
  • cancer is a condition associated with deregulated apoptosis, resulting in cells displaying increased survival.
  • inducing apoptosis is valuable as a defense mechanism against hyper proliferating cells.
  • the anti-apoptotic proteins of the Bcl-2 family govern the pro-survival pathway and are over expressed in a variety of tumor types such as small cell lung cancer, melanoma, prostate and breast cancer.
  • Cancer treatment is among others aimed in restoring the apoptotic capabilities of cancer cells. Further, inhibitors of Bcl-2 and XIAP anti-apoptotic proteins are needed in order to revert to normal apoptotic processes and thus trigger tumor cell death. As indicated above, the inventors have previously found that upon induction of apoptosis, ARTS binds directly to both XIAP and Bcl-2, acting as a scaffold to bring these proteins together, leading to a UPS mediated degradation of Bcl-2.
  • XIAP was revealed as a new E3-ligase to regulate p53 levels through the UPS ( Figure 4C).
  • p53 and ARTS regulate each other in a positive feedback loop manner: p53 activates ARTS transcription, and in turn, ARTS upregulates p53 protein levels by antagonizing XIAP and preventing its degradation through the UPS.
  • discovering these new players to regulate p53 levels assisted in developing a novel class of dual targeting compounds to promote apoptosis by antagonizing XIAP and upregulating p53 ( Figure 5 and Figure 6).
  • the ARTS mimetic B3 compound disclosed herein (specifically, (S)- N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide) could provide a potential solution for limiting the effect of p53 through developing XIAP-dependent chemotherapy resistant.
  • a first aspect of the present disclosure relates to an effective amount of at least one of apoptosis related protein in the TGF-beta signaling pathway (ARTS), any fragments thereof, or at least one mimetic compound thereof or any vehicle, matrix, nano- or microparticle comprising the same, for use in a method for upregulating p53 levels in a cell.
  • ARTS TGF-beta signaling pathway
  • Tumor suppression is the main function of p53 protein, which is encoded by the TF‘53 gene on human chromosome 17.
  • the p53 protein is posited to inhibit the phenotypic and genomic alterations associated with cancer development through a complex interplay with several signaling pathways known to play critical roles in essential cellular processes such as cell division, maintenance of genomic stability, apoptosis, autophagy, immune response, and regulation of tumor microenvironment.
  • Approximately 50% of human tumors harbor mutations in the TP53 gene, while the remaining malignancies expressing WT p53 display functional inactivation of the p53 pathway by alternative mechanisms implicating viral oncoproteins or negative regulators of p53 such as MDM2 or MDM4.
  • inactivation of the TP53 gene usually occurs through the acquisition of loss of function mutations or negative regulation of wild-type p53 proteins. Inactivation of the TP.53 gene drives invasion, proliferation, and cell survival, thereby facilitating cancer progression and metastasis. More than 75% of TP53 gene mutations result in loss of wild-type p53's activities. Mutated p53 proteins might act either as dominant negative over wild-type p53 action, or gain novel turnorigenic properties counteracting the protective function of wildtype p53. p53 acts mainly as a transcription factor that is activated in response to multiple stressors to regulate the expression of genes controlling proliferation, DNA repair, senescence and cell death.
  • binding of wild-type p53 protein to specific DNA response elements induces the expression of a wide array of genes that ultimately guard against cancer development and progression.
  • exposure of cells to different stress signals activates the p53 signaling pathway, allowing the cells to activate several transcriptional programs including cell cycle arrest, DNA repair, senescence, and apoptosis leading to suppression of tumor growth.
  • p53 refers to the human p53 as denoted by Genebank Accession No. NC_.000017.l l. Still further, in some embodiments, p53 as used herein is the human p53 that comprises the amino acid sequence as denoted by NP_000537.3. Still further, in some embodiments, p53 as used herein comprises the amino acid sequence as denoted by SEQ ID NO: 7, or any variants and derivatives thereof. In yet some further embodiments, p53 as used herein is the human p53, encoded by the nucleic acid sequence comprising the nucleic acids sequence of SEQ ID NO: 8, or any homologs or variants thereof.
  • the disclosed methods may upregulate the Wild type p53, specifically, p53 that display no mutations.
  • p53 as indicated herein may alternatively refer to p53 that comprise mutations that do not affect its function.
  • upregulation of p53 by the disclosed ARTS and/or fragments and/or mimetics thereof restores p53 function.
  • the present disclosure further encompasses the use of the ARTS polypeptide and/or any fragments or peptides thereof.
  • the present disclosure provides uses of the ARTS and fragments thereof in methods for upregulating p53 levels in cells, or as disclosed herein after, in a subject in need thereof.
  • the methods may comprise the step of contacting said cells with an effective amount of ARTS, any fragments thereof or any composition comprising the same.
  • ARTS'' apoptosis-related protein in the TGF-P signaling pathway
  • ARTS acts as a tumor suppressor protein that functions as an antagonist of XIAP and thereby promotes apoptosis.
  • ARTS protein refers to the human ARTS (as denoted by SEQ ID NO. 1). More specifically, the human ARTS protein comprises an amino acid sequence of 274 amino acid residues as denoted by GenBank Accession No. AF176379, encoded by a nucleic acid sequence of SEQ ID NO. 2. As indicated above, any fragment of ARTS, specifically, any functional fragment that may lead directly or indirectly to upregulation in p53 levels, may be used. In certain embodiments, any ARTS fragment that leads to reduction in the levels of at least one E3 ligase, for example, XIAP, and/or MDM2, may be applicable in the disclosed uses.
  • E3 ligase for example, XIAP, and/or MDM2
  • peptides derived from ARTS C'-terminal domain may be applicable in the disclosed uses and methods.
  • the ARTS C terminus peptide applicable in the disclosed uses and methods may comprise the amino acid sequence of: YGPSLRLLAPPGAVKGTGQEHQGQGCH, as denoted by SEQ ID NO. 14.
  • such ARTS fragments or peptides may comprise any peptide derived from the ARTS C terminal 27 amino acid residues.
  • such peptides applicable in the disclosed uses and methods may comprise the amino acid sequence of any one of YGPSLRLLA, as denoted by SEQ ID NO. 15, PPGAVKGTG, as denoted by SEQ ID NO. 16, and QEHQGQGCH, as denoted by SEQ ID NO. 17.
  • ARTS fragments derived from its N-terminus may be used in the disclosed uses and methods, as described above.
  • such ARTS fragments may include ARTS BH3-like domain.
  • such BH3-like domain ARTS fragments may comprise the amino acid sequence of residues 1-128, 1-148, 106-148, 106-133, 106-128, 112-148, 112- 133 and 112-128 of ARTS N'-terminus, as denoted by any one of SEQ ID NO. 18 to 25, respectively.
  • the upregulation of p53 levels may comprise reduction of p53 ubiquitylation by at least one E3 ligase.
  • XIAP serves as an E3 ligase for p53, thereby leading to ubiquitylation and UPS mediated degradation thereof.
  • ARTS, fragments thereof, as well as any mimetic compounds thereof, specifically, the "B3" and the “A4" compounds of the present disclosure lead to UPS degradation of XIAP, and in some embodiments, the UPS mediated degradation of other E3 ligases, thereby inhibiting and attenuating ubiquitylation of p53, that results in upregulation of p53 levels.
  • ARTS, fragments thereof and/or any mimetic compounds thereof, specifically, the "B3 compound” are provided in the present disclosure for use in methods, compositions and kits for upregulating p53 levels.
  • the upregulation is a result of UPS-mediated induction of degradation of E3 ligases such as XIAP and/or MDM2.
  • E3 ligases such as XIAP and/or MDM2.
  • the down regulation in E3 ligases results in a clear decrease in p53 ubiquitylation, thereby upregulating the levels of p53.
  • ubiquitin proteasome system denotes a multi component system that identifies and degrades unneeded, damaged or misfolded proteins by breaking peptide bonds (proteolysis) of the protein in the cytoplasm of cells.
  • degradation of a protein via the UPS involves two discrete and successive steps. In the first step, proteins are tagged for degradation with a small protein called ubiquitin. The tagging reaction is catalyzed by enzymes called ubiquitin ligases. Once a protein is tagged with a single ubiquitin molecule, this is a signal to other ligases to attach additional ubiquitin molecules.
  • conjugation of ubiquitin a highly evolutionarily conserved 76 amino acid residue polypeptide, to the protein substrate proceeds via a three-step cascade mechanism involving El , E2 and E3 enzymes.
  • ubiquitin moieties By successively adding activated ubiquitin moieties to internal lysine residues on the previously conjugated ubiquitin molecule, a polyubiquitin chain is synthesized that is subsequently recognized by the downstream 26S proteasome complex.
  • protease complex a large, protease complex, referred to as the 26S proteasome that does not recognize nonmodified substrates.
  • the proteasomes are multicatalytic protease protein complexes found in all cells that degrades polyubiquitinated proteins to short peptides by breaking peptide bonds (proteolysis). Following degradation of the substrate, short peptides derived from the substrate are released, along with reusable ubiquitin.
  • ubiquitin-proteasome system plays a central and complex role in regulating apoptosis by directly targeting key cell death proteins, including caspases.
  • E3 ligases are the largest and most studied group of the Ubiquitin Proteasome System (UPS), which is responsible for the regulated degradation of intracellular proteins.
  • UPS Ubiquitin Proteasome System
  • Ubiquitylation is the post -translational conjugation of a ubiquitin protein, which tags proteins destined for degradation via the 26S proteasome
  • the ubiquitylation cascade requires a ubiquitin activating enzyme (El), ubiquitin conjugating enzymes (E2) and the ubiquitin ligases (E3).
  • the El enzyme forms an ATP-dependent thioester linkage with the carboxyl-tenninus of ubiquitin; El is not substrate-specific.
  • the E2 enzyme receives the activated ubiquitin from El, which in turn transfers the ubiquitin to the E3 ligase.
  • Different E2 enzymes can regulate a single E3 ligase.
  • the E3 ligases are substratespecific and are essential for the final transfer of the activated ubiquitin from the E2 enzyme to the lysine residue onto the target protein.
  • E3 ligases can be classified into three major groups: (1) the RING E3 ligase family (largest group), (2) the HECT family (homologous to Human Papilloma virus E6 Carboxyl Terminal domain), and (3) the RBR (RING between RING fingers) E3 ligase family.
  • such E3 ligase may be XIAP.
  • by downregulating the levels of XIAP, specifically, increasing the UPS mediated degradation of XIAP, the ARTS, fragments thereof and the ARTS mimetic compounds, specifically the "B3" compound as disclosed herein after, used by the present disclosure lead to upregulation of p53.
  • the inventors assume that ARTS, ARTS fragments and the ARTS mimetic compounds disclosed herein may affect p53 activity also by translocating p53 into the nucleus ( Figure 3) and/or by other p53-dependent signaling pathway means.
  • ARTS binds to XIAP through a domain comprising 27 residues covering the C-terminus of ARTS. This interaction induces auto degradation of XIAP.
  • the ARTS mimetic compound/s of the invention target BIR3 domain of XIAP mimicking the ability of ARTS to enhance XIAP degradation.
  • IAPs denotes a family of proteins that harbor between one to three copies of a baculovirus IAP repeat (BIR) domain that enable interaction with activated caspases. It was previously suggested that the BIR domains of certain IAPs, in particular XIAP, have the ability to directly inhibit caspase activity in vitro.
  • BIR baculovirus IAP repeat
  • X-linked inhibitor of apoptosis protein also known as inhibitor of apoptosis protein 3 (IAP3) and baculoviral IAP repeat-containing protein 4 (BIRC) denotes a protein known to stop an apoptotic process and thus inhibit cell death.
  • XIAP is produced by a gene named XIAP gene located on the X chromosome.
  • XIAP is also called human lAP-like Protein (hILP), because it is not as well conserved as the human IAPS: hIAP-1 and hIAP-2 XIAP are the most potent human IAP proteins currently identified.
  • XIAP belongs to a family of apoptotic suppressor proteins. Members of this family share a conserved motif termed, baculovirus IAP repeat (BIR domain), which is necessary for their anti-apoptotic function. XIAP acts as a direct caspase inhibitor by directly binding to the active site pocket of CASP3 and CASP7 and obstructing substrate entry. It further inactivates CASP9 by keeping it in a monomeric, inactive state.
  • BIR domain baculovirus IAP repeat
  • the invention relates to the human XIAP protein (GenBank Accession Nos. NP_001158, NP_001191330, as denoted by SEQ ID NO. 9) encoded by the XIAP gene (GenBank Accession Nos. NM_001167, NM_001204401, as denoted by SEQ ID NO. 10).
  • the ARTS, fragments thereof and the ARTS mimetic compounds, specifically the "B3" compound, used by the present disclosure may lead to down-regulation of MDM2 levels, specifically, via UPS, thereby leading to upregulation of p53 levels.
  • MDM2 refers to the human MDM2, that is an E3 ligase that acts on p53.
  • MDM2 is the human MDM2, that comprises the amino acid sequence as denoted by UNIPROT accession number Q00987.
  • MDM2, as used herein may be the human MDM2, encoded by a nucleic acid sequence comprising the sequence as denoted by GeneBank accession number NM_001145337.3.
  • the MDM2 as disclosed herein may comprise the amino acid sequence as denoted by SEQ ID NO. 12, or any variants or derivatives thereof.
  • the human MDM2 as disclosed herein may be encoded by a nucleic acid sequence comprising the nucleic aid sequence as denoted by SEQ ID NO. 13.
  • the present invention relates to the ARTS mimetic compounds of the invention that act as antagonist/s of XIAP and other E3 ligases (e.g., MDM2).
  • An antagonist is a compound that competes with a specific protein, a ligand for example, on binding to another protein, a receptor for example. Such binding usually, induces a specific biological response or action that is blocked by the competing antagonist.
  • Antagonists have affinity but no efficacy for their cognate binding protein and binding will disrupt the interaction and inhibit the function of such cognate protein.
  • XIAP degradation and optionally, any other relevant E3 ligase
  • ARTS fragments thereof and the ARTS mimetic compound/s of the present disclosure
  • inhibit the anti-apoptotic effect of XIAP protein The terms “inhibition”, “moderation” or “attenuation” as referred to herein, relate to the retardation, restraining or reduction of the anti-apoptotic activity of a the XIAP protein, and specifically, the E3 ligase activity thereof on p53.
  • Such inhibition may be of about 1% to 99.9%, specifically, about 1% to about 95%, about 5% to 90%, about 10% to 85%, about 15% to 80%, about 20% to 75%, about 25% to 70%, about 30% to 65%, about 35% to 60%, about 40% to 55%, about 45% to 50%. More specifically, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ,99.9% or 100%.
  • ARTS ARTS, fragments thereof and/or the ARTS mimetic compounds of the present disclosure upregulate p53, and/or induce or enhances apoptosis.
  • the ARTS, fragments thereof and/or the ARTS mimetic compounds of the present disclosure, specifically, B3, as well as any of the compositions and methods of the invention described herein after may lead to upregulation, an increase, enhancement, induction or elevation in p53 levels in the treated cells, and/or in apoptosis of treated cells
  • the upregulation, increase, induction or elevation of apoptosis may be an increase by about 1% to 99.9%, specifically, about 1% to about 95%, about 5% to 90%, about 10% to 85%, about 15% to 80%, about 20% to 75%, about 25% to 70%, about 30% to 65%, about 35% to 60%, about 40% to 55%, about 45% to 50%.
  • 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, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 etc., respectively.
  • the ARTS mimetic compound for use in the preset disclosure is a compound having the general formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof, wherein formula (I) is: wherein
  • R 1 , R2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is L 1 '-R 3 '-L 1 ”-R 3 ” and R2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R 2 is L 2 '-R 4 '-L 2 ”-R 4 ”; or
  • R 1 is L 1 '-R 3 '-L 1 ”-R 3 ” and R 2 is L 2 '-R 4 '-L 2 ”-R 4 ”; wherein R 3 ’, R 3 ”, R4’, R4” is each independently from each other, absent or H, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, aryl, arylalkyl, heteroaryl, heterocycloalkyl, a ring system containing five to twelve atoms, each optionally substituted;
  • the ARTS mimetic compound for use is having the general formula (II)
  • the ARTS mimetic compound for use in accordance with the present disclosure is a compound having the general formula (lib):
  • the R1 is L1’-R 3 ’-L1”-R 3 ”.
  • the R1 is at least one of:
  • L1’, L1”and R 3 ” are each absent and R 3 ’ is an optionally substituted
  • the ARTS mimetic compound for use of the present disclosure is a compound having the general formula (IIIc), (Illd) or (Ille):
  • L1 and R3 are each as defined above, wherein R is one or more of H, OH,
  • the ARTS mimetic compound for use in accordance with the present disclosure is a compound having the general formula (life), or (life):
  • the ARTS mimetic compound for use in accordance with the present disclosure is a compound having the formula (3.1), (3.2), (3.3); N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide ;
  • the present disclosure provides the use of 1 ,2-di-carbonyl compounds that serve herein as ARTS mimetics, in upregulating p53 levels.
  • the present disclosure provides a compound comprising at least one oxalamide moiety.
  • the present disclosure also encompasses pharmaceutically acceptable salt, solvate, hydrate or any stereoisomer of the compounds described herein.
  • the present disclosure provides the use of a compound having the general formula (I): or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof; wherein
  • R 1 , R 2 are each, independently from each other, absent or H, alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is L 1 ’-R 3 ’-L 1 ”-R 3 ” and R 2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R 2 is L 2 '-R 4 '-L 2 ”-R 4 ”; or
  • each one of L1’, L1”, L2’ and L2” is each independently from each other, may be optionally substituted by one or more of C 1 -C 5 alkoxy, C 1 -C 5 carboxylic acid, -(CH 2 ) m -OH, -(CH 2 ) m -SH, -(CH 2 ) m -NH 2 , -(CH 2 ) m -halogen and m is an integer selected from 0, 1, 2, 3, 4, 5.
  • R 1 , R2 are each, independently from each other, a ring system containing five to twelve atoms.
  • a ring system containing five to twelve atoms may be substituted with one or more substituents, in certain embodiments one, two, three or four substituents as further described herein.
  • R 1 , R2 are each, independently from each other, a monocyclic or polycyclic ring system containing five to twelve atoms, optionally substituted with one or more substitutes.
  • each one of R 3 ’, R 3 ”, R 4 ’ and R4 may be independently from each other a ring system containing five to twelve atoms, each optionally substituted.
  • each one of R 3 ’ , R 3 ” , R 4 ’ , R 4 ” is independently from each other, a monocyclic or polycyclic ring system containing five to twelve atoms, optionally substituted with one or more substitutes.
  • the ring system of each one of R 3 ’ , R 3 ” , R 4 ’ , R 4 ” may be independently from each other an aryl, heteroaryl or aliphatic ring (non- aromatic ring).
  • each one of R 3 ’ , R 3 ” , R 4 ’ , R 4 ” may be independently from each other C 5 -C 12 saturated cycloalkyl, C 5 -C 12 saturated cycloalkylene, C 5 -C 12 aryl, C 5 - C 12 heteroaryl or C 5 -C 12 arylene.
  • the ring system of each one of R 3 ’ , R 3 ” , R 4 ’ , R 4 ” may independently from each other contain at least two carbon atoms and may include at least one heteroatom ring.
  • each one of R 3 ’ , R 3 ” , R 4 ’ , R 4 ” may be independently from each other heteroaryl, heteroarylene, heterocycloalkylene or heterocycloalkyl.
  • each one of R 3 ’ , R 3 ” , R 4 ’ , R 4 ” may be independently from each other C 2 -C 12 heterocycloalkyl ring, C 2 -C 12 heteroaryl or C 2 -C 12 heteroarylene.
  • the heteroatom in a heteroaryl ring may be N, O, S.
  • the heteroatom in a heteroaryl ring may be N, O.
  • the heteroatom in a heteroaryl ring may be N.
  • each one of R 3 ’ , R 3 ” , R 4 ’ , R 4 ” is absent, H, an optionally substituted aryl or an optionally substituted heteroaryl.
  • each one of R 3 ’ , R 3 ” , R 4 ’ , R 4 ” is absent, an optionally substituted aryl or an optionally substituted heteroaryl.
  • each one of R 3 ’ , R 3 ” , R 4 ’ , R 4 ” is absent or may be independently from each other H, an optionally substituted aromatic ring or an optionally substituted heteroaromatic ring.
  • each one of R 3 ’ , R 3 ” , R 4 ’ , R 4 ” is absent or may be independently from each other an aromatic or heteroaromatic five to eleven membered ring.
  • each one of R 3 ’ , R 3 ” , R 4 ’ , R 4 ” is absent or may be independently from each other H, phenyl, 1 -naphthyl, 2-naphthyl, and 4-biphenyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl (furanyl), quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4- thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimid
  • each one of R 3 ’ , R 3 ” , R 4 ’ , R 4 ” is absent or may be independently from each other H, phenyl, 1-methyl-1H-benzo[d]imidazole, benzoimidazole, pyridine, pyrrole, 1 -methyl-1H-imidazole or IH-imidazole.
  • each one L1’, L1”, L2’ and L2” may be substituted by one or more of C 1 -C 5 alkoxy, C 1 -C 5 carboxylic acid, -(CH 2 ) m -OH, -(CH 2 ) m -SH, -(CH 2 ) m -NH 2 , or -(CH 2 ) m -halogen;
  • each one of L2’, L2” may be optionally substituted with -(CH 2 ) m -OH.
  • n may be 0 to 3
  • at times n may be 1 to 3
  • at times n may be 2 to 3
  • m may be 1 to 3.
  • R 1 is L 1 ’-R 3 ’-L 1 ”-R 3
  • L 1 ’ and L 1 are each absent
  • R 3 ’ is an optionally substituted aromatic or heteroaromatic five to eleven membered ring and R 3 ” is absent.
  • R 1 is L 1 ’-R 3 ’-L 1 ”-R 3
  • L 1 ’ and L 1 are each absent
  • R 3 ’ is an optionally substituted aryl or optionally substituted heteroaryl and R 3 ” is absent.
  • R 1 is L 1 ’-R 3 ’-L 1 ”-R 3
  • L 1 ’ and L 1 are each absent
  • R 3 ’ is an optionally substituted phenyl, benzoimidazole, 1-methyl-1H-benzo[d]imidazole, pyridine, pyrrole, 1-methyl-1H-imidazole or IH-imidazole and R 3 ” is absent.
  • R 1 is L 1 ’-R 3 ’-L 1 ”-R 3
  • L 1 ’ and L 1 are each absent
  • R1 is L1’-R 3 ’-L1”-R 3
  • L1’ and L1 are each absent
  • R1 is L1’-R 3 ’-L1”-R 3
  • L1’ and L1 are each absent
  • R 3 ’ is 1 -methyl- lH-benzo[d] imidazole, optionally substituted with halogen, or CF 3
  • R3 is absent.
  • R1 is L1’- R 3 ’-L1”- R 3 ”, L1’ and L1”are each absent, R 3 ’ is 1 -methyl- lH-benzo[d] imidazole, optionally substituted with, CF 3 , and R3” is absent.
  • R 1 is L1’- R 3 ’-L1”-R 3
  • L1’ is absent
  • R1 is L1’-R 3 ’-L1”-R 3
  • L1’ is absent
  • R1 is L1’-R 3 ’-L1”-R 3
  • L1’ is absent
  • R1 is L1’- R 3 ’-L1”- R 3
  • L1’ is absent
  • R1 is L1’- R 3 ’-L1”- R 3
  • L1’ is absent
  • R1 is L1’- R 3 ’-L1”- R 3
  • L1’ is absent
  • R1 is L1’- R 3 ’-L1”- R 3
  • L1’ is absent
  • the compound of the present disclosure having general formula (I) have the general formula (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (li), (Ij) (Ik) or (II):
  • R3 is an optionally substituted aryl or an optionally substituted heteroaryl.
  • R3 is an optionally substituted 1 -methyl-1H-imidazole.
  • R3 is 1 -methyl-1H-imidazole.
  • R 2 is L2’-R 4 ’-L2”-R4”.
  • L2 is an optionally substituted -(CH 2 ) n , n is an integer selected from 1, 2, 3, 4, 5, R4’ is an optionally substituted aryl or an optionally substituted heteroaryl and L2” and R4” are each absent.
  • L2 is an optionally substituted -(CH 2 ) n , n is an integer selected from 1, 2, 3, 4, 5, R4’ is an optionally substituted phenyl and L2” and R4” are each absent.
  • L2 is an optionally substituted -(CH 2 ) 2
  • R4’ is an optionally substituted aryl or an optionally substituted heteroaryl
  • L2” and R4” are each absent.
  • L2 is an optionally substituted -(CH 2 ) 2
  • R4’ is an optionally substituted phenyl and L2” and R4” are each absent.
  • R2 is L2’-R4’-L2”-R4
  • L2’ is -(CH 2 ) 2 substituted with OH
  • R4’ is an optionally substituted aryl or an optionally substituted heteroaryl and L2” and R4” are each absent.
  • R2 is L2’-R4’-L2”-R4
  • L2’ is -(CH 2 ) 2 substituted with OH
  • R4’ is an optionally substituted phenyl and L2” and R4” are each absent.
  • R4 is a phenyl optionally substituted with OH.
  • a compound of the invention has the general formula (II), L2’ is (CH 2 )n-, optionally substituted by one or more of C 1 -C 5 alkoxy, C 1 -C 5 carboxylic acid, -(CH 2 ) m -OH, -(CH 2 ) m -SH, -(CH 2 ) m -NH 2 , or -(CH 2 ) m -halogen, n is an integer selected from any one of 0, 1, 2, 3, 4, 5 and m is an integer selected from 0, 1, 2, 3, 4, 5.
  • a compound of the invention has the general formula (II), L2’ is (CH 2 )n-, substituted by OH, n is an integer selected from any one of 0, 1, 2, 3, 4, 5.
  • a compound of the invention has the general formula (II), L2’ is (CH 2 ) n -, substituted by OH, n is 2.
  • a compound of the invention has the general formula (Ila) or (lib):
  • R1 is L1’- R 3 ’-L1”- R 3 ” as defined above.
  • R1 is L1’- R 3 ’-L1”- R 3 ” as defined above.
  • R1 is L1’- R 3 ’-L1”- R 3 ”, L1’, L1”and R 3 ” are each absent and R 3 ’ is an optionally
  • R1 is L1’- R 3 ’-L1”- R 3 ”, L1’, L1”and R 3 ” are each absent and R 3 ’ is an optionally substituted: ring linked to formula (I), (II), (Ila), or (lib).
  • R1 is L1’- R 3 ’-L1”- R 3 ”, L1’, L1”and R 3 ” are each absent and R 3 ’ is: , y ne indicate bond to formula (I), (II), (Ila), or (lib).
  • R1 is L1’-R 3 ’-L1”-R3
  • R 3 ’ is an optionally substituted phenyl
  • R 3 ” is an optionally substituted:
  • R1 is L1’-R 3 ’-L1”-R 3 ”, L1’is absent R 3 ’ is an optionally substituted phenyl, R 3 ” is an optionally substituted:
  • specific examples of compounds or pharmaceutically acceptable salts or hydrates of the compounds of Formula I, II include, without limitation: N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(1-methyl-2-(trifluoromethyl)-1H- benzo [d] imidazol-5 -yl)oxalamide
  • the present disclosure provides 1,5-di-carbonyl compounds.
  • the compounds contain one or more nitrogen atoms.
  • the compounds contain at least one ring structure.
  • the ring structure containing at least one nitrogen atom.
  • the compounds contain a heterocyclic ring structure containing five to twelve atoms, the ring structure containing at least two carbon atoms and at least one heteroatom being N, O or S.
  • the present disclosure provides in accordance with the second aspect, a compound having the general formula (VIII): or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof ; wherein Ro and R 10 may be the same or may be different and may be independently selected from each other from a ring system containing five to twelve atoms, each optionally substituted;
  • Rn may be independently selected from H, straight or branched C 1 -C 12 alkyl, straight or branched C 2 -C 12 alkenyl, straight or branched C2-Ci2alkynyl;
  • each one of R 9 and R 10 being a ring system containing five to twelve atoms may be substituted with one or more substituents, in certain embodiments one, two, three or four substituents,
  • R9 and R 10 may be a ring system containing five to twelve atoms. In some further embodiments, the ring system of R9 and R 10 may contain at least two carbon atoms. In some other embodiments, the ring system of R9 and R 10 may be an aromatic ring, non-aromatic ring, fused ring or the like. In some further embodiments, R9 and R 10 may be C 5 -C 12 saturated cycloalkyl, C 5 -C 12 saturated cycloalkylene, C 5 -C 12 aryl or C 5 -C 12 arylene. In some other embodiments, the ring system of R9 and R 10 may include at least one heteroatom ring.
  • R9 and R 10 may be heteroaryl or heterocycloalkyl. In some further embodiments, R9 and R 10 may be C 2 -C 12 hetero cycloalkyl or C 2 -C 12 hetero aromatic ring (aryl or arylene). It should be noted that according with some embodiments, R9 and R 10 may be independently from each other contain different carbon atoms. In some embodiments, the heteroatom may be N, O, S. In some embodiments, R 9 and R 10 may be independently from each other selected from C 5 -C 12 aryl. In some embodiments, R9 and R 10 may be C6 aryl, optionally substituted. In some embodiments, L5 and L6, may be -(CH 2 ) P -.
  • the present disclosure provides compounds containing one or more nitrogen atoms.
  • the compounds contain at least one ring structure.
  • the ring structure containing at least one nitrogen atom.
  • the compounds contain a heterocyclic ring structure containing five to twelve atoms, the ring structure containing at least two carbon atoms and at least one heteroatom being N, O or S. in some other embodiments, the compounds contain an amide group.
  • the present disclosure provides a compound having a general formula: or a pharmaceutically acceptable salt or hydrate thereof; wherein:
  • X, Y independently may be each independently selected from each other from NH, CH;
  • R 14 and R 15 may be the same or may be different and may be independently from each other selected from a ring system containing five to twelve atoms, each optionally substituted;
  • R 1 4 and R 1 s may be a ring system containing five to twelve atoms.
  • the ring system of R 14 and R 15 may contain at least two carbon atoms.
  • the ring system of R 14 and R 15 may be an aromatic ring or a non-aromatic ring.
  • R 14 and R 15 may be C 5 -C 12 saturated cycloalkyl, C 5 -C 12 saturated cycloalkylene, C 5 -C 12 aryl or C 5 -C 12 arylene.
  • the ring system of R 14 and R 15 may include at least one heteroatom ring.
  • R 14 and R 15 may be C 2 -C 12 hetero cycloalkyl or C 2 -C 12 hetero aromatic ring. It should be noted that according with some embodiments, R 14 and R 15 may be independently from each other contain different carbon atoms. In some embodiments, the heteroatom may be N, O, S. In some embodiments,
  • R 14 and R 15 may be independently from each other selected from C 5 -C 12 aryl optionally substituted. In some embodiments, R 14 and R 15 may be independently from each other selected from isoquinoline or phyel each independently from the other optionally substituted.
  • L8 may be -(CH 2 ) q -.
  • each of q may be 0.
  • L8 may be a bond.
  • a ring system containing five to twelve atoms may be substituted with one or more substituents, in certain embodiments one, two, three or four substituents.
  • each variable can be a different moiety selected from the Markush group defining the variable.
  • the two R groups can represent different moieties selected from the Markush group defined for R.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, as used herein refers to a linear (straight), branched saturated hydrocarbon and can have a number of carbon atoms optionally designated (i.e., C 1 -C 6 means one to six carbons).
  • C1-C12 alkyl or "C1-C12 alkylene” refers to a linear (straight), branched saturated hydrocarbon having from 1 to 12 carbon atoms, in some embodiments, contain from 2 to 8 carbons, in yet some embodiments from 2 to 5 carbons, in yet some further embodiments, from 1 to 3 carbon atoms. It should be noted that alkyl refers to an alkyl end chain and alkylene refers to a middle chain alkyl.
  • Representative C 1 -C 12 alkyl groups include, but are not limited to, methyl, ethyl, propyl, n-propyl, isopropyl, cyclopropyl, n- butyl, butyl, sec-butyl, iso-butyl, tert-butyl, cyclobutyl, n-pentyl, pentyl, iso-pentyl, neopentyl, tert-pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, cycloheptyl, octyl, sec-octyl (1 -methylheptyl), and cyclooctyl as well as homologs and isomers of, for example, n- pentyl, n-hexyl, and the like.
  • haloalkyl as used herein can include alkyl structures that are substituted with one or more halo groups or with combinations thereof, for example, “C1-C12 haloalkyl” refers to a C 1 -C 12 alkyl as defined above, with one or more hydrogens substituted by halogen atoms.
  • alkenyl refers to a linear (straight), branched unsaturated hydrocarbon having from 2 to 20 carbon atoms and at least one carbon-carbon double bond.
  • C 2 -C 12 alkenyl or "C 2 -C 12 alkenylene'' as used herein refers to a linear, branched unsaturated hydrocarbon having from 2 to 12 carbon atoms and at least one carbon-carbon double bond, in some embodiments from 3 to 8 carbons, in yet some further embodiments, from 3 to 5 carbon atoms and at least one double bond.
  • alkenyl refers to an alkyl end chain and alkenylene refers to a middle chain alkyl.
  • alkenyl groups include, but are not limited to, groups such as ethenyl (i.e., vinyl), prop-l-enyl (i.e., allyl), but-l-enyl, pent-l-enyl, penta- 1,4-dienyl, and the like.
  • C 2 -C 12 haloalkenyl refers to a C 2 -C 12 alkenyl as defined above, with one or more hydrogens substituted by halogen atoms.
  • alkynyl refers to a linear (straight), branched unsaturated hydrocarbon having from 2 to 20 carbon atoms and at least one carbon-carbon triple bond.
  • C 2 -C 12 alkynyl or “C 2 -C 12 alkynylene” as used herein refers to a linear, branched unsaturated hydrocarbon having from 2 to 12 carbon atoms in certain embodiments, from 3 to 8 carbons, and at least one triple bond (at least one carbon-carbon triple bond).
  • alkynyl refers to an alkyl end chain and alkynylene refers to a middle chain alkyl.
  • alkynyl groups include, but are not limited to, groups such as ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.
  • C 2 -C 12 haloalkynyl refers to a C 2 -C 12 alkynyl as defined above, with one or more hydrogens substituted by halogen atoms.
  • alkoxy refers to an alkyl group bonded to an oxygen atom.
  • C 1 -C 12 alkoxy!” as used herein refers to a C 1 -C 12 alkyl group linked to an oxygen.
  • the alkyl group may include one to twelve carbon atoms, at times between one to eight carbon atoms, at times one to five carbon atoms and at times one to three carbon atoms.
  • alkoxy groups include, but are not limited to, groups such as methoxy, ethoxy, propyloxy, isopropyloxy, butyloxy, isobutyloxy, tert-butyloxy, pentyloxy, or hexyloxy, and the like.
  • halo or “halogen” (halide) independently or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • halide by itself or as part of another substituent, refers to a fluoride, chloride, bromide, or iodide atom.
  • a ring system containing five to twelve atoms refers to a mono- or multi- cyclic ring system having 5 to 12 atoms.
  • the ring system containing five to twelve atoms may be saturated, unsaturated or aromatic rings and the like including for example cycloalkyl, heterocycloalkyl, aryl, arylene, aromatic, heteroaromatic rings.
  • a ring system containing five to twelve atoms may contain two rings (bicyclic, etc.), for example aromatic rings and in such case the aromatic rings of the aryl group may be joined at a single point (e.g., biphenyl), or fused (e.g., naphthyl).
  • a ring system containing five to twelve atoms is a carbocyclic ring or heterocyclic ring.
  • the term “carbocyclic ring” refers to cyclic compounds containing only carbon atoms.
  • the carbocyclic ring may be optionally substituted by one or more substituents, and may be saturated, unsaturated or aromatic.
  • the term “heterocyclic ring” refers to cyclic compounds where one or more carbons are substituted by heteroatoms. Exemplary heteroatoms include, but not limited to, nitrogen, sulfur, and oxygen.
  • the heterocyclic ring may be optionally substituted, and may be saturated, unsaturated or aromatic.
  • saturated' as used herein means that the compound does not contain double or triple bonds.
  • the term “unsaturated' as used herein means that the compound contains at least one double or triple bond.
  • aromatic as used herein means that the compound contains alternating double and single bonds.
  • aryl refers to polyunsaturated, aromatic ring systems having between 5 to 12 atoms which can be a single ring or multiple rings (e.g., 1 to 2 rings) which are fused together or linked covalently.
  • aryl refers to cyclic, aromatic hydrocarbon groups that have 1 to 2 aromatic rings, including monocyclic or bicyclic groups having between 5 to 12 atoms. Non-limiting examples include phenyl, biphenyl or naphthyl.
  • the aryl group may be optionally substituted by one or more substituents, e.g., 1 to 5 substituents, at any point of attachment. The substituents can themselves be optionally substituted.
  • C 5 -C 12 aromatic refers to aromatic ring systems having 5 to 12 carbon atoms, such as phenyl, naphthalene and the like.
  • heteroaryl refers to aryls as defined above where one or more carbons are substituted by heteroatoms.
  • exemplary heteroatoms include, but not limited to, nitrogen, sulfur, and oxygen.
  • heteromatic refers to refers to a monocyclic or multi-cyclic (fused) aromatic ring system, where one or more of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur.
  • heteroaryl used interchangeably with the term “heteroaryl” denotes a heterocyclic aromatic ring systems containing 5 to 12 atoms, with at least one, preferably two carbon atoms and one or more heteroatoms selected from nitrogen, oxygen and sulfur.
  • Non-limiting examples include furan, thipohene, pyrrole, oxazole, thiazole, imidazole, pyrazole, isoxazole, thiazolem benzofurna, indole, benzothiophene, benzoimidazole, indazole, benzoxazole, benzoisoxazole, benzothiazole, isobenzfuran, isoidole, purine, pyridine, pyrazine, pyrimidine, pyrisazine, quinoline, quinozaline, quinazoline, isoquinoline, furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4- triazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl,
  • C 5 -C 12 saturated cycloalkyl refers to a saturated mono- or multi- cyclic ring system having 5 to 12 carbon atoms, preferably having 5 to 7 carbon atoms.
  • Example of "C 5 -C 12 cycloalkyl” groups include, but are not limited to cyclopentyl, cyclohexyl and cycloheptyl.
  • heterocycloalkyl or “heterocyclyl” or the term “heterocyclic” refers to a monocyclic or multi-cyclic non-aromatic ring system having 5 to 12 members, preferably having 5 to 7 carbon atoms, where one or more, in certain embodiments, 1 to 3, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur.
  • heteroalkyl examples include, but are not limited to, pyran, 1,4-dioxane, 1,3-dioxane, piperidine, pyrrolidine, morpholine, tetrahydrothiopyran, tetrahydrothiophene, and the like.
  • heterocycloalkyl also encompasses non-aromatic ring being unsaturated or having one or more degrees of unsaturation containing one or more heteroatomic substitutions selected from S, SO, SO2, O, or N.
  • heterocyclic ring(s) or cycloalkyl ring(s).
  • heterocyclic examples include, but are not limited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3- dioxane, piperidine, pyrrolidine, morpholine, tetrahydrothiopyran, tetrahydrothiophene, and the like.
  • N-containing group is used herein a chemical group containing a nitrogen atom for example as amino group.
  • amino as used herein encompass primary, secondary, tertiary or quaternary amines where the point of attachment is through the nitrogen atom which is substituted.
  • the "N-containing group” include N, NH, NH2, tertiary amine (tertiary alkyl amine), quaternary ammonium (quaternary alkyl ammonium).
  • the nitrogen atom may be substituted with alkyl.
  • the substituent may be the same or may be different.
  • bonds denotes a covalent bond.
  • the bond may be between two similar atoms or between different atoms.
  • Non-limiting examples include C-C, C-S, C- O, C-N. S-O, S-N, N-0 and the like. It should be noted that a bond as defined above, for example, C-S encompasses both C-S and S-C and this holds for the bonds as defined herein.
  • substituted refers to substitution with the named substituent or substituents, multiple degrees of substitution being allowed unless otherwise stated.
  • each R a is independently hydrogen or alkyl.
  • carbon number refers to the carbon backbone and carbon branching, but does not include carbon atoms of the substituents, such as alkoxy substitutions and the like.
  • the invention also embraces solvates, pharmaceutically acceptable prodrugs, pharmaceutically active metabolites, and pharmaceutically acceptable salts of compounds of the formula (I) or any variations detailed herein.
  • the present disclosure also includes any or all of the stereochemical forms, including any enantiomeric or diastereomeric forms, and any tautomers or other forms of the compounds described.
  • solvate refers to an aggregate of a molecule with one or more solvent molecules, such as hydrate, alcoholate (aggregate or adduct with alcohol), and the like.
  • salts refers to salts derived from organic and inorganic acids of a compound described herein.
  • Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, hydrochloride, bromide, hydrobromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, camphorsulfonate, napthalenesulfon
  • pharmaceutically acceptable salt also includes hydrates of a salt of a compound described herein.
  • hydrate refers to a compound formed by the addition of water. The hydrates may be obtained by any known method in the art by dissolving the compounds in water and recrystallizing them to incorporate water into the crystalline structure.
  • the compounds of the present invention may have the ability to crystallize in more than one form, a characteristic, which is known as polymorphism, and it is understood that such polymorphic forms (“polymorphs”) are within the scope of formulae (I).
  • Polymorphism generally can occur as a response to changes in temperature or pressure or both and can also result from variations in the crystallization process. Polymorphs can be distinguished by various physical characteristics known in the art such as x-ray diffraction patterns, solubility, and melting point.
  • the compounds described herein may contain one or more chiral atoms, or may otherwise be capable of existing as two enantiomers or as two or more diastereomers. Accordingly, the compounds of this invention include mixtures of enantiomers as well as purified enantiomers or enantiomerically enriched mixtures. Furthermore, the compounds of this invention include mixtures of diastereomers, as well as purified stereoisomers or diastereomerically enriched mixtures. Also included within the scope of the invention are the individual isomers of the compounds of the invention, as defined above, as well as any wholly or partially mixtures thereof. The present invention also covers the individual isomers of the compounds represented by the formulas above as mixtures with isomers thereof in which one or more chiral centers are inverted.
  • stereoisomer as used herein is meant to encompass an isomer that possess identical constitution as a corresponding stereoisomer, but which differs in the arrangement of its atoms in space from the corresponding stereoisomer.
  • stereoisomers may be enantiomers, diastereomers and/or cis-trans (E/Z) isomers.
  • a composition comprising a fatty acid amide of the invention may comprise single enantiomers, single diastereomers as well as mixtures thereof at any ratio (for example racemic mixtures, non racemic mixtures, mixtures of at least two diastereomers and so forth).
  • the invention encompasses any stereoisomer of a fatty acid amide of the invention achieved through in vivo or in vitro metabolism, or by any type of synthetic rout.
  • Certain of the compounds described herein may contain one or more chiral atoms, or may otherwise be capable of existing as two enantiomers or as two or more diastereomers.
  • the compounds of this invention include mixtures of enantiomers as well as purified enantiomers or enantiomerically enriched mixtures.
  • the compounds of this invention include mixtures of diastereomers, as well as purified stereoisomers or diastereomerically enriched mixtures.
  • the individual isomers of the compounds of the invention as defined above, as well as any wholly or partially mixtures thereof.
  • the present invention also covers the individual isomers of the compounds represented by the formulas above as mixtures with isomers thereof in which one or more chiral centers are inverted.
  • the compounds of the present invention may form tautomers. It is understood that all tautomers and mixtures of tautomers of the compounds of the present invention, are included within the scope of the compounds of the present invention.
  • solvate refers to an aggregate of a molecule with one or more solvent molecules, such as hydrate, alcoholate (aggregate or adduct with alcohol), and the like.
  • physiologically functional derivative used herein relates to any physiologically acceptable derivative of a compound as described herein.
  • the physiologically functional derivatives also include prodrugs of the compounds of the invention.
  • prodrugs may be metabolized in vivo to a compound of the invention.
  • These prodrugs may or may not be active themselves and are also an object of the present invention.
  • a “pharmaceutically acceptable prodrug” is a compound that may be converted under physiological conditions to the specified compound or to a pharmaceutically acceptable salt of such compound.
  • a “pharmaceutically active metabolite” is a pharmacologically active product produced through metabolism in the body of a specified compound or salt thereof. Metabolites of a compound may be identified using routine techniques known in the art and their activities determined using tests such as those described herein. Prodrugs and active metabolites of a compound may be identified using routine techniques known in the art. It should be noted that in some embodiments, the ARTS mimetic compound used in the methods, composition and kits of the present disclosure may comprise a compound having the structure of formula (3.2). In some embodiments, this compound (as well as derivatives thereof may be referred to herein as "B3" or "B3 ARTS mimetic compound”.
  • the present disclosure provides the uses, compositions and methods of any ARTS mimetic compound.
  • the present disclosure provides the uses of different ARTS mimetic compounds that specifically mimic the C domain of ARTS, specifically in binding thereof to its binding site within the BIR3 domain of XIAP.
  • the ARTS mimetic compound used in the methods, kits and compositions disclosed herein may be a compound having the Formula (X), or any analogs or derivative thereof including any stereoisomer or salt thereof.
  • the compound may be referred to herein by the present disclosure as “A4" or "ARTS mimetic A4 small molecule” or the like. It should be also noted that when referring to A4, the compound may include any stereoisomer or salt thereof, for example the stereoisomer having the structure:
  • the ARTS or at least one mimetic compound as disclosed herein is for use in a method for upregulating p53 levels in at least one cell of a subject suffering from at least one pathologic disorder.
  • the present disclosure provides ARTS or at least one mimetic compound for use in a method for upregulating p53 levels in at least one cell of a subject suffering from at least one p53-associated disorder.
  • the p53-associated disorder comprises at least one of: at least one proliferative disorder and at least one metabolic disorder.
  • the ARTS or at least one mimetic compound thereof for use in a method as disclosed herein is at least one neoplastic malignant disorder.
  • the present disclosure provides ARTS or at least one mimetic compound thereof for use in a method for upregulating the levels of p53 in at least one cell in a subject suffering from at least one pathologic disorder.
  • the method comprising administering to the subject a therapeutically effective amount of the ARTS or at least one mimetic compound thereof.
  • ARTS As shown herein in the following Examples, the use of ARTS, fragments thereof and/or mimetic compounds thereof, lead to upregulation of p53 levels, and to increased apoptosis, as also reflected by elevation in apoptotic markers such as cleaved caspases and PARP.
  • apoptosis refers to a regulated network of biochemical events which lead to a selective form of cell suicide and is characterized by readily observable morphological and biochemical phenomena. Cells undergoing apoptosis show characteristic morphological and biochemical features.
  • apoptosis is a tightly controlled form of active cell death that is necessary for development and organismal homeostasis. Death by the apoptotic pathway is achieved among others, by the activation of a family of highly potent and specific proteases, termed caspases (for cysteine- aspartate protease).
  • caspases The activity of caspases is tightly regulated and the cell maintains several “checkpoints” to control their activity.
  • the first level of regulation is intrinsic to caspases themselves. Caspases are initially transcribed as weakly active zymogens, which only upon proper stimulation are cleaved to form the active enzyme.
  • the second level of caspase regulation is achieved by inhibitors, namely the family of proteins called IAPS (Inhibitor of Apoptosis Protein) as described above.
  • the preset disclosure encompasses uses of ARTS and any fragments and peptides thereof, in methods for upregulating p53 levels.
  • the present disclosure thus provides peptides and polypeptides, as well as any derivatives and variants thereof.
  • polypeptide refers to amino acid residues, connected by peptide bonds.
  • a polypeptide sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group.
  • amino acid molecule is the order in which amino acid residues connected by peptide bonds, lie in the chain in peptides and proteins.
  • sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing amide.
  • Amino acid sequence is often called peptide, protein sequence if it represents the primary structure of a protein, however one must discern between the terms "Amino acid sequence” or “peptide sequence” and “protein”, since a protein is defined as an amino acid sequence folded into a specific three-dimensional configuration and that had typically undergone post-translational modifications, such as phosphorylation, acetylation, glycosylation, manosylation, amidation, carboxylation, sulfhydryl bond formation, cleavage and the like.
  • Amino acids refer to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine.
  • amino acid analogs refers to compounds that have the same fundamental chemical structure as a naturally occurring amino acid, i.e., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • 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 present disclosure further encompasses any derivatives, analogues, variants or homologues of any of the peptides.
  • 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.
  • derivative it is also referred to homologues, variants and analogues thereof, as well as covalent modifications of a polypeptides made according to the present disclosure.
  • polypeptides according to the disclosure can be produced 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 (either the ARTS protein or any fragment or peptide derived therefrom according to the present disclosure), polypeptides that have deletions, substitutions, inversions or additions.
  • derivatives refer to polypeptides, which differ from the polypeptides specifically defined in the present disclosure 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 present disclosure, 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 present disclosure may occur in any position of the modified peptide, as well as in any of the N' or C termini thereof.
  • the peptides of the present disclosure 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 cyclizations, extension or other chemical modifications.
  • polypeptides of the present disclosure can be coupled (conjugated) through any of their residues to another peptide or agent.
  • the polypeptides of the present disclosure can be coupled through their N-terminus to a lauryl-cysteine (LC) residue and/or through their C-terminus to a cysteine (C) residue.
  • LC lauryl-cysteine
  • C cysteine
  • the peptides 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.
  • 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.
  • the peptides may be extended by aromatic amino acid residue/s, which may be naturally occurring or synthetic amino acid residue/s, for example, a specific aromatic amino acid residue may be tryptophan.
  • 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 peptide may be extended at the N- terminus and/or C- terminus thereof with an N-acetyl group.
  • this disclosure includes the corresponding retro-inverse sequence wherein the direction of the peptide chain has been inverted and wherein all the amino acids belong to the D- series.
  • the present disclosure also encompasses any homologues of the polypeptides (either the ARTS protein or any fragments or peptides thereof) specifically defined by their amino acid sequence according to the present disclosure.
  • the term "homologues” is used to define amino acid sequences (polypeptide) which maintain a minimal homology to the amino acid sequences defined by the present disclosure, e.g.
  • polypeptides preferably have at least about 65%, more preferably at least about 75%, even more preferably at least about 85%, most preferably at least about 95% overall sequence homology with the 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 NO:1 and SEQ ID Nos: 18 to 25.
  • 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.
  • the present disclosure also encompasses polypeptides which are variants of, or analogues to, the polypeptides specifically defined in the present disclosure 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. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues, and alleles and analogous peptides of the present disclosure.
  • substitutions may be made wherein an aliphatic amino acid (G, A, I, L, or V) is substituted with another member of the group, or substitution such as the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.
  • substitutions may be made wherein an aliphatic amino acid (G, A, I, L, or V) is substituted with another member of the group, or substitution such as the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.
  • substitutions may be made wherein an aliphatic amino acid (G, A, I, L, or V) is substituted with another member of the group, or substitution such as the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.
  • substitutions may be made wherein an
  • 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 amino acid 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.
  • nonpolar “hydrophobic” amino acids are selected from the group consisting of Valine (V), Isoleucine (I), Leucine (L), Methionine (M), Phenylalanine (F), Tryptophan (W), Cysteine (C), Alanine (A), Tyrosine (Y), Histidine (H), Threonine (T), Serine (S), Proline (P), Glycine (G), Arginine (R) and Lysine (K); “polar” amino acids are selected from the group consisting of Arginine (R), Lysine (K), Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q); “positively charged” amino acids are selected form the group consisting of Arginine (R), Lysine (K) and Histidine (H) and wherein “acidic” amino acids are selected from the group consisting of Aspartic acid (D), Asparagine (N), Glutamic acid (E) and Glutamine (
  • the peptide compounds of the present disclosure may comprise one or more amino acid residue surrogate.
  • An “amino acid residue surrogate” as herein defined is an amino acid residue or peptide employed to produce mimetics of critical function domains of peptides.
  • amino acid surrogate examples include, but are not limited to chemical modifications and derivatives of amino acids, stereoisomers and modifications of naturally occurring amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically modified amino acids, and the like. Examples also include dimers or multimers of peptides.
  • An amino acid surrogate may also include any modification made in a side chain moiety of an amino acid. This thus includes the side chain moiety present in naturally occurring amino acids, side chain moieties in modified naturally occurring amino acids, such as glycosylated amino acids.
  • the present disclosure further encompass any of the peptides of the present disclosure 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 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, benzensulf onate, p-toluenesulf onate and pamoate (i.e., 1 , l'-methylene- bis-(2 -hydroxy-3 -naphthoate)) salts.
  • Certain compounds of the present disclosure can form pharmaceutically acceptable salts with various amino acids.
  • Suitable base salts include, but are not limited to,
  • the present disclosure further encompasses any peptidomimetic compound mimicking the C'-terminus derived peptides used by the present disclosure.
  • peptidomimetics what is meant is a compound that mimics the conformation and desirable features of a particular natural peptide but avoids the undesirable features, e.g., flexibility and bond breakdown.
  • peptidomimetics can have a structure without any peptide bonds, nevertheless, the compound is peptidomimetic due to its chemical properties and not due to chemical structure.
  • Peptidoinimetics both peptide and non-peptidyl analogues
  • peptidomimetics may or may not have similar two-dimensional chemical structures, but share common three-dimensional structural features and geometry. Each peptidomimetic may further have one or more unique additional binding elements.
  • compositions comprising effective amount of at least one of ARTS, any fragments thereof, at least one mimetic compound thereof or any vehicle, matrix, nano- or micro-particle comprising the same, for use in a method for upregulating p53 levels in a cell.
  • the composition optionally further comprises at least one of pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
  • the present disclosure provides a composition for use in a method for upregulating p53 levels in a cell.
  • the upregulating p53 levels comprises reduction of p53 ubiquitylation by at least one E3 ligase.
  • the ARTS mimetic compound of the composition for use in accordance with the present disclosure is a compound having the general formula (I), wherein formula (I) is: wherein
  • R 1 , R2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is L 1 '-R 3 '-L 1 ”-R 3 ” and R2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R 2 is L 2 '-R 4 '-L 2 ”-R 4 ”; or
  • the ARTS mimetic compound of the composition for use, as disclosed herein is having the general formula (IIIc), or (Ille):
  • the ARTS mimetic compound is having the formula (3.1), (3.2), (3.3); N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide ;
  • compositions of the present invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already affected by any p53-associated disorder, for example, a proliferative disorder (e.g., carcinoma, specifically breast carcinoma), and/or any metabolic disorder in an amount sufficient to cure or at least partially arrest the condition and its complications.
  • a proliferative disorder e.g., carcinoma, specifically breast carcinoma
  • An amount adequate to accomplish this is defined as a “therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the condition and the general state of the patient's own immune system, but generally range from about 0.001 to about 1000 mg/Kg.
  • Single or multiple administrations on a daily, weekly or monthly schedule can be carried out with dose levels and pattern being selected by the treating physician.
  • the administration of the compositions of the invention may be periodic, for example, the periodic administration may be affected twice daily, three time daily, or at least one daily for at least about three days to three months.
  • the advantages of lower doses are evident to those of skill in the art. These include, inter alia, a lower risk of side effects, especially in long-term use, and a lower risk of the patients becoming desensitized to the treatment.
  • treatment using the compositions of the present disclosure may be affected following at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 30, 60, 90 days of treatment, and proceeding on to treatment for life.
  • the effective amount of the disclosed ARTS mimetic compounds, and specifically of the B3 compound may range from about 0.1 ⁇ M to about lOO ⁇ M. Specifically, from 0.5 ⁇ M to about 100 ⁇ M, from 1 ⁇ M to about 100 ⁇ M, 1 ⁇ M to 90, 1 ⁇ M to 80 ⁇ M, 1 ⁇ M to 70 ⁇ M, 1 ⁇ M to 80 ⁇ M, 1 ⁇ M to 70 ⁇ M, 1 ⁇ M to 60 ⁇ M, 1 ⁇ M to 50 ⁇ M, 1 ⁇ M to 40 ⁇ M, 1 ⁇ M to 30 ⁇ M, 1 ⁇ M to 20 ⁇ M, 1 ⁇ M to 10 ⁇ M, specifically, 1 ⁇ M or less, 2, 3, 4, 5, 6, 7., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ⁇ M or more. In some embodiments, an effective amount of the "B3" compound may be 20 ⁇ M.
  • p53-associated disorders e.g., proliferative conditions and/or metabolic disorders
  • treatment of different p53-associated disorders may indicate the use of different doses or different time periods, these will be evident to the skilled medical practitioner.
  • the compositions of the invention may include a prophylactic effective amount of the active ingredient.
  • prophylactically effective amount is intended to mean that amount of a pharmaceutical composition that will prevent or reduce the risk of occurrence or recurrence of the biological or medical event that is sought to be prevented in a tissue, a system, animal or human by a researcher, veterinarian, medical doctor or other clinician.
  • the compositions of the invention are administered to a patient who is at risk of developing the disease state to enhance the patient's resistance. Such an amount is defined to be a “prophylactically effective dose”. In this use, the precise amounts again depend upon the patient's state of health and general level of immunity, but generally range from 0.001 to 1000 mg per dose.
  • the ARTS, functional fragments thereof, and ARTS mimetic compounds of the present disclosure may be formulated in any vehicle, matrix, nano- or micro-particle, or composition.
  • formulations of the ARTS mimetic compounds adapted for use as a nano- or microparticles.
  • Nanoscale drug delivery systems using micellar formulations, liposomes and nanoparticles are emerging technologies for the rational drug delivery, which offers improved pharmacokinetic properties, controlled and sustained release of drugs and, more importantly, lower systemic toxicity.
  • a particularly desired solution allows for externally triggered release of encapsulated compounds.
  • NP nanoparticle
  • DDS Controlled drug delivery systems
  • nanostructures including micellar formulations, liposomes, polymers, dendrimers, silicon or carbon materials, polymeric nanoparticles and magnetic nanoparticles, as carriers in drug delivery systems.
  • nanostructure or “nanoparticle” is used herein to denote any microscopic particle smaller than about 100 nm in diameter.
  • the carrier is an organized collection of lipids.
  • micellar formulations or liposomes it is to be understood to mean any biocompatible lipid that can assemble into an organized collection of lipids (organized structure).
  • the lipid may be natural, semi-synthetic or fully synthetic lipid, as well as electrically neutral, negatively or positively charged lipid.
  • the lipid may be a naturally occurring phospholipid.
  • the ARTS, functional fragments thereof, and ARTS mimetic compounds of the present disclosure may be associated with any of the nanostructures described above, specifically, any of the micellar formulations, liposomes, polymers, dendrimers, silicon or carbon materials, polymeric nanoparticles and magnetic nanoparticles disclosed herein above.
  • association may be used interchangeably with the term “entrapped', “attachment” , “linked', “embedded' , “absorbed' and the like, and contemplates any manner by which the at least one ARTS mimetic compounds of the disclosure is held.
  • the compositions provided by the present disclosure optionally further comprise at least one pharmaceutically acceptable excipient or carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.
  • compositions used in the methods and kits of the invention, described herein after may be adapted for administration by systemic, 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.
  • compositions provided by the disclosure optionally further comprise at least one pharmaceutically acceptable excipient or carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.
  • compositions provided by the disclosure comprise an effective amount of any of the ARTS, functional fragments thereof (e.g. any of the peptides of SEQ ID NO: 14 to 25), and any mimetic compounds thereof, specifically, the B3 compound of the disclosure, specifically, the A4 compound including any stereoisomer or salt thereof, as well as any vehicle, matrix, nano- or micro-particle comprising the same.
  • compositions used in the methods and kits of the disclosure, described herein after may be adapted for administration by systemic, 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).
  • systemic administration means the administration of a compound, drug or other material other than directly into the central blood system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • the compounds of the present disclosure which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present disclosure, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
  • the pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions used to treat subjects in need thereof generally comprise a buffering agent, an agent who adjusts the osmolarity thereof, and optionally, one or more pharmaceutically acceptable carriers, excipients and/or additives as known in the art.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • the carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Local administration to the area in need of treatment may be achieved by, for example, local infusion during surgery, topical application, direct injection into the specific organ, etc.
  • compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, solid solution, liposome, films, ovules, sprays or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions used to treat subjects in need thereof according to the disclosure 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 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 disclosure 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 disclosure also include, but are not limited to, emulsions and liposome-containing formulations.
  • formulations may also include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
  • Another aspect of the present disclosure relates to methods for upregulating the levels of p53 in a cell. More specifically, the disclosed method comprises the step of contacting the cell with an effective amount of ARTS, any fragments thereof, or at least one mimetic compound thereof or any vehicle, matrix, nano- or micro-particle comprising the same, or any composition thereof.
  • upregulating p53 levels comprises reduction of p53 ubiquitylation by at least one E3 ligase.
  • the ARTS mimetic compound is having the general formula (I), wherein formula (I) is: wherein
  • R 1 , R2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is L 1 '-R 3 '-L 1 ”-R 3 ” and R2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R 2 is L 2 '-R 4 '-L 2 ”-R 4 ”; or R 1 is L 1 '-R 3 '-L 1 ”-R 3 ” and R 2 is L 2 '-R 4 '-L 2 ”-R 4 ”; wherein R 3 ’, R 3 ”, R4’, R4” is each independently from each other, absent or H, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, aryl, arylalkyl, heteroaryl, heterocycloalkyl, a ring system containing five to twelve atoms, each optionally substituted; L1’, L1”, L2’ and L2” is each independently from each other, absent or -(CH 2 ) n ,
  • the ARTS mimetic compound is having the general formula (IIIc), or (Ille):
  • the ARTS mimetic compound is having the formula (3.1), (3.2), (3.3); N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide ;
  • the disclosed methods upregulate p53 levels in a cell that may be of a subject suffering from at least one pathologic disorder.
  • the disclosed methods upregulate p53 levels in a cell that may be of a subject suffering from at least one p53-associated disorder.
  • the p53-associated disorder may comprise at least one of: at least one proliferative disorder and at least one metabolic disorder.
  • the disclosed methods upregulate p53 levels in a cell that may be of a subject suffering from at least one proliferative disorder, in more specific embodiments, the disorder may be at least one neoplastic malignant disorder.
  • the disclosed method is for upregulating the levels of p53 in at least one cell in a subject suffering from at least one pathologic disorder.
  • the contacting step may comprise administering to the subject a therapeutically effective amount of said ARTS or at least one mimetic compound thereof.
  • Another aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a pathological disorder in a subject in need thereof, by upregulating p53 levels in at least one cell of the subject.
  • the method comprising administering to the subject a therapeutically effective amount of at least one of ARTS, any fragments thereof, or at least one mimetic compound thereof or any vehicle, matrix, nano- or micro-particle thereof or of a composition comprising the same.
  • the disorder is at least one p53-associated disorder.
  • p53-associated disorder and "p53-mediated disorder” refer to pathological and disease conditions in which a p53 protein is downregulated, not functioning, or display altered function. Moreover, this term also encompasses conditions in which p53 plays a role. Such roles can be directly related to the pathological condition or can be indirectly related to the condition. The feature common to this class of conditions is that they can be ameliorated by elevating, upregulating the expression of, activity of, function of, or association with p53.
  • disorders displaying “downregulation” or “non-function” of p53 refer to disorders which demonstrate at least 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90%, 95%, 100% or more, or 1.1 fold, 1.2 fold, 1.4 fold, 1.6 fold, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 fold, or more, reduction, downregulation, decrease in p53 levels and/or function, and/or stability, relative to a control sample.
  • a p53-associated pathological disorder is meant in some embodiments, a disorder characterized by low levels, loss of function, altered function of p53 in a subject or in a diseased tissue of a subject as compared to a healthy subject or a healthy tissue of the same subject.
  • the invention provides methods for treating disorders associated with, or related to p53. It is understood that the interchangeably used terms "associated” 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.
  • Wild type p53 associated metabolic functions in muscle cells include mitochondrial biogenesis and respiration, ATP production, reactive oxygen species (ROS) production, fatty acid oxidation (FAO), pentose phosphate pathway (PPP) and glucose utilization.
  • ROS reactive oxygen species
  • PPP pentose phosphate pathway
  • the consequences of their deregulations on human disease beyond cancer may include for example cardiovascular diseases.
  • the metabolic functions include amino-acid catabolism, gluconeogenesis, FAO, mitochondrial respiration, glucose utilization, anti-oxidant defenses, insulin signaling, cholesterol metabolism and ureagenesis and their deregulation may result in liver diseases such as steatosis and nonalcoholic steatohepatitis (NASH) as well as diabetes.
  • p53 metabolic functions include insulin secretion and mitochondrial respiration and in adipose tissue include energy expenditure, fatty acid synthesis and storage and mitochondrial respiration, all of which may result in obesity in case of p53 deregulation.
  • p53-associated and/or related- and/or mediated- disorders as used herein, encompass any disorder or condition affected by disfunction of p53, and/or altered function of p53, and/or reduced function of p53, loss-of function of p53, caused by at least one of degradation of p53, instability of p53, reduced expression of p53, mutations in p53, altered conformation of p53, reduced ratio between mutated and nonmutated p53, and any combinations thereof.
  • the subject suffering from a p53-associated disorder is heterozygous for mutant p53.
  • the p53-associated disorder treated by the therapeutic methods disclosed herein may comprise at least one of: at least one proliferative disorder and at least one metabolic disorder.
  • the proliferative disorder may be at least one neoplastic malignant disorder.
  • proliferative disorder is a disorder displaying hyper proliferation. This term means cell division and growth that is not part of normal cellular turnover, metabolism, growth, or propagation of the whole organism. Unwanted proliferation of cells is seen in tumors and other pathological proliferation of cells, does not serve normal function, and for the most part will continue unbridled at a growth rate exceeding that of cells of a normal tissue in the absence of outside intervention.
  • hypo proliferative disease A pathological state that ensues because of the unwanted proliferation of cells is referred herein as a "hyper proliferative disease” or “hyper proliferative disorder.”
  • proliferative disorder cancer”, “tumor” and “malignancy” all relate equivalently to a hyperplasia of a tissue or organ.
  • the methods of the invention may be applicable for treating malignant, pre-malignant disorders and/or cancer.
  • the compositions and methods of the present invention may be used in the treatment of non-solid and solid tumors.
  • the therapeutic method of the invention may be particularly effective for a subject suffering from any one of a pre-malignant condition and carcinoma.
  • Carcinoma 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. In terms of solid tumors, this group of cancers may include, among others, carcinomas of the breast, lung, bladder as well as gastric, colorectal, ovarian and uterine carcinomas.
  • carcinomas of the breast, lung, bladder as well as gastric, colorectal, ovarian and uterine carcinomas.
  • the term "carcinoma” refers herein to any tumor tissue derived from putative epithelial cells, or cells of endodermal or ectodermal germ layer during embryogenesis, that become transformed and begin to exhibit abnormal malignant properties.
  • the therapeutic methods of the invention may be applicable for subjects suffering from a breast carcinoma.
  • Breast cancer is one of the leading causes of cancer death in women in the Western world. Though current therapies are effective, a considerable population will relapse, rendering the essential need for improved and new avenues of targeted therapies.
  • Gene expression profiling can be used to distinguish breast cancers into distinct molecular subtypes with prognostic significance, based upon phenotypic diversity in biological factors such as histological grade, estrogen receptor (ER) status, progesterone receptor (PgR) status, and HER2/ne « expression (HER2).
  • breast cancer When presently referring to breast cancer, is meant any type of cancer originating from breast tissue, including ductal and lobular carcinomas.
  • the present context also encompasses genetic or hereditary breast cancers (5-10% of all cases) developing from predisposing mutations in BRCA1 and BRCA2 genes and also other relevant mutations in p53 (Li- Fraumeni syndrome), PTEN (Cowden syndrome), and STK11 (Peutz-Jeghers syndrome), CHEK2, ATM, BRIP1, and PALB2 genes.
  • the present context also encompasses all breast cancer classifications, including those using histopathology (e.g. mammary ductal carcinoma, carcinoma in situ, invasive carcinoma or inflammatory breast cancer), grade (e.g.
  • malignancy as contemplated in the present disclosure may be any one of lymphomas, leukemias, carcinomas, melanomas, myeloma and sarcomas. Therefore, in certain embodiments, the ARTS mimetic compound/s, compositions and methods of the invention may be further relevant for other malignancies such as lymphomas, leukemia, melanomas, myeloma and sarcomas.
  • Lymphoma is a cancer in the lymphatic cells of the immune system.
  • 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.
  • 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).
  • 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.
  • 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.
  • malignancies that may find utility in the present invention can comprise but are not limited to hematological malignancies (including lymphoma, leukemia and myeloproliferative disorders), 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. More particularly, the malignant disorder may be lymphoma.
  • cancers treatable according to the invention include hematopoietic malignancies such as all types of lymphomas, leukemia, e.g.
  • ALL acute lymphocytic leukemia
  • AML acute myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • MDS myelodysplastic syndrome
  • mast cell leukemia hairy cell leukemia
  • Hodgkin's disease non-Hodgkin's lymphomas
  • Burkitt's lymphoma multiple myeloma
  • 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, extrahepatic bile ducts, ampulla of vater, exocrine pancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma, carcinoma and malignant melanom
  • the invention relates to any neurological tumor, for example, neuroblastoma, astrocytoma, CNS lymphoma, neuroma, glioma, Chordoma, medulloblastoma, Oligodendroglioma, Craniopharyngioma, and any mixed neurological tumor.
  • the therapeutic methods disclosed herein may be applicable for at least one metabolic disorder.
  • such metabolic disorder may comprise at least one cardiovascular disorder, liver diseases, diabetes and metabolic syndrome.
  • Metabolic disease is any of the diseases or disorders that disrupt normal metabolism, the process of converting food to energy on a cellular level. Thousands of enzymes participating in numerous interdependent metabolic pathways carry out this process. Metabolic diseases negatively affect the ability of the cell to perform critical biochemical reactions that involve the processing or transport of proteins (amino acids), carbohydrates (sugars and starches), or lipids (fatty acids). Numerous molecular pathways and thus, several organs, can be affected. Many of the metabolic diseases are caused by genetic mutations or by a combination of genetic and environmental factors.
  • metabolic diseases or diseases associated with metabolic disorders include for example: diabetes (or diabetes mellitus), a group of common endocrine diseases characterized by sustained high blood sugar levels affecting nearly every major bodily organ; non-alcoholic steatohepatitis (NASH), a liver inflammation and damage caused by a buildup of fat in the liver; steatosis (or fatty change), an abnormal retention of fat (lipids) within a cell or organ, most often affects the liver, but can also occur in other organs, including the kidneys, heart, and muscle; obesity, a medical condition, in which excess body fat has accumulated to such an extent that it may negatively affect health; dyslipidemia, is the imbalance of lipids such as cholesterol, low-density lipoprotein cholesterol, (LDL-C), triglycerides, and high-density lipoprotein (HDL); cardiovascular diseases, a group of disorders of the heart and blood vessels and include coronary heart disease, cerebrovascular disease, rheumatic heart disease and other conditions.
  • diabetes or diabetes
  • upregulating p53 levels may comprise reduction of p53 ubiquitylation by at least one E3 ligase.
  • such E3 ligase may be XIAP. More specifically, as firstly shown by the present disclosure, XIAP serves as an E3 ligand for p53.
  • XIAP serves as an E3 ligand for p53.
  • the ARTS, fragments thereof and the ARTS mimetic compounds, specifically the "B3" compound, used by the therapeutic methods disclosed herein lead to down-regulation of MDM2 levels, specifically, via UPS, thereby leading to upregulation of p53 levels.
  • the ARTS mimetic compound of the therapeutic methods disclosed herein is having the general formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof, wherein formula (I) is: wherein
  • R 1 , R 2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is L 1 '-R 3 '-L 1 ”-R 3 ” and R 2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R 2 is L 2 ’-R4’-L 2 ”-R4”; or
  • the ARTS mimetic compound is having the general formula (lib):
  • the R1 is L1’-R 3 ’-L1”-R3”.
  • the R1 is at least one of:
  • the ARTS mimetic compound of the therapeutic methods disclosed herein is having the general formula (IIIc), (Illd) or (Ille):
  • L1 and R3 are each as defined above, wherein R is one or more of H, OH,
  • the ARTS mimetic compound of the therapeutic methods disclosed herein is having the general formula (life), or (life):
  • the ARTS mimetic compound of the therapeutic methods disclosed herein is having the formula (3.1), (3.2), (3.3); N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide ;
  • the methods provided herein involve administration of the ARTS mimetic compound/s of the invention in a therapeutically effective amount.
  • effective amount is that determined by such considerations as are known to the man of skill in the art. The amount must be sufficient to prevent or ameliorate tissue damage caused by proliferative disorders and/or metabolic disorders. Dosing is dependent on the severity of the symptoms and on the responsiveness of the subject to the active drug, specifically, the antagonist of the invention. Medically trained professionals can easily determine the optimum dosage, dosing methodology and repetition rates. In any case, the attending physician, taking into consideration the age, sex, weight and state of the disease of the subject to be treated, will determine the dose.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. In general, dosage is calculated according to body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the compositions and combined composition of the invention in bodily fluids or tissues.
  • ARTS, functional fragments thereof, and/or ARTS mimetic compounds used by the method of the present disclosure is administered in maintenance doses, once or more daily.
  • therapeutically effective amount means an amount of the ARTS, functional fragments thereof, and/or ARTS mimetic compounds, a composition comprising the same which provides a medical benefit as noted by the clinician or other qualified observer. Regression of a tumor in a patient is typically measured with reference to the diameter of a tumor. Decrease in the diameter of a tumor indicates regression. Complete regression is also indicated by failure of tumors to reoccur after treatment has stopped.
  • 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, proliferative disorder symptoms or undesired side effects of such proliferative disorder related disorders. More specifically, treatment or prevention 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.
  • “disease”, “disorder”, “condition” 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.
  • the present invention relates to the treatment of subjects, or patients, in need thereof.
  • patient or “subject in need” it is meant any organism who may be affected by the above- mentioned conditions, and to whom the treatment methods herein described are desired, including humans, domestic and non-domestic mammals such as canine and feline subjects, bovine, simian, equine and murine subjects, rodents, domestic birds, aquaculture, fish and exotic aquarium fish. It should be appreciated that the treated subject may be also any reptile or zoo animal. More specifically, the methods and compositions of the invention are intended for mammals.
  • mammalian subject is meant any mammal for which the proposed therapy is desired, including human, equine, canine, and feline subjects, most specifically humans.
  • the method of the invention may be performed using administration via injection, drinking water, feed, spraying, oral gavage and directly into the digestive tract of subjects in need thereof. It should be further noted that particularly in case of human subject, administering of the compositions of the invention to the patient includes both self-administration and administration to the patient by another person.
  • kits comprising:
  • ARTS In one component (a), ARTS, or any fragments thereof, or at least one mimetic compound thereof, optionally, in first dosage form.
  • the ARTS mimetic compound of the disclosed kits may have the general formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof, wherein formula (I) is: wherein
  • R 1 , R2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is L 1 '-R 3 '-L 1 ”-R 3 ” and R2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R 2 is L 2 '-R 4 '-L 2 ”-R 4 ”; or
  • kits further comprise at least one therapeutic compound, optionally, in a second dosage form.
  • the present disclosure thus provides in some aspects thereof combined therapy, combining tire disclosed ARTS, fragments thereof or any mimetic thereof, specifically, the B3 mimetic, with any therapeutic compound, and specifically, with modulators of p53, that lead to upregulation of p53 levels.
  • kits, combined composition and combined therapeutic methods combining the disclosed ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with at least one therapeutic agent.
  • the additional therapeutic agent or compound may be any compound used to treat the disclosed p53-associated condition.
  • the compound may be any compound that modulates p53 levels. Specifically, any compound that upregulates the levels of p53.
  • aminoglycoside antibiotic gentamicin and its derivatives such as G418 and the new-generation synthetic derivative NB124
  • inhibition of the NMD process as an example NMD 14 which targets a structural pocket of SMG7, a key component of the NMD machinery.
  • proteasome inhibitors such as Bortezomib (Velcade, PS341), as well as its second-generation derivative Carfilzomib (Kyprolis) and Ixazomib (MLN9708, Ninlaro) may be applicable in the combined therapy disclosed herein.
  • the combined therapy disclosed by the present disclosure may combine the ARTS, fragments thereof, and/or any mimetics thereof, with gene therapy.
  • p53-based virus-like vectors such as Gendicine, advexin and SCH-58500, Nanoparticles,’ selectively restore p53 expression in cancer cells, for example, SGT-53, a cationic liposome carrying wtp53-encoding DNA that homes selectively to tumour cells via an anti-transferrin single -chain antibody fragment and p53 mRNA-nanoparticle formulations.
  • the disclosed combinations may further use small interfering RNA (siRNA) oligonucleotides that target specific mutations within p53 mRNA and CRISPR--Cas9 base editing technologies.
  • siRNA small interfering RNA
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with CP31398, that restores wild-type-like, P53 conformation and prevents its degradation through inhibition of its ubiquitylation.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with PRJ.MA-1 (p53 reactivation with induction of massive apoptosis 1 ) - compound that restored wtp53 conformation and function upon binding to mutp53. Still further, in some embodiments, the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with MIRA-1 and STIMA-1, a 2-styrylquinazolin-4(3H)-one -related derivative. Like PRIMA-1, bod's MIRA-1 and STIMA-1 also possess Michael acceptor activity, and can potentially modify cysteines in the p53 protein to stabilize die wild-type conformation and prevent mutp53 unfolding.
  • PRJ.MA-1 p53 reactivation with induction of massive apoptosis 1
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof
  • the present disclosure provide a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with mutp53-reactivating small peptides (pCAPs), that bind preferentially to the wtp53 conformation: when a mutp53 molecule assumes transiently a wild-type-like conformation, the peptide binds and stabilizes it, gradually shifting the dynamic equilibrium of the p53 population in that direction.
  • pCAPs mutp53-reactivating small peptides
  • ATO arsenic trioxide
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with APR-246 and pCAPs), that target a broad spectrum of p53 mutants to restore a wtp53-like structure, thus enabling p53 target gene activation.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with ReACp53 or ADH-6, that act by inhibiting mutp53 aggregation, restoring wtp53-like structure and activating p53 target gene.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with PhiKan083, that was found to bind the thermodynamically stabilize p53( ⁇ 220C), shifting it towards a wtp53-like state.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with RG7112, that was the first MDM2 inhibitor tested in clinical trials. Still further, in some embodiments, the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with APG-115, an orally bioavailable potent MDM2 inhibitor.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with ALRN-6924, that blocks both MDM2-p53 and MDM4-p53 interactions.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with AMG 232, that is another orally bioavailable MDM2 inhibitor, shown to promote wtp53 functionality and tumour regression in osteosarcoma cells.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with any additional MDM2 inhibitors, including siremadlin and milademetan.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with p53 -activating proteolysis targeting chimeras (PROTACs), that work by targeting MDM2 for ubiquitylation by particular' E3 ligases, resulting in MDM2 degradation.
  • PROTACs proteolysis targeting chimeras
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with SAH-p53-8, that is capable of blocking the interactions of both MDM2 and MDM4 with p53.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with ALRN-6924, that demonstrated high efficacy against multiple breast cancer cell lines with wtp53.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with RETRA (reactivation of transcriptional reporter activity) and/or NSC59984, which inhibit the interaction of mutp53 with p73, unleash p73 and enable it to enter the nucleus and transactivate target genes that partly overlap with p53 target genes.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with RITA, that display reactivation of p53 and induction of tumour cell apoptosis by blocking E6-P53 interaction.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with compound 12 - A new inhibitor of the E6-p53 interaction.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with XI-011 (NSC146109), a small-molecule MDM4 inhibitor, exerted antiproliferative effects in HPV-positive cervical cancer cell lines, suggesting a potential utility of MDM4 inhibitors for treating HPV-positive cervical cancer.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with aminoglycoside antibiotic gentamicin and its derivatives, such as G418 and the new-generation synthetic derivative NB124.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with NMD14, which targets a structural pocket of SMG7, a key component of the NMD machinery.
  • the present disclosure provides a combined therapy comprising ARTS, fragments thereof and/or mimetic compounds thereof, specifically, the B3 compound, with ataluren.
  • compositions comprising, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
  • 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.
  • compositions comprising, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”. This term encompasses the terms “consisting of” and “consisting essentially of”.
  • Consisting essentially of means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.
  • 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.
  • 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.
  • 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.
  • ARTS mimetics small molecules specifically, B3 and A4, were purchased from eMolecules, suspended in DMSO and resuspended in PBS as per the manufacturer’s instructions.
  • ARTS mouse monoclonal anti-ARTS antibody, specifically targeting the unique c- terminal sequence of ARTS (but not other Septin 4 splice variants) at a dilution of 1: 1000 (Sigma A4471).
  • XIAP mouse monoclonal anti-XIAP antibody (BD cat#610717) at a dilution of 1:4000.
  • XIAP Rabbit monoclonal anti-XIAP antibody (Cell signaling cat#CS 14334) at a dilution of 1:3000.
  • mouse monoclonal anti-actin antibody (ImmunoTM cat#08691002) at a dilution of 1:50,000.
  • Cleaved PARP cl.PARP
  • rabbit monoclonal anti-cl.PARP antibody Cell signaling cat#CS5625
  • Tubulin monoclonal rat anti-tubulin (Abeam cat#YOL1/34) at a dilution of 1:6000.
  • p53 rabbit monoclonal anti- p53 antibody (Cell signaling cat#CS32532) at a dilution of 1:4000.
  • p53 mouse monoclonal anti- p53 antibody (Cell signaling cat#CS2524) for immunoprecipitation.
  • p53 mouse monoclonal anti- p53 antibody (Santacruz DO-1 cat#SC-126) at dilution of 1:1000.
  • p53 goat polyclonal anti-p53 antibody (R&D cat#AF1355).
  • MDM2 mouse monoclonal anti- MDM2 antibody (abeam cat#ab 16895).
  • GAPDH goat polyclonal anti-GAPDH antibody (abacm cat#ab9483).
  • Etoposide (abeam cat#abl20227) - 200 ⁇ M, for three hours or as indicated.
  • Nocodazole (sigma cat#ml404) - 200ng/ml for 1 hour.
  • Cycloheximide - (sigma cat#C7698) 200 ⁇ M was induced for up to 180min.
  • HCT 116 (WT, p53 KO and XIAP KO) cells were grown in complete McCoy’s medium (1% sodium pyruvate, 1% L-glutamate, 1% Pen-strep, and 10% fetal bovine/calf serum).
  • MEFs (WT, Sept4/ARTS KO, and XIAP KO) cells and A375 cells were grown in a complete DMEM medium (1% sodium pyruvate, 1% L-glutamate, 1% non-essential amino acids, 1% Pen-strep, 10% fetal bovine/calf serum, and 0.1% [3- mercaptoethanol).
  • A549 cells were grown in DMEM/F12 complete medium (1% sodium pyruvate, 1% L- glutamate, 1% Pen-strep, 10% fetal bovine/calf serum).
  • Radioimmunoprecipitation assay (RIP A) buffer (Tris- HC1 pH 7.5 50 mM, NaCl 150 mM, NP-40 (Igepal) 0.3%) containing protease inhibitor cocktail (Complete, Roche) and 100 ⁇ g/ml PMSF.
  • Antibodies were used at 5 ⁇ g per 1000 ⁇ g protein and incubated overnight, rotating at 4°C. The next day, agarose beads conjugated to protein A/G (Santa Cruz Biotechnology) were added for 4 h with rotation at 4°C. Samples were centrifuged at 4°C for 5 min and washed five times with RIPA buffer. Proteins were eluted from beads by 10 min of boiling in sample buffer and separated on 12% SDS-PAGE gel, followed by Western blot analysis.
  • RIP A radioimmunoprecipitation assay
  • the cells were harvested and lysed using RIPA buffer (Tris-HCl pH 7.5 50 mM, NaCl 150 mM, NP-40 (Igepal) 0.3%) containing protease inhibitor cocktail (Complete, Roche), 100 ⁇ g/ml PMSF, 5 mM N-ethylmaleimide, and 5 mM iodoacetamide to preserve ubiquitin chains. Following 15 min of centrifugation (10,000 x g, 4 °C), the supernatant was transferred into a clean Eppendorf tube.
  • a ubiquitination assay using immunoprecipitation with anti-p53 antibody (CS2524) (endogenous p53 was pulled down) was performed as described above with 1:500 antibody per 1000 ⁇ g protein. Poly- ubiquitylated forms of p53 were detected using an anti-p53 antibody (DO-1, sc-126).
  • Bacterially expressed His-ARTS was purified using fast protein liquid chromatography (FPLC), and bacterially expressed GST-XIAP was purified using glutathione beads.
  • P53 recombinant protein was purchased from R&D (cat#SP-450). Recombinant p53 and GST-XIAP were incubated. El, UbcH5b, ubiquitin (Ub), and the appropriate E3 in conjugation buffer (20 mM Tris-Cl [pH 7.6], 100 mM KC1, 5 mM MgC12, and 1 mM DTT) containing 2 mM ATP at 37C for 1 hour.
  • the assay was done using Chromata ChIP Kit (Novus Bio cat#NBPl-71709). In brief, cells were exposed to UV irradiation for 5, 10, and 30 min. Cross-linking was done using 1% formaldehyde for 10 min. The reaction was stopped by incubation with 125nM Glycine for 10 min at RT. Cells were washed twice with PBS and lysed on ice for 30 min with SDS-Lysis Buffer containing PI and PMSF. To achieve the appropriate length of DNA fragments (between 1000 and 200 bp) the samples were sonicated for 5 min (30sec on/off).
  • Samples were centrifuged for 10 min at 15000 rpm at 4 °C, and the supernatant was transferred into a fresh tube.
  • 50pl was taken from each sonicated sample reverse cross-linked, cleaned, and run on 1 % agarose gel.
  • the samples were pre-cleared with 20,u l/samplc of A/G agarose beads for Ih at 4°C. 10% of pre-cleared lysate was taken from all the samples as an INPUT control and stored at -20°C. The remained sample was split into three aliquots.
  • p21(sc-51689) IgG (abl8421), and p53 (ab1101 ChIP grade).
  • p21 sample was used as a positive control
  • IgG sample was used as a negative control. All the samples were gently rotated at 4°C for 2h.
  • A/G agarose beads (40 ⁇ l/sample) were washed, re-suspended in IP buffer, and added to the samples for overnight rotation at 4°C. The cells were washed twice with IP washing buffer, then washed twice with Lithium buffer and TE buffers. Protein- DNA complexes were eluted with Elution buffer.
  • the samples were then reverse cross-linked with I ⁇ M NaCL for 4h at 65°C, 1 ⁇ g/ml RNase A for 30 min at 37°C, and 20 ⁇ g/ml of Proteinase K for over night at 55°C.
  • the samples were cleaned with a PCR purification kit.
  • the samples were checked for the presence of the DNA fragments containing the Sept4/ARTS gene using PCR reaction with the following primers : -'5GAGACGAGAGTGGCCTGAACCGA-3' (Forwards), for forward sequencing of ARTS, as denoted by SEQ ID NO: 3; -5’ AACAGGAACCTGTGACCACCTGC-3’ (Reverse), for reverse sequencing of ARTS as denoted by SEQ ID NO: 4;
  • Bimolecular fluorescence complementary assay (BiFC)
  • the split-Venus BiFC system was used to evaluate close proximity indicating possible direct binding between pairs of proteins.
  • the proteins were fused either to the N-terminal part of the Venus YFP (yellow fluorescence protein) (VN) or the C-terminal domain (VC). All Venus fragments were fused to the C-terminal sequences of these proteins.
  • the Jun and bFos pair was used as a positive control (p.c.), and the Jun and bFosdeltaZIP pair was used as a negative control (n.c.).
  • a vector encoding dsRed was used as a transfection efficiency marker.
  • Microscale thermophoresis (MST) binding assays were performed by CreLux, a WuXi AppTech company in Germany, using recombinant ARTS and XIAP proteins. Specifically, for performing experiments with untagged XIAP, a fluorescent label (NT650) was covalently attached to the protein (Maleimide coupling). The labeling was performed in a buffer containing 50 mM HEPES pH 7.0, 150 mM NaCl, and 0.005% Tween-20. A detailed description is provided in the Supplementary Materials and methods.
  • the B3 small molecule (MW 406.43gr/mol as powder, SMILES: (CC22H22N4O4) was purchased from eMolecules, Inc., eMolecule ID: 30500827 (Supplier InterBioScreen STOCK 6S-95262).
  • DMSO dimethyl sulfoxide
  • B3 suspension was incubated in a 37 °C bath for 1 min, mixed thoroughly by pipetting, and spun down again.
  • B3 stock solution was aliquoted in Eppendorf tubes (7-10 ⁇ l/tube) and stored at -80 °C.
  • ARTS is a transcriptional target of p53
  • P53 induces apoptosis mainly through its transcriptional induction of target genes (Aylon and Oren 2007 Cell 130 597-600, Levine and Oren 2009 Nat Rev Cancer 9 749-758). Sequence- specific DNA binding of p53 is a prerequisite for trans-activating target genes (Aubrey et al. 2018 Cell Death Differ 25 104-113, Aylon and Oren 2007 Cell 130 597- 600, Hassin and Oren 2022 Nat Rev Drug Discov 1-18, Levine and Oren 2009 Nat Rev Cancer 9 749-758).
  • ARTS is a splice variant of the human Sept4 gene located on human chromosome 17q22-23 (Mandel-Gutfreund et al. 2011 Biological Chemistry 392 783).
  • This motif contains two half-sites that strongly resemble the consensus DNA sequence sufficient for p53 binding (el-Deiry et al. 1992 Nat Genet 1 45-49).
  • ChIP chromatin immunoprecipitation assay
  • Figures ID(i) and ID(ii) show similar increase in ARTS mRNA in the presence of p53, within 2hrs.
  • RNA levels of ARTS are upregulated during apoptosis in p53 -containing cells, while no upregulation of ARTS is seen in P53 KO cells.
  • HCT116 WT cells and HCT116 p53 KO cells were treated with 50mM Etoposide for the indicated time points. Cells were harvest and RNA was extracted using RNA isolation kit. RT PCR was performed in order to get cDNA, after which PCR was done, using specific primers for ARTS and GAPDH for amplification. GAPDH served as a loading control. The samples were separated using agarose gel.
  • Quantitation of ARTS and GAPDH levels were done using ImageJ analysis software (Quantification of bands from DNA films. The quantification reflects the relative amounts as a ratio of each protein band relative to the lane’s loading control). The graphs represent the ratio numbers of ARTS/GAPDH. . These results suggest that p53 acts as a transcriptional regulator of ARTS.
  • sept4/ARTS KO MEFs The inventors suspected that p53 low levels observed in sept4/ARTS KO MEFs (Figure 2A(i and ii), Figure 2B (i and ii) and Figure 2C (i and ii)) might be a result of excessive degradation via the UPS, and increased levels of XIAP and MDM2 in ARTS KO cells. Therefore, the sept4/ARTS KO MEFs were treated with the proteasome inhibitor MG-132, 20 ⁇ M for 6 hours ( Figure 2A and Figure 2B) or with 200ng/ml nocodazole for Ih ( Figure 2C).
  • ARTS solely significantly upregulated p53 protein levels compared to treatment with 200ng/ml Nocodazole (NOC) for 1 hour (Figure 2C). This indicates that ARTS regulates p53 levels through the UPS.
  • a bimolecular fluorescence complementary assay (BiFC) was conducted. The inventors found that ARTS and p53 bind each other upon 6 hours of 200 ⁇ M of Etoposide treatment (Figure 2D). In their previous published data (Bornstein et al. 2011 Apoptosis 16 869-881; Edison et al.
  • ARTS serves as a physiological antagonist of XIAP and that sept4/ARTS KO MEFs exhibit high levels of XIAP. This suggests another link in the regulatory network between ARTS and p53.
  • Figure 3A shows that ARTS accelerates the translocation of p53 to nucleus in WT MEFs cells ( Figure 3A(i)) that express ARTS as compared to cells that do not express ARTS (sept4/ARTS KO MEFs) ( Figure 3A(ii)).
  • Figure 3B shows that ARTS is important for the localization of p53 to the nucleus, since in sept4/ARTS KO MEFs cells, p53 display predominant mitochondrial localization.
  • Figure 3C further shows intense nuclear localization of p53 upon exposure to UV forlO minutes.
  • the localization is cytoplasmic upon UV exposure.
  • P53 translocate from mitochondria to the nucleus upon induction of apoptosis.
  • MEF WT cells were treated with UV radiation for the indicated time points. With no UV treatment, p53 was localized at the mitochondria and translocated to the nucleus upon UV-induced apoptosis. Nuclei were stained blue with Dapi staining, mitochondria were stained red with Mitotracker.
  • Sept4/ARTS KO MEFS show inhibition of p53 translocation to the nucleus.
  • SEPT4/ARTS KO MEFs were treated with UV radiation for the indicated time points.
  • XIAP acts as an E3 ligase for p53
  • XIAP regulates the levels of many pro-apoptotic proteins through the UPS, including ARTS (Abbas and Larisch 2020 Cells 9; Abbas and Larisch 2021 Cells 10).
  • ARTS regulate XIAP levels by bringing XIAP into close proximity with the E3 ligase SIAH and by initiating the auto-Ubiquitylation of XIAP (Bornstein et al. 2011 Apoptosis 16 869-881; Edison et al. 2012 Clin Cancer Res 18 2569-2578; Garrison et al. 2011 Mol Cell 41 107-116).
  • the inventors examined the possibility that ARTS regulates p53 protein levels through its effect on XIAP.
  • XIAP KO MEFs The inventors tested whether in the absence of XIAP (XIAP KO MEFs), higher levels of p53 are observed compared to MEFs WT. Indeed, p53 protein levels are elevated in XIAP KO MEFs compared to WT MEFs ( Figure 4A(i)-(ii)), that express XIAP. Furthermore, immunoprecipitation of XIAP with anti-XIAP antibodies followed by Western blot confirmed that XIAP binds p53, and upon treatment with 200 ⁇ M of ETP for 3 hours the levels of XIAP were downregulated, and the levels of p53 were upregulated thereby the binding between XIAP and p53 was affected ( Figure 4B).
  • XIAP is overexpressed in leukemia, lung, colon, melanoma, ovarian, bladder, renal, breast, prostate, and thyroid carcinomas (Dubrez et al. 2013 Onco Targets Ther 9 1285-1304; Krajewska et al. 2003 Clin Cancer Res 9 4914-4925; Tamm et al. 2000 Clin Cancer Res 6 1796-1803).
  • XIAP has become an attractive target for developing anti-cancer drugs (Abbas and Larisch 2020 Cells 9; Abbas and Larisch 2021 Cells 10; Jost and Vucic 2019 Cold Spring Harb Perspect Biol; Mamriev et al. 2020 Cell Death Dis 11 483).
  • IBM (Smac) mimetics Bossi et al. 2003 Curr Opin Cell Biol 15 717-724; Chai et al. 2000 Nature 406 855-862; Corti et al. 2018 FEBS J 285 3286-3298; Li et al. 2004 Science 305 1471-1474; Oost et al.
  • a major goal of the pharmaceutical industry is to develop potent and specific small molecules that selectively degrade XIAP (Bornstein et al. 2012 Int J Biochem Cell Biol 44 489-495; Eai and Crews 2017 Nat Rev Drug Discov 16 101-114).
  • the inventors generated small-molecule ARTS mimetics that can bind directly to the unique recognition sequence of ARTS in the BIR3 domain of XIAP (as denoted by SEQ ID NO: 11), but not to cIAPs. These compounds promote XIAP ubiquitylation and degradation via the UPS (Mamriev et al.
  • the inventors identified 100 molecules with the highest affinity scores of dockings to the unique binding site of ARTS in BIR3/XIAP ( Figure 5A).
  • MST microscale thermophoresis
  • ARTS mimetic small molecule upregulates p53 through its effect on XIAP, and promote apoptosis
  • B3 can mimic the function of ARTS
  • the B3 compound has been shown as inducing apoptosis as reflected by elevation in cleaved PARP and Caspase 3 (Figure 6A), while reducing the levels of the anti-apoptotic proteins XIAP and Bcl-2.
  • Figure 6B ARTS mimetic small molecules B3 and A4, upregulate p53 levels and downregulates XIAP.
  • Figure 6C specifically demonstrates that the ARTS mimetic compound B3 reduces the binding between ARTS and p53, as shown by Co-IP between ARTS and p53.
  • Figures 6D-6F show dose response of the ARTS mimetic B3 compound that leads to elevation in p53, and cP ARP or caspase3, and reduction in E3 ligases of p53, MDM2 and XIAP in either A375 cells (Figure 6D(i)-(vi)), HCT116 cells ( Figure 6E), or MEF cells ( Figure 6F (i)-(viii)).
  • ARTS and its mimetic small molecules, specifically, B3 act as negative regulators of E3 ligases that act on p53, and thus function as a strong agonist of p53.
  • ARTS and B3 tested the effect of ARTS and B3 on p53 Ubiquitylation levels by using HCT 116 WT and HCT 116 XIAP KO cells.
  • ARTS overexpression and 20 ⁇ M of B3 both managed to inhibit the Ubiquitylation of p53 ( Figure 6G).
  • HCT 116 XIAP KO cells and the overexpression of ARTS in HCT 116 WT show the same Ubiquitylation levels of p53 ( Figure 6G).
  • ARTS mimetic compounds upregulate p53 through its effect on XIAP.

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Abstract

La présente divulgation concerne une protéine associée à l'apoptose dans la voie de signalisation TGF-beta (ARTS), tout fragment de celle-ci, ou au moins un composé mimétique de celle-ci, régulant à la hausse les taux de p53 dans une cellule. La présente divulgation concerne en outre des compositions et des méthodes thérapeutiques pour le traitement d'affections liées à p53.
PCT/IL2023/050212 2022-03-01 2023-03-01 Protéine associée à l'apoptose dans la voie de signalisation tgf-beta (arts) et composés à petites molécules mimétiques de arts pour réguler à la hausse le niveau p53 WO2023166507A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015035051A1 (fr) * 2013-09-04 2015-03-12 Board Of Regents Of The University Of Texas System Procédés et compositions pour une cancérothérapie sélective et ciblée
WO2017077535A1 (fr) * 2015-11-02 2017-05-11 Carmel-Haifa University Economic Corporation Ltd. Composés mimétiques de protéine liée à l'apoptose dans la voie de signalisation de tgf-beta (arts), compositions, méthodes et utilisations correspondants dans l'induction de la différentiation et/ou de l'apoptose de cellules précancéreuses et malignes, rétablissant ainsi leur phénotype de type normal

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015035051A1 (fr) * 2013-09-04 2015-03-12 Board Of Regents Of The University Of Texas System Procédés et compositions pour une cancérothérapie sélective et ciblée
WO2017077535A1 (fr) * 2015-11-02 2017-05-11 Carmel-Haifa University Economic Corporation Ltd. Composés mimétiques de protéine liée à l'apoptose dans la voie de signalisation de tgf-beta (arts), compositions, méthodes et utilisations correspondants dans l'induction de la différentiation et/ou de l'apoptose de cellules précancéreuses et malignes, rétablissant ainsi leur phénotype de type normal

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PANAYOTIS C THEODOROPOULOS, GONZALES STEPHEN S, WINTERTON SARAH E, RODRIGUEZ-NAVAS CARLOS, MCKNIGHT JOHN S, MORLOCK LORRAINE K, HA: "Discovery of tumor-specific irreversible inhibitors of stearoyl CoA desaturase", NATURE CHEMICAL BIOLOGY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 12, no. 4, New York, pages 218 - 225, XP055357027, ISSN: 1552-4450, DOI: 10.1038/nchembio.2016 *
R. LOTAN: "Regulation of the Proapoptotic ARTS Protein by Ubiquitin-mediated Degradation", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, vol. 280, no. 27, 8 July 2005 (2005-07-08), pages 25802 - 25810, XP055080816, ISSN: 00219258, DOI: 10.1074/jbc.M501955200 *
SARAH E. WINTERTON, EMANUELA CAPOTA, XIAOYU WANG, HONG CHEN, PREMA L. MALLIPEDDI, NOELLE S. WILLIAMS, BRUCE A. POSNER, DEEPAK NIJH: "Discovery of Cytochrome P450 4F11 Activated Inhibitors of Stearoyl Coenzyme A Desaturase", JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 61, no. 12, 28 June 2018 (2018-06-28), US , pages 5199 - 5221, XP055698788, ISSN: 0022-2623, DOI: 10.1021/acs.jmedchem.8b00052 *

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