WO2021091871A1 - Antagonistes peptidiques à haute affinité et à double spécificité de mdm2 et de mdmx pour l'activation de p53 - Google Patents

Antagonistes peptidiques à haute affinité et à double spécificité de mdm2 et de mdmx pour l'activation de p53 Download PDF

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WO2021091871A1
WO2021091871A1 PCT/US2020/058669 US2020058669W WO2021091871A1 WO 2021091871 A1 WO2021091871 A1 WO 2021091871A1 US 2020058669 W US2020058669 W US 2020058669W WO 2021091871 A1 WO2021091871 A1 WO 2021091871A1
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peptide
mdm2
mdmx
pmi
cell
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PCT/US2020/058669
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English (en)
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Wuyuan Lu
Xiang Li
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University Of Maryland, Baltimore
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • ASCII text file A sequence listing in electronic (ASCII text file) format is filed with this application and incorporated herein by reference.
  • the name of the ASCII text file is “2020_2650A_ST25.txt”; the file was created on November 3, 2020; the size of the file is 12 KB.
  • the present invention generally relates to peptide antagonists of MDM2 and MDMX, and to the inhibition of p53-MDM2/MDMX interactions.
  • the invention is further related to pharmaceutical compositions comprising the antagonists, and methods of treating conditions such as cancer using the antagonists.
  • p53 is a tumor suppressor that transcriptionally regulates, in response to cellular stresses such as DNA damage or oncogene activation, the expression of various target genes that mediate cell-cycle arrest, DNA repair, senescence or apoptosis [10,12] .
  • Loss of p53 activity - either by somatic mutation of the TP53 gene or by functional inhibition of the p53 protein - is a common feature of human tumors. It is estimated that 50% of human tumors carry loss-of- function mutations in TP53, many of which are associated with malignant progression, poor prognosis and resistance to treatment
  • p53 is present in its wild-type form.
  • high levels of negative regulators of p53 such as the E3 ubiquitin ligase MDM2 and its homolog MDMX (also known as MDM4) impede p53- induced growth inhibitory and apoptotic responses
  • MDM2 primarily controls p53 stability by targeting the tumor suppressor protein for ubiqui tin-mediated constitutive degradation by the proteasome [30]
  • MDMX mainly functions as an effective transcriptional antagonist of p53 that blocks its ability to regulate expression of responsive genes [33] .
  • peptides that bind with high affinity to MDM2 or MDMX, or to both MDM2 and MDMX. These peptides act as antagonists of MDM2 and/or MDMX, and they can be used in methods based on blocking inhibition of p53 activity by MDM2 and/or MDMX. Such methods can be used in the treatment of diseases, such as cancer.
  • the present invention is directed to each of the following peptide antagonists:
  • (v) targeted conjugates comprising any of (i)-(iv) conjugated to a targeting moiety, where the peptide antagonists bind with high affinity (a K d value of less than 1 mM) to MDM2 and/or MDMX.
  • the peptide antagonists have a nanomolar binding affinity for MDM2 or MDMX. In certain aspects of the invention, the peptide antagonists have a nanomolar binding affinity for both MDM2 and MDMX.
  • the peptide antagonists have a picomolar binding affinity for MDM2 or MDMX. In certain aspects of the invention, the peptide antagonists have a picomolar binding affinity for both MDM2 and MDMX.
  • the peptide antagonists have a nanomolar binding affinity for MDM2 and a picomolar binding affinity for MDMX.
  • the peptide antagonists have a picomolar binding affinity for MDM2 and a nanomolar binding affinity for MDMX.
  • the peptide antagonists block inhibition of p53 activity by MDM2 or MDMX. In certain aspects of the invention, the peptide antagonists block inhibition of p53 activity by both MDM2 and MDMX.
  • the peptides include those dodecapeptides set forth in SEQ ID NOs:l-27 and provided in Table 1.
  • the peptide (i) is the PMI-M3 peptide (LTFLEYWAQLMQ) set forth in SEQ ID NO:l.
  • Each of the peptides (i) binds with high affinity (a K d value of less than 1 mM) to MDM2, or MDMX, or both MDM2 and MDMX.
  • the variants include sequence variants of the peptides (i) that have one, two, three or four amino acid changes, where the changes are independently selected from additions, deletions, and substitutions.
  • the sequence variants include variants of the PMI-M3 peptide set forth in SEQ ID NO:l and variants of the PMI peptide (TSFAEYWNLLSP; SEQ ID NO:2).
  • the sequence variants include the PMI-2K peptide (KTSFAEYWNLLSPK; SEQ ID NO:51) and the M3-2K peptide (KLTFLE YW AQLMQK; SEQ ID NO:52).
  • Each of the sequence variants (ii) binds with high affinity (a K d value of less than 1 mM) to MDM2, or MDMX, or both MDM2 and MDMX.
  • the one, two, three, or four amino acid changes of a sequence variant may be made at any of the 12 positions in one of the peptides set forth in SEQ ID NOs:l-27, or at any of positions 1, 2, 4, 5, 6, 8, 9, 10, 11, and 12, or at any of positions 1, 2,
  • the changes may be individually selected from additions, deletions and substitutions.
  • lysine residues are added at the amino- and carboxy -termini of the peptides, such as at the amino- and carboxy-termini of any of the peptides set forth in SEQ ID NOs: 1-27.
  • the peptide antagonist is a sequence variant of the PMI-M3 peptide set forth in SEQ ID NO: 1 or the PMI peptide set forth in SEQ ID NO :2.
  • the D-peptide variants have one or more (including all) of the L-amino acids of the wild-type peptide, i.e. the peptides (i) set forth in SEQ ID NOs: 1-27 or the sequence variants (ii), replaced by a corresponding D-amino acid.
  • Each of the D-peptide variants (iii) binds with high affinity (a K d value of less than 1 mM) to MDM2, or MDMX, or both MDM2 and MDMX.
  • the side-chain stapled variants are prepared using the peptides (i) set forth in SEQ ID NOs: 1-27, the sequence variants (ii), or the D-peptide variants (iii) of the present invention.
  • Each of the side-chain stapled variants (iv) binds with high affinity (a K d value of less than 1 mIUI) to MDM2, or MDMX, or both MDM2 and MDMX.
  • the targeted conjugates are prepared using one or more of the peptides (i) set forth in SEQ ID NOs:l-27, the sequence variants (ii), the D-peptide variants (iii), or the side-chain stapled variants (iv) of the present invention.
  • Each of the targeted conjugates (v) binds with high affinity (a K d value of less than 1 mM) to MDM2, or MDMX, or both MDM2 and MDMX.
  • the targeted conjugates comprising an antibody as a targeting moiety, such as a humanized antibody.
  • the targeting moiety is an antibody having binding specificity for CD33.
  • the present invention also relates to pharmaceutical compositions comprising one or more of the peptide antagonists as defined herein and a pharmaceutically carrier and/or excipient.
  • the peptide antagonists are the peptides (i), the sequence variants (ii), the D- peptide variants (iii), the side-chain stapled variants (iv), and the targeted conjugates (v).
  • the carrier is a PEGylated liposome.
  • the carrier is a PEGylated liposome coated via a PEG spacer with a cyclic RGD peptide c(RGD°YK).
  • the present invention also relates to uses for the peptide antagonists and pharmaceutical compositions of the invention, such as in methods for interfering with the interactions between p53 and MDM2 and/or MDMX, and methods of treatment based thereon.
  • the peptide antagonists and pharmaceutical compositions may be used in methods for inhibiting binding of p53 by MDM2 and/or MDMX.
  • Such methods comprise contacting MDM2 and/or MDMX with one or more of the peptide antagonists of the invention in an amount sufficient to inhibiting binding of p53 by MDM2 and/or MDMX, thereby inhibiting binding of p53 by MDM2 and/or MDMX.
  • Such methods may be practiced in vitro , ex vivo , or in vivo.
  • the peptide antagonists and pharmaceutical compositions may be used in methods for activating p53. Such methods comprise contacting p53 bound by MDM2 and/or MDMX with one or more of the peptide antagonists of the invention in an amount sufficient to permit p53 activation, thereby activating p53. Such methods may be practiced in vitro , ex vivo , or in vivo. [0025] In a related aspect, the peptide antagonists and pharmaceutical compositions may be used in methods for blocking inhibition of p53 activity by MDM2 and/or MDMX.
  • Such methods comprise contacting MDM2 and/or MDMX with one or more of the peptide antagonists of the invention in an amount sufficient to block inhibition of p53 activity by MDM2 and/or MDMX, thereby blocking inhibition of p53 activity by MDM2 and/or MDMX.
  • Such methods may be practiced in vitro , ex vivo , or in vivo.
  • the peptide antagonists and pharmaceutical compositions may be used in methods for treating a disease or condition characterized by p53 dysregulation.
  • Such methods comprise administering a therapeutically effective amount of one or more of the peptide antagonists of the invention to a subject having a disease or condition characterized by p53 dysregulation, thereby treating a disease or condition characterized by p53 dysregulation in a subject.
  • Such methods also comprise administering a therapeutically effective amount of one or more of the pharmaceutical compositions of the invention to a subject having a disease or condition characterized by p53 dysregulation, thereby treating a disease or condition characterized by p53 dysregulation in a subject.
  • the cells of the disease or condition being treated lack TP 53 gene mutations. In other instances, the cells of the disease or condition being treated possess at least one TP53 gene mutation. In some instances, the cells of the disease or condition being treated over-express MDM2 and/or MDMX. In other instances, the cells of the disease or condition being treated possess elevated levels of MDM2 and/or MDMX.
  • Diseases and conditions characterized by p53 dysregulation include, but are not limited to, cancer, Li-Fraumeni syndrome, human papillomavirus (HPV) infections, and warts.
  • HPV human papillomavirus
  • the methods of the invention comprise administering a therapeutically effective amount of one or more of the peptide antagonists of the invention to a subject having cancer, thereby treating cancer in a subject. Such methods also comprise administering a therapeutically effective amount of one or more of the pharmaceutical compositions of the invention to a subject having cancer, thereby treating cancer in a subject.
  • the pharmaceutical compositions of the invention comprise one or more of the peptide antagonists of the invention and a pharmaceutically acceptable excipient and/or carrier.
  • carcinomas that may be treated used the methods of the invention include, but are not limited to, carcinoma - such as adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, and cancer of the skin, breast, prostate, bladder, vagina, cervix, uterus, liver, kidney, pancreas, spleen, lung, trachea, bronchi, colon, small intestine, stomach, esophagus, gall bladder; sarcoma - such as chondrosarcoma, Ewing’s sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcoma, and cancers of bone, cartilage, fat, muscle, vascular, and hematopoietic tissues; lymphoma and leukemia - such as acute myeloid leukemia (AML), mature B cell ne
  • the cells of the cancer being treated lack TP53 gene mutations. In other instances, the cells of the cancer being treated possess at least one TP53 gene mutation. In some instances, the cells of the cancer being treated over-express MDM2 and/or MDMX. In other instances, the cells of the cancer being treated possess elevated levels of MDM2 and/or MDMX.
  • the present invention also relates to (a) polynucleotides encoding the peptides (i), (b) polynucleotides encoding the sequence variants (ii), (c) polynucleotides encoding the D-peptide variants (iii), (d) polynucleotides encoding the side-chain stapled variants (iv), and (e) polynucleotides encoding some or all of the targeted conjugates (v).
  • the present invention also relates to vectors comprising one or more of the polynucleotides of the present invention.
  • the vectors are cloning vectors or expression vectors.
  • the present invention also relates to host cells comprising one or more of the vectors of the present invention.
  • the present invention provides methods of producing one or more of the peptides (i), the sequence variants (ii), the D-peptide variants (iii), the side-chain stapled variants (iv), or the targeted conjugates (v), comprising culturing one or more of the host cells under conditions promoting expression of the one or more peptides (i), the sequence variants (ii), the D-peptide variants (iii), the side-chain stapled variants (iv), or the targeted conjugates (v).
  • FIGURE 1 Quantification of the interactions of (25-109)MDM2 and (24-108)MDMX with representative PMI analogs by SPR- (A) and FP -based (B) competition assays.
  • A MDM2 at 50 nM or MDMX at 100 nM was incubated at 25 °C, pH 7.4, with varying concentrations of PMI-L10A or PMI-1-5, and the concentrations of unbound protein were quantified by SPR on a p53 transactivation domain (TAD) peptide-immobilized CM5 sensor chip.
  • TAD p53 transactivation domain
  • FIGURE 2 Pearson correlation analysis of MDM2 and MDMX interactions with PMI analogs.
  • A Plot of Ki ratios of 22 peptide analogs relative to PMI-L10A versus corresponding K d ratios, yielding correlation coefficients, r, of 0.878 and 0.951 for MDM2 and MDMX, respectively.
  • B Plot of relative Ki or K d ratios for MDMX versus those for MDM2, yielding respective correlation coefficients, r, of 0.806 for Ki and 0.938 for Kd.
  • FIGURE 3 Quantification of the interactions of MDM2 and MDMX with PMI and representative analogs by SPR- and FP -based competition assays.
  • A MDM2 at 50 nM with PMI, PMI-L10A, PMI-M2, PMI-M3, PMI-M4, and PMI-M5, and
  • B MDMX at 50 or 100 nM with various PMI peptides as quantified by SPR.
  • C MDM2, and
  • D MDMX at 50 nM with various PMI peptides as quantified by FP.
  • the experimental details were essentially as described in the legend of Figure 1 and the Experimental Section. Each curve is the mean of three independent measurements.
  • FIGURE 5 Crystal structure of MDM2 and MDMX in complex with PMI-M3.
  • the electrostatic potential is displayed over the molecular surfaces of MDM2/MDMX colored red for negative, blue for positive and white for apolar.
  • the PMI-M3 peptide is shown in a ribbon and stick representation. Residues mutated in PMI-M3 (as compared to PMI sequence) are shown in yellow. 12 and 11 residues of PMI-M3 are resolved in the MDM2-PMI-M3 and MDMX-PMI- M3 crystal structures, respectively.
  • FIGURE 6 PMI-M3 versus PMI binding to MDM2 and MDMX.
  • A The MDM2- PMI-M3/PMI and MDMX-PMI-M3 /PMI complex interfaces.
  • the PMI-M3 and PMI peptides are shown as ribbon-ball-stick representations. For clarity only side chains of some residues of MDM2 and MDMX are shown as ball-sticks.
  • the PMI-M3 binding doesn’t involve V49, L102 and L106 of MDMX, which are engaged in PMI binding.
  • a new contact to Lys50 of MDMX is formed to accommodate Metl 1 of PMI-M3.
  • There are also two direct protein-peptide H-bonds formed at the MDMX-PMI-M3 contact interface (Q71 Oe ⁇ to F3 N, M53 O to W3 Ne ⁇ ) as compared to three formed at the MDMX-PMI interface (Q71 Oe ⁇ to F3 N, M53 O to W3 Ne ⁇ and Y99 (OH) to SI 1 O).
  • B Analysis of the peptide-binding interface.
  • FIGURE 7 Relative positioning of PMI -M3 and PMI peptides within the MDM2 and MDMX binding pockets. The complex structures were superimposed based on MDM2 (left) and MDMX (right). Only the backbones of the PMI-M3 and PMI peptides are shown with side chains as ball-stick representations for interacting residues. Molecular surfaces are displayed for the MDM2/MDMX molecules. The distances between main chain atoms of corresponding N-, C- terminal residues, Phe3, Trp7 and LeulO of PMI-M3 and PMI were measured and shown in blue. [0042] FIGURE 8.
  • Targeted molecular therapy is superior to genotoxic chemotherapy as the former aims to kill tumor cells while sparing normal cells by targeting specific proteins or signaling pathways that either promote or suppress tumorigenesis.
  • One of the most promising molecular targets for anticancer drug discovery is p53 [10,11] , a transcription factor that induces powerful growth inhibitory and apoptotic responses to cellular stresses such as DNA damage and oncogene activation, playing a pivotal role in maintaining genetic stability and preventing damaged cells from becoming cancerous [12,13] .
  • impairment of the p53 pathway is a hallmark of almost all human cancers where either the TP53 gene is mutated (in -50% cases) or the wild-type p53 protein is functionally inactivated predominantly by the E3 ubiquitin ligase MDM2 and/or its homolog MDMX 0 4 - 24]
  • the human form of MDM2 is 491 amino acids and comprises three major domains, including (1) an N-terminal domain that binds the N-terminal transactivation peptide of p53 to block p53 trans-activating expression of responsive genes [25,26] , (2) a central domain rich in acidic residues that provides a second binding site for p53 to enhance MDM2 -mediated p53 ubiquitination and (3) a C-terminal Zn 2+ -binding RING (really interesting wewgene) domain that functions as an E3 ubiquitin ligase to facilitate the transfer of E2 -conjugated ubiquitin molecules to Lys residues of p53 [29 32] .
  • an N-terminal domain that binds the N-terminal transactivation peptide of p53 to block p53 trans-activating expression of responsive genes [25,26]
  • a central domain rich in acidic residues that provides a second binding site for p53 to enhance MDM2 -
  • MDMX (also known as MDM4) was first discovered as a p53 -binding protein in cells. Structurally related to MDM2, human MDMX consists of 490 amino acids and possesses domain structures arranged similarly to MDM2 [33] . In particular, the N-terminal p53-binding domains of MDM2 and MDMX are highly homologous with an over 50% sequence identity. MDMX inhibits p53 transactivation as an effective transcriptional antagonist in the nucleus [33] . Unlike MDM2, however, MDMX lacks E3 ubiquitin ligase activity and is not transcriptionally activated by p53 [23] .
  • MDM2 and MDMX function as a heterodimer formed via their RING domains to augment p53 degradation as the heterodimer is thought to be a more productive structure than an MDM2 homodimer for E2 recruitment 0 4 - 37]
  • MDM2 and MDMX are amplified and/or over-expressed, directly contributing to p53 inactivation and tumor development and progression23 24
  • Nu erous studies have demonstrated that tumor cells with wild-type p53 status are highly susceptible to antagonists of MDM2/MDMX, validating MDM2/MDMX antagonism as a viable therapeutic paradigm 117421 .
  • MDM2 and MDMX are non-redundant negative regulators of p53 l
  • the present invention is directed to peptide antagonists of MDM2 and MDMX.
  • the peptide antagonists of the invention include antagonists of MDM2 or MDMX, as well as dual-specificity antagonists where the same peptide is an antagonist of both MDM2 and MDMX.
  • These peptide antagonists can be used in methods based on blocking inhibition of p53 activity by MDM2 and/or MDMX. Such methods can be used in the treatment of diseases, such as cancer.
  • the “peptide antagonists” of the invention encompass the peptides (i), sequence variants (ii), D-peptide variants (iii), side-chain stapled variants (iv), and targeted conjugates (v) as further defined below.
  • Each of the peptide antagonists of the invention binds with high affinity to MDM2, or MDMX, or both MDM2 and MDMX.
  • the peptide antagonists of the invention include each of the dodecapeptides shown in Table 1 and set forth in SEQ ID NOs:l-27. As shown in the Examples, each of these peptides exhibits high affinity binding to either MDM2 or MDMX, or to both MDM2 and MDMX. Table 1 [0051] One of the dodecapeptides provided in Table 1 shows particularly high affinity for both MDM2 and MDMX, namely the PMI-M3 peptide (LTFLEYWAQLMQ; SEQ ID NO:l).
  • PMI- M3 binds to the p53 -binding domains of MDM2 ( 25 109 MDM2) and MDMX ( 24 108 MDMX) at affinities of 21 and 253 pM, respectively, as determined by surface plasmon resonance (SPR)- based competition binding assays.
  • SPR surface plasmon resonance
  • Structural analysis by X-ray crystallography of PMI in complex with MDM2 and MDMX reveals that the peptide antagonist adopts an a-helical conformation, burying four critical hydrophobic residues, namely Phe3, Tyr6, Trp7 and LeulO, in the p53-binding cavity of MDM2 or MDMX.
  • PMI-M3 peptide is thus an excellent example of a peptide antagonist of the invention that has dual-specificity.
  • the peptide antagonists of the invention include sequence variants of the dodecapeptides shown in Table 1 and set forth in SEQ ID NOs:l-27.
  • the sequence variants of the invention include variants having one, two, three, or four amino acid changes, in comparison to the amino acid sequence of the wild-type peptide (i.e., one of SEQ ID NOs:l-27) upon which they are based, where the changes are independently selected from additions, deletions, and substitutions.
  • Each of the sequence variants binds with high affinity to MDM2, or MDMX, or both MDM2 and MDMX.
  • Each of the amino acid substitutions may independently be: (i) a change in the enantiomeric configuration of the amino acid (i.e., D or L), (ii) an amino acid substitution, such as a conservative amino acid substitution or a non-conservative amino acid substitution, where the substituted amino acid has the same enantiomeric configuration, or (iii) an amino acid substitution, such as a conservative amino acid substitution or a non-conservative amino acid substitution, where the substituted amino acid has a different enantiomeric configuration.
  • the deletions may be deletions of one or more consecutive amino acids from the amino terminus or the carboxy terminus of the peptide, or one or more consecutive amino acids from within the peptide.
  • each of the amino acid additions may have the same or different enantiomeric configuration (i.e., D or L) in comparison to the majority configuration of the peptide.
  • the one, two, three, or four amino acid changes of a sequence variant may be made at any of the 12 positions in one of the peptides set forth in SEQ ID NOs:l-27.
  • the peptide is the PMI peptide set forth in SEQ ID NO:2.
  • the changes are individually selected from additions, deletions and substitutions. The substitutions may be conservative or non-conservative substitutions.
  • the one, two, three, or four amino acid changes of a sequence variant may be made at any of positions 1, 2, 4, 5, 6, 8, 9, 10, 11, and 12 in one of the peptides set forth in SEQ ID NOs:l-27.
  • the peptide is the PMI peptide set forth in SEQ ID NO:2.
  • the one, two, three, or four amino acid changes of a sequence variant may be made at any of positions 1, 2, 4, 5, 8, 9, 11, and 12 in one of the peptides set forth in SEQ ID NOs:l-27.
  • the peptide is the PMI peptide set forth in SEQ ID NO:2.
  • the one, two, three, or four amino acid changes of a sequence variant may be made at any of positions 1, 2, 4, 8, 9, 10, 11, and 12 in one of the peptides set forth in SEQ ID NOs:l-27.
  • the peptide is the PMI peptide set forth in SEQ ID NO:2.
  • the one, two, three, or four amino acid changes of a sequence variant may be made at any of positions 1, 2, 4, 8, 9, 11, and 12 in one of the peptides set forth in SEQ ID NOs:l-27.
  • the peptide is the PMI peptide set forth in SEQ ID NO:2.
  • the one, two, three, or four amino acid changes of a sequence variant may be made at any of positions 2, 4, 5, 8, 9, 11, and 12 in one of the peptides set forth in SEQ ID NOs:l-27.
  • the peptide is the PMI peptide set forth in SEQ ID NO:2.
  • the one, two, three, or four amino acid changes of a sequence variant may be made at any of positions 1, 2, 5, 8, 9, 11, and 12 in one of the peptides set forth in SEQ ID NOs:l-27.
  • the peptide is the PMI peptide set forth in SEQ ID NO:2.
  • the one, two, three, or four amino acid changes of a sequence variant may be made at any of positions 1, 2, 4, 5, 8, 9, and 12 in one of the peptides set forth in SEQ ID NOs: 1-27.
  • the peptide is the PMI peptide set forth in SEQ ID NO:2.
  • single amino acids are added to the amino- and/or carboxy-termini of any of the peptides set forth in SEQ ID NOs: 1-27.
  • the peptide is the PMI-M3 set forth in SEQ ID NO: 1 or the PMI peptide set forth in SEQ ID NO:2.
  • Exemplary amino acids to be added include lysine residues.
  • the sequence variants include the PMI-2K peptide (KTSFAEYWNLLSPK; SEQ ID NO:51) and the M3-2K peptide (KLTFLEYWAQLMQK; SEQ ID NO:52).
  • Mirror image phage display is a technique developed by Kim and co-workers that screens a phage-displayed L-peptide library against the D-enantiomeric form of a natural protein target for the discovery of proteolysis-resistant D-peptide ligands [7,8] . After enantiomeric inversion, the resultant D-peptide ligand, for reasons of symmetry, binds specifically to the native L-protein with the same affinity.
  • Mirror image phage display takes advantage of vast biodiversity presented by a phage library and affords an elegant and powerful tool for the discovery of potent and proteolysis-resistant D-peptide for therapeutic applications [50,57,58] .
  • D-peptide composed entirely of D-amino acids are completely resistant to proteolytic degradation due to an exceedingly high energy -barrier, afforded by steric incompatibility, to the transition state of the enzyme -substrate complex.
  • D-peptide drugs are fully stable in vivo , orally administrable, and much less immunogenic due to full resistance to proteasomal processing for antigen presentation [9] .
  • the peptide antagonists of the invention include D-peptide variants of the peptides shown in Table 1 (and set forth in SEQ ID NOs: 1-27) and the sequence variants thereof as defined herein. Each of the D-peptide variants binds with high affinity to MDM2, or MDMX, or both MDM2 and MDMX.
  • the D-peptide variants of the invention have at least one D-amino acid in place of the corresponding L-amino acid found at the corresponding position in the peptide or sequence variant upon which the D-peptide variant is based.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or all 12 of the L-amino acids in a peptide or sequence variant of the invention is replaced by a corresponding D-amino acid.
  • each of the L-amino acids in a peptide or sequence variant of the invention is replaced by a corresponding D-amino acid.
  • “corresponding” amino acid means the amino acid of the same identity, i.e. leucine, but the other enantiomeric version of that amino acid.
  • D-leucine “corresponds” to L-leucine
  • L- leucine corresponds” to D-leucine.
  • SAH-p53-8 The stapled p53 peptide, termed SAH- p53-8, bound MDM2 at an affinity of 55 nM as determined by fluorescence polarization, and induced p53-dependent apoptosis in SJSA-1 cells over-expressing MDM2 [1] . More recently, Bernal et al. reported that SAH-p53-8 bound MDMX with a K d value of 2.3 nM, a 25-fold greater binding preference for MDMX over MDM2 [6] .
  • the SAH-p53-8 peptide effectively induced p53-dependent killing of tumor cells by targeting MDM2, MDMX, or both, and significantly suppressed tumor growth in experimental animals bearing JEG-3 xenografts - an MDMX-expressing and Nutlin-3 -resistant cancer [6] .
  • the peptide antagonists of the invention include side-chain stapled variants of the peptides shown in Table 1 (and set forth in SEQ ID NOs:l-27), the sequence variants thereof, and the D-peptide variants thereof, as defined herein.
  • Each of the side-chain stapled variants binds with high affinity to MDM2, or MDMX, or both MDM2 and MDMX.
  • the side-chain stapled variants have a core comprised of amino acids linked by amide bonds, but with two of the amino acids substituted for by non-classical amino acids.
  • the positions in which the non-classical amino acids are used are positions 5+9, 5+12, 6+9, 6+12, 8+12 or 9+12.
  • the non-classical amino acids that may be used in the production of the side- chain stapled variants are any that contains an olefmic side chain.
  • Suitable non-classical amino acids include, but are not limited to, (S)-2-(7’-octenyl)alanine, (R)-2-(7’-octenyl)alanine, (S)-2- (4’-pentenyl)alanine, (R)-2-(4’-pentenyl)alanine, and D-omithine, and amino acids with olefmic side chains.
  • Peptide antagonists of MDM2/MDMX must traverse the cell membrane to activate p53 in vitro and in vivo. In the absence of a suitable delivery vehicle, however, cellular uptake of peptides may be inefficient and can constitute a functional obstacle limiting their therapeutic value. Liposomes and micelles can serve as a delivery vehicle for peptide drugs, but they are non-specific and inefficient.
  • Arg-rich cell -penetrating peptides are capable of promoting cellular uptake of covalently attached proteins and peptides to the cytoplasm and nuclei of many cell types [ 60 65] , they are not only non-specific and inefficient but may be cytotoxic when conjugated peptides are activators of p53 l55 - w, , - ,xl Lack of tumor-targeting specificity is also shared by hydrocarbon-stapled peptides despite their ability to traverse the cell membrane. Further, peptides alone, even proteolytically stable in vivo , may not have a sufficiently long circulation half-life due to their small size. A clinically proven solution to overcoming these obstacles is targeting moiety-drug conjugates.
  • the peptide antagonists of the invention thus include targeted conjugates comprising
  • each of the targeted conjugates binds with high affinity to MDM2, or MDMX, or both MDM2 and MDMX.
  • the targeting moieties that may be used in the targeted conjugates are only limited in that they have the ability to bind and/or induce entry of the peptide antagonists of the invention into a target cell, such as a cancer cell or other cell that exhibits p53 dysregulation.
  • Acceptable targeting moieties include antibodies and functional fragments thereof, ligands, aptamers, nucleic acids, polynucleotides, and other protein and peptide species. Suitable types of antibodies include humanized antibody, chimeric antibodies, and fully human antibodies.
  • Suitable functional fragments are those that retain the binding specificity of the antibody from which they are derived and they include, but are not limited to, Fab fragments, F(ab')2 fragments, single chain Fv (scFv) antibodies, and fragments produced by a Fab expression library, as well as bi specific antibody and triple-specific antibodies.
  • Humanized antibodies are those antibodies where a human antibody has been engineered to contain non-human complementarity-determining regions (CDRs) derived from an antibody produced in a non-human host against a selected antigen.
  • CDRs complementarity-determining regions
  • Chimeric antibodies are those where an antigen binding region (e.g., F(ab’)2 or hypervariable region) of a non-human antibody is transferred into the framework of a human antibody by recombinant DNA techniques. Techniques developed for the production of such antibodies include the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity. Such techniques are also well known [81 83] .
  • an antigen binding region e.g., F(ab’)2 or hypervariable region
  • Antibody fragments such as F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity [84] .
  • the targeting moiety is an antibody having binding specificity for CD33.
  • AML acute myelogenous leukemia
  • CD33 is not expressed on normal hematopoietic pluripotent stem cells, making the antigen an attractive candidate for targeted therapy of AML [51,52] .
  • anti-CD33 mAbs rapidly internalize upon binding to AML cells, they have been used as a delivery vehicle for conjugated cytotoxic drugs to treat AML patients [ 51 , 53 , 54 ]
  • p re ii minai y studies show the efficiency of an anti-CD33 mAh in delivery of peptide activators of p53 into AML cells.
  • the peptide antagonists and the targeting moieties may be conjugated through means typically used to conjugate peptides with the noted types of targeting moieties.
  • means typically used to conjugate peptides with the noted types of targeting moieties A variety of robust chemistries are available to conjugate peptide antagonists and the targeting moieties [69] .
  • Suitable means include non-covalent intermolecular interactions, for example through hydrogen bonding, electrostatic interactions, hydrophobic and van der Waals forces.
  • disulfide bind formation may be used.
  • a peptide antagonist is linked to antibody, such as the anti-CD33 antibody mentioned above, the S-S bond is readily cleavable in the reducing intracellular environment, ensuring the release of the peptide cargo from an endocytosed peptide-antibody conjugate.
  • An extra N-terminal Cys residue, preceding a GlyGly spacer, can be linked to the peptide antagonist of interest via solid phase peptide synthesis, and then a disulfide-linked peptide dimer can be prepared via air or DMSO oxidation in aqueous buffer, followed by HPLC purification to homogeneity.
  • the dissociation constant (K d ) is commonly used to describe the affinity between two binding partners, such as a drug and the protein targeted by the drug. It is a measure of how tightly the drug binds to its cognate protein. Affinities between the two partners are influenced by non-covalent intermolecular interactions, such as hydrogen bonding, electrostatic interactions, hydrophobic and van der Waals forces.
  • the dissociation constant is expressed as molar units (M), which corresponds to the concentration of drug when the binding site on a particular protein is half occupied, i.e. the concentration of drug when the concentration of drug-bound protein is equal to the concentration of unbound protein.
  • M molar units
  • the smaller the dissociation constant the greater the affinity of the drug for the protein. For example, a drug with a nanomolar (nM) dissociation constant binds more tightly to a particular protein than a drug with a micromolar (mM) dissociation constant.
  • Each of the peptide antagonists of the invention binds with high affinity to MDM2, or MDMX, or both MDM2 and MDMX.
  • high affinity means a K d value of less than 1 mM, i.e., K d value in the nanomolar range or lower.
  • K d values for the peptide antagonists of the invention include a K d value of less about 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 60 nM, 80 nM, 100 nM, 120 nM, 140 nM, 160 nM, 180 nM, 200 nM, 220 nM, 240 nM, 260 nM, 280 nM, 300 nM, 320 nM, 340 nM, 360 nM, 380 nM, 400 nM, 420 nM, 440 nM, 460 nM, 480 nM, 500 nM, 520 nM, 540 nM, 560 nM, 580 nM, 600 nM, 620 nM, 640 nM, 660 nM, 680 nM,
  • K d values for the peptide antagonists of the invention thus also include a K d value of less about 1 pM, 5 pM, 10 pM, 25 pM, 50 pM, 60 pM, 80 pM, 100 pM, 120 pM, 140 pM, 160 pM, 180 pM, 200 pM, 220 pM, 240 pM, 260 pM, 280 pM, 300 pM, 320 pM, 340 pM, 360 pM, 380 pM, 400 pM, 420 pM, 440 pM, 460 pM, 480 pM, 500 pM, 520 pM, 540 pM, 560 pM, 580 pM, 600 pM,
  • the high affinity of the peptide antagonists may also be understood in terms of a range, and includes a K d value from about 10 to 500 nM, from about 20 to 400 nM, from about 25 to 300 nM, from about 30 to 220 nM, from about 10 to 1000 pM, from about 10 to 500 pM, from about 20 to 400 pM, from about 25 to 300 pM, from about 30 to 220 pM, from about 10 to 100 pM, from about 1 to 25 pM, and from about 1 to 15 pM.
  • the peptide antagonists have a nanomolar binding affinity for MDM2 or MDMX. In certain aspects of the invention, the peptide antagonists have a nanomolar binding affinity for both MDM2 and MDMX.
  • the peptide antagonists have a picomolar binding affinity for MDM2 or MDMX. In certain aspects of the invention, the peptide antagonists have a picomolar binding affinity for both MDM2 and MDMX.
  • the peptide antagonists have a nanomolar binding affinity for MDM2 and a picomolar binding affinity for MDMX.
  • the peptide antagonists have a picomolar binding affinity for MDM2 and a nanomolar binding affinity for MDMX.
  • the peptide antagonists block inhibition of p53 activity by MDM2 or MDMX. In certain aspects of the invention, the peptide antagonists block inhibition of p53 activity by both MDM2 and MDMX.
  • the peptide antagonists of the present invention preferably bind to MDM2 in the p53 binding pocket, which is located at the N-terminus of MDM2 and encompasses, approximately, amino acids 17-124 of the human MDM2 amino acid sequence [73] .
  • the peptide antagonists of the present invention also preferably bind to MDMX in the p53 binding pocket, which is located at the N-terminus of MDMX and encompasses, approximately, amino acids 1-185 of the human MDMX amino acid sequence [74] .
  • the present invention also encompasses: (i) polynucleotide sequences encoding peptide antagonists of the present invention, (ii) vectors into which the polynucleotide sequences are inserted, (iii) host cells genetically engineered (transduced, transformed, or transfected) with the vectors, (iv) methods of culturing the host cells under conditions promoting production of the peptide antagonists encoded by the polynucleotide sequences, and (v) methods of isolating the expressed peptide antagonists from the culture media and host cells.
  • the polynucleotide sequences may be in the form of RNA or DNA, where DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encodes the peptide antagonists may vary due to the redundancy or degeneracy of the genetic code, yet encode the same peptide.
  • the present invention also includes polynucleotide sequences wherein the coding sequence for the peptide antagonist may be fused in the same reading frame to a polynucleotide which encodes a peptide or protein that aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a peptide from the cell.
  • the leader sequence is cleaved by the host cell to form the mature form of the peptide antagonist.
  • the polynucleotide sequences of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of a peptide antagonist of the invention.
  • the marker sequence may be, but is not limited to, a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature peptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein [78] .
  • the vector into which the polynucleotide sequence is inserted may any one of a variety of cloning vectors or expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, non -chromosomal and synthetic DNA sequences, e.g, derivatives of SV40, bacterial plasmids, phage DNA, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. Any other plasmid or vector may be used so long as it is replicable and viable in the host cell.
  • the vector containing the polynucleotide sequence may contain an appropriate promoter or control sequence.
  • the vector may contain at least one selectable marker gene to provide a phenotypic trait for selection of transformed host cells.
  • markers include dihydrofolate reductase (DHFR) or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance for culturing in E. coli and other bacteria.
  • Representative examples of appropriate host cells include but are not limited to: bacterial cells, such as E.
  • Salmonella typhimurium a group consisting of Salmonella typhimurium , Salmonella typhimurium , fungal cells, such as yeast, insect cells, such as Drosophila S2 and Spodoptera Sfl9, animal cells such as CHO, COS, and Bowes melanoma; and plant cells.
  • the peptide antagonists can be recovered and purified from host cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. It is preferred to have low concentrations (approximately 0.1 15 mM) of calcium ion present during purification [75] . Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. High performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • the peptide antagonists of the invention may also be synthetically produced by conventional peptide synthesizers [76,77] . Furthermore, such techniques allow the introduction of non-classical amino acids or chemical amino acid analogs into the peptides, thus producing the variants of the present invention.
  • Non-classical amino acids include, but are not limited to, the D- amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2- amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b- alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general, and unnatural amino acids with olefmic
  • the invention encompasses peptide antagonists that are modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBEE acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.
  • Additional post-translational modifications encompassed by the invention include, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends, attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression.
  • the peptide antagonists may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the peptide.
  • the present invention also relates to pharmaceutical compositions comprising the peptide antagonists of the present invention.
  • the peptide antagonists may be used in combination with any suitable pharmaceutical excipient or carrier.
  • Such pharmaceutical compositions thus comprise one or more of the peptide antagonists of the invention and a pharmaceutically acceptable excipient(s) and/or carrier(s).
  • the specific formulation will suit the mode of administration.
  • Excipients included in the pharmaceutical compositions have different purposes depending, for example on the nature of the peptide antagonist and the mode of administration.
  • Examples of generally-used excipients include, without limitation: saline, buffered saline, dextrose, water-for-infection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, lubricating agents (such as talc or silica, and fats, such as vegetable stearin, magnesium stearate or stearic acid), emulsifiers, suspending or viscosity agents, inert diluents, fillers (such as cellulose, dibasic calcium phosphate, vegetable fats and oils, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, and magnesium stearate), disintegrating agents (such as crosslinked polyvinyl pyrrol
  • Carriers are compounds and substances that improve and/or prolong the delivery of an active ingredient to a subject in the context of a pharmaceutical composition.
  • Carrier may serve to prolong the in vivo activity of a peptide antagonist or slow the release of the peptide antagonist in a subject, using controlled-release technologies. Carriers may also decrease metabolism in a subject and/or reduce the toxicity of the antagonist. Carrier can also be used to target the delivery of the peptide antagonist to particular cells or tissues in a subject.
  • Common carriers include fat emulsions, lipids, PEGylated phospholids, PEGylated liposomes, PEGylated liposomes coated via a PEG spacer with a cyclic RGD peptide C(RGD D YK), liposomes and lipospheres, microspheres (including those made of biodegradable polymers or albumin), polymer matrices, biocompatible polymers, protein-DNA complexes, protein conjugates, erythrocytes, vesicles, nanoparticles, and side-chains for hydro-carbon stapling.
  • the aforementioned carriers can also be used to increase cell membrane permeability of the peptide antagonists of the invention.
  • carriers may also be used in compositions for other uses, such as research uses in vitro (e.g., for delivery to cultured cells) and/or in vivo.
  • compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions).
  • Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof.
  • Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.
  • excipients which may be used include for example water, polyols and sugars.
  • suspensions oils e.g. vegetable oils
  • delayed release preparations may be advantageous and compositions which can deliver the peptide antagonists in a delayed or controlled release manner may also be prepared.
  • Prolonged gastric residence brings with it the problem of degradation by the enzymes present in the stomach and so enteric-coated capsules may also be prepared by standard techniques in the art where the active substance for release lower down in the gastro-intestinal tract.
  • compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • the active ingredient may be delivered from the patch by iontophoresis as described [98] .
  • compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.
  • the active ingredient When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base.
  • the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
  • Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.
  • Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.
  • compositions adapted for rectal administration may be presented as suppositories or enemas.
  • compositions adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.
  • compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators.
  • Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
  • compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti -oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • Excipients which may be used for injectable solutions include water-for-injection, alcohols, polyols, glycerine and vegetable oils, for example.
  • compositions may be presented in unit-dose or multi -dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water or saline for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • the pharmaceutical compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. They may also contain therapeutically-active agents in addition to the substance of the present invention.
  • the pharmaceutical compositions may be administered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramuscular, intratumor, subcutaneous, intranasal or intradermal routes.
  • the pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication.
  • the pharmaceutical compositions are administered in an amount of comprising at least about 0.1 mg of peptide antagonist per kg body weight to about 100 mg/kg body weight. In most cases, the dosage is from about 1 mg/kg to about 10 mg/kg body weight, taking into account the routes of administration, symptoms, etc.
  • the present invention also relates to practical uses for the peptide antagonists of the invention. As discussed below, such uses include in vitro , ex vivo , and in vivo uses.
  • the peptide antagonists may be used in methods for inhibiting binding of p53 by MDM2 and/or MDMX. Such methods comprise contacting MDM2 and/or MDMX with one or more of the peptide antagonists of the invention in an amount sufficient to inhibiting binding of p53 by MDM2 and/or MDMX. Such methods may be practiced in vitro , ex vivo , or in vivo.
  • the peptide antagonists may be used in methods for activating p53. Such methods comprise contacting p53 bound by MDM2 and/or MDMX with one or more of the peptide antagonists of the invention in an amount sufficient to permit p53 activation. Such methods may be practiced in vitro , ex vivo , or in vivo.
  • the peptide antagonists may be used in methods for blocking inhibition of p53 activity by MDM2 and/or MDMX. Such methods comprise contacting MDM2 and/or MDMX with one or more of the peptide antagonists of the invention in an amount sufficient to block inhibition of p53 activity by MDM2 and/or MDMX. Such methods may be practiced in vitro, ex vivo , or in vivo.
  • the peptide antagonists may be used in methods for treating a disease or condition characterized by p53 dysregulation.
  • Such methods comprise administering a therapeutically effective amount of one or more of the peptide antagonists of the invention to a subject having a disease or condition characterized by p53 dysregulation, thereby treating a disease or condition characterized by p53 dysregulation in a subject.
  • Such methods also comprise administering a therapeutically effective amount of one or more of the pharmaceutical compositions of the invention to a subject having a disease or condition characterized by p53 dysregulation, thereby treating a disease or condition characterized by p53 dysregulation in a subject.
  • the pharmaceutical compositions of the invention comprise one or more of the peptide antagonists of the invention and a pharmaceutically acceptable excipient and/or carrier.
  • the cells of the disease or condition being treated lack TP53 gene mutations. In other instances, the cells of the disease or condition being treated possess at least one TP53 gene mutation. In some instances, the cells of the disease or condition being treated over-express MDM2 and/or MDMX. In other instances, the cells of the disease or condition being treated possess elevated levels of MDM2 and/or MDMX.
  • Diseases and conditions characterized by p53 dysregulation include, but are not limited to, cancer, Li-Fraumeni syndrome, human papillomavirus (HPV) infections, and warts.
  • the methods of the invention comprise administering a therapeutically effective amount of one or more of the peptide antagonists of the invention to a subject having cancer, thereby treating cancer in a subject. Such methods also comprise administering a therapeutically effective amount of one or more of the pharmaceutical compositions of the invention to a subject having cancer, thereby treating cancer in a subject.
  • the pharmaceutical compositions of the invention comprise one or more of the peptide antagonists of the invention and a pharmaceutically acceptable excipient and/or carrier.
  • cancer is intended to be broadly interpreted and it encompasses all aspects of abnormal cell growth and/or cell division.
  • carcinoma including but not limited to adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, and cancer of the skin, breast, prostate, bladder, vagina, cervix, uterus, liver, kidney, pancreas, spleen, lung, trachea, bronchi, colon, small intestine, stomach, esophagus, gall bladder; sarcoma, including but not limited to chondrosarcoma, Ewing’s sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcoma, and cancers of bone, cartilage, fat, muscle, vascular, and hematopoietic tissues; lymphoma and leukemia, including but not limited to acute myeloid leukemia (AML), mature B cell neoplasms, such as chronic lymphocytic leukemia
  • the cells of the cancer being treated lack TP53 gene mutations. In other instances, the cells of the cancer being treated possess at least one TP53 gene mutation. In some instances, the cells of the cancer being treated over-express MDM2 and/or MDMX. In other instances, the cells of the cancer being treated possess elevated levels of MDM2 and/or MDMX.
  • a therapeutically effective amount of a peptide antagonist or pharmaceutical composition will vary between wide limits, depending upon the location, source, identity, extent and severity of the cancer, or other disease or condition, as well as the age and condition of the individual to be treated, etc. A physician will ultimately determine appropriate dosages to be used. However, a therapeutically effective amount is an amount sufficient to treat the disease or condition, such as cancer, in the subject receiving treatment.
  • Therapeutically effective amounts include from about 0.1 mg of peptide antagonist per kg body weight to about 100 mg/kg body weight.
  • Other therapeutically effective amounts include from about 0.1 mg/kg to about 10 mg/kg body weight; from about 1 mg/kg to about 10 mg/kg body weight; and from about 10 mg/kg to about 100 mg/kg body weight.
  • administer and “administering” are used to mean introducing at least one peptide antagonist, or a pharmaceutical composition comprising at least one peptide antagonist, into a subject.
  • the peptide antagonist is administered at, or after the diagnosis of an abnormal cell growth, such as a tumor.
  • the therapeutic administration of the peptide antagonist serves to inhibit cell growth of the tumor or abnormal cell growth.
  • dose refers to physically discrete units that contain a predetermined quantity of active ingredient (e.g., a peptide antagonist) calculated to produce a desired therapeutic effect (e.g., death of cancer cells).
  • active ingredient e.g., a peptide antagonist
  • desired therapeutic effect e.g., death of cancer cells.
  • the terms “treat”, “treating”, and “treatment” have their ordinary and customary meanings, and include one or more of: blocking, ameliorating, or decreasing in severity and/or frequency a symptom of a disease or condition, such as cancer, in a subject, and/or inhibiting the growth, division, spread, or proliferation of cells, including cancer cells, or progression of cancer (e.g., emergence of new tumors) in a subject.
  • Treatment means blocking, ameliorating, decreasing, or inhibiting by about 1% to about 100% versus a subject to which a peptide antagonist has not been administered.
  • the blocking, ameliorating, decreasing, or inhibiting is about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%,
  • an amount sufficient to inhibiting binding of p53 by MDM2 and/or MDMX means an amount sufficient to inhibit binding by about 1% to about 100% versus inhibiting binding in the absence of a peptide antagonist of the invention.
  • inhibiting is about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1% versus binding between the binding partners in the absence of a peptide antagonist.
  • an amount sufficient to permit p53 activation means an amount sufficient to permit p53 activation by about 1% to about 100% versus permitting p53 activation in the absence of a peptide antagonist of the invention.
  • permitting is about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1% versus p53 activation in the absence of a peptide antagonist.
  • an amount sufficient to block inhibition of p53 activity by MDM2 and/or MDMX means an amount sufficient to block inhibition of p53 activity by MDM2 and/or MDMX by about 1% to about 100% versus blocking inhibition of p53 activity in the absence of a peptide antagonist of the invention.
  • blocking is about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1% versus blocking inhibition of p53 activity in the absence of a peptide antagonist.
  • the peptide antagonists may also be employed in accordance with the present invention by expression of the antagonists in vivo , i.e., via gene therapy.
  • the use of the peptides or compositions in a gene therapy setting is also considered to be a type of “administration” of the peptides for the purposes of the present invention.
  • the subject receiving treatment is a human or non-human animal, e.g., a non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal.
  • the subject is a human.
  • the invention also provides a kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention, such as a container filled with a pharmaceutical composition comprising a peptide antagonist and a carrier or excipient.
  • a container filled with a pharmaceutical composition comprising a peptide antagonist and a carrier or excipient Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the pharmaceutical compositions may be employed in conjunction with other therapeutic compounds.
  • concentration of unbound MDM2 or MDMX in solution was deduced, based on p53 -association RU values, from a calibration curve established by RU measurements of different concentrations of MDM2/MDMX injected alone. Three independent experiments, each in duplicate, were performed.
  • FP Fluorescence polarization
  • a FP -based competitive binding assay was established using 25 109 MDM2, 24 108 MDMX and a fluorescently tagged PMI peptide.
  • Unlabeled PMI competed with TAMRA-PMI for MDM2/MDMX binding, based on which the K d values of TAMRA-PMI with MDM2 and MDMX were determined by changes in FP to be 0.62 and 0.72 nM, respectively.
  • the binding of Nutlin-3 to MDM2 and MDMX was quantified, yielding respective Ki values of 5.1 nM and 1.54 M, similar to the values reported in the literature.
  • ITC Isothermal titration calorimetry
  • Direct protein-peptide interactions were quantified at 25 °C in PBS, pH 7.4, using a MicroCal 2000 microcalorimeter (GE Healthcare).
  • a typical experiment involved 20 stepwise injections of 2 pL of 100 pM peptide solution into an ITC cell containing 10-20 pM protein solution.
  • a reference set of injections of peptide was made in a separate experiment into the buffer alone.
  • Data were analyzed using the Microcal Origin program, yielding the binding affinity, stoichiometry and other thermodynamic parameters. Two independent experiments were performed.
  • micro crystals were then reproduced and optimized using the hanging-drop vapor diffusion method (drops of 0.5 pi of protein and 0.5 pi of precipitant solution equilibrated against 700 m ⁇ of reservoir solution).
  • Diffraction quality crystals for MDM2-PMI-M3 crystals were obtained from a solution containing 0.1 M sodium cacodylate, pH 5.5, 25% PEG (w/v) 4000. Prior to being frozen, the crystals were transferred into a crystallization solution containing 20% (v/v) glycerol.
  • Crystals of MDMX-PMI-M3 were grown from 14% (v/v) 2-propanol, 70 mM sodium acetate/hydrochloric acid pH 4.6, 140 mM calcium chloride, 30% (v/v) glycerol and soaked in mother liquor supplemented with 20% 2 -methyl-2, 4- pentanediol (MPD) and 20% glycerol prior to being frozen for data collection.
  • the data for both complexes were processed and scaled with HKL2000 package [96] . Structures were solved by molecular replacement with Phaser [97] from the CCP4 suite based on the coordinates extracted from the structure of PMI-MDM2 complex (PDB code: 3EQS) and PMI- MDMX complex (PDB code: 3EQY).
  • amino acid substitutions at each position were determined based on the following criteria: (1) compatibility with parent residues at structural and chemical levels, (2) previous selection by phage display as non-consensus residues, (3) presence in wild type p53 from some other animal species, (4) high helix propensity as internal residues, and/or (5) favourable charge-dipole interaction as terminal residues.
  • a fluorescence polarization (FP)-based competitive binding assay was used
  • 22 peptide analogs out of 94 were found stronger in binding to MDM2 and MDMX than the control peptide, i.e., PMI-L10A.
  • Their amino acid sequences are tabulated in Table 3.
  • MDM2 were also highly correlated to those for MDMX (Fig. 2B), suggesting that mutations good for MDM2 generally improve peptide binding to MDMX as well.
  • the TIL mutation augmented peptide binding to MDM2 by 7.3 -fold (K d ) and 6.6-fold (Ki), and to
  • AAG(ni, ..., 3 ⁇ 4 , .. h) XAAG(m), where DDO represents the free energy change relative to wild type; n, stands for position i where single mutation occurs; ( , ..., 3 ⁇ 4, . . n j ) stands for positions 1 to j where multiple mutations occur.
  • the most potent peptide antagonist of MDM2 or MDMX can be readily constructed by combining the best mutations at individual positions into one sequence.
  • the amino acid sequence of a potent peptide antagonist of MDM2 could be created by introducing 7 mutations (best for MDM2) into PMI-L10A, i.e., TIL, S2T, A4L, N8A, L9Q, SI 1M and P12Q, yielding LTFLEYWAQAMQ (SEQ ID NO:4) termed PMI-M2 (Table 4).
  • PMI was a severely non-additive system with respect to MDM2/MDMX binding and posed a challenge to the design of ultrahigh-affmity and dual-specific peptide antagonists of both MDM2 and MDMX.
  • PMI-M2 bound to MDM2/MDMX at low single-digit nanomolar affinities, several fold stronger for MDMX than the parent peptide PMI (Table 4).
  • the reversion of Ala 10 in PMI-M2 to Leu dramatically improved, as expected, peptide binding to MDM2 and MDMX to the extent that reliable K d or Ki values could no longer be determined by the SPR and FP techniques (Fig. 3 and Table 4).
  • Isothermal titration calorimetry techniques were used to quantify the ultrahigh affinity interaction between PMI -M3 and MDM2/MDMX. As shown in Fig.
  • Non-additivity is known to arise from factors such as peptide conformational flexibility and interacting side chains [90,91] , contributing to a significant deviation of AAG(ni, ..., 3 ⁇ 4 , ..., n j ) from NAAG(n )
  • TIL T1L/S2T/A4L-PMI-L10A
  • PMI-M5 N8A/L9Q/S11M/P12Q-PMI-L10A
  • Missing in the crystallographic electron density map is Glnl2 of PMI-M3 in each of four copies of the MDMX-PMI-M3 complex in the asymmetric unit, indicating that the C- terminal residue is disordered and does not directly contribute to MDMX binding.
  • the Ca-Ca distance propagates progressively starting from Trp 7 and reaches a maximum of 1.6 A at residue 11 (Metl 1 in PMI-M3 versus Seri 1 in PMI).
  • PMI-M3 buries 1195 A 2 at the complex interface as compared with a buried surface area (BSA) of 1140 A 2 of PMI in the MDM2-PMI complex (Fig. 6B).
  • BSA buried surface area
  • the marginally increased BSA is contributed mainly by Metl 1 of PMI-M3, which, alone, buries 94.2 A 2 at the interface and contributes solvation energy of -1.5 kcal/mol (as opposed to 60 A 2 and -0.2 kcal/mol by Seri 1 of PMI).
  • Met 11 of PMI-M3 also establishes a new H-bond to Lys51 of MDM2 to replace the less favorable water-mediated H-bond formed by Seri 1 of PMI (Fig. 6 A).
  • two more H-bonds are formed at the MDM2-PMI-M3 interface, which are absent at the MDM2-PMI interface, including a water-mediated H-bond between Gln9 Oe ⁇ of peptide and His96 Ne3 and Val93 O of MDM2 and an elongated but direct H-bond (3.7 A) involving Tyr6 OH of PMI-M3 and Lys54 Ne ⁇ of MDM2.
  • the new H-bonding pattern along with an increased BSA seen with PMI-M3 may provide a structural explanation for its higher binding affinity for MDM2.
  • PMI-M3 in the p53-binding pocket of MDMX as compared with PMI.
  • the entire PMI-M3 backbone is shifted in relation to PMI with distances of 1.0 to 1.3 A, 2.4 A and 5 A between equivalent main-chain atoms of the Phe3-Trp7-Leul0 triads, the N-termini and C-termini, respectively (Figs. 6 and 7).
  • Leul in PMI-M3 shifts forward in the binding pocket to maximize hydrophobic contacts to MDMX (54.3 A 2 of BSA and -0.8 kcal/mol of A‘G, (Fig.
  • the added BSA and solvation energy by Leul and Metl 1 in the PMI-M3 peptide may adequately compensate for the backbone shift and the loss of solvation energy contributed by Prol2 in PMI.
  • the BSA of 1185 A 2 for MDMX-PMI-M3 is 90 A 2 larger than that of PMI-MDMX (1095 A 2 ) and increased solvation energies of Leul and Metl 1 provide a structural basis for PMI-M3’s higher affinity for MDMX than PMI (Fig 6B).
  • PMI-2K KTSFAEYWNLLSPK (SEQ ID NO:51)
  • M3-2K M3-2K
  • KLTFLEYWAQLMQK (SEQ ID NO: 52) were assayed for activity against cell lines U87MG and U251 in vitro and the results are provided in Figs. 8 A and 8B.
  • U87MG and U251 cells were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in DMEM medium supplemented with 10% FBS (Gibco). Cells were seeded at a density of 9 c 10 3 per well in 96-well plates. After overnight culture, cells were treated with M3-2K and PMI-2K at various concentrations, followed by incubation of 72 h.
  • mice models were prepared using BALB/c-nu mice. 10 6 U87MG cells (200 pi solution in PBS) were subcutaneously implanted into the right hind leg of the mice. Treatment began once tumors reached sizes of 5-10 mm in diameter. Mice were randomly divided into three treatment groups (six mice per group): PMI-2K, M3-2K, and PBS. Drugs were intravenously injected into the mice at the dose of 141 pmol/kg of free linear peptides.

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Abstract

L'invention concerne des antagonistes peptidiques de MDM2 et de MDMX. Les antagonistes peptidiques comprennent des antagonistes de MDM2 ou de MDMX, ainsi que des antagonistes à double spécificité selon lesquels le même peptide est un antagoniste de MDM2 et de MDMX à la fois. Les antagonistes peptidiques ont au moins une affinité de liaison nanomolaire pour MDM2 et/ou pour MDMX et, dans certains cas, une affinité de liaison picomolaire. Les antagonistes peptidiques peuvent être utilisés dans des méthodes basées sur un blocage d'inhibition de l'activité de p53 par MDM2 et/ou par MDMX. Ces méthodes peuvent être utilisées pour le traitement de maladies, telles que le cancer.
PCT/US2020/058669 2019-11-04 2020-11-03 Antagonistes peptidiques à haute affinité et à double spécificité de mdm2 et de mdmx pour l'activation de p53 WO2021091871A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120328692A1 (en) * 2011-06-24 2012-12-27 University Of Maryland, Baltimore Potent d-peptide antagonists of mdm2 and mdmx for anticancer therapy
US20150051155A1 (en) * 2012-02-15 2015-02-19 Aileron Therapeutics Peptidomimetic macrocycles
US20170349638A1 (en) * 2016-03-21 2017-12-07 Aileron Therapeutics, Inc. Companion diagnostic tool for peptidomimetic macrocycles

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120328692A1 (en) * 2011-06-24 2012-12-27 University Of Maryland, Baltimore Potent d-peptide antagonists of mdm2 and mdmx for anticancer therapy
US20150051155A1 (en) * 2012-02-15 2015-02-19 Aileron Therapeutics Peptidomimetic macrocycles
US20170349638A1 (en) * 2016-03-21 2017-12-07 Aileron Therapeutics, Inc. Companion diagnostic tool for peptidomimetic macrocycles

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