AU2016210709B2 - Stabilized p53 peptides and uses thereof - Google Patents

Abstract

Cross-linked peptides related to human p53 and bind to HMD2 or a family member of HDM2 useful for promoting apoptosis, e.g., in the treatment of and identifying therapeutic agents that binding to HMD2 or a family member of HDM2. See Figure 1. Pph 3 FmocHN 0FmocHN OH cl-S Me' O Me Pph 3 Fmoc-R8 Fmoc-S5

Description

The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with reduced p53 activity. This is because the polypeptides are expected to act as inhibitors of p53 binding to HDM2 and/or HDMX. As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient^ or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecule^ peptides, antibodies, ribozymes and antisense oligonucleotides.

[0098] The polypeptides described herein ean be used to treat, prevent, and/or diagnose cancers and neoplastic conditions. As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.

[0099] Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, or metastatic disorders. The compounds (i.e., polypeptides) can act as novel therapeutic agents for controlling osteosarcomas, colon cancer, breast cancer, T cell cancers and B cell cancer. The polypeptides may

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2016210709 04 Aug 2016 also be useful for treating mucoepidermoid carcinoma, retinoblastoma and medulloblastoma. The compounds can be used to treat disorders associated with unwanted proliferation of cells having reduced activity and/or expression of p53, particularly where the cells produce at least some active p53.

[0100] Examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Exemplary disorders include: acute leukemias, e.g., erythroblastic leukemia and acute mcgakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes Blineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), multiple mylenoma, hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

[0101] Examples of cellular proliferative and/or differentiative disorders of the breast include, but are not limited to, proliferative breast disease including, e.g., epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget’s disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and

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2016210709 04 Aug 2016 invasive papillary carcinoma, and miscellaneous malignant neoplasms. Disorders in the male breast include, but are not limited to, gynecomastia and carcinoma.

[0102] Pharmaceutical Compositions and Routes of Administration

As used herein, the compounds of this invention, including the compounds of formulae described herein, are defined to include pharmaceutically acceptable derivatives or prodrugs thereof. A “pharmaceutically acceptable derivative or prodrug” means any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention. Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Preferred prodrugs include derivatives where a group which enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein.

[0103] The compounds of this invention may be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological compartment (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.

[0104] Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, bexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,

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2016210709 04 Aug 2016 nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate, trifluoromethylsulfonate, and undecanoate. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)4⁺ salts. This invention also envisions the quatemization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quatemization.

[0105] The compounds of the formulae described herein can, for example, be administered by injection, intravenously, intraarterially, subdermally, intraperitoneally, intramuscularly, or subcutaneously; or orally, buccally, nasally, transmucosally, topically, in an ophthalmic preparation, or by inhalation, with a dosage ranging from about 0.001 to about 100 mg/kg of body weight, or according to the requirements of the particular drug. The methods herein contemplate administration of an effective amount of compound or compound composition to achieve the desired or stated effect. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations contain from about 20% to about 80% active compound.

[0106] Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient’s disposition to the disease, condition or symptoms, and the judgment of the treating physician.

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2016210709 04 Aug 2016 [0107] Upon improvement of a patient’s condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

[0108] Pharmaceutical compositions of this invention comprise a compound of the formulae described herein or a pharmaceutically acceptable salt thereof; an additional agent including for example, morphine or codeine; and any pharmaceutically acceptable carrier, adjuvant or vehicle. Alternate compositions of this invention comprise a compound of the formulae described herein or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier, adjuvant or vehicle. The compositions delineated herein include the compounds of the formulae delineated herein, as well as additional therapeutic agents if present, in amounts effective for achieving a modulation of disease or disease symptoms.

The term “pharmaceutically acceptable carrier or adjuvant” refers to a carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

[0109] Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,

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2016210709 04 Aug 2016 polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropyleneblock polymers, polyethylene glycol and wool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, may also be advantageously used to enhance delivery of compounds of the formulae described herein.

[0110] The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or administration by injection. The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0111] The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example. Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain

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2016210709 04 Aug 2016 a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens or Spans and/or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

[0112] The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried com starch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

[0113] The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

[0114] The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

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When the compositions of this invention comprise a combination of a compound of the formulae described herein and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. The additional agents may be administered separately, as part of a multiple dose regimen, from the compounds of this invention. Alternatively, those agents may be part of a single dosage form, mixed together with the compounds of this invention in a single composition.

[0115] Modification of Polypeptides

The stapled polypeptides can include a drug, a toxin, a derivative of polyethylene glycol; a second polypeptide; a carbohydrate, etc. Where a polymer or other agent is linked to the stapled polypeptide is can be desirable for the composition to be substantially homogeneous.

[0116] The addition of polyethelene glycol (PEG) molecules can improve the pharmacokinetic and pharmacodynamic properties of the polypeptide. For example, PEGylation can reduce renal clearance and can result in a more stable plasma concentration. PEG is a water soluble polymer and can be represented as linked to the polypeptide as formula:

XO-(CH₂CH₂O)_n-CH2CH2-Y where n is 2 to 10,000 and X is H or a terminal modification, e.g., a Cm alkyl; and Y is an amide, carbamate or urea linkage to an amine group (including but not limited to, the epsilon amine of lysine or the Nterminus) of the polypeptide. Y may also be a maleimide linkage to a thiol group (including but not limited to, the thiol group of cysteine). Other methods for linking PEG to a polypeptide, directly or indirectly, are known to those of ordinary skill in the art. The PEG can be linear or branched. Various forms of PEG including various functionalized derivatives are commercially available.

[0117] PEG having degradable linkages in the backbone can be used. For example, PEG can be prepared with ester linkages that are subject to hydrolysis. Conjugates

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2016210709 04 Aug 2016 having degradable PEG linkages are described in WO 99/34833; WO 99/14259, and U.S. 6,348,558.

[0118] In certain embodiments, macromolecular polymer (e.g., PEG) is attached to an agent described herein through an intermediate linker. In certain embodiments, the linker is made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art. In other embodiments, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Non-peptide linkers are also possible. For example, alkyl linkers such as -NH(CH₂)nC(O)-, wherein n = 2-20 can be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., Ci-Ct) lower acyl, halogen (e.g., Cl, Br),.CN, NH2, phenyl, etc. U.S. Pat. No. 5,446,090 describes a bifunctional PEG linker and its use in forming conjugates having a peptide at each of the PEG linker termini.

[0119] Screening Assays

The invention provides methods (also referred to herein as “screening assays”) for identifying polypeptides, small molecules, or bifunctional derivatives which bind to HDM2 and/or HDMX.

[0120] The binding affinity of polypeptides that bind HDM2 and/or HDMX can be measured using the methods described herein, for example, by using a titration binding assay. HDM2 and/or HDMX can be exposed to varying concentrations of a candidate compound (i.e., polypeptide) (e.g., 1 nM, 10 nM, 100 nM, 1 μΜ, 10 μΜ, 100 μΜ, 1 mM, and 10 mM) and binding can be measured using surface plasmon resonance to determine the Kd for binding. Additionally, the binding interactions of fluorescently-labeled SAH-p53 peptides to HDM2 and/or HDMX can be used in a competitive binding assay to screen for and identify peptides, small molecules, or bifunctional derivatives thereof that compete with FITC-SAH-p53 peptides, and

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2016210709 04 Aug 2016 further calculate Ki values for binding competition. Candidate compounds could also be screened for biological activity in vivo. Cell permeability screening assays in which fluorescently labeled candidate compounds are applied to intact cells, which are then assayed for cellular fluorescence by microscopy or high-throughput cellular fluorescence detection can also be used.

[0121] The assays described herein can be performed with individual candidate compounds or can be performed with a plurality of candidate compounds. Where the assays are performed with a plurality of candidate compounds, the assays can be performed using mixtures of candidate compounds or can be run in parallel reactions with each reaction having a single candidate compound. The test compounds or agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art.

[0122] Thus, one can expose HDM2 (e.g., purified MDM2) or HDMX (e.g., purified MDM2) purified to a test compound in the presence of a stapled p53 peptide and determining whether the test compound reduces (inhibits) binding of the stapled p53 peptide to MDM2 or MDMX. A test compound that inhibits binding is a candidate inhibitor of the interaction between p53 and MDM2 or MDMX (or both). Test compounds can be tested for their ability to inhibit binding to MDM2 and MDMX in order to identify compounds that are relatively selective for inhibit p53 binding. In some cases, nutlin-3 (CAS 548472-68-0) can be used as a control since nutlin-3 is a selective inhibitor of p53 binding to HMD2 [0123] Other applications

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

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2016210709 04 Aug 2016 [0124] References (1) Kastan, Μ. B.; Onyekwere, O.; Sidransky, D.; Vogelstein, B.; Craig, R. W., Cancer Res. 1991, 51, 6304-6311.

(2) Wu, X.; Bayle, J. H.; Olson, D.; Levine, A. J., Genes Dev. 1993, 7,11261132; Yonish-Rouach, E.; Resnftzky, D.; Lotem, J.; Sachs, L.; Kimchi, A.; Oren, M., Nature 1991, 352, 345-347; Momand, J.; Zambetti, G. P.; Olson, D. C.; George, D.; Levine, A. J., Cell 1992, 69, 1237.

(3) Levine, A. J.; Hu, W.; Feng, Z., Cell Death Differ 2006, 13, 1027; Honda, R.; Tanaka, H.; Yasuda, H„ FEBS Lett. 1997,420,25; Tao, W.; Levine, A. Proc. Nat. Acad. Sci. U.S.A. 1999, 96, 3077-3080.

(4) Li, M.; Brooks, C. L.; Wu-Baer, F.; Chen, D.; Baer, R.; Gu, W., Science

2003.302, 1972-1975.

(5) Hollstein, M.; Sidransky, D,; Vogelstein, B.; Harris, C. C., Science 1991, 253,49-53.

(6) Chene, P., Nat. Rev. Cancer 2003,3, 102-109.

(6a) Toledo, F.; Wahl, G.M, Int. J. Biochem. Cell Biol. 2007, 39,1476-82.

(7) Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine, A. J.; Pavletich, N. P., Science 1996, 274, 948-953.

(8) Sakurai, K.; Chung, H. S.; Kahne, D., J. Am. Chem. Soc. 2004, 126, 1628816289; Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.; Filipovic, Z.; Kong, N.; Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi, N.; Liu, E. A., Science

2004.303, 844-848.

(9) Lin, J.; Chen, J.; Elenbaas, B.; Levine, A. J., Genes Dev. 1994, 8, 12351246.

(10) Schafmeister, C. E.; Po, J.; Verdine, G. L, J. Am. Chem. Soc. 2000, 122, 5891-5892; Walensky, L.D.; Kung, A.L.; Escher, I.; Malia, T.J.; Barbuto, S.; Wright, R.D.; Wagner, G.; Verdine,G.L.; Korsmeyer, S.J.; Science, 2004,305, 1466-1470.

(11) Vives, E.; Lebleu, B., The Tat-Derived Cell-Penetrating Peptide. In CellPenetrating Peptides: Processes and Applications, Langel, ϋ., Ed. CRC Press: Boca Raton, 2002; pp 3-21.

(12) Zhang, Y.; Xiong, Y., Science 2001,292, 1910-1915.

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2016210709 04 Aug 2016 (13) Barak, Y,; Juven, T.; Haffner, R.; Oren, M., EMBO J. 1993,12, 461-468; Juven, T.; Barak, Y.; Zauberman, A.; George, D. L.; Oren, M., Oncogene 1993, 8, 3411-3416.

(14) Dulic, V.; Kaufmann, W. K.; Wilson, S. J.; Tisty, T. D.; Lees, E.; Harper, J. W.; Elledge, S. J.; Reed, S. 1., Cell 1994,76, 1013; El-Deiry, W. S.; Tokino, T.; Velculescu, V. E.; Levy, D. B.; Parsons, R.; Trent, J. M.; Lin, D.; Mercer, W. E.; Kinzler, K. W.; Vogelstein, B., Cell 1993, 75, 817.

(15) Pochampally, R.; Fodera, B.; Chen, L.; Lu, W.; Chen, J., J. Biol. Chem. 1999,274, 15271-15277.

(16) Duncan, S. J.; Gruschow, S.; Williams, D. H.; McNicholas, C.; Purewal, R.; Hajek, M.; Gerlitz, M.; Martin, S.; Wrigley, S. K.; Moore, M., J. Am. Chem. Soc. 2001, 123, 554-560; Chene, P.; Fuchs, J.; Bohn, J.; Garcia-Echeverria, C.; Furet, P.; Fabbro, D., J. Mol. Biol. 2000,299,245; Sakurai, K.; Schubert, C.; Kahne, D., J. Am. Chem. Soc. 2006, 128, 11000-11001; Kritzer, J. A.; Hodsdon, Μ. E.;

Schepartz, A., J. Am. Chem. Soc. 2005, 127,4118^-4119; Kritzer, J. A.; Lear, J. D.; Hodsdon, Μ. E.; Schepartz, A., J. Am. Chem. Soc. 2004,126, 9468-9469; Wasylyk, C.; Salvi, R.; Argentini, M.; Dureuil, C.; Delumeau, I.; Abecassis, J.; Debussche, L.; Wasylyk, B., Oncogene 1999,18, 1921-1934; Garcia-Echeverria, C.; Chene, P.; Blommers, M. J. J.; Furet, P., J. Med. Chem. 2000,43, 3205-3208; Grasberger, B. L., et al., J. Med. Chem. 2005, 48, 909-912.

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Claims (34)

1. A polypeptide of Formula (I):

[Xaa]_v

Formula (I) or a pharmaceutically-acceptable salt thereof, wherein:

each Ri and R₂ is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;

each R3 is independently alkylene, alkenylene, alkynylene, or [R4-K-R4’]_n substituted with 0-6 R5;

each R4 and R4’ is independently alkylene, alkenylene, or alkynylene;

each R5 is independently halo, alkyl, OR6, N(R6)₂, SR6, SOR6, SO₂R6, CO₂R6, R6, a fluorescent moiety, or a radioisotope;

each K is independently O, S, SO, SO₂, CO, CO₂, CONR6, or ;

each R6 is independently H, alkyl, or a therapeutic agent; each n is independently an integer from 1-4;

each x is independently 6, 7, 8, 9, or 10;

each y and w is independently an integer from 0-100;

z is an integer from 1-10; and each Xaa is independently an amino acid;

wherein the polypeptide comprises an amino acid sequence Xaai Gln₂ Glm Thr4 Alas Xaa6 Asp₇ Leus Trp9 Argio Leun Leui₂ Xaan Glui4 Asms, wherein each Xaai, Xaa6, Xaai 3 is independently an amino acid.

2016210709 04 Apr 2018

2. The polypeptide of claim 1, wherein the polypeptide binds to HDM2.

3. The polypeptide of claim 1 or 2, wherein the polypeptide binds to HDMX.

4. The polypeptide of any one of claims 1-3, wherein the polypeptide is permeable to a cell membrane.

5. The polypeptide of any one of claims 1-4, wherein the polypeptide comprises a helix.

6. The polypeptide of any one of claims 1-5, wherein the polypeptide comprises an a-helix.

7. The polypeptide of any one of claims 1-6, wherein at least one Rs extends across a length of one helical turn.

8. The polypeptide of any one of claims 1-6, wherein at least one R3 extends across a length of two helical turns.

9. The polypeptide of any one of claims 1-8, further comprising an amino-terminal fatty acid.

10. The polypeptide of any one of claims 1-9, further comprising a targeting moiety.

11. The polypeptide of any one of claims 1-10, wherein the polypeptide does not have a net negative charge at pH 7.

12. The polypeptide of any one of claims 1-11, wherein the polypeptide comprises at least one amino acid that (i) has a positive charge at pH 7 and (ii) is located either: at (a) amino terminal to the amino acid Xaai or (b) carboxy terminal to Asms.

13. The polypeptide of any one of claims 1-12, wherein each R3 is independently an alkylene.

14. The polypeptide of any one of claims 1-13, wherein each R3 is independently a Cs alkylene.

15. The polypeptide of any one of claims 1-13, wherein each R3 is independently a Cn alkylene.

16. The polypeptide of any one of claims 1-13, wherein each R3 is independently an

2016210709 04 Apr 2018 alkenylene.

17. The polypeptide of any one of claims 1-13 and 16, wherein each R3 is independently an alkenylene containing a no greater than one double bond, and both Ri and R2 are H.

18. The polypeptide of any one of claims 1-12, 16, and 17, wherein each R3 is independently a Cs alkenylene.

19. The polypeptide of any one of claims 1-12, 16, and 17, wherein each R3 is independently a Cn alkenylene.

20. The polypeptide of any one of claims 1-16, 18, and 19, wherein each Ri and R2 is independently H or C1-C6 alkyl.

21. The polypeptide of any one of claims 1-16 and 18-20, wherein each Ri and R2 is independently C1-C3 alkyl.

22. The polypeptide of any one of claims 1-16 and 18-21, wherein each Ri and R2 is methyl.

23. The polypeptide of any one of claims 1-16 and 18-20, wherein each Ri and R2 is H.

24. The polypeptide of any one of claims 1-23, wherein each y and w is independently an integer from 1 to 15.

25. The polypeptide of any one of claims 1-24, wherein each w is independently an integer from 3 to 15.

26. The polypeptide of any one of claims 1-25, wherein each y is independently an integer from 3 to 15.

27. The polypeptide of any one of claims 1-26, wherein z is 1.

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29.

30.

29.

30.

A polypeptide of Formula (I), [Xaa]_v

Formula (I) or a pharmaceutically-acceptable salt thereof, wherein:

each Ri and R2 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;

each R3 is independently alkylene, alkenylene, alkynylene, or [R4-K-R4’]_n substituted with 0-6 R5;

each R₄ and R₄’ is independently alkylene, alkenylene, or alkynylene;

each R5 is independently halo, alkyl, OR6, N(R6)2, SR6, SOR6, SO2R6, CO2R6, R6, a fluorescent moiety, or a radioisotope;

each K is independently O, S, SO, SO2, CO, CO2, CONR6, or ;

each R6 is independently H, alkyl, or a therapeutic agent; each n is independently an integer from 1-4;

each x is independently 6, 7, 8, 9, or 10;

each y and w is independently an integer from 0-100;

z is an integer from 1-10; and each Xaa is independently an amino acid;

wherein the polypeptide exhibits a binding affinity for HDM2 that is from about 0.75 nM to about 110 nM.

The polypeptide of claim 28, wherein the polypeptide binds to HDMX.

The polypeptide of claim 28 or 29, wherein the polypeptide is permeable to a cell membrane.

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31. The polypeptide of any one of claims 28-30, wherein the polypeptide comprises a helix.

32. The polypeptide of any one of claims 28-31, wherein the polypeptide comprises an ahelix.

33. The polypeptide of any one of claims 28-32, wherein at least one R3 extends across a length of one helical turn.

34. The polypeptide of any one of claims 28-32, wherein at least one R3 extends across a length of two helical turns.

35. The polypeptide of any one of claims 28-34, further comprising an amino-terminal fatty acid.

36. The polypeptide of any one of claims 28-35, further comprising a targeting moiety.

37. The polypeptide of any one of claims 28-36, wherein the polypeptide does not have a net negative charge at pH 7.

38. The polypeptide of any one of claims 28-37, wherein each R3 is independently an alkylene.

39. The polypeptide of any one of claims 28-38, wherein each R3 is independently a Cg alkylene.

40. The polypeptide of any one of claims 28-38, wherein each R3 is independently a C11 alkylene.

41. The polypeptide of any one of claims 28-37, wherein each R3 is independently an alkenylene.

42. The polypeptide of any one of claims 28-37 and 41, wherein each R3 is independently an alkenylene containing a no greater than one double bond, each both Ri and R2 is H.

43. The polypeptide of any one of claims 28-37 and 41-42, wherein each R3 is independently a Cs alkenylene.

44. The polypeptide of any one of claims 28-37 and 41-42, wherein each R3 is independently

2016210709 04 Apr 2018 a Cn alkenylene.

45. The polypeptide of any one of claims 28-41 and 43-44, wherein each Ri and R₂ is independently H or C1-C6 alkyl.

46. The polypeptide of any one of claims 28-41 and 43-45, wherein each Ri and R₂ is independently C1-C3 alkyl.

47. The polypeptide of any one of claims 28-41 and 43-46, wherein each Ri and R₂ is methyl.

48. The polypeptide of any one of claims 28-41 and 43-45, wherein each Ri and R₂ is H.

49. The polypeptide of any one of claims 28-48, wherein each y and w is independently an integer from 1 to 15.

50. The polypeptide of any one of claims 28-49, wherein each w is independently an integer from 3 to 15.

51. The polypeptide of any one of claims 28-50, wherein each y is independently an integer from 3 to 15.

52. The polypeptide of any one of claims 28-51, wherein z is 1.

53. A method of treating a disorder associated with disrupted regulation of a p53 pathway in a subject, comprising administering to the subject the polypeptide of any one of claims 152.

54. The method of claim 53, wherein the disorder is a proliferative disorder.

55. The method of claim 53 or 54, wherein the disorder is a cancer.

56. The method of claim 55, wherein the cancer is carcinoma, sarcoma, metastatic disorder, osteosarcomas, colon cancer, breast cancer, T cell cancer, or B cell cancer.

57. The method of any one of claims 53-56, wherein the subject is a mammal.

58. The method of claim 57, wherein the mammal is a human.

59. The polypeptide of any one of claims 1-12, wherein R3 is a straight chain alkylene,

2016210709 04 Apr 2018 alkenylene, or alkynylene.

60. The polypeptide of any one of claims 29-38, wherein each R3 is independently a straight chain alkylene, alkenylene, or alkynylene.

2016210709 04 Aug 2016

1/23

FIG. 1A

2016210709 04 Aug 2016

2/23

cell death 1 1 1 1 1 1 1 1 +· 1 1 cell permeable 5 iJ³ ·<- co CD T- o · σ> σ oo η ο ν- co co +ι Ώ 2 i- ο τ- ο τ- +1 -Η +ι ο Α Λ τ- τ- Ο -Η oooogig+ι+ι+ισ ο t-OOCM^l'NcCOOlOxt- Ο xt- τ— M-t-OOLOLOlOA a helicity V© ν»Ο >© χ© ·χθ X© χ© χ© χθ χθ χθ σ> ©** ο^· o'* ©** ο^· ο*» ο*“ 0^s· ο^· ©** v-mocMCDO^coina) co v-Oit-T-tOCNT^-OCOCO CO charge at pH 7.4 + + + tn ω II « 00 o* Ξ5 CT ω co ^z Z H z Z 1 2 z z 1 ω ω § § g § f a σ a g 5?SS§SMSII 8 8 8 8 £ E i £ i Ϊ fe LL-tLULutP-uZh-l— A H fct^S-^σασαα σσσσσσσ^οσ σ £ωωωωωω«ωωω Λώόόώώώόούο 4έ<<<<<<<<< < compound . £ τΟ. ^CNOxtlCCDSCOCO CO » ι ι ι ι > t > ι _L cococococooococOco Γ2 miOiommmiOLOio uj 0.0.0.(10.0.0.0.0. O. . χχχχΐχχχχ x t<c<c<c<c<<<cc<c < ^ωωωωίοωωωω z>

2016210709 04 Aug 2016 [hDM2], M

FIG. 1D

2016210709 04 Aug 2016 [hDM2], M

FIG. 1F

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5/23

Treatment

Time post-Tx WB: p53

WB: hDM2

WB: p21

WB: actin

veh. WT (20μΜ) SAH-p53-8 (20 μΜ) SAH-p53-8 fi9a (20μΜ) _C JZ .C JZ r cm oo o 00 T— r- CM CO JZ -£= -C JZ JZ C\j CO Μ· O CO ¹ T— V- CM co _cz jz x: _c r CM oo NT o co x- x- cm co ,γτ^-t vV.sFi.rPtrii'Wrs·.·· irir ^'^.‘‘·Ζ·Δδ! &iZi.'.2lNF±- • *5* '·. a ,’'·· ϊ7-Όί'’>ί;·’Λ? V..' ϋ·.··τ?' v< .*4 jtfrm/iMw'v.· * *' ' A#* '•ίΐίϊ-ύ'. ·... .·„ /,.,... . .·· .,♦ ·'' \* ^fcT.'’T=.‘’·?-'·'~^r’.

FIG. 2

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FIG. 3B

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7/23

Ο co

Ο l_L

Add

2016210709 04 Aug 2016

8/23

300

Z> 200 <

E

100

-100

0 5 10 15 20 25

Min

FIG. 4A

100 mAU

1070.9

714.2

1427.6

1081.4 I

M⁺

2140.7

500 1000 1500 2000 2500

Min

FIG. 4B

2016210709 04 Aug 2016

FIG. 6

10/23

2016210709 04 Aug 2016

TRITC-dextran • SAH-p53-5 ' I. · . . . ·.- · X· , . . ··.·' · . ··' ' 20μτη ' . 20μτη . dapi ί . · Overlay . i »«, J ; . ' /<“j«Μ*. · .» ·ξ>4 I i · . i Νι... .:,/--1- ' ' '· ’·«. . ί . . . '_ ί ' ' 20μΠΊ '.ίζΟμητ*

FIG. 7A

FIG. 7B

11/23

2016210709 04 Aug 2016

FIG. 7C

12/23

2016210709 04 Aug 2016

FIG. 7D-1

13/23

2016210709 04 Aug 2016

FIG. 7D-2

14/23

2016210709 04 Aug 2016

FIG. 7D-3 θ 15/23

2016210709 04 Aug χ χΧ'^ yX^ <S%<5° <^V°' <6°

-c^ -c>® x5< . >y „ 'Y , sy v<r . xy . xY , >y <<5^V <?^v g$>^v <?^V g$>^V h$)^V hDM2

FITC

Peptide

WB: ct-hDM2

WB: a-FITC

FIG. 8

16/23

2016210709 04 Aug 2016

FIG. 9

2016210709 04 Aug 2016

17/23

Fluorescence Polarization Binding Assay SAH-p53-4 Mutants and Truncations

FIG. 10

18/23

2016210709 04 Aug 2016

Cell Viability Assay SJSA-1 Osteosarcoma Cell Line Effects of SAH-p.53-8 Alone or Combined

FIG. 11

19/23

2016210709 04 Aug 2016

Inhibition of p53(14-29) Binding to hMDM2(17-125)

100 o

co

CT?

CM

I

CO in ex

I o

SAH-P53-1 -n- SAH-p53-2

2.0 2.5 3.0

Log [Ac-p53(14-29)], nM

4.0

FIG. 12

2016210709 04 Aug 2016

20/23

Caspase-3 Fluorometric Assay 24 h Tx, SJSA-1 Cell Line

250

200

150

Ο

LL.

££ 100

r' > 1 _t 1 ^Z1 1 y 1 Si 1 S J } ____.________________ί__ 1 1 1 1 1 1 > 1 1 4 ! 7 i > > / ’ > 1 1 1 X ί 1 1 1 1 1 / i / [ i 1 1 .....7......-...........Γ / J z Z 1 1 1 Ϊ Z ’ z _ Z z 5 +z ί z 1 >Z 1 ........ < 1 / -¹* 1 Λ, ' _------- .Li #·-)— ------------ --- J- -J 1 1 1 1 1 1 1 I 1 1 1 1 1 I 1 1 1 1

i-1 I i i i i I i i i i i i i i I ¹ ι > i

0.1 1 10

Dose, μΜ

— Δ - - NoTx —□— WT - SAH-p53-6 — —o-- SAH-p53-7 ---V--- SAH-p53-8_{F1 gA} --0-- SAH-p53-8 --*-- SAH-p53-8 + DEVD-CHO

FIG. 13

21/23

2016210709 04 Aug 2016

FIG. 14A

1 meepqsdpsv epplsqetfs dlwkllpenn vlsplpsqam ddlmlspddi eqwftedpgp 61 deaprmpeaa prvapapaap tpaapapaps wplsssvpsq ktyqgsygfr Igflhsgtak 121 svtctyspal nkmfcqlakt cpvqlwvdst pppgtrvram aiykqsqhmt evvrrcphhe 181 rcsdsdglap pqhlirvegn Irveylddrn tfrhsvvvpy eppevgsdct tihynymcns 241 scmggmnrrp iltiitleds sgnllgrnsf evrvcacpgr drrteeenlr kkgephhelp 301 pgstkralpn ntssspqpkk kpldgeyftl qirgrerfem frelnealel kdaqagkepg 361 gsrahsshlk skkgqstsrh kklmfktegp dsd

FIG. 15

22/23

2016210709 04 Aug 2016

1. Cap-Linker-L-*-Q-E-T-F-S-D-*-W-K-L-L-P-E-N-NH₂

2. Cap-Linker-L-S-Q-*-T-F-S-D-L-W-*-L-L-P-E-N-NH₂

3. Cap-Linker-L-S-Q-E-*-F-S-D-L-W-K-*-L-P-E-N-NH₂

4. Cap-Linker-L-S-Q-E-T-*-S-D-L-W-K-L-*-P-E-N-NH₂

5. Cap-Linker-L-S-Q-E-T-F-*-D-L-W-K-L-L-*-E-N-NH₂ (SAH-p53-4)

6. Cap-Linker-L-*-Q-E-T-F-S-*-L-W-K-L-L-P-*-N-NH₂

7. Cap-Linker-L-S-Q-E-T-F-S-D-*-W-K-L-L-P-E-*-NH₂ • Cap denotes Ac (acetyl) or FITC (fluorescein thiocarbomoyl); Linker denotes β-alanine or no linker: indicates the amino acid pairs R₅-S₈ Or

R_e-S₅ in either uncross-linked (unstapled, unmetathesized) or cross-linked (stapled, metathesized) form

8. Cap-Linker-L-S-Q-Q-T-F-*-D-L-W-K-L-L-*~E-N-NH₂

9. Cap-Linker-L-S-Q-E-T-F-*-D-L-W-K-L-L-*-Q-N-NH₂

10. Cap-Linker-L-S-Q-Q-T-F-*-D-L-W-K-L-L-*-Q-N-NH₂

11. Cap-Linker-L-S-Q-E-T-F-*-N-L-W-K-L-L-*-Q-N-NH₂

12. Cap-Linker-L-S-Q-Q-T-F-*-N-L-W-K-L-L-*-Q-N-NH₂

13. Cap-Linker-L-S-Q-Q-T-F-*-N-L-W-R-L-L-*-Q-N-NH₂

14. Cap-Linker-Q-S-Q-Q-T-F-*-N-L-W-K-L-L-*-Q-N-NH₂

15. Cap-Linker-Q-S-Q-Q-T-F-*-N-L-W-R-L-L-*-Q-N-NH₂ (SAH-p53-8)

16. Cap-Linker-Q-S-Q-Q-T-A-*-N-L-W-R-L-L-*-Q-N-NH₂ (SAH-p53-8_F19A) • Cap denotes Ac (acetyl), FITC (fluorescein thiocarbamoyl). DOTA (cryptand capable of chelating radaoctive In), lauroyl, heptanoyl, and myristoyl: Linker denotes β-alanine or no linker: indicates the amino acid pairs R₅-S₈ or R₈-S₅ in either uncross-linked (unstapled, unmetathesized) or cross-linked (stapled, metathesized) form

17. Cap-Linker-Q-Q-T-F-*-D-L-W-R-L-L-*-E-N.-NH₂

18. Cap-Linker-Q-Q-T-F-*-D-L-W-R-L-L-*-NH₂

19. Cap-Linker-L-S-Q-Q-T-F-*-D-L-W-*-L-L-NH₂

20. Cap-Linker-Q-Q-T-F-*-D-L-W-*-L-L-NH

21. Cap-Linker-Q-Q-T-A-*-D-L-W-R-L-L-*-E-N-NH₂ • Cap denotes Ac (acetyl), FITC (fluorescein thiocarbamoyl). lauroyl, heptanoyl, and myristoyl: Linker denotes β-alanine or no linker; indicates the amino acid pairs R₈-S₅ (peptides 17. 18. and-21) or S₅-S₅ (peptides 19 and 20) in either uncross-linked (unstapled, unmetathesized) or cross-linked (stapled, metathesized) forms

22. Cap-K(Myr)-Linker-Q-S-Q-Q-T-F-*-N-L-W-R-L-L-*-Q-N-NH₂

23. Cap-K(Biotin)-Linker-Q-S-Q-Q-T-F-*-N-L-W-R-L-L-*-Q-N-NH₂

24. Cap-K(PEG3)-Linker-Q-S-Q-Q-T-F-*-N-L-W-R-L-L-*-Q-N-NH₂

25. Cap-Linker-Q-S-Q-Q-T-F-*-N-L-W-R-L-L-*-Q-N-NH₂ diol

26. Cap-Linker-Q-S-Q-Q-T-A-*-N-L-W-R-L-L-*-Q-N-NH₂ diol • Cap denotes Ac (acetyl) or FITC (fluorescein thiocarbamoyl): Linker denotes B-alanine or no linker: indicates the amino acid pair R₈-S₅ in either uncross-linked (unstapled, unmetathesized) or cross-linked (stapled, metathesized) form: diol indicates a di hydroxylated cross-link ol ef i n

FIG. 16-1

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2016210709 04 Aug 2016

27. Cap-Linker-Q-S-Q-Q-T-F-*-D-L-W-R-L-L-*-Q-N-NH₂ (SAH-p53-10)

28. Cap-Linker-Q-T-F-*-N-L-W-R-L-L-*-NH₂ (SAH-p53-ll)

29. Cap-Linker-Q-S-Q-Q-T-F-*-N-L-W-*-L-L-P-Q-N-NH₂ (SAH-p53-8S_A)

30. Cap-Linker-Q-S-*-Q-T-F-*-N-L-W-R-L-L-P-Q-N-NH₂ (SAH-p53-8S_B)

31. Cap-LTnker-*-T-F-S-*-L-W-K-L-L-NH₂ (SAH-p53-12)

32. Cap-Linker-E-T-F-*-D-L-W-*-L-L-NH₂ (SAH-p53-13)

33. Cap-Linker-Q-T-F-*-N-l_-W-*-L-L-NH₂ (SAH-p53-14)

34. Cap-Linker-*-S-Q-E-*-F-S-N-L-W-K-L-L-NH₂ (SAH-p53-15)

FIG. 16-2