DK2603600T3 - Peptidomimetiske makrocyklusser - Google Patents

Description

DESCRIPTION

BACKGROUND OF THE INVENTION

[0001] The human transcription factor protein p53 induces cell cycle arrest and apoptosis in response to DNA damage and cellular stress, and thereby plays a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase HDM2 negatively regulates p53 function through a direct binding interaction that neutralizes the p53 transactivation activity, leads to export from the nucleus of p53 protein, and targets p53 for degradation via the ubiquitylation-proteasomal pathway. Loss of p53 activity, either by deletion, mutation, or HDM2 overexpression, is the most common defect in human cancers. Tumors that express wild type p53 are vulnerable to pharmacologic agents that stabilize or increase the concentration of active p53. In this context, inhibition of the activities of HDM2 has emerged as a validated approach to restore p53 activity and resensitize cancer cells to apoptosis in vitro and in vivo. HDMX (HDM4) has more recently been identified as a similar negative regulator of p53, and studies have revealed significant structural homology between the p53 binding interfaces of HDM2 and HDMX.

[0002] The p53-HDM2 and p53-HDMX protein-protein interactions are mediated by the same 15-residue alpha-helical transactivation domain of p53, which inserts into hydrophobic clefts on the surface of HDM2 and HDMX. Three residues within this domain of p53 (F19, W23, and L26) are essential for binding to HDM2 and HDMX. The present invention provides p53-based peptidomimetic macrocycles that modulate the activities of p53 by inhibiting the interactions between p53 and HDM2, p53 and HDMX, or p53 and both HDM2 and HDMX proteins, and that may be used for treating diseases including but not limited to cancer and other hyperproliferative diseases. WO 2009/126292 and WO 2010/033617 disclose peptidomimetic macrocycle compound derived from p53 binding to HDM2 and/or HDMX. Bernal, F. et al., J. Am. Chem. Soc. (2007), 129, pages 2456-2457, discloses p53 derived macrocycle peptides and their pro-apoptotic activity.

SUMMARY OF THE INVENTION

[0003] Described below are stably cross-linked peptides related to a portion of human p53 ("p53 peptidomimetic macrocycles"). These cross-linked peptides contain at least two modified amino acids that together form an intramolecular cross-link that can help to stabilize the alpha-helical secondary structure of a portion of p53 that is thought to be important for binding of p53 to HDM2 and for binding of p53 to HDMX. Accordingly, a cross-linked polypeptide described herein can have improved biological activity relative to a corresponding polypeptide that is not cross-linked. The p53 peptidomimetic macrocycles are thought to interfere with binding of p53 to HDM2 and/or of p53 to HDMX, thereby liberating functional p53 and inhibiting its destruction. The p53 peptidomimetic macrocycles described herein can be used therapeutically, for example to treat cancers and other disorders characterized by an undesirably low level or a low activity of p53, and/or to treat cancers and other disorders characterized by an undesirably high level of activity of HDM2 or HDMX. The p53 peptidomimetic macrocycles may also be useful for treatment of any disorder associated with disrupted regulation of the p53 transcriptional pathway, leading to conditions of excess cell survival and proliferation such as cancer and autoimmunity, in addition to conditions of inappropriate cell cycle arrest and apoptosis such as neurodegeneration and immunedeficiencies. In some instances, the p53 peptidomimetic macrocycles bind to HDM2 (e.g., GenBank®) Accession No.: 228952; GL228952) and/or HDMX (also referred to as HDM4; GenBank® AccessionNo.: 88702791; GL88702791).

[0004] The present invention provides a peptidomimetic macrocycle which interferes with binding between p53 and HDM2 or HDMX, and which has an amino acid sequence at least 60% identical to:

Leu Thr Phe $-) Glu Tyr Trp Ala Gin Leu $2 Ala; wherein $j, is an a, a-disubstituted amino acid in the R configuration, wherein one substituent is a methyl group and the other substituent is a linker group to $2; and wherein $2 is an a, a-disubstituted amino acid in the S configuration, wherein one substituent is a methyl group and the other substituent is the linker group to $j; and wherein the linker group, starting from $j is -(CH2)6-CH=CH-(CH2)3-. casecase [0005] Additionally, disclosed is a pharmaceutical composition comprising the peptidomimetic macrocycle of the invention. Also provided is a peptidomimetic macrocycle or a pharmaceutical composition according to the invention for use in treating cancer in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIGURE 1 describes the synthesis of Fmoc-Me-6-Chloro-Tryptophan & Fmoc-6-Chloro-Tryptophan. FIGURE 2 shows an LC-MS trace of Me-6-Chloro-(Boc)Tryptophan-Ni-S-BPB. FIGURE 3 shows a 1H-NMR spectrum of Me-6-Chloro-(Boc)Tryptophan-Ni-S-BPB. FIGURE 4 shows an LC-MS trace of Fmoc-Me-6-Chloro-(Boc)Tryptophan. FIGURE 5 shows a 1H-NMR spectrum of Fmoc-Me-6-Chloro-(Boc)Tryptophan. FIGURES 6a-f describe the results of a cell viability assay, a competition ELISA assay, GRIP assay, Kd data, p21 activation assay, fluorescence polarization competition binding and circular helicity data for exemplary peptidomimetic macrocycles of the disclosure. FIGURES 7A-D provide data from a variety of macrocycles. FIGURES 8A-B provide data from a variety of macrocycles.

DETAILED DESCRIPTION

[0007] The invention is set forth in the appended claims.

[0008] As used herein, the term "macrocycle" refers to a molecule having a chemical structure including a ring or cycle formed by at least 9 covalently bonded atoms.

[0009] As used herein, the term "peptidomimetic macrocycle" or "crosslinked polypeptide" refers to a compound comprising a plurality of amino acid residues joined by a plurality of peptide bonds and at least one macrocycle-forming linker which forms a macrocycle between a first naturally-occurring or non-naturally-occurring amino acid residue (or analog) and a second naturally-occurring or non-naturally-occurring amino acid residue (or analog) within the same molecule. Peptidomimetic macrocycle include cases where the macrocycle-forming linker connects the a carbon of the first amino acid residue (or analog) to the a carbon of the second amino acid residue (or analog). The peptidomimetic macrocycles optionally include one or more non-peptide bonds between one or more amino acid residues and/or amino acid analog residues, and optionally include one or more non-naturally-occurring amino acid residues or amino acid analog residues in addition to any which form the macrocycle. A "corresponding uncrosslinked polypeptide" when referred to in the context of a peptidomimetic macrocycle is understood to relate to a polypeptide of the same length as the macrocycle and comprising the equivalent natural amino acids of the wild-type sequence corresponding to the macrocycle.

[0010] As used herein, the term "stability" refers to the maintenance of a defined secondary structure in solution by a peptidomimetic macrocycle of the invention as measured by circular dichroism, NMR or another biophysical measure, or resistance to proteolytic degradation in vitro or in vivo. Examples of secondary structures are a-helices, β-turns, and β-pleated sheets.

[0011] As used herein, the term "helical stability" refers to the maintenance of a helical structure by a peptidomimetic macrocycle of the disclosure as measured by circular dichroism or NMR. For example, in some cases, the peptidomimetic macrocycles of the disclosure exhibit at least a 1.25, 1.5, 1.75 or 2-fold increase in α-helicity as determined by circular dichroism compared to a corresponding uncrosslinked macrocycle.

[0012] The term "α-amino acid" or simply "amino acid" refers to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated the α-carbon. Suitable amino acids include, without limitation, both the D-and L-isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes. Unless the context specifically indicates otherwise, the term amino acid, as used herein, is intended to include amino acid analogs.

[0013] The term "naturally occurring amino acid" refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, Η, I, L, K, M, F, P, S, T, W, Y and V.

[0014] The term "amino acid analog" or "non-natural amino acid" refers to a molecule which is structurally similar to an amino acid and which can be substituted for an amino acid in the formation of a peptidomimetic macrocycle. Amino acid analogs include, without limitation, compounds which are structurally identical to an amino acid, as defined herein, except for the inclusion of one or more additional methylene groups between the amino and carboxyl group (e.g., a-amino β-carboxy acids), or for the substitution of the amino or carboxy group by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution of the carboxy group with an ester). Non-natural amino acids include structures according to the following:

[0015] A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide without abolishing or substantially altering its essential biological or biochemical activity (e.g., receptor binding or activation). An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of the polypeptide, results in abolishing or substantially abolishing the polypeptide's essential biological or biochemical activity.

[0016] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predicted nonessential amino acid residue in a polypeptide, for example, is preferably replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g. norleucine for methionine) or other properties (e.g. 2-thienylalanine for phenylalanine).

[0017] The term "capping group" refers to the chemical moiety optionally occurring at either the carboxy or amino terminus of the polypeptide chain of the subject peptidomimetic macrocycle. The capping group of a carboxy terminus includes an unmodified carboxylic acid (ie -COOH) or a carboxylic acid with a substituent. For example, the carboxy terminus may be substituted with an amino group to yield a carboxamide at the C-terminus. Various substituents include but are not limited to primary and secondary amines, including pegylated secondary amines. Representative secondary amine capping groups for the C-terminus include:

[0018] The capping group of an amino terminus includes an unmodified amine (ie -NhQ) or an amine with a substituent. For example, the amino terminus may be substituted with an acyl group to yield a carboxamide at the N-terminus. Various substituents include but are not limited to substituted acyl groups, including C-|-Cg carbonyls, C7-C30 carbonyls, and pegylated carbamates. Representative capping groups for the N-terminus include:

[0019] The term "member" as used herein in conjunction with macrocycles or macrocycle-forming linkers refers to the atoms that form or can form the macrocycle, and excludes substituent or side chain atoms. By analogy, cyclodecane, 1,2-difluoro-decane and 1,3-dimethyl cyclodecane are all considered ten-membered macrocycles as the hydrogen or fluoro substituents or methyl side chains do not participate in forming the macrocycle.

[0020] The term "amino acid side chain" refers to a moiety attached to the α-carbon in an amino acid. For example, the amino acid side chain for alanine is methyl, the amino acid side chain for phenylalanine is phenylmethyl, the amino acid side chain for cysteine is thiomethyl, the amino acid side chain for aspartate is carboxymethyl, the amino acid side chain for tyrosine is 4-hydroxyphenylmethyl, etc. Other non-naturally occurring amino acid side chains are also included, for example, those that occur in nature (e.g., an amino acid metabolite) or those that are made synthetically (e.g., an a, na di-substituted amino acid).

[0021] The term "a, na di-substituted amino" acid refers to a molecule or moiety containing both an amino group and a carboxyl group bound to a carbon (the α-carbon) that is attached to two natural or non-natural amino acid side chains.

[0022] The term "polypeptide" encompasses two or more naturally or non-naturally-occurring amino acids joined by a covalent bond (e.g., an amide bond). Polypeptides as described herein include full length proteins (e.g., fully processed proteins) as well as shorter amino acid sequences (e.g., fragments of naturally-occurring proteins or synthetic polypeptide fragments).

[0023] The term "macrocyclization reagent" or "macrocycle-forming reagent" as used herein refers to any reagent which may be used to prepare a peptidomimetic macrocycle of the disclosure by mediating the reaction between two reactive groups. Reactive groups may be, for example, an azide and alkyne, in which case macrocyclization reagents include, without limitation, Cu reagents such as reagents which provide a reactive Cu(l) species, such as CuBr, Cul or CuOTf, as well as Cu(ll) salts such as Cu(CO2CH3)2, CUSO4, and CuCl2 that can be converted in situ to an active Cu(l) reagent by the addition of a reducing agent such as ascorbic acid or sodium ascorbate. Macrocyclization reagents may additionally include, for example, Ru reagents known in the art such as Cp*RuCI(PPh3)2, [Cp*RuCI]4 or other Ru reagents which may provide a reactive Ru(ll) species. In other cases, the reactive groups are terminal olefins. In such cases, the macrocyclization reagents or macrocycle-forming reagents are metathesis catalysts including, but not limited to, stabilized, late transition metal carbene complex catalysts such as Group VIII transition metal carbene catalysts. For example, such catalysts are Ru and Os metal centers having a +2 oxidation state, an electron count of 16 and pentacoordinated. Additional catalysts are disclosed in Grubbs et al., "Ring Closing Metathesis and Related Processes in Organic Synthesis" Acc. Chem. Res. 1995, 28, 446-452, and U.S. Pat. No. 5,811,515.

[0024] In some cases, the compounds contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. In some cases, the compounds are also represented in multiple tautomeric forms (e.g., if alkylation of a ring system results in alkylation at multiple sites, the invention includes all such reaction products). All crystal forms of the compounds described herein are included in the present disclosure unless expressly provided otherwise.

[0025] As used herein, the terms "increase" and "decrease" mean, respectively, to cause a statistically significantly (i.e., p < 0.1) increase or decrease of at least 5%.

[0026] As used herein, unless specifically indicated otherwise, the word "or" is used in the inclusive sense of "and/or" and not the exclusive sense of "either/or." [0027] The term "on average" represents the mean value derived from performing at least three independent replicates for each data point.

[0028] The term "biological activity" encompasses structural and functional properties of a macrocycle of the disclosure. Biological activity is, for example, structural stability, alpha-helicity, affinity for a target, resistance to proteolytic degradation, cell penetrability, intracellular stability, in vivo stability, or any combination thereof.

[0029] In some cases, the peptide sequences are derived from the p53 protein.

[0030] Described below are suitable peptide sequences derived from p53 which may be used to make macrocyclic peptides. TABLE 1

[0031] In Table 3 and elsewhere, "Aib" represents a 2-aminoisobutyric acid residue, while "Ac3c" represents a aminocyclopropane carboxylic acid residue.

Preparation of Peptidomimetic Macrocycles [0032] Peptidomimetic macrocycles may be prepared by any of a variety of methods known in the art. For example, any of the residues indicated by "X" in Tables 1, 2, 3, or 4 may be substituted with a residue capable of forming a crosslinker with a second residue in the same molecule ora precursor of such a residue.

[0033] Various methods to effect formation of peptidomimetic macrocycles are known in the art. For example, the preparation of peptidomimetic macrocycles of Formula I is described in Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Schafmeister &amp; Verdine, J. Am. Chem. Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); US Patent No. 7,192,713 and PCT application WO 2008/121767. The α,α-disubstituted amino acids and amino acid precursors disclosed in the cited references may be employed in synthesis of the peptidomimetic macrocycle precursor polypeptides. For example, the "S5-olefin amino acid" is (S)-a-(2'-pentenyl) alanine and the "R8 olefin amino acid" is (R)-a-(2'-octenyl) alanine. Following incorporation of such amino acids into precursor polypeptides, the terminal olefins are reacted with a metathesis catalyst, leading to the formation of the peptidomimetic macrocycle. In various cases, the following amino acids may be employed in the synthesis of the peptidomimetic macrocycle:

[0034] Additional methods of forming peptidomimetic macrocycles include those disclosed by Mustapa, M. Firouz Mohd et al., J. Org. Chem (2003), 68, pp. 8193-8198; Yang, Bin et al. Bioorg Med. Chem. Lett. (2004), 14, pp. 1403-1406; U.S. Patent No. 5,364,851; U.S. Patent No. 5,446,128; U.S. Patent No. 5,824,483; U.S. Patent No. 6,713,280; and U.S. Patent No. 7,202,332. In such cases, aminoacid precursors are used

containing an additional substituent R- at the alpha position. Such aminoacids are incorporated into the macrocycle precursor at the desired positions, which may be at the positions where the crosslinker is substituted or, alternatively, elsewhere in the sequence of the macrocycle precursor. Cyclization of the precursor is then effected according to the indicated method.

Assays [0035] The properties of the peptidomimetic macrocycles are assayed, for example, by using the methods described below. In some cases, a peptidomimetic macrocycle has improved biological properties relative to a corresponding polypeptide lacking the substituents described herein.

Assay to Determine a-helicity.

[0036] In solution, the secondary structure of polypeptides with a-helical domains will reach a dynamic equilibrium between random coil structures and α-helical structures, often expressed as a "percent helicity". Thus, for example, alpha-helical domains are predominantly random coils in solution, with α-helical content usually under 25%. Peptidomimetic macrocycles with optimized linkers, on the other hand, possess, for example, an alpha-helicity that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide. In some cases, macrocycles will possess an alpha-helicity of greater than 50%. To assay the helicity of peptidomimetic macrocyles, the compounds are dissolved in an aqueous solution (e.g. 50 mM potassium phosphate solution at pH 7, or distilled H2O, to concentrations of 25-50 μΜ). Circular dichroism (CD) spectra are obtained on a spectropolarimeter (e.g., Jasco J-710) using standard measurement parameters (e.g. temperature, 20°C; wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The α-helical content of each peptide is calculated by dividing the mean residue ellipticity (e.g. [<t>]222obs) by the reported value for a model helical decapeptide (Yang etal. (1986), Methods Enzymol. 130:208)).

Assay to Determine Melting Temperature (Tm).

[0037] A peptidomimetic macrocycle of the invention comprising a secondary structure such as an a-helix exhibits, for example, a higher melting temperature than a corresponding uncrosslinked polypeptide. Typically peptidomimetic macrocycles of the invention exhibit Tm of > 60°C representing a highly stable structure in aqueous solutions. To assay the effect of macrocycle formation on melting temperature, peptidomimetic macrocycles or unmodified peptides are dissolved in distilled H2O (e.g. at a final concentration of 50 μΜ) and the Tm is determined by measuring the change in ellipticity over a temperature range (e.g. 4 to 95 °C) on a spectropolarimeter (e.g., Jasco J-710) using standard parameters (e.g. wavelength 222nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; temperature increase rate: 1°C/min; path length, 0.1 cm).

Protease Resistance Assay.

[0038] The amide bond of the peptide backbone is susceptible to hydrolysis by proteases, thereby rendering peptidic compounds vulnerable to rapid degradation in vivo. Peptide helix formation, however, typically buries the amide backbone and therefore may shield it from proteolytic cleavage. The peptidomimetic macrocycles of the present invention may be subjected to in vitro trypsin proteolysis to assess for any change in degradation rate compared to a corresponding uncrosslinked polypeptide. For example, the peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide are incubated with trypsin agarose and the reactions quenched at various time points by centrifugation and subsequent HPLC injection to quantitate the residual substrate by ultraviolet absorption at 280 nm. Briefly, the peptidomimetic macrocycle and peptidomimetic precursor (5 meg) are incubated with trypsin agarose (Pierce) (S/E —125) for 0, 10, 20, 90, and 180 minutes. Reactions are quenched by tabletop centrifugation at high speed; remaining substrate in the isolated supernatant is quantified by HPLC-based peak detection at 280 nm. The proteolytic reaction displays first order kinetics and the rate constant, k, is determined from a plot of ln[S] versus time (k=-1Xslope).

Ex Vivo Stability Assay.

[0039] Peptidomimetic macrocycles with optimized linkers possess, for example, an ex vivo half-life that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide, and possess an ex vivo half-life of 12 hours or more. For ex vivo serum stability studies, a variety of assays may be used. For example, a peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide (2 meg) are incubated with fresh mouse, rat and/or human serum (2 mL) at 37°C for 0, 1,2, 4, 8, and 24 hours. To determine the level of intact compound, the following procedure may be used: The samples are extracted by transferring 100 μΙ of sera to 2 ml centrifuge tubes followed by the addition of 10 pL of 50 % formic acid and 500pL acetonitrile and centrifugation at 14,000 RPM for 10 min at 4 ± 2°C. The supernatants are then transferred to fresh 2 ml tubes and evaporated on Turbovap under N2 < 10 psi, 37°C. The samples are reconstituted in 100pL of 50:50 acetonitrile:water and submitted to LC-MS/MS analysis.

In vitro Binding Assays.

[0040] To assess the binding and affinity of peptidomimetic macrocycles and peptidomimetic precursors to acceptor proteins, a fluorescence polarization assay (FPA) isused, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g. FITC-labeled peptides bound to a large protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g. FITC- labeled peptides that are free in solution).

[0041] For example, fluoresceinated peptidomimetic macrocycles (25 nM) are incubated with the acceptor protein (25- 1000nM) in binding buffer (140mM NaCI, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Binding activity is measured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, CA). A peptidomimetic macrocycle of the invention shows, in some instances, similar or lower Kd than a corresponding uncrosslinked polypeptide.

In vitro Displacement Assays To Characterize Antagonists of Peptide-Protein Interactions.

[0042] To assess the binding and affinity of compounds that antagonize the interaction between a peptide and an acceptor protein, a fluorescence polarization assay (FPA) utilizing a fluoresceinated peptidomimetic macrocycle derived from a peptidomimetic precursor sequence is used, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g. FITC-labeled peptides bound to a large protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g. FITC-labeled peptides that are free in solution). A compound that antagonizes the interaction between the fluoresceinated peptidomimetic macrocycle and an acceptor protein will be detected in a competitive binding FPA experiment.

[0043] For example, putative antagonist compounds (1 nM to 1 mM) and a fluoresceinated peptidomimetic macrocycle (25 nM) are incubated with the acceptor protein (50 nM) in binding buffer (140mM NaCI, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Antagonist binding activity is measured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, CA).

[0044] Any class of molecule, such as small organic molecules, peptides, oligonucleotides or proteins can be examined as putative antagonists in this assay.

Assay for Protein-ligand binding by Affinity Selection-Mass Spectrometry [0045] To assess the binding and affinity of test compounds for proteins, an affinity-selection mass spectrometry assay is used, for example. Protein-ligand binding experiments are conducted according to the following representative procedure outlined for a system-wide control experiment using 1 μΜ peptidomimetic macrocycle plus 5 μΜ hMDM2. A1 pL DMSO aliquot of a 40 μΜ stock solution of peptidomimetic macrocycle is dissolved in 19 pL of PBS (Phosphate-buffered saline: 50 mM, pH 7.5 Phosphate buffer containing 150 mM NaCI). The resulting solution is mixed by repeated pipetting and clarified by centrifugation at 10 OOOg for 10 min. To a 4 pL aliquot of the resulting supernatant is added 4 pL of 10 pM hMDM2 in PBS. Each 8.0 pL experimental sample thus contains 40 pmol (1.5 pg) of protein at 5.0 μΜ concentration in PBS plus 1 μΜ peptidomimetic macrocycle and 2.5% DMSO. Duplicate samples thus prepared for each concentration point are incubated for 60 min at room temperature, and then chilled to 4 °C prior to size-exclusion chromatography-LC-MS analysis of 5.0 pL injections. Samples containing a target protein, protein-ligand complexes, and unbound compounds are injected onto an SEC column, where the complexes are separated from non-binding component by a rapid SEC step. The SEC column eluate is monitored using UV detectors to confirm that the early-eluting protein fraction, which elutes in the void volume of the SEC column, is well resolved from unbound components that are retained on the column. After the peak containing the protein and protein-ligand complexes elutes from the primary UV detector, it enters a sample loop where it is excised from the flow stream of the SEC stage and transferred directly to the LC-MS via a valving mechanism. The (M + 3H)3+ ion of the peptidomimetic macrocycle is observed by ESI-MS at the expected m/z, confirming the detection of the protein-ligand complex.

Assay for Protein-ligand Kd Titration Experiments.

[0046] To assess the binding and affinity of test compounds for proteins, a protein-ligand Kd titration experiment is performed, for example. Protein-ligand Κ$ titrations experiments are conducted as follows: 2 pL DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (5, 2.5, ..., 0.098 mM) are prepared then dissolved in 38 pL of PBS. The resulting solutions are mixed by repeated pipetting and clarified by centrifugation at 10 OOOg for 10 min. To 4.0 pL aliquots of the resulting supernatants is added 4.0 pL of 10 μΜ hMDM2 in PBS. Each 8.0 pL experimental sample thus contains 40 pmol (1.5 pg) of protein at 5.0 μΜ concentration in PBS, varying concentrations (125, 62.5, ..., 0.24 μΜ) of the titrant peptide, and 2.5% DMSO. Duplicate samples thus prepared for each concentration point are incubated at room temperature for 30 min, then chilled to 4 °C prior to SEC-LC-MS analysis of 2.0 pL injections. The (M + H)1+, (M + 2H)2+, (M + 3H)3+, and/or (M + Na)1+ ion is observed by ESI-MS; extracted ion chromatograms are quantified, then fit to equations to derive the binding affinity Κ$ as described in "A General Technique to Rank Protein-Ligand Binding Affinities and Determine Allosteric vs. Direct Binding Site Competition in Compound Mixtures." Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. R; Nash, Η. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in "ALIS: An Affinity Selection-Mass Spectrometry System for the Discovery and Characterization of Protein-Ligand Interactions" D. A. Annis, C.-C. Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Hofner G: Wiley-VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series Editors): Methods and Principles in Medicinal Chemistry.

Assay for Competitive Binding Experiments by Affinity Selection-Mass Spectrometry [0047] To determine the ability of test compounds to bind competitively to proteins, an affiinity selection mass spectrometry assay is performed, for example. A mixture of ligands at 40 μΜ per component is prepared by combining 2 pL aliquots of 400 μΜ stocks of each of the three compounds with 14 pL of DMSO. Then, 1 pL aliquots of this 40 pM per component mixture are combined with 1 pL DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (10, 5, 2.5, ..., 0.078 mM). These 2 pL samples are dissolved in 38 pL of PBS. The resulting solutions were mixed by repeated pipetting and clarified by centrifugation at 10 OOOg for 10 min. To 4.0 pL aliquots of the resulting supernatants is added 4.0 pL of 10 μΜ hMDM2 protein in PBS. Each 8.0 pL experimental sample thus contains 40 pmol (1.5 pg) of protein at 5.0 μΜ concentration in PBS plus 0.5 μΜ ligand, 2.5% DMSO, and varying concentrations (125, 62.5, ..., 0.98 μΜ) of the titrant peptidomimetic macrocycle. Duplicate samples thus prepared for each concentration point are incubated at room temperature for 60 min, then chilled to 4 °C prior to SEC-LC-MS analysis of 2.0 pL injections. Additional details on these and other methods are provided in "A General Technique to Rank Protein-Ligand Binding Affinities and Determine Allosteric vs. Direct Binding Site Competition in Compound Mixtures." Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. R; Nash, Η. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in "ALIS: An Affinity Selection-Mass Spectrometry System for the Discovery and Characterization of Protein-Ligand Interactions" D. A. Annis, C.-C. Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Hofner G: Wiley-VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series Editors): Methods and Principles in Medicinal Chemistry.

Binding Assays in Intact Cells.

[0048] It is possible to measure binding of peptides or peptidomimetic macrocycles to their natural acceptors in intact cells by immunoprecipitation experiments. For example, intact cells are incubated with fluoresceinated (FITC-labeled) compounds for 4 hrs in the absence of serum, followed by serum replacement and further incubation that ranges from 4-18 hrs. Cells are then pelleted and incubated in lysis buffer (50mM Tris [pH 7.6], 150 mM NaCI, 1% CHAPS and protease inhibitor cocktail) for 10 minutes at 4°C. Extracts are centrifuged at 14,000 rpm for 15 minutes and supernatants collected and incubated with 10 pi goat anti-FITC antibody for 2 hrs, rotating at 4°C followed by further 2 hrs incubation at 4°C with protein A/G Sepharose (50 pi of 50% bead slurry). After quick centrifugation, the pellets are washed in lysis buffer containing increasing salt concentration (e.g., 150, 300, 500 mM). The beads are then re-equilibrated at 150 mM NaCI before addition of SDS-containing sample buffer and boiling. After centrifugation, the supernatants are optionally electrophoresed using 4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-P membranes. After blocking, blots are optionally incubated with an antibody that detects FITC and also with one or more antibodies that detect proteins that bind to the peptidomimetic macrocycle.

Cellular Penetrability Assays.

[0049] A peptidomimetic macrocycle is, for example, more cell penetrable compared to a corresponding uncrosslinked macrocycle. Peptidomimetic macrocycles with optimized linkers possess, for example, cell penetrability that is at least two-fold greater than a corresponding uncrosslinked macrocycle, and often 20% or more of the applied peptidomimetic macrocycle will be observed to have penetrated the cell after 4 hours.To measure the cell penetrability of peptidomimetic macrocycles and corresponding uncrosslinked macrocycle, intact cells are incubated with fluoresceinated peptidomimetic macrocycles or corresponding uncrosslinked macrocycle (10 μΜ) for 4 hrs in serum free media at 37°C, washed twice with media and incubated with trypsin (0.25%) for 10 min at 37°C. The cells are washed again and resuspended in PBS. Cellular fluorescence is analyzed, for example, by using either a FACSCalibur flow cytometer or Cellomics' KineticScan ® HCS Reader.

Cellular Efficacy Assays.

[0050] The efficacy of certain peptidomimetic macrocycles is determined, for example, in cell-based killing assays using a variety of tumorigenic and non-tumorigenic cell lines and primary cells derived from human or mouse cell populations. Cell viability is monitored, for example, over 24-96 hrs of incubation with peptidomimetic macrocycles (0.5 to 50 μΜ) to identify those that kill at EC50<10 μΜ. Several standard assays that measure cell viability are commercially available and are optionally used to assess the efficacy of the peptidomimetic macrocycles. In addition, assays that measure Annexin V and caspase activation are optionally used to assess whether the peptidomimetic macrocycles kill cells by activating the apoptotic machinery. For example, the Cell Titer-gio assay is used which determines cell viability as a function of intracellular ATP concentration.

In Vivo Stability Assay.

[0051] To investigate the in vivo stability of the peptidomimetic macrocycles, the compounds are, for example,administered to mice and/or rats by IV, IP, PO or inhalation routes at concentrations ranging from 0.1 to 50 mg/kg and blood specimens withdrawn at O', 5', 15', 30', 1 hr, 4 hrs, 8 hrs and 24 hours post-injection. Levels of intact compound in 25 pL of fresh serum are then measured by LC-MS/MS as above.

In vivo Efficacy in Animal Models.

[0052] To determine the anti-oncogenic activity of peptidomimetic macrocycles of the invention in vivo, the compounds are, for example, given alone (IP, IV, PO, by inhalation or nasal routes) or in combination with sub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide, doxorubicin, etoposide). In one example, 5 x 106 RS4;11 cells (established from the bone marrow of a patient with acute lymphoblastic leukemia) that stably express luciferase are injected by tail vein in NOD-SCID mice 3 hrs after they have been subjected to total body irradiation. If left untreated, this form of leukemia is fatal in 3 weeks in this model. The leukemia is readily monitored, for example, by injecting the mice with D-luciferin (60 mg/kg) and imaging the anesthetized animals (e.g., Xenogen In Vivo Imaging System, Caliper Life Sciences, Hopkinton, MA). Total body bioluminescence is quantified by integration of photonic flux (photons/sec) by Living Image Software (Caliper Life Sciences, Hopkinton, MA). Peptidomimetic macrocycles alone or in combination with sub-optimal doses of relevant chemotherapeutics agents are, for example, administered to leukemic mice (10 days after injection/day 1 of experiment, in bioluminescence range of 14-16) by tail vein or IP routes at doses ranging from 0.1 mg/kg to 50 mg/kg for 7 to 21 days. Optionally, the mice are imaged throughout the experiment every other day and survival monitored daily for the duration of the experiment. Expired mice are optionally subjected to necropsy at the end of the experiment. Another animal model is implantation into NOD-SCID mice of DoHH2, a cell line derived from human follicular lymphoma, that stably expresses luciferase. These in vivo tests optionally generate preliminary pharmacokinetic, pharmacodynamic and toxicology data.

Clinical Trials.

[0053] To determine the suitability of the peptidomimetic macrocycles for treatment of humans, clinical trials are performed. For example, patients diagnosed with cancer and in need of treatment are selected and separated in treatment and one or more control groups, wherein the treatment group is administered a peptidomimetic macrocycle, while the control groups receive a placebo or a known anti-cancer drug. The treatment safety and efficacy of the peptidomimetic macrocycles can thus be evaluated by performing comparisons of the patient groups with respect to factors such as survival and quality-of-life. In this example, the patient group treated with a peptidomimetic macrocyle show improved long-term survival compared to a patient control group treated with a placebo.

Pharmaceutical Compositions and Routes of Administration [0054] The peptidomimetic macrocycles of the invention also include pharmaceutically acceptable salt, which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention.

[0055] In some cases, the peptidomimetic macrocycles are modified by covalently or non-covalently joining appropriate functional groups to enhance selective biological properties. Such modifications 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.

[0056] 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, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate 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.

[0057] For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers include either solid or liquid carriers. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances, which also acts as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA.

[0058] In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

[0059] Suitable solid excipients are carbohydrate or protein fillers include, but are not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents are added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

[0060] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.

[0061] The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

[0062] When the compositions of this invention comprise a combination of a peptidomimetic macrocycle 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. In some cases, the additional agents are administered separately, as part of a multiple dose regimen, from the compounds of this invention. Alternatively, those agents are part of a single dosage form, mixed together with the compounds of this invention in a single composition.

Methods of Use [0063] In one aspect, the present disclosure provides novel peptidomimetic macrocycles that are useful in competitive binding assays to identify agents which bind to the natural ligand(s) of the proteins or peptides upon which the peptidomimetic macrocycles are modeled. For example, in the p53/HDMX system, labeled peptidomimetic macrocycles based on p53 can be used in a HDMX binding assay along with small molecules that competitively bind to HDMX. Competitive binding studies allow for rapid in vitro evaluation and determination of drug candidates specific for the p53/HDMX system. Such binding studies may be performed with any of the peptidomimetic macrocycles disclosed herein and their binding partners.

[0064] The disclosure further provides for the generation of antibodies against the peptidomimetic macrocycles. In some cases, these antibodies specifically bind both the peptidomimetic macrocycle and the precursor peptides, such as p53, to which the peptidomimetic macrocycles are related. Such antibodies, for example, disrupt the native protein-protein interaction, for example, binding between p53 and HDMX.

[0065] In other aspects, the present disclosure 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 aberrant (e.g., insufficient or excessive) expression or activity of the molecules including p53, HDM2 or HDMX.

[0066] In another case, a disorder is caused, at least in part, by an abnormal level of p53 or HDM2 or HDMX, (e.g., over or under expression), or by the presence of p53 or HDM2 or HDMX exhibiting abnormal activity. As such, the reduction in the level and/or activity of p53 or HDM2 or HDMX, or the enhancement of the level and/or activity of p53 or HDM2 or HDMX, by peptidomimetic macrocycles derived from p53, is used, for example, to ameliorate or reduce the adverse symptoms of the disorder.

[0067] Described herein are methods for treating or preventing a disease including hyperproliferative disease and inflammatory disorder by interfering with the interaction or binding between binding partners, for example, between p53 and HDM2 or p53 and HDMX. These methods comprise administering an effective amount of a compound of the invention to a warm blooded animal, including a human. In some cases, the administration of the compounds of the present disclosure induces cell growth arrest or apoptosis.

[0068] 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.

[0069] The peptidomimetics macrocycles of the invention may 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. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of breast, lung, liver, colon and ovarian origin. "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. Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, or metastatic disorders. In some embodiments, the peptidomimetics macrocycles are novel therapeutic agents for controlling breast cancer, ovarian cancer, colon cancer, lung cancer, metastasis of such cancers and the like.

[0070] Examples of cancers or neoplastic conditions include, but are not limited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular cancer, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi sarcoma.

[0071] 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. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic 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 (1991), Crit Rev. Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), 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-Stemberg disease.

[0072] 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 invasive papillary carcinoma, and miscellaneous malignant neoplasms. Disorders in the male breast include, but are not limited to, gynecomastia and carcinoma.

[0073] Examples of cellular proliferative and/or differentiative disorders of the lung include, but are not limited to, bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, and metastatic tumors; pathologies of the pleura, including inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.

[0074] Examples of cellular proliferative and/or differentiative disorders of the colon include, but are not limited to, non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.

[0075] Examples of cellular proliferative and/or differentiative disorders of the liver include, but are not limited to, nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma of the liver and metastatic tumors.

[0076] Examples of cellular proliferative and/or differentiative disorders of the ovary include, but are not limited to, ovarian tumors such as, tumors of coelomic epithelium, serous tumors, mucinous tumors, endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma, Brenner tumor, surface epithelial tumors; germ cell tumors such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-theca cell tumors, thecomafibromas, androblastomas, hill cell tumors, and gonadoblastoma; and metastatic tumors such as Krukenberg tumors.

[0077] In other or further cases, the peptidomimetics macrocycles described herein are used to treat, prevent or diagnose conditions characterized by overactive cell death or cellular death due to physiologic insult, etc. Some examples of conditions characterized by premature or unwanted cell death are or alternatively unwanted or excessive cellular proliferation include, but are not limited to hypocellular/hypoplastic, acellular/aplastic, or hypercellular/hyperplastic conditions. Some examples include hematologic disorders including but not limited to fanconi anemia, aplastic anemia, thalaessemia, congenital neutropenia, and myelodysplasia.

Examples

Example 1: Synthesis of 6-chlorotryptophan Fmoc amino acids [0078]

[0079] Tert-butyl 6-chloro-3-formyl-1 H-indole-1-carboxylate, 1. To a stirred solution of dry DMF (12 mL) was added dropwise POCI3 (3.92 mL, 43 mmol, 1.3 equiv) at 0 °C under Argon. The solution was stirred at the same temperature for 20 min before a solution of 6-chloroindole (5.0 g, 33 mmol, 1 eq.) in dry DMF (30 mL) was added dropwise. The resulting mixture was allowed to warm to room temperature and stirred for an additional 2.5h. Water (50 mL) was added and the solution was neutralized with 4M aqueous NaOH (pH ~ 8). The resulting solid was filtered off, washed with water and dried under vacuum. This material was directly used in the next step without additional purification. To a stirred solution of the crude formyl indole (33 mmol, 1 eq.) in THF (150 mL) was added successively BOC2O (7.91 g, 36.3 mmol, 1.1 equiv) and DMAP (0.4 g, 3.3 mmol, 0.1 equiv) at room temperature under N2. The resulting mixture was stirred at room temperature for 1.5h and the solvent was evaporated under reduced pressure. The residue was taken up in EtOAc and washed with 1N HCI, dried and concentrated to give the formyl indole 1 (9 g, 98 % over 2 steps) as a white solid. 1H NMR (CDCI3) δ: 1.70 (s, Boc, 9H); 7.35 (dd, 1H); 8.21 (m, 3H); 10.07 (s, 1H).

[0080] Tert-butyl 6-chloro-3-(hydroxymethyl)-1H-indole-1-carboxylate, 2. To a solution of compound 1 (8.86g, 32 mmol, 1 eq.) in ethanol (150 mL) was added NaBH4 (2.4g, 63 mmol, 2 eq.). The reaction was stirred for 3 h at room temperature. The reaction mixture was concentrated and the residue was poured into diethyl ether and water. The organic layer was separated, dried over magnesium sulfate and concentrated to give a white solid (8.7g, 98%). This material was directly used in the next step without additional purification. 1H NMR (CDCI3) δ: 1.65 (s, Boc, 9H); 4.80 (s, 2H, CH2); 7.21 (dd, 1H); 7.53 (m, 2H); 8.16 (bs, 1H).

[0081] Tert-butyl 3-(bromomethyl)-6-chloro-1H-indole-1-carboxylate, 3. To a solution of compound 2 (4.1g, 14.6 mmol, 1 eq.) in dichloromethane (50 mL) under argon was added a solution of triphenylphosphine (4.59g, 17.5 mmol, 1.2 eq.) in dichloromethane (50 mL) at -40°C. The reaction solution was stirred an additional 30 min at 40°C. Then NBS (3.38g, 19 mmol, 1.3 eq.) was added. The resulting mixture was allowed to warm to room temperature and stirred overnight. Dichloromethane was evaporated, Carbon Tetrachloride (100 mL) was added and the mixture was stirred for 1 h and filtrated. The filtrate was concentrated, loaded in a silica plug and quickly eluted with 25% EtOAc in Hexanes. The solution was concentrated to give a white foam (3.84g, 77%). 1H NMR(CDCI3) δ: 1.66 (s, Boc, 9H); 4.63 (s, 2H, CH2); 7.28 (dd, 1H); 7.57 (d, 1H); 7.64 (bs, 1H); 8.18 (bs, 1H).

[0082] aMe-6CI-Trp(Boc)-Ni-S-BPB, 4. To S-Ala-Ni-S-BPB (2.66g, 5.2 mmol, 1 eq.) and KO-fBu (0.87g, 7.8 mmol, 1.5 eq.) was added 50 mL of DMF under argon. The bromide derivative compound 3 (2.68g, 7.8 mmol, 1.5 eq.) in solution of DMF (5.0 mL) was added via syringe. The reaction mixture was stirred at ambient temperature for 1h. The solution was then quenched with 5 % aqueous acetic acid and diluted with water. The desired product was extracted in dichloromethane, dried and concentrated. The oily product 4 was purified by flash chromatography (solid loading) on normal phase using EtOAc and Hexanes as eluents to give a red solid (1.78g, 45% yield). aMe-6CI-Trp(Boc)-Ni-S-BPB, 4: M+H calc. 775.21, M+H obs. 775.26; 1H NMR (CDCI3) δ: 1.23 (s, 3H, aMe); 1.56 (m, 11H, Boc + CH2); 1.82-2.20 (m, 4H, 2CH2); 3.03 (m, 1H, CHa); 3.24 (m, 2H, CH2); 3.57 and 4.29 (AB system, 2H, CH2 (benzyl), J= 12.8Hz); 6.62 (d, 2H); 6.98 (d, 1H); 7.14 (m, 2H); 7.23 (m, 1H); 7.32-7.36 (m, 5H); 7.50 (m, 2H); 7.67 (bs, 1H); 7.98 (d, 2H); 8.27 (m, 2H).

[0083] Fmoc-aMe-6CI-Trp(Boc)-OH, 6. To a solution of 3N HCI/MeOH (1/3, 15 mL) at 50°C was added a solution of compound 4 (1.75g, 2.3 mmol, 1 eq.) in MeOH (5 ml) dropwise. The starting material disappeared within 3-4 h. The acidic solution was then cooled to 0°C with an ice bath and quenched with an aqueous solution of Na2CC>3 (1.21 g, 11.5 mmol, 5 eq.). Methanol was removed and 8 more equivalents of Na2CC>3 (1.95g, 18.4 mmol) were added to the suspension. The Nickel scavenging EDTA disodium salt dihydrate (1.68g, 4.5 mmol, 2 eq.) was then added and the suspension was stirred for 2h. A solution of Fmoc-OSu (0.84g, 2.5 mmol, 1.1 eq.) in acetone (50 mL) was added and the reaction was stirred overnight. Afterwards, the reaction was diluted with diethyl ether and 1N HCI. The organic layer was then dried over magnesium sulfate and concentrated in vacuo. The desired product 6 was purified on normal phase using acetone and dichloromethane as eluents to give a white foam (0.9g, 70% yield). Fmoc-aMe-6CI-Trp(Boc)-OH, 6: M+H calc. 575.19, M+H obs. 575.37; 1H NMR (CDCI3) 1.59 (s, 9H, Boc); 1.68 (s, 3H, Me); 3.48 (bs, 2H, CH2); 4.22 (m, 1H, CH); 4.39 (bs, 2H, CH2); 5.47 (s, 1H, NH); 7.10 (m, 1H); 7.18 (m, 2H); 7.27 (m, 2H); 7.39 (m, 2H); 7.50 (m, 2H); 7.75 (d, 2H); 8.12 (bs, 1H).

[0084] 6CI-Trp(Boc)-Ni-S-BPB, 5. To Gly-Ni-S-BPB (4.6g, 9.2 mmol, 1 eq.) and KO-fBu (1.14g, 10.1 mmol, 1.1 eq.) was added 95 mL of DMF under argon. The bromide derivative compound 3 (3.5g, 4.6 mmol, 1.1 eq.) in solution of DMF (10 mL) was added via syringe. The reaction mixture was stirred at ambient temperature for 1h. The solution was then quenched with 5 % aqueous acetic acid and diluted with water. The desired product was extracted in dichloromethane, dried and concentrated. The oily product 5 was purified by flash chromatography (solid loading) on normal phase using EtOAc and Hexanes as eluents to give a red solid (5g, 71% yield). 6CI-Trp(Boc)-Ni-S-BPB, 5: M+H calc. 761.20, M+H obs. 761.34; 1H NMR (CDCI3) δ: 1.58 (m, 11H, Boc + CH2); 1.84 (m, 1H); 1.96 (m, 1H); 2.24 (m, 2H, CH2); 3.00 (m, 1H, CHa); 3.22 (m, 2H, CH2); 3.45 and 4.25 (AB system, 2H, CH2 (benzyl), J= 12.8Hz); 4.27 (m, 1H, CHa); 6.65 (d, 2H); 6.88 (d, 1H); 7.07 (m, 2H); 7.14 (m, 2H); 7.28 (m, 3H); 7.35-7.39 (m, 2H); 7.52 (m, 2H); 7.96 (d, 2H); 8.28 (m, 2H).

[0085] Fmoc-6CI-Trp(Boc)-OH, 7. To a solution of 3N HCI/MeOH (1/3, 44 mL) at 50°C was added a solution of compound 5 (5g, 6.6 mmol, 1 eq.) in MeOH (10 ml) dropwise. The starting material disappeared within 3-4 h. The acidic solution was then cooled to 0°C with an ice bath and quenched with an aqueous solution of Na2CO3 (3.48g, 33 mmol, 5 eq.). Methanol was removed and 8 more equivalents of Na2CO3 (5.57g, 52 mmol) were added to the suspension. The Nickel scavenging EDTA disodium salt dihydrate (4.89g, 13.1 mmol, 2 eq.) and the suspension was stirred for 2h. A solution of Fmoc-OSu (2.21g, 6.55 mmol, 1.1 eq.) in acetone (100 mL) was added and the reaction was stirred overnight. Afterwards, the reaction was diluted with diethyl ether and 1N HCI. The organic layer was then dried over magnesium sulfate and concentrated in vacuo. The desired product 7 was purified on normal phase using acetone and dichloromethane as eluents to give a white foam (2.6g, 69% yield). Fmoc-6CI-Trp(Boc)-OH, 7: M+H calc. 561.17, M+H obs. 561.37; 1H NMR (CDCI3) 1.63 (s, 9H, Boc); 3.26 (m, 2H, CH2); 4.19 (m, 1H, CH); 4.39 (m, 2H, CH2); 4.76 (m, 1H); 5.35 (d, 1H, NH); 7.18 (m, 2H); 7.28 (m, 2H); 7.39 (m, 3H); 7.50 (m, 2H); 7.75 (d, 2H); 8.14 (bs, 1H).

Example 2: Peptidomimetic macrocycles of the invention [0086] Peptidomimetic macrocycles were synthesized, purified and analyzed as previously described and as described below (Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Schafmeister &amp; Verdine, J. Am. Chem. Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); and US Patent No. 7,192,713). Peptidomimetic macrocycles were designed by replacing two or more naturally occurring amino acids with the corresponding synthetic amino acids. Substitutions were made at i and i+4, and i and i+7 positions. Peptide synthesis was performed either manually or on an automated peptide synthesizer (Applied Biosystems, model 433A), using solid phase conditions, rink amide AM resin (Novabiochem), and Fmoc main-chain protecting group chemistry. For the coupling of natural Fmoc-protected amino acids (Novabiochem), 10 equivalents of amino acid and a 1:1:2 molar ratio of coupling reagents HBTU/HOBt (Novabiochem)/DIEA were employed. Non-natural amino acids (4 equiv) were coupled with a 1:1:2 molar ratio of HATU (Applied Biosystems)/HOBt/DIEA. The N-termini of the synthetic peptides were acetylated, while the C-termini were amidated.

[0087] Purification of cross-linked compounds was achieved by high performance liquid chromatography (HPLC) (Varian ProStar) on a reverse phase C18 column (Varian) to yield the pure compounds. Chemical composition of the pure products was confirmed by LC/MS mass spectrometry (Micromass LCT interfaced with Agilent 1100 HPLC system) and amino acid analysis (Applied Biosystems, model 420A).

[0088] Table 4 shows a list of prepared peptidomimetic macrocycles.

Table 4

i SP i Seq i Exact Mass M+2 i Observed mass ISP-765 ÅC-LTFSr8ÅCou2WAQ LSS-N H 2.........................|.......1‘697.92..........849.96 .................................850.82 I SP-766~] Dm^cTrF$r84^^ I sp-767 Hexac-LTFSr8AYWAQLSS-NH2 | 1587.91 794.96 795.11 ] |SP-768 Napac-L^ |'~^657O9~™ 82495 83o'36 | SP-769 ] Pam-LTF$r8AYWAQL$S-N | 1728.06 865Ό3 86545] ISP-770 Ac-LT2NalSr8HYAAQLSS-NH2 | 1532.84 767.42 767.611 | sp-772 Ac-LT2NalSr8HYFAQLSS-NH2 | 1608.87 805.44 8049] I sP-7'7'3" AcTkT^ "^155486™" ^778.93^ 779.08^ ISP-774 AcZT2Nal$r8HYAW^ | 164788 82494 Ί 825Ό4Ϊ | sP-775 IAc-LT2NalSr8HYAAQWSS-NH2 | 160483 803.92 804.05 I s'p-776 TZ^W$r8HY\^^L$S^NHT™™™^" ' 8Ϊ944 ] ^SP-777 Ac-LT1NalSr8HYWAQLSS-NH2 | 1647.88 824.94 825.41 [0089] In the sequences shown above and elsewhere, the following abbreviations are used: "Nle" represents norleucine, "Aib" represents 2-aminoisobutyric acid, "Ac" represents acetyl, and "Pr" represents propionyl. Amino acids represented as "$" are alpha-Me S5-pentenyl-alanine olefin amino acids connected by an allcarbon i to i+4 crosslinker comprising one double bond. Amino acids represented as "$r5" are alpha-Me R5-pentenyl-alanine olefin amino acids connected by an all-carbon i to i+4 crosslinker comprising one double bond. Amino acids represented as "$s8" are alpha-Me S8-octenyl-alanine olefin amino acids connected by an all-carbon i to i+7 crosslinker comprising one double bond. Amino acids represented as "$r8" are alpha-Me R8-octenyl-alanine olefin amino acids connected by an all-carbon i to i+7 crosslinker comprising one double bond. "Ahx" represents an aminocyclohexyl linker. The crosslinkers are linear all-carbon crosslinker comprising eight or eleven carbon atoms between the alpha carbons of each amino acid. Amino acids represented as "$/" are alpha-Me S5-pentenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as "$/r5" are alpha-Me R5-pentenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as "$/s8" are alpha-Me S8-octenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as "$/r8" are alpha-Me R8-octenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as "Amw" are alpha-Me tryptophan amino acids. Amino acids represented as "Ami" are alpha-Me leucine amino acids. Amino acids represented as "2ff" are 2-fluoro-phenylalanine amino acids. Amino acids represented as "3ff" are 3-fluoro-phenylalanine amino acids. Amino acids represented as "St" are amino acids comprising two pentenyl-alanine olefin side chains, each of which is crosslinked to another amino acid as indicated. Amino acids represented as "St//" are amino acids comprising two pentenyl-alanine olefin side chains that are not crosslinked. Amino acids represented as "%St" are amino acids comprising two pentenyl-alanine olefin side chains, each of which is crosslinked to another amino acid as indicated via fully saturated hydrocarbon crosslinks.

[0090] For example, the compounds represented as SP-72, SP-56 and SP-138 have the following structures: 2 SP72

X-Gln-Se\^ ^f^Gln-Thr+Phés. .>C-.Asn-Leu-Trp-Arg-Leu-Leu ^f^GIn-Asn-NHS Ο Η I H J Η T SP-56:

Example 3: Competition Binding ELISA (HDM2 &amp; HDMX) [0092] p53-His6 protein (30 nM/well) is coated overnight at room temperature in the wells of a 96-well Immulon plates. On the day of the experiment, plates are washed with IX PBS-Tween 20 (0.05%) using an automated ELISA plate washer, blocked with ELISA Micro well Blocking for 30 minutes at room temperature; excess blocking agent is washed off by washing plates with IX PBS-Tween 20 (0.05%). Peptides are diluted from 10 mM DMSO stocks to 500 μΜ working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. The peptides are added to wells at 2X desired concentrations in 50 μΙ volumes, followed by addition of diluted GST-HDM2 or GST-HMDX protein (final

concentration: 10nM). Samples are incubated at room temperature for 2h, plates are washed with PBS-Tween 20 (0.05%) prior to adding 100 μΙ of HRP-conjugated anti-GST antibody [Hypromatrix, INC] diluted to 0.5 pg/ml in HRP-stabilizing buffer. Post 30 min incubation with detection antibody, plates are washed and incubated with 100 μΙ per well of TMB-E Substrate solution up to 30 minutes; reactions are stopped using 1M HCL and absorbance measured at 450 nm on micro plate reader. Data is analyzed using Graph Pad PRISM software.

Example 4: SJSA-1 Cell Viability assay [0093] SJSA1 cells are seeded at the density of 5000 cells/ 100 μΙ/well in 96-well plates a day prior to assay. On the day of study cells are washed once with Opti-MEM Media and 90μΙ_ of the Opti-MEM Media is added to cells. Peptides are diluted from 10 mM DMSO stocks to 500 μΜ working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. The final concentration range μΜ will be 50, 25, 12.5, 6.25, 3.1, 1.56, 0.8 and 0 μΜ in 100 pL final volume per well for peptides. Final highest DMSO concentration is 0.5% and will be used as the negative control. Cayman Chemicals Cell-Based Assay (-)-Nutlin-3 (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides 10 pi of 10X desired concentrations is added to the appropriate well to achieve the final desired concentrations. Cells are then incubated with peptides for 20-24h at 37°C in humidified 5% CO2 atmosphere. Post-incubation period, cell viability is measured using Promega Cell Titer-Gio reagents according to manufacturer' instructions.

Example 5: SJSA-1 p21 up-regulation assay [0094] SJSA1 cells are seeded at the density of 0.8 million cells/ 2 ml/well in 6-well plates a day prior to assay. On the day of study cells are washed once with Opti-MEM Media and 1350pLofthe Opti-MEM Media is added to cells.Peptides are diluted from 10 mM DMSO stocks to 500 pM working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. Final highest DMSO concentration is 0.5% and is used as the negative control. Cayman Chemicals Cell-Based Assay (-)-Nutlin-3 (10 mM) is used as positive control. Nutlin is diluted using the same dilution scheme as peptides 150 pi of 10X desired concentrations is added to the appropriate well to achieve the final desired concentrations. Cells are then incubated with peptides for 18-20 h at 37°C in humidified 5% CO2 atmosphere. Post-incubation period, cells are harvested, washed with IX PBS (without Ca++/Mg++) and lysed in IX Cell lysis buffer (Cell Signaling technologies 10X buffer diluted to IX and supplemented with protease inhibitors and Phosphatase inhibitors) on ice for 30 min.Lysates are centrifuged at 13000 rpm speed in a microfuge at 40C for 8 min; clear supernatants are collected and stored at -80 0C till further use. Total protein content of the lysates is measured using BCA protein detection kit and BSA standards from Thermofisher. 25 pg of the total protein is used for p21 detection ELISA assay. Each condition is set in triplicate for ELISA plate. The ELISA assay protocol is followed as per the manufacturer's instructions. 25 pg total protein used for each well, and each well is set up in triplicate. Data is analyzed using Graph Pad PRISM software.

Example 6: p53 GRIP assay [0095] Thermo Scientific* Biolmage p53-Hdm2 Redistribution Assay monitors the protein interaction with Hdm2 and cellular translocation of GFP-tagged p53 in response to drug compounds or other stimuli. Recombinant CHO-hIR cells stably express human p53(1-312) fused to the C-terminus of enhanced green fluorescent protein (EGFP) and PDE4A4-Hdm2(1-124), a fusion protein between PDE4A4 and Hdm2(1-124). They provide a ready-to-use assay system for measuring the effects of experimental conditions on the interaction of p53 and Hdm2. Imaging and analysis is performed with a HCS platform.

[0096] CHO-hIR cells are regularly maintained in Ham's F12 media supplemented with 1% Penicillin-Streptomycin, 0.5 mg/ml Geneticin, 1 mg/ml Zeocin and 10% FBS. Cells seeded into 96-well plates at the density of 7000 cells/100 μΙ per well 18-24 hours prior to running the assay using culture media. The next day, media is refreshed and PD177 is added to cells to the final concentration of 3μΜ to activate foci formation. Control wells are kept without PD-177 solution. 24h post stimulation with PD177, cells are washed once with Opti-MEM Media and 50 μΙ_ of the Opti-MEM Media supplemented with PD-177(6 μΜ) is added to cells. Peptides are diluted from 10 mM DMSO stocks to 500 μΜ working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. Final highest DMSO concentration is 0.5% and is used as the negative control. Cayman Chemicals Cell-Based Assay (-)-Nutlin-3 (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides.50 μΙ of 2X desired concentrations is added to the appropriate well to achieve the final desired concentrations. Cells are then incubated with peptides for 6 h at 37°C in humidified 5% CO2 atmosphere. Post-incubation period, cells are fixed by gently aspirating out the media and adding 150 μΙ of fixing solution per well for 20 minutes at room temperature. Fixed cells are washed 4 times with 200 μΙ PBS per well each time. At the end of last wash, 100 μΙ of 1 μΜ Hoechst staining solution is added. Sealed plates incubated for at least 30 min in dark, washed with PBS to remove excess stain and PBS is added to each well. Plates can be stored at 4°C in dark up to 3 days. The translocation of p53/HDM2 is imaged using Molecular translocation module on Cellomics Arrayscan instrument using 10x objective, XF-100 filter sets for Hoechst and GFP. The output parameters was Mean-CircRINGAvelntenRatio (the ratio of average fluorescence intensities of nucleus and cytoplasm, (we 11 average)). The minimally acceptable number of cells per well used for image analysis was set to 500 cells.

Example 7: Direct binding assay hDM2 with Fluorescence polarization (FP) [0097] The assay was performed according to the following general protocol: 1.1. Dilute hDM2 (In-house, 41 kD) into FP buffer (High salt buffer-200mM Nacl,5mM CHAPS, pH 7.5) to make 10μΜ working stock solution. 2.2. Add 30μΙ of 10μΜ of protein stock solution into A1 and B1 well of 96-well black HE microplate (Molecular Devices). 3. 3. Fill in 30μΙ of FP buffer into column A2 to A12, B2 to B12, C1 to C12, and D1 to D12. 4. 4. 2 or 3 fold series dilution of protein stock from A1, B1 into A2, B2; A2, B2 to A3, B3; ... to reach the single digit nM concentration at the last dilution point. 5. 5. Dilute 1mM (in 100% DMSO) of FAM labeled linear peptide with DMSO to 100μΜ (dilution 1: 10). Then, dilutefrom 100μΜ to 10μΜ with water (dilution 1:10) and then dilute with FP buffer from 10μΜ to 40nM (dilution 1:250). This is the working solution which will be a 10nM concentration in well (dilution 1:4). Keep the diluted FAM labeled peptide in the dark until use. 6. 6. Add 10μΙ of 10nM of FAM labeled peptide into each well and incubate, and read at different time points. Kd with 5-FAM-BaLTFEHYWAQLTS-NH2 is -13.38 nM.

Example 8: Competitive Fluorescence polarization assay forhDM2 [0098] The assay was performed according to the following general protocol: 1.1. Dilute hDM2 (In-house, 41 kD) into FP buffer (High salt buffer-200mM Nacl,5mM CHAPS, pH 7.5) to make 84nM (2X) working stock solution. 2. 2. Add 20μΙ of 84nM (2X) of protein stock solution into each well of 96-well black HE microplate (Molecular Devices) 3. 3. Dilute 1mM (in 100% DMSO) of FAM labeled linear peptide with DMSO to 100μΜ (dilution 1: 10). Then, dilute from 100μΜ to 10μΜ with water (dilution 1:10) and then dilute with FP buffer from 10μΜ to 40nM (dilution 1:250). This is the working solution which will be a 10nM concentration in well (dilution 1:4). Keep the diluted FAM labeled peptide in the dark until use. 4. 4. Make unlabeled peptide dose plate with FP buffer starting with 1μΜ (final) of peptide and making 5 fold serial dilutions for 6 points using following dilution scheme.

Dilute 10mM (in 100% DMSO) with DMSO to 5mM (dilution 1: 2). Then, dilute from 5mM to 500μΜ with H2O (dilution 1:10) and then dilute with FP buffer from 500μΜ to 20μΜ (dilution 1:25). Making 5 fold serial dilutions from 4μΜ (4X) for 6 points. 5. 5. Transfer 10μΙ of serial diluted unlabeled peptides to each well which is filled with 20μΙ of 84nM of protein. 6. 6. Add 10μΙ of 10nM (4X) of FAM labeled peptide into each well and incubate for 3hr to read.

[0099] Results of Examples 7 and 8 are provided in HDM2 data in Figures 7A-D.

Example 9: Direct binding assay hDMXwith Fluorescence polarization (FP) [0100] The assay was performed according to the following general protocol: 1.1. Dilute hDMX (In-house, 40kD) into FP buffer (High salt buffer-200mM Nacl,5mM CHAPS, pH 7.5) to make 10μΜ working stock solution. 2.2. Add 30μΙ of 10μΜ of protein stock solution into A1 and B1 well of 96-well black HE microplate (Molecular Devices). 3. 3. Fill in 30μΙ of FP buffer into column A2 to A12, B2 to B12, C1 to C12, and D1 to D12. 4. 4. 2 or 3 fold series dilution of protein stock from A1, B1 into A2, B2; A2, B2 to A3, B3; ... to reach the single digit nM concentration at the last dilution point. 5. 5. Dilute 1mM (in 100% DMSO) of FAM labeled linear peptide with DMSO to 100μΜ (dilution 1: 10). Then, dilute from 100μΜ to 10μΜ with water (dilution 1:10) and then dilute with FP buffer from 10μΜ to 40nM (dilution 1:250). This is the working solution which will be a 10nM concentration in well (dilution 1:4). Keep the diluted FAM labeled peptide in the dark until use. 6. 6. Add 10μΙ of 10nM of FAM labeled peptide into each well and incubate, and read at different time points.

Kd with 5-FAM-BaLTFEHYWAQLTS-NH2 is ~51 nM.

Example 10: Competitive Fluorescence polarization assay for hDMX

[0101] The assay was performed according to the following general protocol: 1.1. Dilute hDMX (In-house, 40kD) into FP buffer (High salt buffer-200mM Nacl,5mM CHAPS, pH 7.5.) to make 300nM (2X) working stock solution. 2. 2. Add 20μΙ of 300nM (2X) of protein stock solution into each well of 96-well black HE microplate (Molecular Devices) 3. 3. Dilute 1mM (in 100% DMSO) of FAM labeled linear peptide with DMSO to 100μΜ (dilution 1: 10). Then, dilute from 100μΜ to 10μΜ with water (dilution 1:10) and then dilute with FP buffer from 10μΜ to 40nM (dilution 1:250). This is the working solution which will be a 10nM concentration in well (dilution 1:4). Keep the diluted FAM labeled peptide in the dark until use. 4. 4. Make unlabeled peptide dose plate with FP buffer starting with 5μΜ (final) of peptide and making 5 fold serial dilutions for 6 points using following dilution scheme. 5. 5. Dilute 10mM (in 100% DMSO) with DMSO to 5mM (dilution 1: 2). Then, dilute from 5mM to 500μΜ with H2O (dilution 1:10) and then dilute with FP buffer from 500μΜ to 20μΜ (dilution 1:25). Making 5 fold serial dilutions from 20μΜ (4X) for 6 points. 6. 6. Transfer 10μΙ of serial diluted unlabeled peptides to each well which is filled with 20μΙ of 300nM of protein. 7. 7. Add 10μΙ of 10nM (4X) of FAM labeled peptide into each well and incubate for 3hr to read.

[0102] Results of Examples 9 and 10 are provided in HDMX data in Figures 7A-D.

Example 11: Cell Viability assay [0103] The assay was performed according to the following general protocol:

Cell Plating: Trypsinize, count and seed cells at the pre-determined densities in 96-well plates a day prior to assay. Following cell densities are used for each cell line in use: • SJSA-1: 7500 cells/well • RKO: 5000 cells/well • RKO-E6: 5000 cells/well . HCT-116: 5000 cells/well . SW-480: 2000 cells/well • MCF-7: 5000 cells/well [0104] On the day of study, replace media with fresh media with 11% FBS (assay media) at room temperature. Add 180pL of the assay media per well. Control wells with no cells, receive 200 μΙ media.

[0105] Peptide dilution: all dilutions are made at room temperature and added to cells at room temperature. • Prepare 10 mM stocks of the peptides in DMSO. Serially dilute the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01mM solutions using DMSO as diluents. Dilute the serially DMSO-diluted peptides 33.3 times using sterile water. This gives range of 10X working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for control wells. • Thus the working stocks concentration range μΜ will be 300, 100, 30, 10, 3, 1, 0.3 and 0 μΜ. Mix well at each dilution step using multichannel. • Row H has controls. H1 - H3 will receive 20 ul of assay media. H4-H9 will receive 20 ul of 3% DMSO-water vehicle. H10-H12 will have media alone control with no cells. • Positive control: HDM2 small molecule inhibitor, Nutlin-3a (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides.

[0106] Addition of working stocks to cells: • Add 20 μΙ of 10X desired concentration to appropriate well to achieve the final concentrations in total 200 μΙ volume in well. (20 μΙ of 300 μΜ peptide + 180 μΙ of cells in media = 30 μΜ final concentration in 200 μΙ volume in wells). Mix gently a few times using pipette. Thus final concentration range used will be 30, 10, 3, 1,0.3, 0.1,0.03 &amp; 0 μΜ (for potent peptides further dilutions are included). • Controls include wells that get no peptides but contain the same concentration of DMSO as the wells containing the peptides, and wells containing NO CELLS. • Incubate for 72 hours at 37°C in humidified 5% CO2 atmosphere. • The viability of cells is determined using MTT reagent from Promega. Viability of SJSA-1, RKO, RKO-E6, HCT-116 cells is determined on day 3, MCF-7 cells on day 5 and SW-480 cells on day 6. At the end of designated incubation time, allow the plates to come to room temperature. Remove 80 μΙ of assay media from each well. Add 15 μΙ of thawed MTT reagent to each well. • Allow plate to incubate for 2h at 37°C in humidified 5% CO2 atmosphere and add 100 μΙ solubilization reagent as per manufacturer's protocol. Incubate with agitation for 1h at room temperature and read on Synergy Biotek multiplate reader for absorbance at 570nM. • Analyze the cell viability against the DMSO controls using GraphPad PRISM analysis tools.

[0107] Reagents: • Invitrogen cell culture Media i.Falcon 96-well clear cell culture treated plates (Nunc 353072) • DMSO (Sigma D 2650) • RPMI 1640 (Invitrogen 72400) • MTT (Promega G4000)

Instruments: Multiplate Reader for Absorbance readout (Synergy 2) [0108] Results of Example 11 are provided in SJSA-1 EC50 data in Figures 7A-D.

Example 12. P21 ELISA assay [0109] The assay was performed according to the following general protocol:

Cell Platimg: [0110] • Trypsinize, count and seed SJSA1 cells at the density of 7500 cells/ 100 μΙ/well in 96-well plates a day prior to assay. • On the day of study, replace media with fresh RPMI-11% FBS (assay media). Add 90pL of the assay media per well. Control wells with no cells, receive 100 μΙ media.

Peptide dilution: [0111] • Prepare 10 mM stocks of the peptides in DMSO. Serially dilute the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01mM solutions using DMSO as diluents. Dilute the serially DMSO- diluted peptides 33.3 times using sterile water This gives range of 10X working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for control wells. • Thus the working stocks concentration range μΜ will be 300, 100, 30, 10, 3, 1, 0.3 and 0 μΜ. Mix well at each dilution step using multichannel. • Row H has controls. H1 - H3 will receive 10 ul of assay media. H4-H9 will receive 10 ul of 3% DMSO-water vehicle. H10-H12 will have media alone control with no cells. • Positive control: HDM2 small molecule inhibitor,Nutlin-3a (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides.

Addition of working stocks to cells: [0112] • Add 10 μΙ of 10X desired concentration to appropriate well to achieve the final concentrations in total 100 μΙ volume in well. (10 μΙ of 300 μΜ peptide + 90 μΙ of cells in media = 30 μΜ final concentration in 100 μΙ volume in wells). Thus final concentration range used will be 30, 10, 3, 1,0.3&amp; 0 μΜ. • Controls will include wells that get no peptides but contain the same concentration of DMSO as the wells containing the peptides, and wells containing NO CELLS. • 20h-post incubation, aspirate the media; wash cells with 1X PBS (without Ca++/Mg++) and lyse in 60 μΙ of IXCell lysis buffer (Cell Signaling technologies 10X buffer diluted to 1Xand supplemented with protease inhibitors and Phosphatase inhibitors) on ice for 30 min. • Centrifuge plates in at 5000 rpm speed in at 4°C for 8 min; collect clear supernatants and freeze at -80 °C till further use.

Protein Estimation: [0113] • Total protein content of the lysates is measured using BCA protein detection kit and BSA standards from Thermofisher. Typically about 6-7 pg protein is expected per well. • Use 50 μΙ of the lysate per well to set up p21 ELISA.

[0114] Human Total p21 ELISA: The ELISA assay protocol is followed as per the manufacturer's instructions. 50 μΙ lysate is used for each well, and each well is set up in triplicate.

Reagents: [0115] • -Cell-Based Assay (-)-Nutlin-3 (10 mM): Cayman Chemicals, catalog # 600034 • - OptiMEM, Invitrogen catalog # 51985 • -Cell Signaling Lysis Buffer (10X), Cell signaling technology, Catalog # 9803 • -Protease inhibitor Cocktail tablets(mini), Roche Chemicals, catalog # 04693124001 • -Phosphatase inhibitor Cocktail tablet, Roche Chemicals, catalog # 04906837001 • -Human total p21 ELISA kit, R&amp;D Systems, DYC1047-5 • -STOP Solution (1M HCL), Cell Signaling Technologies, Catalog # 7002

Instruments: Micro centrifuge- Eppendorf 5415D and Multiplate Reader for Absorbance readout (Synergy 2) [0116] Results of Example 12 are provided in p21 data in Figures 7A-D.

Example 13: Caspase 3 Detection assay: [0117] The assay was performed according to the following general protocol:

Cell Platimg: Trypsinize, count and seed SJSA1 cells at the density of 7500 cells/ 100 μΙ/well in 96-well plates a day prior to assay. On the day of study, replace media with fresh RPMI-11% FBS (assay media). Add 180pL of the assay media per well. Control wells with no cells, receive 200 μΙ media.

Peptide dilution: [0118] • Prepare 10 mM stocks of the peptides in DMSO. Serially dilute the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33, 0.11, 0.03, 0.01mM solutions using DMSO as diluents. Dilute the serially DMSO-diluted peptides 33.3 times using sterile water This gives range of 10X working stocks. Also prepare DMSO/sterile water (3% DMSO) mix for control wells. • Thus the working stocks concentration range μΜ will be 300, 100, 30, 10, 3, 1, 0.3 and 0 μΜ. Mix well at each dilution step using multichannel. Add 20 ul of 10X working stocks to appropriate wells. • Row H has controls. H1 - H3 will receive 20 ul of assay media. H4-H9 will receive 20 ul of 3% DMSO-water vehicle. H10-H12 will have media alone control with no cells. • Positive control: HDM2 small molecule inhibitor,Nutlin-3a (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides.

Addition of working stocks to cells: [0119] • Add 10 μΙ of 10X desired concentration to appropriate well to achieve the final concentrations in total 100 μΙ volume in well. (10 μΙ of 300 μΜ peptide + 90 μΙ of cells in media = 30 μΜ final concentration in 100 μΙ volume in wells). Thus final concentration range used will be 30, 10, 3, 1, 0.3&amp; 0 μΜ. • Controls will include wells that get no peptides but contain the same concentration of DMSO as the wells containing the peptides, and wells containing NO CELLS. • 48 h-post incubation, aspirate 80 μΙ media from each well; add 100 μΙ Caspase3/7Glo assay reagent (Promega Caspase 3/7 gio assay system, G8092)per well, incubate with gentle shaking for 1h at room temperature. • read on Synergy Biotek multiplate reader for luminescence. • Data is analyzed as Caspase 3 activation over DMSO-treated cells.

[0120] Results of Example 13 are provided in p21 data in Figures 7A-D.

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

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Claims (4)

1. Peptidomimetisk makrocyklus, der interfererer med binding mellem p53 og HDM2 eller HDMX, og som har en aminosyresekvens, der er mindst 60 % identisk med: Leu Thr Phe $1 Glu Tyr Trp Ala Gin Leu $2 Ala; hvor $1 er en a, a-disubstitueret aminosyre i R-konfigurationen, hvor én substituent er en methylgruppe og den anden substituent er en linker-gruppe til $2; og hvor $2 er en a, a-disubstitueret aminosyre i S-konfigurationen, hvor én substituent er en methylgruppe og den anden substituent er linker-gruppen til $1; og hvor linker-gruppen, der starter fra $1, er -(CH2)6-CH=CH-(CH2)3-.

2. Farmaceutisk sammensætning, der omfatter the peptidomimetisk makrocyklus ifølge krav 1.

3. Peptidomimetisk makrocyklus ifølge krav 1, til anvendelse i behandling af cancer hos et individ.

4. Farmaceutisk sammensætning ifølge krav 2, til anvendelse i behandling af cancer hos et individ.