P53 peptidomimetic macrocycles

Abstract

Disclosed are p53 peptidomimetic macrocycles, wherein each p53 peptidomimetic macrocycle comprises an i,i+4 olefin staple and a polypeptide tail covalently attached to the p53 peptidomimetic macrocycle; an i,i+7 olefin staple and a polypeptide tail covalently attached to the p53 peptidomimetic macrocycle; or an i,i+7 dialkyne staple and an optional polypeptide tail covalently attached to the p53 peptidomimetic macrocycle, wherein the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, the polypeptide tail comprises 3-9 amino acids, and each amino acid in the polypeptide tail independently has the D- or L-configuration. The p53 peptidomimetic macrocycles are protease resistant and cell permeable without inducing membrane disruption, and activate p53 in cells by binding to MDM2 and MDMX, thereby antagonizing the binding of MDM2 and MDMX to p53. These p53 peptidomimetic macrocycles may be useful in anti-cancer therapy, especially in combination with chemotherapy or radiation therapy.

Description

[Technical field]

[0001] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated by reference in its entirety. The XML file created on October 3, 2022 is named 25346WOPCT_SL.XML and has a size of 104 bytes.

[0002] (1) Field of the invention The present invention provides p53 peptidomimetic macrocycles, wherein each p53 peptidomimetic macrocycle comprises an i,i+4 olefin staple and a polypeptide tail covalently attached to the p53 peptidomimetic macrocycle; an i,i+7 olefin staple and a polypeptide tail covalently attached to the p53 peptidomimetic macrocycle; or an i,i+7 dialkyne staple and an optional polypeptide tail covalently attached to the p53 peptidomimetic macrocycle, wherein the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, the polypeptide tail comprises 3-9 amino acids, and each amino acid in the polypeptide tail independently has the D- or L-configuration. The p53 peptidomimetic macrocycles are protease resistant and cell permeable without inducing membrane disruption, and activate p53 in cells by binding to MDM2 and MDMX, thereby antagonizing the binding of MDM2 and MDMX to p53. [Background technology]

[0003] (2) Description of related technology p53 is an important tumor suppressor protein that primarily functions as a DNA transcription factor. It is commonly repressed in cancer (hereafter referred to as cancer) and plays a crucial role in protecting cells in response to various stress signals through induction of cell cycle arrest, apoptosis or senescence

[46] . The mechanism that frequently leads to p53 inactivation and tumorigenesis involves enhanced expression of MDM2 and MDMX (also known as MDM4), which are negative regulators of p53. Both MDM2 and MDMX are known to directly interact with p53 and to mediate the expression of related activators (e.g., dTAFs) that are required for transcription. II , hTAF II ) to reduce p53 function. Also, they are both E3 ligase components and target p53 for proteasome-mediated degradation. Unlike MDM2, MDMX does not have intrinsic E3 ubiquitin ligase activity. Instead, MDMX forms a heterodimeric complex with MDM2, thereby stimulating the ubiquitin activity of MDM2. As a result, p53 activity and protein levels are acutely suppressed by overexpression of MDM2 and MDMX. Therefore, the development of inhibitors that block p53 interaction with either or both MDM2 or MDMX is highly desirable, because it would prevent p53 degradation and restore p53-dependent transcriptional antitumor responses [47,48].

[0004] The structural boundary of the p53 MDM2 / MDMX complex is characterized by an α-helix from the N-terminal transactivation domain of p53 that binds within a hydrophobic groove on the surface of the N-terminal domain of both MDM2 and MDMX. Three hydrophobic residues in p53, namely Phe 19 , Trp 23 and Leu 26The p53 peptide is a key determinant of this interaction and protrudes deeply into the MDM2 / MDMX interaction groove [see Figure 1A]. Isolated p53 peptides are highly disordered and distort into an α-helical conformation upon binding. There are several examples of small molecules, peptides, and biologics that mimic these interactions and compete for MDM2 / MDMX binding to liberate p53

[49] . However, the majority of small molecules developed show little affinity and activity for MDMX. MDMX has several distinct structural differences in the p53 peptide binding groove compared to MDM2. Although several MDM2-specific molecules have entered early clinical trials, most of them have shown dose-limiting toxicity in patients

[49] . It has been demonstrated that overexpression of MDMX in tumors reduces the efficacy of MDM2-specific compounds. This is likely due to the maintenance of a heterodimeric complex of MDM2 and MDMX that inhibits and targets p53 for proteasomal degradation. MDM2-selective inhibitors can also induce higher levels of MDMX, highlighting the importance of simultaneously targeting both proteins to achieve efficient activation of p53 for optimal therapeutic responses.

[0005] Protein-protein interactions (PPIs) are central to most biological processes and are frequently dysregulated in disease [1,2]. Thus, PPIs are attractive therapeutic targets for drug discovery. However, in contrast to the deep protein cavities that typically accommodate small molecules, PPI surfaces are generally large and flat, which contributes to the limited success of developing small molecule inhibitors against PPI targets [3]. The realization that 40% of all PPIs are caused by relatively short peptide motifs has raised the possibility of developing peptide-based inhibitors that orthosterically compete for the interface between the ligand-target cognate partners [4]. Such peptides, when synthesized removed from the context of a protein ligand, are often unstructured and intrinsically disordered, but can achieve their biologically relevant conformation upon binding to a protein target [4]. However, in the case of intracellular targets, peptide modalities can be challenging due to proteolytic susceptibility, low conformational stability (resulting in weak affinity and off-target effects) and low cell permeability (further limiting function to intracellular targets and oral bioavailability) [5-11]. To address these issues, several strategies have been pursued, including macrocyclization and modification of the peptide backbone to obtain molecules with improved activity and pharmacokinetic properties, as well as constraining peptides to biologically relevant conformations so that they bind to their targets [5-13]. First, by biasing the peptide towards its binding conformation, the entropic penalty upon binding is reduced, improving the binding constant and possibly reducing the possibility of undesired off-target effects. Second, macrocyclization can provide varying degrees of proteolytic resistance by modifying the central backbone and / or side chain structural moieties within the peptide. Third, macrocyclization can enhance cell permeability. This is because, for example, the enhanced stability of intramolecular hydrogen bonds reduces the desolvation penalty that would otherwise occur in the transport of peptides across non-polar cell membranes.Among the several cyclization techniques described, stapling by metathesis using non-proteinogenic amino acids, e.g., alpha-methylalkenyl side chains, has proven to be very effective, especially when the desired secondary structure of the peptide macrocycle is helical [13-18]. Stapling requires the incorporation of suitable non-natural amino acid precursors, positioned at appropriate positions along the peptide sequence so that they do not interfere with the binding face of the helix. Although they have been primarily used to stabilize helical conformations, recent studies have also applied ring-closing metathesis (RCM) methods to non-helical peptides [19,20].

[0006] The stapled peptide approach has been successfully applied to inhibit several PPIs with therapeutic potential, including: BCL-2 family-BH3 domain [21-24], β-catenin-TCF

[25] , Rab-GTPase-effector

[26] , ERα-coactivator protein

[27] , Cullin3-BTB

[28] , VDR-coactivator protein

[29] , eIf4E

[30] , ATSP-7041 [see WO2013123266], SAH-p53-8 [Bernal et al., Cancer Cell 18:411-422 (2010)] and p53-MDM2 / MDMX [31-34]. Of note, in the case of p53-MDM2 / MDMX, a dual-selective stapled peptide (ALRN-6924; Aileron Therapeutics, Inc.) has been successfully advanced into phase II clinical trials [35-37]. Although this example is certainly encouraging for stapled peptides to be advanced into the clinic, challenges still remain. Among these, it is crucial to design molecules with sufficient proteolytic stability for sustained target binding and cellular activity. Indeed, although stapling of L-amino acid peptides can provide resistance to protease-mediated degradation, the effect is often not complete and can affect residues located outside the macrocyclic ring [38-40].

[0007] On the other hand, all-D α-amino acid peptides are ultrastable to proteolysis because most proteases are chiral and can discriminate between L- and D-enantiomers of substrates, and as a result, all-D α-amino acid peptides can resist protease activity. All-D α-amino acid peptides have been designed to have strong binding affinity to various targets, including p53-MDM2 [41-42], VEGF-VEGF receptor

[43] , PD-1 - PD-L1

[44] , and human immunodeficiency virus type 1 (HIV-1) entry

[45] . Unfortunately, although all-D α-amino acid peptides are inherently ultrastable to proteolysis, they generally lack membrane permeability and cellular activity.

[0008] for example, D PMI-δ is an all-D configuration α-amino acid linear peptide (PMI stands for p53-MDM2 / MDMX inhibitor) derived from a mirror-image phage display screen reported by Liu et al.

[41] and in US Patent Publication No. 20120328692. However, this peptide lacked cell permeability, but activated p53 in cells when delivered using nanocarriers

[42] .

[0009] Peptide-based inhibitors hold great promise for targeted modulation of intracellular protein-protein interactions (PPIs) because they have access to a broad range of chemical space, including a multitude of different secondary and tertiary structures and sequence diversity. However, drawbacks including low conformational stability, proteolytic susceptibility, and cell permeability currently impede the development of peptide therapeutics. Also, it is difficult to convert these in vitro binders into intracellularly and in vivo active compounds with on-target selectivity and specificity. Several modern peptide design strategies address these issues from different angles. Strategic macrocyclization with optimally placed chemically reinforcing structures such as olefinic hydrocarbon bridges, commonly referred to as staples, may address these issues by i) restricting conformational freedom to improve target affinity, ii) improving proteolytic resistance, and iii) enhancing cell permeability. Conversely, molecules constructed entirely from D-amino acids are ultra-resistant to proteolytic cleavage, but generally lack conformational stability and membrane permeability. Summary of the Invention

[0010] Overview of the Invention The inventors have herein developed p53 peptidomimetic macrocycles that solve many of the problems associated with peptide inhibitors identified in the art. The present invention provides p53 peptidomimetic macrocycles, wherein each p53 peptidomimetic macrocycle comprises an i,i+4 olefin staple and a polypeptide tail covalently attached to the p53 peptidomimetic macrocycle; an i,i+7 olefin staple and a polypeptide tail covalently attached to the p53 peptidomimetic macrocycle; or an i,i+7 dialkyne staple and an optional polypeptide tail covalently attached to the p53 peptidomimetic macrocycle, wherein the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration (D-amino acids), the polypeptide tail comprises 3 to 9 amino acids, and each amino acid in the polypeptide tail independently has the D-configuration or the L-configuration, or in certain embodiments, each amino acid in the polypeptide tail has the D-configuration. These p53 peptidomimetic macrocycles are (i) protease resistant, (ii) conformationally stable, (iii) cell permeable without inducing membrane disruption, and (iv) have low or no cytotoxicity (the disclosed p53 peptidomimetic macrocycles have reduced cellular activity in counter screen activity and lactate dehydrogenase (LDH) release.) Additionally, the p53 peptidomimetic macrocycles of the invention bind to mouse double minute 2 (MDM2; also known as E3 ubiquitin-protein ligase) and MDMX (also known as MDM4), and activate p53 in cells by binding to MDM2 and MDMX and thereby antagonizing the binding of MDM2 and MDMX to p53.

[0011] In certain embodiments, the invention provides a p53 peptidomimetic macrocycle comprising an i,i+4 olefin staple and a polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle; an i,i+7 olefin staple and a polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle; or an i,i+7 dialkyne staple and an optional polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle, wherein the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, and the polypeptide tail comprises from 3 to 9 amino acids, and each amino acid in the polypeptide tail independently has the D-configuration or the L-configuration, or in certain embodiments, each amino acid in the polypeptide tail has the D-configuration.

[0012] In certain embodiments, the p53 peptidomimetic macrocycle comprises an i,i+4 olefin staple and a polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle, where the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, and where the polypeptide tail comprises 3 to 9 amino acids, and where each amino acid in the polypeptide tail independently has the D-configuration or the L-configuration, or in certain embodiments, each amino acid in the polypeptide tail has the D-configuration.

[0013] In certain embodiments, the p53 peptidomimetic macrocycle comprises an i,i+7 olefin staple and a polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle, where the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, and the polypeptide tail comprises 3 to 9 amino acids, and each amino acid in the polypeptide tail independently has the D-configuration or the L-configuration, or in certain embodiments, each amino acid in the polypeptide tail has the D-configuration. In certain embodiments, the olefin staple is an alkyne staple.

[0014] In certain embodiments, the p53 peptidomimetic macrocycle comprises an i,i+7 dialkyne staple and a polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle, where the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, and where the polypeptide tail comprises 3 to 9 amino acids, and where each amino acid in the polypeptide tail independently has the D-configuration or the L-configuration, or in certain embodiments, each amino acid in the polypeptide tail has the D-configuration.

[0015] In a further embodiment, the invention provides a p53 peptidomimetic macrocycle comprising an i,i+4 olefin staple formed between the α-carbons of two α,α-disubstituted amino acids located at amino acid positions 6 and 10 of the p53 peptidomimetic macrocycle (such a staple is referred to as a "6-10 olefin staple") and a polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle, wherein the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, the polypeptide tail comprises 3 to 9 amino acids, and each amino acid of the polypeptide tail independently has the D-configuration or the L-configuration, or in certain embodiments, each amino acid in the polypeptide tail has the D-configuration. In certain embodiments, the α,α-disubstituted amino acids at positions 6 and 10 comprise (R)-2-amino-2-methylhept-6-enoic acid.

[0016] In a further embodiment, the invention provides a p53 peptidomimetic macrocycle comprising 12 amino acids, an i,i+4 olefin staple formed between the α-carbons of two α,α-disubstituted amino acids located at amino acid positions 6 and 10 of the p53 peptidomimetic macrocycle, and a polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle, wherein the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, the polypeptide tail comprises 3 to 9 amino acids, and each amino acid of the polypeptide tail independently has the D-configuration or the L-configuration, or in certain embodiments, each amino acid of the polypeptide tail has the D-configuration. In certain embodiments, the α,α-disubstituted amino acids at amino acid positions 6 and 10 comprise (R)-2-amino-2-methylhept-6-enoic acid.

[0017] In certain embodiments of the p53 peptidomimetic macrocycle comprising a 6-10 olefin staple, the p53 peptidomimetic macrocycle further comprises D-6-fluoro-tryptophan at position 3 and Dp-CF3-phenylalanine at position 7.

[0018] In yet another embodiment of the p53 peptidomimetic macrocycle comprising a 6-10 olefin staple, the amino acid at position 1 comprises a threonine, the amino acid at position 2 comprises an alanine, the amino acid at position 4 comprises a tyrosine, the amino acid at position 5 comprises an alanine, the amino acid at position 8 comprises a glutamic acid, the amino acid at position 9 comprises a lysine or glutamine, the amino acid at position 11 comprises a leucine, and the amino acid at position 12 comprises an arginine or serine.

[0019] In yet another embodiment of the p53 peptidomimetic macrocycle containing a 6-10 olefin staple, the amino acid at position 9 comprises a glutamine and the amino acid at position 12 comprises a serine.

[0020] In a further embodiment, the invention provides a p53 peptidomimetic macrocycle comprising a 5-12 dialkyne staple formed between the α-carbons of two α,α-disubstituted amino acids located at amino acid positions 5 and 12 of the p53 peptidomimetic macrocycle, and an optional polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle, wherein the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, the polypeptide tail comprises 3 to 9 amino acids, and each amino acid of the polypeptide tail independently has the D-configuration or the L-configuration, or in certain embodiments, each amino acid of the polypeptide tail has the D-configuration.

[0021] In certain embodiments of p53 peptidomimetic macrocycles comprising a 5-12 dialkyne staple, the α,α-disubstituted amino acid at amino acid position 5 comprises (S)-2-amino-2-methylhept-6-ynoic acid and the α,α-disubstituted amino acid at amino acid position 12 comprises (R)-2-amino-2-methyloct-7-ynoic acid.

[0022] In certain embodiments of a p53 peptidomimetic macrocycle comprising a 5-12 dialkyne staple, the p53 peptidomimetic macrocycle is covalently attached at its C-terminal amino acid to the N-terminus of a polypeptide tail, wherein the polypeptide tail comprises 3 to 9 amino acids and each amino acid in the polypeptide tail independently has the D-configuration or the L-configuration, or in certain embodiments, each amino acid in the polypeptide tail has the D-configuration.

[0023] In a further embodiment, the invention provides a p53 peptidomimetic macrocycle comprising 12 amino acids, an i,i+7 dialkyne staple formed between the α-carbons of two α,α-disubstituted amino acids located at amino acid positions 5 and 12 of the p53 peptidomimetic macrocycle (such a staple is referred to as a "5-12 dialkyne staple"), and an optional polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle, wherein the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, and the polypeptide tail comprises from 3 to 9 amino acids, and each amino acid of the polypeptide tail, independently, has the D- or L-configuration.

[0024] In certain embodiments of p53 peptidomimetic macrocycles comprising a 5-12 dialkyne staple, the α,α-disubstituted amino acid at amino acid position 5 comprises (S)-2-amino-2-methylhept-6-ynoic acid and the α,α-disubstituted amino acid at amino acid position 12 comprises (R)-2-amino-2-methyloct-7-ynoic acid.

[0025] In certain embodiments of a p53 peptidomimetic macrocycle comprising a 5-12 dialkyne staple, the p53 peptidomimetic macrocycle is covalently attached at the C-terminal amino acid to the N-terminus of a polypeptide tail, the polypeptide tail comprising from 3 to 9 amino acids, each amino acid of the polypeptide tail independently having the D-configuration or the L-configuration, or in certain embodiments, each amino acid in the polypeptide tail has the D-configuration.

[0026] In certain embodiments of the p53 peptidomimetic macrocycle comprising a 5-12 dialkyne staple, the p53 peptidomimetic macrocycle comprising a 5-12 dialkyne staple further comprises D-6-fluorotryptophan at amino acid position 3 and Dp-CF3-phenylalanine at amino acid position 7.

[0027] In yet another embodiment of the p53 peptidomimetic macrocycle containing a 5-12 dialkyne staple, the amino acid at position 1 comprises a threonine, the amino acid at position 2 comprises an alanine, the amino acid at position 4 comprises a tyrosine, the amino acid at position 6 comprises an asparagine, the amino acid at position 8 comprises a glutamic acid, the amino acid at position 9 comprises a lysine or glutamine, the amino acid at position 10 comprises a leucine, and the amino acid at position 11 comprises a leucine. In another embodiment, the amino acid at position 9 comprises a glutamine.

[0028] In a further embodiment, the invention provides a p53 peptidomimetic macrocycle comprising an i,i+7 olefin staple formed between the α-carbons of two α,α-disubstituted amino acids located at amino acid positions 5 and 12 of the p53 peptidomimetic macrocycle (such a staple is a "5-12 olefin staple") and an optional polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle, wherein the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, the polypeptide tail comprises from 3 to 9 amino acids, each amino acid of the polypeptide tail independently has the D-configuration or the L-configuration, or in a particular embodiment, each amino acid in the polypeptide tail has the D-configuration, and wherein the α,α-disubstituted amino acid at amino position 5 is (S)-2-amino-2-methyldec-9-enoic acid and the α,α-disubstituted amino acid at amino acid position 12 is (R)-2-amino-2-methylhept-6-enoic acid.

[0029] In certain embodiments of a p53 peptidomimetic macrocycle comprising a 5-12 olefinic bond, the p53 peptidomimetic macrocycle is covalently attached at the C-terminal amino acid to the N-terminus of a polypeptide tail, wherein the polypeptide tail comprises 3 to 9 amino acids and each amino acid of the polypeptide tail independently has the D- or L-configuration, or in certain embodiments, each amino acid in the polypeptide tail has the D-configuration.

[0030] In a further embodiment, the invention provides a p53 peptidomimetic macrocycle comprising 12 amino acids, an i,i+7 olefin staple formed between the α-carbons of two α,α-disubstituted amino acids located at amino acid positions 5 and 12 of the p53 peptidomimetic macrocycle, and an optional polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle, wherein the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, the polypeptide tail comprises from 3 to 9 amino acids, each amino acid of the polypeptide tail independently has the D-configuration or the L-configuration, and wherein the α,α-disubstituted amino acid at amino position 5 is 2-amino-2-methyldec-9-enoic acid and the α,α-disubstituted amino acid at amino acid position 12 is (R)-2-amino-2-methylhept-6-enoic acid.

[0031] In certain embodiments of a p53 peptidomimetic macrocycle comprising a 5-12 olefinic bond, the p53 peptidomimetic macrocycle is covalently attached at the C-terminal amino acid to the N-terminus of a polypeptide tail, wherein the polypeptide tail comprises 3 to 9 amino acids and each amino acid of the polypeptide tail independently has the D- or L-configuration, or in certain embodiments, each amino acid in the polypeptide tail has the D-configuration.

[0032] In certain embodiments of a p53 peptidomimetic macrocycle comprising a 5-12 olefinic bond, the p53 peptidomimetic macrocycle further comprises D-6-fluoro-tryptophan at amino acid position 3 and Dp-CF3-phenylalanine at amino acid position 7.

[0033] In yet another embodiment of the p53 peptidomimetic macrocycle containing a 5-12 olefinic bond, the amino acid at position 1 comprises a threonine, the amino acid at position 2 comprises an alanine, the amino acid at position 4 comprises a tyrosine, the amino acid at position 6 comprises an asparagine, the amino acid at position 8 comprises a glutamic acid, the amino acid at position 9 comprises a lysine or glutamine, the amino acid at position 10 comprises a leucine, and the amino acid at position 11 comprises a leucine. In another embodiment, the amino acid at position 9 comprises a glutamine.

[0034] In certain embodiments of the p53 peptidomimetic macrocycles disclosed herein, one substituent of the α,α-disubstituted amino acid is alkenyl and the other substituent of the α,α-disubstituted amino acid is methyl. In certain embodiments, the alkenyl is C 3-10 Alkenyl, C 4-7 Alkenyl or C 5-6 It is alkenyl.

[0035] The invention further provides a method for converting a p53 peptidomimetic macrocycle and i,i+4 olefinic bond into a p53 peptidomimetic macrocycle with improved pharmacological properties of low or undetectable toxicity, conformational stability and membrane permeability, comprising covalently attaching a polypeptide tail comprising 3-9 amino acids to the C-terminal amino acid of the p53 peptidomimetic macrocycle, wherein each amino acid of the polypeptide tail independently has the D- or L-configuration, or in certain embodiments, each amino acid in the polypeptide tail has the D-configuration. In another embodiment, the α-carbons of two α,α-disubstituted amino acids at positions 6 and 10 of the p53 peptidomimetic macrocycle are linked by an i,i+4 olefinic bond. In certain embodiments, the α,α-disubstituted amino acids at amino acid positions 6 and 10 comprise (R)-2-amino-2-methylhept-6-enoic acid.

[0036] The invention further provides a method for converting a p53 peptidomimetic macrocycle and an i,i+7 olefinic or i,i+7 dialkyne bond to a p53 peptidomimetic macrocycle with improved pharmacological properties of low or undetectable toxicity, conformational stability and membrane permeability, comprising attaching a polypeptide tail comprising 3-9 amino acids to the C-terminal amino acid of the p53 peptidomimetic macrocycle, wherein each amino acid of the polypeptide tail independently has the D- or L-configuration, or in certain embodiments, each amino acid in the polypeptide tail has the D-configuration. In another embodiment, the α-carbons of two α,α-disubstituted amino acids at positions 5 and 12 of the p53 peptidomimetic macrocycle are linked by an i,i+7 olefinic bond. In certain embodiments, the α,α-disubstituted amino acid at amino acid position 5 comprises (S)-2-amino-2-methylhept-6-ynoic acid and the α,α-disubstituted amino acid at amino acid position 12 comprises (R)-2-amino-2-methyloct-7-ynoic acid.

[0037] In another embodiment of the method, a p53 peptidomimetic macrocycle is converted to a p53 peptidomimetic macrocycle with improved pharmacological properties of low or undetectable toxicity, conformational stability and membrane permeability by joining the α-carbons of two α,α-disubstituted amino acids located at amino acid positions 5 and 12 of the p53 peptidomimetic macrocycle with an olefinic bond and covalently attaching a polypeptide tail comprising 3-9 amino acids to the C-terminal amino acid of the p53 peptidomimetic macrocycle, wherein each amino acid in the polypeptide tail independently has the D-configuration or the L-configuration, or in a specific embodiment, each amino acid in the polypeptide tail has the D-configuration, wherein the α,α-disubstituted amino acid at amino position 5 is (S)-2-amino-2-methyldec-9-enoic acid and the α,α-disubstituted amino acid at amino acid position 12 is (R)-2-amino-2-methylhept-6-enoic acid.

[0038] In embodiments directed to improving binding of p53 peptidomimetic macrocycles to MDM2 and / or MDMX and improving cellular activity in p53 cellular reporter gene assays, the amino acids at each position of the polypeptide tail comprising 3-9 amino acids are independently selected from natural or unnatural L- or D-amino acids. In certain embodiments of the disclosed p53 peptidomimetic macrocycles, the amino acids at each position of the polypeptide tail comprise alanine. In another embodiment, the amino acids at positions 3 and 6 of the polypeptide tail are each independently selected from alkyl or aromatic amino acids. In certain embodiments of the polypeptide tail, the amino acids at positions 3 and 6 of the polypeptide tail are each independently selected from alkyl or phenylalanine. In certain embodiments of the polypeptide tail, the polypeptide tail comprises or consists of 6 amino acids. In certain embodiments of the polypeptide tail, the alkyl amino acid of the polypeptide tail comprises alanine. In certain embodiments of the polypeptide tail, the polypeptide tail comprises 6 amino acids. In further embodiments of the polypeptide tail, each amino acid of the polypeptide tail is in the D configuration.

[0039] In embodiments aimed at improving the amphipathicity, solubility and / or cellular activity of p53 peptidomimetic macrocycles, the amino acids at each position of a polypeptide tail comprising 3-9 amino acids are independently selected from natural or non-natural negatively charged L- or D-amino acids, so long as at least one amino acid is negatively charged. In another embodiment of a polypeptide tail, the amino acid at position 1 of the polypeptide tail is independently selected from alkyl, glutamic acid or gamma-carboxyl glutamic acid (Gla), the amino acid at position 3 of the polypeptide tail is independently selected from alkyl or glutamic acid, and the amino acid at position 5 of the polypeptide tail is independently selected from alkyl and alpha-methyl glutamic acid, with the proviso that at least one of the amino acids at positions 1, 3, or 5 comprises glutamic acid or alpha-methyl glutamic acid. In certain embodiments of the polypeptide tail, the polypeptide tail comprises or consists of 6 amino acids. In a further embodiment of the polypeptide tail, the alkyl amino acid of the polypeptide tail comprises alanine. In a further embodiment of the polypeptide tail, each amino acid of the polypeptide tail is in the D configuration.

[0040] The present invention further comprises: TIFF2024546110000002.tif17160 [where X 3 are D-Trp, D-6-fluoro-Trp, D-6-chloro-Trp, D-6-bromo-Trp, D-6-iodo-Trp, D-6-methyl-Trp, D-6-cyano-Trp, D-6-hydroxy-Trp, D-6-NO2-Trp, D-7-fluoro-Trp, D-7-chloro-Trp, D-7-bromo-Trp, D-7-iodo-Trp, D-7-methyl- Trp, D-7-cyano-Trp, D-7-hydroxy-Trp, D-7-NO2-Trp, D-6,7-fluoro-Trp, D-6,7-chloro-Trp, D-6,7-bromo-Trp, D-6,7-iodo-Trp, D-6,7-methyl-Trp, D-6,7-cyano-Trp, D-6,7-hydroxy-Trp or D-6,7-NO2-Trp; X 6is (R)-2-amino-2-methylhept-6-enoic acid; X 7 is Dp-fluoro-Phe, Dp-chloro-Phe, Dp-bromo-Phe, Dp-iodo-Phe, Dp-methyl-Phe, Dp-cyano-Phe, Dp-hydroxy-Phe, Dp-NO2-Phe or Dp-CF3-Phe; X 10 is (R)-2-amino-2-methylhept-6-enoic acid; X 11 is D-Leu;X 12 is D-Arg or D-Ser;X 13 is D-Ala, D-Glu, or D-Gla (γ-carboxyl glutamic acid); X 14 is D-Ala;X 15 is D-Ala, D-Phe, or D-Glu; X 16 is D-Ala or absent;X 17 is D-Ala, D-α-methyl-Glu, or absent; X 18 is D-Ala or absent;X 19 is D-Ala or absent;X 20 is D-Ala or absent;X 21 is D-Ala or absent; the N-terminal amino group of Thr at position 1 is linked to an acyl group having the formula RCO-; R is a n H 2n+1 n is an integer from 1 to 10; the C-terminal amino acid may optionally contain an amino group; and the staple is X 6 and X 10 is an olefin obtained by ring-closing metathesis between The present invention provides a p53 peptidomimetic macrocycle comprising:

[0041] In certain embodiments, amino acids 1-12 are all D-amino acids, and amino acids 13-21 are each independently an L-amino acid or a D-amino acid, or each is a D-amino acid.

[0042] In another embodiment of the p53 peptidomimetic macrocycle, X 12 is D-Ser. In a further embodiment of the p53 peptidomimetic macrocycle, X 3 is D-6-fluoro-Trp; X 7 is Dp-CF3-Phe; or X 3 is D-6-fluoro-Trp, and X 7 is Dp-CF3-Phe. In another embodiment, the acyl group is an acetyl group.

[0043] The present invention relates to TIFF2024546110000003.tif14162 [where X 3 are D-Trp, D-6-fluoro-Trp, D-6-chloro-Trp, D-6-bromo-Trp, D-6-iodo-Trp, D-6-methyl-Trp, D-6-cyano-Trp, D-6-hydroxy-Trp, D-6-NO2-Trp, D-7-fluoro-Trp, D-7-chloro-Trp, D-7-bromo-Trp, D-7-iodo-Trp, D-7-methyl- Trp, D-7-cyano-Trp, D-7-hydroxy-Trp, D-7-NO2-Trp, D-6,7-fluoro-Trp, D-6,7-chloro-Trp, D-6,7-bromo-Trp, D-6,7-iodo-Trp, D-6,7-methyl-Trp, D-6,7-cyano-Trp, D-6,7-hydroxy-Trp or D-6,7-NO2-Trp; X 5 is (S)-2-amino-2-methylhept-6-ynoic acid; X 7 is Dp-fluoro-Phe, Dp-chloro-Phe, Dp-bromo-Phe, Dp-iodo-Phe, Dp-methyl-Phe, Dp-cyano-Phe, Dp-hydroxy-Phe, Dp-NO2-Phe or Dp-CF3-Phe; X 9 is D-Lys or D-Gln; X 11 is D-Leu;X 12 is (R)-2-amino-2-methyloct-7-ynoic acid; 13 is D-Ala, D-Glu, D-Gla (γ-carboxyl glutamic acid), or absent; X14 is D-Ala or absent;X 15 is D-Ala, D-Phe, or D-Glu, or is absent; X 16 is D-Ala or absent;X 17 is D-Ala, D-α-methyl-Glu, or absent; X 18 is D-Ala or absent;X 19 is D-Ala or absent;X 20 is D-Ala or absent;X 21 is D-Ala or absent; the N-terminal amino group of Thr at position 1 is linked to an acyl group having the formula RCO-; R is a n H 2n+1 n is an integer from 1 to 10; the C-terminal amino acid may optionally contain an amino group; and the staple is X 5 and X 12 is a dialkyne obtained by alkyne cross-coupling between The present invention provides a p53 peptidomimetic macrocycle comprising:

[0044] In certain embodiments, amino acids 1-12 are all D-amino acids, and amino acids 13-21 are each independently an L-amino acid or a D-amino acid, or each is a D-amino acid.

[0045] In another embodiment of the p53 peptidomimetic macrocycle, X 9 is D-Gln. In another embodiment of the p53 peptidomimetic macrocycle, X 3 is D-6-fluoro-Trp; X 7 is Dp-CF3-Phe; or X 3 is D-6-fluoro-Trp, and X 7 is Dp-CF3-Phe. In another embodiment, the acyl group is an acetyl group.

[0046] The present invention relates to TIFF2024546110000004.tif16160 [where X 3 are D-Trp, D-6-fluoro-Trp, D-6-chloro-Trp, D-6-bromo-Trp, D-6-iodo-Trp, D-6-methyl-Trp, D-6-cyano-Trp, D-6-hydroxy-Trp, D-6-NO2-Trp, D-7-fluoro-Trp, D-7-chloro-Trp, D-7-bromo-Trp, D-7-iodo-Trp, D-7-methyl- Trp, D-7-cyano-Trp, D-7-hydroxy-Trp, D-7-NO2-Trp, D-6,7-fluoro-Trp, D-6,7-chloro-Trp, D-6,7-bromo-Trp, D-6,7-iodo-Trp, D-6,7-methyl-Trp, D-6,7-cyano-Trp, D-6,7-hydroxy-Trp or D-6,7-NO2-Trp; X 5 is (S)-2-amino-2-methyldec-9-enoic acid; 7 is Dp-fluoro-Phe, Dp-chloro-Phe, Dp-bromo-Phe, Dp-iodo-Phe, Dp-methyl-Phe, Dp-cyano-Phe, Dp-hydroxy-Phe, Dp-NO2-Phe or Dp-CF3-Phe; X 9 is D-Lys or D-Gln; X 11 is D-Leu;X 12 is (R)-2-amino-2-methylhept-6-enoic acid; X 13 are D-Ala, D-Glu, and D-Gla (γ-carboxyl glutamic acid); 14 is D-Ala;X 15 is D-Ala, D-Phe, or D-Glu; X 16 is D-Ala;X 17 is D-Ala or D-α-methyl-Glu; X 18 is D-Ala;X 19 is D-Ala or absent;X 20 is D-Ala or absent;X 21 is D-Ala or absent; the N-terminal amino group of Thr at position 1 is linked to an acyl group having the formula RCO-; R is a n H2n+1 n is an integer from 1 to 10; the C-terminal amino acid may optionally contain an amino group; amino acids 1 to 12 are D-amino acids; and the staple is X 5 and X 12 is an olefin obtained by ring-closing metathesis between The present invention provides a p53 peptidomimetic macrocycle comprising:

[0047] In certain embodiments, amino acids 1-12 are all D-amino acids, and amino acids 13-21 are each independently an L-amino acid or a D-amino acid, or each is a D-amino acid.

[0048] In another embodiment of the p53 peptidomimetic macrocycle, X 9 is D-Gln. In a further embodiment of the p53 peptidomimetic macrocycle, X 3 is D-6-fluoro-Trp; X 7 is Dp-CF3-Phe; or X 3 is D-6-fluoro-Trp, and X 7 is Dp-CF3-Phe. In another embodiment, the acyl group is an acetyl group.

[0049] The present invention relates to TIFF2024546110000005.tif9157 [where X 3are D-Trp, D-6-fluoro-Trp, D-6-chloro-Trp, D-6-bromo-Trp, D-6-iodo-Trp, D-6-methyl-Trp, D-6-cyano-Trp, D-6-hydroxy-Trp, D-6-NO2-Trp, D-7-fluoro-Trp, D-7-chloro-Trp, D-7-bromo-Trp, D-7-iodo-Trp, D-7-methyl- Trp, D-7-cyano-Trp, D-7-hydroxy-Trp, D-7-NO2-Trp, D-6,7-fluoro-Trp, D-6,7-chloro-Trp, D-6,7-bromo-Trp, D-6,7-iodo-Trp, D-6,7-methyl-Trp, D-6,7-cyano-Trp, D-6,7-hydroxy-Trp or D-6,7-NO2-Trp; X 5 is (S)-2-amino-2-methylhept-6-ynoic acid; X 7 is Dp-fluoro-Phe, Dp-chloro-Phe, Dp-bromo-Phe, Dp-iodo-Phe, Dp-methyl-Phe, Dp-cyano-Phe, Dp-hydroxy-Phe, Dp-NO2-Phe or Dp-CF3-Phe; X 9 is D-Lys or D-Gln; X 11 is D-Leu;X 12 is (R)-2-amino-2-methyloct-7-ynoic acid; the N-terminal amino group of Thr at position 1 is linked to an acyl group having the formula RCO-; R is a group of the formula C n H 2n+1 n is an integer from 1 to 10; the C-terminal amino acid may optionally contain an amino group; amino acids 1 to 12 are D-amino acids; and the staple is X 5 and X 12 is a dialkyne obtained by alkyne cross-coupling between The present invention provides a p53 peptidomimetic macrocycle comprising:

[0050] In another embodiment of the p53 peptidomimetic macrocycle, X 9 is D-Gln. In another embodiment of the p53 peptidomimetic macrocycle, X 3is D-6-fluoro-Trp; X 7 is Dp-CF3-Phe; or X 3 is D-6-fluoro-Trp, and X 7 is Dp-CF3-Phe. In another embodiment, the acyl group is an acetyl group.

[0051] In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO: 10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:9.

[0052] The present invention further provides a composition comprising a p53 peptidomimetic macrocycle disclosed herein and a pharma- ceutically acceptable carrier. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9.

[0053] The present invention further provides a method of treating cancer in a subject in need thereof comprising administering to the subject a p53 peptidomimetic macrocycle as disclosed herein or a composition comprising the p53 peptidomimetic macrocycle. The present invention further provides the use of a peptidomimetic macrocycle as disclosed herein for the manufacture of a medicament for the treatment of cancer. The present invention further provides a peptidomimetic macrocycle as disclosed herein for the treatment of cancer. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:9.

[0054] In further embodiments of any of the disclosed methods, uses, or compositions for use, the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin's lymphoma, renal cancer, Hodgkin's lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary gland cancer, pancreatic cancer, bronchial cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral cavity or pharynx cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, and cancer of the blood tissue.

[0055] The present invention further provides a method of modulating the activity of p53 and / or MDM2 and / or MDMX in a subject comprising administering to the subject a p53 peptidomimetic macrocycle as disclosed herein or a composition comprising said p53 peptidomimetic macrocycle.The present invention further provides the use of a peptidomimetic macrocycle as disclosed herein for the manufacture of a medicament for modulating said activity.The present invention further provides a peptidomimetic macrocycle as disclosed herein for modulating said activity.

[0056] In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO: 10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:9.

[0057] The present invention further provides a method of antagonizing the interaction between p53 and MDM2 and / or between p53 and MDMX in a subject comprising administering to the subject a p53 peptidomimetic macrocycle as disclosed herein or a composition comprising said p53 peptidomimetic macrocycle.The present invention further provides the use of a p53 peptidomimetic macrocycle as disclosed herein for the manufacture of a medicament for antagonizing the interaction between p53 and MDM2 and / or between p53 and MDMX.

[0058] The present invention further provides peptidomimetic macrocycles as disclosed herein for antagonizing the interaction between p53 and MDM2 and / or between p53 and MDMX. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9.

[0059] The present invention further provides a combination therapy for treating cancer comprising administering to a subject a therapeutically effective amount of a p53 peptidomimetic macrocycle or a composition comprising the p53 peptidomimetic macrocycle disclosed herein and a therapeutically effective amount of a chemotherapeutic agent or radiation. In another embodiment, the chemotherapeutic agent or radiation is administered to the subject and then the p53 peptidomimetic macrocycle; the p53 peptidomimetic macrocycle is administered to the subject and then the chemotherapeutic agent or radiation; or the chemotherapeutic agent or radiation is administered to the subject simultaneously with administration of the p53 peptidomimetic macrocycle. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9.

[0060] The present invention further provides a combination therapy for treating cancer comprising a therapeutically effective amount of a p53 peptidomimetic macrocycle disclosed herein or a composition comprising said p53 peptidomimetic macrocycle and a therapeutic dose of a chemotherapeutic agent or radiation. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:9.

[0061] In certain embodiments, the chemotherapeutic agent is actinomycin, all-trans-retinoic acid, alitretinoin, azacitidine, azathioprine, bexarotene, bleomycin, bortezomib, carmofur, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, Selected from the group consisting of idarubicin, imatinib, ixabepilone, irinotecan, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, nitrosourea, oxaliplatin, paclitaxel, pemetrexed, romidepsin, tegafur, temozolomide (oral dacarbazine), teniposide, thioguanine, topotecan, utiderone, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, and vorinostat.

[0062] The present invention further provides a combination therapy for treating cancer comprising administering to a subject a therapeutically effective amount of a p53 peptidomimetic macrocycle or a composition comprising the p53 peptidomimetic macrocycle disclosed herein and a therapeutically effective amount of a checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor is an anti-PD1 antibody or an anti-PD-L1 antibody. In another embodiment, the combination therapy further comprises administering to the subject a therapeutically effective amount of a chemotherapeutic agent or radiation. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:

[0063] The present invention further provides a method for treating cancer comprising administering to a subject having cancer a vector comprising a nucleic acid molecule encoding wild-type p53 or a p53 mutant or analog having transcriptional activation activity, followed by one or more administrations of a therapeutically effective amount of a p53 peptidomimetic macrocycle or a composition comprising said p53 peptidomimetic macrocycle as disclosed herein. In certain embodiments, the vector is a plasmid, a retrovirus, an adenovirus, or an adeno-associated virus. In certain embodiments, the subject is administered chemotherapy or radiation therapy prior to or after administering the vector to the subject. In a further embodiment, the therapy further comprises administering a checkpoint inhibitor to the subject. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9. [Brief description of the drawings]

[0064] [Figure 1] FIG. 1A shows the crystal structure of the native human p53 peptide:MDM2 [Protein Data Bank (PDB) ID: 1YCR] complex (Baek et al., JACS 134:103-106 (2012)). MDM2 is shown as a surface and the bound native p53 peptide (SEQ ID NO: 15) is shown as a ribbon drawing, where interacting residues L-Phe19, L-Trp23 and L-Leu26 of the p53 peptide are highlighted as sticks. Hydrogen bond interactions are shown as dotted lines. The amino acid numbers of the p53 peptide correspond to amino acids 15-29 of native human p53, which has the amino acid sequence set forth in SEQ ID NO: 16. H96, K94, Q72 and Y100 are amino acids in MDM2.

[0065] Figure 1B DThe crystal structure of the PMI-δ p53 peptidomimetic peptide:MDM2 (PDB ID: 3PTX) complex is shown (Zhan et al., J. Med. Chem. 55:6237-6241 (2012)). MDM2 is shown as a face, with the bound D The PMI-δ peptidomimetic macrocycle (SEQ ID NO: 11) is shown as a ribbon drawing, where the interacting residues are D Leu 11 , pCF3- D Phe 7 and 6-F- D Trp 3 are highlighted as sticks. Hydrogen bond interactions are shown as dotted lines. H96, K94, Q72 and Y100 are amino acids in MDM2. [Diagram 2] Figure 2 shows a structural depiction of a representative DPMI-δ-(5-12) olefin-stapled p53 peptidomimetic macrocycle:MDM2 complex obtained from molecular dynamics (MD) simulations. MDM2 is shown as a surface and the bound DPMI-δ-(5-12) olefin-stapled peptidomimetic macrocycle is shown as a stick figure, where interacting residues are highlighted as sticks. The hydrocarbon olefin linker is the light portion of the peptide indicated by the arrow. Hydrogen bond interactions are shown as dotted lines. H96, K94, and Q72 are amino acids in MDM2. [Diagram 3] Figure 3 shows the conformation of a representative DPMI-δ-(6-10) olefin-stapled p53 peptidomimetic macrocycle linked to a six amino acid polypeptide tail containing the amino acid sequence AFAAAA (SEQ ID NO:21) in complex with MDM2 sampled during molecular dynamics simulations. MDM2 is shown as a surface / picture, and the bound p53 peptidomimetic macrocycle is shown pictorially with the three key binding residues 6-F-DTrp3, pCF3-DPhe7 and DLeu11;DPhe14 (in the polypeptide tail) and the olefin staple highlighted. [Figure 4]Figure 4 shows a representative DPMI-δ-(5-12) dialkyne-stapled p53 peptidomimetic macrocycle conformation sampled during molecular dynamics simulations. The p53 peptidomimetic macrocycle is shown pictorially, with the three key binding residues and the dialkyne highlighted as sticks.

[0066] Detailed Description of the Invention definition As used herein, "administration" and "administering" refer to the introduction of at least one p53 peptidomimetic macrocycle or a pharmaceutical composition comprising at least one p53 peptidomimetic macrocycle into a subject. When administration is for therapeutic purposes, the substance is administered at or after diagnosis of abnormal cell growth, such as a tumor. The therapeutic administration of the substance serves to inhibit cell growth or abnormal cell growth of the tumor.

[0067] As used herein, "α-amino acid" or simply "amino acid" refers to a molecule that contains both an amino group and a carboxyl group bonded to a carbon, referred to as the α-carbon, which is bonded to a side chain (the R group) and a hydrogen atom, and can be represented by the following formulae shown for (R) α-amino acids and (S) α-amino acids: [ka]

[0068] Generally, L-amino acids have the (S) configuration, with the exception of cysteine, which has the (R) configuration, and glycine, which is achiral. Suitable α-amino acids for the all-D amino acid configuration peptides disclosed herein include only the D isomers of the naturally occurring amino acids and their analogs, as well as only the D isomers of non-naturally occurring amino acids produced by organic synthesis or other metabolic routes, except that α,α-disubstituted amino acids can be L, D or achiral. As used herein, the term amino acid is intended to include amino acid analogs, unless the context specifically indicates otherwise. As used herein, D-amino acids (or D-configured amino acids) are referred to by the superscript "D" (e.g.,D Leu) and L-amino acids are designated by "L" (e.g., L-Leu) or without the L identifier (e.g., Leu). As used herein, the terms D-amino acids and D-configured amino acids are used interchangeably.

[0069] As used herein, "α,α-disubstituted amino acid" refers to a molecule or moiety that contains both an amino group and a carboxyl group attached to an α carbon that is attached to two natural or unnatural amino acid side chains (or a combination thereof). Exemplary α,α-disubstituted amino acids are shown below. These α,α-disubstituted amino acids contain a side chain with a terminal olefin reactive group. [ka]

[0070] These α,α-disubstituted amino acids contain a side chain that has a terminal alkyne reactive group. [ka]

[0071] As used herein, "amino acid analog" or "unnatural amino acid" refers to a molecule structurally similar to an amino acid that can substitute for the amino acid in forming a p53 peptidomimetic macrocycle. Amino acid analogs include, but are not limited to, compounds that are structurally identical to the amino acids defined herein except that they contain one or more additional methylene groups between the amino and carboxyl groups (e.g., α-amino, β-carboxy acids), or the amino or carboxy group is replaced with a group of similar reactivity (e.g., a primary amine is replaced with a secondary or tertiary amine, or a carboxy group is replaced with an ester).

[0072] As used herein, "amino acid side chain" refers to the moiety attached to the alpha carbon of an amino acid. For example, the amino acid side chain of alanine is methyl, the amino acid side chain of phenylalanine is phenylmethyl, the amino acid side chain of cysteine ​​is thiomethyl, the amino acid side chain of aspartic acid (aspartate) is carboxymethyl, the amino acid side chain of tyrosine is 4-hydroxyphenylmethyl, etc. Also included are other amino acid side chains that do not occur in nature, such as those that are synthetically produced (e.g., alpha,alpha-disubstituted amino acids) or that occur naturally (e.g., amino acid metabolites).

[0073] As used herein, an "acyl group" refers to a moiety having the formula RCO-, where R is of the formula C n H 2n+1 It is an alkane having the formula:

[0074] As used herein, "capping group" refers to a chemical moiety present at either the carboxy or amino terminus of the polypeptide chain of a subject p53 peptidomimetic macrocycle. Carboxy-terminal capping groups include unmodified carboxylic acid (i.e., -COOH), or carboxylic acid with a substituent. For example, the carboxy terminus can be substituted with an amino group to generate a carboxamide at the C-terminus. Various substituents include, but are not limited to, primary amines, and secondary amines, such as pegylated secondary amines. Amino-terminal capping groups include unmodified amines (i.e., -NH2), or amines with a substituent. For example, the amino terminus can be substituted with an acyl group to generate a carboxamide at the N-terminus. Various substituents include substituted acyl groups, such as C1-C6 carbonyl, C7-C8 carbonyl, C9-C10 carbonyl, C11-C12 carbonyl, C12-C14 carbonyl, C13-C15 carbonyl, C14-C16 carbonyl, C15-C17 carbonyl, C16-C18 carbonyl, C17-C19 carbonyl, C18-C20 carbonyl, C19-C21 carbonyl, C19-C22 carbonyl, C18-C23 carbonyl, C19-C24 carbonyl, C19-C25 carbonyl, C20-C26 carbonyl, C21-C27 carbonyl, C22-C28 carbonyl, C23-C24 carbonyl, C24-C25 carbonyl, C25-C26 carbonyl, C25-C27 carbonyl, C26-C28 carbonyl, C27-C28 carbonyl, C28-C29 carbonyl, C29-C30 carbonyl, C29-C31 carbonyl, C29-C32 carbonyl, C29-C3 30 Including, but not limited to, carbonyl and pegylated carbamates.

[0075] As used herein, "co-administration" means that at least two different biologically active compounds are each administered to a subject within a time frame in which their respective periods of biological activity overlap. Thus, the term includes sequential and co-extensive administration. When co-administration is used, the route of administration does not have to be the same. Biologically active compounds include p53 peptidomimetic macrocycles, as well as other compounds useful in the treatment of cancer, such as, but not limited to, vinca alkaloids, nucleic acid inhibitors, platinum agents, interleukin 2, interferons, alkylating agents, antimetabolites, corticosteroids, DNA intercalating agents, anthracyclines, and ureas. In addition to those exemplified herein, examples of specific agents include hydroxyurea, 5-fluorouracil, anthramycin, asparaginase, bleomycin, dactinomycin, dacabazine, cytarabine, busulfan, thiotepa, lomustine, mechlorethamine, cyclophosphamide, melphalan, mechlorethamine, chlorambucil, carmustine, 6-thioguanine, methotrexate, etc. One of skill in the art will appreciate that two different p53 peptidomimetic macrocycles may be co-administered to a subject, or a p53 peptidomimetic macrocycle and an agent such as one of the foregoing agents may be co-administered to a subject.

[0076] As used herein, "combination therapy" refers to treatment of a human or animal individual that involves administering to the individual, either sequentially or simultaneously, a first therapeutic agent and a second therapeutic agent. Generally, the first and second therapeutic agents are administered to the individual separately, rather than as a mixture, although there can be embodiments in which the first and second therapeutic agents are mixed prior to administration.

[0077] As used herein, "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.), in which case the change can often be made without changing the biological activity of the protein. In general, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially change biological activity (see, for example, Watson et al. Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co., p.224(4th Ed.)(1987)). Also, substitution of structurally or functionally similar amino acids is unlikely to impair biological activity. Exemplary conservative substitutions are shown in Table 1. [Table 1]

[0078] As used herein, "dose," "dosage," "unit dose," "unit dosage," "effective dose," and related terms refer to a physically discrete unit containing a predetermined quantity of active ingredient (e.g., a p53 peptidomimetic macrocycle) calculated to produce a desired therapeutic effect (e.g., cancer cell death). These terms are synonymous with a therapeutically effective amount and an amount sufficient to achieve the stated objectives of the methods disclosed herein.

[0079] As used herein, D "PMI-δ p53 peptidomimetic peptide" refers to a linear p53 peptidomimetic peptide having the amino acids set forth in SEQ ID NO: 11, including all amino acids that are in the D configuration, as indicated by the superscript "D."

[0080] As used herein, D PMI-δ(6-10) Olefin-stapled p53 Peptidomimetic Macrocycles” Modified DEach of the amino acids at amino acid positions 6 and 10 is replaced with an α,α-disubstituted amino acid containing an amino acid side chain with a terminal alkenyl group to obtain a PMI-δ p53 peptidomimetic peptide. D PMI-δ means a p53 peptidomimetic peptide, wherein the modification is by ring-closing metathesis between two α,α-disubstituted amino acids thereof D Macrocyclization of PMI-δ peptide D To generate PMI-δ(6-10) olefin-stapled p53 peptidomimetic macrocycles, in certain embodiments, the other side chain of the α,α-disubstituted amino acid is a methyl substituent. D The PMI-δ(6-10) olefin-stapled p53 peptidomimetic macrocycles may contain further amino acid modifications, insertions, substitutions or additions at the N-terminus or C-terminus, or non-amino acid additions at the N-terminus, e.g., acylation, or non-amino acid additions at the C-terminus, e.g., amidation.

[0081] As used herein, D PMI-δ(5-12) Olefin-stapled p53 Peptidomimetic Macrocycles” Modified D Each of the amino acids at amino acid positions 5 and 12 is replaced with an α,α-disubstituted amino acid containing an amino acid side chain with a terminal alkenyl group to obtain a PMI-δ p53 peptidomimetic peptide. D PMI-δ means a p53 peptidomimetic peptide, wherein the modification is by ring-closing metathesis between two α,α-disubstituted amino acids thereof D Macrocyclization of PMI-δ peptide D To generate PMI-δ(6-10) olefin-stapled p53 peptidomimetic macrocycles, in certain embodiments, the other side chain of the α,α-disubstituted amino acid is a methyl substituent. D The PMI-δ(5-12) olefin-stapled p53 peptidomimetic macrocycles may contain further amino acid modifications, insertions, substitutions or additions at the N-terminus or C-terminus, or non-amino acid additions at the N-terminus, e.g., acylation, or non-amino acid additions at the C-terminus, e.g., amidation.

[0082] As used herein, D PMI-δ(5-12) Dialkyne-stapled p53 Peptidomimetic Macrocycles Modified D Each of the amino acids at amino acid positions 5 and 12 is replaced with an α,α-disubstituted amino acid containing an amino acid side chain with a terminal alkynyl group to obtain a PMI-δ p53 peptidomimetic peptide. D PMI-δ means a p53 peptidomimetic peptide, wherein the modification is by alkyne cross-coupling between two α,α-disubstituted amino acids thereof. D Macrocyclization of PMI-δ peptide D Generate the PMI-δ(6-12) dialkyne-stapled p53 peptidomimetic macrocycle. In certain embodiments, the other side chain of the α,α-disubstituted amino acid is a methyl substituent. D The PMI-δ(5-12) dialkyne stapled p53 peptidomimetic macrocycle may include further amino acid modifications, insertions, substitutions or additions at the N-terminus or C-terminus, or non-amino acid additions at the N-terminus, e.g., acylation, or non-amino acid additions at the C-terminus, e.g., amidation.

[0083] "Helical stability" as used herein refers to the maintenance of α-helical structure by the staples or stitches of the p53 peptidomimetic macrocycles of the invention as measured by circular dichroism or NMR. For example, in some embodiments, the p53 peptidomimetic macrocycles of the invention exhibit at least a 1.25, 1.5, 1.75 or 2-fold increase in α-helicity as measured by circular dichroism compared to the corresponding unbridged macrocycle.

[0084] As used herein, "macrocycle" refers to a molecule having a chemical structure that includes a ring or cycle formed by at least nine covalently bonded atoms.

[0085] As used herein, "macrocyclization reagent" or "macrocycle-forming reagent" refers to any reagent that can be used to produce the p53 peptidomimetic macrocycles of the present invention by inducing a reaction between two reactive groups on an α,α-disubstituted amino acid, where the reactive groups on the α,α-disubstituted amino acid can be, for example, azide and alkyne, where macrocyclization reagents include, but are not limited to, Cu reagents, e.g., reagents that provide reactive Cu(I) species, such as CuBr, CuI, or CuOTf, as well as Cu(II) salts, such as Cu(CO2CH3)2, CuSO4, and CuCl2, that can be converted in situ to active Cu(I) reagents by the addition of a reducing agent, such as ascorbic acid or sodium ascorbate.

[0086] Macrocyclization reagents can also include, for example, Ru reagents known in the art, such as Cp * RuCl(PPh3)2, [Cp * RuCl]4, or other Ru reagents capable of providing reactive Ru(II) species. In other cases, the reactive group is a terminal olefin. In such embodiments, the macrocyclization or macrocycle-forming reagent is a metathesis catalyst, 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 with a +2 oxidation state, 16 electron count, and pentacoordination. 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. In still other cases, the reactive group is a thiol group. In such embodiments, the macrocyclization reagent is a linker functionalized with two thiol-reactive groups, such as, for example, halogen groups.

[0087] As used herein, "p53" refers to the tumor protein P53, also known as cellular tumor antigen p53 (UniProt name), phosphoprotein p53, tumor suppressor p53, antigen NY-CO-13 or transformation-related protein 53 (TRP53), or any isoform of the protein encoded by homologous genes in various organisms (e.g., the TP53 gene in humans).

[0088] As used herein, "MDM2" refers to mouse double minute 2 protein, also known as E3 ubiquitin-protein ligase. MDM2 is a protein encoded by the MDM2 gene in humans. The MDM2 protein is a key negative regulator of the p53 tumor suppressor. The MDM2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal transactivation domain (TAD) of the p53 tumor suppressor and as an inhibitor of p53 transcriptional activation. As used herein, the term MDM2 refers to the human homolog. See GenBank Accession No. 228952; GI:228952.

[0089] As used herein, "MDMX" or "MDM4" refers to mouse double minute X or 4, a protein that shows significant structural similarity to MDM2. MDMX or MDM4 interacts with p53 through a binding domain located in the N-terminal region of the MDMX or MDM4 protein. As used herein, the terms MDMX or MDM4 refer to the same human homologue. See GenBank Accession No.: 88702791; GI:88702791.

[0090] "Transcriptional activation activity" as used herein with respect to p53 or a mutant or analogue thereof means the ability of p53 or a mutant or analogue thereof to activate transcription from a p53-dependent promoter. The activation ability of p53 or a mutant or analogue thereof can be measured in a transcriptional assay that allows the expression of a reporter gene operably linked to a p53-dependent promoter to be detected and measured.

[0091] As used herein, "member" ("member") with respect to a macrocycle or macrocycle-forming linker means an atom that forms or can form a macrocycle, and does not include atoms of the substituents or side chains. By analogy, cyclodecane, 1,2-difluorodecane, and 1,3-dimethylcyclodecane are all considered 10-membered macrocycles because the hydrogen or fluoro substituents or methyl side chains do not participate in the formation of the macrocycle.

[0092] As used herein, "naturally occurring amino acid" or "natural amino acid" means any of the twenty amino acids commonly found in naturally synthesized peptides and which are designated by the one-letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. Naturally occurring amino acids can have the D-configuration (D-amino acids) or the L-configuration (L-amino acids).

[0093] As used herein, "non-naturally occurring amino acid" or "unnatural amino acid" refers to an amino acid analogue that is not found in nature.

[0094] A "non-essential" amino acid residue is one that can be modified from the wild-type sequence of a polypeptide without eliminating or substantially altering an essential biological or biochemical activity of the polypeptide (e.g., receptor binding or activation). An "essential" amino acid residue is one that, when modified from the wild-type sequence of the polypeptide, eliminates or substantially alters an essential biological or biochemical activity of the polypeptide.

[0095] As used herein, a "peptidomimetic macrocycle" or "bridged polypeptide" refers to a compound that includes multiple amino acid residues linked by multiple peptide bonds and at least one macrocycle-forming linker that 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 macrocycles include embodiments in which the macrocycle-forming linker connects the α-carbon of a first α,α-disubstituted amino acid residue (or analog) to the α-carbon of a second α,α-disubstituted amino acid residue (or analog). Peptidomimetic macrocycles may optionally include one or more non-peptide bonds between one or more amino acid residues and / or amino acid analog residues, and may optionally include one or more non-naturally occurring amino acid residues or amino acid analog residues in addition to those that form the macrocyclic ring (macrocycle). A "corresponding non-crosslinked polypeptide" when referred to in the context of a peptidomimetic macrocycle is understood to refer to a polypeptide of the same amino acid sequence as the peptidomimetic macrocycle, with the exception of the amino acids involved in the staple or stitch crosslinks.

[0096] As used herein, a "dialkyne" refers to two alkynes separated by a single bond, e.g., the structure [ka]

[0097] It is represented by:

[0098] Unless otherwise stated, compounds and structures referred to herein are also intended to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, a hydrogen has been replaced by deuterium or tritium, or a carbon atom has been replaced by 13 C or 14Compounds having this structure in which a C-enriched carbon is substituted or a carbon atom is substituted by silicon are within the scope of the present invention. The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may contain tritium ( 3 H), Iodine-125( 125 I) or carbon-14 ( 14 C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

[0099] As used herein, "pharmacologically acceptable derivative" refers to any pharma- ceutically acceptable salt, ester, salt of an ester, prodrug or other derivative of the p53 peptidomimetic macrocycles disclosed herein that, when administered to an individual, can (directly or indirectly) produce the p53 peptidomimetic macrocycles disclosed herein. Particularly preferred pharma- ceutical acceptable derivatives are those that, when administered to an individual, enhance the bioavailability of the p53 peptidomimetic macrocycles disclosed herein (e.g., by enhancing absorption of orally administered p53 peptidomimetic macrocycles disclosed herein into the blood) or enhance delivery of the active compound to a biological compartment (e.g., the brain or lymphatic system) compared to the parent species. Some pharma- ceutical acceptable derivatives contain chemical groups that enhance water solubility or active transport across the gastrointestinal mucosa.

[0100] As used herein, a "polypeptide" comprises two or more naturally occurring or non-naturally occurring amino acids linked by a covalent bond (e.g., an amide bond). The polypeptides 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).

[0101] "Stability" as used herein means the maintenance of a constant secondary structure in solution or resistance to proteolysis in vitro or in vivo by the p53 peptidomimetic macrocycles of the invention as measured by circular dichroism, NMR or other biophysical means. Non-limiting examples of secondary structures contemplated in the present invention include α-helices, β-turns and β-pleated sheets.

[0102] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a particular substance sufficient to achieve a desired effect in a treated subject. For example, this may be the amount of the p53 peptidomimetic macrocycle of the present invention required to activate p53 by inhibiting p53 binding to MDM2 and MDMX. It may also refer to the amount or dose of chemotherapeutic agent or radiation administered to a subject with cancer that is typically administered to the subject to treat the cancer.

[0103] As used herein, "treat" or "treatment" refers to the administration, internally or externally, of a therapeutic agent, such as a composition containing any of the p53 peptidomimetic macrocycles of the present invention, to a subject or patient having or suspected of having one or more disease symptoms for which the agent has therapeutic or prophylactic activity. Typically, the therapeutic agent is administered in an amount effective to reduce one or more disease symptoms in the subject or population being treated, by inducing regression of such symptoms or inhibiting the progression of such symptoms by any clinically measurable degree. The amount of a therapeutic agent effective to reduce any particular disease symptom may vary depending on factors such as the condition, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject. Whether a disease symptom has been reduced may be assessed by any clinical scale typically used by a physician or other skilled health care provider to assess the severity or progression of that symptom. The term further includes delaying the onset of symptoms associated with the disorder and / or reducing the severity of the symptoms of such disorder. The term further includes the amelioration of existing uncontrolled or undesirable symptoms, the prevention of additional symptoms, and the amelioration or prevention of the underlying causes of such symptoms. Thus, the term indicates that a beneficial result has been produced in a human or animal subject having a disorder, disease or condition, or having a potential for developing such a disorder, disease or condition.

[0104] As used herein, "treatment" as applied to a human or veterinary individual means a therapeutic procedure comprising contacting a human or animal individual in need of treatment with a p53 peptidomimetic macrocycle of the invention, where the p53 peptidomimetic macrocycle has therapeutic or prophylactic activity.

[0105] P53 peptidomimetic macrocycles Peptide-based inhibitors hold great promise for targeted modulation of intracellular protein-protein interactions (PPIs) because they have access to a broad range of chemical space, including a multitude of different secondary and tertiary structures and sequence diversity. Currently, drawbacks that impede the development of peptide therapeutics include poor conformational stability, proteolytic susceptibility, and cell permeability. Also, it is difficult to convert these in vitro peptide inhibitors into intracellularly and in vivo active compounds with on-target selectivity and specificity. Several modern peptide design strategies address these issues from different angles. Strategic macrocyclization with optimally placed chemical reinforcing structures, such as olefinic hydrocarbon bridges, commonly referred to as staples, may address these issues by (i) restricting conformational freedom to improve target affinity, (ii) improving proteolytic resistance, and (iii) enhancing cell permeability. Conversely, molecules constructed entirely from D-amino acids are ultra-resistant to proteolytic cleavage, but generally lack conformational stability and membrane permeability. The present inventors have found that all-D-configured α-helical stapled peptides (hereafter referred to as peptide macrocycles) can be designed by using specific staples, and that the addition of a 3-9 amino acid polypeptide tail to the C-terminus of the peptide macrocycle exhibits improved pharmacological properties. These improvements in pharmacological properties include protease resistance, conformational stability, cell permeability in the absence of induction of membrane disruption, and low or no cytotoxicity (e.g., p53 peptidomimetic macrocycles exhibited reduced cellular activity in counter screen activity and LDH release measured using the assays disclosed in "General Methods" below). Furthermore, the p53 peptidomimetic macrocycles of the present invention bind to mouse double minute 2 (MDM2; also known as E3 ubiquitin-protein ligase) and MDMX (also known as MDM4), and activate p53 in cells by binding to MDM2 and MDMX and antagonizing the binding of MDM2 and MDMX to p53.

[0106] The present inventors have D Our findings have been embodied in the present invention by appending a polypeptide tail containing 3-9 amino acids to the C-terminal amino acid of a p53 peptidomimetic macrocycle in an all-D configuration derived from the PMI-δ peptide (disclosed in Liu et al.

[41] and in US Patent Publication No. 20120328692; SEQ ID NO:11) containing an olefin staple between the α-carbons of two α,α-disubstituted amino acids (6-10 olefin staple) located at amino acid positions 6 and 10 of the peptide disclosed in WO2020257153, to obtain a p53 peptidomimetic macrocycle with improved pharmacological properties. Further improvements can be achieved by substituting the amino acid at position 9 with glutamine and the amino acid at position 12 with serine.

[0107] The present inventors have also developed a method for preparing a peptide that includes an olefin staple between amino acid positions 5 and 12 (5-12 olefin staple). D We have also found that improvements in the pharmacological properties of PMI-δ peptides can be achieved by replacing the 5-12 olefin staple with a 5-12 dialkyne staple and, optionally, adding a polypeptide tail of 3 to 9 amino acids to the C-terminus. Further improvements can be achieved by replacing the amino acid at position 9 with glutamine.

[0108] The invention provides a p53 peptidomimetic macrocycle comprising: (a) an i,i+4 olefin staple and a polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle; (b) an i,i+7 olefin staple and a polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle; or (c) an i,i+7 dialkyne staple and an optional polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle, wherein the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, and the polypeptide tail comprises from 3 to 9 amino acids, and each amino acid in the polypeptide tail independently has the D-configuration or the L-configuration, or in certain embodiments, each amino acid in the polypeptide tail has the D-configuration.

[0109] In another embodiment of the p53 peptidomimetic macrocycle, the p53 peptidomimetic macrocycle comprises an i,i+7 alkyne staple, covalently attached to the N-terminus of the polypeptide tail at the C-terminal amino acid.

[0110] In another embodiment of the p53 peptidomimetic macrocycle, the p53 peptidomimetic macrocycle comprises 12 amino acids, an i,i+4 olefin staple formed between the α-carbons of two α,α-disubstituted amino acids located at amino acid positions 6 and 10 of the p53 peptidomimetic macrocycle, and a polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle, wherein the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, the polypeptide tail comprises 3 to 9 amino acids, and each amino acid of the polypeptide tail independently has the D-configuration or the L-configuration, or in certain embodiments, each amino acid of the polypeptide tail has the D-configuration.

[0111] In another embodiment of the p53 peptidomimetic macrocycle, the α,α-disubstituted amino acids at amino acid positions 6 and 10 of the p53 peptidomimetic macrocycle comprise (R)-2-amino-2-methylhept-6-enoic acid.

[0112] In another embodiment of the p53 peptidomimetic macrocycle, the p53 peptidomimetic macrocycle further comprises D-6-fluoro-tryptophan at amino acid position 3 and Dp-CF3-phenylalanine at amino acid position 7.

[0113] In another embodiment of the p53 peptidomimetic macrocycle, the p53 peptidomimetic macrocycle further comprises a threonine at amino acid position 1, an alanine at amino acid position 2, a tyrosine at amino acid position 4, an alanine at amino acid position 5, a glutamic acid at amino acid position 8, a lysine or glutamine at amino acid position 9, a leucine at amino acid position 11, and an arginine or serine at amino acid position 12.

[0114] In another embodiment of the p53 peptidomimetic macrocycle, the p53 peptidomimetic macrocycle contains a glutamine at amino acid position 9 and a serine at amino acid position 12.

[0115] In another embodiment of the p53 peptidomimetic macrocycle, the p53 peptidomimetic macrocycle comprises 12 amino acids, an i,i+7 dialkyne staple formed between the α-carbons of two α,α-disubstituted amino acids located at amino acid positions 5 and 12 of the p53 peptidomimetic macrocycle, and an optional polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle, wherein the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, the polypeptide tail comprises from 3 to 9 amino acids, and each amino acid of the polypeptide tail independently has the D-configuration or the L-configuration.

[0116] In another embodiment of the p53 peptidomimetic macrocycle, the α,α-disubstituted amino acid at amino acid position 5 of the p53 peptidomimetic macrocycle comprises (S)-2-amino-2-methylhept-6-ynoic acid and the α,α-disubstituted amino acid at amino acid position 12 of the p53 peptidomimetic macrocycle comprises (R)-2-amino-2-methyloct-7-ynoic acid.

[0117] In another embodiment of the p53 peptidomimetic macrocycle, the p53 peptidomimetic macrocycle further comprises D-6-fluoro-tryptophan at amino acid position 3 of the p53 peptidomimetic macrocycle and Dp-CF3-phenylalanine at amino acid position 7 of the p53 peptidomimetic macrocycle.

[0118] In another embodiment of the p53 peptidomimetic macrocycle, the p53 peptidomimetic macrocycle further comprises a threonine at amino acid position 1, an alanine at amino acid position 2, a tyrosine at amino acid position 4, an asparagine at amino acid position 6, a glutamic acid at amino acid position 8, a lysine or glutamine at amino acid position 9, a leucine at amino acid position 10, and a leucine at amino acid position 11.

[0119] In another embodiment of the p53 peptidomimetic macrocycle, the p53 peptidomimetic macrocycle comprises a glutamine at amino acid position 9.

[0120] In another embodiment of the p53 peptidomimetic macrocycle, the p53 peptidomimetic macrocycle comprises 12 amino acids, an i,i+7 olefin staple formed between the α-carbons of two α,α-disubstituted amino acids located at amino acid positions 5 and 12 of the p53 peptidomimetic macrocycle, and an optional polypeptide tail covalently attached at its N-terminus to the C-terminal amino acid of the p53 peptidomimetic macrocycle, wherein the p53 peptidomimetic macrocycle comprises amino acids that are all in the D-configuration, the polypeptide tail comprises from 3 to 9 amino acids, and each amino acid of the polypeptide tail independently has the D- or L-configuration.

[0121] In another embodiment of the p53 peptidomimetic macrocycle, the α,α-disubstituted amino acid at amino acid position 5 of the p53 peptidomimetic macrocycle comprises (S)-2-amino-2-methyldec-9-enoic acid and the α,α-disubstituted amino acid at amino acid position 12 of the p53 peptidomimetic macrocycle comprises (R)-2-amino-2-methylhept-6-enoic acid.

[0122] In another embodiment of the p53 peptidomimetic macrocycle, the p53 peptidomimetic macrocycle further comprises D-6-fluoro-tryptophan at amino acid position 3 of the p53 peptidomimetic macrocycle and Dp-CF3-phenylalanine at amino acid position 7 of the p53 peptidomimetic macrocycle.

[0123] In another embodiment of the p53 peptidomimetic macrocycle, the p53 peptidomimetic macrocycle further comprises a threonine at amino acid position 1, an alanine at amino acid position 2, a tyrosine at amino acid position 4, an asparagine at amino acid position 6, a glutamic acid at amino acid position 8, a lysine or glutamine at amino acid position 9, a leucine at amino acid position 10, and a leucine at amino acid position 11.

[0124] In another embodiment of the p53 peptidomimetic macrocycle, the p53 peptidomimetic macrocycle comprises a glutamine at amino acid position 9.

[0125] In another embodiment of the p53 peptidomimetic macrocycle, the polypeptide tail comprises 3 to 9 amino acids, and each amino acid in the polypeptide tail independently has the D- or L-configuration, or in a particular embodiment, each amino acid in the polypeptide tail has the D-configuration.

[0126] In another embodiment of the p53 peptidomimetic macrocycle, the polypeptide tail comprises six amino acids, and each amino acid in the polypeptide tail independently has the D- or L-configuration, or in a particular embodiment, each amino acid in the polypeptide tail has the D-configuration.

[0127] In another embodiment of the disclosed p53 peptidomimetic macrocycle, the polypeptide tail comprises the amino acid sequence set forth in SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23.

[0128] The present invention further comprises: TIFF2024546110000011.tif16161

[0129] [where X 3 are D-Trp, D-6-fluoro-Trp, D-6-chloro-Trp, D-6-bromo-Trp, D-6-iodo-Trp, D-6-methyl-Trp, D-6-cyano-Trp, D-6-hydroxy-Trp, D-6-NO2-Trp, D-7-fluoro-Trp, D-7-chloro-Trp, D-7-bromo-Trp, D-7-iodo-Trp, D-7-methyl- Trp, D-7-cyano-Trp, D-7-hydroxy-Trp, D-7-NO2-Trp, D-6,7-fluoro-Trp, D-6,7-chloro-Trp, D-6,7-bromo-Trp, D-6,7-iodo-Trp, D-6,7-methyl-Trp, D-6,7-cyano-Trp, D-6,7-hydroxy-Trp or D-6,7-NO2-Trp; X 6 is (R)-2-amino-2-methylhept-6-enoic acid; X 7 is Dp-fluoro-Phe, Dp-chloro-Phe, Dp-bromo-Phe, Dp-iodo-Phe, Dp-methyl-Phe, Dp-cyano-Phe, Dp-hydroxy-Phe, Dp-NO2-Phe or Dp-CF3-Phe; X 10 is (R)-2-amino-2-methylhept-6-enoic acid; X 11 is D-Leu;X 12 is D-Arg or D-Ser;X 13 is D-Ala, D-Glu, or D-Gla (γ-carboxyl glutamic acid); X 14 is D-Ala;X 15 is D-Ala, D-Phe, or D-Glu; X16 is D-Ala or absent;X 17 is D-Ala, D-α-methyl-Glu, or absent; X 18 is D-Ala or absent;X 19 is D-Ala or absent;X 20 is D-Ala or absent;X 21 is D-Ala or absent; the N-terminal amino group of Thr at position 1 is linked to an acyl group having the formula RCO-; R is a n H 2n+1 n is an integer from 1 to 10; the C-terminal amino acid may optionally contain an amino group; and the staple is X 6 and X 10 is an olefin obtained by ring-closing metathesis between The present invention provides a p53 peptidomimetic macrocycle comprising:

[0130] In certain embodiments, amino acids 1-12 are all D-amino acids, and amino acids 13-21 are each independently an L-amino acid or a D-amino acid, or each is a D-amino acid.

[0131] In another embodiment of the p53 peptidomimetic macrocycle, X 12 is D-Ser. In another embodiment of the p53 peptidomimetic macrocycle, X 3 is D-6-fluoro-Trp; X 7 is Dp-CF3-Phe; or X 3 is D-6-fluoro-Trp, and X 7 is Dp-CF3-Phe. In another embodiment, the acyl group is an acetyl group.

[0132] The present invention relates to TIFF2024546110000012.tif15159

[0133] [where X 3are D-Trp, D-6-fluoro-Trp, D-6-chloro-Trp, D-6-bromo-Trp, D-6-iodo-Trp, D-6-methyl-Trp, D-6-cyano-Trp, D-6-hydroxy-Trp, D-6-NO2-Trp, D-7-fluoro-Trp, D-7-chloro-Trp, D-7-bromo-Trp, D-7-iodo-Trp, D-7-methyl- Trp, D-7-cyano-Trp, D-7-hydroxy-Trp, D-7-NO2-Trp, D-6,7-fluoro-Trp, D-6,7-chloro-Trp, D-6,7-bromo-Trp, D-6,7-iodo-Trp, D-6,7-methyl-Trp, D-6,7-cyano-Trp, D-6,7-hydroxy-Trp or D-6,7-NO2-Trp; X 5 is (S)-2-amino-2-methylhept-6-ynoic acid; X 7 is Dp-fluoro-Phe, Dp-chloro-Phe, Dp-bromo-Phe, Dp-iodo-Phe, Dp-methyl-Phe, Dp-cyano-Phe, Dp-hydroxy-Phe, Dp-NO2-Phe or Dp-CF3-Phe; X 9 is D-Lys or D-Gln; X 11 is D-Leu;X 12 is (R)-2-amino-2-methyloct-7-ynoic acid; 13 is D-Ala, D-Glu, D-Gla (γ-carboxyl glutamic acid), or absent; X 14 is D-Ala or absent;X 15 is D-Ala, D-Phe, or D-Glu, or is absent; X 16 is D-Ala or absent;X 17 is D-Ala, D-α-methyl-Glu, or absent; X 18 is D-Ala or absent;X 19 is D-Ala or absent;X 20 is D-Ala or absent;X 21 is D-Ala or absent; the N-terminal amino group of Thr at position 1 is linked to an acyl group having the formula RCO-; R is an H 2n+1 n is an integer from 1 to 10; the C-terminal amino acid may optionally contain an amino group; and the staple is X 5 and X 12 is a dialkyne obtained by alkyne cross-coupling between The present invention provides a p53 peptidomimetic macrocycle comprising:

[0134] In certain embodiments, amino acids 1-12 are all D-amino acids, and amino acids 13-21 are each independently an L-amino acid or a D-amino acid, or each is a D-amino acid.

[0135] In another embodiment of the p53 peptidomimetic macrocycle, X 9 is D-Gln. In another embodiment of the p53 peptidomimetic macrocycle, X 3 is D-6-fluoro-Trp; X 7 is Dp-CF3-Phe; or X 3 is D-6-fluoro-Trp, and X 7 is Dp-CF3-Phe. In another embodiment, the acyl group is an acetyl group.

[0136] The present invention relates to TIFF2024546110000013.tif16160

[0137] [where X 3are D-Trp, D-6-fluoro-Trp, D-6-chloro-Trp, D-6-bromo-Trp, D-6-iodo-Trp, D-6-methyl-Trp, D-6-cyano-Trp, D-6-hydroxy-Trp, D-6-NO2-Trp, D-7-fluoro-Trp, D-7-chloro-Trp, D-7-bromo-Trp, D-7-iodo-Trp, D-7-methyl- Trp, D-7-cyano-Trp, D-7-hydroxy-Trp, D-7-NO2-Trp, D-6,7-fluoro-Trp, D-6,7-chloro-Trp, D-6,7-bromo-Trp, D-6,7-iodo-Trp, D-6,7-methyl-Trp, D-6,7-cyano-Trp, D-6,7-hydroxy-Trp or D-6,7-NO2-Trp; X 5 is (S)-2-amino-2-methyldec-9-enoic acid; 7 is Dp-fluoro-Phe, Dp-chloro-Phe, Dp-bromo-Phe, Dp-iodo-Phe, Dp-methyl-Phe, Dp-cyano-Phe, Dp-hydroxy-Phe, Dp-NO2-Phe or Dp-CF3-Phe; X 9 is D-Lys or D-Gln; X 11 is D-Leu;X 12 is (R)-2-amino-2-methylhept-6-enoic acid; X 13 are D-Ala, D-Glu, and D-Gla (γ-carboxyl glutamic acid); 14 is D-Ala;X 15 is D-Ala, D-Phe, or D-Glu; X 16 is D-Ala;X 17 is D-Ala or D-α-methyl-Glu; X 18 is D-Ala;X 19 is D-Ala or absent;X 20 is D-Ala or absent;X 21 is D-Ala or absent; the N-terminal amino group of Thr at position 1 is linked to an acyl group having the formula RCO-; R is a n H 2n+1n is an integer from 1 to 10; the C-terminal amino acid may optionally contain an amino group; and the staple is X 5 and X 12 is an olefin obtained by ring-closing metathesis between The present invention provides a p53 peptidomimetic macrocycle comprising:

[0138] In certain embodiments, amino acids 1-12 are all D-amino acids, and amino acids 13-21 are each independently an L-amino acid or a D-amino acid, or each is a D-amino acid.

[0139] In another embodiment of the p53 peptidomimetic macrocycle, X 9 is D-Gln. In another embodiment of the p53 peptidomimetic macrocycle, X 3 is D-6-fluoro-Trp; X 7 is Dp-CF3-Phe; or X 3 is D-6-fluoro-Trp, and X 7 is Dp-CF3-Phe. In another embodiment, the acyl group is an acetyl group.

[0140] The present invention relates to TIFF2024546110000014.tif9155

[0141] [where X 3are D-Trp, D-6-fluoro-Trp, D-6-chloro-Trp, D-6-bromo-Trp, D-6-iodo-Trp, D-6-methyl-Trp, D-6-cyano-Trp, D-6-hydroxy-Trp, D-6-NO2-Trp, D-7-fluoro-Trp, D-7-chloro-Trp, D-7-bromo-Trp, D-7-iodo-Trp, D-7-methyl- Trp, D-7-cyano-Trp, D-7-hydroxy-Trp, D-7-NO2-Trp, D-6,7-fluoro-Trp, D-6,7-chloro-Trp, D-6,7-bromo-Trp, D-6,7-iodo-Trp, D-6,7-methyl-Trp, D-6,7-cyano-Trp, D-6,7-hydroxy-Trp or D-6,7-NO2-Trp; X 5 is (S)-2-amino-2-methylhept-6-ynoic acid; X 7 is Dp-fluoro-Phe, Dp-chloro-Phe, Dp-bromo-Phe, Dp-iodo-Phe, Dp-methyl-Phe, Dp-cyano-Phe, Dp-hydroxy-Phe, Dp-NO2-Phe or Dp-CF3-Phe; X 9 is D-Lys or D-Gln; X 11 is D-Leu;X 12 is (R)-2-amino-2-methyloct-7-ynoic acid; the N-terminal amino group of Thr at position 1 is linked to an acyl group having the formula RCO-; R is a group of the formula C n H 2n+1 n is an integer from 1 to 10; the C-terminal amino acid may optionally contain an amino group; amino acids 1 to 12 are D-amino acids; and the staple is X 5 and X 12 is a dialkyne obtained by alkyne cross-coupling between The present invention provides a p53 peptidomimetic macrocycle comprising:

[0142] In another embodiment of the p53 peptidomimetic macrocycle, X 9 is D-Gln. In another embodiment of the p53 peptidomimetic macrocycle, X 3is D-6-fluoro-Trp; X 7 is Dp-CF3-Phe; or X 3 is D-6-fluoro-Trp, and X 7 is Dp-CF3-Phe. In another embodiment, the acyl group is an acetyl group.

[0143] In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO: 10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:9.

[0144] The present invention further comprises a structure [ka]

[0145] The present invention provides a p53 peptidomimetic macrocycle having the formula:

[0146] The present invention further comprises a structure [ka]

[0147] The present invention provides a p53 peptidomimetic macrocycle having the formula:

[0148] The present invention further comprises a structure [ka]

[0149] The present invention provides a p53 peptidomimetic macrocycle having the formula:

[0150] The present invention further comprises a structure [ka]

[0151] The present invention provides a p53 peptidomimetic macrocycle having the formula:

[0152] The present invention further comprises a structure [ka]

[0153] The present invention provides a p53 peptidomimetic macrocycle having the formula:

[0154] The present invention further comprises a structure [ka]

[0155] The present invention provides a p53 peptidomimetic macrocycle having the formula:

[0156] The present invention further comprises a structure [ka]

[0157] The present invention provides a p53 peptidomimetic macrocycle having the formula:

[0158] The present invention further comprises a structure [ka]

[0159] The present invention provides a p53 peptidomimetic macrocycle having the formula:

[0160] The present invention further comprises a structure [ka]

[0161] The present invention provides a p53 peptidomimetic macrocycle having the formula:

[0162] The present invention further provides a composition comprising a p53 peptidomimetic macrocycle disclosed herein and a pharma- ceutically acceptable carrier. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9.

[0163] The present invention further provides a method of treating cancer in a subject in need thereof comprising administering to the subject a p53 peptidomimetic macrocycle as disclosed herein or a composition comprising the p53 peptidomimetic macrocycle. The present invention further provides the use of a peptidomimetic macrocycle as disclosed herein for the manufacture of a medicament for the treatment of cancer. The present invention further provides a peptidomimetic macrocycle as disclosed herein for the treatment of cancer. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:9.

[0164] In another embodiment, the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin's lymphoma, renal cancer, Hodgkin's lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary gland cancer, pancreatic cancer, bronchial cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral cavity or pharynx cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, and cancer of the blood tissue.

[0165] The present invention further provides a method of modulating the activity of p53 and / or MDM2 and / or MDMX in a subject comprising administering to the subject a p53 peptidomimetic macrocycle as disclosed herein or a composition comprising said p53 peptidomimetic macrocycle. The present invention further provides the use of a p53 peptidomimetic macrocycle as disclosed herein for the manufacture of a medicament for modulating said activity. The present invention further provides a p53 peptidomimetic macrocycle as disclosed herein for modulating said activity. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:9.

[0166] The present invention further provides a method of antagonizing the interaction between p53 and MDM2 and / or between p53 and MDMX in a subject comprising administering to the subject a p53 peptidomimetic macrocycle as disclosed herein or a composition comprising said p53 peptidomimetic macrocycle. The present invention further provides the use of a p53 peptidomimetic macrocycle as disclosed herein for the manufacture of a medicament for antagonizing the interaction between p53 and MDM2 and / or between p53 and MDMX. The present invention further provides a p53 peptidomimetic macrocycle as disclosed herein for antagonizing the interaction between p53 and MDM2 and / or between p53 and MDMX. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:9.

[0167] The present invention further provides a combination therapy for treating cancer comprising administering to a subject a therapeutically effective amount of a p53 peptidomimetic macrocycle or a composition comprising the p53 peptidomimetic macrocycle disclosed herein and a therapeutically effective amount of a chemotherapeutic agent or radiation. In another embodiment, the chemotherapeutic agent or radiation is administered to the subject and then the p53 peptidomimetic macrocycle; the p53 peptidomimetic macrocycle is administered to the subject and then the chemotherapeutic agent or radiation; or the chemotherapeutic agent or radiation is administered to the subject simultaneously with administration of the p53 peptidomimetic macrocycle. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9.

[0168] The present invention further provides a combination therapy for treating cancer comprising a therapeutically effective amount of a p53 peptidomimetic macrocycle disclosed herein or a composition comprising said p53 peptidomimetic macrocycle and a therapeutic dose of a chemotherapeutic agent or radiation. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:9.

[0169] In certain embodiments, the chemotherapeutic agent is actinomycin, all-trans-retinoic acid, alitretinoin, azacitidine, azathioprine, bexarotene, bleomycin, bortezomib, carmofur, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, Selected from the group consisting of idarubicin, imatinib, ixabepilone, irinotecan, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, nitrosourea, oxaliplatin, paclitaxel, pemetrexed, romidepsin, tegafur, temozolomide (oral dacarbazine), teniposide, thioguanine, topotecan, utiderone, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, and vorinostat.

[0170] The present invention further provides a combination therapy for treating cancer comprising administering to a subject in need of such treatment a therapeutically effective amount of a p53 peptidomimetic macrocycle or a composition comprising the p53 peptidomimetic macrocycle disclosed herein and a therapeutically effective amount of a checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor is an anti-PD1 antibody or an anti-PD-L1 antibody. In another embodiment, the combination therapy further comprises administering to the subject a therapeutically effective amount of a chemotherapeutic agent or radiation. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9.

[0171] The present invention further provides a method for treating cancer comprising administering to a subject having cancer a vector comprising a nucleic acid molecule encoding wild-type p53 or a p53 mutant or analog having transcriptional activation activity, followed by one or more administrations of a therapeutically effective amount of a p53 peptidomimetic macrocycle or a composition comprising said p53 peptidomimetic macrocycle as disclosed herein. In certain embodiments, the vector is a plasmid, a retrovirus, an adenovirus, or an adeno-associated virus. In certain embodiments, the subject is administered chemotherapy or radiation therapy prior to or after administering the vector to the subject. In a further embodiment, the therapy further comprises administering a checkpoint inhibitor to the subject. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10. In another embodiment, the p53 peptidomimetic macrocycle comprises the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9.

[0172] Pharmaceutical Compositions The present invention also provides pharmaceutical compositions comprising the p53 peptidomimetic macrocycles of the invention. The p53 peptidomimetic macrocycles may be used in combination with any suitable pharmaceutical carrier or excipient. Such pharmaceutical compositions comprise a therapeutically effective amount of one or more p53 peptidomimetic macrocycles and a pharma- ceutically acceptable excipient and / or carrier. The particular formulation will suit the mode of administration. In certain embodiments, the pharma- ceutically acceptable carrier may be water or a buffered solution.

[0173] The excipients contained in pharmaceutical compositions have various purposes, for example, depending on the nature of the drug and the mode of administration. Examples of commonly used excipients include, but are not limited to, saline, buffered saline, dextrose, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonicity agents, bulking agents, lubricants (e.g., talc or silica, and fats such as vegetable stearin, magnesium stearate or stearic acid), emulsifiers, suspending agents or thickeners, inert diluents, fillers (e.g., cellulose, dibasic calcium phosphate, vegetable fats and oils, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate and magnesium stearate), disintegrants (e.g., cross-linked polyvinylpyrrolidone, sodium starch glycolate, cross-linked sodium carboxymethylcellulose), binders (e.g., starch, gelatin, cellulose, methylcellulose, or modified celluloses such as microcrystalline cellulose, hydroxypropylcellulose, sugars such as sucrose and lactose, or sugar alcohols such as xylitol, sorbitol or maltitol, polyvinylpyrrolidone and polyethylene glycols), humectants, antimicrobial agents, chelating agents, coatings (e.g., cellulose film coatings, synthetic polymers, shellac, corn protein zein or other polysaccharides, and gelatin), preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate and selenium, cysteine, methionine, citric acid and sodium citrate, and synthetic preservatives such as methylparaben and propylparaben), sweeteners, fragrances, flavorings, coloring agents, administration aids, and combinations thereof.

[0174] Carriers are compounds and substances that, in the case of pharmaceutical compositions, improve and / or prolong the delivery of an active ingredient to a subject. Carriers may act to prolong the in vivo activity of a drug or to delay the release of a drug in a subject using controlled release technology. Carriers may also reduce drug metabolism and / or reduce drug toxicity in a subject. Carriers may also be used to target the delivery of a drug to specific cells or tissues in a subject. Common carriers (both hydrophilic and hydrophobic) include fat emulsions, lipids, PEGylated phospholipids, PEGylated liposomes, cyclic RGD peptide c (RGD DYK Examples of suitable carriers include PEGylated liposomes coated with PEG via a PEG spacer, liposomes and lipospheres, microspheres (including those composed of biodegradable polymers or albumin), polymer matrices, biocompatible polymers, protein-DNA complexes, protein conjugates, red blood cells, vesicles, nanoparticles, and side chains for carbohydrate stapling. The carriers may also be used to enhance cell membrane permeability of the p53 peptidomimetic macrocycles of the invention. In addition to their use in the pharmaceutical compositions of the invention, carriers may also be used in compositions for other applications, such as in vitro (e.g., delivery to cultured cells) and / or in vivo research applications.

[0175] Pharmaceutical compositions suitable for oral administration may be provided as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids); or as edible foams or whips; or as emulsions. Suitable excipients for tablets or hard gelatin capsules include lactose, corn starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients used in soft gelatin capsules include, for example, vegetable oils, waxes, fats, semi-solids, or liquid polyols. For the preparation of solutions and syrups, excipients that may be used include, for example, water, polyols, and sugars. For the preparation of suspensions, oils, such as vegetable oils, may be used to obtain oil-in-water or water-in-oil suspensions. In certain circumstances, delayed release formulations may be advantageous. Compositions may also be prepared that can deliver p53 peptidomimetic macrocycles in a delayed or controlled release manner. Prolonged retention in the stomach poses the problem of degradation by enzymes present in the stomach; therefore, enteric coated capsules, in which the active substance is released lower in the gastrointestinal tract, may also be prepared by standard techniques in the art.

[0176] Pharmaceutical compositions suitable for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis, as generally described in Pharmaceutical Research, 3(6):318 (1986).

[0177] Pharmaceutical compositions suitable for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. When formulated in ointments, the active ingredient can be used with a paraffinic or water-miscible ointment base. Alternatively, the active ingredient can be formulated in a cream using an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions suitable for topical administration to the eye include eye drops, in which the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent. Pharmaceutical compositions suitable for topical administration in the mouth include lozenges, troches and mouthwashes.

[0178] Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas.

[0179] Pharmaceutical compositions suitable for intranasal administration where the carrier is a solid include coarse powders having a particle size of, for example, 20 to 500 microns, which are administered by nasal inhalation, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose. Suitable compositions where the carrier is a liquid, for administration as a nasal spray or nasal drops, include aqueous or oily solutions of the active ingredient.

[0180] Pharmaceutical compositions suitable for administration by inhalation include fine particle dusts or mists, which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators.

[0181] Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0182] Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation substantially isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents. Excipients that may be used for injectable solutions include, for example, water for injection, alcohols, polyols, glycerin, and vegetable oils. The compositions may be provided in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid carrier, such as water for injection or physiological saline, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Pharmaceutical compositions may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, salts (the substances of the present invention themselves may be provided in the form of pharma-ceutically acceptable salts), buffers, coating agents, or antioxidants. They may also contain therapeutically active substances in addition to the substances according to the invention.

[0183] The pharmaceutical composition may be administered in any convenient manner, such as by topical, intravenous, intraperitoneal, intramuscular, intratumoral, subcutaneous, intranasal or intradermal routes. The pharmaceutical composition is administered in an amount effective for the treatment and / or prevention of a particular indication. In general, the pharmaceutical composition is administered in an amount of at least about 0.1 mg / kg body weight to about 100 mg / kg body weight. In most cases, the dosage is about 10 mg / kg body weight to about 1 mg / kg body weight per day, taking into account the route of administration, symptoms, etc.

[0184] The dosage of the p53 peptidomimetic macrocycles of the invention can vary over a wide range, depending on the site, origin, type, spread and severity of the cancer, the age and condition of the individual being treated, etc. The physician will ultimately determine the appropriate dosage to be used.

[0185] The p53 peptidomimetic macrocycles may also be used in accordance with the present invention to express the antagonist in vivo, i.e., by gene therapy. Use of the peptide or composition in a gene therapy context is also considered, for the purposes of the present invention, to be a form of "administration" of the peptide.

[0186] Thus, the present invention also relates to a method of treating a subject with cancer comprising administering to a subject in need of treatment a pharma- ceutical effective amount of one or more of the p53 peptidomimetic macrocycles of the invention, or a pharmaceutical composition comprising one or more of said antagonists. The term "cancer" is intended to be interpreted broadly, as it includes all aspects of abnormal cell growth and / or cell division. Examples include, but are not limited to, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, and cancers of the skin, breast, prostate, bladder, vagina, cervix, uterus, liver, kidney, pancreas, spleen, lung, trachea, bronchus, colon, small intestine, stomach, esophagus, and gallbladder; sarcomas, such as (but are not limited to), chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcoma, and cancers of bone, cartilage, fat, muscle, blood vessels, and hematopoietic tissue; lymphomas and leukemias, such as (but are not limited to), mature B-cell neoplasms, such as tumors, such as chronic lymphocytic leukemia / small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphomas and plasma cell neoplasms, mature T-cell and natural killer (NK) cell neoplasms, such as T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK cell leukemia and adult T-cell leukemia / lymphoma, Hodgkin's lymphoma, and immunodeficiency-associated lymphoproliferative disorders; germ cell tumors, such as, but not limited to, testicular cancer and ovarian cancer; blastomas, such as, but not limited to, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma. The term also includes benign tumors.

[0187] In each of the embodiments of the invention, the individual or subject to be treated is a human or a non-human animal, such as a non-human primate, bird, horse, cow, goat, sheep, companion animal, such as a dog, cat or rodent, or other mammal. In some embodiments, the subject is a human.

[0188] The invention also provides kits comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention, e.g., a pharmaceutical composition comprising a p53 peptidomimetic macrocycle of the invention and a pharma- ceutically acceptable carrier or diluent. Such containers may bear a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products. The notice indicates approval by that agency of the manufacture, use, or sale for administration to humans. The pharmaceutical compositions may also be used in combination with other therapeutic compounds.

[0189] Combination therapy including chemotherapy The p53 peptidomimetic macrocycles of the invention may be administered to an individual with cancer in combination with chemotherapy. The individual may receive chemotherapy at the same time that the p53 peptidomimetic macrocycle is administered to the individual. The individual may receive chemotherapy after the individual has completed a course of treatment with the p53 peptidomimetic macrocycle. The individual may receive the p53 peptidomimetic macrocycle after the individual has completed a course of treatment with a chemotherapeutic agent. The combination therapy of the invention may be administered to an individual with recurrent or metastatic cancer with disease progression or recurrence who is receiving or has completed chemotherapy.

[0190] The chemotherapy includes a chemotherapeutic agent selected from the following group: (i) Alkylating agents, including but not limited to, bifunctional alkylating agents, cyclophosphamide, mechlorethamine, chlorambucil, and melphalan; (ii) monofunctional alkylating agents, such as (but not limited to) dacarbazine, nitrosoureas and temozolomide (oral dacarbazine); (iii) Anthracyclines, including but not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone and valrubicin; (iv) cytoskeletal disrupting agents (taxanes), including but not limited to, paclitaxel, docetaxel, abraxane, and taxotere; (v) Epothilones, including but not limited to, ixabepilone and utiderone; (vi) histone deacetylase inhibitors, such as (but not limited to), vorinostat and romidepsin; (vii) inhibitors of topoisomerase I, including but not limited to, irinotecan and topotecan; (viii) topoisomerase II inhibitors, such as (but not limited to) etoposide, teniposide, and tafluposide; (ix) Kinase inhibitors, such as (but not limited to) bortezomib, erlotinib, gefitinib, imatinib, vemurafenib and vismodegib; (x) Nucleotide analogues and precursor analogues, including but not limited to, azacitidine, azathioprine, fluoropyrimidines (e.g., capecitabine, carmofur, doxifluridine, fluorouracil, and tegafur), cytarabine, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine (formerly thioguanine); (xi) peptide antibiotics, such as, but not limited to, bleomycin and actinomycin; platinum-based agents, such as, but not limited to, carboplatin, cisplatin, and oxaliplatin; (xii) retinoids, including but not limited to, tretinoin, alitretinoin, and bexarotene; and (xiii) Vinca alkaloids and derivatives, including but not limited to, vinblastine, vincristine, vindesine, and vinorelbine.

[0191] The choice of the dose of the chemotherapeutic agent for chemotherapy depends on several factors, including the serum or tissue turnover rate of the substance, the severity of symptoms, the immunogenicity of the substance, and the accessibility of the target cell, tissue or organ in the individual being treated.The dose of the additional therapeutic agent should be an amount that produces an acceptable level of side effects.Therefore, the dose and frequency of administration of each additional therapeutic agent depends in part on the individual therapeutic agent, the severity of the cancer being treated, and the characteristics of the patient.Guidelines are available for choosing the appropriate dose of antibodies, cytokines, and small molecules. See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 344:783-792. Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed); Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002).The determination of an appropriate dosing regimen can be made by the clinician using, for example, parameters or factors known or suspected in the art to affect treatment or expected to affect treatment, and will depend, for example, on the individual's clinical history (e.g., previous treatments), the type and stage of the cancer being treated, and biomarkers of response to one or more of the therapeutic agents in the combination therapy.

[0192] The present invention contemplates embodiments of combination therapy that include chemotherapy steps that include platinum-containing chemotherapy, pemetrexed and platinum chemotherapy, or carboplatin and paclitaxel and nab-paclitaxel. In certain embodiments, combination therapy with chemotherapy steps can be used to treat at least NSCLC and HNSCC.

[0193] The combination therapy can be used to treat any proliferative disease, particularly cancer. In certain embodiments, the combination therapy of the present invention can be used to treat melanoma, non-small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin's lymphoma, renal cancer, Hodgkin's lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, or salivary gland cancer.

[0194] In another embodiment, the combination therapy may be used to treat pancreatic cancer, bronchial cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

[0195] In certain embodiments, the combination therapy may be used to treat one or more cancers selected from melanoma (metastatic or unresectable), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, MSIHC, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (including advanced cancer), and cutaneous squamous cell carcinoma.

[0196] Additional combination therapy The p53 peptidomimetic macrocycles disclosed herein may be used in combination with other therapies. For example, combination therapy may include compositions comprising p53 peptidomimetic macrocycles co-formulated and / or co-administered with one or more additional therapeutic agents, such as hormonal therapy, vaccines, and / or other immunotherapies. In other embodiments, the p53 peptidomimetic macrocycles are administered in combination with other therapeutic modalities, including surgery, radiation therapy, cryosurgery, and / or hyperthermia. Such combination therapy may advantageously utilize lower dosages of the administered therapeutic agents, avoiding possible toxicities or complications associated with various monotherapies.

[0197] "In combination with" does not imply that the therapies or therapeutic agents must be administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope described herein. The p53 peptidomimetic macrocycle may be administered simultaneously with, prior to, or after one or more other additional therapies or therapeutic agents. The p53 peptidomimetic macrocycle and other agents or treatment protocols may be administered in any order. Generally, each agent will be administered at a dose and / or time schedule determined for that agent. It will further be understood that additional therapeutic agents used in the combination may be administered together in a single composition or may be administered separately in different compositions. In general, it is expected that additional therapeutic agents used in combination will be used at levels that do not exceed the levels at which they are used individually. In some embodiments, the levels used in combination will be lower than the levels at which they are used individually.

[0198] In certain embodiments, the p53 peptidomimetic macrocycles described herein are administered in combination with one or more checkpoint inhibitors or antagonists of the programmed death receptor 1 (PD-1) or its ligands PD-L1 and PD-L2. The inhibitors or antagonists can be antibodies, antigen-binding fragments, immunoadhesins, fusion proteins, or oligopeptides. In some embodiments, the anti-PD-1 antibody is selected from nivolumab (OPDIVO, Bristol Myers Squibb, New York, New York), pembrolizumab (KEYTRUDA, Merck Sharp & Dohme Corp, Kenilworth, NJ USA), setiplimab (Regeneron, Tarrytown, NY), or pidilizumab (CT-011). In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin that includes an extracellular or PD-1-binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 inhibitor is AMP-224. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody, such as durvalumab (IMFINZI, Astrazeneca, Wilmingon, DE), atezolizumab (TECENTRIQ, Roche, Zurich, CH) or avelumab (BAVENCIO, EMD Serono, Billerica, MA). In some embodiments, the anti-PD-L1 binding antagonist is selected from YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

[0199] The following examples are intended to facilitate a further understanding of the invention.

[0200] Common methods Peptide synthesis All peptides were obtained from CPC Scientific. Peptide purity and identity were confirmed by analytical HPLC and mass spectrometry. All final peptides were 90% or higher purity. All peptides were dissolved in neat dimethyl sulfoxide (DMSO) as 10 mM stock solutions and diluted for subsequent experiments.

[0201] Peptides were synthesized using Rink Amide 4-methylbenzhydrylamine (MBHA) resin and Fmoc-protected amino acids, where couplings were performed sequentially using N,N'-diisopropylcarbodiimide / hydroxybenzotrizole (DIC / HOBt) activating agents. Double coupling reactions were performed at the first amino acid and also at the stapling positions. At these latter positions, the activating reagent was switched to N,N-diisopropylethylamine / hexafluorophosphate azabenzotriazole tetramethyluronium (DIEA / HATU) to improve coupling efficiency. Ring-closing metathesis reactions were performed by first washing the resin three times with DCM (dichloromethane) and then adding first generation Grubbs catalyst (20 mol% in DCM) and reacting for 2 hours. All steps using Grubbs catalyst were performed in the dark. RCM (ring-closing metathesis) reactions were repeated to ensure complete reaction. After completion of RCM, test cleavage was performed to ensure sufficient yield. The peptide was cleaved and then purified as a mixture of cis-trans isomers by reversed-phase high performance liquid chromatography (RP-HPLC). The Glaser alkyne cross-coupling reaction was carried out on the resin as follows: the resin was first washed three times with DCM, then tetrahydrofuran (THF), N,N-diisopropylethylamine (DIPEA), Pd(PPh3)2Cl2 were added, followed by CuI, sonication, and then heating at 30°C for 16 hours. The mixture was filtered and washed with dimethylformamide (DMF).

[0202] Production of MDM2 protein The human MDM2 1-125 sequence was cloned into the pNIC-GST vector for use in peptide binding assays. The TEV (Tobacco Etch Virus) cleavage site was changed from ENLYFQS (SEQ ID NO: 13) to ENLYFQG (SEQ ID NO: 14) resulting in a fusion protein with the following sequence:

[0203] TIFF2024546110000024.tif92161

[0204] The corresponding plasmids were transformed into BL21(DE3) Rosetta T1R Escherichia coli cells and grown under kanamycin selection. A 750 mL bottle of Terrific Broth supplemented with the appropriate antibiotic and 100 μL of antifoam 204 (Sigma-Aldrich, St. Louis, MO, USA) was inoculated with a 20 mL seed culture grown overnight. The culture was incubated at 37 °C in a LEX system (Harbinger Biotech, Toronto, Canada) with aeration and agitation by blowing filtered air through the culture. When the culture reached an OD600 of 2, the temperature of the LEX system was reduced to 18 °C and the culture was induced with 0.5 mM IPTG 600 minutes later. Protein expression was allowed to continue overnight. Cells were harvested by centrifugation at 4000 × g for 10 min at 15 °C. The supernatant fraction was discarded and the cell pellet was resuspended in cell lysis buffer (1.5 mL per gram of cell pellet). The cell suspension was stored at -80°C before purification was performed.

[0205] The resuspended cell pellet suspension was thawed and sonicated on ice for 3 min at 70% amplitude, 3 s on / off (Sonics Vibra-Cell, Newtown, CO, USA). The lysate was clarified by centrifugation at 47,000×g for 25 min at 4° C. The supernatant fraction was filtered through a 1.2 μm syringe filter and loaded onto an AKTA Xpress system (GE Healthcare, Fairfield, CO, USA). The purification method is briefly described below.

[0206] The lysate was loaded onto a 1 mL Ni-NTA Superflow column (Qiagen, Valencia, CA, USA) equilibrated with 10 column volumes of Wash 1 buffer. Overall buffer conditions were as follows: Immobilized metal affinity chromatography (IMAC) wash 1 buffer - 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 500 mM NaCl, 10 mM imidazole, 10% (v / v) glycerol, 0.5 mM TCEP (tris(2-carboxyethyl)phosphine), pH 7.5; IMAC wash 2 buffer - 20 mM HEPES, 500 mM NaCl, 25 mM imidazole, 10% (v / v) glycerol, 0.5 mM TCEP, pH 7.5; IMAC elution buffer - 20 mM HEPES, 500 mM NaCl, 500 mM imidazole, 10% (v / v) glycerol, 0.5 mM TCEP, pH 7.5. Samples were loaded at 0.8 mL / min until air was detected by the air sensor. The column was then washed with 20 column volumes (CV) of Wash 1 buffer, followed by 20 CV of Wash 2 buffer. Protein was eluted with 5 CV of Elution Buffer. Eluted protein was collected in a sample loop on the system, stored, and then injected into a gel filtration (GF) column. Elution peaks were collected in 2 mL fractions and analyzed on SDS-PAGE gels. The entire purification was carried out at 4°C. Relevant peaks were pooled and TCEP was added to a total concentration of 2 mM. Protein samples were concentrated to approximately 15 mg / mL in Vivaspin 20® filter concentrators (VivaScience, Littleton, MA, USA) at 15°C (<18 kDa - 5K MWCO, 19-49 kDa - 10K MWCO, >50 kDa - 30K MWCO). The final protein concentration was assessed by measuring absorbance at 280 nm with a Nanodrop ND-1000® (Thermo Fisher, Waltham, MA, USA). The final protein purity was assessed on an SDS-PAGE gel. The final protein batch was then aliquoted into smaller fractions, frozen in liquid nitrogen, and stored at −80° C.

[0207] For X-ray crystallography, MDM2(6-125) was cloned as a GST fusion protein using the pGEX-6P-1 GST expression vector (GE Healthcare). The GST-fused MDM2(6-125) construct was then transformed into E. coli BL21(DE3)pLysS (Thermo Fisher, Waltham, MA, USA) competent cells. Cells were grown in Luria-Bertani (LB) medium at 37°C and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16°C to an OD600nm of 0.6. After overnight induction, cells were harvested by centrifugation, resuspended in binding buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl), and lysed by sonication. After centrifugation at 19,000×g for 60 min at 4° C., the cell lysate was applied to a 5 mL GSTrap® FF column (GE Healthcare) pre-equilibrated with wash buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM DTT). GST-fused MDM2 (amino acids 6-125) was then cleaved on-column by PreScission® protease (GE Healthcare) overnight at 4° C. and eluted from the column with wash buffer. The protein sample was then dialyzed into buffer A solution (20 mM Bis-Tris (pH 6.5), 1 mM DTT) using a HiPrep® 26 / 10 desalting column and loaded onto a cation exchange Resource S 1 mL column (GE Healthcare) pre-equilibrated in buffer A. The column was then washed with 6 CV of buffer A and bound protein was eluted with a linear gradient in a buffer containing 1 M NaCl, 20 mM BisTris (pH 6.5) and 1 mM DTT over 30 column volumes. Protein purity was approximately 95% as assessed by SDS-PAGE. Protein was concentrated using Amicon-Ultra (3 kDa MWCO) concentrators (Millipore, Burlington, MA, USA). Protein concentration was determined using absorbance measurements at 280 nm.

[0208] Production of MDM4 protein MDM4 protein was cloned into pNIC-GST vector and expressed in the LEX system (Harbinger Biotech) at the Protein Production Platform (PPP) of NTU School of Biological Sciences. The glycerol stock was used to start an inoculum culture in 20 mL of Terrific Broth containing 8 g / L glycerol supplemented with kanamycin. The culture was incubated overnight at 37 °C and 200 rpm. The following morning, the culture was inoculated into a bottle of 750 mL of Terrific Broth containing 8 g / L glycerol supplemented with kanamycin and 100 μL of antifoam 204 (Sigma-Aldrich). The culture was incubated at 37 °C in the LEX system with aeration and agitation by blowing filtered air through the culture. Once the OD600 reached 2, the temperature was reduced to 18 °C and the culture was induced with 0.5 mM IPTG after 30–60 min. Protein expression was continued overnight. The next morning, cells were harvested by centrifugation at 4200×g for 10 min at 15° C. The supernatant fraction was discarded and cells were resuspended in cell lysis buffer [100 mM HEPES, 500 mM NaCl, 10 mM imidazole, 10% glycerol, 0.5 mM TCEP, pH 8.0, containing 250 U / μL Merck Calbiochem™ Protease Inhibitor Cocktail Set III without EDTA (diluted 1:1000 in cell lysis buffer) and benzonase (4 μL per 750 mL culture, obtained from Calbiochem)] at 200 rpm for approximately 30 min at 4° C. and stored at −80° C. The resuspended cell pellet suspension was thawed and sonicated (Sonics Vibra-Cell) on ice for 3 min at 70% amplitude, 3 sec on / off. Lysates were clarified by centrifugation at 47,000×g for 25 min at 4° C. The supernatant was filtered through a 1.2 μm syringe filter and loaded onto an AKTA Xpress system (GE Healthcare) with a 1 mL Ni-NTA Superflow (Qiagen) IMAC column.The column was washed with 20 column volumes (CV) of wash buffer 1 (20 mM HEPES, 500 mM NaCl, 10 mM imidazole, 10% (v / v) glycerol, 0.5 mM TCEP, pH 7.5) and 20 CV of wash buffer 2 (20 mM HEPES, 500 mM NaCl, 25 mM imidazole, 10% (v / v) glycerol, 0.5 mM TCEP, pH 7.5) or until a stable baseline for 3 min and a delta base of 5 mAU (0.8 mL / min) were obtained, respectively. MDM4 protein was eluted with elution buffer (20 mM HEPES, 500 mM NaCl, 500 mM imidazole, 10% (v / v) glycerol, 0.5 mM TCEP, pH 7.5) and the elution peak (collection start: >50 mAU, gradient >200 mAU / min, collection stop: <50 mAU, stable plateau at 0.5 min, delta plateau 5 mAU) was collected and stored in a sample loop on the system and then injected into an equilibrated gel filtration (GF) column (HiLoad 16 / 60 Superdex 200 prep grade (GE Healthcare)) and eluted with 20 mM HEPES, 300 mM NaCl, 10% (v / v) glycerol, 0.5 mM TCEP, pH 7.5 at a flow rate of 1.2 mL / min. The elution peak (collection start: >20mAU, gradient >10mAU / min, collection end: <20mAU, gradient >10mAU / min, minimum peak width 0.5 min) was collected in 2mL fractions. The entire purification was performed at 4°C. Relevant peaks were pooled and TCEP was added to a final concentration of 2mM. Protein samples were concentrated to approximately 15mg / mL in Vivaspin 20 filter concentrators (VivaScience) at 15°C. Final protein concentration was assessed by measuring absorbance at 280nm with a Nanodrop™ ND-1000 (Nano-Drop Technologies). Final protein purity was assessed by SDS-PAGE and purified MDM4 protein was frozen in liquid nitrogen and stored at -80°C.

[0209] Competitive fluorescence anisotropy assay (MDM2 and MDM4) Purified MDM2(1-125) protein was titrated against 50 nM carboxyfluorescein (FAM)-labeled 12 / 1 peptide 13 [9-mer FAM-RFMDYWEGL-NH2 (SEQ ID NO: 26) as disclosed in Fischer, Intl. J. Pept. Res. Ther. 12:3-19 (2006)]. The dissociation constants of MDM2 and MDM4 titration against the FAM-labeled 12 / 1 peptide were determined by fitting the experimental data to the 1:1 binding model equation shown below.

[0210] Equation 1:

number

[0211] [P] is the protein concentration (MDM2), [L] is the labeled peptide concentration, r is the measured anisotropy, r0 is the anisotropy of the free peptide, and r b is the anisotropy of the MDM2-FAM-labeled peptide complex, and K d is the dissociation constant, [L] t is the total FAM-labeled peptide concentration, [P] t is the total MDM2 concentration. The apparent K d The values ​​were determined to be 13.0 nM and 4.0 nM, respectively. These values ​​were then used to calculate the apparent K of each competing ligand in subsequent competition assays in fluorescence anisotropy experiments. d The Kd values ​​were determined. Competition experiments for MDM2 and MDM4 were carried out in the presence of 50 nM FAM-labeled 12 / 1, with the respective concentrations kept constant at 250 nM and 75 nM. The competitor molecules were then titrated against the FAM-labeled peptide-protein complex. The apparent Kd values ​​were determined by fitting the experimental data to the equation shown below.

number

[0212] [L] st and [L]t denote the input concentrations of labeled ligand and total unlabeled ligand, respectively. K d2 is the dissociation constant of the interaction between the unlabeled ligand and the protein. In all competition experiments, [P] t > [L] st If this is not the case, a significant amount of free labeled ligand will always be present and will interfere with the measurement. d1 is the apparent K of the labeled peptide used d and was experimentally determined as described in the previous paragraph. FAM-labeled peptides were dissolved in dimethyl sulfoxide (DMSO) at 1 mM and diluted in the experiment buffer. Readings were performed using an Envision Multilabel Reader (PerkinElmer). Experiments were performed in PBS (2.7 mM KCl, 137 mM NaCl, 10 mM Na2HPO4, 2 mM KH2PO4 (pH 7.4)) and 0.1% Tween-20 buffer. All titrations were performed in triplicate. Curve fitting was performed using Prism 4.0 (GraphPad®). To validate the fitting of the 1:1 binding model, it was carefully confirmed that the starting anisotropy values ​​of the direct titration between MDM2 and FAM-labeled peptide were not significantly different from the anisotropy values ​​observed with the free fluorescently labeled peptide. A negative control titration of the ligand being tested was also performed using the fluorescently labeled peptide (in the absence of MDM2) to ensure that no interaction occurred between the ligand and the FAM-labeled peptide. Also, the final baseline in the competitive titration was not allowed to fall below the anisotropy value of the free FAM-labeled peptide. Otherwise, it would indicate unintended interactions between the FAM-labeled peptide and the ligand that should be removed from the MDM2 binding site. Measurements were performed at least three times (biological replicates). Values ​​are shown as the geometric mean of the replicates.

[0213] Stability of whole cell homogenates Peptides at a concentration of 1 μM were incubated with HCT116 whole cell homogenates prepared from 1 million lysed cells / mL at 37°C. The reactions were stopped with organic solvent at 0, 1, 2, 4 and 22 hours, followed by centrifugation. The resulting supernatants were injected into LC / MS for detection of the test peptides. The remaining percentage of each compound was normalized to the amount at time 0 and reported.

[0214] Plasma Stability Peptides were incubated with human plasma at a concentration of 1 mM for 1, 2, 3, and 4 h at 37°C. At the indicated time points, the incubation was stopped by the addition of organic solvent, followed by centrifugation. The parent compound in the supernatant was analyzed by LC / MS. The remaining percentage of peptide was calculated relative to the amount of compound at time 0.

[0215] p53 beta-lactamase reporter gene cellular functional assay HCT116 cells were stably transfected with a p53-responsive β-lactamase reporter and grown in McCoy's 5A medium containing 10% fetal bovine serum (FBS), blasticidin and penicillin / streptomycin, then transferred to 1.5 mL cryovials and stored under liquid nitrogen in growth medium containing 5% DMSO. One day before the assay, vials of banked cells were harvested in cell culture flasks and incubated for 24 hours, then the cell growth medium was removed and replaced with Opti-MEM containing 2% FBS. Cells were then seeded at a density of 8000 cells / well in 384-well plates. Peptides were then dispensed into each well using a liquid handler ECHO555 and incubated for 16 hours. The final working concentration of DMSO was 0.5%. β-lactamase activity was detected using ToxBLAzer Dual Screen (Invitrogen) according to the manufacturer's instructions. Measurements were performed using an Envision multiplate reader (PerkinElmer). Maximum p53 activity was defined as the amount of β-lactamase activity induced by 50 μM azido-ATSP-7041. It was determined from titrations in HCT116 cells as the highest amount of p53 activity induced by azido-ATSP-7041. Measurements were performed at least three times (biological replicates). Values ​​are shown as the geometric mean of the replicates.

[0216] Lactate dehydrogenase (LDH) release assay HCT116 cells were stably transfected with a p53-responsive β-lactamase reporter and grown in McCoy's 5A medium containing 10% fetal bovine serum (FBS), blasticidin and penicillin / streptomycin, then transferred to 1.5 mL cryovials and stored under liquid nitrogen in growth medium containing 5% DMSO. One day prior to the assay, a vial of banked cells was harvested in a cell culture flask, incubated for 24 hours, and then the cell growth medium was removed and replaced with Opti-MEM containing 2% FBS. The cells were then seeded at a density of 8000 cells / well in 384-well plates. Peptides were then dispensed into each well using a liquid handler ECHO555 and incubated for 16 hours. The final working concentration of DMSO was 0.5%. Lactate dehydrogenase release was detected using the CytoTox-ONE Homogenous Membrane Integrity Assay Kit (Promega) according to the manufacturer's instructions. Measurements were performed using a Tecan plate reader. Maximum LDH release was defined as the amount of LDH release induced by a cytolytic peptide (iDNA79) and was used to normalize the results. Measurements were performed at least three times (biological replicates). Values ​​are presented as the geometric mean of the replicates.

[0217] Tetracycline-beta-lactamase reporter gene cell assay (CounterScreen) The assay is based on Jump-In™ T-REx™ CHO-K1 BLA cells containing stably integrated β-lactamase under the control of an inducible cytomegalovirus (CMV) promoter. Cells were maintained in Dulbecco's Minimum Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), blasticidin and penicillin / streptomycin, then transferred to 1.5 mL cryovials and stored under liquid nitrogen in growth medium containing 5% DMSO. One day before the assay, vials of banked cells were harvested in cell culture flasks, incubated for 24 hours, then the cell growth medium was removed and replaced with Opti-MEM containing 2% FBS. Cells were seeded in 384-well plates at a density of 4000 cells per well. Peptides were then dispensed into each well using the liquid handler ECHO555 and incubated for 16 hours. The final working concentration of DMSO was 0.5%. β-lactamase activity was detected using ToxBLAzer Dual Screen (Invitrogen) according to the manufacturer's instructions. Measurements were performed using an Envision multiplate reader (PerkinElmer). Counterscreen activity was defined as the amount of β-lactamase activity induced by tetracycline. Measurements were performed at least three times (biological replicates). Values ​​are presented as the geometric mean of the replicates.

[0218] HCT-116 Western blot analysis Preparation of compound stock and working solutions: 10 mM or 1 mM stock solutions of compounds were prepared in 100% DMSO. Each compound was then serially diluted in 100% DMSO and further diluted 10-fold in HPLC grade sterile water to prepare a 10× working solution of each compound in 10% DMSO / water. Compounds were added according to the required volume used in the relevant assay to obtain the final concentrations shown in the relevant figures, with a residual DMSO concentration of 1% v / v.

[0219] HCT116 cells (Thermo Fisher Scientific) were cultured in DMEM cell medium supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin. All cell lines were maintained in a humidified incubator at 37°C with a 5% CO2 atmosphere. HCT116 cells were seeded in 96-well plates at a density of 60,000 cells per well and incubated overnight. Cells were also maintained in DMEM cell medium supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin. Cell medium was then removed and replaced with cell medium containing the indicated concentrations of various compounds / vehicle controls in DMEM cell medium containing 2% FCS. After the indicated incubation times (4 or 24 hours), cells were rinsed with PBS and then harvested in 100 μL of 1× NuPAGE LDS sample buffer (NP0008) supplied by Invitrogen. Samples were then sonicated, heated at 90°C for 5 min, sonicated twice for 10 s, and centrifuged at 13,000 rpm for 5 min. Protein concentration was measured by BCA assay (Pierce). Samples were separated on Tris-Glycine 4-20% gradient gels (BIORAD) according to the manufacturer's protocol. Western transfer was performed on Immuno-blot PVDF membranes (Bio-Rad) using the Trans-Blot Turbo system (BIORAD). Western blot staining was then performed using antibodies against actin (AC-15, Sigma) as a loading control, p21 (118 mouse monoclonal antibody), MDM2 (2A9 mouse monoclonal antibody), and p53 (DO-1 mouse monoclonal antibody).

[0220] Isothermal titration calorimetry (ITC) Overnight dialysis of proteins and peptides was performed in a buffer containing 1x phosphate-buffered saline (PBS) (pH 7.2), 3% DMSO and 0.001% Tween-20. Approximately 100-200 μM peptides were titrated into 20 μM purified recombinant human MDM2 protein (amino acids 1-125) over 40 injections of 1 μL each. For highly concentrated and insoluble peptides, reverse ITC (200 μM MDM2 protein titrated into 20 μM peptide) was performed. All experiments were performed in duplicate using a MicroCal PEAQ-ITC Automated system. Data analysis was performed using MicroCal PEAQ-ITC analysis software.

[0221] Circular dichroism (CD) A total of 5 μL of 10 mM stock peptide was mixed with 45 μL of 100% methanol and dried in a SpeedVac™ concentrator (Thermo Scientific) for 2 h. The dried peptide was reconstituted to a concentration of 1 mM in buffer (1 mM Hepes, pH 7.4 and 5% methanol). Peptide samples were placed in quartz cuvettes with a path length of 0.2 cm. Peptide concentrations were measured by the absorbance of the peptide at 280 nM. CD spectra were recorded from 300 to 190 nm at 25 °C using a Chirascan-plus qCD instrument (Applied Photophysics, Surrey, UK). All experiments were performed in duplicate. CD spectra were converted to mean residual ellipticity before deconvolution and estimation of secondary structure components of the peptides using CDNN software (distributed by Applied Photophysics). Measurements were performed at least twice and expressed as arithmetic means.

[0222] Example 1 had low cytotoxicity and both LDH and counterscreen activity D Improvement of PMI-δ(6-10) olefin-stapled p53 peptidomimetic macrocycles WO2020257153A1 describes a series of peptides that exhibit improved cellular activity compared to the parent peptide. D have described a PMI-δ olefin-stapled peptide (see also Chem Sci. 2020, 11, 5577). We focused on the DPM-δ(6-10) olefin-stapled p53 peptidomimetic macrocycle because it had intrinsic ultrastability, verified binding to MDM2 and amphiphilicity. However, it showed low cellular and cellular activity drawbacks in both LDH and counterscreen assays (compound 1, Table 1, parts 1-3). Those drawbacks could be due to the three positive charges of the peptide with a free N-terminus and two basic residues (D-lysine at position 9 and D-arginine at position 12). Using ATSP-7041 as an amphiphilic reference substance with good cell permeability and activity, we focused on the presence of Ser and Gln residues on its polar helical face. Thus, D To eliminate the positive charge of the PMI-δ(6-10) olefin-stapled p53 peptidomimetic macrocycle, D-glutamine was introduced at position 9 and D-serine at position 12, and the N-terminus was acetylated. These modifications successfully eliminated LDH release and counterscreen activity (both EC50>50μM), and improved the functional cellular activity of the p53 reporter gene three-fold to 3.6μM in 0% serum (compound 2). However, the compound showed weak cellular efficacy (>50μM) under 10% serum conditions.

[0223] Example 2 DPMI TM (6-10) Addition of a C-terminal tail to olefin-stapled p53 peptidomimetic macrocycles results in improved cell renewal and physicochemical properties Introduction of a polypeptide tail containing several D-alanine residues could further enhance the cellular activity of our DPMI-δ(6-10) olefin-stapled p53 peptidomimetic macrocycles, presumably by modulating the helical properties of the added poly-D-alanine tail. This could increase solubility and alter the dynamics of the solution-state peptide oligomers by favoring α-helical conformations by alleviating β-sheet-based peptide aggregation. Also, enhanced aqueous phase helicity could enhance permeability by decreasing the entropic penalty associated with the accommodation of high helical content typically induced in the low dielectric constant of cell membrane lipid bilayers. We generated a series of compound 2 analogs with the addition of polypeptide tails containing various numbers of D-alanine residues (compounds 3-5, Table 1, parts 1-3). Of the three tail lengths evaluated, the 6 amino acid length yielded the best profile (compound 4). Addition of a 6×(D-Ala) C-terminal tail improved cellular activity to 0.3 μM in 10% serum, a 100-fold overall improvement compared to the original parent peptide. Interestingly, in the case of compound 2, further extending the length of the polyalanine tail to 9×(D-Ala) yielded a compound 2 analog (compound 5) with lower cellular potency.

[0224] Comparison of the physicochemical properties of these analogs with different polyalanine size lengths provides a possible explanation for why compound 5 is much less soluble than the analogs with 3x(D-Ala) or 6x(D-Ala) polypeptide tails (16 μM vs. 140 μM or 150 μM, respectively) and less soluble than the parent compound 2 (16 μM vs. 167 μM) (Table 2). Similarly, compound 5 also exhibited a higher measured HPLC logD compared to compounds 2-4, which could result in poorer solubility behavior, including aggregation.

[0225] We aimed to further enhance cellular potency while avoiding off-target effects by optimizing the polypeptide tail sequence. Our multi-faceted approach included optimizing MDM2(X) binding through additional binding interactions in the tail, and optimizing solubility and dissolved-state behavior by enhancing amphipathicity to balance hydrophobic properties. Examination of a helical wheel showed that placement of a non-polar residue at position 14 enhanced MDM2 binding. Furthermore, molecular modeling suggested that phenylalanine might be a suitable residue for that position (Figure 3). Therefore, compound 6 was generated, which has a polyalanine tail in which D-Ala at position 14 was replaced with D-Phe. This peptide exhibited about 65% helicity in aqueous solution.

[0226] Compound 6 was found to retain some cellular activity compared to parent compound 2, which lacks the polypeptide tail, but was comparable to compound 4. This result could be related to the high measured HPLC logD measurements for the compound (Table 1, parts 1-3 and Table 2). To enhance the solubility of the peptide, amino acid positions that tolerate polar residues without compromising cellular activity were also examined. Examination of the helical wheel (see Chem Sci. 2020, 11, 5577 for an explanation of the helix structure) showed that placement of a non-polar residue at amino acid position 16 could be tolerated. Thus, compound 7 was made with a polypeptide tail of six D-Ala, where the D-Ala at amino acid position 16 was replaced with D-Glu. This peptide improved cellular activity compared to compound 6 in 0% and 10% serum (1.41 μM and 0.79 μM vs. 9.65 μM and 1.75 μM). Although its cellular activity is slightly shifted to the right compared to compound 4, glutamic acid may offer advantages in a less soluble scaffold.

[0227] Example 3 DUsing specific bridges (i,i+7 bisalkynes at positions 5 and 12) instead of olefins in PMI-δ-(5-12) olefin-stapled p53 peptidomimetic macrocycles resulted in improved rigidity and α-helicity We also used our α-aminobutyric acid (ABA) complex to stabilize the helical conformation as a means to improve counter-screen (off-target) activity and solubility. D We also considered introducing a different staple type into the PMI-δ series. D The PMI-δ-(5-12) olefin-stapled p53 peptidomimetic macrocycle (see WO2020257153 and Chem Sci.2020,11,5577) was a good candidate because it showed moderate cellular activity and some cellular activity properties in the counterscreen assay. Attempts to determine the solubility and HPLC logD were also unsuccessful, likely due to its low solubility (compound 8, Table 1, parts 1-3 and Table 2). For the i,i+7 olefin staple, a dialkyne staple substitution was performed because such a substitution could provide optimal distance between the β carbons of the i,i+7 amino acid side chains to give a stabilized α-helical structure (see Chembiochem 2018;19,1031). The dialkyne staple provided a single conformation to the helix, significantly stabilizing the structure (Figure 4). The predicted helicity also showed improvement (Figure 4): this peptide showed an increased helicity of about 60% (the corresponding hydrocarbon staple showed a helicity of about 32%).

[0228] Example 4 D Combination of PMI-.DELTA.-(5-12) dialkyne-stapled p53 peptidomimetic macrocycles with certain other specific amino acid substitutions and polypeptide tails improved cell renewal and physiochemical properties The inventors DThe PMI-δ-(5-12) dialkyne-stapled p53 peptidomimetic macrocycle was modified by: (i) D Similar to our modifications in the PMI-δ-(6-10) olefin-stapled p53 peptidomimetic macrocycle, (ii) the introduction of D-Gln at amino acid position 9 to remove the positive charge along with N-terminal acetylation, and (iii) the attachment of a 6×(D-Ala) polypeptide tail to the C-terminus to optimize solubility and helicity. Overall, the modifications produced compound 9, which exhibited significantly improved cellular activity up to 0.3 μM in 10% serum, as a 100-fold overall improvement compared to the parent peptide compound 8 (Table 1, parts 1-3). This compound was also cleaner than the parent compound in counter screen assays (Table 1, parts 1-3) and it was possible to measure solubility (Table 2). Compound 9 demonstrates the applicability of the modifications described in this invention.

[0229] Example 5 By applying the above modifications, very clean compounds can be obtained in counter screens. To further establish the impact of the above modifications in creating cleaner "drug"-like compounds, a set of compounds of the present invention was profiled in cell proliferation assays (Table 3). Reference compound 2 showed moderate potency at 9.1 μM in the HTC116 positive control cell line, and also showed some level of activity in the p53 null negative control cell line, represented by 88% inhibition at the maximum concentration. In contrast, compound 4 improved cell potency by more than 6-fold, reducing activity in the p53 null negative control cell line to 66% inhibition at the maximum concentration. Compound 9 showed submicromolar cell activity in the HTC116 positive control cell line (on-target assay) and lack of defects in the p53 null negative control cell line (off-target assay) (0.65 μM and -0.5% inhibition at the maximum concentration, respectively).

[0230] The compounds described herein are a promising candidate for a novel method for obtaining potent, cytoactive and clean p53 peptidomimetic macrocycles. D 1 illustrates the applicability of various modifications disclosed herein to PMI-δ p53 peptidomimetic peptides. [Table 2]

[0231] TIFF2024546110000028.tif186168

[0232] TIFF2024546110000029.tif197168

[0233] TIFF2024546110000030.tif208168

[0234] TIFF2024546110000031.tif105167

[0235] summary Modern display technologies such as phage display and mRNA display readily provide binding agents to proteins of interest (POIs) in vitro. It is difficult to convert these in vitro binding agents into compounds that are intracellularly and in vivo active with on-target selectivity and specificity.

[0236] The present invention relates to D We demonstrate that toxic (poor cellular activity with counterscreen activity and LDH release) p53 peptidomimetic macrocycles such as the PMI-δ-(6-10) olefin-stapled p53 peptidomimetic macrocycle can be converted into all-D-configured peptides with improved pharmacological properties by adding a polypeptide tail to the C-terminus of the macrocycle. Further improvements were achieved by replacing D-Lys at amino acid position 9 with D-Gln and D-Arg at amino acid position 12 with D-Ser.

[0237] The present invention also provides a method for the preparation of macrocycles comprising the steps of: (a) synthesizing a macrocycle comprising: D We also demonstrated the improvement of the PMI-δ-(5-12) olefin-stapled p53 peptidomimetic macrocycle, which resulted in an improved compound with a much cleaner off-target profile. Further improvement was achieved by replacing D-Lys at amino acid position 9 with D-Gln. [Table 3]

[0238] TIFF2024546110000033.tif241166

[0239] TIFF2024546110000034.tif246166

[0240] TIFF2024546110000035.tif246165

[0241] TIFF2024546110000036.tif241165

[0242] TIFF2024546110000037.tif241166

[0243] TIFF2024546110000038.tif249166

[0244] TIFF2024546110000039.tif247166

[0245] TIFF2024546110000040.tif140166

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While the present invention has been described herein with reference to exemplary embodiments, it should be understood that the invention is not limited thereto. Those skilled in the art having access to the teachings herein will recognize additional modifications and embodiments within the scope of the present invention. Accordingly, the present invention is limited only by the scope of the claims appended hereto.

Claims

1. (a) C of i,i+4 olefin staples and p53 peptide-mimicking macrocyclic molecules A polypeptide tail covalently bonded to a terminal amino acid at its N-terminus; (b) C of i,i+7 olefin staples and p53 peptide-mimicking macrocyclic molecules A polypeptide tail covalently bonded to a terminal amino acid at its N-terminus; or (c) C of i,i+7 dialkyne staples and p53 peptide-mimicking macrocyclic molecules A polyp that may be present if desired, covalently bonded to the terminal amino acid at its N-terminus. petite tail A macrocyclic molecule that mimics p53 peptides, Here, the p53 peptide-mimicking macrocyclic molecule contains amino acids that are all in the D configuration, and the polyp The butylated tail contains 3 to 9 amino acids, and each amino acid in the polypeptide tail can function independently. Having a D configuration or an L configuration, or each amino acid in the polypeptide tail having a D configuration The aforementioned p53 peptide-mimicking macrocyclic molecule.

2. The p53 peptide mimetic macrocyclic molecule is composed of i,i+7 dialkyne staples and p53 peptide A polypeptide tail covalently bonded at its N-terminus to the C-terminal amino acid of a do-mimicking macrocyclic molecule. A macrocyclic p53 peptide mimic according to claim 1, comprising the part.

3. The p53 peptide mimetic macrocyclic molecule consists of 12 amino acids and the p53 peptide mimetic macrocyclic component The two α,α-disubstituted amino acids located at amino acid positions 6 and 10 have a shape between their α-carbons. The resulting i,i+4 olefin staples and the C-terminus of the p53 peptide-mimicking macrocyclic molecule It comprises an amino acid and a polypeptide tail covalently bonded at its N-terminus, where p5 The three peptide-mimicking macrocyclic molecules contain amino acids that are all in the D configuration, and the polypeptide tail has three It contains up to 9 amino acids, and each amino acid in the polypeptide tail is independently in a D configuration or L configuration. In some configurations, or in certain embodiments, each amino acid in the polypeptide tail is D-configured A macrocyclic p53 peptide mimic according to claim 1, having a position.

4. α,α-disubstituted amino acids at amino acid positions 6 and 10 of the p53 peptide mimetic macrocyclic molecule The p53pe according to claim 3, which contains (R)-2-amino-2-methylhepta-6-enoic acid. A macrocyclic molecule mimicking a ptide.

5. The p53 peptide-mimicking macrocyclic molecule contains D-6-fluorotryptophan at amino acid position 3. and D-p-CF at amino acid position 7 ３ - The claim 4 further comprises phenylalanine. p53 is a macrocyclic molecule that mimics the peptide.

6. The p53 peptide mimetic macrocyclic molecule has threonine at amino acid position 1 and A at amino acid position 2. Lanine, tyrosine at amino acid position 4, alanine at amino acid position 5, and amino acid position 8 Glutamic acid is added to amino acid position 9, lysine or glutamine to amino acid position 11, and ro Claim 5 further comprises isine and arginine or serine at amino acid position 12. A macrocyclic molecule mimicking the p53 peptide.

7. The p53 peptide mimetic macrocyclic molecule has glutamine at amino acid position 9, and at amino acid position A macrocyclic p53 peptide mimic according to claim 6, comprising serine in 12.

8. The p53 peptide mimetic macrocyclic molecule consists of 12 amino acids and the p53 peptide mimetic macrocyclic component The two α,α-disubstituted amino acids located at amino acid positions 5 and 12 have a shape between their α-carbons. The resulting i,i+7 dialkyne staples and the C-terminus of the p53 peptide-mimicking macrocyclic molecule A polypeptide which may be present if desired, covalently bonded to an amino acid at its N-terminus. It includes a tail portion, where the p53 peptide-mimicking macrocyclic molecule consists of amino acids in the D configuration. It contains, and the polypeptide tail contains 3 to 9 amino acids, and each amino acid in the polypeptide tail However, independently having a D configuration or an L configuration, the p53 peptide mimetic macrocyclic according to claim 1. molecule.

9. The α,α-disubstituted amino acid at amino acid position 5 of the p53 peptide mimetic macrocyclic molecule is (S)- It contains 2-amino-2-methylhepta-6-ic acid and is a p53 peptide mimetic macrocyclic molecule. The α,α-disubstituted amino acid at position 12 of the amino acid is (R)-2-amino-2-methylocta-7 - A macrocyclic molecule mimicking the p53 peptide according to claim 8, comprising ic acid.

10. The p53 peptide mimetic macrocyclic molecule has amino acid position 3 of the p53 peptide mimetic macrocyclic molecule. Amino acid position 7 of -6-fluorotryptophan and p53 peptide-mimicking macrocyclic molecules D-p-CF ３ - A p53 peptide mimic according to claim 9, further comprising phenylalanine. Cyclic molecule.

11. The p53 peptide mimetic macrocyclic molecule has threonine at amino acid position 1 and A at amino acid position 2. Lanine, tyrosine at amino acid position 4, asparagine at amino acid position 6, amino acid position Glutamic acid at position 8, lysine or glutamine at amino acid position 9, and amino acid position 10 The p53 peptide-mimicking macrocyclic molecule according to claim 10, further comprising leucine at amino acid position 11.

12. Claim 11, a p53 peptide-mimicking macrocyclic molecule containing glutamine at amino acid position 9. A macrocyclic molecule mimicking the p53 peptide.

13. The p53 peptide mimetic macrocyclic molecule consists of 12 amino acids and the p53 peptide mimetic macrocyclic component The two α,α-disubstituted amino acids located at amino acid positions 5 and 12 have a shape between their α-carbons. The i,i+7 olefin staples formed and the optionally present polyp It includes a cydos tail, and here, all p53 peptide-mimicking macrocyclic molecules are in the D configuration amino acid. It contains acid, and the polypeptide tail contains 3 to 9 amino acids, and each amino acid in the polypeptide tail The p53 peptide mimetic macroring according to claim 1, wherein the acid independently has a D configuration or an L configuration. shaped molecules.

14. The α,α-disubstituted amino acid at amino acid position 5 of the p53 peptide mimetic macrocyclic molecule is (S)- It contains 2-amino-2-methyldeca-9-enoic acid, and is a p53 peptide mimetic macrocyclic molecule. The α,α-disubstituted amino acid at position 12 is (R)-2-amino-2-methylhepta-6- A macrocyclic p53 peptide mimic according to claim 13, comprising enoic acid.

15. The p53 peptide mimetic macrocyclic molecule has amino acid position 3 of the p53 peptide mimetic macrocyclic molecule. Amino acid position 7 of -6-fluorotryptophan and p53 peptide-mimicking macrocyclic molecules D-p-CF ３ - The p53 peptide mimicry according to claim 14, further comprising phenylalanine. Macrocyclic molecule.

16. The p53 peptide mimetic macrocyclic molecule has threonine at amino acid position 1 and A at amino acid position 2. Lanine, tyrosine at amino acid position 4, asparagine at amino acid position 6, amino acid position Glutamic acid at position 8, lysine or glutamine at amino acid position 9, and amino acid position 10 p53 according to claim 15, further comprising leucine at amino acid position 11 and leucine at amino acid position 11. A macrocyclic molecule that mimics a peptide.

17. Claim 16, a p53 peptide-mimicking macrocyclic molecule containing glutamine at amino acid position 9. A macrocyclic molecule mimicking the p53 peptide.

18. The polypeptide tail contains 3 to 9 amino acids, and each amino acid in the polypeptide tail is unique It stands upright and has a D configuration or an L configuration, or each amino acid in the polypeptide tail is D A macrocyclic p53 peptide mimic according to claim 1 or 2, having the configuration.

19. The polypeptide tail contains 3 to 9 amino acids, and each amino acid in the polypeptide tail A macrocyclic p53 peptide mimic according to claim 1 or 2, wherein the p53 peptide has a D configuration.

20. The polypeptide tail contains six amino acids, and each amino acid in the polypeptide tail is independent. The molecule has a D configuration or an L configuration, or each amino acid in the polypeptide tail has a D configuration. A macrocyclic p53 peptide mimic according to claim 1 or 2, comprising the features of the p53 peptide mimic macrocyclic molecule according to claim 1 or 2.

21. The polypeptide tail corresponds to SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23 A p53 peptide mimetic macroring according to claim 1 or 2, comprising the amino acid sequence described above. shaped molecules.

22. [Here, X ３ D-Trp, D-6-fluoro-Trp, D-6-chloro-Trp, D -6-bromo-Trp, D-6-iodo-Trp, D-6-methyl-Trp, D-6-cy Ano-Trp, D-6-hydroxy-Trp, D-6-NO ２ -Trp, D-7-Fluoro Low-Trp, D-7-chloro-Trp, D-7-bromo-Trp, D-7-iod-Tr p, D-7-methyl-Trp, D-7-cyano-Trp, D-7-hydroxy-Trp, D-7-NO ２ -Trp, D-6,7-fluoro-Trp, D-6,7-chloro-Trp D-6,7-bromo-Trp, D-6,7-iodo-Trp, D-6,7-methyl-T rp, D-6,7-cyano-Trp, D-6,7-hydroxy-Trp or D-6,7 - NO ２ -Trp; X ６ (R)-2-amino-2-methylhepta-6-enoic acid There is; X ７ is D-p-fluoro-Phe, D-p-chloro-Phe, D-p-bromo-P he, D-p-iodo-Phe, D-p-methyl-Phe, D-p-cyano-Phe, D -p-hydroxy-Phe, D-p-NO ２ -Phe or Dp-CF ３ -Phe dea ru; X １０ (R)-2-amino-2-methylhepta-6-enoic acid; X １１ is D- Leu is; X １２ is D-Arg or D-Ser; X １３ is D-Ala, D- It is Glu or D-Gla (γ-carboxyl glutamic acid); X １４ is D-Ala is; X １５ is D-Ala, D-Phe, or D-Glu; X １６ is D-Ala is or does not exist; X １７ These are D-Ala, D-α-methyl-Glu, and It does not exist; X １８ is D-Ala or does not exist; X １９ is D-Ala to be, or not to exist; X ２０ is D-Ala or does not exist; X ２１ is D-A la is or is absent; the N-terminal amino group is desired to be an acyl group having the formula RCO-. They may be joined by formula C ｎ H ２ｎ＋１ It is an alkane having; n is 1 to It is an integer of 10; the C-terminal amino acid may optionally contain an amino group; No acids 1-21 are D amino acids; and staples are X ６ and X １０ Closed-ring meta between [This is an olefin obtained by cesis.] A macrocyclic molecule mimicking the p53 peptide, containing the peptide.

23. X ３ is D-6-fluoro-Trp; or X ７ D-p-CF ３ - It is Phe ; or X ３ is D-6-fluoro-Trp, and X ７ D-p-CF ３ - It is Phe , the p53 peptide-mimicking macrocyclic molecule according to claim 22.

24. The p53 peptide-mimicking macrocyclic molecule according to claim 22, wherein the acyl group is an acetyl group.

25. [Here, X ３ D-Trp, D-6-fluoro-Trp, D-6-chloro-Trp, D -6-bromo-Trp, D-6-iodo-Trp, D-6-methyl-Trp, D-6-cy Ano-Trp, D-6-hydroxy-Trp, D-6-NO ２ -Trp, D-7-Fluoro Low-Trp, D-7-chloro-Trp, D-7-bromo-Trp, D-7-iod-Tr p, D-7-methyl-Trp, D-7-cyano-Trp, D-7-hydroxy-Trp, D-7-NO ２ -Trp, D-6,7-fluoro-Trp, D-6,7-chloro-Trp D-6,7-bromo-Trp, D-6,7-iodo-Trp, D-6,7-methyl-T rp, D-6,7-cyano-Trp, D-6,7-hydroxy-Trp or D-6,7 - NO ２ -Trp; X ５ (S)-2-amino-2-methylhepta-6-ic acid There is; X ７ These are D-p-fluoro-Phe, D-p-chloro-Phe, and D-p-bromo-P. he, D-p-iodo-Phe, D-p-methyl-Phe, D-p-cyano-Phe, D -p-hydroxy-Phe, D-p-NO ２ -Phe or Dp-CF ３ -Phe dea ru; X ９ is D-Lys or D-Gln; X １１ is D-Leu; X １２ teeth( R) is 2-amino-2-methylocta-7-ic acid; X １３ D-Ala, D-G lu, D-Gla (γ-carboxyl glutamic acid), or absent; X １４ is D-Ala or does not exist; X １５ is D-Ala, D-Phe or D- Glu is or does not exist; X １６ is D-Ala or does not exist; X １ ７ is D-Ala, D-α-methyl-Glu, or is absent; X １８ is D-Al a is, or does not exist; X １９ is D-Ala or does not exist; X ２０ teeth D-Ala is or does not exist; X ２１ is D-Ala, or does not exist; The N-terminus may optionally be bonded to an acyl group having the formula RCO-; R is C ｎ H ２ｎ＋１ It is an alkane having; n is an integer from 1 to 10; the C-terminal amino acid is They may optionally contain an amino group; amino acids 1 to 21 are D amino acids; and The staples are X ５ and X １２ The dia obtained by alkyne cross coupling between [It is Lukin] A macrocyclic molecule mimicking the p53 peptide, containing the p53 peptide.

26. X ９ The macrocyclic p53 peptide mimicry molecule according to claim 25, wherein is D-Gln.

27. X ３ is D-6-fluoro-Trp; or X ７ D-p-CF ３ - It is Phe ; or X ３ is D-6-fluoro-Trp, and X ７ D-p-CF ３ - It is Phe The p53 peptide-mimicking macrocyclic molecule according to claim 25.

28. The p53 peptide-mimicking macrocyclic molecule according to claim 25, wherein the acyl group is an acetyl group.

29. [Here, X ３ D-Trp, D-6-fluoro-Trp, D-6-chloro-Trp, D -6-bromo-Trp, D-6-iodo-Trp, D-6-methyl-Trp, D-6-cy Ano-Trp, D-6-hydroxy-Trp, D-6-NO ２ -Trp, D-7-Fluoro Low-Trp, D-7-chloro-Trp, D-7-bromo-Trp, D-7-iod-Tr p, D-7-methyl-Trp, D-7-cyano-Trp, D-7-hydroxy-Trp, D-7-NO ２ -Trp, D-6,7-fluoro-Trp, D-6,7-chloro-Trp D-6,7-bromo-Trp, D-6,7-iodo-Trp, D-6,7-methyl-T rp, D-6,7-cyano-Trp, D-6,7-hydroxy-Trp or D-6,7 - NO ２ -Trp; X ５ (S)-2-amino-2-methyldeca-9-enoic acid ru; X ７ These are D-p-fluoro-Phe, D-p-chloro-Phe, and D-p-bromo-Ph e, D-p-iodo-Phe, D-p-methyl-Phe, D-p-cyano-Phe, D- p-hydroxy-Phe, D-p-NO ２ -Phe or Dp-CF ３ - It is Phe ;X ９ is D-Lys or D-Gln; X １１ is D-Leu; X １２ is (R )-2-amino-2-methylhepta-6-enoic acid; X １３ is D-Ala, D-Gl u, D-Gla (γ-carboxyl glutamic acid); X １４ is D-Ala; X １５ is D-Ala, D-Phe, or D-Glu; X １６ is D-Ala; X １７ is D-Ala or D-α-methyl-Glu; X １８ is D-Ala; X １９ is D-Ala or does not exist; X ２０ is D-Ala, or exists Do not; X ２１ is D-Ala or absent; the N-terminus has the formula RCO- It may optionally be bonded to an acyl group; R is of formula C ｎ H ２ｎ＋１ Alkanes that have There is; n is an integer from 1 to 10; the C-terminal amino acid optionally contains an amino group. It may be; amino acids 1-21 are D amino acids; and staples are X ５ and X １２ and This is an olefin obtained by ring-closing metathesis during the process. A macrocyclic molecule mimicking the p53 peptide, containing the p53 peptide.

30. X ９ The p53 peptide-mimetic macrocyclic molecule according to claim 29, wherein ９ is D-Gln.

31. X ３ is D-6-fluoro-Trp; or X ７ D-p-CF ３ - It is Phe ; or X ３ is D-6-fluoro-Trp, and X ７ D-p-CF ３ - It is Phe The p53 peptide-mimicking macrocyclic molecule according to claim 29.

32. The p53 peptide-mimicking macrocyclic molecule according to claim 29, wherein the acyl group is an acetyl group.

33. [Here, X ３ is D-Trp, D-6-fluoro-Trp, D-6-chloro-Trp, D -6-bromo-Trp, D-6-iodo-Trp, D-6-methyl-Trp, D-6-cy Ano-Trp, D-6-hydroxy-Trp, D-6-NO ２ -Trp, D-7-Fluoro Low-Trp, D-7-chloro-Trp, D-7-bromo-Trp, D-7-iod-Tr p, D-7-methyl-Trp, D-7-cyano-Trp, D-7-hydroxy-Trp, D-7-NO ２ -Trp, D-6,7-fluoro-Trp, D-6,7-chloro-Trp D-6,7-bromo-Trp, D-6,7-iodo-Trp, D-6,7-methyl-T rp, D-6,7-cyano-Trp, D-6,7-hydroxy-Trp or D-6,7 - NO ２ -Trp; X ５ (S)-2-amino-2-methylhepta-6-ic acid There is; X ７ These are D-p-fluoro-Phe, D-p-chloro-Phe, and D-p-bromo-P. he, D-p-iodo-Phe, D-p-methyl-Phe, D-p-cyano-Phe, D -p-hydroxy-Phe, D-p-NO ２ -Phe or Dp-CF ３ -Phe dea ru; X ９ is D-Lys or D-Gln; X １１ is D-Leu; X １２ teeth( It is R)-2-amino-2-methylocta-7-ic acid; the N-terminus has the formula RCO-. It may optionally be bonded to an acyl group; R is of formula C ｎ H ２ｎ＋１ Alkanes containing n is an integer from 1 to 10; the C-terminal amino acid optionally contains an amino group. It may be; amino acids 1-21 are D amino acids; and staple is X ５ and X １２ This is a dialkyne obtained by alkyne cross coupling between the two. A macrocyclic molecule mimicking the p53 peptide, containing the p53 peptide.

34. X ９ The macrocyclic p53 peptide mimicry molecule according to claim 33, wherein is D-Gln.

35. X ３ is D-6-fluoro-Trp; or X ７ D-p-CF ３ - It is Phe ; or X ３ is D-6-fluoro-Trp, and X ７ D-p-CF ３ - It is Phe , the p53 peptide-mimicking macrocyclic molecule according to claim 33.

36. The p53 peptide-mimicking macrocyclic molecule according to claim 33, wherein the acyl group is an acetyl group.

37. Sequence ID 2, Sequence ID 3, Sequence ID 4, Sequence ID 5, Sequence ID 6, Sequence ID 7, Sequence Number p53 peptide-mimicking macrocyclic containing the amino acid sequence described in No. 9 or SEQ ID NO. 10 molecule.

38. The p53 peptide mimetic macrocyclic molecule is sequence number 4, sequence number 6, sequence number 7 or sequence number A macrocyclic p53 peptide mimic according to claim 37, comprising the amino acid sequence described in claim 9. molecule.

39. A composition comprising the p53 peptide-mimicking macrocyclic molecule described in claim 1 and a pharmaceutically acceptable carrier.

40. The composition according to claim 39 for the treatment of cancer in subjects requiring cancer treatment.

41. Use of the p53 peptide mimetic macrocyclic molecule according to claim 1 or the composition according to claim 39 for the manufacture of a pharmaceutical product for the treatment of cancer.

42. A macrocyclic p53 peptide mimetic molecule according to claim 1 or a composition according to claim 39 for the treatment of cancer.

43. The composition according to claim 40, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin lymphoma, kidney cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary gland cancer, pancreatic cancer, bronchial cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, adrenal cancer, osteosarcoma, chondrosarcoma, and hematological cancer.

44. The composition according to claim 39 for regulating the activity of p53 and / or MDM2 and / or MDMX in a subject.

45. Use of the p53 peptide mimetic macrocyclic molecule according to claim 1 or the composition according to claim 39 for the manufacture of a pharmaceutical for modulating the activity of p53 and / or MDM2 and / or MDMX.

46. A macrocyclic p53 peptide mimetic molecule according to claim 1 or a composition according to claim 39 for regulating the activity of p53 and / or MDM2 and / or MDMX.

47. The composition according to claim 39 for antagonizing the interaction between p53 and MDM2 and / or between p53 and MDMX in a subject.

48. Use of the p53 peptide-mimicking macrocyclic molecule according to claim 1 or the composition according to claim 39 for the manufacture of a pharmaceutical product that antagonizes the interaction between p53 and MDM2 and / or between p53 and MDMX.

49. A macrocyclic p53 peptide mimetic molecule according to claim 1 or a composition according to claim 39 for antagonizing the interaction between p53 and MDM2 and / or between p53 and MDMX.

50. A composition according to claim 39 for combination therapy for treating cancer, wherein the combination therapy comprises administering a therapeutically effective amount of the composition according to claim 39 and a therapeutically effective amount of a chemotherapeutic agent or radiation to a target.

51. The composition according to claim 50, wherein in the combination therapy, a chemotherapeutic agent or radiation is administered to the target, followed by the administration of a p53 peptide mimetic macrocyclic molecule; the p53 peptide mimetic macrocyclic molecule is administered to the target, followed by the administration of a chemotherapeutic agent or radiation; or the chemotherapeutic agent or radiation is administered to the target simultaneously with the administration of the p53 peptide mimetic macrocyclic molecule.

52. A kit for combination therapy to treat cancer, comprising a therapeutically effective amount of the p53 peptide mimetic macrocyclic molecule described in claim 1 or the composition described in claim 39 and a therapeutic dose of a chemotherapeutic agent or radiation.

53. The chemotherapy agents include actinomycin, all-trans retinoic acid, alitretinoin, azacitidine, azathioprine, bexarotene, bleomycin, bortezomib, carmofur, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epotilon, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, and imatinib. The kit according to claim 52, selected from the group consisting of ixabepyrone, irinotecan, mechloretamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, nitrosourea, oxaliplatin, paclitaxel, pemetrexed, romidepsin, tegafur, temozolomide (oral dacarbazine), teniposide, thioguanine, topotecan, uchideron, barrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, and vorinostat.

54. A composition according to claim 39 for combination therapy for treating cancer, wherein the combination therapy comprises administering the composition according to claim 39 and a therapeutically effective amount of a checkpoint inhibitor to a subject requiring treatment for cancer.

55. The kit according to claim 53, wherein the checkpoint inhibitor is an anti-PD1 antibody or an anti-PD-L1 antibody.

56. The kit according to claim 53, wherein the combination therapy further comprises administering a therapeutically effective dose of a chemotherapeutic agent or radiation.

57. A composition according to claim 39 for the treatment of cancer, wherein the treatment comprises administering to a subject having cancer a vector comprising a nucleic acid molecule encoding wild-type p53 or a p53 variant or analog having transcriptional activating activity, and then administering the composition according to claim 39 once or more times.

58. The composition according to claim 57, wherein the vector is a plasmid, retrovirus, adenovirus or adeno-associated virus.

59. The composition according to claim 57, wherein the subject is subjected to chemotherapy or radiotherapy before or after administering the vector to the subject.

60. The composition according to claim 57, administered to checkpoint inhibitors.

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