Stapled peptide proteolysis targeting chimeras (SP-protacs) and methods of use

Abstract

This disclosure relates to chimeras comprising a stapled peptide conjugated to a compound that binds a protein (e.g., a protein that is involved in or that is causative of disease, e.g., cancer) (e.g., a compound that binds to a BET protein, e.g., JQ1), also referred to herein as Stapled Peptide-PROteolysis Targeting Chimeras (SP-PROTACs), and related compositions and uses thereof, e.g., in the treatment of a disease (e.g., a cancer or other disease driven by or related to the protein to which the compound binds). The chimeras act as combined protein degradation-inducing and protein targeting and moieties, e.g., stapled peptide to recruit the degradation protein such as HDM2 (and target HDMX to maximally reactivate p53) and a small molecule to engage a protein (e.g., a protein that is involved in or that is causative of a disease, e.g., a cancer).

Description

STAPLED PEPTIDE PROTEOLYSIS TARGETING CHIMERAS (SP-PROTACS) AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 650,785, filed on May 22, 2024, which is hereby incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety.Said XML copy, created on May 20, 2025, is named 00530-0423W01_SL.xml and is 131,406 bytes in size.TECHNICAL FIELD

[0003] This disclosure relates to chimeras comprising a stapled peptide conjugated to a compound that binds a protein (e.g., a protein that is involved in or that is causative of disease, e.g., cancer) (e.g., a compound that binds to a BET protein, e.g., JQ1) also referred to herein as Stapled Peptide-PROteolysis Targeting Chimeras (SP-PROTACs), and related compositions and uses thereof, e.g., in the treatment of a disease (e.g., a cancer or other disease driven by or related to the protein to which the compound binds). The chimeras act as combined protein targeting and protein degradation-inducing moieties, e.g., stapled peptide to recruit the degradation protein such as HDM2 (and target HDMX to maximally reactivate p53) and small molecule to engage a protein (e.g., a protein that is involved in or that is causative of disease, e.g., cancer).BACKGROUND

[0004] The p53 tumor suppressor protein plays a pivotal role in the control of a wide variety of cellular functions, including as a tumor suppressor. The cellular availability of p53 is regulated by the oncoproteins HDM2 and HDMX. HDM2 and HDMX latch ontoa single coiled domain of p53 to either destroy or sequester it. HDM2 targets p53 for proteasomal degradation by ubiquitylation, while HDMX sequesters p53 and blocks its transcriptional activity.

[0005] Bromodomain and extraterminal domain (BET) proteins, e.g., bromodomain 2 (BRD2), BRD3, and BRD4, regulate cancer-related genes and are associated with cancer progression. There remains an unmet need to develop efficient therapeutics that target and degrade cancer proteins such as BET proteins.SUMMARY

[0006] Featured herein is a new form of the SP-PROTAC that represents a platform technology to simultaneously (1) bind and recruit HDM2 for degrading a disease-causing protein (while also disrupting the p53 / HDM2 interaction to reactivate p53); (2) bind and inhibit HDMX (to inhibit p53 / HDMX interactions to maximally reactivate p53); and (3) link the latter two to a compound that binds a protein (e.g., a protein that is involved in or that is causative of disease, e.g., cancer), which can be degraded by HDM2. All of these events happen at about the same time in the same cell with the same pharmacokinetics and pharmacodynamics to achieve a new form of disease therapy - essentially a “3-in-l” effect.

[0007] Provided herein are chimeras, specifically SP-PROTACs, that can be used to target HDMX, HDM2, and a protein (e.g., a protein that is involved in or that is causative of a disease, e.g., a cancer, e.g., a BET protein, e.g., BRD2, BRD3, or BRD4). These chimeras comprise a stapled peptide (which binds to HDM2 and HDMX) linked to a compound (which binds to a protein (e.g., a protein that is involved in or that is causative of a disease, e.g., a cancer, e.g., a BET protein, e.g., BRD4). Such chimeras are useful for treating a disease (e.g., a cancer), wherein the protein is involved in or is causative of the disease).

[0008] In one aspect, provided herein is a chimera or a pharmaceutically acceptable salt thereof, wherein the chimera or the pharmaceutically acceptable salt thereof comprises a stapled peptide conjugated to a compound, wherein the stapled peptidecomprises (i) AC-LTFXIX2YWAX3X4X5X6AAX7-NH2(SEQ ID NO: 19), wherein: Xi is an a, a-disubstituted non-natural amino acid with an olefinic side chain cross-linked to Xs, X2is E or A, X3 is: (1) an amino acid (e.g., any amine containing amino acid, e.g., diaminopropionic acid, diaminobutanoic acid, ornithine, lysine, homolysine), (2) (NH- (CH2)m-CO)n, wherein m is 1-11 and n is 0-5, or (3) NH-(CH2-CH2-O)n-CH2-CH2-CO, wherein n is 1-5, conjugated to the compound, optionally via a linker, optionally wherein X3 is lysine conjugated to the compound via the linker, X4 is leucine or cyclobutylalanine, X5 is an a, a-disubstituted non-natural amino acid with an olefinic side chain cross-linked to Xi, Xe is S or A, X7 is absent or AAXs, wherein X8 is D-alanine, and Ac is an acetyl group, or (ii) SEQ ID NO: 19 with 1 or 2 amino acid substitutions, wherein the 1 or 2 amino acid substitutions are not at Xi, X3, or X5, and wherein the stapled peptide binds to HDMX and HDM2; and wherein the compound binds to a protein (e.g., a BET protein, e.g., BRD4) (e.g., the protein binds to a BET protein and the compound is JQ1). In some instances Xi is (R)-2-(7’-octenyl)alanine cross-linked to X5, and wherein X5 is (S)-2-(4’-pentenyl)alanine cross-linked to Xi. In some instances, X3 is an amino acid (e.g., any amine containing amino acid, e.g., diaminopropionic acid, di aminobutanoic acid, ornithine, lysine, homolysine) conjugated to the compound, optionally via the linker. In some instances, X3 is (NH-(CH2)m-C0)n, wherein m is 1-11 and n is 0-5, conjugated to the compound, optionally via the linker. In some instances, X3 is NH-(CH2-CH2-O)n-CH2-CH2-CO, wherein n is 1-5, conjugated to the compound, optionally via the linker. In some instances, X3 is lysine conjugated to the compound via the linker. In some instances, the compound is conjugated to X3 via the linker. In some instances, X3 is conjugated to the compound via the linker, optionally wherein the linker is 5 to 13 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13) atoms in length from C-alpha of X3. In some instances, the linker comprises (P-alanine)n, (ethylene glycol)n, PEG, or (y-Aminobutyric acid)n, wherein n is 1 to 5. In some instances, the linker is (P-alanine)n, wherein n is 1 to 5. In some instances, the linker is P-alanine. In some instances, the chimera or the pharmaceutically acceptable salt thereof comprises or consists of Ac- LTFXIAYWAX2LX3AAAAAX4-NH2, wherein Xi is (R)-2-(7’-octenyl)alanine crosslinked to X3, X2 is lysine conjugated to the compound via the linker, X3 is (S)-2-(4’- pentenyl)alanine cross-linked to Xi, X4 is D-alanine, the linker is P-alanine, and Ac is an acetyl group (SEQ ID NO:200).

[0009] In some instances, the compound of the chimera is a compound that binds a BET protein (e g., BRD2, BRD3, or BRD4). In some instances, the compound that binds a BET protein is JQ1, ABBV-075 (Mivebresib), I-BET151, 1-BET726, OTX015(Birabresib), or PFI-1, whose structures are provided below:I-BET726:OTX015 (Birabresib):PFI-1 :

[0010] In some instances, the compound of the chimera is JQ1 (i.e., (6S)-4-(4- chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f]triazolo[4,3-a]diazepine-6-acetic acid, 1,1- dimethylethyl ester). In some cases, the JQ1 that is coupled to the stapled peptide lacks the 1,1 -di methyl ethyl ester. For example, in some cases, the 1 ,1 -dimethylethyl ester is replaced by an amide. In some instances, the stapled peptide comprises or consists of Ac- LTFX1AYWAX2LX3AAAAAX4-NH2, wherein Xi is (R)-2-(7’-octenyl)alanine cross- linked to X3, X2 is lysine conjugated to JQ1 via the linker, X3 is (S)-2-(4’- pentenyl)alanine cross-linked to Xi, X4 is D-alanine, the linker is β-alanine, and Ac is an acetyl group (SEQ ID NO: 14).

[0011] In some instances, JQ1 attached to the linker has the structure of Formula I:(Formula I).

[0012] Also provided herein is a chimera comprising the structure of Formula II(SEQ ID NO: 14):(Formula II) or a pharmaceutically acceptable salt thereof. In one instance, the chimera comprises the structure of Formula II. The disclosure encompasses compositions comprising of the chimera comprising the structure of Formula II (SEQ ID NO: 14) and a pharmaceutically acceptable salt thereof.

[0013] Also provided herein is a pharmaceutical composition comprising any one of the foregoing chimeras or the pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In some instances, the pharmaceutical composition further comprises a second therapeutic agent. In some instances, the second therapeutic agent is a nuclear export inhibitor. In some instances, the nuclear export inhibitor is selinexor. In some cases, the foregoing chimeras or the pharmaceutically acceptable salt thereof and the nuclear export inhibitor are in a single composition. In other instances, the foregoing chimeras or the pharmaceutically acceptable salt thereof and the nuclear export inhibitor are in separate compositions.

[0014] Also provided herein is a method of treating a disease (e.g., a cancer) in a subject (e.g., human) in need thereof, the method comprising administering (e.g., subcutaneously, intravenously, intramuscularly) to the subject a therapeutically effective amount of any one of the foregoing chimeras or pharmaceutically acceptable salts thereof. The diseases that may be treated with the chimeras of the disclosure are driven by or related to the protein to which the compound (e.g., JQ1) of the chimera binds. For instance, a chimera described herein comprising a compound (e.g., JQ 1) that binds to a BET protein (e.g., BRD4) may be used to treat a cancer (e.g., a cancer expressing or encoding a BET protein, e.g., BRD4). In some instances, the disease expresses wild type p53. In some instances, the disease expresses or encodes HDMX. In some instances, the disease expresses or encodes HDM2. In some instances, the disease expresses or encodes a BET protein (e.g., BRD2, BRD3, or BRD4) and the compound of the chimera binds the BET protein. In some instances, the disease expresses BRD4 and the compound of the chimera binds BRD4. In some instances, the disease expresses wild type or functional p53 and HDMX. In certain instances, the disease expresses wild type or functional p53, HDMX, and BRD4 and the compound of the chimera binds BRD4. In some instances, the disease is a cancer. In some cases, the cancer is a solid cancer. In other cases, the cancer is a liquid cancer. In some cases, the cancer is a hematological cancer (e.g., a leukemia, a lymphoma). In some instances, the cancer is a sarcoma (such as an osteosarcoma), an ovarian cancer, a kidney cancer, a soft tissue cancer, a brain cancer, askin cancer, an endometrial cancer, a blood cancer, or a urinary tract cancer. In some instances, the cancer is a pediatric cancer. In some cases, the pediatric cancer is pediatric ALL.

[0015] In some instances, provided herein is a method of treating a cancer or tumor that expresses wild type or functional p53 in a subject (e g., human) in need thereof, the method comprising administering (e.g., subcutaneously, intravenously, intramuscularly) to the subject a chimera comprising the structure of formula II, or a pharmaceutically acceptable salt thereof. In other cases, the method comprises administering a pharmaceutical composition comprising a chimera comprising the structure of formula II, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In some cases, the cancer or tumor that expresses wild type or functional p53 is a solid cancer. In other cases, the cancer or tumor that expresses wild type or functional p53 is a liquid cancer. In some cases, the cancer or tumor that expresses wild type or functional p53 is a hematological cancer (e.g., a leukemia, a lymphoma). In some instances, the cancer or tumor that expresses wild type or functional p53 is a sarcoma (such as an osteosarcoma), an ovarian cancer, a kidney cancer, a soft tissue cancer, a brain cancer, a skin cancer, an endometrial cancer, a blood cancer, or a urinary tract cancer. In some instances, the cancer or tumor that expresses wild type or functional p53 is a pediatric cancer. In some cases, the pediatric cancer is pediatric ALL. In some cases, in all of the above instances, the cancer or tumor encodes or expresses functional p53, functional p53 and HDM2, functional p53 and HDMX, or functional p53, HDM2, and HDMX.

[0016] In some instances, the method of treating a disease further comprises administering (e g., subcutaneously, intravenously, intramuscularly) to the subject a therapeutically effective amount of a second therapeutic agent. In some instances, the second therapeutic agent is a nuclear export inhibitor. In some instances, the nuclear export inhibitor is selinexor.

[0017] The disclosure also features a method of selecting a subject (e.g., human) for treatment with a therapeutically effective amount of any one of the foregoing chimeras orpharmaceutically acceptable salts thereof. The method involves determining whether a diseased (e.g., cancerous) cell from the subject expresses wild type or functional p53. If the diseased (e.g., cancerous) cell expresses wild type or functional p53, the subject is selected for treatment with a therapeutically effective amount of any one of the foregoing chimeras or pharmaceutically acceptable salts thereof. In certain instances, the method further involves assessing whether the diseased (e.g., cancerous) cell expresses one or both of HDMX and HDM2. If the diseased (e.g., cancerous) cell expresses HDMX and HDM2, the subject is selected for treatment with a therapeutically effective amount of any one of the foregoing chimeras or pharmaceutically acceptable salts thereof. In certain instances, the method further involves assessing whether the diseased (e.g., cancerous) cell expresses the protein to which the compound of the chimera binds. If the diseased (e.g., cancerous) cell expresses the protein to which the compound of the chimera binds, the subject is selected for treatment with a therapeutically effective amount of any one of the foregoing chimeras or pharmaceutically acceptable salts thereof. In certain instances, the method further involves assessing whether the diseased (e.g., cancerous) cell expresses p53, HDM2, HDMX, and the protein to which the compound of the chimera binds. If the diseased (e.g., cancerous) cell expresses p53, HDM2, HDMX, and the protein to which the compound of the chimera binds, the subject is selected for treatment with a therapeutically effective amount of any one of the foregoing chimeras or pharmaceutically acceptable salts thereof. In certain instances, the method further involves assessing whether the cancer cell contains complexes of HDM2 / p53 and / or complexes of HDMX / p53. If the diseased (e.g., cancerous) cell contains such complexes, the subject is selected for treatment with a therapeutically effective amount of any one of the foregoing chimeras or pharmaceutically acceptable salts thereof. In certain instances, the subject is also administered a therapeutically effective amount of a second therapeutic agent, e.g., a nuclear export inhibitor, e.g., selinexor.

[0018] Also provided herein is a method of making any one of the foregoing chimeras or the pharmaceutically acceptable salts thereof. In some instances, the method comprises: (a) providing a peptide comprising the sequence: (i) Ac-LTFX1X2YWAX3X4X5X6AAX7-NH2 (SEQ ID N0:300), wherein: Xi is an a, a- disubstituted non-natural amino acid with an olefinic side chain cross-linked to X5, X2 is E or A, X3 is an orthogonally protected amino acid, optionally wherein X3 is orthogonally protected lysine, X4 is leucine or cyclobutylalanine, Xs is an α, α-di substituted non- natural amino acid with an olefinic side chain cross-linked to Xi, X6 is S or A, X7 is absent or AAXs, wherein X8 is D-alanine, and Ac is an acetyl group; or (ii) SEQ ID NO:300 with 1 or 2 amino acid substitutions, wherein the 1 or 2 amino acid substitutions are not at Xi, X3, or Xs, and wherein the peptide binds to HDMX and HDM2; (b) incorporating a linker onto X3, (c) coupling a compound to the linker, and (d) cross- linking the peptide, thereby making the chimera. In some instances, the method further comprises purifying the chimera. In some instances, the method further comprises formulating the purified chimera as a sterile pharmaceutical composition.

[0019] Also provided herein is a method of making any one of the foregoing chimeras or the pharmaceutically acceptable salts thereof. In some instances, the method comprises: (a) providing a peptide comprising the sequence: (i) Ac- LTFX1X2YWAX3X4X5X6AAX7-NH2 (SEQ ID NO:20), wherein: Xi is an a, a- disubstituted non-natural amino acid with an olefinic side chain cross-linked to X5, X2 is E or A, X3 is an orthogonally protected amino acid, optionally wherein X3 is orthogonally protected lysine, X4 is leucine or cyclobutylalanine, X5 is an a, a-di substituted non- natural amino acid with an olefinic side chain cross-linked to Xi, X6 is S or A, X7 is absent or AAX8, wherein X8 is D-alanine, and Ac is an acetyl group; or (ii) SEQ ID NO:20 with 1 or 2 amino acid substitutions, wherein the 1 or 2 amino acid substitutions are not at Xi, X3, or X5, and wherein the peptide binds to HDMX and HDM2; (b) incorporating a linker onto X3, (c) coupling JQ1 to the linker, and (d) cross- linking the peptide, thereby making the chimera. In some instances, the method further comprises purifying the chimera. In some instances, the method further comprises formulating the purified chimera as a sterile pharmaceutical composition.

[0020] Also provided herein is a pharmaceutical composition comprising: (a) a means for treating a disease (e.g., a cancer) in a human subject in need thereof, and (b) a pharmaceutically acceptable carrier.

[0021] Also provided herein is a pharmaceutical composition comprising: (a) a means for treating a disease (e.g., a cancer) in a human subject in need thereof, (b) a means for inhibiting nuclear export, and (c) a pharmaceutically acceptable carrier.

[0022] Also provided herein is a pharmaceutical composition comprising: (a) a means for binding HDM2, HDMX, and a protein (e.g., a BET protein, e.g., BRD4), and (b) a pharmaceutically acceptable carrier.

[0023] Also provided herein is a pharmaceutical composition comprising: (a) a means for binding HDM2, HDMX, and a protein (e.g., a BET protein, e.g., BRD4), (b) a means for inhibiting nuclear export, and (c) a pharmaceutically acceptable carrierBRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 presents a schematic overview of how a cancer-causing E3 ligase, HDM2, binds and degrades p53, a critical tumor suppressor protein. However, targeting HDM2 with a chimera (referred to as stapled peptide (SP) PROTAC (SP-PROTAC) in FIG. 1) described herein prevents HDM2 from degrading p53 and instead is repurposed to degrade a cancer-causing protein. In this manner, the chimera described herein transforms a cancer-causing protein into a cancer-killing protein.

[0025] FIG. 2 presents exemplary chimeras that contain a stapled peptide that targets HDM2 and HDMX, a linker comprised of beta-alanine residue(s), and JQ1, which targets BET proteins. Top: the linker-JQl moiety is attached to a lysine at position 25 (numbered according to SEQ ID NO: 1) (SEQ ID NO:2). Bottom: the linker-JQl moiety is attached to the N-terminus of the stapled peptide (SEQ ID NO: 301). Position 21 (numbered according to SEQ ID NO: 1) may be E or A and position 28 (numbered according to SEQ ID NO:1) may be S or A. Lower case “a” is D-alanine. 8 is (R)-2-(7’- octenyljalanine; X is (S)-2-(4’-pentenyl)alanine. 8 is cross-linked to X.

[0026] FIG. 3 is a depiction of a crystal structure of SP645 (SEQ ID NO:4) complexed with HDMX. L17 and Q25 (numbered according to SEQ ID NO: 1) are identified sites for installing the linker-small molecule moiety.

[0027] FIG. 4 shows graphs depicting TP53 status as the single most predictive genetic determinant for susceptibility to the chimera SP6924-E21 A / Q25K-PAla-JQl (SEQ ID NO: 14), consistent with the drug’s mechanism of action; dam = damaging mutations; hs = hotspot mutations; wt is wild type. The left-hand graph displays the effect size (x-axis) and -loglO q value (y-axis) of each feature (mutation or gene) at a given dose (including logzAUC and log2lC50). The right-hand graph plots the log2AUC for TP53 (y-axis), where each point is a cell line in the box plots with different genotypes (x-axis) and the dashed line represents the average log2AUC across all cell lines.

[0028] FIG. 5 shows that there is a correlation between expression of BRD4, a BET protein target of the SP-PROTAC, and HDMX but not HDM2, highlighting the importance of multi-modal targeting that includes HDMX.

[0029] FIG. 6A shows that there is no advantage of treating an HDM2- overexpressing osteosarcoma cell line SJSA-1 with a SP-PROTAC (chimera, JQ1- (PAla)2-SP645) compared to the combination treatment of its components - the stapled p53 peptide SP645 and the small molecule JQ1 (IC50s, JQ1 0.29 uM, SP645 3.40 uM, JQ1+SP645 0.84 uM, SP-PROTAC 0.46 uM). In contrast, FIG. 6B shows that upon dual expression of HDM2 and HDMX, the SJSA-X cell line is notably more susceptible to the SP-PROTAC (chimera, JQl-(PAla)2-SP645) by 6-fold compared to SJSA-1 and 8.5-fold more susceptible to the SP-PROTAC (chimera, JQl-(PAla)2-SP645) as compared to combined treatment of the individual components SP645 and JQ1. (IC50s, JQ1 0.25 uM, SP645 >10 uM, JQ1+SP645 0.64 uM, SP-PROTAC 0.075 uM). FIG. 6C shows that a small molecule PROTAC comprised of a selective HDM2 inhibitor molecule, RG-7388, and JQ1 (Al 874) has no advantage over the combination of individual components upon treating the SJSA-1 or SJSA-X cell line. (IC50s, JQ1 0.42 uM, RG7388 0.067 uM, JQ1+RG7388 0.043 uM, A1874 0.10 uM). FIG. 6D shows that the added expression ofHDMX in the SJSA-X cell line weakens the activity of the small molecule HDM2 inhibitor RG-7388 and Al 874, with Al 874 activity tracking with single agent JQ1 activity (IC50s, JQ1 0.25 uM, RG7388 3.73 uM, JQ1+RG7388 0.16 uM, A1874 0.25 uM).

[0030] FIG. 7A shows that E21A mutagenesis in the context of the SP-PROTAC SP6924 Q25K-pAla-JQl enhances cytotoxicity of the SJSA-X osteosarcoma cell line by 2.6-fold (IC50s, SP-PROTAC SP6924 Q25K-pAla-JQl 116 nM vs. SP-PROTAC SP6924-E21A Q25K-PAla-JQl 44 nM). FIG. 7B shows that E21A mutagenesis in the context of the SP-PROTAC JQl-(PAla)2-SP6924 enhances cytotoxicity of the SJSA-X osteosarcoma cell line by 10.3-fold (IC50s, JQl-(PAla)2-SP6924 350 nM, JQl-(PAla)2- SP6924-E21A 34 nM).

[0031] FIG. 8A demonstrates the specificity of action of SP-PROTAC SP6924- E21A / Q25K-pAla-JQl in killing SJSA-X osteosarcoma cells in that F19A mutagenesis impairs cytotoxicity by 47.3-fold (IC50s, SP6924-E21A / Q25K-PAla-JQl 44 nM, SP6924-F19A / E21A / Q25K-pAla-JQl 2080 nM). FIG. 8B demonstrates the specificity of action of SP-PROTAC JQl-(PAla)2-SP6924-E21A in killing SJSA-X osteosarcoma cells in that F19A mutagenesis, which impairs SP6924 binding to HDM2 / HDMX, blunts cytotoxicity by 16.4-fold (IC50s, JQl-(pAla)2-SP6924-E21A 34 nM, JQl-(pAla)2- SP6924-F19A / E21A 557 nM).

[0032] FIG. 9A shows that the SP-PROTAC JQl-(PAla)2-SP6924-E21 A effectively kills U2OS osteosarcoma cells that express wild-type p53 (IC50, 61 nM), with the F19A point mutation markedly decreasing cytotoxic activity (IC50, 3.3 uM), consistent with the mutation impairing SP6924 binding to HDM2 / HDMX. Figure discloses SEQ ID NOS 302-303, respectively, in order of appearance. In contrast, FIG. 9B shows that in the context of a p53-mutant osteosarcoma cell line, SAOS2, the SP-PROTAC JQl-(PAla)2- SP6924-E21A has relatively weak cytotoxic activity (IC50, 1.1 uM) that is similar to its F19A mutant, consistent with cytotoxicity that derives from BET inhibition by JQ1 alonein the absence of an intact p53 pathway. Figure discloses SEQ ID NOS 302-303, respectively, in order of appearance.

[0033] FIG. 10A shows that the three-in-one mechanism (HDM2 / HDMX / BET) of SP6924-E21A / Q25K-PAla-JQl is marginally more effective than the two-in-one mechanism (HDM2 / BET) of Al 874 in SJSA-1 osteosarcoma cells that overexpress HDM2, with Fl 9A point mutagenesis markedly blunting the activity of the SP-PROTAC as a specificity control (IC50s, SP-PRO2.3 23 nM, A1874 84 nM, SP-PRO2.3 F19A 2 uM). Notably, FIG. 10B shows that, upon added HDMX expression, SP6924- E21A / Q25K-0Ala-JQl is markedly more potent than A1874 in killing the SJSA-X osteosarcoma cells, with F19A point mutagenesis markedly blunting the activity of the SP-PROTAC as a specificity control (IC50s, SP-PRO2.3 4.2 nM, Al 874200 nM, SP- PRO2.3 F19A 1.3 uM). FIG. 10C shows that in the context of mutant p53, SP6924- E21 A / Q25K-PAla-JQl and A1874 are as ineffective as the F19A point mutant control, such that cytotoxicity only derives from BET inhibition by JQ1 in the absence of an intact p53 pathway, resulting in all the compounds now having the same weakened activity.

[0034] FIG. 11 shows the cytotoxicity of SP6924-E21 A / Q25K-P Ala- JQ 1 in U2OS osteosarcoma cells that retain wild-type p53 (IC50, 174 nM). F19A point mutagenesis abrogates cytotoxicity, highlighting the specificity of action of the SP-PROTAC.

[0035] FIG. 12 shows that the SP-PROTAC SP6924-E21A / Q25K-pAla-JQl (IC50, 87 nM) is markedly more potent than Al 874 (IC50, >5 uM) in killing p53 wild-type BT869 diffuse intrinsic pontine glioma (DIPG) neurospheres, with Fl 9A point mutagenesis markedly blunting the activity (IC50, >5 uM) of the SP-PROTAC as a specificity control.

[0036] FIG. 13 shows that the SP-PROTAC JQl-(pAla)2-SP6924-E21A (IC50, 8.7 nM) is markedly more potent than Al 874 in killing p53 wild-type BT869 DIPG neurospheres, with Fl 9A point mutagenesis blunting the activity of the SP-PROTAC (by~100-fold to a similar IC50 observed for A1874) as a specificity control. Figure discloses SEQ ID NOS 302-303, respectively, in order of appearance.

[0037] FIG. 14 shows that the SP-PROTAC SP6924-E21A / Q25K-pAla-JQl (IC50, 21 nM) is more potent than A1874 (IC50, 143 nM) in killing JEG3 choriocarcinoma cells, which express wild-type p53, HDM2 and HDMX.

[0038] FIG. 15 shows that the SP-PROTAC SP6924-E21A / Q25K-pAla-JQl (IC50, 12 nM) is more potent than Al 874 (IC50, 180 nM) in killing EOL1 leukemia cells, which express wild-type p53, HDM2 and HDMX.

[0039] FIG. 16 shows that the SP-PROTAC SP6924-E21A / Q25K-pAla-JQl is more potent than Al 874 by 4-fold and 8-fold in killing A2780 (left) and OVTOKO (right) ovarian cancer cells, respectively, with F19A point mutagenesis markedly blunting the activity of the SP-PROTAC as a specificity control (IC50s A2780: SP-PRO2.3 84 nM, A1874 340 nM, SP-PRO2.3 F19A 1.8 uM; OVTOKO: SP-PRO2.3 35 nM, A1874 274 nM, SP-PRO2.3 F19A 22 uM).

[0040] FIG. 17 shows that the SP-PROTAC SP6924-E21A / Q25K-pAla-JQl is more potent than A1874 by 6-fold and 4-fold in killing TOV21G (left) and PAI (right) ovarian cancer cells, respectively, with Fl 9A point mutagenesis markedly blunting the activity of the SP-PROTAC as a specificity control (IC50s TOV21G: SP-PRO2.3 29 nM, Al 874 178 nM, SP-PRO2.3 F19A 151 uM; PAI : SP-PRO2.3 122 nM, A1874463 nM, SP- PRO2.3 F19A 17 uM)..

[0041] FIG. 18 shows that the SP-PROTAC SP6924-E21A / Q25K-pAla-JQl is more potent than A1874 by 3-fold in killing LOVO colon cancer cells, with F19A point mutagenesis markedly blunting the activity of the SP-PROTAC as a specificity control (IC50s, LOVO: SP-PRO2.3 103 nM, A1874 295 nM, SP-PRO2.3 F19A 2.8 uM).

[0042] FIG. 19 is a series of graphs depicting the amount of killing T-ALL leukemic blasts in the peripheral blood mononuclear cells (PBMCs) isolated from the peripheral blood of newly diagnosed pediatric patients exposed to SP6924-E21A / Q25K-pAla-JQl (SEQ ID NO: 14; grey bottom line in each graph) or Al 874 (black top line in each graph).

[0043] FIG. 20 is a graph depicting ex vivo plasma stability at the indicated times post-treatment with the indicated chimeras. Sequences: SP6924-E21A / Q25K-pAla-JQl (SEQ ID NO: 14), IQl-(PAla)2-SP6924-E21A (SEQ ID NO: 17).

[0044] FIG. 21 are graphs showing the plasma concentration of SP6924-E21 A / Q25K-PAla-IQl (SEQ ID NO: 14) over time upon IV (top row) or IP (bottom row) administration at 3 mg / kg dosing to three mice per experimental arm.

[0045] FIG. 22 is a graph depicting the plasma concentrations of SP6924- E21A / Q25K-PAla-JQl (SEQ ID NO: 14) over time upon IV (circles) or IP (squares) administration at 3 mg / kg dosing to three mice per experimental arm.

[0046] FIG. 23A is a graph showing the plasma concentrations of SP6924- E21A / Q25K-pAla-JQl (SEQ ID NO: 14) over time upon IV or IP administration at 3 mg / kg dosing, 10 mg / kg dosing, and 30 m / kg dosing to three mice per experimental arm. FIG. 23B is a graph depicting the data of FIG. 23A, with an expanded y axis so that the IP data and lower dose IV data can be visualized.

[0047] FIG. 24 is a series of graphs showing the relative preservation of mouse body weight of SP-PRO2.3 -treated mice relative to vehicle-treated mice for animals bearing SJSA-X xenografted tumors as plotted by daily weight (top) and percent daily weight change relative to day 1 body weight (bottom). N=5 mice per arm with p<0.0025 for days 2-6 of treatment. SP-PRO 2.3 is SP6924-E21A / Q25K-pAla-JQl (SEQ ID NO: 14).

[0048] FIG. 25 is a series of graphs showing tumor regression in SP-PRO2.3 -treated mice and tumor progression in vehicle-treated mice as monitored by daily caliper measurement of tumors and plotted by daily tumor volume (top) and percent change in daily tumor volume relative to day 1 tumor volume. N=5 mice per arm with p<0.0062 for days 4-7 of treatment. SP-PRO 2.3 is SP6924-E21A / Q25K-pAla-JQl (SEQ ID NO: 14).

[0049] FIG. 26 is a graph showing tumor regression in SP-PRO2.3 -treated mice and tumor progression in vehicle-treated mice as monitored by daily caliper measurement of tumors and body weight and plotted by tumor volume / body weight. N=5 mice per armwith p<0.0035 for days 2-7 of treatment. SP-PRO 2.3 is SP6924-E21 A / Q25K-pAla-JQl (SEQ ID NO: 14).

[0050] FIG. 27 is a series of graphs showing that combining SP-PRO2.3 with Selinexor, a nuclear export inhibitor, enhances anti-cancer activity in SJSA-1 (top), SJSA-X (middle), and MCF-7 (bottom) cells. SP-PRO 2.3 is SP6924-E21A / Q25K-pAla- JQ1 (SEQ ID NO: 14).

[0051] FIG. 28 shows that the enhanced cytotoxicity observed upon combining SP- PRO2.3 and Selinexor is dependent on an intact p53 pathway and is thus selective, as reflected by the absence of a combinatorial effect in cells that lack p53, whether eliminated genetically (SAOS-2; top) or by protein degradation (HeLa [HPV D6]; bottom). SP-PRO 2.3 is SP6924-E21A / Q25K-PAla-JQl (SEQ ID NO: 14).

[0052] FIG. 29 shows SP-PRO2.3 effectively generated ternary complexes between the BD1 domain of BRD4 and HDM2 (left) and HDMX (right). Control elution profiles are shown for the individual proteins alone, including BRD4, HDM2, and HDMX, and their combinations, including BRD4 and HDM2 and BRD4 and HDMX.

[0053] FIG. 30 is an in vitro ubiquitylation assay demonstrating the natural selectivity of HDM2 for p53, as evidenced by time-dependent laddering of p53 but not BRD4 (left 4 lanes). However, in the presence of SP-PRO2.3, the primary target of HDM2 is switched from p53 to BRD4, which exhibits newfound laddering at the expense of p53 (right 4 lanes).

[0054] FIG. 31 shows that upon treatment of SISA-X cells with 100 nM SP-PRO2.3, we observed prompt degradation of BRD4 within 4 hours, coinciding with time- dependent upregulation of p53, which peaked at 12 hours and triggered both a surge in p21 and counter-elevation of HDM2 and HDMX by 24 hours. The reduction of p53 levels observed between 12 and 24 hours is consistent with the characteristic negative feedback loop of the p53 pathway.

[0055] FIG. 32A shows a quantitative proteomics analysis revealing that SP-PRO2.3 treatment (1 pM, 24 h) of SJSA-X cells caused a striking reduction of BET protein levels(e.g., BRD2-4) and marked upregulation of p53 pathway proteins (e.g., p53, p21, HDM2, HDMX).

[0056] FIG. 32B shows a quantitative proteomics analysis revealing that SP-PRO2.3 treatment (500 nM, 18 h) of primary pediatric T-ALL cells caused a striking reduction of BET protein levels (e.g., BRD2,4) and marked upregulation of p53 protein.

[0057] FIG. 33A shows images of tumors removed after 7 days of treatment from each of 3 euthanized mice that received either vehicle or SP-PRO2.3 (10 mg / kg / day IV), demonstrating the striking anti-tumor effect of SP-PRO2.3.

[0058] FIG. 33B shows a quantitative proteomics (TMTproTM 18-plex) analysis of the tumor specimens in FIG. 33 A, demonstrating marked downregulation of BRD4 and persistent upregulation of HDMX, in SP-PRO2.3- vs. vehicle-treated mice at day 7.

[0059] FIG. 34A shows an in vivo efficacy experiment whereby mice bearing SJSA- X osteosarcoma tumors received reduced daily dosing levels of 3.0, 1.0, or 0.3 mg / kg / day IV (mean tumor volume ± SD of 216 ± 47 mm3on day 1 of treatment) and correspondingly demonstrated dose-responsive anti-tumor activity and associated prolongation of survival. Data are mean tumor volume ± SEM as measured daily (n=5 mice per arm).

[0060] FIG. 34B shows an in vivo efficacy experiment whereby mice bearing SISA- X osteosarcoma tumors received reduced frequency of 3 mg / kg IV dosing on either Mon / Wed / Fri or Mon / Thurs (mean tumor volume ± SD of 100± 25 mm3on day 1 of treatment). Data are mean tumor volume ± SEM as measured daily (n=6-7 mice per arm).

[0061] FIG. 35 shows a high-throughput analysis of cancer cell susceptibility to SP- PRO2.3 treatment, as assessed by PRISM assay. The heat map reflects the relative viability of >900 genetically characterized cancer cells, as measured on triplicate samples after 5-day treatment with an 8-dose, 3-fold serial dilution of compound starting at 5pM.

[0062] FIG. 36 is a correlative mutagenesis plot revealing that disruptive p53 mutations were far and away the most predictive indicator of relative resistance to SP-PRO2.3, consistent with the critical role of p53 reactivation in the compound’s mechanism of action.

[0063] FIG. 37 show the tumor subtypes with greatest susceptibility to SP-PRO2.3, such as skin (including melanoma), kidney, soft tissue, ovarian, and hematopoietic cancers.

[0064] FIG. 38 shows that SP-PRO2.3 sensitivity correlated with cancer cell dependency on a series of p53-pathway related genes, including HDM2 (mechanistic target of SP-PRO2.3), and key p53 transcriptional targets implicated in pro-apoptotic signaling.

[0065] FIG. 39 shows GO analysis of the quantitative proteomics dataset derived from SP-PRO2.3 vs. vehicle treatment (1 pM, 24 h) of SJSA-X cells, again highlighting a series of enriched and mechanistically-relevant pathway signatures, with DNA damage response, signal transduction by p53 class mediator emerging as the top hit.

[0066] FIG. 40 shows the dose-response curves upon SP-PRO2.3 treatment of an exemplary series of susceptible cancer cells, including melanoma, kidney, ovarian, soft tissue, and hematopoietic cancers, as demonstrated by the negative log-fold change (LFC) plots. Data are mean LFC for experiments performed in technical triplicate.

[0067] FIG. 41 shows that SP-PRO2.3 exhibits no dose-responsive cytotoxic effect on non-transformed renal and endothelial cell lines, RPTEC and HUVEC, highlighting the therapeutic window / selectivity of SP-PRO2.3.

[0068] FIG. 42 shows that immunocompromised NSG mice bearing EOL-1 leukemia and treated with either vehicle or SP-PRO2.3 at 2 mg / kg / day IV, demonstrated marked suppression of leukemic growth by SP-PRO2.3, as demonstrated by CBC analysis performed prior to EOL-1 leukemia injection (baseline values) and then either at time of death (TOD) or termination of the experiment (TOE) after 7 days of treatment. The mean ± SD peripheral white blood cell (WBC) counts (xl03 / mL) for vehicle- and SP-PRO2.3- treated mice were respectively 3.82 ± 1.05 and 4.15 ± 2.48 at baseline and 286.4 ± 210.6 at TOD (vehicle-treated mice) and 33.2 ± 24.2 at TOE (SP-PRO2.3-treated mice). Asignificant increase in WBC count was observed in the vehicle group from baseline to endpoint (paired t-test, p=0.037), with the SP-PRO2.3- treated group showing significantly lower WBC counts at the experimental endpoint compared to the vehicle group (independent t-test, p=0.027). Plotted data are mean ± SD for WBC counts performed on n=4 animals per arm.

[0069] FIG. 43 shows the mean ± SD peripheral blast percentages for vehicle- and SP-PRO2.3 -treated mice were respectively 0.65 ± 0.59% and 1.2 ± 0.73% at baseline, and 33.3 ± 28.1% at TOD (vehicle-treated mice) and 3.48 ± 1.58% at TOE (SP-PRO2.3- treated mice). Blast percentage significantly increased in the vehicle group from baseline to endpoint (paired t-test, p=0.05), with the SP-PRO2.3 -treated group showing significantly lower percentage blasts compared to the vehicle group (independent t-test, p=0.039). Plotted data are mean ± SD for percentage blast counts performed on n=4 animals per arm.

[0070] FIG. 44 shows that SP-PRO2.3 -treated mice maintained a similar percentage of neutrophils (mean ± SD of 36.8 ± 18.9% and 47.6 ± 14.5% at baseline and at TOE, respectively), whereas vehicle-treated mice experienced a significant drop in neutrophil percentage (mean ± SD of 36.1 ± 13.6% and 6.33 ± 3.91% at baseline and at TOD, respectively; paired t-test, p=0.015), consistent with their high peripheral blast percentage. Plotted data are mean ± SD for percentage neutrophil counts performed on n=4 animals per arm.

[0071] FIG. 45 shows the relative suppression of leukemic growth by SP-PRO2.3 resulted in a notable survival advantage, with all vehicle-treated mice expiring between treatment days 4 and 6, in contrast to the survival of all SP-PRO2.3 -treated mice through the 7-day treatment period. Kaplan-Meier plot comparing vehicle- and SP-PRO2.3- treated groups (n=4 per arm) demonstrates a statistically significant difference in survival curves, as determined by the log-rank (Mantel-Cox) test (p=0.0058).

[0072] FIG. 46 shows that immunocompromised NSG mice bearing EOL-1 leukemia (leukemia injection on day 0) and treated with either vehicle or SP-PRO2.3 at 2mg / kg / day IV on days 4-7, demonstrated marked suppression of leukemic growth by SP- PRO2.3, as demonstrated by CBC analysis performed prior to EOL-1 leukemia injection (baseline values) and then within 24 hours of day 7 treatment. The mean ± SD peripheral white blood cell (WBC) counts (xlO3 / mL) for vehicle- and SP-PRO2.3-treated mice were respectively 6.36 ± 1.36 and 6.93 ± 2.82 at baseline and 66.3 ± 27.3 and 42.1 ± 8.2 at TOE, reflecting a relatively lower WBC count in the SP-PRO2.3 -treated group after 7 days of treatment (independent t-test, p=0.03). Plotted data are mean ± SD for WBC counts performed on n=6 animals per arm.

[0073] FIG. 47 shows that the mean ± SD peripheral blast and neutrophil percentages for vehicle- and SP-PRO2.3 -treated mice were respectively 26.5 ± 0.20.7% and 9.4 ± 2.5%, and 12.2 ± 6.2% and 17.9 ± 4.6%, after 7 days of treatment (independent t-tests, percentage blasts, p=0.03; percentage neutrophils, p=0.05), reflecting relative suppression of blasts and preservation of neutrophils by SP-PRO2 3 Plotted data are mean ± SD for percentage blast and neutrophil counts performed on n=6 animals per arm at TOE.DETAILED DESCRIPTIONCHIMERAS AND METHODS OF MAKING THE SAME

[0074] HDM2 and HDMX are two negative regulators of the tumor suppressor protein p53. Whereas HDM2 binds and destroys p53, HDMX binds and sequesters p53, thereby blocking the anti -cancer activity of p53 and promoting the development, maintenance and chemoresistance of diverse subtypes of adult and pediatric cancers.

[0075] An exemplary amino acid sequence of human p53 is shown below (GenBank Accession No. CAA26306) (the transactivation domain is in bold): MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWF TEDPGPDEAPRMPEAAPPVAP AP AAPTP AAP APAPS WPL S S S VP SQKT YQGS YGF RLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIY KQ SQHMTEVVRRCPHHERC SD SDGL APPQHLIRVEGNLRVEYLDDRNTFRHS VV VPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTS S SPQPKKKPLDGEYF TLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKK LMFKTEGPDSD (SEQ ID NO: 1). The transactivation domain of human p53 consists of amino acids 14 to 29 of SEQ ID NO: 1 , i.e., the amino acid sequence LSQETFSDLWKLLPEN (SEQ ID NO: 100).

[0076] An exemplary amino acid sequence of human HDM2 is shown below (GenBank Accession No. NP_002383.2):MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVL FYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLV VVNQQESSDSGTSVSENRCHLEGGSDQKDLVQELQEEKPSSSHLVSRPSTSSRRR AISETEENSDELSGERQRKRHKSDSISLSFDESLALCVIREICCERSSSSESTGTPSN PDLDAGVSEHSGDWLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEV YQVTVYQAGESDTDSFEEDPEISLADYWKCTSCNEMNPPLPSHCNRCWALREN WLPEDKGKDKGEISEKAKLENSTQAEEGFDVPDCKKTIVNDSRESCVEENDDKIT QASQSQESEDYSQPSTSSSIIYSSQEDVKEFEREETQDKEESVESSLPLNAIEPCVIC QGRPKNGCIVHGKTGHLMACFTCAKKLKKRNKPCPVCRQPIQMIVLTYFP (SEQ ID NO:101).

[0077] An exemplary amino acid sequence of human HDMX is shown below (GenBank Accession No. NP_002384.2):MTSFSTSAQCSTSDSACRISPGQINQVRPKLPLLKILHAAGAQGEMFTVKEVMHY LGQ YIM VKQL YDQQEQHMVYCGGDLLGELLGRQ SF S VKDP SPL YDMLRKNL VT LATATTDAAQTLALAQDHSMDIPSQDQLKQSAEESSTSRKRTTEDDIPTLPTSEH KCIHSREDEDLIENLAQDETSRLDLGFEEWDVAGLPWWFLGNLRSNYTPRSNGS TDLQTNQDVGTAIVSDTTDDLWFLNESVSEQLGVGIKVEAADTEQTSEEVGKVS DKKVIEVGKNDDLEDSKSLSDDTDVEVTSEDEWQCTECKKFNSPSKRYCFRCW ALRKDWYSDCSKLTHSLSTSDITAIPEKENEGNDVPDCRRTISAPVVRPKDAYIK KENSKLFDPCNSVEFLDLAHSSESQETISSMGEQLDNLSEQRTDTENMEDCQNLLKPCSLCEKRPRDGNIIHGRTGHLVTCFHCARRLKKAGASCPICKKEIQLVIKVFIA (SEQ ID NO: 102).

[0078] Featured herein is a new form of the SP-PROTAC that represents a platform technology to simultaneously (1) bind and recruit HDM2 for degrading a disease-causing protein (while also disrupting the p53 / HDM2 interaction to reactivate p53); (2) bind and inhibit HDMX (to inhibit p53 / HDMX interactions to maximally reactivate p53); and (3) link the latter two to a compound that binds a protein (e.g., a protein that is involved in or that is causative of disease, e.g., cancer), which can be degraded by HDM2. All of these events happen at about the same time in the same cell with the same pharmacokinetics and pharmacodynamics to achieve a new form of disease therapy - essentially a “3-in-l” effect.

[0079] Provided herein are chimeras (also referred to as SP-PROTACs) that act as protein degradation-inducing moieties. The chimeras provided herein comprise a stapled peptide (which binds to HDM2 and HDMX) conjugated to a compound (which binds a protein (e.g., a protein that is involved in or that is causative of a disease, e.g., a cancer)) (e.g., a compound that binds to a BET protein, e.g., JQ1). Without being bound by any particular theory, the chimeras described herein bind HD M2 and HDMX, preventing or reducing HDM2 -mediated degradation of p53 and repurposing HDM2 to mediate degradation of a protein that is involved in or that is causative of a disease (e.g., a cancer) transforming the pathological protein into a disease-killing protein. See, e.g., FIG. 1, bottom panel.

[0080] The stapled peptide of the chimeras described herein are based on SP645 (SEQ ID NO:4) and SP6924 (SEQ ID NO:6) (e.g., comprising 1, 2, 3, 4, or 5 substitutions relative to SEQ ID NO:4 or 6, wherein the substitutions are not at positions 4 and 11). The sequence of SP645 is LTFX1EYWAQLX2SAA, wherein Xi is (R)-2-(7’- octenyl)alanine, X2 is (S)-2-(4’-pentenyl)alanine, and Xi is cross-linked to X2 (SEQ ID NO: 4). The sequence of SP6924 is LTFX1EYWAQLX2SAAAAX3, wherein Xi is (R)-2- (7’-octenyl)alanine, X2 is (S)-2-(4’-pentenyl)alanine, X3 is D-alanine, and Xi is cross-linked to X2 (SEQ ID NO:6). Exemplary stapled peptides based on SP645 and SP6924 are provided in Table 1 and in the chimeras of Table 2 in the working examples.

[0081] The compound (e.g. JQ1) of the chimera is attached to the stapled peptide of the chimeras described herein at the position corresponding to Q9 of SEQ ID NO:4 or SEQ ID NO:6 (corresponding to position 25 of SEQ ID NO:1). The position corresponding to Q9 of SEQ ID NO:4 or SEQ ID NO: 6 may be substituted to attach the compound. For example, the position corresponding to Q9 of SEQ ID NO:4 or SEQ ID NO:6 is substituted with another amino acid, e.g., any amine containing amino acid, e.g., diaminopropionic acid, diaminobutanoic acid, ornithine, lysine, homolysine, optionally wherein the substituted amino acid is conjugated to the compound via a linker. In some instances, the position corresponding to Q9 of SEQ ID NON or SEQ ID NO:6 is substituted with (NH-(CH2)m-C0)n, wherein m is 1-11 and n is 0-5, conjugated to the compound. In some instances, the position corresponding to Q9 of SEQ ID NON or SEQ ID NO:6 is NH-(CH2-CH2-O)n-CH2-CH2-CO, wherein n is 1-5, conjugated to the compound. For example, in some instances the position corresponding to Q9 of SEQ ID NON or SEQ ID NO:6 is substituted with lysine, and the substituted lysine is attached to the compound (e.g., JQ 1 ) via a linker. See, e.g., Formula II below as an example of a Q9K substitution (numbered according to SEQ ID NON or SEQ ID NO:6, corresponding to position 25 of SEQ ID NO: 1) conjugated to the compound (specifically, JQ 1 ) via a linker(specifically, P-alanine). Linkers for use in conjugating a compound (e.g., JQ 1) to the stapled peptide of the chimeras described herein include (P-alanine)n, (ethylene glycol)n, and (y-Aminobutyric acid)n, wherein n is 1 to 5. In some instances, the linker is (P-alanine)n, wherein n is 1 to 5. In some instances, the linker is (P-alanine)n, wherein n is 2. In some instances, the linker is (P-alanine). In some instances, the linker is a linker depicted in FIG. 2. In some instances, the linker is 5-13 atoms (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13) in length from the C-alpha of position 9 (numbered according to SEQ ID NON or SEQ ID NO:6) to the compound (e.g., JQ 1). For example, in some instances, the position corresponding to Q9 of SEQ ID NON or SEQ ID NO:6 is substituted with a lysine and the lysine is attached to a linker of 5-13 atoms in length from the C-alpha of Q9K(numbered according to SEQ ID NO:4 or SEQ ID NO:6) to the compound (e.g., JQ1). In some instances, the linker is 5 atoms in length from the C-alpha of position 9 (numbered according to SEQ ID NO:4 or SEQ ID NO:6) to the compound (e g., JQ1). In some instances, the linker is 7 atoms in length from the C-alpha of position 9 (numbered according to SEQ ID NO:4 or SEQ ID NO:6) to the compound (e g., JQ1). In some instances, the linker is 11 atoms in length from the C-alpha of position 9 (numbered according to SEQ ID NO:4 or SEQ ID NO:6) to the compound (e g., JQ1). In some instances in which the compound is JQ1, the linker may be appended to JQ1 by replacing the t-butyl ester moiety of JQ1 with an amide linker (see FIG. 2 and Formula II). Where discussed herein in respect to the disclosed chimeras, it is apparent to one skilled in the art that JQ1 attached to the linker has the structure of Formula I:(Formula I), i.e., wherein the tert-butyl ester is replaced by an amide bond or an ester bond with the linker (e.g., as in Formula II).

[0082] “Peptide stapling” is a term coined from a synthetic methodology wherein two olefin-containing side-chains (e.g., a, a-di substituted non-natural amino acids with olefinic side chains) present in a peptide chain are covalently joined (“stapled together”) using a ring-closing metathesis (RCM) reaction to form a cross-linked ring (see, e.g., Blackwell etal., J. Org. Chem., 66: 5291-5302, 2001; Angew et al., Chem. Int. Ed. 37:3281, 1994, each of which is incorporated by reference herein in their entireties). “Peptide stapling” includes the joining of two double bond-containing side-chains, triplebond-containing side-chains, or a double bond-containing side chain and a triple bond- containing side chain, which may be present in a peptide chain, using any number of reaction conditions and / or catalysts to facilitate such a reaction, to provide a singly “stapled” peptide. Additional description regarding making and use of hydrocarbon stapled peptides can be found, e.g., in U.S. Patent Publication Nos. 2012 / 0172285, 2010 / 0286057, and 2005 / 0250680, the contents of all of which are incorporated by reference herein in their entireties. Exemplary amino acid pairs that may be used for stapling include (i) (R)-2-(7'-octenyl)alanine and (S)-2-(4'-pentenyl)alanine; and (ii) (R)- 2-(4'-pentenyl)alanine and (S)-2-(7'-octenyl)alanine.

[0083] In some instances, the chimera or the pharmaceutically acceptable salt thereof comprises a stapled peptide conjugated to a compound, wherein the stapled peptide comprises or consists of AC-LTFX1X2YWAX3X4X5X6AAX7-NH2 (SEQ ID NO: 19), wherein Xi is an a, a-di substituted non-natural amino acid with an olefinic side chain cross-linked to Xs, X2 is E or A, X3 is: (1) an amino acid (e.g., any amine containing amino acid, e.g., diaminopropionic acid, diaminobutanoic acid, ornithine, lysine, homolysine), (2) (NH-(CH2)m-C0)n, wherein m is 1-11 and n is 0-5, or (3) NH-(CH2- CH2-O)n-CH2-CH2-CO, wherein n is 1-5, conjugated to the compound, optionally via the linker, optionally wherein X3 is lysine conjugated to the compound via the linker, X4 is leucine or cyclobutylalanine, X5 is an a, a-di substituted non-natural amino acid with an olefinic side chain cross-linked to Xi, Xr> is S or A, X7 is absent or AAXs, wherein Xs is D-alanine, and Ac is an acetyl group. In some instances, the stapled peptide comprises the sequence of SEQ ID NO: 19. In some instances, the stapled peptide is 14 to 19 (e.g., 14, 15, 16, 17, 18, 19) amino acids in length. In some instances, the stapled peptide consists of the sequence of SEQ ID NO: 19. Stapling amino acids that can be used at positions Xi and X5 of SEQ ID NO: 19 are known in the art and include a,a-disubstituted non-natural amino acids with olefinic side chains that can be cross-linked, optionally by ring closing metathesis reaction, such as (R)-2-(7'-octenyl)alanine and (S)-2-(4'- pentenyl)alanine, respectively, or (R)-2-(4'-pentenyl)alanine and (S)-2-(7'- octenyl)alanine, respectively. In some instances, Xi is (R)-2-(7’-octenyl)alanine and Xsis (S)-2-(4’-pentenyl)alanine. In some instances, X2 is E. In some instances, X2 is A. In some instances, X3 is an amino acid (e.g., any amine containing amino acid, e.g., diaminopropionic acid, diaminobutanoic acid, ornithine, lysine, homolysine) conjugated to the compound, optionally via the linker. In some instances, X3 is (NH-(CH2)m-C0)n, wherein m is 1-11 and n is 0-5, conjugated to the compound, optionally via the linker. In some instances, X3 is NH-(CH2-CH2-O)n-CH2-CH2-CO, wherein n is 1-5, conjugated to the compound, optionally via the linker. In some instances, X3 is lysine conjugated to the compound via the linker. In some instances, X4 is leucine. In some instances, X4 is cyclobutylalanine. In some instances, Xe is S. In some instances, Xe is A. In some instances, X7 is absent. In some instances, X7 is AAXx, wherein X8 is D-alanine. In some instances, the compound binds to a BET protein (e.g., BRD4). In some instances, the compound is JQ1.

[0084] In some instances, the stapled peptide comprises Ac- LTFXIX2YWAX3X4X5X6AAX7-NH2(SEQ ID NO: 19), wherein Xi is (R)-2-(7'- octenyl)alanine cross-linked to X5, X2 is A, X3 is a lysine conjugated to the compound via a linker (see, e.g., FIG. 2 and Formula II), X4 is leucine, X5 is (S)-2-(7'-octenyl)alanine, Xe is A, X7 is AAXs, wherein X8 is D-alanine, and Ac is an acetyl group. In some instances, the stapled peptide consists of the sequence of SEQ ID NO: 19), wherein Xi is (R)-2-(7'-octenyl)alanine cross-linked to X5, X2 is A, X3 is a lysine conjugated to the compound via a linker (see, e.g., FIG. 2 and Formula II), X4 is leucine, X5 is (S)-2-(7 - octenyl)alanine, Xe is A, X7 is AAXs, wherein X8 is D-alanine, and Ac is an acetyl group. In some instances, the stapled peptide is 14 to 19 amino acids in length. In some instances, the stapled peptide consists of the sequence of SEQ ID NO: 19. In some instances, the compound binds to a BET protein (e.g., BRD4). In some instances, the compound is JQ1.

[0085] In some instances, the stapled peptide comprises the sequence of SEQ ID NO: 19 with 1 or 2 amino acid substitutions, wherein the 1 or 2 amino acid substitutions are not at Xi, X3, or X5, and wherein the stapled peptide binds to HDMX and HDM2. In some instances, the stapled peptide comprises the sequence of SEQ ID NO: 19, with 1 or2 amino acid substitutions. In some instances, the stapled peptide is 14 to 19 amino acids in length. In some instances, the stapled peptide consists of the sequence of SEQ ID NO: 19, with 1 or 2 amino acid substitutions. Stapling amino acids that can be used at positions Xi and Xs of SEQ ID NO: 19 are known in the art and include a, a-di substituted non-natural amino acids with olefinic side chains that can be cross-linked, optionally by ring closing metathesis reaction, such as (R)-2-(7'-octenyl)alanine and (S)-2-(4'- pentenyl)alanine, respectively, or (R)-2-(4'-pentenyl)alanine and (S)-2-(7'- octenyl)alanine, respectively. In some instances, Xi is (R)-2-(7’-octenyl)alanine and Xs is (S)-2-(4’-pentenyl)alanine. In some instances, X2 is E. In some instances, X2 is A. In some instances, X3 is an amino acid (e.g., any amine containing amino acid, e.g., diaminopropionic acid, diaminobutanoic acid, ornithine, lysine, homolysine) conjugated to the compound, optionally via the linker. In some instances, X is (NH-(CH2)m-C0)n, wherein m is 1-11 and n is 0-5, conjugated to the compound, optionally via the linker. In some instances, X3 is NH-(CH2-CH2-O)n-CH2-CH2-CO, wherein n is 1-5, conjugated to the compound, optionally via the linker. In some instances, X is lysine conjugated to the compound via the linker. In some instances, X4 is leucine. In some instances, X4 is cyclobutylalanine. In some instances, Xe is S. In some instances, Xe is A. In some instances, X7 is absent. In some instances, X7 is AAXx, wherein X8 is D-alanine. In some instances, the compound binds to a BET protein (e.g., BRD4). In some instances, the compound is JQ1.

[0086] In some instances, the 1 or 2 amino acid substitutions are conservative amino acid substitutions. In some instances, the 1 or 2 amino acid substitutions are non- conservative amino acid substitutions. In some instances, the 2 amino acid substitutions are a conservative amino acid substitution and a non-conservative amino acid substitution. Conservative amino acid substitutions suitable for inclusion in the stapled peptides disclosed herein are discussed below can include substitutions in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0087] In some instances, the chimera comprises or consists of Ac- LTFX1X2YWAX3X4X5X6AAX7-NH2 (SEQ ID NO: 19), wherein Xi is an a, a- disubstituted non-natural amino acid with an olefinic side chain cross-linked to X5, X2 is E or A, X3 is an amino acid conjugated to the compound via a linker, optionally wherein X3 is lysine conjugated to the compound via a linker, X4 is leucine or cyclobutylalanine, X5 is an a, a-di substituted non-natural amino acid with an olefinic side chain cross-linked to Xi, Xe is S or A, X7 is absent or AAXs, wherein X8 is D-alanine, and Ac is an acetyl group. In some instances, the stapled peptide consists of the sequence of SEQ ID NO: 19. In some instances, the linker comprises (P-alanine)n, (ethylene glycol)n, or (y- Aminobutyric acid)n, wherein n is 1 to 5. In some instances, linker is P-alanine.

[0088] In some instances, the stapled peptide of the chimera comprises the following sequence: (i) AC-LTFX1EYWAKLX2SAA-NH2, wherein Xi is (R)-2-(7’-octenyl)alanine, X2 is (S)-2-(4’-pentenyl)alanine, Xi is cross-linked to X2, and Ac is an acetyl group (SEQ ID NO:5); (ii) AC-LTFX1EYWAKLX2SAAAAX3-NH2, wherein Xi is (R)-2-(7’- octenyl)alanine, X2 is (S)-2-(4’-pentenyl)alanine, X3 is D-alanine, Xi is cross-linked to X2, and Ac is an acetyl group (SEQ ID NO:7); or (iii) Ac- LTFX1AYWAKLX2AAAAAX3-NH2, wherein Xi is (R)-2-(7’-octenyl)alanine, X2 is (S)- 2-(4’-pentenyl)alanine, X3 is D-alanine, Xi is cross-linked to X2, and Ac is an acetyl group (SEQ ID NO:9); wherein the compound (e.g., JQ 1) of the chimera is attached to position K9 of SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 (see, e g., FIG. 2 and Formula II), optionally via a linker. In some instances, position K9 of SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 is substituted with ornithine, di aminobutanoic acid, or diaminopropionic acid and conjugated to the compound, optionally via a linker. In someinstances, the linker comprises (P-alanine)n, (ethylene glycol)n, or (y-Aminobutyric acid)n, wherein n is 1 to 5. In some instances, linker is (P-alanine).

[0089] In some instances, the stapled peptide comprises or consists of Ac- LTFX1AYWAX2LX3AAAAAX4-NH2, wherein Xi is (R)-2-(7’-octenyl)alanine cross- linked to X3, X2 is lysine conjugated to the compound via a linker (see, e.g., FIG. 2 and Formula II), X3 is (S)-2-(4’-pentenyl)alanine cross-linked to Xi, X4 is D-alanine, the linker is P-alanine, and Ac is an acetyl group (SEQ ID NO:200). In some instances, the stapled peptide consists of the sequence of SEQ ID NO:200.

[0090] In some instances, the stapled peptide comprises or consists of Ac- LTFX1AYWAX2LX3AAAAAX4-NH2, wherein Xi is (R)-2-(7’-octenyl)alanine cross- linked to X3, X2 is lysine conjugated to JQ1 via a linker (see, e.g., FIG. 2 and Formula II), X3 is (S)-2-(4’-pentenyl)alanine cross-linked to Xi, X4 is D-alanine, the linker is P- alanine, and Ac is an acetyl group (SEQ ID NO: 14). In some instances, the stapled peptide consists of the sequence of SEQ ID NO: 14.

[0091] The stapled peptide of the chimera can be N-terminal acetylated and / or C- terminal amidated. N-terminal acetylation and C-terminal amidation create modified proteins that mimic the native protein by reducing the overall charge of a peptide, thus increasing the metabolic stability of peptides and the ability to resist enzymatic degradation. N-terminal acetylation is a post-translational modification in which an acetyl group is appended to the N-terminal amino group of a peptide, altering the charge, hydrophobicity, and size of the N-terminus. N-terminal acetylation is catalyzed by N“- acetyltransferases, which accept acetyl-CoA as the donor for the transfer of the activated acetyl moiety to the N“-terminus of the protein (Linster et al., J Exp Bot. 2018 Aug 31;69(19):4555-4568). In some embodiments, an acetylation reaction includes deprotection of an Fmoc group, followed by reaction with an esterification agent (e.g., neat acetic anhydride) and an organic compound e.g., N,N-Diisopropylethylamine (DIPEA)). C-terminal amidation is a post-translational modification to include an amide group at the C-terminus. In some embodiments, the modified amino acid is followed by aglycine, which provides the amide group. During C-terminal amidation, the glycine is oxidized to form alpha-hydroxy-glycine. The oxidized glycine cleaves into the C- terminally amidated peptide and an N-glyoxylated peptide. In some instances, for peptide synthesis the synthesis is started with RINK-AMIDE resin, which renders the most C- terminal residue “amidated”.

[0092] In some instances, the chimera comprises or consists of the structure ofFormula II (SEQ ID NO: 14):(Formula II), or a pharmaceutically acceptable salt thereof.

[0093] In some instances, the chimera comprises or consists of the structure of Formula II, or a pharmaceutically acceptable salt thereof, except for 1 to 6 amino acid substitutions or deletions, wherein the 1 to 6 amino acid substitutions or deletions are not at the internal cross-link or at K9-|3-Alanine-JQ1, and wherein the 1 to 6 amino acid substitutions or deletions are selected from: A5E, LlOCyclobutylalanine, A12S, deletion of A15, deletion of A16, and deletion of D-alaninel7 (all numbered according to SEQ ID NO: 14).

[0094] In some instances, the chimera comprises a stapled peptide described in the working examples herein. In some instances, the chimera is a chimera described in theworking examples described herein. In some instances, the chimera is a chimera described in Table 2, except wherein JQ1 is replaced with a different compound (e.g., a different BET-binding compound, e.g., ABBV-075 (Mivebresib), I-BET151, 1-BET726, OTX015 (Birabresib), PFI-1).

[0095] The compounds of the chimeras described herein bind a protein, e g., a protein that is involved in or that is causative of disease, e.g., cancer. In some instances, the protein to which the compound of the chimera binds is a BET protein. In some instances, the BET protein is BRD2, BRD3, or BRD4. In some instances, the compound of the chimera binds BRD4. Nonlimiting examples of compounds that bind a BET protein are JQ1, ABBV-075 (Mivebresib), 1-BET151, 1-BET726, OTX015 (Birabresib), and PFI-1.

[0096] JQ1 is a small molecule that binds to a BET protein (e.g., BRD2, BRD3, and / or BRD4) (see Filippakopoulos etal., Nature. 2010;468(7327):1067-1073; WO 2011 / 143669, each of which is incorporated by reference herein in its entirety). The structure of JQ1 is:(Compound 1).

[0097] The chimeras described herein include pharmaceutically acceptable salts thereof (e.g., hydrochloride, sodium, sulfate, acetate, trifluoroacetate, phosphate or diphosphate, chloride, potassium, maleate, calcium, citrate, mesylate, nitrate, tartrate, aluminum, gluconate, or any combination thereof).

[0098] Also provided herein is a method of making any one of the foregoing chimeras or the pharmaceutically acceptable salts thereof. In some instances, the methodcomprises: (a) providing a peptide comprising the sequence: (i) Ac- LTFX1X2YWAX3X4X5X6AAX7-NH2 (SEQ ID NO OO), wherein: Xi is an a, a- disubstituted non-natural amino acid with an olefinic side chain cross-linked to X5, X2 is E or A, X3 is an orthogonally protected amino acid, optionally wherein X3 is orthogonally protected lysine, X4 is leucine or cyclobutylalanine, X5 is an a, a-di substituted non- natural amino acid with an olefinic side chain cross-linked to Xi, Xr> is S or A, X7 is absent or AAXs, wherein X8 is D-alanine, and Ac is an acetyl group; or (ii) SEQ ID NO:300 with 1 or 2 amino acid substitutions, wherein the 1 or 2 amino acid substitutions are not at Xi, X3, or X5, and wherein the peptide binds to HDMX and HDM2; (b) incorporating a linker onto X3, (c) coupling a compound to the linker, and (d) cross- linking the peptide, thereby making the chimera. In some instances, the method further comprises purifying the chimera. In some instances, the method further comprises formulating the purified chimera as a sterile pharmaceutical composition.

[0099] Also provided herein is a method of making any one of the foregoing chimeras or the pharmaceutically acceptable salts thereof. In some instances, the method comprises: (a) providing a peptide comprising the sequence: (i) Ac- LTFX1X2YWAX3X4X5X6AAX7-NH2 (SEQ ID NO: 20), wherein: Xi is an a, a- disubstituted non-natural amino acid with an olefinic side chain cross-linked to X5, X2 is E or A, X3 is an orthogonally protected amino acid, optionally wherein X3 is orthogonally protected lysine, X4 is leucine or cyclobutylalanine, X5 is an a, a-di substituted non- natural amino acid with an olefinic side chain cross-linked to Xi, Xe is S or A, X7 is absent or AAXs, wherein X8 is D-alanine, and Ac is an acetyl group; or (ii) SEQ ID NO:20 with 1 or 2 amino acid substitutions, wherein the 1 or 2 amino acid substitutions are not at Xi, X3, or X5, and wherein the peptide binds to HDMX and HDM2; (b) incorporating a linker onto X3, (c) coupling JQ1 to the linker, and (d) cross- linking the peptide, thereby making the chimera. In some instances, the method further comprises purifying the chimera. In some instances, the method further comprises formulating the purified chimera as a sterile pharmaceutical composition.

[0100] Methods suitable for obtaining (e.g., synthesizing), stapling, and purifying the peptides disclosed herein that can be incorporated into the chimeras of this disclosure are also known in the art (see, e.g., Bird et. Al., Methods in Enzymol., 446:369-386 (2008); Bird et al, Current Protocols in Chemical Biology, 2011 ; Walensky et al., Science, 305: 1466-1470 (2004); Schafmeister et al., J. Am. Chem. Soc., 122:5891-5892 (2000); U.S. Patent Application No. 12 / 525,123, filed March 18, 2010; and U.S. Patent No. 7,723,468, issued May 25, 2010, each of which are hereby incorporated by reference in their entirety).

[0101] More specifically, the chimeras described herein can be synthesized according to the following methods. Hydrocarbon-stapled peptides can be synthesized, purified, and quantitated using previously reported methods using Fmoc protected amino acids and HATU as the coupling reagent (see, e.g., Bird et al., Methods Enzymol., 446:369-86 (2008); Bird et al., Curr. Protoc. Chem. Biol., 3(3):99-l 17 (2011), each of which is incorporated by reference herein in its entirety). An orthogonally protected lysine (or an alternatively functionalized lysine analog such as ornithine, di aminobutanoic acid, or diaminopropionic acid) is incorporated into the peptide at position 25 (numbered according to SEQ ID NO:1, i.e., position 9 numbered according to SEQ ID NO:6), where the orthogonal protecting group is, for example, Mtt or ivDde. Once the peptide synthesis is complete, the N-terminus is deprotected (e.g., using piperidine) and, in some instances, an acetyl cap is incorporated at the N-terminus. The orthogonal protecting group is removed (e.g., using hydrazine treatment to remove the Dde group from lysine). After deprotection, a linker is incorporated onto the lysine side chain (or lysine analog), followed by stapling using Grubbs catalyst (generation 1) and coupling of JQ1 to the liberated amine of the linker. The peptides are then cleaved (e.g., for 1.5 hours) with, e.g., TFA and purified (e.g., by LCMS).

[0102] In some embodiments, the chimeras are substantially free of non-stapled peptide contaminants or are isolated. Methods for purifying peptides and chimeras include, for example, synthesizing the peptide or chimera on a solid-phase support. Following cyclization, the solid-phase support may be isolated and suspended in asolution of a solvent such as DMSO, DMSO / dichloromethane mixture, or DMSO / NMP mixture. The DMSO / dichloromethane or DMSO / NMP mixture may comprise about 30%, 40%, 50% or 60% DMSO. In a specific embodiment, a 50% / 50% DMSO / NMP solution is used. The solution may be incubated for a period of, e.g., 1, 6, 12 or 24 hours, following which the resin may be washed, for example with di chloromethane or NMP. In one embodiment, the resin is washed with NMP. Shaking and bubbling an inert gas into the solution may be performed.

[0103] Properties and functional activities of the chimeras described herein can be assayed, for example, using the methods described below and, e.g., in WO 2019 / 118893, which is hereby incorporated by reference in its entirety.

[0104] Binding of Chimeras to Protein Targets. Competitive fluorescence polarization assays can be performed to monitor the capacity of (1) the stapled peptide portion of a chimera described herein to retain binding affinity for its protein target and (2) the IQ1 component to retain binding affinity for its protein target. Exemplary fluorescence polarization methods for stapled peptides and molecular degrons include Pitter et al., Methods Enzymol 446: 387-408 (2008) and Nowak et al., Nat Chem Biol 14:706-714 (2018), each of which is incorporated by reference in its entirety. In celhilo degradation assays using GFP-labeled target protein substrate (e.g., GFP-BRD4) likewise can be used to confirm the capacity of chimeras described herein to penetrate intact cells and compete with positive control chimeras to inhibit induced degradation. Exemplary methods for such competitive cellular degradation assays can be found in, e.g., Nowak et al, Nat Chem Biol 14:706-714 (2018).

[0105] Monitoring Ubiquitination of Recombinant Protein Targets as Induced byChimeras. To monitor ubiquitination of protein targets (e.g., BRD4) in vitro, a commercial Mdm2 / HDM2 Ubiquitin Ligase Kit (R&D Systems, Cat. No. K-200B) can be employed. Briefly, chimera (10 pM), recombinant full length MDM2 (GST-tagged, 1 pM), El enzyme (UBE1, 50 nM), E2 enzyme (UBE2D3, 1 pM), ubiquitin (100 pM), ATP (1 mM), and recombinant target protein (100 nM) are combined in a 1.5 mb microtube inreaction buffer. The mixture is incubated at 37 degrees Celsius for six hours. Subsequently, 20 uL of reaction mixture is assayed using standard western blotting techniques whereby antibodies raised against the target protein are used to visualize the upward band shifts due to ubiquitination.

[0106] Monitoring Native Protein Degradation in Cellulo as Induced by Chimeras.To assay for intracellular protein degradation, cancer cells (e.g., SJSA-1, SJSA-X, U2OS) are passaged at 37 degrees Celsius in a humidity-controlled, CCh-equilibrated incubator in DMEM (Life Technologies, Grand Island, NY) culture medium (CM) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Pen Strep). One day before treatment, cells are passaged and plated in six well plates at a density of 100,000 cells / mL. After 24 hours, cells are treated with a stapled peptide degron chimera (e.g., 10 pM) for 0, 2, 4 and 6 hours after which they were harvested and lysed. Cell lysates are then assayed using standard western blotting techniques using an antibody raised against the target protein to assess protein level and an actin antibody for the loading control.

[0107] Monitoring the Impact of Target Protein Degradation Induced by Chimeras on Cancer Cell Viability. Chimeras that retain binding to both protein targets (i.e., HDM2 / HDMX and a BET protein, e.g., BRD4), achieve cellular uptake, and can access their dual targets in cellulo to induce targeted protein degradation can be assessed for their cytotoxic effect on cancer cells using established cell viability and apoptosis assays, including CELLTITER-GLO (Promega)and caspase 3 / 7 activation assays, performed as reported (e.g., Labelle et al., J Clin Invest 122:2018-31 (2012); Wachter et al., Oncogene, 36:2184-2190 (2017); Guerra et al., Cell Reports 24:3393-3403 (2018), each of which is incorporated by reference herein in its entirety). Specificity of action controls studies can be performed using, for example, point mutant peptides that cannot engage their protein target and / or cell lines that do not express the target protein and / or degrader protein of interest.PHARMACEUTICAL COMPOSITIONS

[0108] One or more of the chimeras described herein can be formulated for use as or in pharmaceutical compositions. Thus, provided herein are pharmaceutical compositions comprising a chimera described herein. Such compositions can be formulated or adapted for administration to a subject (e.g., human) via any route, e.g., any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA’s CDER Data Standards Manual, version number 004 (which is available at fda.give / cder / dsm / DRG / drg00301.htm).

[0109] In some instances, pharmaceutical compositions can include a therapeutically effective amount of one or more chimeras described herein. In some instances, the therapeutically effective amount is an amount or a concentration of the one or more chimeras described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome e.g., treatment of a cancer).

[0110] Pharmaceutical compositions of this invention can include one or more chimeras and a pharmaceutically acceptable carrier and / or vehicle. In some instances, pharmaceutical compositions described herein can further include one or more additional therapeutic agents in amounts effective for achieving a modulation of disease or disease symptoms (e.g., a cancer). In some instances, the pharmaceutical composition further comprises a nuclear export inhibitor (e.g., a therapeutically effective amount of the nuclear export inhibitor). Non-limiting examples of nuclear export inhibitors include selinexor and xpovio. In some instances, the nuclear export inhibitor is selinexor.

[0111] The term “pharmaceutically acceptable carrier or adjuvant” refers to a carrier or adjuvant that may be administered to a patient, together with a chimera of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutically effectiveamount of the chimera. Pharmaceutically acceptable carriers and adjuvants are known in the art.METHODS OF USE

[0112] The chimeras (and pharmaceutical compositions comprising the same) disclosed herein can facilitate degradation of a protein (e.g., a BET protein, e.g., BRD2, BRD3, or BRD4) to which the compound of the chimera binds. In some instances, the protein to which the compound of the chimera binds is a protein that is involved in or that is causative of a disease, e.g., a cancer). In certain instances, the protein is a BET protein (e.g., BRD2, BRD3, or BRD4). In certain instances, the protein is BRD2. In certain instances, the protein is BRD3. In certain instances, the protein is BRD4. Thus, in some instances, provided herein is a method of degrading a protein (e.g., a BET protein, e.g., BRD2, BRD3, and / or BRD4), comprising administering a therapeutically effective amount of a chimera (or a pharmaceutical composition comprising the chimera) described herein to a subject (e.g., a human) in need thereof, wherein the compound of the chimera binds the protein.

[0113] The disclosure also features methods of using any of the chimeras (or pharmaceutical compositions comprising the chimeras) described herein for the prophylaxis and / or treatment of a disease (e.g., a cancer or other disease driven by or related to the protein to which the compound of the chimera binds). The terms “treat” or “treating” include alleviating, inhibiting, or ameliorating the disease or condition (e.g., a cancer) from which the subject (e.g., a human) is suffering. Thus, also provided herein is a method of treating a disease (e.g., a cancer or other disease driven by or related to the protein to which the compound of the chimera binds) in a subject (e.g., human) in need thereof, the method comprising administering to the subject (e.g., human) a therapeutically effective amount of a chimera (or pharmaceutical composition comprising the same) described herein. The diseases that may be treated with the chimeras of the disclosure are driven by or related to the protein to which the compound (e.g., JQ1) of thechimera binds. For instance, a chimera described herein comprising a compound (e.g., JQ1) that binds to a BET protein (e.g., BRD4) may be used to treat a cancer (e.g., a cancer expressing or encoding a BET protein, e g., BRD4). In some instances, the disease is a cancer. In some instances, the cancer is a sarcoma, a brain cancer, a skin cancer, an endometrial cancer, a blood cancer, or a urinary tract cancer. In some instances, the cancer is a melanoma, a leukemia, lymphoma, or other hematologic malignancy or solid tumor. In some instances, the solid tumor is a melanoma, a breast cancer or a lung cancer. In some instances, the cancer is a pediatric cancer. In some instances, the method comprises administering (e.g., subcutaneously, intravenously, intramuscularly) to the subject (e.g., a human) a chimera comprising the structure of Formula II (SEQ ID NO: 14)(Formula II), or a pharmaceutically acceptable salt thereof.

[0114] In some instances, the disease (e.g., a cancer) expresses or encodes functional or wild type p53. In some instances, the disease (e.g., a cancer) expresses wild type p53. In some instances, functional p53 includes a wild type Tp53 gene or a Tp53 gene having one or more mutations, as compared to the corresponding wild type Tp53 gene, that do not result in complete loss of any essential function in the protein encoded by the functional gene, as compared to the p53 protein encoded by the corresponding wild typeTp53 gene. In some instances, functional p53 protein is a wild type p53 protein or a p53 protein having one or more amino acid changes, as compared to the corresponding wild type p53 protein (e.g., SEQ ID NO: 1), that do not result in complete loss of any essential function in the functional p53 protein, as compared to the corresponding wild type protein. In some instances, a wild type Tp53 gene refers to a germ-line Tp53 gene having a nucleic acid sequence that occurs in non-cancerous, somatic cells. See, e.g., http: / / p53.iarc.fr / p53Sequences.aspx and http: / / p53.iarc.fr / p53Sequence.aspx for exemplary human p53 wild type gene sequences. In some instances, a wild type p53 protein refers to a protein encoded by a wild-type Tp53 gene, or by a Tp53 gene with one or more silent mutations or polymorphisms. An exemplary wild type human p53 has the amino acid sequence of SEQ ID NO: 1. Methods of determining whether a disease (e.g., a cancer) expresses or encodes functional or wild type p53 (gene or protein) and of evaluating p53 activity are known in the art, see, e.g., WO 2017 / 165617, which is incorporated by reference herein in its entirety. In some instances, the present disclosure features methods of treating a cancer or tumor that expresses wild type p53 in a human subject in need thereof, comprising administering (e.g., subcutaneously, intravenously, intramuscularly) to the human subject a chimera comprising the structure of Formula II (SEQ ID NO: 14)(Formula II), or a pharmaceutically acceptable salt thereof.

[0115] In some instances, the disease (e.g., a cancer) expresses or encodes HDM2. In some instances, the disease (e.g., a cancer) expresses or encodes HDMX. In some instances, the disease (e.g., a cancer) expresses wild type or functional p53 and HDMX. In some instances, the disease (e.g., a cancer) expresses or encodes the protein to which the compound (e.g., JQ1) of the chimera binds. For instance, in some instances in which the compound of the chimera binds to a BET protein (e.g., in which the compound is JQ1), the cancer expresses or encodes a BET protein (e g., BRD2, BRD3, and / or BRD4). In some instances in which the compound of the chimera binds to a BET protein, the disease (e.g., a cancer) expresses BRD4. In certain instances in which the compound of the chimera binds to a BET protein, the cancer expresses wild type or functional p53, HDMX, and BET proteins. In certain instances, in which the compound of the chimera binds to a BET protein, the disease (e.g., a cancer) expresses wild type or functional p53, HDM2, and BET proteins. In certain instances in which the compound of the chimera binds to a BET protein, the disease (e.g., a cancer) expresses wild type or functional p53, HDMX, HDM2, and BET proteins.

[0116] In some instances, the method further comprises administering to the subject (e.g., human) a therapeutically effective amount of a second therapeutic agent. In someinstances, the second therapeutic agent is a nuclear export inhibitor. Non-limiting examples of nuclear export inhibitors include selinexor and xpovio. In some instances, the nuclear export inhibitor is selinexor.

[0117] In some instances, the methods of treating further include selecting a subject and administering to the subject a therapeutically effective amount of one or more of the chimeras herein (or a pharmaceutical composition comprising the same), and optionally repeating administration as required for the prophylaxis or treatment of a disease or disorder described herein (e.g., a cancer). A subject can be selected for treatment based on, e.g., determining that the subject has a disease (e.g., a cancer) that expresses HDM2 and / or HDMX.

[0118] A therapeutically effective amount of a chimera described herein (or a pharmaceutical composition comprising the same) can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a chimera (z.e., an effective dosage) depends on the chimera selected. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the chimera described herein can include a single treatment or a series of treatments. For example, therapeutically effective amounts can be administered at least once.

[0119] Specific dosage and treatment regimens for any particular subject will depend upon a variety of factors, including the activity of the specific chimera (or pharmaceutical composition comprising the same) employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the subject’s disposition to the disease, condition or symptoms, and the judgment of the treating physician.

[0120] Also provided herein is use of a chimera (or a pharmaceutical composition comprising the same) described herein in the preparation of a medicament for treating a disease (e.g., a cancer) in a subject (e.g., human) in need thereof.

[0121] The disclosure also features a method of selecting a subject (e.g., human) for treatment with a therapeutically effective amount of any one of the foregoing chimeras or pharmaceutically acceptable salts thereof. The method involves determining whether a diseased (e.g., cancerous) cell from the subject expresses wild type or functional p53. If the diseased (e.g., cancerous) cell expresses wild type or functional p53, the subject is selected for treatment with a therapeutically effective amount of any one of the foregoing chimeras or pharmaceutically acceptable salts thereof. In certain instances, the method further involves assessing whether the diseased (e.g., cancerous) cell expresses one or both of HDMX and HDM2. If the diseased (e.g., cancerous) cell expresses HDMX and HDM2, the subject is selected for treatment with a therapeutically effective amount of any one of the foregoing chimeras or pharmaceutically acceptable salts thereof. In certain instances, the method further involves assessing whether the diseased (e.g., cancerous) cell expresses the protein to which the compound of the chimera binds. If the diseased (e.g., cancerous) cell expresses the protein to which the compound of the chimera binds, the subject is selected for treatment with a therapeutically effective amount of any one of the foregoing chimeras or pharmaceutically acceptable salts thereof. In certain instances, the method further involves assessing whether the diseased (e.g., cancerous) cell expresses p53, HDM2, HDMX, and the protein to which the compound of the chimera binds. If the diseased (e.g., cancerous) cell expresses p53, HDM2, HDMX, and the protein to which the compound of the chimera binds, the subject is selected for treatment with a therapeutically effective amount of any one of the foregoing chimeras or pharmaceutically acceptable salts thereof. In certain instances, the method further involves assessing whether the cancer cell contains complexes of HDM2 / p53 and / or complexes of HDMX / p53. If the diseased (e.g., cancerous) cell contains such complexes, the subject is selected for treatment with a therapeutically effective amount of any one of the foregoing chimeras or pharmaceutically acceptable salts thereof. In certain instances,the subject is also administered a therapeutically effective amount of a second therapeutic agent, e.g., a nuclear export inhibitor, e.g., selinexor.

[0122] In some instances, the subject of the methods described herein is a human.EXAMPLES

[0123] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art can develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.EXAMPLE 1: DESIGN AND SYNTHESIS OF OPTIMIZED SP-PROTACS

[0124] PROteolysis Targeting Chimeras with Stabilized Peptides (also referred to herein as “SP-PROTACs”) were generated to achieve a 3-in-l mechanism of action for killing cancer cells by targeting HDM2, HDMX, and a BET protein (such as BRD2, BRD3, and / or BRD4) and converting the oncogene, HDM2, into a tumor suppressor by diverting its degradation of p53 to degradation of cancer protein BET (see FIG. 1 and FIG. 2). Exemplary chimeras were designed as depicted in FIG. 2 and incorporated stapled p53 peptides having distinct branch points for appending the JQ1 molecule (e.g., N-terminus or a lysine installed at position 25, numbered according to SEQ ID NO: 1, i.e., position 9 numbered according to SEQ ID NO:6).

[0125] Table 1 shows exemplary stapled peptide (SP) sequences that were incorporated into the chimeras, including mutants (Q25K, for appending the linker-small molecule moiety; E21 A, for improving potency of action; and F19A, for impairing binding activity and, thus, providing specificity-of-action negative control compounds). The stapled peptides of Table 1 can be N-terminally acetylated and C-terminallyamidated. Table 2 shows exemplary chimera sequences. FIG. 3 shows positions L17 and Q25 (numbered according to SEQ ID NO: 1, i.e., positions 1 and 9, respectively, numbered according to SEQ ID NO:6) for installing the linker-JQl moiety on the SP645 stapled peptide.

[0126] Table 1.

[0127] Table 2.

[0128] The chimeras were synthesized according to the following methods. Hydrocarbon-stapled peptides were synthesized, purified, and quantitated using previously reported methods using Fmoc protected amino acids and HATU as the coupling reagent (Bird et al., Methods Enzymol., 446:369-86 (2008); Bird et al., Curr. Protoc. Chem. BioL, 3(3):99-l 17 (2011), each of which is incorporated by reference herein in its entirety). When JQ1 was appended at position Q25K (position 9 numbered according to SEQ ID NO:6), an orthogonally Dde protected lysine (N-£-l-(4,4-dimethyl- 2,6-dioxocyclohex-l-ylidene)ethyl-L-lysine) was incorporated into the sequence. The N- terminus was deprotected using piperidine and either an acetyl cap (for JQ1 linkage at position Q25K) or linker (for JQ1 linkage at N-terminus) was incorporated. Hydrazine treatment to remove the Dde group from lysine followed acetylation, and stapling using Grubbs catalyst (generation 1) followed the incorporation of an N-terminal linker. After Dde deprotection, an Fmoc protected linker was incorporated onto the lysine side chainfollowed by stapling using Grubbs catalyst (generation 1) and then coupling of JQ1 to the piperidine liberated amine. Likewise, the last step for the N-terminal linker was also JQ1 incorporation via the piperidine liberated amine. The peptides were then cleaved for 1.5 hours with TFA and purified by LCMS.EXAMPLE 2: CANCER CELL SUSCEPTIBILITIES TO SP-PROTACS

[0129] Over 950 genetically characterized human cancer cell lines were treated with SP6924-E21A / Q25K-|3Ala-JQl (SEQ ID NO: 14) using Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) analysis (C. Yu et al. (2016) Nat. Biotechnol. 34;419-423, which is incorporated by reference herein in its entirety). SP6924-E21A / Q25K- PAla-JQl (SEQ ID NO: 14) showed robust dose-responsive and differential cytotoxicity across a panel of over 950 cancer cell lines representing a large diversity of cancer subtypes. Intriguingly, analysis of mutation data demonstrated that the presence of functional p53 was the most significant predictive factor for the efficacy of SP6924- E21A / Q25K-pAla-JQl (SEQ ID NO: 14) (Table 3 and FIG. 4), with cell lines harboring either damaging or hotspot mutations in p53 exhibiting far less sensitivity.

[0130] Table 3

[0131] Tabulation of the most sensitive cell lines to SP6924-E21A / Q25K-PAla-JQl (SEQ ID NO: 14) showed cancer subtypes of diverse origins, such as sarcomas (specifically, rhabdomyosarcoma and uterine sarcoma), as well as cancers originating in the brain (glioblastoma multiforme), skin (melanoma), endometrial tissue, blood (leukemia, lymphoma), and urinary tract (Table 4). Table 4 shows the top 15 most susceptible cancer cell lines among the more than 950 genetically characterized human cancer cell lines in the PRISM experiment.

[0132] Table 4

[0133] Correlation analysis using gene expression data from the Dependency Map (DepMap, Broad Institute) established a link between the expression levels of BRD4, a bromodomain and extraterminal (BET) protein that is a degradation target of the chimera, and HDMX, but not HDM2, underscoring the therapeutic significance of targeting HDMX in conjunction with BRD4 (FIG. 5).

[0134] Given the presence of sarcomas in the top 15 most susceptible cancer cell lines and the correlation between BRD4 and HDMX but not HDM2 in the PRISM experiment (Table 4 and FIG. 5), the findings were validated in an orthogonal model of two osteosarcoma cell lines that overexpress HDM2 and vary only by HDMX expression:SJSA-1 (CVCL 1697), and HDMX-overexpressing SJSA-X (M. Wade et al. (2008) Cell Cycle 7; 1973-1982) (Table 5).

[0135] Table 5

[0136] There was no marked advantage of treating the HDM2-overexpressing cell line SJSA-1 with SP-PROTAC chimera (JQl-(PAla)2-SP645 (SEQ ID NO: 12); IC50 0.46 uM) compared to combination treatment with the individual components (JQ1 and SP645 (SEQ ID NO:4); 0.84 uM), but SP-PROTAC (JQl-(PAla)2-SP645 (SEQ ID NO: 12))- treatment of the dual HDM2- and HDMX-expressing SJSA-X cell line was more efficacious by 6-fold (IC50, 0.075 uM) compared to SJSA-1 (IC50, 0.46 uM) (FIGs. 6A- 6B) and 8.5-fold more susceptible to the SP-PROTAC (JQl-(PAla)2-SP645 (SEQ ID NO: 12)) as compared to combined treatment of the individual components (JQ1 and SP645 (SEQ ID NO:4) in SJSA-X cells (IC50, 0.64 uM) (Table 5).

[0137] Upon evaluation of small molecule PROTAC Al 874 (comprised of a selective HDM2 inhibitor molecule RG-7388 and JQ1; J. Hines et al. (2019) Cancer Res. 79; 251- 262.) in the same cellular system, Al 874 had no advantage over the combination of individual components upon treating either the SJSA-1 (IC50, 0. 1 uM vs. 0.043 uM) or SJSA-X (IC50, 0.25 uM vs. 0.16 uM) cell lines, and the added expression of HDMX inthe SJSA-X cell line further weakened the activity of both RG-7388 (IC50, 0.067 uM vs. 3.73 uM) and A1874 (IC50, 0.1 uM vs. 0.25 uM), with A1874 activity tracking with single agent JQ1 activity (Table 6, FIGs. 8C-8D).

[0138] To further evaluate the activity and specificity of SP-PROTAC chimeras, mutations, Q25K, E21A, and F19A (numbered according to SEQ ID NO:1, i.e., positions Q9, E5, and F3 numbered according to SEQ ID NO:6) were introduced and the length and location of the P-alanine (PAla) linker relative to the stapled peptide was varied and cytotoxicity was evaluated.

[0139] E21A mutagenesis of the chimera SP6924 Q25K-PAla-JQl enhanced cytotoxicity of the SJSA-X osteosarcoma cell line by 2.6-fold (IC50s, 44 nM vs. 116 nM; Table 6; FIG. 7A). E21A mutagenesis of the chimera JQl-(PAla)2-SP6924 enhanced cytotoxicity of the SJSA-X osteosarcoma cell line by 10.3-fold (IC50s, 34 nM vs. 350 nM; Table 7; FIG. 7B). F19A point mutagenesis, which impairs SP6924 binding to HDM2 / HDMX, blunted cytotoxicity of SP-PROTACs SP6924-E21A / Q25K-pAla-JQl and JQl-(pAla)2-SP6924-E21A by 47.3-fold (IC50s, 2080 nM vs. 44 nM; Table 8) and 16.4-fold (IC50s, 557 nM vs. 34 nM; Table 8), respectively (FIGs. 8A-8B). In each case, the specificity-of-action of the chimeras was confirmed by impairment of cytotoxicity upon F19A mutagenesis (Table 8; FIGs. 8A-8B).

[0140] Table 6

[0141] Table 7

[0142] Table 8

[0143] Because wild-type p53 status was the single most predictive genetic determinant to SP6924-E21 A / Q25K-pAla-JQl (SEQ ID NO: 14) in the PRISM experiment (Table 1, FIG. 4), the finding was validated in an orthogonal cell culture model of two osteosarcoma cell lines that differed in p53 mutation status: p53 wild-type U-2 OS (CVCL_0042), and p53 mutant SAOS-2 (CVCL_0548). SP6924 E21A N-term JQl-|3Ala2 (SEQ ID NO: 17) effectively killed wild-type U-2 OS (IC50, 61 nM), with F19A mutagenesis again impairing cytotoxic activity (IC50, 3.3 uM) (FIG. 9A). Conversely, in the context of a p53-mutant SAOS-2 osteosarcoma cell line, SP6924 E21A N-term JQl-pAla2 (SEQ ID NO: 17) had relatively weak cytotoxic activity (IC50, 1.1 uM) akin to its F19A mutant specificity control (SEQ ID NO: 18), consistent with cytotoxicity that derives from BET inhibition by JQ1 alone in the absence of an intact p53 pathway (FIG. 9B).

[0144] As observed for the relatively less potent JQl-(PAla)2-SP645 (SEQ ID NO: 12) (FIGs. 6A-6B), the 3-in-l mechanism (HDM2 / HDMX / BET) of SP6924- E21A / Q25K-pAla-JQl (SEQ ID NO: 14) was only marginally more effective than the 2- in-one mechanism (HDM2 / BET) of A1874 in SJSA-1 osteosarcoma cells that overexpress HDM2, with Fl 9A point mutagenesis markedly blunting the activity of the SP-PROTAC, highlighting specificity of action (IC50s, SP-PRO2.3 23 nM, Al 874 84 nM, SP-PRO2.3 F19A 2 uM) (FIG. 10A). However, upon added HDMX expression inSJSA-X osteosarcoma cells, SP6924-E21A / Q25K-pAla-JQl (SEQ ID NO: 14) was 50x more potent than Al 874 in killing the SJSA-X cells (IC50s, 4.2 nM vs. 200 nM), with F19A point mutagenesis markedly blunting the activity of the SP-PROTAC (IC50s, SP- PRO2.3 4.2 nM, Al 874 200 nM, SP-PRO2.3 F19A 1 .3 uM) (FIG. 10B). In contrast to A1874, SP6924-E21A / Q25K-pAla-JQl (SEQ ID NO: 14) was more active in SJSA-X cells than SJSA-1 cells (IC50s, 4.2 nM vs. 23 nM). In the context of mutant p53- expressing SAOS-2 cells, SP6924-E21A / Q25K-pAla-JQl (SEQ ID NO: 14) and A1874 were as ineffective as the SP-PROTAC F19A point mutant specificity control (SEQ ID NO: 15), such that cytotoxicity only derives from BET inhibition by JQ1 in the absence of an intact p53 pathway, resulting in all compounds demonstrating the same weakened activity. In p53 wild-type U-2 OS osteosarcoma cells, SP6924-E21A / Q25K-pAla-JQl (SEQ ID NO: 14) demonstrated cytotoxicity (IC50, 174 nM) that was abrogated by the F19A point mutant control (SEQ ID NO: 15; IC50 > 10 uM), highlighting the specificity of action of the SP-PROTAC and lack of efficacy of isolated BET inhibition in this cellular context (FIG. 11).

[0145] Since cancers of the brain, placenta, and blood were in the top 15 most susceptible cancer cell lines to SP6924-E21A / Q25K-pAla-JQl (SEQ ID NO: 14) in the PRISM experiment (Table 4), these findings were validated by expanding the cell line testing to include BT869 diffuse intrinsic pontine glioma (DIPG) neurospheres (CVCL C1MH), JEG3 choriocarcinoma cells (CVCL 0363), and EOL1 leukemia cells (CVCL_0258), respectively. When tested against p53 wild-type BT869 neurospheres, both SP6924-E21A / Q25K-pAla-JQl (SEQ ID NO: 14; IC50, 87 nM) and JQl-(PAla)2- SP6924-E21A (SEQ ID NO: 17; IC50, 8.7 nM) were markedly more potent than Al 874 (IC50, >5 uM), with the respective F19A point mutants (SEQ ID NOs: 15 and 18) blunting the activity of the SP-PROTAC (IC50, >5 uM), again highlighting the specificity of action (FIGs. 12-13). JEG3 choriocarcinoma cells, which express wild- type p53, HDM2 and HDMX, were 7-fold more sensitive to SP6924-E21 A / Q25K-PAla- JQ1 (SEQ ID NO: 14; IC50, 21 nM) than A1874 (IC50, 143 nM) (FIG. 14). Similarly, EOL1 leukemia cells, which express wild-type p53, HDM2 and HDMX, were 15-foldmore sensitive to SP6924-E21A / Q25K-pAla-JQl (SEQ ID NO: 14; IC50, 12 nM) than Al 874 (IC50, 180 nM) (FIG. 15).

[0146] Additional PRISM analyses identified particular ovarian and colon cancer cell lines as distinctively susceptible to SP-PROTACs. Thus, cell line testing was expanded to include p53 wild-type ovarian cancer cell lines A2780 (CVCL 0134), OVTOKO (CVCL_3117), TOV21G (CVCL_3613), and PAI (CVCL_0479), and the colon cancer cell line LOVO (CVCL 0399). In all cases, SP6924-E21A / Q25K-pAla-JQl (SEQ ID NO: 14) was 3-fold to 8-fold more potent than A1874, with F19A point mutagenesis blunting the activity of the SP-PROTAC, highlighting the specificity of action (Table 9;FIGs. 16-18).

[0147] Table 9EXAMPLE 3: CYTOTOXIC ACTIVITY OF SP-PROTACS IN T-ALL LEUKEMIC BLASTS ISOLATED FROM NEWLY DIAGNOSED PEDIATRIC PATIENTS

[0148] Cancers of the blood were in the top 15 most susceptible cancer cell lines in the PRISM experiment (Table 4). To evaluate anti-cancer cell activity of SP-PROTACs beyond immortalized cell lines, SP6924-E21A / Q25K-PAla-JQl (SEQ ID NO: 14) wastested against peripheral blood mononuclear cells (PBMCs) purified from the peripheral blood of pediatric patients newly diagnosed with T-cell acute lymphoblastic leukaemia (T-ALL). Samples were obtained as part of an approved clinical protocol at the Dana- Farber Cancer Institute (06-078) and typically prior to initiation of treatment. SP6924- E21A / Q25K-pAla-JQl (SEQ ID NO: 14) was more potent than A1874 in killing primary pediatric T-ALL leukemia cells across 3 individual patient samples (FIG. 19).EXAMPLE 4: SP-PROTAC STABILITY AND PHARMACOKINETICS[00149J Ex vivo plasma stability testing revealed striking persistence of SP6924- E21A / Q25K-pAla-JQl (SEQ ID NO: 14) and JQl-(PAla)2-SP6924-E21A (SEQ ID NO: 17), with no evidence of degradation after 4-hour incubation at 37°C (FIG. 20). Pharmacokinetic studies were performed in mice with SP6924-E21 A / Q25K-PAla-JQl (SEQ ID NO: 14) administered at 3 mg / kg IV (intravenous) or IP (intraperitoneal), 10 mg / kg IV or IP, or 30 mg / kg IP in the indicated formulations (see Methods below). Plasma concentration values for 3 mg / kg IV and IP dosing are plotted in FIG. 21, with the composite data plotted in FIG. 22 demonstrating similar overall coverage and elimination profiles. The derived PK parameters are tabulated in Table 10 and indicate a half-life of 5 hours, Cmax of 2.1 and 0.94 uM and AUC of 24955 and 21671 hr*ng / mL for IV and IP administration, respectively. The 10 mg / kg and 30 mg / kg dosing levels yielded dose-responsively higher plasma concentrations, as plotted in FIG. 23. Strikingly, the 30 mg / kg IP dose showed only a modest decrease in plasma level at 24 hours from Cmax (5 to 3.5 uM), and the 10 mg / kg IV and IP dosing maintained supra- IC90 levels out to 24 hours. AUCs were 148827, 76140, and 168699 hr*ng / mL for 10 mg / kg IV, 10 mg / kg IP, and 30 mg / kg IP, respectively (Table 11). These data highlight the favorable stability and pharmacokinetic properties of SP SP6924-E21 A / Q25K-PAla- JQ1 (SEQ ID NO: 14).30-0423W01 / DFCI 3391.WO1WO30-0423W01 / DFCI 3391.WO1WO30-0423W01 / DFCI 3391.WO1WOEXAMPLE 5: SP-PROTAC IN VIVO EFFICACY

[0152] Eight-week old female NSG mice (N=5 per arm) were inoculated subcutaneously with 5xl06SJSA-X cells in 200 mb with Matrigel. Treatment was initiated at tumor sizes of -800 mm3with either 100 mL IV of vehicle (5% DMSO, 5% PEG400, 0.1% Tween80, 0.4% methylcellulose low viscosity) or SP6924-E21A / Q25K- PAla-JQl (SEQ ID NO: 14) in vehicle at 10 mg / kg / dose daily for 7 days. Animal weights and caliper measurements of tumor were obtained daily for each animal prior to administration of vehicle or treatment, such that Day 1 measurements reflect the weights and tumor volumes of animals just prior to initiation of therapy.

[0153] FIG. 24 is a series of graphs depicting the relative preservation of mouse body weight of SP6924-E21A / Q25K-PAla-JQl (SEQ ID NO:14)-treated mice relative to vehicle-treated mice for animals bearing SJSA-X xenografted tumors as plotted by daily weight (top) and percent daily weight change relative to day 1 body weight (bottom).N=5 mice per arm with p<0.0025 for days 2-6 of treatment.

[0154] FIG. 25 is a series of graphs showing tumor regression in SP6924- E21A / Q25K-PAla-JQl (SEQ ID NO: 14)-treated mice and tumor progression in vehicle- treated mice as monitored by daily caliper measurement of tumors and plotted by daily tumor volume (top) and percent change in daily tumor volume relative to day 1 tumor volume. N=5 mice per arm with p<0.0062 for days 4-7 of treatment. Mice treated with SP6924-E21A / Q25K-PAla-JQl showed striking reduction in tumor volume, especially given the large starting tumor size (FIG. 25).

[0155] FIG. 26 shows tumor regression in SP6924-E21A / Q25K-pAla-JQl (SEQ ID NO: 14)-treated mice and tumor progression in vehicle-treated mice as monitored by daily caliper measurement of tumors and body weight and plotted by tumor volume / body weight. N=5 mice per arm with p<0.0035 for days 2-7 of treatment.EXAMPLE 6: COMBINATION TREATMENT OF SP-PRO2.3.

[0156] Combining SP6924-E21 A / Q25K-0Ala-JQl (SEQ ID NO: 14) with additional therapeutic modalities further enhanced anti-cancer activity. Cultured SJSA-1, SJSA-X, and MCF-7 cells (CVCL 0031) treated with SP6924-E21A / Q25K-pAla-JQl (SEQ ID NO: 14) in combination with Selinexor resulted in enhanced cytotoxicity compared to each treatment alone (FIG. 27). Without being bound by any particular theory, Selinexor is a selective inhibitor of nuclear export, which in addition to its independent anti-cancer activity, can potentially increase the effective dose of SP6924-E21A / Q25K- Ala-JQl (SEQ ID NO: 14) in the nucleus, the cellular compartment of its mechanism of action, by preventing its nuclear export. The specificity of action of this combination treatment was highlighted by a complete lack of supplementary activity when SP6924-E21A / Q25K- PAla-JQl (SEQ ID NO: 14) was administered with Selinexor in cells that lack p53, whether eliminated genetically (SAOS-2 cells) or by protein degradation (HeLa [HPV D6]) (FIG. 28).EXAMPLE 7: IN VITRO DEMONSTRATION OF SP-PRO2.3 COMPLEX FORMATION, TARGET PROTEIN UBIQUITINATION, AND TIME DEPENDENCE OF BRD4 DEGRADATION AND P53 REACTIVATION AND QUANTITATIVE PROTEOMIC ANALYSIS IN TREATED SJSA-X CELLS

[0157] Size exclusion chromatography analyses revealed the capacity of SP-PRO2.3 to form a ternary complex, as reflected by a shift in the elution profile of the two proteins (whether BRD4BDI and HDM2 or BRD4BDI and HDMX) upon incubation with the stapled peptide-small molecule chimera (FIG. 29). Whereas HDM2 effectively polyubiquitinates p53, but not BRD4BDI, in vitro, the addition of SP-PRO2.3 switched the targeting specificity of HDM2, resulting in polyubiquitylation of BRD4BDI rather than p53 (FIG. 30). These data demonstrate the capacity of SP-PRO2.3 to not only nucleate the desired ternary complexes, but also effectively transform HDM2 from an oncoprotein that targets p53 to a tumor suppressor that instead polyubiquitylates a BET oncoprotein. To confirm that the negative impact of SP-PRO2.3 treatment on cancer cell viability relates to alteration of BET and / or p53 pathway protein levels, we first treated SJSA-Xcells with 100 nM SP-PRO2.3 followed by time-dependent monitoring of BRD4, p53, p21, HDM2, and HDMX levels by Western blotting analysis. We observed that (1) BRD4 becomes undetectable by 8 hours after treatment, (2) p53 protein levels gradually increase over time and peak at 12 hours followed by normalization by 24 hours (consistent with its negative feedback loop), (3) the p53 transcriptional targets p21 and HDM2 surge by 24 hours, and (4) HDMX levels increase and remain elevated (FIG. 31). These protein dynamics are remarkably consistent with the anticipated mechanism of action of SP-PRO2.3. To expand the analysis to the entire proteome, we performed quantitative proteomic analyses on SJSA-X cells treated with 1 mM SP-PRO2.3 and then prepared lysates at 24 hours to measure protein levels. Strikingly, the BET proteins BRD2-4 are the most downregulated and the p53 pathway proteins p53, p21, HDM2, and HDMX are the most upregulated, as compared to the proteome of vehicle-treated cells (FIG. 32A). Quantitative proteomics analysis of SP-PRO2.3 treated primary pediatric T- ALL cells (06078-840, FIG. 19) likewise caused a striking decrease in BRD proteins (e.g., BRD2, BRD4) and increase in p53 protein (FIG. 32B), as evaluated at 18 hours after 500 nM treatment.EXAMPLE 8: IN VIVO EFFICACY OF SP-PRO2.3 DOSING LEVEL AND FREQUENCY IN A MOUSE MODEL OF SJSA-X OSTEOSARCOMA

[0158] Prior to day 1 treatment with 10 mg / kg / day IV, SJSA-X tumors (n=5 NSG mice per treatment arm) averaged 400 mm3in size and grew larger each day in the vehicle-treated mice but regressed daily throughout the 7-day SP-PRO2.3 treatment period, as tracked by tumor volume measurement (FIG. 25). Excised tumors from each of three mice exposed to 10 mg / kg IV daily dosing for 7 days showed the stark contrast in size between vehicle- and SP-PRO2.3 -treated tumors (FIG. 33A). Quantitative proteomic analysis of the specimens revealed BRD4 as one of the most downregulated tumor proteins in vivo, with HDMX remaining upregulated (FIG. 33B). To assess whether we could achieve disease control at lower dosing regimens and in a dose-responsive fashion, we repeated the experiment with 0.3, 1, and 3 mg / kg daily dosing IV (n=5 mice per arm), and rather than sacrifice the animals at day 7, we extended treatment to assess for prolonged tolerance and efficacy. This 20-day experimental set up revealed dose-responsive efficacy in suppressing SJSA-X tumor growth in vivo and a corresponding impact on animal survival, with all vehicle and 0.3 mg / kg treated animals, and all 1 mg / kg treated animals, having expired by days 12 and 17 of treatment, respectively (FIG. 34A). Reducing the interval of 3 mg / kg IV dosing to 3 or even 2 times per week (Mon / Wed / Fri or Mon / Thurs) still resulted in striking suppression of SJSA-X tumor growth in vivo (FIG. 34B). Importantly, the effective dosing levels of SP-PRO2.3 are at least 1 to 2 orders of magnitude lower than what has been reported for JQ1 and stapled p53 peptide monotherapy in suppressing tumor growth in murine models.EXAMPLE 9: BROAD SPECTRUM ACTIVITY OF SP-PRO2.3 IN WT P53 CANCER CELLS

[0159] To expand our analysis of SP-PRO2.3 activity across the diversity of human cancer cells, we performed Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) analysis which tests the effects of compounds on the viability of >900 genetically characterized cancer cell lines, revealing relative susceptibilities and potential biomarkers. Our first observation was that SP-PRO2.3 exhibited striking potency across a diversity of cancer cell lines (FIG. 35), with melanoma, skin, kidney, soft tissue, hematopoietic, and ovarian cancers among the most susceptible tumor types (FIG. 37), as exemplified by IC50s ranging from 4 to 436 nM. The far and away greatest predictor of cancer cell susceptibility to SP-PRO2.3 was p53 status (FIG. 36), with cancer cells that retain WT p53 demonstrating an average area under the (viability) curve (AUC) of 0.66, whereas those with mutant or deleted p53 having an average AUC of 0.90 (FIG. 4). Further bioinformatic analyses revealed that the topmost genes required for cancer cell vulnerability to SP-PRO2.3 were a series of p53 transcriptional targets critical to its pro- apoptotic function, including EDA2R, FDXR, CDKN1 A (p21), BAX, and BBC3 (PUMA), and HDM2 itself, which has been transformed by SP-PRO2.3 from an oncogene into a tumor suppressor (FIG. 38). GO analysis of the quantitative proteomicsdataset derived from SP-PRO2.3 vs. vehicle treatment (1 pM, 24 h) of SJSA-X cells, again highlighted a series of enriched and mechanistically-relevant pathway signatures, with DNA damage response, signal transduction by p53 class mediator emerging as the top hit (FIG. 39).EXAMPLE 10: SELECTIVITY OF SP-PRO2.3 FOR CANCER CELLS OVER NON-TRANSFORMED CELLS

[0160] PRISM analysis highlighted dose-responsive cytotoxic activity of SP-PRO2.3 across many cancer cell lines, including melanoma, kidney, ovarian, soft tissue, and hematopoietic cancers, as demonstrated by the negative log-fold change (LFC) plots (FIG. 40). In contrast, SP-PRO2.3 has comparatively little effect on the viability of non- transformed human cells, such as RPTEC and HUVEC (FIG. 41).EXAMPLE 11: IN VIVO EFFICACY OF SP-PRO2.3 IN A MOUSE MODEL OF EOL-1 LEUKEMIA

[0161] We extended our in vivo studies to include a liquid tumor model, specifically EOL-1 acute myeloid leukemia, which scored by PRISM analysis (FIG. 40) as a susceptible hematologic malignancy bearing wild-type p53 and expressing HDM2, HDMX, and BET proteins. Baseline complete blood counts (CBC) were drawn followed by tail vein injection of NSG mice (n=4 per arm) with EOL1 cells (2xl06) on day 0, and then daily treatment consisting of vehicle or SP-PRO2.3 at 2 mg / kg IV was initiated on day 7. Repeat CBCs were performed at time of death (vehicle-treated mice) or after completing 7 days of treatment (SP-PRO2.3-treated mice). Whereas vehicle-treated mice exhibited total white blood cell (WBC) counts above 100K at time of death (range, 127- 585K), SP-PRO2.3 -treated animals maintained WBCs below the 100K (range, 6.72- 64.8K) (FIG. 42). Correspondingly, SP-PRO2.3 treatment resulted in marked suppression of percent blasts and preservation of percent neutrophils compared to vehicle-treated mice (mean 3.5% vs. 33% blasts and 48% vs. 6.3% neutrophils for SP- PRO2.3- and vehicle-treated mice, respectively) (FIG. 43-44). Ultimately, all vehicle- treated mice expired between days 4 and 6 of treatment but all SP-PRO2.3-treated micesurvived the 7-day treatment period (FIG. 45). To compare endpoint counts at the same time, we repeated the study (n=6 per arm) but initiated treatment 3 days earlier (day 4 post cellular injection) to decrease the overall duration of the experiment (and thus tumor burden), with the goal of preserving evaluable vehicle-treated mice after 7 days of treatment. Again, we observed relative suppression of total WBC counts for SP-PRO2.3 vs. vehicle-treated animals (mean, 42K vs. 66K, respectively) at this earlier timing of leukemia progression (as compared to the above study), and relative suppression of percent blasts and preservation of percent neutrophils (mean, 9.4% vs. 26.5% and 18.0% vs. 12.2%, respectively) (FIG. 46-47). Thus, SP-PRO2.3 demonstrated therapeutic activity in both solid and liquid tumor models of WT p53 human cancer.METHODS USED IN WORKING EXAMPLES 1-11

[0162] Synthesis of Stapled Peptides PROTACs. Hydrocarbon-stapled peptides corresponding to the HDM2 and HDMX targeting alpha-helical p53 peptide were synthesized according to established methods. The small molecule ligand was placed at discrete positions relative to the peptide sequence, such as at the N-terminus or position Q25K (numbered according to SEQ ID NO: 1, i.e., position 9 of SEQ ID NO:6). To append the small molecule at a particular position along the peptide backbone, the molecule was linked to the peptide using an orthogonally protected lysine, wherein the orthogonal protecting group was ivDde or Dde. Once the peptide synthesis was complete, olefin metathesis was performed and the N-term or side chain amino group was unmasked and a linker incorporated between the stapled peptide and small molecule ligand.

[0163] High Throughput Cancer Cell Line Screening by PRISM. SP6924- E21A / Q25K-pAla-JQl (SEQ ID NO: 14) was screened at an 8-point dosing regimen (3- fold dilution) against 950 genomically characterized cancer cell lines by Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) assay at the Broad Institute of Harvard and MIT. Cells were treated for 5 days in triplicate, with each treatment plate containing positive (bortezomib) and negative (0.1% DMSO) controls. For correlation ofsensitivity, univariate associations between the PRISM sensitivity profile of SP6924- E21 A / Q25K-pAla-JQl (SEQ ID NO: 14) and genomic features such as gene expression, gene dependency, and mutation status were determined by computing Pearson correlations and associated p values.

[0164] Cancer Cell Lines Viability Assay. Cells were maintained in DMEM supplemented with 10% (v / v) FBS, 100 U / ml penicillin, 100 mg / ml streptomycin, and 10 mM HEPES and seeded (1-5,000 / well) in 384-well opaque plates. Cells were then incubated in quadruplicate with vehicle or a serial dilution of the indicated drug treatments in DMEM with 5% FBS at 37 °C in a final volume of 40 pL. Cell viability was assayed at 48-72 hr by addition of CELLTITER-GLO reagent according to the manufacturer’s protocol (Promega) and luminescence was measured using a SpectraMax M5 microplate reader (Molecular Devices).

[0165] Primary Patient Cancer Cell Lines Viability Assay. Whole blood from pediatric T-ALL patients was obtained under Dana-Farber Cancer Institute Protocol 06- 078 protocol. Peripheral blood mononuclear cells (PBMCs) were purified using Ficoll- paque separation according to the manufacturer’s protocol and then viably frozen in fetal bovine serum (FBS) containing 10% dimethyl sulfoxide (DMSO). PBMCs (2xl07) were thawed for 2 minutes at 37 °C and resuspended in a total volume of 10 mL RPMI, with 10% FBS added in a drop-wise fashion. PBMCs were centrifuged at 1300 RPM for 10 minutes, resuspended in 5 mL RPMI 10% FBS, counted using Trypan blue and rested at 37 °C, 5% CCh for 90 minutes. Then, PBMCs were centrifuged, resuspended in 5 mL RPMI 5% FBS, and counted as above. For the viability assay, 5xl04viable PBMCs in RPMI 5% FBS were seeded per well in a 96-well white plate and treated with a 10 mM top dose, 7-point, 2-fold dilution series of SP6924 Q25K-PAla-JQl or Al 874 (Selleckchem) in technical triplicate, or DMSO vehicle control in technical triplicate in a total volume of 100 mL at 37 °C, 5% CO2 for 19 hours. The plate and its contents were equilibrated at room temperature for approximately 20 minutes, followed by addition of 100 mL CellTiter-Glo reagent (Promega) and mixing on an orbital shaker for 10 minutes to induce cellular lysis. Luminescence was recorded after a brief incubation withoutshaking to stabilize the luciferase signal. Data is represented for each patient sample by normalizing the relative light units (RLUs) from each well to the mean RLUs of the DMSO vehicle control and multiplied by 100 to obtain a percentage value. For proteomic analysis, viably frozen PBMCs were revived with high viability and plated at 2.5 x 106viable cells per well in a 6-well plate format. Cells were treated with 500 nM SP-PRO2.3 (ICso in viability assay of 473 nM) — or with a DMSO vehicle control (0.005% final concentration). Each condition was performed in biological triplicate (n = 3 wells per condition) and incubated overnight for 18 hours. A total of 55 pg of protein per sample was submitted for proteomic analysis. Data analysis was conducted using a minimum fold change threshold of 1.5, a p-value cutoff of 0.01, and multiple hypothesis correction using the Benjamini -Hochberg false discovery rate (FDR) method.

[0166] Formulation and Pharmacokinetics for SP6924-E21A / Q25K-pAla-JQl (SEQ TD NO: 14) Pharmacokinetics were tested in male C57B1 / 6J mice, 7-9 weeks of age, with body weights between 21-26 g. Doses were free base equivalent, factoring out the contribution of the TFA salt. SP-PRO2.3 was formulated in 5% DMSO / 95% water at final concentrations of 0.3 mg / ml and injected at a volume of 10 ml / g body weight for a 3 mg / kg IV and IP dose. Higher doses of SP-PRO2.3 were formulated in 5% DMSO, 5% PEG400, and 0.1% Tween-80, diluted with 0.4% w:v methylcellulose at a final concentration of 1 and 3 mg / ml for 10 and 30 mg / kg doses, respectively. Triplicate mice were dosed via the lateral tail vein or via intraperitoneal injection. Repeated blood collection of approximately 20 pl was obtained at 5, 15, 30, 60, 120, 240, 360, 480, and 1440 minutes using a micro-sampling technique where blood was collected in capillary hematocrit tubes to reduce the total blood taken from the mouse. Plasma was generated using a microcentrifuge with a hematocrit rotor, resulting in approximately 10 pl of plasma, which was immediately frozen. Plasma samples were thawed, and 5 pl of plasma was deprotonated / extracted with the addition of 75 pl of acetonitrile, containing carbamazepine as an internal standard. Standards were prepared in blank mouse plasma and processed in the same analytical run. Samples were centrifuged through a Millipore Multiscreen Solvinter 0.45 micron low binding PTFE hydrophilic filter plate andanalyzed using a Sciex 6500 mass spectrometer with multiple reaction monitoring, following the mass transition of 776.1 to 427.7 Da corresponding to the triple charged ion. Pharmacokinetic parameters were calculated using a non-compartmental model (Phoenix WinNonlin, Pharsight Inc ).

[0167] In Vivo Efficacy Testing. SJSA-X xenografts were established by injecting 2xl06cells subcutaneously with matrigel into the flanks of 7-8 week old female NSG mice (Jackson Labs, 005557). Mice (n=5 per arm) were treated with vehicle (5% DMSO, 5% PEG400, 0.4% methylcellulose solution, 0.1% Tween-80) or SP-PRO2.3 at 10, 3, 1, or 0.3 mg / kg in vehicle, once daily, by intravenous injection, or at the 3 mg / kg dose at two (Mon / Thurs) or three (Mon / Wed / Fri) times per week. For the 10 mg / kg dosing study (starting tumor volumes of -400 mm3), animals were euthanized after 7 days of treatment and tumors from 3 vehicle and 3 SP-PRO2.3 -treated mice were isolated and processed for quantitative proteomics. For the dose-responsive study (starting tumor volumes of -200 mm3), treatments were extended beyond 7 treatment days to monitor for extended tolerance and efficacy. Tumor size was measured with calipers daily to calculate and track tumor volume. EOL-1 xenografts were established by injecting 2xl06cells intravenously into 8 week old female NSG mice (n=4) on day 0. Starting on day 7 post-injection, animals were treated with either vehicle (as above) or 2 mg / kg SP-PRO2.3 IV. Complete blood counts (CBC) with differential were drawn prior to EOL-1 cell injection, at time of death, or upon termination of the experiment after 7 days of treatment, and samples processed at the Beth Israel / Deaconess Preclinical Murine Pharmacogenetics Core. The study above was repeated in 10 week old female NSG mice (n=6 per arm) with treatment initiated at day 4 post cellular injection and repeat CBCs drawn within 24 hours after 7 days of treatment, upon termination of the experiment. Tumor volume (SJSA-X) and CBC (EOL-1) data were analyzed and plotted using PRISM software (GraphPad). All animal studies were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Dana-Farber Cancer Institute under approved protocol #06-004.

[0168] Recombinant protein production

[0169] Recombinant HDM2 (residues 17-125) bearing an N-terminal hexahistidine tag and a thrombin cleavage site was cloned into the pET28a vector, expressed in BL21(DE3) E. coll. and purified using sequential Ni-affinity and size-exclusion chromatography (SEC). Protein expression was induced with 1 mM isopropyl P-D-l- thiogalactopyranoside (IPTG; Gold Biotechnology) for 4 hours at 30°C. Bacterial pellets were resuspended in lysis buffer (20 mM Tris, pH 8, 500 mM NaCl, 2 protease inhibitor tablets [Roche]) and lysed by two passes through a microfluidizer (M-l 10L, Microfluidics) chilled to 4°C. Insoluble debris was removed by centrifugation (20,000 rpm, 45 min, 4°C). The supernatant was applied to a Ni-NTA (Qiagen) column pre- equilibrated with lysis buffer. The column was washed with lysis buffer and wash buffer (20 mM Tris, pH 8, 300 mM NaCl), and proteins were eluted using an imidazole gradient (5 to 300 mM) in wash buffer. Eluted protein fractions were dialyzed, concentrated, and further purified on a Superdex S-75 (GE Healthcare) gel filtration column equilibrated with FPLC buffer (20 mM HEPES, pH 7.2, 300 mM NaCl). Protein purity and identity were confirmed by Coomassie staining and Western blotting analysis.

[0170] Humanized zebrafish HDMX (zHDMX) was cloned, generated, and purified as previously described1. Briefly, zHDMX (residues 15-106, L46V / V95L double mutation) bearing an N-terminal hexahistidine tag and enterokinase recognition sequence (pET15b vector, Novagen) was expressed in BL21(DE3) E. coli using 0.2 mM IPTG for 4 hours at 30°C. The protein was purified by sequential Ni-affinity chromatography and SEC. Bacterial pellets were resuspended in lysis buffer (20 mM Tris, pH 8, 500 mM NaCl, 2 protease inhibitor tablets [Roche]) and lysed by two passes through a microfluidizer (M-l 10L, Microfluidics) chilled to 4°C. Insoluble debris was removed by centrifugation (20,000 rpm, 45 min, 4°C). The supernatant was applied to a Ni-NTA (Qiagen) column pre-equilibrated with lysis buffer. The column was washed with lysis buffer and wash buffer (20 mM Tris, pH 8, 300 mM NaCl), and proteins were eluted using an imidazole gradient (5 to 300 mM) in wash buffer. Eluted protein fractions were dialyzed, concentrated, and further purified on a Superdex S-75 (GE Healthcare) gel filtration column equilibrated with FPLC buffer (50 mM NaPCL, pH 8, 150 mM NaCl, 2mM TCEP). Protein purity and identity were confirmed by Coomassie staining and Western blotting analysis.

[0171] The human BRD4 BD1 domain (residues 48-168) in the pNIC28Bsa4 vector (Addgene) was expressed in BL21(DE3) E. coli in LB medium containing 50 mg / mL kanamycin. Cells were grown at 37°C to an OD of 0.8, induced with 0.5 mM IPTG, incubated overnight at 17°C, collected by centrifugation, and stored at -80°C. Cell pellets were lysed using a microfluidizer in buffer Al (25 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 20 mM imidazole, 7 mM BME) supplemented with 0.1% IGEPAL and 1 mM PMSF. The lysate was centrifuged at 20,000xg for 40 min. Ni-NTA beads (Qiagen) were mixed with the lysate supernatant for 2 hours at 4°C and washed with 10 column volumes of buffer Al, followed by 5 column volumes of buffer A2 (25 mM HEPES, pH 7.5, 2000 mM NaCl, 5% glycerol, 20 mM imidazole). Protein was eluted with an imidazole gradient (20 to 400 mM) in buffer A2. Further purification was performed by SEC in buffer A3 (25 mM HEPES, pH 7.5, 200 mM NaCl, 5% glycerol, 0.5 mM TCEP, 1 mM DTT) using a Superdex-200 10 / 300 column (GE Healthcare).

[0172]

[0173] Complex formation analysis by size exclusion chromatography.Recombinant HDM2, zHDMX, and BRD4 BD1 proteins, described above, were diluted to 20 p.M in 500 mL of assay buffer (300 mM NaCl, 20 mM HEPES, pH 7.5). Protein samples were incubated individually, in combination, or in combination with added SP- PRO2.3 for 1 hour at 30°C. The proteins and their complexes were evaluated by SEC using a Superdex S-75 column (GE Healthcare). Min-max normalization of the data was performed with RStudio (version 4.1.2) and visualized using ggplot2 (version 3.4.4). SDS-PAGE analysis was conducted on peak-containing fractions to further confirm the presence of the indicated proteins and complexes.

[0174] In vitro ubiquitylation assay. An in vitro ubiquitylation system was established using ubiquitylation assay buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 1 mM TCEP) and the following components (R&D Systems): 1 mM Human GST-HDM2(E3-202-050), 100 nM UBEl (E-305-025), 1 mM UbcH5c / UBE2D3 (E2-627-100), 10 mM MgATP (B-20), and 100 mM human ubiquitin protein (U-100H-10M). Recombinant FLAG-p53 (20 mM, BPS Bioscience, 100412) and recombinant 6xHis-BRD4 bearing BD1 and BD2 domains (20 mM, BPS Bioscience, 31045) were added as substrates for the ubiquitylation assay, in the absence or presence of SP-PRO2.3 (60 pM), and incubated for 0, 1, 2, or 4 hours. Reactions were stopped with LDS buffer (Invitrogen) and boiled at 95°C for 5 minutes. Samples were run on a 4-12% Bis-Tris gel in lx MES buffer (Invitrogen) and transferred to a nitrocellulose membrane, which was blocked for 1 hour at room temperature in 3% BSA. Rabbit His-Tag Antibody (Cell Signaling, 2365S) was incubated overnight at 4°C to detect 6xHis-BRD4, and Rabbit FLAG-Tag Antibody (Cell Signaling, 2368S) was used to detect FLAG-p53. Membranes were washed in PBST and incubated at room temperature with goat anti -rabbit poly-HRP (Invitrogen, 32260). After washing with PBST, membranes were imaged by chemiluminescence for the presence of ubiquitin-based protein laddering.[00175J Protein dynamics. SJSA-X cells were treated with 100 nM SP-PRO2.3 for 0, 1, 2, 4, 8, 12, and 24 hours in DMEM supplemented with 5% FBS, 1% Penicillin / Streptomycin, and 2 mM glutamine. To prepare samples for Western blotting analysis, cells were first rinsed with PBS and subsequently lysed on ice for 20 minutes using RIPA lysis buffer (Sigma #20-188) supplemented with cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche). Insoluble material was separated by centrifugation at 8000 rpm for 10 min in a table-top centrifuge and protein concentration of the isolated supernatant was measured by BCA protein assay kit (Thermo Fisher Scientific, #23227). Protein samples (10 mg) were separated by SDS-PAGE using 4-12% Bis-Tris Protein Gels (Thermo Fisher Scientific) and transferred to nitrocellulose membranes using the iBlot™ 2 Dry Blotting System (Thermo Fisher Scientific). The membranes were blocked in a solution of 5% milk prepared in PBS-T (Phosphate Buffered Saline, 0.01% Tween) and incubated with primary antibodies overnight at 4 °C, 1 : 1000 in 3% BSA (p53 [Millipore #OP43-100UG, RRID:AB_213402], p21 [Cell Signaling #2947, RRID: AB 823586], HDM2 [Millipore, #OP46-100UG, RRID:AB_10681293], HDMX [FortisLife Sciences, #A300-287A, (RRID: AB_263407], actin [Sigma, #A1978, RRID:AB_476692]) or 5% milk prepared in PBS-T (BRD4 [Cell Signaling Technologies, #13440, RRID: AB_2687578). The membranes were then washed with PBS-T and treated with secondary HRP-conjugated antibodies 1 :2000 in 3% BSA (p53, HDM2 membranes: anti-mouse, Bio-Rad #1706516, RRID :AB_2921252; p21, HDMX membranes: anti-rabbit, Cell Signaling #7074, RRID:AB 2099233) or in 5% milk prepared in PBS-T (BRD4 membrane; anti-rabbit, Cell Signaling #7074, RRID:AB_2099233) for 90 min at room temperature. After three additional 5 min washes with PBS-T, protein detection was carried out using Fischer Scientific Cytiva Amersham™ ECL™ Prime Western Blotting Detection Reagent (#45-002-401) and an AI600 Chemiluminescent Imager.

[0176] Quantitative proteomics

[0177] Sample Preparation. Tandem mass tag (TMT) proteomics was performed as described in Adhikary, U. et al. Cell Rep 42, 113176 (2023). Briefly, SISA-X cells were treated with 1 mM TAPTAC1 for 24 hours and then whole cell extracts were prepared by lysis in IX RIPA lysis buffer (Thermo Fisher Scientific). Lysate protein was quantified Bradford assay (Pierce). For quantitative proteomics of tumor specimens, whole tumors (n=3) were resected en bloc from mice treated with either vehicle or SP- PRO2.3 (10 mg / kg / d) for 7 days and immediately flash frozen. Each frozen tumor was homogenized into a fine powder using a Retsch Cryomill and then suspended in lysis buffer (8 M urea, 200 mM EPPS pH 8.5, lx Roche Protease Inhibitors, lx Roche PhosSTOP phosphatase inhibitors). The homogenate was centrifuged at 21,000 x g for 5 minutes, and the resulting supernatant was transferred to a fresh tube. Protein concentrations were measured using the Bradford assay (Pierce). For each sample, 100 mg of protein was aliquoted for reduction and alkylation steps. Proteins underwent disulfide bond reduction using 5 mM tris(2-carboxyethyl)phosphine for 30 minutes at room temperature, followed by alkylation with 10 mM iodoacetamide for 30 minutes in the dark at room temperature. To quench excess iodoacetamide, 10 mM dithiothreitol was added, and the mixture was incubated for 15 minutes in the dark at room temperature.Proteins were precipitated using a chloroform-methanol method before digestion. Specifically, 4 volumes of pure methanol were added to the sample and vortexed, followed by the addition of 1 volume of chloroform and vortexing, and then 3 volumes of water with vortexing. The mixture was centrifuged at 4,000 rpm for 15 minutes at room temperature, washed twice with 100% methanol, and dried by vacuum centrifugation.

[0178] Trypsin and LysC digestion. The dried samples were resuspended in 200 mM EPPS, pH 8.5, and digested for 3 hours at room temperature using Lys-C protease at a 100: 1 protein-to-protease ratio. Trypsin was subsequently added at the same ratio, and the reaction was incubated for 6 hours at 37°C.

[0179] Tandem Mass Tag (TMT) Labeling. TMTpro™ 18-plex reagents (0.8 mg) were dissolved in anhydrous acetonitrile (40 mL) of which 5 mL was added to the peptides (50 mg) with 15 mL of acetonitrile to achieve a final concentration of -30% (v / v). Following incubation at room temperature for 1 h, the reaction was quenched with hydroxylamine to a final concentration of 0.3% (v / v). TMT4abeled samples were pooled at a 1 : 1 ratio across all samples. For each experiment, the pooled sample was vacuum centrifuged to near dryness and subjected to C18 solid-phase extraction (SPE) (Sep-Pak, Waters).

[0180] Off-line basic pH reversed-phase (BPRP) fractionation (whole proteome).The pooled TMT-labeled peptide (specifically, the unbound and wash from the phosphopeptide enrichment) was fractionated by BPRP HPLC using an Agilent 1260 pump. Peptides were subjected to a 560 min linear gradient from 5% to 35% acetonitrile in 10 mM ammonium bicarbonate pH 8 at a flow rate of 0.25 mL / min over an Agilent 300Extend C18 column (3.5 mm particles, 2.1 mm ID and 25 cm long). The peptide mixture was fractionated into a total of 96 fractions, which were consolidated into 24 super-fractions and subjected to FAIMS-MS / MS. These fractions were subsequently acidified with 1% formic acid and vacuum centrifuged to near dryness. Each fraction was also desalted via StageTip, dried via vacuum centrifugation, and reconstituted in 5% acetonitrile, 5% formic acid for LC-MS / MS.

[0181] Mass spectrometric data collection (SJSA-X cellular lysates). Mass spectrometry data were collected using an Orbitrap Astral mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled to a Neo Vanquish liquid chromatograph. We used the Orbitrap detector of MS2 analysis. Peptides were separated on a 1 10 cm uPAC C18 column (Thermo Fisher Scientific). For each analysis, ~0.5 mg was loaded onto the column. Peptides were separated using a 60 min gradient of 5 to 29% acetonitrile in 0.125% formic acid with a flow rate of 300 nL / min. The scan sequence began with an MSI spectrum (Orbitrap analysis, resolution 60,000, 350-1350 Th, AGC target set to 100%, maximum injection time set to “auto”). Data were acquired for 90 minutes per fraction. The hrMS2 stage consisted of fragmentation by higher energy collisional dissociation (HCD, normalized collision energy 35%) and analysis using the Orbitrap (AGC 200%, maximum injection time 86 ms, isolation window 0.5 Th, resolution 45,000). Data were acquired using the FAIMSpro interface with the dispersion voltage (DV) set to 5,000V and the compensation voltages (CVs) set at -30V, -50V, and -70V or -40V, -60V, and -80V. The TopSpeed parameter was set at 1 sec per CV.

[0182] Mass spectrometric data collection (tumor specimens) . Mass spectrometric data were collected on an Orbitrap Eclipse mass spectrometer coupled to a Vanquish Neo UHPLC. Approximately 1 mg of peptide was separated at a flow rate of 450 nL / min on a 100 mm capillary column that was packed with 35 cm of Accucore 150 resin (2.6 mm, 150A; ThermoFisher Scientific). The scan sequence began with an MSI spectrum (Orbitrap analysis, resolution 60,000, 350-1350 Th, automatic gain control [AGC] target set to 100%, maximum injection time set to “auto”). Data were acquired for 90 minutes per fraction. The hrMS2 stage consisted of fragmentation by higher energy collisional dissociation (HCD, normalized collision energy 36%) and analysis using the Orbitrap (AGC 200%, maximum injection time 120ms, isolation window 0.6 Th, resolution 30,000 Turbo TMT). Data were acquired using the FAIMSpro interface with the dispersion voltage (DV) set to 5,000V and the compensation voltages (CVs) set at -30V, - 50V, and -70V or -40V, -60V, and -80V. The TopSpeed parameter was set at 1 sec per CV.

[0183] Mass spectrometric data analysis. Spectra were converted to mzXML using MSconvert. Database searching included all entries from the human UniProt reference database (downloaded: June 2022). The database was concatenated with one composed of all protein sequences for that database in the reversed order. Searches were performed using a 50-ppm precursor ion tolerance for total protein level profiling. The product ion tolerance was set to 0.03 Da. These wide mass tolerance windows were chosen to maximize sensitivity in conjunction with Comet searches and linear discriminant analysis. TMTpro labels on lysine residues and peptide N-termini (+304.207 Da), as well as carbamidomethylation of cysteine residues (+57.021 Da) were set as static modifications, while oxidation of methionine residues (+15.995 Da) was set as a variable modification. Peptide-spectrum matches (PSMs) were adjusted to a 1% false discovery rate (FDR). PSM filtering was performed using a linear discriminant analysis, as described previously and then assembled further to a final protein-level FDR of 1%. Proteins were quantified by summing reporter ion counts across all matching PSMs, also as described previously. Reporter ion intensities were adjusted to correct for the isotopic impurities of the different TMTpro reagents according to manufacturer specifications. The signal-to-noise (S / N) measurements of peptides assigned to each protein were summed and these values were normalized so that the sum of the signal for all proteins in each channel was equivalent to account for equal protein loading. Finally, each protein abundance measurement was scaled, such that the summed signal-to-noise for that protein across all channels equals 100, thereby generating a relative abundance (RA) measurement.OTHER EMBODIMENTS

[0184] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A chimera or a pharmaceutically acceptable salt thereof, wherein the chimera or the pharmaceutically acceptable salt thereof comprises a stapled peptide conjugated to a compound, wherein the stapled peptide comprises(i) AC-LTFXIX2YWAX3X4X5X6AAX7-NH2 (SEQ ID NO: 19), wherein:Xi is an a, a-di substituted non-natural amino acid with an olefinic side chain cross-linked to Xs,X2 is E or A,X3 is: (1) an amino acid conjugated to the compound, optionally via a linker, (2) (NH-(CH2)m-C0)n, wherein m is 1-11 and n is 0-5, conjugated to the compound, optionally via a linker, or (3) NH-(CH2-CH2-O)n-CH2-CH2-CO, wherein n is 1-5, conjugated to the compound, optionally via a linker, optionally wherein X3 is lysine conjugated to the compound via a linker,X4is leucine or cyclobutylalanine,Xs is an a, a-di substituted non-natural amino acid with an olefinic side chain cross-linked to Xi,Xe is S or A,X7 is absent or AAXs, wherein X8 is D-alanine, andAc is an acetyl group, or(ii) SEQ ID NO: 19 with 1 or 2 amino acid substitutions, wherein the 1 or 2 amino acid substitutions are not at Xi, X3, or X5, and wherein the stapled peptide binds to HDMX and HDM2, andwherein the compound binds a protein.

2. The chimera or the pharmaceutically acceptable salt thereof of claim 1, wherein the compound binds a bromodomain and extraterminal domain (BET) protein, optionally wherein the BET protein is bromodomain 2 (BRD2), BRD3, or BRD4.

3. The chimera or the pharmaceutically acceptable salt thereof of claim 2, wherein the compound is JQ1.

4. The chimera or the pharmaceutically acceptable salt thereof of claim 3, wherein JQ1 is conjugated to X3 via a linker.

5. The chimera or the pharmaceutically acceptable salt thereof of claim 4, wherein the linker is attached to JQ1 as set forth in Formula I:(Formula I).

6. The chimera or the pharmaceutically acceptable salt thereof of any one of claims 1 to 5, wherein Xi is (R)-2-(7’-octenyl)alanine cross-linked to X5, and wherein X5 is (S)- 2-(4’-pentenyl)alanine cross-linked to Xi.

7. The chimera or the pharmaceutically acceptable salt thereof of claim 6, wherein X3 is conjugated to the compound via a linker, optionally wherein the linker is 5 to 13 atoms in length from C-alpha of X3.

8. The chimera or the pharmaceutically acceptable salt thereof of claim 7, wherein the linker is (P-alanine)n, in particular P-alanine.

9. The chimera or the pharmaceutically acceptable salt thereof of claim 1, wherein the chimera or the pharmaceutically acceptable salt thereof comprises or consists of Ac- LTFX1AYWAX2LX3AAAAAX4-NH2, wherein Xi is (R)-2-(7’-octenyl)alanine cross- linked to X3, X2 is lysine conjugated to the compound via the linker, X3 is (S)-2-(4’- pentenyl)alanine cross-linked to Xi, X4 is D-alanine, the linker is P-alanine, and Ac is an acetyl group (SEQ ID NO:200).

10. The chimera or the pharmaceutically acceptable salt thereof of claim 1, wherein the compound is JQ1, and wherein the chimera or the pharmaceutically acceptable salt thereof comprises or consists of AC-LTFX1AYWAX2LX3AAAAAX4-NH2, wherein Xi is (R)-2-(7’-octenyl)alanine cross-linked to X3, X2 is lysine conjugated to JQ1 via the linker, X3 is (S)-2-(4’-pentenyl)alanine cross-linked to Xi, X4 is D-alanine, the linker is P-alanine, Ac is an acetyl group (SEQ ID NO: 14), and wherein IQ1 attached to the linker has the structure of Formula I:

11. A chimera comprising the structure of Formula II (SEQ ID NO: 14):(Formula II) or a pharmaceutically acceptable salt thereof.

12. A pharmaceutical composition comprising the chimera or the pharmaceutically acceptable salt thereof of any one of clams 1 to 11 and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition of claim 12, further comprising a nuclear export inhibitor, optionally wherein the nuclear export inhibitor is selinexor.

14. A method of treating a disease in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of the chimera or the pharmaceutically acceptable salt thereof of any one of claims 1 to 11, wherein the disease is driven by or related to the protein to which the compound of the chimera binds, and optionally wherein the disease encodes or expresses functional p53, functional p53 and HDM2, functional p53 and HDMX, or functional p53, HDM2, and HDMX.

15. The method of claim 14, wherein disease is a cancer, optionally a liquid tumor or a solid tumor, further optionally wherein the cancer is a sarcoma (such as osteosarcoma), an ovarian cancer, a kidney cancer, a soft tissue cancer, a brain cancer, a skin cancer, an endometrial cancer, a hematopoietic cancer (such as a leukemia or a lymphoma), or a urinary tract cancer, and yet further optionally wherein the cancer expresses or encodes functional p53.

16. The method of claim 14 or 15, wherein the method further comprises administering to the human subject a therapeutically effective amount of a second therapeutic agent, optionally wherein the second therapeutic agent is a nuclear export inhibitor, optionally wherein the nuclear export inhibitor is selinexor.

17. A method of making the chimera or the pharmaceutically acceptable salt thereof of any one of claims 1 to 11, the method comprising: (a) providing a peptide comprising the sequence:(i) AC-LTFX1X2YWAX3X.IX5X6AAX7-NH2 (SEQ ID NO:300), wherein:Xi is an a, a-disubstituted non-natural amino acid with an olefinic side chain cross-linked to Xs,X2 is E or A,X3 is an orthogonally protected amino acid, optionally wherein X3 is orthogonally protected lysine,X4 is leucine or cyclobutylalanine,Xs is an a, a-disubstituted non-natural amino acid with an olefinic side chain cross-linked to Xi,Xe is S or A,X7 is absent or AAXs, wherein X8 is D-alanine, andAc is an acetyl group; or(ii) SEQ ID NO:300 with 1 or 2 amino acid substitutions, wherein the 1 or 2 amino acid substitutions are not at Xi, X3, or X5, and wherein the peptide binds to HDMX and HDM2;(b) incorporating a linker onto X3, (c) coupling a compound to the linker, and (d) cross- linking the peptide, thereby making the chimera; optionally wherein the compound is JQ1, optionally wherein the method further comprises purifying the chimera; and optionally wherein the method further comprises formulating the purified chimera as a sterile pharmaceutical composition.

18. A pharmaceutical composition comprising: (a) a means for treating a disease in a human subject in need thereof, and (b) a pharmaceutically acceptable carrier, optionally wherein the disease is a cancer.

19. A pharmaceutical composition comprising: (a) a means for treating a disease in a human subject in need thereof, (b) a means for inhibiting nuclear export, and (c) a pharmaceutically acceptable carrier, optionally wherein the disease is a cancer.

20. A pharmaceutical composition comprising: (a) a means for binding HDM2, HDMX, and a protein, and (b) a pharmaceutically acceptable carrier, optionally wherein the protein is a BET protein, optionally wherein the BET protein is a BRD4 protein.

21. A pharmaceutical composition comprising: (a) a means for binding HDM2, HDMX, and a protein, (b) a means for inhibiting nuclear export, and (c) a pharmaceutically acceptable carrier, optionally wherein the protein is a BET protein, optionally wherein the BET protein is a BRD4 protein.

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