Stabilized peptide-mediated targeted protein degradation

Abstract

The invention relates to stable peptide-mediated targeted protein degradation. Specifically, the invention relates to a chimera, and more specifically relates to a nailing peptide degradation determinant chimera. The present invention relates to a protein fusion protein that acts as a protein degradation inducing moiety by binding a nailing peptide that binds to a disease-related protein to a small molecule degradation determinant (e.g., a cereblone-or VHL-binding small molecule as a degradation determinant) or a polypeptide sequence degradation determinant (e.g., a Cop1-binding Trib peptide as a degradation determinant); or by binding a stapling peptide degradation determinant to a peptide (e.g., a stapling peptide) or a small molecule bound to a disease-associated protein to act as a protein degradation inducing moiety. The present application also relates to methods of targeted degradation of endogenous proteins by using a stapling peptide degradation determinant chimera that can be used to treat proliferative diseases or other conditions, where elimination of a disease-causing or disease-associated protein may have a therapeutic benefit. The invention also provides a method for preparing the compounds and intermediates thereof.

Description

[0001] This application is a divisional application. The international application number of its original application is PCT / US2018 / 065784, the international filing date is December 14, 2018, the Chinese national application number is 201880088640.9, and the date of entry into China is August 05, 2020. The invention title is "Stable Peptide-Mediated Targeted Protein Degradation".

[0002] Cross-reference to related applications

[0003] This application claims the priority of U.S. Provisional Application No. 62 / 599,608, filed on December 15, 2017, the entire content of which is incorporated herein by reference. Technical field

[0004] The present disclosure relates to stable peptides and small molecule chimeric compounds, referred to as stapled peptide-degrader conjugates, which act as a protein targeting moiety for binding (through a stapled peptide or molecular moiety) and a moiety for inducing protein degradation (through another stapled peptide or molecular moiety). The chimeras include a stapled peptide fused to any of the following: (i) a small molecule degrader (e.g., a cereblon- or VHL-binding small molecule as a degrader), (ii) a polypeptide sequence degrader, (iii) a stapled peptide as a degrader, or (iv) a small molecule as a protein targeting compound (with a stapled peptide as a degrader), and methods of using the same. Background art

[0005] A degron is a part of a protein that plays a major role in its degradation. Degrons are typically short amino acid sequences and can be located anywhere in the protein sequence (Cho et al., Genes & Development, 24(5): 438–442 (2010); Fortmann et al., Journal of Molecular Biology, 427(17): 2748–2756 (2015); Dohmen et al., Science, 263(5151): 1273-1276 (1994); Varshavsky, Proceedings of National Academy of Sciences, 93(22): 12142–12149 (1996)). In fact, some proteins have multiple degrons. Degrons have been identified in both prokaryotes and eukaryotes. Although there are several types of degrons and despite the high degree of variability between these groups, degrons are all very similar in regulating the rate of protein degradation. Degradation may or may not involve ubiquitin. Ubiquitin-dependent degrons contain specific sequences that are recognized by homologous ubiquitin E3 ligases.

[0006] The ubiquitin-proteasome pathway (UPP) is the main pathway for regulating key regulatory proteins and degrading misfolded or abnormal proteins. The covalent attachment of ubiquitin to specific protein substrates is achieved through the action of E3 ubiquitin ligases. For example, Cereblon (CRBN) interacts with damaged DNA-binding protein 1 (DDB1) and forms an E3 ubiquitin ligase complex with Cullin 4 (CUL4A), and then it acts as a substrate receptor, enabling the proteins recognized by CRBN to be ubiquitinated and degraded by the proteasome. It has also been determined that CRBN can bind immunomodulatory drugs (IMiDs), such as thalidomide. Such binding is related to the mechanism of teratogenicity and the cytotoxicity of IMiDs (such as lenalidomide), which are used to treat multiple myeloma. Summary of the Invention

[0007] The present disclosure relates to the synthesis and characterization of bifunctional stable peptide-small molecule (e.g., thalidomide degrader) conjugates, stable peptide-peptide (e.g., primary degrader sequence) conjugates, stable peptide-stable peptide (e.g., primary degrader sequence) conjugates, and small molecule-stable peptide (e.g., primary degrader sequence) conjugates (these conjugates are also referred to as "chimeras"). For example, these conjugates can be used to target proteins involved in or causing diseases. The targeted proteins can be of viral, bacterial, animal, or human origin. In certain cases, the conjugates can be used to target proteins that cause or are associated with diseases. Such stable peptide conjugates can be used to treat diseases driven by such pathological proteins. The ability of stapled peptides to target large and generally undruggable protein interaction surfaces, combined with degrader functionality, extends the scope of application of degrader technology beyond what can be achieved with small molecules and can enhance the bioactivity of stapled peptides.

[0008] In a first aspect, the present disclosure features a peptide-small molecule fusion comprising a protein-targeting stapled peptide and a small molecule degrader moiety (e.g., a thalidomide moiety or a Von Hippel-Lindau (VHL) moiety).

[0009] In some embodiments, a small molecule degrader stator (e.g., a thalidomide moiety or a VHL moiety) is conjugated to the N-terminus of a protein-targeting stapled peptide. In some cases, a small molecule degrader stator (e.g., a thalidomide moiety or a VHL moiety) is conjugated to the C-terminus of a protein-targeting stapled peptide. In certain cases, a small molecule degrader stator (e.g., a thalidomide moiety or a VHL moiety) is included in a non-natural amino acid inserted into the peptide sequence between the N- and C-termini of the protein fragment stapled peptide. In some cases, the stapled peptide binds a disease-causing protein. In some cases, the stapled peptide binds an intracellular protein. In some cases, the stapled peptide binds an extracellular protein. In some cases, the stapled peptide binds a cell surface protein (e.g., a receptor). In some cases, the stapled peptide binds a killer protein (e.g., BAX, BAK) or a protein harmful to cells or causing neurodegeneration (e.g., IgG, β-amyloid, tau, α-synuclein, TDP-43, hemoglobin (sickle cell), superoxide dismutase, Notch3, FUS, GFAP). In some cases, the stapled peptide binds a protein selected from the group consisting of: BCL2, BCLX L , MCL-1, BFL-1, BCL-w, BCL-B, EZH2, HDM2 / HDMX, KRAS / NRAS / HRAS, MYC, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI, MLL fusion protein, receptor tyrosine kinase, HOX homolog, JUN, Cyclin D, Cyclin E, BRAF, CRAF, CDK4, CDK2, HPV-E6 / E7, Aurora kinase, MITF, Wnt1, PD-1, BCR, and CCR5. In some cases, the stapled peptide binds a bacterial protein. In some cases, the stapled peptide binds a viral protein. In certain cases, the thalidomide moiety comprises the structures provided below:

[0010]

[0011] In some cases, the thalidomide moiety, when conjugated to the N-terminus of a stabilizing peptide, comprises the structures provided below:

[0012]

[0013] In some cases, the thalidomide moiety, when conjugated to the C-terminus of a stabilizing peptide, comprises the structures provided below:

[0014]

[0015] In some cases, the VHL moiety comprises the following structures:

[0016]

[0017] In some cases, the VHL portion comprises the following structure:

[0018]

[0019] In a second aspect, the present disclosure features a method of treating a disease or disorder driven by a pathological peptide or protein in a human subject in need thereof. The method comprises administering to the human subject a therapeutically effective amount of a peptide-small molecule fusion as described herein.

[0020] In a third aspect, the invention features a peptide degrader that binds to a WD40 repeat protein, wherein the WD40 repeat protein is a substrate adaptor of an E3 ubiquitin ligase. The peptide comprises a modified form of a native binding sequence or a native binding consensus sequence of an amino acid sequence that binds to the WD40 repeat protein. The modified form comprises at least one amino acid substitution, at least one amino acid deletion, at least one amino acid insertion, or any combination thereof within the native binding consensus sequence of the amino acid sequence that binds to the WD40 repeat protein. Exemplary peptides comprising modified forms of native binding sequences or native binding consensus sequences of amino acid sequences that bind to WD40 repeat proteins are provided in SEQ ID NOs: 26-30 and 106-118. In an illustrative example, the present disclosure provides a peptide that binds to the Constitutive Photomorphogenic 1 (Cop1) protein. The peptide comprises a modified form of the amino acid sequence DQIVPEY (SEQ ID NO: 25). The modified form comprises at least one amino acid substitution, at least one amino acid deletion, at least one amino acid insertion, or any combination thereof in SEQ ID NO: 25. If the modified form consists of a single amino acid substitution, the amino acid substitution is not A or R at any position from 1 to 7 in SEQ ID NO: 25, or V at position 4 in SEQ ID NO: 25.

[0021] In some embodiments, the peptide comprises the amino acid sequence set forth in SEQ ID NO:25, except that it has at least one amino acid substitution. In some cases, the peptide comprises the amino acid sequence set forth in SEQ ID NO:25, except that it has at least one amino acid deletion. In some cases, the peptide comprises the amino acid sequence set forth in SEQ ID NO:25, except that it has at least one amino acid substitution and at least one amino acid deletion. In certain cases, the peptide comprises the amino acid sequence set forth in SEQ ID NO:25, except that it has 1 to 6 amino acid substitutions. In some cases, positions 4 (V) and / or 5 (P) of SEQ ID NO:25 are unsubstituted. In some cases, one or more of positions 1 (D), 2 (Q), 3 (I), and 6 (E) of SEQ ID NO:25 are substituted. In some cases, the peptide comprises the amino acid sequence set forth in SEQ ID NO:25, except that it has 1 amino acid deletion. In certain cases, position 7 (Y) of the amino acid sequence set forth in SEQ ID NO:25 is deleted. In some cases, the peptide comprises the amino acid sequence set forth in SEQ ID NO:25, except that it has 1 to 6 amino acid substitutions and at least one amino acid deletion. In some cases, the peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs:26 to 30. In one case, the peptide has the amino acid sequence set forth in SEQ ID NO:30.

[0022] In certain embodiments, the peptide is 4 to 10 amino acids in length.

[0023] In some cases, the peptide binds to Cop1 with a binding affinity of 1 nM to 300 nM. In some cases, the peptide binds to Cop1 with a binding affinity of 1 nM to 1000 nM. In some cases, the peptide binds to Cop1 with a binding affinity of 10 nM to 300 nM. In some cases, the peptide binds to Cop1 with a binding affinity of 100 nM to 300 nM. In some cases, the peptide binds to Cop1 with a binding affinity of 200 nM to 300 nM. In some cases, the peptide binds to Cop1 with a binding affinity of 200 nM to 1000 nM.

[0024] In a fourth aspect, the present disclosure relates to a chimeric fusion polypeptide comprising a protein-targeting stapled peptide and a Trib1 peptide degrader or a variant thereof.

[0025] In some embodiments, the stapled peptide binds to an intracellular protein. In some embodiments, the stapled peptide binds to an extracellular protein. In some embodiments, the stapled peptide binds to a cell surface protein (e.g., a receptor). In some embodiments, the stapled peptide binds to a disease-causing or disease-related protein. In some embodiments, the stapled peptide binds to a killer protein (e.g., BAX, BAK) or a protein that is harmful to cells or causes neurodegeneration (e.g., IgG, β-amyloid, tau, α-synuclein, TDP-43, HbS, superoxide dismutase, Notch3, FUS, GFAP). In some embodiments, the stapled peptide binds to a protein selected from the group consisting of: BCL2, BCLXL, MCL-1, BFL-1, BCL-w, BCL-B, EZH2, HDM2 / HDMX, KRAS / NRAS / HRAS, MYC, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion protein, MLL fusion protein, receptor tyrosine kinase, HOX homolog, JUN, Cyclin D, Cyclin E, BRAF, CRAF, CDK4, CDK2, HPV-E6 / E7, Aurora kinase, MITF, Wnt1, PD-1, BCR, and CCR5. In some embodiments, the stapled peptide binds to a bacterial protein. In some embodiments, the stapled peptide binds to a viral protein.

[0026] In a fifth aspect, the present disclosure features a chimeric polypeptide comprising a stapled peptide and a peptide that binds to a WD40 repeat protein, wherein the WD40 repeat protein is a substrate adaptor of an E3 ubiquitin ligase. The peptide comprises a modified form of a natural binding sequence or natural binding consensus sequence of amino acids that bind to the WD40 repeat protein. The modified form comprises at least one amino acid substitution, at least one amino acid deletion, at least one amino acid insertion, or any combination thereof within the natural binding consensus sequence of amino acids that bind to the WD40 repeat protein.

[0027] In some embodiments, the WD40 repeat protein that is a substrate adaptor of an E3 ubiquitin ligase is selected from the group consisting of: MDM2, SKP2-CKS1, FBXW1, FBXW2, FBXW4, FBXW5, FBXW7, FBXW8, FBXW9, FBXW10, FBXW11, FBXW12, SPOP, VHL, ITCH, KEAP1, KLHL2, KLHL3, KLHL7, KLHL12, KLHL13, KLHL15, KLHL20, KLHL21, KLHL24, KLHL40, KLHL42, COP1, TRAF7, RFWD3, DCAF1, DCAF2, DCAF3, DCAF4, DCAF5, DCAF6, DCAF7, DCAF8, DCAF9, DCAF10, DCAF11, DCAF12, DCAF13, DCAF14, DCAF15, DCAF16, DCAF17, DCAF19, SIAH1, TRPC4AC, DET1, WSB1, WSB2, HERC1, DDB2, CSA, CBL, CDC20, and FZR1.

[0028] In some embodiments, the native binding sequence or native binding consensus sequence is a sequence selected from the group consisting of: SEQ ID NO.: 25, 31-46, and 65-105. In some cases, the native binding consensus sequence is SEQ ID NO: 25. In other cases, the native binding consensus sequence is SEQ ID NO: 46.

[0029] In some embodiments, the peptide comprises the amino acid sequence set forth in SEQ ID NO:25, except that it has at least one amino acid substitution. In some cases, the peptide comprises the amino acid sequence set forth in SEQ ID NO:25, except that it has at least one amino acid deletion. In some cases, the peptide comprises the amino acid sequence set forth in SEQ ID NO:25, except that it has at least one amino acid substitution and at least one amino acid deletion. In certain cases, the peptide comprises the amino acid sequence set forth in SEQ ID NO:25, except that it has 1 to 6 amino acid substitutions. In some cases, positions 4 (V) and / or 5 (P) of SEQ ID NO:25 are unsubstituted. In some cases, one or more of positions 1 (D), 2 (Q), 3 (I), and 6 (E) of SEQ ID NO:25 are substituted. In some cases, the peptide comprises the amino acid sequence set forth in SEQ ID NO:25, except that it has 1 amino acid deletion. In certain cases, position 7 (Y) of the amino acid sequence set forth in SEQ ID NO:25 is deleted. In some cases, the peptide comprises the amino acid sequence set forth in SEQ ID NO:25, except that it has 1 to 6 amino acid substitutions and at least one amino acid deletion. In some cases, the peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs:26 to 30. In one case, the peptide has the amino acid sequence set forth in SEQ ID NO:30.

[0030] In certain embodiments, the peptide is 4 to 30 amino acids in length. In certain embodiments, the peptide is 4 to 20 amino acids in length. In certain embodiments, the peptide is 4 to 15 amino acids in length. In certain embodiments, the peptide is 5 to 20 amino acids in length.

[0031] In certain embodiments, the peptide binds to Cop1 with a binding affinity of 1 nM to 300 nM; 10 nM to 300 nM; 100 nM to 300 nM; or 200 nM to 300 nM. In certain embodiments, the peptide binds to Cop1 with a binding affinity of 1 nM to 1000 nM. In certain embodiments, the peptide binds to Cop1 with a binding affinity of 200 nM to 1000 nM.

[0032] In some embodiments, the stapled peptide binds to an intracellular protein. In some embodiments, the stapled peptide binds to an extracellular protein. In some embodiments, the stapled peptide binds to a cell surface protein (e.g., a receptor). In some embodiments, the stapled peptide binds to a disease-causing or disease-related protein. In some embodiments, the stapled peptide binds to a killer protein (e.g., BAX, BAK) or a protein that is harmful to cells or causes neurodegeneration (e.g., IgG, β-amyloid, tau, α-synuclein, TDP-43, HbS (hemoglobin-sickle cell), superoxide dismutase, Notch3, FUS, GFAP). In some embodiments, the stapled peptide binds to a protein selected from the group consisting of: BCL2, BCLXL, MCL-1, BFL-1, BCL-w, BCL-B, EZH2, HDM2 / HDMX, KRAS / NRAS / HRAS, MYC, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion protein, MLL fusion protein, receptor tyrosine kinase, HOX homolog, JUN, Cyclin D, Cyclin E, BRAF, CRAF, CDK4, CDK2, HPV-E6 / E7, Aurora kinase, MITF, Wnt1, PD-1, BCR, and CCR5. In some embodiments, the stapled peptide binds to a bacterial protein. In some embodiments, the stapled peptide binds to a viral protein. In certain cases, the stapled peptide targets protein aggregates that cause neurodegeneration (e.g., β-amyloid).

[0033] In a sixth aspect, the present disclosure features a modified protein of a first protein that includes a structurally disordered region. The modified protein differs from the first protein in that the structurally disordered region includes a peptide that binds to a WD40 repeat protein, which is a substrate adaptor of an E3 ubiquitin ligase. The peptide includes a modified form of a natural binding consensus sequence, wherein the modified form includes at least one amino acid substitution, at least one amino acid deletion, at least one amino acid insertion, or any combination thereof within the natural binding consensus sequence.

[0034] In certain embodiments, the WD40 repeat protein that serves as a substrate adaptor for an E3 ubiquitin ligase is selected from the group consisting of: MDM2, SKP2-CKS1, FBXW1, FBXW2, FBXW4, FBXW5, FBXW7, FBXW8, FBXW9, FBXW10, FBXW11, FBXW12, SPOP, VHL, ITCH, KEAP1, KLHL2, KLHL3, KLHL7, KLHL12, KLHL13, KLHL15, KLHL20, KLHL21, KLHL24, KLHL40, KLHL42, COP1, TRAF7, RFWD3, DCAF1, DCAF2, DCAF3, DCAF4, DCAF5, DCAF6, DCAF7, DCAF8, DCAF9, DCAF10, DCAF11, DCAF12, DCAF13, DCAF14, DCAF15, DCAF16, DCAF17, DCAF19, SIAH1, TRPC4AC, DET1, WSB1, WSB2, HERC1, DDB2, CSA, CBL, CDC20, and FZR1. In some cases, the native binding consensus sequence is a sequence selected from the group consisting of: SEQ ID NO.: 25, 31-46, and 65-105. In some cases, the native binding consensus sequence is SEQ ID NO: 25. In some cases, the native binding consensus sequence is SEQ ID NO: 46.

[0035] In some embodiments, the peptide comprises the amino acid sequence shown in SEQ ID NO:25, except that there is at least one amino acid substitution. In some cases, the peptide comprises the amino acid sequence shown in SEQ ID NO:25, except that there is at least one amino acid deletion. In some cases, the peptide comprises the amino acid sequence shown in SEQ ID NO:25, except that there is at least one amino acid substitution and at least one amino acid deletion. In some cases, the peptide comprises the amino acid sequence shown in SEQ ID NO:25, except that there is 1 to 6 amino acid substitutions. In some cases, SEQ ID NO:25 4 (V) and / or 5 (P) are not substituted. In some cases, SEQ ID NO:25 1 (D), 2 (Q), 3 (I) and 6 (E) of one or more are substituted. In some cases, the peptide comprises the amino acid sequence shown in SEQ ID NO:25, except that there is 1 amino acid deletion. In some cases, SEQ ID NO:25 amino acid sequence 7 (Y) is missing. In some cases, the peptide comprises the amino acid sequence shown in SEQ ID NO:25, except that there is 1 to 6 amino acid substitutions and at least one amino acid deletion. In some cases, the peptide has an amino acid sequence selected from the group consisting of: SEQ ID NO.: 26 to 30. In one case, the peptide has an amino acid sequence set forth in SEQ ID NO:30.

[0036] In certain embodiments, the peptide is 4 to 10 amino acids in length.

[0037] In certain embodiments, the peptide binds to Cop1 with an affinity of 1 nM to 300 nM; 10 nM to 300 nM; 100 nM to 300 nM; or 200 nM to 300 nM. In certain embodiments, the peptide binds to Cop1 with an affinity of 1 nM to 1000 nM. In certain embodiments, the peptide binds to Cop1 with an affinity of 200 nM to 1000 nM.

[0038] In a seventh aspect, the disclosure features a method of treating a disease or condition driven by a pathological peptide or protein in a human subject in need thereof. The method comprises administering to the human subject a therapeutically effective amount of a chimeric fusion polypeptide described herein.

[0039] In an eighth aspect, the disclosure features a peptide degron selected from the group consisting of: SEQ ID NOs.: 106-118.

[0040] In some embodiments, these peptides are linked to stabilizing peptides.

[0041] In a ninth aspect, the present disclosure provides stable peptide-peptide degrader stator chimeras selected from the group consisting of SEQ ID NOs: 119 to 126.

[0042] In certain embodiments of all of the above aspects, the stable peptide-degrader stator chimeras are used to target the degradation of one or more of the following: BCL2, BCLXL L , BCL W , MCL-1, BFL-1, BAX, MDM2 or MDMX.

[0043] In a tenth aspect, the present disclosure provides stable peptide-stable peptide degrader stator chimeras that comprise two stable peptides - a first stable peptide and a second stable peptide, wherein the first stable peptide binds to a first protein that is a protein target to be degraded, and the second stable peptide binds to a second protein that is a degrader protein. In certain embodiments, the first stable peptide binds to a disease-related protein that is a degradation target, and the second stable peptide binds to a degrader protein such as an E3 ligase (e.g., MDM2).

[0044] In some embodiments, the first protein is an intracellular protein. In some embodiments, the first protein is an extracellular protein. In some embodiments, the first protein is a cell surface protein (e.g., a receptor). In some embodiments, the first protein is a disease-causing or disease-related protein. In some embodiments, the first protein is a killer protein (e.g., BAX, BAK) or a protein that is harmful to cells or causes neurodegeneration (e.g., IgG, beta-amyloid, tau, alpha-synuclein, TDP-43, HbS (hemoglobin-sickle cell), superoxide dismutase, Notch3, FUS, GFAP). In some embodiments, the first protein is a protein selected from the group consisting of: BCL2, BCLXL, MCL-1, BFL-1, BCL-w, BCL-B, EZH2, HDM2 / HDMX, KRAS / NRAS / HRAS, MYC, beta-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion protein, MLL fusion protein, receptor tyrosine kinase, HOX homolog, JUN, Cyclin D, Cyclin E, BRAF, CRAF, CDK4, CDK2, HPV-E6 / E7, Aurora kinase, MITF, Wnt1, PD-1, BCR, and CCR5. In some embodiments, the first protein is a bacterial protein. In some embodiments, the first protein is a viral protein. In certain cases, the first protein is a protein aggregate that causes neurodegeneration (e.g., beta-amyloid).

[0045] In certain embodiments, the first stabilizing peptide has the amino acid sequence shown in any one of SEQ ID NOs: 1-24 and 134 or a variant thereof. In certain embodiments, the second stabilizing peptide has the amino acid sequence shown below: SEQ ID NO: 6 or a variant thereof. In certain embodiments, the second stabilizing peptide has the amino acid sequence shown below: SEQ ID NO: 18 or a variant thereof.

[0046] In certain embodiments, the second protein is a degrader protein, such as an E3 ubiquitin ligase or a substrate adaptor of an E3 ubiquitin ligase. In certain embodiments, the second protein is an E3 ubiquitin ligase. In some embodiments, the second protein binds to an E3 ligase (e.g., MDM2) or a protein complexed with an E3 ligase, such as MDMX that binds MDM2. In certain embodiments, the E3 ubiquitin ligase is a RING E3 ubiquitin ligase (e.g., Mdm2-MdmX, TRIM5α, c-CBL, cIAP, RNF4, BIRC7, IDOL, BRCA1-BARD1, RING1B-Bmi1, E4B, CHIP, Prp19). In certain embodiments, the E3 ubiquitin ligase is a HECT E3 ubiquitin ligase (e.g., Smurf1, Smurf2, Itch, E6AP). In certain embodiments, the E3 ubiquitin ligase is a RBR E3 ubiquitin ligase (e.g., Parkin, Parc, RNF144(A / B), HOIP, HHARI). For example, non-limiting examples of E3 ubiquitin ligases are described in Morreale and Walden, Cell 165, 2016 DOI http: / dx.doi.org / 10.1016 / j.cell.2016.03.003.

[0047] In certain embodiments, the second stabilizing peptide moiety of the chimera has the amino acid sequence shown below: SEQ ID NO: 134 or a variant thereof. In certain embodiments, the second stabilizing peptide has the amino acid sequence shown below: SEQ ID NO: 6 or a variant thereof. In certain embodiments, the second stabilizing peptide has the amino acid sequence shown below: SEQ ID NO: 18 or a variant thereof.

[0048] In an eleventh aspect, the present disclosure provides a small molecule-stabilizing peptide degrader chimera comprising: a small molecule and a stabilizing peptide, wherein the small molecule binds to a first protein that is a protein target to be degraded; and the stabilizing peptide binds to a second protein that is a degrader protein. In certain embodiments, the stabilizing peptide binds and recruits a degrader protein, such as an E3 ligase (e.g., MDM2) or a degrader protein complex, such as the MDM2 / MDMX complex.

[0049] In some embodiments, the first protein is an intracellular protein. In some embodiments, the first protein is an extracellular protein. In some embodiments, the first protein is a cell surface protein (e.g., a receptor). In some embodiments, the first protein is a disease-causing or disease-related protein. In some embodiments, the first protein is a killer protein (e.g., BAX, BAK) or a protein harmful to cells or causing neurodegeneration (e.g., IgG, amyloid-β, tau, α-synuclein, TDP-43, HbS (hemoglobin-sickle cell), superoxide dismutase, Notch3, FUS, GFAP). In some embodiments, the first protein is a protein selected from the group consisting of: BCL2, BCLXL, MCL-1, BFL-1, BCL-w, BCL-B, EZH2, HDM2 / HDMX, KRAS / NRAS / HRAS, MYC, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion protein, MLL fusion protein, receptor tyrosine kinase, HOX homolog, JUN, Cyclin D, Cyclin E, BRAF, CRAF, CDK4, CDK2, HPV-E6 / E7, Aurora kinase, MITF, Wnt1, PD-1, BCR, and CCR5. In some embodiments, the first protein is a bacterial protein. In some embodiments, the first protein is a viral protein. In certain cases, the first protein is a protein aggregate (e.g., amyloid-β).

[0050] In certain embodiments, the second protein is a degrader protein, such as an E3 ubiquitin ligase or a substrate adaptor of an E3 ubiquitin ligase. In certain embodiments, the second protein is an E3 ubiquitin ligase. In some embodiments, the second protein binds to an E3 ligase (e.g., MDM2) or a protein complexed with an E3 ligase, such as MDMX that binds MDM2. In certain embodiments, the E3 ubiquitin ligase is a RING E3 ubiquitin ligase (e.g., Mdm2-MdmX, TRIM5α, c-CBL, cIAP, RNF4, BIRC7, IDOL, BRCA1-BARD1, RING1B-Bmi1, E4B, CHIP, Prp19). In certain embodiments, the E3 ubiquitin ligase is a HECT E3 ubiquitin ligase (e.g., Smurf1, Smurf2, Itch, E6AP). In certain embodiments, the E3 ubiquitin ligase is a RBR E3 ubiquitin ligase (e.g., Parkin, Parc, RNF144(A / B), HOIP, HHARI). For example, non-limiting examples of E3 ubiquitin ligases are described in Morreale and Walden, Cell 165, 2016 DOI http: / dx.doi.org / 10.1016 / j.cell.2016.03.003.

[0051] In a twelfth aspect, the present disclosure provides a chimera comprising: a first portion that binds to a second portion; wherein the first portion binds a first protein targeted for degradation and the second portion binds a second protein; wherein the second protein is a protein degrader.

[0052] In certain aspects, the first portion and the second portion are covalently linked to each other. In certain aspects, the first portion and the second portion are linked to each other through a linker.

[0053] In some embodiments, the first protein is an intracellular protein. In some embodiments, the first protein is an extracellular protein. In some embodiments, the first protein is a cell surface protein (e.g., a receptor). In some embodiments, the first protein is a disease-causing or disease-related protein. In some embodiments, the first protein is a killer protein (e.g., BAX, BAK) or a protein harmful to cells or causing neurodegeneration (e.g., IgG, amyloid-β, tau, α-synuclein, TDP-43, HbS (hemoglobin-sickle cell), superoxide dismutase, Notch3, FUS, GFAP). In some embodiments, the first protein is a protein selected from the group consisting of: BCL2, BCLXL, MCL-1, BFL-1, BCL-w, BCL-B, EZH2, HDM2 / HDMX, KRAS / NRAS / HRAS, MYC, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion protein, MLL fusion protein, receptor tyrosine kinase, HOX homolog, JUN, Cyclin D, Cyclin E, BRAF, CRAF, CDK4, CDK2, HPV-E6 / E7, Aurora kinase, MITF, Wnt1, PD-1, BCR, and CCR5. In some embodiments, the first protein is a bacterial protein. In some embodiments, the first protein is a viral protein. In certain cases, the first protein is a protein aggregate (e.g., amyloid-β).

[0054] In certain cases, the first moiety comprises a first stapled peptide that binds to the first protein to be targeted for degradation. In certain cases, the first moiety comprises a small molecule that binds to the first protein to be targeted for degradation. In certain cases, the second moiety comprises a second stapled peptide that binds to a second protein, such as a proteolytic agent. In certain cases, the second moiety comprises a small molecule that binds to a second protein, such as a proteolytic agent. In certain cases, the second moiety comprises a peptide degrader that binds to a proteolytic agent. In certain cases, the first moiety comprises a first stapled peptide that binds to the first protein, and the second moiety comprises a second stapled peptide that binds to the second protein. In certain cases, the first moiety comprises a first stapled peptide that binds to the first protein, and the second moiety comprises a small molecule that binds to the second protein. In certain cases, the first moiety comprises a first stapled peptide that binds to the first protein, and the second moiety comprises a peptide degrader that binds to a proteolytic agent. In certain cases, the first moiety comprises a small molecule that binds to the first protein, and the second moiety comprises a stapled peptide that binds to the second protein.

[0055] In certain cases where the first moiety is a stapled peptide, the stapled peptide does not include a Bcl-2 homology 3 (BH3) domain polypeptide. In certain cases where the first moiety is a stapled peptide, the stapled peptide does not include: (a) the Bcl-2 homology 3 domain of MCL-1, (b) the stable α-helix of MCL-1 of the BCL2 domain, or (c) MCL-1SAHBD.

[0056] In certain cases where the first moiety is a stapled peptide, the second moiety binds to the N-terminus of the first moiety. In certain cases where the first moiety is a stapled peptide, the second moiety binds to the C-terminus of the first moiety. In certain cases where the first moiety is the first stapled peptide, the second moiety binds to an internal amino acid position of the first moiety.

[0057] In certain cases where the second moiety is a stapled peptide, the first moiety binds to the N-terminus of the second moiety. In certain cases where the second moiety is a stapled peptide, the first moiety binds to the C-terminus of the second moiety. In certain cases where the second moiety is a stapled peptide, the first moiety binds to an internal amino acid position of the second moiety.

[0058] In some aspects, the proteolytic agent degrades the first protein to be targeted for degradation.

[0059] In a thirteenth aspect, the present disclosure provides a method of treating a disease or disorder driven by a pathological peptide or protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the chimera described herein.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present application (including definitions) will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0061] Other features and advantages of the present invention will become apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1A series of representative peptide sequences and degradation determinant types / positions on stapled peptide sequences are provided. The amino acid sequences in column 1 are designated as SEQ ID NO.: 1-24. # = N-terminal Ac or degradation determinant Ahx, as indicated; % = C-terminal Lys (ivdde) or Lys (degradation determinant), as indicated; X = S-pentenylalanine; 8 = R-octenylalanine; B = norleucine; * (within the amino acid sequence) = cyclobutanealanine (ivdde = 1-(4,4-dimethyl-2,6-dioxocyclohex-1-yl)-3-methylbutyl)

[0063] Figure 2 Shown is the coupling of the carboxyl degron thalidomide moiety to a resin-bound primary amine to generate Figure 1 Stapled peptide degrons are shown.

[0064] Figure 3 The portion containing the C-terminal degron is shown, which is a side chain conjugated lysine attached to the peptide.

[0065] Figure 4 The portion containing the N-terminal degron is shown, which is the aminocaproic acid to which the peptide is linked.

[0066] Figure 5 The structure of diaminobutyric acid ("DAB") is shown along with the chemical structures of THAL and VHL ligands used for coupling to acids or amines.

[0067] Figure 6 The structures of various linkers (Gly, βAla, and linkers 1-8) are shown.

[0068] Figure 7 A series of stapled peptide degron chimeras are shown, in which diaminobutyric acid was incorporated into the stapled peptide to link the small molecule degron, and linkers of various compositions and lengths were installed to separate the stapled peptide from the small molecule or peptide degron (THAL, TRIB, or VHL). Figure 7 The stapled peptide sequences shown are: IWIA%ELRXIGDXFNAYYARR (SEQ ID NO: 127), IWIAQELRXIGDXFN%YYARR (SEQ ID NO: 128), LTF8%YWAQLXSAA (SEQ ID NO: 129), LTF8EYWAQLX%AA (SEQ ID NO: 130); and %TF8EYWAQLXSAA (SEQ ID NO: 131), wherein % is as shown, 8 is (R)-2-(7-octenyl)alanine, and X is (S)-2-(4-pentenyl)alanine.

[0069] Figure 8The ability of stapled peptide degron chimeras consisting of a stapled peptide linked to a thalidomide degron to retain binding to recombinant cereblon is shown, monitored by a competitive fluorescence polarization assay. Lenalidomide was a positive control for this experiment. Sequences: #QLTAARLKXLGDXLHQRTBWR% (SEQ ID NO: 11); #AELEVESATQLRXFGDXLNFRQKLL% (SEQ ID NO: 12); and #RRFFGIXLTNXLKTEEGN% (SEQ ID NO: 3), where X is (S)-2-(4-pentenyl)alanine, and # and % are as described in the accompanying figures. "N-terminal Ac" = N-terminal acetylation; Lys(ivDde) ​​= N-α-Fmoc-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-yl)-3-methylbutyl-L-lysine; "Lys-degron" = Figure 3 Structure shown; "N-terminal degron Ahx" = Figure 4 The structure shown.

[0070] Figures 9A-9I It was shown that the stapled peptide degron chimera can enter cells to compete with dBET6 for interaction with cereblon and inhibit the induced degradation of GFP-BRD4.

[0071] #RRFFGIXLTNXLKTEEGN%(SEQ ID NO:3);

[0072] #FSSNRXKILXRTQILNQEWKQRRIQPV%(SEQ ID NO:2);

[0073] #NLWAAQRYGRELRXBSDXFVDSFKK%(SEQ ID NO:10);

[0074] #LSQEQLEHRERSLXTLRXIQRBLF%(SEQ ID NO:5); and

[0075] #NLWAAQRYGRELRXBDDXFVDSFKK% (SEQ ID NO: 9), wherein X is (S)-2-(4-pentenyl)alanine, and # and % are as described in the accompanying figure. "N-terminal Ac" = N-terminal acetylation; Lys(ivDde) ​​= N-α-Fmoc-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohexylene-1-yl)-3-methylbutyl-L-lysine; "Lys-degradant" = Figure 3 Structure shown; "N-terminal degron Ahx" = Figure 4 The structure shown.

[0076] Figure 10 It was shown that BIM-C-terminal degron induced MCL-1 degradation when added to A375P melanoma cell line at a concentration of 10 μM. BIM-C-terminal degron is #IWIAQELRXIGDXFNAYYARR% (SEQ ID NO: 134; # = N-terminal Ac; % = Lys-degron (the structure of Lys-degron is shown in Figure 3 )).

[0077] Figure 11 SJSA-1 cells treated with a panel of stapled peptide degron (1 μM) chimeras consisting of an MDM2 / MDMX targeting stapled peptide ("ATSP") and a thalidomide degron moiety were shown to have lower MDM2 levels in cancer cells compared to cells treated with ATSP-7041 alone, as assessed by anti-MDM2 Western blot analysis. Actin represents a loading control. Sequences: LTF8EYWAQLX%AA (SEQ ID NO: 130) and LTF8EYWAQ#XSAA (SEQ ID NO: 6). Linkers 1, 2, 3, and 5 are shown in Table 1. Figure 6 shown.

[0078] Figure 12 It is shown that treatment of SJSA-1 cells with a set of stapled peptide degron chimeras consisting of an MDM2 / MDMX targeting stapled peptide (ATSP), different linkers, and thalidomide degron impairs the cell viability of cancer cells to varying degrees, with "5-L5" (LTF8EYWAQLX%AA (SEQ ID NO: 130), where % is DAB / LINKER5 / THAL) showing the strongest cytotoxic activity (left). Certain compositions with different linkers showed no cell activity (right). Linker 1-5 as shown Figure 5 shown.

[0079] Figure 13 Expression of MDM2 p60 chimeric constructs using Myc tags in 293T cells shows how genetic modification of MDM2 p60 isoforms with sequences from Trib1 results in Cop1-mediated degradation. The native peptide sequence GFDVPD (SEQ ID NO:26) in MDM2 was replaced with the indicated sequences (lane 3: SEQ ID NO:27; lane 4: SEQ ID NO:28; lane 5: SEQ ID NO:29; and lane 6: SEQ ID NO:30). Replacement of the native sequence with the mutant sequence DQIVPD (SEQ ID NO:30) results in disruption of the p60 chimeric protein by Cop1 protein. Bottom: Cop1 loading control.

[0080] Figure 14 Shown is binding of Myc-tagged MDM2 p60 mutant constructs to Copl assessed by co-immunoprecipitation in 293T cells.Sequences: GFDVPD (SEQ ID NO:26); GFDAAD (SEQ ID NO:27); GNDVPD (SEQ ID NO:28); PQTVPD (SEQ ID NO:29); and DQIVPD (SEQ ID NO:30).

[0081] Figure 15 SAH+Trib peptide sequences for evaluating activity for targeted Trib-degron mediated protein degradation are provided; stapled peptide chimera sequences are designated as SEQ ID NOs.: 119-126.

[0082] Figure 16 Shown is the structure of a peptide degron modeled after the TRIB sequence and the relevant moieties incorporated therein for coupling to amines or acids.

[0083] Figure 17 Shown, treatment of SJSA-1 or SJSA-X cells with a panel of stapled peptide degron chimeras consisting of an MDM2 / MDMX targeting stapled peptide (e.g., ATSP-7041-like stapled p53 peptide) and a TRIB degron moiety (20 μM) demonstrated varying degrees of reduction in MDM2 levels in cancer cells compared to cancer cells treated with ATSP-7041 alone (1 μM), as assessed by anti-MDM2 Western blot analysis. Actin represents a loading control. Sequences: LTF8EYWAQ#XSAA (SEQ ID NO: 6) and LTF8%YWAQLXSAA (SEQ ID NO: 129).

[0084] Figure 18 It was shown that treatment of SJSA-1 or SJSA-X cells with a panel of stapled peptide degron chimeras consisting of an MDM2 / MDMX targeting stapled peptide (e.g., ATSP-7041-like stapled p53 peptide) and a TRIB degron variably impaired cell viability of cancer cells. Sequence: LTF8%YWAQLXSAA (SEQ ID NO: 129).

[0085] Figure 19Shown, a panel of stapled peptide degron chimeras (1 μM) consisting of an MDM2 / MDMX targeting stapled peptide (e.g., ATSP-7041-like stapled p53 peptide) and a VHL degron portion treated SJSA-1 or SJSA-X cells, as assessed by anti-MDM2 Western blot analysis, exhibited varying degrees of reduction in MDM2 levels in cancer cells compared to cancer cells treated with ATSP-7041 alone. Actin represents a loading control. Sequences: LTF8EYWAQ#XSAA (SEQ ID NO: 6) and LTF8EYWAQLX%AA (SEQ ID NO: 130).

[0086] Figure 20 It was shown that a panel of stapled peptide degron chimeras (1 μM) consisting of an MDM2 / MDMX targeting stapled peptide (e.g., ATSP-7041-like stapled p53 peptide) and a VHL degron portion treated SJSA-1 or SJSA-X cells impaired the cell viability of cancer cells to varying degrees. Sequences: %TF8EYWAQLXSAA (SEQ ID NO: 131) and LTF8EYWAQLX%AA (SEQ ID NO: 130).

[0087] Figure 21 The structure of an exemplary stapled peptide degradation determinant chimera is shown, which comprises a stapled peptide and a second stapled peptide. One stapled peptide targets a protein of interest (e.g., a disease-associated protein of interest) and the second stapled peptide binds a degrader protein (e.g., stapled peptide ATSP-7041 (SEQ ID NO:6, for binding to MDM2). Also described are examples of stapled peptide degradation determinant chimeras comprising two identical stapled peptides, wherein the stapled peptides target a target protein (e.g., a disease-associated target protein) that is a degrader protein, such that the resulting protein dimerization and self-degradation ensures binding of the stapled peptide degradation determinant chimera to the target protein. In each of the examples shown, the two stapled peptides are connected by a linker of variable length (e.g., exemplary linkers are shown in Figure 6 ). Left column (from top to bottom): SEQ ID NOs: 1-5, 132, 7-10, 133 and 12. Right column: SEQ ID NO: 134.

[0088] Figure 22 It was shown that incubation of the ubiquitination machinery (including E1, E2 and recombinant MDM2) with recombinant MCL-1 and stapled peptide degron chimeras that bind to both MDM2 and MCL-1 induced ubiquitination of MCL-1 (amino acids 1-327) by MDM2.

[0089] Figure 23The structure of the stapled peptide degron chimera is shown. A stapled peptide (ATSP-7041 (LTF8EYWAQ#XSAA (SEQ ID NO: 6)) was introduced to bind and recruit a degrader protein (MDM2), and a small molecule (JQ1) was included to bind a disease-associated protein (BRD4).

[0090] Figure 24 It was shown that incubation of the ubiquitination machinery (including E1, E2 and MDM2) with recombinant BRD4 species (e.g., amino acids 342-460; amino acids 49-170) and stapled peptide degradation determinant chimeras that bind (e.g., stapled p53 peptide ATSP-7041) and BRD4 (e.g., small molecule JQ1) induces ubiquitination of BRD4 in two different regions that can be induced by MDM2, where the linker consists of two β-alanine amino acids.

[0091] Figure 25 The use of stapled peptides that bind to MDM2 and small molecules that bind to BRD4, such as JQ1, and Figure 23 Treatment of U2OS cells with stapled peptide degron chimeras with a linker consisting of two β-alanines as shown results in a time-dependent degradation of native BRD4. Actin represents a loading control.

[0092] Figure 26 The top panel shows the chemical structures of exemplary unnatural amino acids used to generate various stapled peptides for insertion into peptides.

[0093] Figure 26 The middle panel shows peptides with stapled peptides of various lengths.

[0094] Figure 26 The bottom panel shows a stapled peptide walk along the peptide sequence.

[0095] Figure 27 is a schematic diagram showing various double and triple stapling strategies and exemplary stapled peptide walks for generating stapled peptides.

[0096] Figure 28 is a schematic diagram showing a stapled peptide walk using various lengths of branched double stapled portions that produce stapled peptides.

[0097] Figure 29 is a schematic diagram showing exemplary chemical alterations used to generate stapled peptides. DETAILED DESCRIPTION

[0098] The present disclosure features stable peptide degradation determinant chimeras that act as protein degradation inducing portions, which combine a stable peptide targeting a disease-associated protein with a cereblon-bound small molecule thalidomide as a "degradation determinant", such as or more generally an alternative small molecule degradation determinant or polypeptide sequence "degradation determinant", including a stable polypeptide sequence "degradation determinant". Stable peptide degradation determinant chimeras also include a stable peptide that binds and recruits a degrader protein in combination with a small molecule or peptide introduced to target a disease-associated protein. By combining the ability of a stable peptide to effectively target a wide range of intracellular proteins (previously inaccessible to small molecules) with a small molecule or peptide degradation determinant portion of a degrader protein that can recruit degradation of the bound protein, or by combining a stable peptide that effectively binds and recruits a degrader protein with a small molecule or peptide targeting a disease-associated protein, this novel stapled peptide degradation determinant chimera expands the potency and breadth of the biological activity of the stapled peptide. The disclosure also relates to methods for targeted degradation of endogenous proteins by using stapled peptide degradation determinant chimeras, which can be used to treat diseases (e.g., proliferative diseases) caused by the presence of disease-related proteins. The application also provides methods for preparing compounds of the application and intermediates thereof.

[0099] Stable peptide

[0100] The peptide helix is ​​an important mediator of key protein-protein interactions that regulate many important biological processes (e.g., apoptosis); however, when this helix is ​​separated from its protein environment and prepared separately, it can unfold and adopt a random helical conformation, resulting in a sharp decrease in biological activity, thereby reducing therapeutic potential. To avoid this problem, structurally stable peptides can be used. In some cases, the structurally stable peptide comprises at least two modified amino acids connected by internal (intramolecular) cross-links (or staples). Stabilized peptides as described herein include stapled peptides, stitched peptides, peptides comprising multiple stitches, peptides comprising multiple staples, or peptides comprising a mixture of staples and stitches, as well as peptides structurally enhanced by other chemical strategies (see, e.g., Balaram P. Cur. Opin. Struct. Biol. 1992; 2:845; Kemp DS et al., J. Am. Chem. Soc. 1996; 118:4240; Orner BP, et al., J. Am. Chem. Soc. 2001; 123:5382; Chin JW et al., Int. Ed. 2001; 40:3806; Chapman RN, et al., J. Am. Chem. Soc. 2004; 126:12252; Horne WS et al., Chem., Int. Ed. 2008; 47:2853; Madden et al., Chem. Commun (Camb). 2009 Oct 7; (37): 5588–5590; Lau et al., Chem. Soc. Rev., 2015, 44: 91-102; and Gunnoo et al., Org. Biomol. Chem., 2016, 14: 8002-8013; all of which are incorporated herein by reference in their entirety).

[0101] In certain embodiments, the polypeptide can be stabilized by peptide stapling (see, e.g., Walensky, J. Med. Chem., 57: 6275-6288 (2014), the contents of which are incorporated herein by reference in their entirety). The peptide is "stable" because it maintains its native secondary structure. For example, stapling allows a polypeptide that tends to have an α-helical secondary structure to maintain its native α-helical conformation. This secondary structure increases the resistance of the polypeptide to proteolytic cleavage and heating, and can also increase target binding affinity, hydrophobicity, and cell permeability. Therefore, the stapled (cross-linked) polypeptides described herein have improved biological activity relative to the corresponding non-stapled (uncross-linked) polypeptides.

[0102] "Peptide stapling" is a term coined by a synthetic method in which two olefin-containing side chains (e.g., cross-linkable side chains) present in a polypeptide chain are covalently linked (e.g., "stapled together") using a ring-closing metathesis (RCM) reaction to form a cross-linked ring (e.g., see Blackwell et al., J. Org. Chem., 66:5291-5302, 2001; Angew et al., Chem. Int. Ed. 37:3281, 1994). As used herein, the term "peptide stapling" includes the joining of two (e.g., at least one pair) of double-bond-containing side chains, triple-bond-containing side chains, or double-bond-containing and triple-bond-containing side chains that may be present in a polypeptide chain, using any number of reaction conditions and / or catalysts to promote such a reaction to provide a single "stapled" polypeptide. The term "multiple-stapled" polypeptide refers to those polypeptides that contain more than one single stapled staple, and may include two, three, or more independent staples with different spacings. In addition, as used herein, the term "peptidestitching" refers to multiple series of "stitching" events in a single polypeptide chain to provide a "stitched" (e.g., series or multiple stapled) polypeptide, wherein, for example, two staples are connected to a common residue. Peptide stitching is disclosed, for example, in WO2008 / 121767 and WO 2010 / 068684, which are incorporated herein by reference in their entirety. In some cases, as used herein, the staples can retain unsaturated bonds or can be reduced.

[0103] In some embodiments, the polypeptide can be stabilized by, for example, hydrocarbon stapling. In some cases, the stapled peptide comprises at least two (e.g., 2, 3, 4, 5, 6) amino acid substitutions, wherein the substituted amino acid is separated by two, three or six amino acids, and wherein the substituted amino acid acid is a non-natural amino acid with an olefin side chain. There are many known non-natural or unnatural amino acids, any of which can be included in the stapled peptide. Some examples of unnatural amino acids are 4-hydroxyproline, desmosine, γ-aminobutyric acid, β-cyanoalanine, norvaline, 4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine, 1-amino-cyclopropanecarboxylic acid, 1-amino-2-phenyl-cyclopropanecarboxylic acid, 1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid, 3-amino-cyclohexanecarboxylic acid, 4-piperidyl acetic acid, 4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminopimelic acid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, phenylalanine substituted at the ortho, meta, and para positions (e.g., with -C(=O)C 6 H 5 ;-CF3 ;-CN;-halogen;-NO 2 ; CH 3 substituted), disubstituted phenylalanine, substituted tyrosine (e.g., further substituted with -C=O)C 6 H 5 ;-CF 3 ;-CN;-halogen;-NO 2 ; CH 3 In addition, the amino acids may be derivatized to include amino acid residues that are hydroxylated, phosphorylated, sulfonated, acylated, or glycosylated.

[0104] Hydrocarbon stapled polypeptides contain one or more tethers (links) between two non-natural amino acids, which significantly enhance the alpha-helical secondary structure of the polypeptide. Typically, the tethers extend across the length of one or two helical turns (i.e., about 3.4 or about 7 amino acids). Therefore, amino acids located at i and i+3; i and i+4; or i and i+7 are ideal candidates for chemical modification and cross-linking. Thus, for example, in the case where a peptide has the sequence ... X1, X2, X3, X4, X5, X6, X7, X8, X9 ..., the cross-linking between X1 and X4, or between X1 and X5, or between X1 and X8 is a useful hydrocarbon stapled form of the peptide, as is the cross-linking between X2 and X5, between X2 and X6, or between X2 and X9, etc. It is also contemplated to use multiple cross-links (e.g., 2, 3, 4 or more). The use of multiple cross-links is very effective in stabilizing and optimizing peptides, especially as the length of the peptide increases. Therefore, the disclosure encompasses incorporating more than one crosslink in a polypeptide sequence to further stabilize the sequence or promote structural stability, proteolytic resistance, acid stability, thermal stability, cell permeability and / or enhanced biological activity of longer polypeptide extensions. Additional descriptions of preparing and using hydrocarbon stapled polypeptides can be found in, for example, U.S. Patent Publication Nos. 2012 / 0172285, 2010 / 0286057 and 2005 / 0250680, the entire contents of which are incorporated herein by reference in their entirety.

[0105] In certain embodiments, when the staples are at residues i and i+3, R-pentenylalanine and S-pentenylalanine; or R-pentenylalanine and S-pentenylalanine replace the amino acids at those positions. In certain embodiments, when the staples are at residues i and i+4, S-pentenylalanine replaces the amino acids at those positions. In certain embodiments, when the staples are at residues i and i+7, S-pentenylalanine and R-octenylalanine replace the amino acids at those positions. In some cases, when the peptide is stitched, the amino acids of the peptide to be involved in the "stitching" are replaced with dipentenylglycine, S-pentenylalanine and R-octenylalanine; or dipentenylglycine, S-octenylalanine and R-octenylalanine.

[0106] The staple or suture position can be changed by testing different staple positions during a staple walk.

[0107] Figure 26 (Top) Exemplary chemical structures of unnatural amino acids that can be used to generate various cross-linked compounds are shown. Figure 26 (Middle) Illustration of peptides with hydrocarbon cross-links between residues at positions i and i+3; i and i+4; or i and i+7. Figure 26 (Bottom) A staple walk along the peptide sequence is shown. Figure 27 Various peptide sequences with double and triple stapling strategies are shown, along with exemplary stapling walks. Figure 28 Exemplary stapling needle runs using branch suture portions of various lengths are shown.

[0108] In one aspect, the stabilized polypeptide has formula (I),

[0109]

[0110] in:

[0111] R 1 and R 2 Each is independently H or C 1 To C 10 Alkyl, alkenyl, alkynyl, aralkyl, cycloalkylalkyl, heteroaralkyl or heterocycloalkyl;

[0112] R 3 is alkyl, alkenyl, alkynyl; [R 4 —K—R 4 ] n ; Each of these substitutions has 0-6 R 5 ;

[0113] R 4 is alkyl, alkenyl or alkynyl;

[0114] R5 Is halogen, alkyl, OR 6 、N(R 6 ) 2 , SR 6 、SOR 6 、SO 2 R 6 , CO 2 R 6 , R 6 , fluorescent moieties or radioactive isotopes;

[0115] K is O, S, SO, SO 2 ,CO,CO 2 ,CONR 6 ,or

[0116]

[0117] R 6 is H, alkyl, or therapeutic agent;

[0118] n is an integer from 1 to 4;

[0119] x is an integer from 2 to 10;

[0120] Each y is independently an integer from 0 to 100;

[0121] z is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

[0122] And each Xaa is independently an amino acid.

[0123] The tether may include an alkyl, alkenyl, or alkynyl moiety (e.g., C 5 , C 8 or C 11 Alkyl, C 5 , C 8 or C 11 Alkenyl or C 5 , C 8 or C 11 The tethered amino acid may be α-disubstituted (e.g., C 1 -C 3 or methyl).

[0124] In some cases, x is 2, 3, or 6. In some cases, each y is independently an integer from 1 to 15 or from 3 to 15. In some cases, R 1 and R 2 Each is independently H or C 1 -C 6 In some cases, R 1 and R 2 Each is independent of C1 -C 3 In some cases, R 1 and R 2 At least one of them is a methyl group. For example, R 1 and R 2 In some cases, R 3 is an alkyl group (e.g., C 8 alkyl) and x is 3. In some cases, R 3 It is C 11 alkyl, and x is 6. In some cases, R 3 is an alkenyl group (e.g., C 8 alkenyl) and x is 3. In some cases, x is 6 and R 3 It is C 11 In some cases, R 3 is a straight chain alkyl, alkenyl or alkynyl group. 3 Yes—CH 2 —CH 2 —CH 2 —CH═CH—CH 2 —CH 2 —CH 2 —.

[0125] In another aspect, both α,α disubstituted stereocenters are in the R configuration or the S configuration (e.g., i,i+4 crosslinking), or one stereocenter is R and the other stereocenter is S (e.g., i,i+7 crosslinking). Thus, if Formula I is represented as:

[0126]

[0127] The C′ and C″ disubstituted stereocenters may both be in the R configuration, or they may both be in the S configuration, for example, when x is 3. When x is 6, the C′ disubstituted stereocenter is in the R configuration and the C″ disubstituted stereocenter is in the S configuration. 3 The double bond can be in the E or Z stereochemical configuration.

[0128] In some cases, R 3 Yes 4 —K—R 4 ] n ; and R 4 It is a straight chain alkyl, alkenyl or alkynyl group.

[0129] In some embodiments, the disclosure features internally cross-linked ("stapled" or "stitched") peptides in which the side chains of two amino acids separated by two, three, or six amino acids are replaced by internal staples; the side chains of three amino acids are replaced by internal stitching; the side chains of four amino acids are replaced by two internal staples, or the side chains of five amino acids are replaced by a combination of internal staples and internal stitching. The stapled / stitched peptides can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.

[0130] In some cases, the stabilizing peptide is a peptide of an intracellular protein. In some cases, the stabilizing peptide is a peptide of a disease-causing or disease-associated protein. In some cases, the stabilizing peptide is a peptide of a bacterial protein. In some cases, the stabilizing peptide is a peptide of a human protein. In some cases, the stabilizing peptide is a peptide of an oncogenic protein. Non-limiting examples of oncogenic proteins include BCL2, BCLX L , MCL-1, BFL-1, BCL-w, BCL-B, EZH2, HDM2 / HDMX, KRAS / NRAS / HRAS, MYC, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion protein, MLL fusion protein, receptor tyrosine kinases, HOX homologs, JUN, Cyclin D, Cyclin E, BRAF, CRAF, CDK4, CDK2, HPV-E6 / E7, Aurora kinase, MITF, Wnt1, PD-1, BCR and CCR5.

[0131] Non-limiting examples of stapled peptides are listed below:

[0132] QWAREIGAQLRX 1 BADX 2 LNAQYERR (SEQ ID NO: 1)-PUMA

[0133] FSSNRX 1 KILX 2 RTQILNQEWKQRRIQPV(SEQ ID NO:2)–EZH2

[0134] RRFFGIX 1 LTNX 2 LKTEEGN (SEQ ID NO: 3)-SOS

[0135] <h2 style=";text-align:left;direction:ltr">RKALETLRRVGDGVX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> RNHX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> TAF(SEQ ID NO:4)–MCL-1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0136] <h2 style=";text-align:left;direction:ltr"> LSQEQLEHRERSLX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> TLRX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> IQRBLF(SEQ ID NO:5)–BCL9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0137] <h2 style=";text-align:left;direction:ltr"> LTF8EYWAQ#XSAA(SEQ ID NO:6)–p53<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0138] <h2 style=";text-align:left;direction:ltr"> DIIRNIARHLAX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> VGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> BDRSI(SEQ ID NO:7)-BID<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0139] <h2 style=";text-align:left;direction:ltr"> IWIAQELRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> IGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> FNAYYARR(SEQ ID NO:8)-BIM<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0140] <h2 style=";text-align:left;direction:ltr"> NLWAAQRYGRELRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> BDDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> FVDSFKK(SEQ ID NO:9)-BAD S153D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0141] <h2 style=";text-align:left;direction:ltr"> NLWAAQRYGRELRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> BSDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> FVDSFKK(SEQ ID NO:10)–BAD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0142] <h2 style=";text-align:left;direction:ltr"> QLTAARLKX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> LGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> LHQRTBWR(SEQ ID NO:11)-HRK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0143] <h2 style=";text-align:left;direction:ltr"> AELEVESATQLRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> FGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> LNFRQKLL(SEQ ID NO:12)-NOXA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0144] <h2 style=";text-align:left;direction:ltr"> QWAREIGAQLRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> BADX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> LNAQYERR(SEQ ID NO:13)-PUMA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0145] <h2 style=";text-align:left;direction:ltr"> FSSNRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> KILX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> RTQILNQEWKQRRIQPV(SEQ ID NO:14)–EZH2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0146] <h2 style=";text-align:left;direction:ltr">RRFFGI X<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> LTNX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> LKTEEGN(SEQ ID NO:15)-SOS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0147] <h2 style=";text-align:left;direction:ltr"> RKALETLRRVGDGVX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> RNHX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> TAF(SEQ ID NO:16)–MCL-1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0148] <h2 style=";text-align:left;direction:ltr"> LSQEQLEHRERSLX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> TLRX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> IQRBLF(SEQ ID NO:17)–BCL9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0149] <h2 style=";text-align:left;direction:ltr"> LTF8EYWAQ#XSAA(SEQ ID NO:18)–p53<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0150] <h2 style=";text-align:left;direction:ltr"> DIIRNIARHLAX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> VGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> BDRSI(SEQ ID NO:19)-BID<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0151] <h2 style=";text-align:left;direction:ltr"> IWIAQELRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> IGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> FNAYYARR(SEQ ID NO:20)-BIM<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0152] <h2 style=";text-align:left;direction:ltr"> NLWAAQRYGRELRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> BDDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> FVDSFKK(SEQ ID NO:21)–BAD-S153D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0153] <h2 style=";text-align:left;direction:ltr"> NLWAAQRYGRELRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> BSDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> FVDSFKK(SEQ ID NO:22)-BAD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0154] <h2 style=";text-align:left;direction:ltr"> QLTAARLKX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> LGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> LHQRTBWR(SEQ ID NO:23)-HRK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0155] <h2 style=";text-align:left;direction:ltr"> AELEVESATQLRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> FGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> LNFRQKLL(SEQ ID NO:24)–NOXA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0156] <h2 style=";text-align:left;direction:ltr"> LTF8EYWAQLXSAA(SEQ ID NO:134)–p53(ATSP-7041#26L),<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0157] wherein 8 = R-octenylalanine; B = norleucine; # = cyclobutylalanine; X = S-pentenylalanine, and in some cases, X 1 and X 2 The same (e.g., S-pentenylalanine).

[0158] In certain embodiments, the stapled polypeptide comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs: 1 to 24 and 134. In certain embodiments, the disclosure features stabilizing peptides that differ from the above-disclosed peptides in that their staple / suture positions are different. In certain embodiments, the disclosure features stabilizing peptides that differ from the above-disclosed peptides in that, unlike the sequences disclosed above, they have 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, 7) amino acid substitutions on the non-interacting face of the α-helix of these peptides. In some cases, the substitutions are conservative. In other cases, the substitutions are non-conservative. In certain embodiments, the disclosure features stabilizing peptides that differ from the above-disclosed peptides in that, unlike the sequences disclosed above, they have 1 to 5 (e.g., 1, 2, 3, 4, 5) amino acid substitutions on the interacting face of the α-helix of these peptides. In some cases, the substitutions are conservative. Figure 29 Exemplary types of changes / modifications to stapled peptides are shown in .

[0159] In certain embodiments, the stapled peptide is not a Bcl-2 homology 3 (BH3) domain polypeptide (e.g., not the BH3 domain of MCL-1, not the MCL-1 stabilized alpha helix (SAHB) of the BCL2 domain, or not the MCL-1 SAHB D ).

[0160] In some embodiments, a stabilizing peptide (e.g., a stapled peptide) directly binds to and recruits a degradation agent protein, such as an ubiquitin E3 ligase MDM2. For example, the E3 ligase MDM2 can be strongly bound by a stapled p53 peptide known in the art, and the known stapled p53 peptides are incorporated herein by reference as a whole. In some cases, the peptide degradation determinant is a stable or stapled peptide that directly binds to and recruits a complex comprising a degradation agent protein, such as a complex between MDMX and an ubiquitin E3 ligase MDM2. In this example, the stapled p53 peptide can effectively bind to MDMX and recruit the MDMX / MDM2 complex, so that MDM2 can be recruited as a degradation agent protein.

[0161] In certain embodiments, the stabilizing peptide directly or indirectly binds to a degrader protein, such as an E3 ubiquitin ligase or a substrate adapter of an E3 ubiquitin ligase. In certain embodiments, the stabilizing peptide directly or indirectly binds to an E3 ubiquitin ligase. In some embodiments, the stabilizing peptide directly or indirectly binds to an E3 ligase (e.g., MDM2) or a protein complexed with an E3 ligase, such as MDMX bound to MDM2. In certain embodiments, the E3 ubiquitin ligase is a RING E3 ubiquitin ligase (e.g., Mdm2-MdmX, TRIM5α, c-CBL, cIAP, RNF4, BIRC7, IDOL, BRCA1-BARD1, RING1B-Bmi1, E4B, CHIP, Prp19). In certain embodiments, the E3 ubiquitin ligase is a HECT E3 ubiquitin ligase (e.g., Smurf1, Smurf2, Itch, E6AP). In certain embodiments, the E3 ubiquitin ligase is a RBR E3 ubiquitin ligase (e.g., Parkin, Parc, RNF144 (A / B), HOIP, HHARI). For example, non-limiting examples of E3 ubiquitin ligases are described in Morreale and Walden, Cell 165, 2016 DOI http: / dx.doi.org / 10.1016 / j.cell.2016.03.003.

[0162] Non-limiting examples of other stabilizing peptides that can be used in the chimeric fusion proteins described herein are found in U.S. Patents 9,834,581; 9,822,165; 9,695,224; 9,617,309; 9,579,395; 9,556,229; 9,556,227; 9,527,896; 9,522,947; 9,517,252; 9,505,816; 9,505,804; 9,505,801; 9,493,510; 9,464,125; 9,485,202; 9,458,189; 9,416,162; 9,408,885; 9,346,868; 9,296,805; 9,227,995; 9,175,047; 9, 175,045; 9,163,330; 9,096,684; 9,079,970; 8,957,026; 8,937,154; 8,933,109; 8,927,500; 8,889,632; 8,592,377; 8,586,707; 8,324,153; and U.S. Patent Application Publication No. 20170 20170240604; 20170212125; 20170165320; 20170066747; 20170015716; 20160376336; and 20160244494, the entire contents of which are incorporated herein by reference in their entireties (particularly the disclosure of stabilized (e.g., stapled or stitched) peptides).

[0163] Although hydrocarbon tethers are common, other tethers can also be used in stable peptides described herein. For example, the tether can include one or more of an ether, a thioether, an ester, an amine or an amide or a triazole moiety. In some cases, naturally occurring amino acid side chains can be incorporated into the tether. For example, the tether can be coupled to a functional group, such as a hydroxyl in serine, a thiol in cysteine, a primary amine in lysine, an acid in aspartic acid or glutamic acid, or an amide in asparagine or glutamine. Therefore, naturally occurring amino acids can be used to produce tethers, rather than using tethers made by coupling two non-natural amino acids. Single non-natural amino acids can also be used together with naturally occurring amino acids. Crosslinking containing triazoles (e.g., 1,4 triazoles or 1,5 triazoles) can be used (see, e.g., Kawamoto et al., 2012 Journal of Medicinal Chemistry 55: 1137; WO 2010 / 060112). Additionally, other methods for performing different types of stapling are well known in the art and may be employed (see, e.g., Lactam stapling: Shepherd et al., J. Am. Chem. Soc., 127:2974–2983 (2005); UV-cycloaddition stapling: Madden et al., Bioorg. Med. Chem. Lett., 21:1472–1475 (2011); Disulfide stapling: Jackson et al., Am. Chem. Soc., 113:9391–9392 (1991); Oxime stapling: Haney et al., Chem. Commun., 47:10915–10917 (2011); Thioether stapling: Brunel and Dawson, Chem. Commun., 552–2554 (2005); Photoswitchable stapling: JR Kumita et al., Proc. Natl. Acad. Sci. USA, 97: 3803–3808 (2000); Double-click stapling: Lau et al., Chem. Sci., 5: 1804–1809 (2014); Bis-lactam stapling: JCPhelan et al., J.Am.Chem.Soc., 119:455–460 (1997); and Bis-arylationstapling: AMSpokoyny et al., J.Am.Chem.Soc., 135:5946–5949 (2013)).

[0164] It is further contemplated that the length of the tether may vary. For example, in situations where it is desired to provide a relatively high degree of constraint to the secondary α-helical structure, a shorter tether may be used, whereas in certain situations where it is desired to provide less constraint to the secondary α-helical structure, a longer tether may be required.

[0165] Additionally, while tethers spanning from amino acids i to i+3, i to i+4, and i to i+7 are common to provide tethers that are primarily located on a single face of the alpha helix, tethers can be synthesized to span any number of amino acid combinations and can be used in combination to install multiple tethers.

[0166] In some cases, the hydrocarbon tethers described herein (i.e., crosslinks) can be further manipulated. In one case, the double bonds of the hydrocarbon alkenyl tethers (e.g., as synthesized using ruthenium-catalyzed ring-closing metathesis (RCM)) can be oxidized (e.g., by epoxidation, aminohydroxylation, or dihydroxylation) to provide one of the following compounds.

[0167]

[0168] One of the epoxy moiety or the free hydroxyl moiety can be further functionalized. For example, the epoxide can be treated with a nucleophilic reagent that provides additional functionality that can be used, for example, to connect a therapeutic agent. Alternatively, this derivatization can be achieved by synthetic manipulation of the amino or carboxyl terminus of the polypeptide or by an amino acid side chain. Other reagents can be connected to the functionalized tether, for example, reagents that promote the entry of the polypeptide into the cell.

[0169] In some cases, α-disubstituted amino acids are used in polypeptides to improve the stability of the α-helical secondary structure. However, instances are also envisioned where α-disubstituted amino acids are not required, and a single α-substituent is used (e.g., in a tethered amino acid).

[0170] The stapled polypeptide may include a drug, a toxin, a derivative of polyethylene glycol; a second polypeptide; a sugar, etc. Where a polymer or other agent is attached to the stapled polypeptide, a substantially homogeneous composition is desirable.

[0171] The addition of polyethylene glycol (PEG) molecules can improve the pharmacokinetic and pharmacodynamic properties of peptides. For example, PEGylation can reduce renal clearance and produce more stable plasma concentrations. PEG is a water-soluble polymer that can be expressed as a peptide linked to the following formula:

[0172] XO--(CH 2 CH 2 O) n --CH 2 CH 2 —Y, wherein n is 2 to 10,000, and X is H or a terminal modification, such as C1-4 alkyl; Y is an amide, carbamate or urea bond to an amine group of a polypeptide (including but not limited to the epsilon amine of lysine or the N-terminus). Y may also be a maleimide bond to a thiol group (including but not limited to the thiol group of cysteine). Other methods of directly or indirectly attaching PEG to a polypeptide are known to those of ordinary skill in the art. PEG may be linear or branched. Various forms of PEG, including various functionalized derivatives, are commercially available.

[0173] PEG with degradable bonds in the backbone can be used. For example, PEG with easily hydrolyzed ester bonds can be prepared. Conjugates with degradable PEG bonds are described in WO 99 / 34833; WO 99 / 14259 and US6,348,558.

[0174] In certain embodiments, a macromolecular polymer (e.g., PEG) is connected to an agent described herein via an intermediate linker. In certain embodiments, the linker is composed of 1 to 20 amino acids connected by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids. As is well known to those skilled in the art, some of these amino acids may be glycosylated. In other embodiments, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker is composed of most amino acids without steric hindrance, such as glycine and alanine. Non-peptide linkers are also possible. For example, an alkyl linker, such as -NH(CH 2 ) n C(O)-, wherein n=2-20. These alkyl linkers may be further substituted with any non-sterically hindering groups, such as lower alkyl (e.g., C 1 -C 6 ), lower acyl, halogen (e.g. Cl, Br), CN, NH 2 , phenyl, etc. US Pat. No. 5,446,090 describes a bifunctional PEG linker and its use in forming a conjugate having a peptide at each end of the PEG linker.

[0175] In some embodiments, the stabilized peptides can also be modified, for example, to further promote cellular uptake or increase in vivo stability. For example, acylation or pegylation of the peptidomimetic macrocycles can facilitate cellular uptake, increase bioavailability, increase blood circulation, alter pharmacokinetics, reduce immunogenicity and / or reduce the required frequency of administration.

[0176] In some embodiments, the stapled peptides disclosed herein have an enhanced ability to penetrate cell membranes (eg, relative to non-stapled peptides).

[0177] Methods for synthesizing stable peptides described herein are known in the art. However, the following exemplary methods can be used. It should be understood that various steps can be performed in an alternative order or sequence to obtain the desired compound. Synthetic chemical transformations and protecting group methods (protection and deprotection) that can be used to synthesize compounds described herein are known in the art, and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. Wuts, Protective Groups in Organic Synthesis, 3d. Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent versions thereof.

[0178] Stable peptides can be prepared by chemical synthesis methods well known to those of ordinary skill. See, for example, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, WH Freeman & Co., New York, NY, 1992, p. 77. Thus, peptides can be synthesized using the automated Merrifield technique of solid phase synthesis, wherein α-NH 2 Side-chain protected amino acids are used by t-Boc or Fmoc chemical protection, for example on an Applied Biosystems model 430A or 431 peptide synthesizer.

[0179] One way to prepare the peptides described herein is to use solid phase peptide synthesis (SPPS). The C-terminal amino acid is connected to a cross-linked polystyrene resin via a linker molecule via an acid-labile bond. The resin is insoluble in the solvent used for the synthesis, so it is relatively simple and fast to wash away excess reagents and by-products. The N-terminus is protected by an Fmoc group, which is stable in acid but can be removed by alkali. Any side chain functional groups are protected by groups that are stable to alkali and unstable to acid.

[0180] By connecting a single synthetic peptide using natural chemistry, longer peptides can be made. Alternatively, longer synthetic peptides can be synthesized by well-known recombinant DNA technology. Such technology is provided in the well-known standard manual with detailed procedures. In order to construct the gene encoding the peptide of the present invention, the amino acid sequence is reverse translated to obtain the nucleic acid sequence encoding the amino acid sequence, preferably for the biological best codon to express the gene therein. Next, synthetic genes are usually prepared by synthesizing oligonucleotides of the coded peptide and any regulatory element (if necessary). The synthetic gene is inserted into a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions suitable for the selected expression system and host. Purify and characterize the peptide by standard methods.

[0181] The peptides can be prepared in a high-throughput combinatorial manner, for example, using a high-throughput multichannel combinatorial synthesizer available from Advanced Chemtech. The peptide bonds can be replaced, for example, with the following bonds to increase the physiological stability of the peptide: retro-inverse bonds (C(O)-NH); reduced amide bonds (NH-CH 2 ); thiomethylene bond (S-CH 2 or CH 2 -S); oxymethylene bond (O-CH 2 or CH 2 -O); ethylidene bond (CH 2 -CH 2 ); sulfamide bond (C(S)-NH); trans olefin bond (CH=CH); fluorinated trans olefin bond (CF=CH); ketomethylene bond (C(O)-CHR) or CHR-C(O), where R is H or CH 3 ; Fluoro-ketomethylene bond (C(O)-CFR or CFR-C(O), where R is H or F or CH 3 .

[0182] The polypeptide can be further modified by acetylation, amidation, biotinylation, cinnamylation, farnesylation, fluorescing, formylation, myristylation, palmitoylation, phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation and sulfonation. As described above, the peptide can be conjugated to, for example, polyethylene glycol (PEG); alkyl (e.g., C1-C20 straight or branched alkyl); fatty acid group; and combinations thereof. α,α-disubstituted non-natural amino acids containing olefin side chains of variable length can be synthesized by known methods (Williams et al. J. Am. Chem. Soc., 113: 9276, 1991; Schafmeister et al., J. Am. Chem Soc., 122: 5891, 2000; and Bird et al., Methods Enzymol., 446: 369, 2008; Bird et al., Current Protocols in Chemical Biology, 2011). For peptides stapled with i and i+7 (stable two-turn helix): a) one S5 amino acid and one R8 were used; or b) one S8 amino acid and one R5 amino acid were used. R8 was synthesized using the same approach, except that the starting chiral auxiliary conferred the R-alkyl-stereoisomer. In addition, 8-iodooctene was used instead of 5-iodopentene. The inhibitors were synthesized on a solid support using solid phase peptide synthesis (SPPS) on MBHA resin (see, e.g., WO 2010 / 148335).

[0183] Fmoc-protected α-amino acids (olefinic amino acids Fmoc-S 5 -OH、Fmoc-R 8 -OH、Fmoc-R 8 -OH、Fmoc-S 8 -OH and Fmoc-R 5 -OH), 2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium hexafluorophosphate (HCTU) and Rink Amide MBHA are commercially available, for example, from Novabiochem (San Diego, CA). Dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), 1,2-dichloroethane (DCE), fluorescein isothiocyanate (FITC) and piperidine are commercially available, for example, from Sigma-Aldrich. The synthesis of olefinic amino acids has been reported in the art (Williams et al., Org. Synth., 80:31, 2003).

[0184] Again, methods suitable for obtaining (e.g., synthesizing), stapling, and purifying the peptides disclosed herein 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, the entire contents of which are incorporated herein by reference).

[0185] In some embodiments, the peptide is substantially free of non-stapled peptide contaminants or is isolated. Methods for purifying peptides include, for example, synthesizing the peptide on a solid support. After cyclization, the solid support can be isolated and suspended in a solution of a solvent, such as DMSO, a DMSO / dichloromethane mixture, or a DMSO / NMP mixture. The DMSO / dichloromethane or DMSO / NMP mixture may contain about 30%, 40%, 50%, or 60% DMSO. In a specific embodiment, a 50% / 50% DMSO / NMP solution is used. The solution can be incubated for 1, 6, 12, or 24 hours, and then the resin can be washed with, for example, dichloromethane or NMP. In one embodiment, the resin is washed with NMP. Inert gas can be shaken and bubbled into the solution.

[0186] The properties of the stabilized (eg, stapled) polypeptides of the invention can be determined, for example, using the methods described below.

[0187] Determination of α-helicity: Compounds were dissolved in aqueous solution (e.g., 5 mM potassium phosphate solution at pH 7 or distilled H 2 0, concentration 25-50 μM). Circular dichroism (CD) spectra were obtained on a spectropolarimeter (e.g., Jasco J-710, Aviv) using standard measurement parameters (e.g., temperature 20°C; wavelength 190-260 nm; step resolution 0.5 nm; speed 20 nm / sec; accumulation: 10; response: 1 sec; bandwidth: 1 nm; path length, 0.1 cm). The α-helical content of each peptide was calculated by dividing the average residue ellipticity by the reported value for a model helical decapeptide (Yang et al., Methods Enzymol. 130: 208 (1986)).

[0188] Determination of melting temperature (Tm): Dissolve the cross-linked or unmodified template peptide in distilled H 2 O or other buffer or solvent (e.g., final concentration of 50 μM) and determine Tm by measuring the change in ellipticity over a temperature range (e.g., 4 to 95°C) on a spectropolarimeter (e.g., Jasco J-710, Aviv) using standard parameters (e.g., wavelength 222 nm; step resolution: 0.5 nm; speed: 20 nm / sec; accumulation: 10; response; 1 sec; bandwidth 1 nm; heating rate: 1°C / min; path length 0.1 cm).

[0189] In vitro protease resistance assay: The amide bonds of the peptide backbone are susceptible to protease hydrolysis, making the peptide compounds susceptible to rapid degradation in vivo. However, the formation of peptide helices usually buries and / or distorts and / or shields the amide backbone, thus preventing or substantially preventing proteolytic cleavage. The peptidomimetic macrocycles of the present invention can be subjected to in vitro enzymatic proteolysis (e.g., trypsin, chymotrypsin, pepsin) to assess any changes in degradation rate compared to the corresponding uncrosslinked or stapled polypeptides. For example, the peptidomimetic macrocycles and the corresponding uncrosslinked polypeptides are incubated with trypsin agarose, and the reaction is quenched at various time points by centrifugation, and the residual substrate is quantified by subsequent HPLC injection with UV absorption at 280 nm. Briefly, the peptidomimetic macrocycles and peptidomimetic precursors (5mcg) are incubated with trypsin agarose (Pierce) (S / E~125) for 0, 10, 20, 90 and 180 minutes. The reaction was quenched by high speed benchtop centrifugation; the remaining substrate in the separated supernatant was quantified by HPLC-based peak detection at 280 nm. The proteolytic reaction exhibited first order kinetics, with the rate constant k determined from a plot of ln[S] versus time.

[0190] Peptidomimetic macrocycles and / or corresponding uncrosslinked polypeptides can be incubated with fresh mouse, rat and / or human serum (e.g., 1-2 mL) at 37°C for 0, 1, 2, 4, 8 and 24 hours, respectively. Samples with different macrocycle concentrations can be prepared by serial dilution with serum. To determine the level of intact compound, the following steps can be used: extract the sample, for example, by transferring 100 μL of serum to a 2 ml centrifuge tube, then adding 10 μL of 50% formic acid and 500 μL of acetonitrile, and centrifuging at 14,000 RPM for 10 minutes at 4+ / -2°C. The supernatant is then transferred to a fresh 2 ml tube and centrifuged on a Turbovap at N 2 <10 psi, evaporate at 37°C. Samples were reconstituted in 100 μL of 50:50 acetonitrile:water and subjected to LC-MS / MS analysis. Equivalent or similar procedures for testing ex vivo stability are known and can be used to determine the stability of macrocycles in serum.

[0191] In vivo protease resistance assay: A key benefit of peptide stapling is the translation of in vitro protease resistance into significantly improved in vivo pharmacokinetics.

[0192] In vitro binding assays: To assess the binding and affinity of peptidomimetic macrocycles and peptidomimetic precursors to receptor proteins, fluorescence polarization assays (FPA) can be used, for example. The FPA technique uses polarized light and fluorescent tracers to measure the orientation and mobility of molecules. When excited with polarized light, fluorescent tracers (such as FITC) attached to molecules with high apparent molecular weights (such as FITC-labeled peptides bound to large proteins) will emit higher levels of polarized fluorescence because their rotation rates are slower than tracers attached to smaller molecules (such as FITC-labeled peptides free in solution).

[0193] Cellular analysis: Cultured cells (e.g., cancer cells) are treated with stapled peptide-degrader chimeras and the target protein levels are monitored over time by protein analysis. Negative control proteins that are not targeted by the chimeric peptides are also monitored to demonstrate targeted degradation specificity. Depending on the specific target, phenotypic outcomes, such as apoptosis induction, are assessed by a combination of viability, annexin V binding, caspase 3 / 7 activation, and mitochondrial cytochrome c release assays.

[0194] Peptide degrader

[0195] The disclosure features peptide degraders that bind to a peptide of a protein that is a substrate adaptor for a ubiquitin E3 ligase. In some cases, the degrader binds to a WD-40 protein, which is a substrate adaptor for a ubiquitin E3 ligase. The degrader binds to a substrate recognition domain of a ubiquitin E3 ligase in a shallow groove and allows for fine processing (i.e., conjugation of a stapled peptide sequence) at the N- or C-terminus. Exemplary substrate adaptors for ubiquitin E3 ligases include MDM2, SKP2-CKS1, FBXW1, FBXW2, FBXW4, FBXW5, FBXW7, FBXW8, FBXW9, FBXW10, FBXW11, FBXW12, SPOP, VHL, ITCH, KEAP1, KLHL2, KLHL3, KLHL7, KLHL12, KLHL13, KLHL15, KLHL20, KLHL21, KLHL24, KLHL40, KLHL42 , COP1, TRAF7, RFWD3, DCAF1, DCAF2, DCAF3, DCAF4, DCAF5, DCAF6, DCAF7, DCAF8, DCAF9, DCAF10, DCAF11, DCAF12, DCAF13, DCAF14, DCAF15, DCAF16, DCAF17, DCAF19, SIAH1, TRPC4AC, DET1, WSB1, WSB2, HERC1, DDB2, CSA, CBL, and FZR1. Although the E3 ligase containing WD40 comprises many E3 ligases, there are other types of E3 ligases and degrons that bind to proteins that are substrate adaptors of the E3 ligases are also encompassed by the present disclosure.

[0196] In some cases, the peptide degron is based on the Trib1 protein sequence: DQIVPEY (SEQ ID NO: 25) or a variant thereof.

[0197] Position 1 2 3 4 5 6 7 Amino acid D Q I V P E Y

[0198] Degraders disclosed herein include variants of SEQ ID NO: 25, wherein the variants include one or more (e.g., 1, 2, 3, 4, 5) amino acid substitutions; one or more (e.g., 1, 2, 3) deletions; one or more (e.g., 1, 2, 3) insertions; or any combination of two or more thereof. In some cases, variants of SEQ ID NO: 25 are based on one or more (e.g., 1, 2, 3, 4, 5) substitutions. In some cases, one or more (e.g., 1, 2, 3, 4, 5, 6, 7) of these substitutions are not substitutions of A, R at any one of positions 1 to 6 of SEQ ID NO: 25. In some cases, these substitutions do not include substitution of position 4 V to I in SEQ ID NO: 25. In some cases, variants of SEQ ID NO: 25 have one or more (e.g., 1, 2, 3) deletions. In some cases, variants of SEQ ID NO: 25 have one or more (e.g., 1, 2, 3) insertions. In some cases, SEQ ID NO:25 variants have one or more (e.g., 1, 2, 3, 4, 5) substitutions and one or more (e.g., 1, 2, 3) deletions. In some cases, SEQ ID NO:25 variants have one or more (e.g., 1, 2, 3, 4, 5) substitutions and one or more insertions (e.g., 1, 2, 3). In some cases, SEQ ID NO:25 variants have one or more (e.g., 1, 2, 3) deletions and one or more (e.g., 1, 2, 3) insertions. In some cases, SEQ ID NO:25 variants have 1-6, 1-5, 1-4, 1-3, 2 or 1 amino acid substitutions in SEQ ID NO:25. In some cases, SEQ ID NO:25 4 (V) and / or 5 (P) are not substituted. In some cases, SEQ ID NO:25 1 (D), 2 (Q), 3 (I) and 6 (E) one or more are substituted. In some embodiments, the peptide degron comprises SEQ ID NO:25, except that any one of positions 1 to 7 is not substituted with alanine. In some embodiments, the peptide degron comprises an amino acid sequence comprising SEQ ID NO:25, except that any one of positions 1 to 7 is not substituted with arginine. In some embodiments, the peptide degron comprises SEQ ID NO:25, except that position 4 is not substituted with isoleucine. In some cases, variants of SEQ ID NO:25 have a deletion. The deletion can be at the C-terminus or N-terminus of SEQ ID NO:25.

[0199] In some cases, the peptide degron is a peptide that binds to the F-box / WD repeat-containing protein 7 (FBXW7) protein. In one embodiment, the peptide degron comprises the amino acid sequence phospho-Ser / phospho-ThrPXXE / phospho-Ser / phospho-Thr (pS / pT-PX a -X b -E / pS / pT) (SEQ ID NO: 46), wherein X a and X b is independently any amino acid. In certain cases, X a =P. In some cases, X b = V, L or Q. In other cases, X a =P and X b =V, L or Q. In certain embodiments, the peptide degron is a variant of SEQ ID NO: 46. Such variants include peptides that are different from SEQ ID NO: 46 in that they have one or more (e.g., 1, 2, 3, 4) amino acid substitutions; one or more deletions (e.g., 1, 2, 3); one or more insertions (e.g., 1, 2, 3); or any combination of two or more thereof. In some cases, the variant of SEQ ID NO: 46 has one or more (e.g., 1, 2, 3, 4) substitutions. In some cases, the variant of SEQ ID NO: 46 has one or more (e.g., 1, 2, 3) deletions. In some cases, the variant of SEQ ID NO: 46 has one or more (e.g., 1, 2, 3) insertions. In some cases, the variant of SEQ ID NO: 46 has one or more (e.g., 1, 2, 3, 4) substitutions and one or more (e.g., 1, 2, 3) deletions. In some cases, the variant of SEQ ID NO: 46 has one or more (e.g., 1, 2, 3, 4) substitutions and one or more (e.g., 1, 2, 3) deletions. In some cases, the variant of SEQ ID NO: 46 has one or more (e.g., 1, 2, 3, 4) substitutions and one or more insertions (e.g., 1, 2, 3). In some cases, the variant of SEQ ID NO:46 has one or more (e.g., 1, 2, 3) deletions and one or more (e.g., 1, 2, 3) insertions. In some cases, the variant of SEQ ID NO:46 has 1-5, 1-4, 1-3, 2 or 1 amino acid substitutions in SEQ ID NO:46. In some cases, position 2 (P) is not substituted. In some cases, position 1 is pS and position 2 is P. In some cases, position 1 is pS, position 2 is P, and position 5 is E. In some cases, position 1 is pS, position 2 is P, and position 5 is pS. In some cases, position 1 is pS, position 2 is P, and position 5 is pT. In some cases, position 1 is pT, position 2 is P, and position 5 is E. In some cases, position 1 is pT, position 2 is P, and position 5 is pS. In some cases, position 1 is pT, position 2 is P, and position 5 is pT. In some cases, position 1 is pT, position 2 is P, and position 5 is E. In some cases, position 1 is pT, position 2 is P, and position 5 is pS. In some cases, position 1 is pT, position 2 is P, and position 5 is pT.

[0200] In some cases, the peptide degron is based on a natural binding consensus sequence of a peptide that binds to a WD40 repeat protein that is a substrate adaptor for an E3 ubiquitin ligase. In some cases, the peptide degron is a variant (e.g., a substitution, deletion, or insertion variant) of a natural binding consensus sequence of a peptide that binds to a WD40 repeat protein that is a substrate adaptor for an E3 ubiquitin ligase. Non-limiting examples of natural binding consensus sequences of peptides that bind to a WD40 repeat protein that is a substrate adaptor for an E3 ubiquitin ligase are provided below (sequences are designated from top to bottom as SEQ ID NOs.: 65 to 92):

[0201]

[0202]

[0203] The motif pattern uses the following terms: '.' specifies any amino acid type, '[X]' specifies the amino acid type allowed at that position, '^X' at the beginning of the pattern specifies that the sequence starts with an amino acid of type X, '[^X]' indicates that the position can have any amino acid other than type X, and numbers are specified as follows 'X{x,y}', where x and y specify the 'X' required at that position

[0204] Minimum and maximum number of amino acid types. The '$' symbol indicates the C-terminus of the protein chain. Conserved residue positions in primary degrons that are known to be post-translationally modified (e.g., phosphorylation and proline hydroxylation) are shown in bold.

[0205] Any other peptide degron known in the art may also be used in the present invention. See, for example, Mészáros et al., Sci. Signal., 10(470):eaak9982 (2017); Guharoy et al., Nature Communications, 7:10239, doi:10.1038 / ncomms10239 (2016); U.S. Patents 9,783,575; 9,297,017; and 9,115,184, all of which are incorporated herein by reference in their entirety.

[0206] In some cases, the peptide degron has the amino acid sequence of the peptide listed below, or a variant thereof:

[0207] FSDLWKLL (SEQ ID NO: 31) - E3 ligase: MDM2;

[0208] SVEQTPKK (SEQ ID NO: 32) - E3 ligase: SKP2-CKS1;

[0209] DSGIHS (SEQ ID NO: 32) - E3 ligase: β-TrCP1;

[0210] LLPTPPLS (SEQ ID NO: 33) - E3 ligase: FBXW7;

[0211] ASSSS (SEQ ID NO: 34) - E3 ligase: SPOP;

[0212] LAPAAGDTIISLDF (SEQ ID NO: 35) - E3 ligase: VHL;

[0213] PFLTPSPE (SEQ ID NO: 36) - E3 ligase: FBXW7;

[0214] PPPY (SEQ ID NO: 37) - E3 ligase: ITCH;

[0215] DEETGE (SEQ ID NO: 38) - E3 ligase: KEAP1;

[0216] QDIDLGV (SEQ ID NO: 39) - E3 ligase: KEAP1;

[0217] LLQPNNYQFC (SEQ ID NO: 40) - E3 ligase: CBL;

[0218] DYR-E3 ligase: CBL;

[0219] RAVENQYSFY (SEQ ID NO: 41) - E3 ligase: CBL;

[0220] QKENS (SEQ ID NO: 42) - E3 ligase: CDH1;

[0221] FDIYMD (SEQ ID NO: 43) - E3 ligase: CDC20 / CDH1;

[0222] PRTALGDIG (SEQ ID NO: 44) - E3 ligase: CDC20 / CDH1;

[0223] DKENG (SEQ ID NO: 45)—E3 ligase: PTTG1;

[0224] HRKHLQEIP (SEQ ID NO:93) - E3 ligase: APC / C;

[0225] SKENV (SEQ ID NO: 94) - E3 ligase: APC / C;

[0226] TRIR (SEQ ID NO: 95) - E3 ligase: APC / C;

[0227] DQIVPEY (SEQ ID NO:96) - E3 ligase: COP1;

[0228] TSMTDFYHSKRRL (SEQ ID NO:97)—E3 ligase: DCAF2;

[0229] SPETGE (SEQ ID NO:98) - E3 ligase: KEAP1;

[0230] EPEEPEADQH (SEQ ID NO:99) - E3 ligase: KLHL3;

[0231] LAPYIPMDDDFQL (SEQ ID NO: 100)—E3 ligase: VHL;

[0232] LTPPQS (SEQ ID NO: 101)—E3 ligase: FBXW7;

[0233] SVEQTPRK (SEQ ID NO: 102) - E3 ligase: SKP2 / CKS1;

[0234] DSGNYS (SEQ ID NO: 103) - E3 ligase: β-TrCP1;

[0235] KPAAVVAPI (SEQ ID NO: 104)—E3 ligase: Siah; or

[0236] ADSST (SEQ ID NO: 105)—E3 ligase: SPOP

[0237] Variants of the above peptides (i.e., SEQ ID NOs.: 31-45 and 93-105) include peptides having one or more (e.g., 1, 2, 3, 4, 5) amino acid substitutions; one or more deletions (e.g., 1, 2, 3); one or more insertions (e.g., 1, 2, 3); or any combination of two or more thereof. Variants that interact with the relevant E3 ligase are selected. In some cases, the selected peptide degradation determinant binds to its relevant E3 ligase with a binding affinity of 1nM to 300nM. In some cases, the selected peptide degradation determinant binds to the relevant E3 ligase with a binding affinity of 10nM to 300nM. In some cases, the selected peptide degradation determinant binds to the relevant E3 ligase with a binding affinity of 50nM to 300nM. In some cases, the selected peptide degradation determinant binds to the relevant E3 ligase with a binding affinity of 100nM to 300nM. In some cases, the selected peptide degron binds to the relevant E3 ligase with an affinity of 200nM to 300nM. In some cases, the selected peptide degron binds to the relevant E3 ligase with an affinity of 200nM to 250nM. In some cases, the selected peptide degron binds to the relevant E3 ligase with an affinity of 1nM to 1000nM. In some cases, the selected peptide degron binds to the relevant E3 ligase with an affinity of 200nM to 1000nM.

[0238] Non-limiting exemplary variations are provided below:

[0239] For FBXW7 E3 ligase: LTPPAS (SEQ ID NO: 106), LTPPSS (SEQ ID NO: 107), LSPPPS (SEQ ID NO: 108), LSPPAS (SEQ ID NO: 109), LSPPLS (SEQ ID NO: 110);

[0240] For β-TrCP1 E3 ligase: DSGIIS (SEQ ID NO: 111), DSGNYT (SEQ ID NO: 112), DSGIDT (SEQ ID NO: 113), DSGIET (SEQ ID NO: 114), DSGVDTS (SEQ ID NO: 115); and

[0241] For DCAF2 E3 ligase: TSMTDFYHSKRRI (SEQ ID NO: 116), TSMTDFYHSKRKL (SEQ ID NO: 117), TSMTDFYHSKRRS (SEQ ID NO: 118).

[0242] In some cases, a peptide degron is 4 to 20 amino acids in length (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0243] In some cases, the peptide degron has an amino acid sequence of a peptide as set forth in any one of SEQ ID NOs.: 26 to 30. In some cases, the peptide degron has an amino acid sequence of a variant of a peptide as set forth in any one of SEQ ID NOs.: 26 to 30. In some cases, the peptide degron has an amino acid sequence of a peptide as set forth in any one of SEQ ID NOs.: 31 to 45. In some cases, the peptide degron has an amino acid sequence of a variant of a peptide as set forth in any one of SEQ ID NOs.: 31 to 45. In some cases, the peptide degron has an amino acid sequence of a peptide as set forth in any one of SEQ ID NOs.: 65 to 118. In some cases, the peptide degron has an amino acid sequence of a variant of a peptide as set forth in any one of SEQ ID NOs.: 65 to 118. Variants include peptide degron having one or more (e.g., 1, 2, 3, 4, 5) amino acid substitutions; one or more deletions (e.g., 1, 2, 3); one or more insertions (e.g., 1, 2, 3); or a combination of any two or more thereof.

[0244] The above-mentioned peptide degraders bind to the substrate adapters of their associated ubiquitin E3 ligases (e.g., Cop1, FBXW7, FBXW8). In some cases, the peptide degraders bind to the substrate adapters of ubiquitin E3 ligases (e.g., Cop1, FBXW7, FBXW8) with a binding affinity of 1nM to 300nM. In some cases, the peptide degraders bind to the substrate adapters of ubiquitin E3 ligases (e.g., Cop1, FBXW7, FBXW8) with a binding affinity of 10nM to 300nM. In some cases, the peptide degraders bind to the substrate adapters of ubiquitin E3 ligases (e.g., Cop1, FBXW7, FBXW8) with a binding affinity of 50nM to 300nM. In some cases, the peptide degron binds to a substrate adaptor of a ubiquitin E3 ligase (e.g., Cop1, FBXW7, FBXW8) with an affinity of 100 nM to 300 nM. In some cases, the peptide degron binds to a substrate adaptor of a ubiquitin E3 ligase (e.g., Cop1, FBXW7, FBXW8) with an affinity of 200 nM to 300 nM. In some cases, the peptide degron binds to a substrate adaptor of a ubiquitin E3 ligase (e.g., Cop1, FBXW7, FBXW8) with an affinity of 200 nM to 250 nM. In some cases, the peptide degron binds to a substrate adaptor of a ubiquitin E3 ligase (e.g., Cop1, FBXW7, FBXW8) with an affinity of 1 nM to 1000 nM. In some cases, the peptide degron binds to a substrate adaptor of a ubiquitin E3 ligase (eg, Cop1, FBXW7, FBXW8) with an affinity of 200 nM to 1000 nM.

[0245] The present disclosure also features a method for selecting a protein degradation determinant. The protein degradation determinant so selected can be used in the chimeric construct of the present disclosure. The method includes contacting a substrate adapter of an ubiquitin E3 ligase with a variant of a naturally occurring peptide degradation determinant, and selecting a degradation determinant that binds to the substrate adapter with a desired affinity. For example, the method includes contacting a WD40 repeat protein as a substrate adapter of an E3 ubiquitin ligase with a variant of an amino acid sequence that binds to a natural binding consensus sequence of a WD40 repeat protein (e.g., Cop1, FBXW7, FBXW8), and then selecting a peptide that binds to the WD40 repeat protein. In some cases, the selected peptide degradation determinant binds to the WD40 repeat protein with an affinity of 1 nM to 1000 nM. In some cases, the selected peptide degradation determinant binds to the WD40 repeat protein with an affinity of 10 nM to 300 nM. In some cases, the selected peptide degradation determinant binds to the WD40 repeat protein with an affinity of 50 nM to 300 nM. In some cases, the selected peptide degron binds to WD40 repeat proteins with a binding affinity of 100nM to 300nM. In some cases, the selected peptide degron binds to WD40 repeat proteins with a binding affinity of 200nM to 300nM. In some cases, the selected peptide degron binds to WD40 repeat proteins with a binding affinity of 200nM to 250nM. In some cases, the selected peptide degron binds to WD40 repeat proteins with a binding affinity of 1nM to 1000nM. In some cases, the selected peptide degron binds to WD40 repeat proteins with a binding affinity of 200nM to 1000nM. In some cases, the selected peptide degron binds to WD40 repeat proteins with a binding affinity of less than 1nM (e.g., about 0.01nM, about 0.05nM, about 0.1nM, about 0.5nM). In some cases, the selected peptide degron binds to a WD40 repeat protein with an affinity greater than 300 nM (eg, about 350 nM, about 400 nM, about 500 nM, about 1000 nM).

[0246] In some cases, the protein that needs targeted degradation is directly modified. The region including the structural disorder region of the protein is checked. The region with a disordered structure is a region where the disorder score calculated by IUPred:iupred.enzim.hu is less than or equal to 0.4. The protein is modified to include the above-mentioned peptide degradation determinant sequence or its variant in the structural disorder region. In some cases, the structural disorder region is at the N- or C- terminal of the protein. In some cases, the structural disorder region is between the N- and C- terminal of the protein. The peptide degradation determinant sequence can be inserted into the structural disorder region. In other cases, the peptide degradation determinant sequence is replaced to replace the amino acid sequence in the structural disorder region of the protein. Such replacement can be performed, for example, by CRISPR / Cas9 modification.

[0247] Small molecule degrader

[0248] The present disclosure features small molecule degrons that can be used in the chimeras described herein. In one embodiment, the small molecule degrons are based on thalidomide having the following structure:

[0249]

[0250] In some cases, when a small molecule degron is conjugated at the N-terminus of a stabilizing peptide, its structure is as follows:

[0251]

[0252] In some cases, when a small molecule degron is conjugated at the C-terminus of a stabilizing peptide, its structure is as follows:

[0253]

[0254] In some cases, the small molecule degrons employed herein bind to the Von Hippel-Lindau ("VHL") protein and have a ligand having the structure provided below (compatible with coupling to acid residues):

[0255]

[0256] In some cases, when a small molecule VHL degron is conjugated to an amine, a carboxylate analogue is used as shown below:

[0257]

[0258] Any small molecule degron known in the art can be used in the chimeras described herein. In some cases, the small molecule degron used herein is any of the degrons described in U.S. Patents 9,694,084; 9,750,816; 9,770,512; 9,821,068; 9,783,575; 9,765,019; 9,632,089 and 9,500,653, the entire contents of which are incorporated herein by reference in their entirety.

[0259] Chimera of stable peptide and degrader

[0260] The present disclosure provides chimeras of stabilized peptides (e.g., stapled, stitched) and degrons (e.g., small molecule degrons, primary sequence degrons, and stable (e.g., stapled) peptide degrons). Such chimeras can effectively target a wide range of proteins that were previously inaccessible to small molecules with small molecules (e.g., cereblon-binding molecules) or other small molecules or peptide degrons that can target the degradation of bound proteins. Similarly, stable peptide degrons that can bind and recruit degradable proteins can be combined with small molecules that bind to multiple proteins to degrade disease-associated proteins. These novel stapled peptide degron chimeras expand the effectiveness and breadth of the biological activity of the stapled peptides. Chimeras comprising more than one (e.g., 2, 3, 4 or more) stabilizing peptides and one degron (e.g., one small molecule degron, one primary sequence degron, or one stable (e.g., stapled) peptide degron) are also contemplated herein. Also contemplated herein are chimeras comprising more than one (e.g., 2, 3, 4, or more) degrons (e.g., more than one small molecule degrons, more than one primary sequence degrons, or more than one stabilizing (e.g., stapled) peptide degrons) and one stabilizing peptide. Also contemplated herein are chimeras comprising more than one (e.g., 2, 3, 4, or more) stabilizing peptides and more than one (e.g., 2, 3, 4, or more) degrons (e.g., more than one small molecule degrons, more than one primary sequence degrons, or more than one stabilizing (e.g., stapled) peptide degrons).

[0261] In certain embodiments, the stapled peptide of the chimera is not a Bcl-2 homology 3 (BH3) domain polypeptide (e.g., not the BH3 domain of MCL-1, not the MCL-1 stabilized alpha helix of the BCL2 domain (SAHB), or not the MCL-1 SAHB D ).

[0262] Stable peptide-peptide degron chimeras

[0263] Provided herein are stable peptide-peptide degron chimeras. These chimeras consist of a stable peptide and a peptide degron, wherein the stable peptide binds to a first protein, the first protein being a protein targeted for degradation, and the peptide degron binds directly or indirectly to a second protein, the second protein being a substrate adaptor for a ubiquitin E3 ligase. Thus, in certain embodiments, the above-mentioned stable peptide is (e.g., stapled, stitched) attached to the above-mentioned peptide degron. Exemplary chimeras are as follows Figure 7 , 15 , 17 and 18.

[0264] The stabilizing peptide can be linked to the degron via any linker of interest (e.g., a peptide linker, a synthetic compound linker). Non-limiting examples of linkers that can be used to link a peptide degron to a stabilizing peptide to form a chimera described herein are described below. Figure 6 A subset is shown in .

[0265] In certain embodiments, the peptide has any of the amino acid sequences shown below: SEQ ID NO.: 1-24 and 134 or variants thereof. In some embodiments, the peptide degron has any of the amino acid sequences shown below: SEQ ID NO.: 25-46, 65-118 or variants thereof. In some cases, the peptide degron is connected to the N-terminus of the stabilizing peptide. In other cases, the peptide degron is connected to the C-terminus of the stabilizing peptide. In some cases, the degron or degron is connected to the N- and C-termini of the stabilizing peptide. In some cases, the degron is connected to the internal amino acid position of the stabilizing peptide (i.e., any amino acid position in the stabilizing peptide other than the N- or C-terminus, e.g., 2, 3, 4, 5, 6, 7, 8, 9, etc.). In some cases, more than one (e.g., 2 or 3) degron is connected to the stabilizing peptide. In some cases where more than one (e.g., 2 or 3) degron is connected to the stabilizing peptide, one degron may be connected to the end of the stabilizing peptide, and one degron may be connected to the internal position of the stabilizing peptide. In some cases where more than one (e.g., 2 or 3) degron is attached to a stabilizing peptide, one degron can be attached to each end of the stabilizing peptide. In some cases where more than one (e.g., 2 or 3) degron is attached to a stabilizing peptide, more than one degron is each attached to an internal position of the stabilizing peptide. Figure 7 Depicted are exemplary chimeras in which the degron is linked to an internal amino acid position of the stabilizing peptide.

[0266] In some cases, the stabilizing peptide-peptide degron chimera has an amino acid sequence of one of SEQ ID NOs.: 119-126.

[0267] In certain embodiments, the stabilizing peptide-peptide degron chimera comprises one or more (e.g., 2, 3, 4 or more) stabilizing peptides and a peptide degron. In certain embodiments, the stabilizing peptide-peptide degron chimera comprises one or more (e.g., 2, 3, 4 or more) peptide degron and a stabilizing peptide. In certain embodiments, the stabilizing peptide-peptide degron chimera comprises one or more (e.g., 2, 3, 4 or more) stabilizing peptides and one or more (e.g., 2, 3, 4 or more) peptide degron.

[0268] This disclosure covers Figure 7 , 15, 17 and 18. The present disclosure covers each chimeric construct listed in Figure 7 , 15 , 17 and 18 listed variants of each chimeric construct.

[0269] In certain embodiments, the chimera is a chimera described in the Examples section below. For non-limiting examples of stable peptide-peptide degron chimeras, see, e.g., Example 6 below.

[0270] Stable peptide-small molecule degron chimeras

[0271] Provided herein are stable peptide-small molecule degron chimeras. These chimeras are composed of a stable peptide and a small molecule degron, wherein the stable peptide binds to a first protein, the first protein is a degradation target, and the small molecule degron binds to a second protein, the second protein is a degrader protein. Therefore, in certain embodiments, the above-mentioned stable peptide is (e.g., stapled, sutured) connected to the above-mentioned small molecule degron. The stable peptide can be connected to the degron by any linker of interest (e.g., a synthetic compound linker). Exemplary chimeras are as follows Figure 1 , 7 , 8, 9, 11, 12, 19 and 20.

[0272] In certain embodiments, the first protein is a target for degradation of the second protein, or a ligand or receptor of the second protein. In some embodiments, the first protein is an intracellular protein. In some embodiments, the first protein is an extracellular protein. In some embodiments, the first protein is a cell surface protein (e.g., a receptor). In some embodiments, the first protein is a disease-causing or disease-associated protein. In some embodiments, the first protein is a killer protein (e.g., BAX, BAK) or a protein that is harmful to cells or causes neurodegeneration (e.g., IgG, beta amyloid protein, tau, alpha-synuclein, TDP-43, HbS (hemoglobin-sickle cell), superoxide dismutase, Notch3, FUS, GFAP). In some embodiments, the first protein is a protein selected from the group consisting of: BCL2, BCLXL, MCL-1, BFL-1, BCL-w, BCL-B, EZH2, HDM2 / HDMX, KRAS / NRAS / HRAS, MYC, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion protein, MLL fusion protein, receptor tyrosine kinase, HOX homolog, JUN, Cyclin D, Cyclin E, BRAF, CRAF, CDK4, CDK2, HPV-E6 / E7, Aurora kinase, MITF, Wnt1, PD-1, BCR and CCR5. In some embodiments, the first protein is a bacterial protein. In some embodiments, the first protein is a viral protein. In some cases, the first protein is a protein aggregate (e.g., β-amyloid) that causes neurodegeneration.

[0273] In certain embodiments, the stabilized peptide has any one of the amino acid sequences shown below: SEQ ID NO.: 1-24 and 134 or variants thereof.

[0274] In certain embodiments, the stabilizing peptide is bound to the small molecule degron via a linker. Non-limiting examples of linkers that can be used to connect the stabilizing peptide and the small molecule to each other to form the chimeras described herein are described below. Figure 6 A subset is depicted in .

[0275] In certain embodiments, the stabilizing peptide is indirectly bound to the small molecule peptide.

[0276] In some cases, a small molecule degron is connected to the N-terminus of a stabilizing peptide. In other cases, a small molecule degron is connected to the C-terminus of a stabilizing peptide. In some cases, a degron or a degron is connected to the N- and C-terminus of a stabilizing peptide. In some cases, a degron is connected to the internal amino acid position of a stabilizing peptide (i.e., any amino acid position in a stabilizing peptide other than the N- or C-terminus, for example, 2, 3, 4, 5, 6, 7, 8, 9, etc.). In some cases, more than one (e.g., 2 or 3) degron is connected to a stabilizing peptide. In some cases where more than one (e.g., 2 or 3) degron is connected to a stabilizing peptide, a degron may be connected to the end of the stabilizing peptide, and a degron may be connected to the internal position of the stabilizing peptide. In some cases where more than one (e.g., 2 or 3) degron is connected to a stabilizing peptide, a degron may be connected to each end of the stabilizing peptide. In some cases where more than one (e.g., 2 or 3) degron is connected to a stabilizing peptide, more than one degron is each connected to the internal position of the stabilizing peptide.

[0277] In some embodiments, the second protein is a degrader protein, such as an E3 ubiquitin ligase or a substrate adaptor of an E3 ubiquitin ligase. In some embodiments, the second protein is an E3 ubiquitin ligase. Non-limiting examples of E3 ubiquitin ligases include VHL, COP1, and MDM2. In some embodiments, the second protein is selected from the group consisting of MDM2, SKP2-CKS1, FBXW1, FBXW2, FBXW4, FBXW5, FBXW7, FBXW8, FBXW9, FBXW10, FBXW11, FBXW12, SPOP, VHL, ITCH, KEAP1, KLHL2, KLHL3, KLHL7, KLHL12, KLHL13, KLHL15, KLHL20, KLHL21, KLHL24, KLHL40, KLHL42 , COP1, TRAF7, RFWD3, DCAF1, DCAF2, DCAF3, DCAF4, DCAF5, DCAF6, DCAF7, DCAF8, DCAF9, DCAF10, DCAF11, DCAF12, DCAF 13. DCAF14, DCAF15, DCAF16, DCAF17, DCAF19, SIAH1, TRPC4AC, DET1, WSB1, WSB2, HERC1, DDB2, CSA, CBL, CDC20 and FZR1. In certain embodiments, the second protein is a protein that binds to a protein selected from the group consisting of: MDM2, SKP2-CKS1, FBXW1, FBXW2, FBXW4, FBXW5, FBXW7, FBXW8, FBXW9, FBXW10, FBXW11, FBXW12, SPOP, VHL, ITCH, KEAP1, KLHL2, KLHL3, KLHL7, KLHL12, KLHL13, KLHL15, KLHL20, KLHL21, KLHL24, KLHL40, KLHL41, KLHL42, KLHL43, KLHL44, KLHL45, KLHL46, KLHL47, KLHL48, KLHL49, KLHL50, KLHL51, KLHL52, KLHL53, KLHL54, KLHL55 In some embodiments, the second protein binds to MDM2 or a protein complexed with MDM2, such as MDMX.

[0278] In certain embodiments, the second protein is a degrader protein, such as an E3 ubiquitin ligase or a substrate adapter of an E3 ubiquitin ligase. In certain embodiments, the second protein is an E3 ubiquitin ligase. In some embodiments, the second protein is bound to an E3 ligase (e.g., MDM2) or a protein complexed with an E3 ligase, such as MDMX bound to MDM2. In certain embodiments, the E3 ubiquitin ligase is a RING E3 ubiquitin ligase (e.g., Mdm2-MdmX, TRIM5α, c-CBL, cIAP, RNF4, BIRC7, IDOL, BRCA1-BARD1, RING1B-Bmi1, E4B, CHIP, Prp19). In certain embodiments, the E3 ubiquitin ligase is a HECT E3 ubiquitin ligase (e.g., Smurf1, Smurf2, Itch, E6AP). In certain embodiments, the E3 ubiquitin ligase is a RBR E3 ubiquitin ligase (e.g., Parkin, Parc, RNF144 (A / B), HOIP, HHARI). For example, non-limiting examples of E3 ubiquitin ligases are described in Morreale and Walden, Cell 165, 2016 DOI http: / dx.doi.org / 10.1016 / j.cell.2016.03.003.

[0279] In certain embodiments, the small molecule degron is based on thalidomide (e.g., see the above structure). In certain embodiments, the small molecule degron is based on a ligand that binds to the Von Hippel-Lindau protein (e.g., see the above structure). In certain embodiments, the small molecule degron is any degron known in the art. In certain embodiments, the small molecule degron used herein is any degron described in U.S. Patents 9,694,084; 9,750,816; 9,770,512; 9,821,068; 9,783,575; 9,765,019; 9,632,089 and 9,500,653, all of which are incorporated herein by reference in their entirety.

[0280] Non-limiting examples of stapled peptide-small molecule degron chimeras are provided below:

[0281] ^QWAREIGAQLRX 1 BAD X 2 LNAQYERR (SEQ ID NO: 1)-PUMA

[0282] ^FSSNRX 1 KILX 2 RTQILNQEWKQRRIQPV(SEQ ID NO:2)–EZH2 <h2 style=";text-align:left;direction:ltr">

[0283] <h2 style=";text-align:left;direction:ltr"> ^RRFFGIX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> LTNX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> LKTEEGN(SEQ ID NO:3)-SOS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0284] <h2 style=";text-align:left;direction:ltr"> ^RKALETLRRVGDGVX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> RNHX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> TAF(SEQ ID NO:4)–MCL-1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0285] <h2 style=";text-align:left;direction:ltr"> ^LSQEQLEHRERSLX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> TLRX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> IQRBLF(SEQ ID NO:5)–BCL9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0286] <h2 style=";text-align:left;direction:ltr"> ^LTF8EYWAQ#XSAA(SEQ ID NO:6)–p53<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0287] <h2 style=";text-align:left;direction:ltr"> ^DIIRNIARHLAX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> VGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> BDRSI(SEQ ID NO:7)-BID<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0288] <h2 style=";text-align:left;direction:ltr"> ^IWIAQELRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> IGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> FNAYYARR(SEQ ID NO:8)-BIM<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0289] <h2 style=";text-align:left;direction:ltr"> ^NLWAAQRYGRELRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> BDDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> FVDSFKK(SEQ ID NO:9)-BAD S153D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0290] <h2 style=";text-align:left;direction:ltr"> ^NLWAAQRYGRELRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> BSDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> FVDSFKK(SEQ ID NO:10)–BAD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0291] <h2 style=";text-align:left;direction:ltr"> ^QLTAARLKX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> LGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> LHQRTBWR(SEQ ID NO:11)-HRK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0292] <h2 style=";text-align:left;direction:ltr"> ^AELEVESATQLRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> FGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> LNFRQKLL(SEQ ID NO:12)-NOXA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0293] <h2 style=";text-align:left;direction:ltr"> QWAREIGAQLRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> BADX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> LNAQYERR&(SEQ ID NO:13)-PUMA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0294] <h2 style=";text-align:left;direction:ltr"> FSSNRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> KILX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> RTQILNQEWKQRRIQPV&(SEQ ID NO:14)–EZH2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0295] <h2 style=";text-align:left;direction:ltr"> RRFFGI X<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> LTNX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> LKTEEGN&(SEQ ID NO:15)-SOS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0296] <h2 style=";text-align:left;direction:ltr"> RKALETLRRVGDGVX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> RNHX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> TAF&(SEQ ID NO:16)–MCL-1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0297] <h2 style=";text-align:left;direction:ltr"> LSQEQLEHRERSLX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> TLRX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> IQRBLF&(SEQ ID NO:17)–BCL9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0298] <h2 style=";text-align:left;direction:ltr"> LTF8EYWAQ#XSAA&(SEQ ID NO:18)–p53<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0299] <h2 style=";text-align:left;direction:ltr"> DIIRNIARHLAX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> VGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> BDRSI&(SEQ ID NO:19)-BID<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0300] <h2 style=";text-align:left;direction:ltr"> IWIAQELRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> IGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> FNAYYARR&(SEQ ID NO:20)-BIM<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0301] <h2 style=";text-align:left;direction:ltr"> NLWAAQRYGRELRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> BDDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> FVDSFKK&(SEQ ID NO:21)–BAD-S153D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0302] <h2 style=";text-align:left;direction:ltr"> NLWAAQRYGRELRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> BSDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> FVDSFKK&(SEQ ID NO:22)-BAD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0303] <h2 style=";text-align:left;direction:ltr"> QLTAARLKX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> LGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> LHQRTBWR&(SEQ ID NO:23)-HRK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0304] <h2 style=";text-align:left;direction:ltr"> AELEVESATQLRX<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> FGDX<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> LNFRQKLL&(SEQ ID NO:24)–NOXA

[0305] IWIA%ELRX 1 IGDX 2 FNAYYARR (SEQ ID NO: 136)–BIM

[0306] IWIAQELRX 1 IGDX 2 FN%YYARR(SEQ ID NO:137)–BIM

[0307] LTF8$YWAQLXSAA (SEQ ID NO: 138)

[0308] LTF8EYWAQLX$AA (SEQ ID NO: 139)

[0309] %TF8EYWAQLXSAA(SEQ ID NO:140)

[0310] LTF8%YWAQLXSAA (SEQ ID NO:141)

[0311] LTF8EYWAQLX%AA (SEQ ID NO:142)

[0312] @TF8EYWAQLXSAA(SEQ ID NO:143),

[0313] wherein 8 = R-octenylalanine; B = norleucine; # = cyclobutylalanine; X = S-pentenylalanine, and in some cases, X 1 and X 2 Same as (e.g., S-pentenylalanine), ^ = thalidomide-aminocaproic acid, & = Lys-ε-amino-thalidomide, % = A 1 , A 2 , A 3 , A 4 , A 5、 A 6 , A 7 , A 8 , A 9 , A 10 or A 11 , where A 1 =DAB-thalidomide, A 2 =DAB-Gly-Thal, A 3 =DAB-βAla-Thal, A 4 =DAB-Linker 1-Thal, A 5 =DAB-Linker 2-Thal, A 6 =DAB-connector 3-Thal, A7 =DAB-Linker 4-Thal, A 8 =DAB-Linker 5-Thal, A 9 =DAB-Linker 6-Thal, A 10 =DAB-Linker 7-Thal, and A 11 =DAB-connector 8-Thal; $=B 1 , B 2 , B 3 , B 4 , B 5 or B 6 , where B 1 =DAB-TRIB formate, B 2 =DAB-Gly-TRIB formate, B 3 =DAB-βAla-TRIB formate, B 4 =DAB-Linker 3-TRIB Formate, B 5 =DAB-linker 5-TRIB formate, and B 6 =DAB-Linker 7-TRIB Formate; @=C 1 , C 2 , C 3 , C 4 , C 5 or C 6 , where C 1 =DAB-VHL formate, C 2 =DAB-Gly-VHL formate, C 3 =DAB-βAla-VHL formate, C 4 =DAB-Linker 3-VHL Formate, C 5 =DAB-linker 5-VHL formate, and C 6 =DAB-Linker 7-VHL Formate,

[0314] Among them, connector 1-connector 8 is as follows Figure 6 In some embodiments, thalidomide-aminocaproic acid (^) or Lys-ε-amino-thalidomide (&) is linked to the stapled peptide via a linker.

[0315] In certain embodiments, the stabilizing peptide-small molecule degron chimera comprises one or more (e.g., 2, 3, 4 or more) stabilizing peptides and a small molecule degron. In certain embodiments, the stabilizing peptide-small molecule degron chimera comprises one or more (e.g., 2, 3, 4 or more) small molecule degron and a stabilizing peptide. In certain embodiments, the stabilizing peptide-small molecule degron chimera comprises one or more (e.g., 2, 3, 4 or more) stabilizing peptides and one or more (e.g., 2, 3, 4 or more) small molecule degron.

[0316] Each of the chimeric constructs listed above and variants thereof are encompassed by the present disclosure. Figure 1 , 7 , 8, 9, 11, 12, 19 and 20. The present disclosure covers Figure 1 , 7 , 8, 9, 11, 12, 19 and 20 listed variants of each chimeric construct.

[0317] In certain embodiments, the chimera is a chimera described in the Examples section below. For example, non-limiting examples of stable peptide-small molecule degron chimeras are provided in Examples 2, 3, 4, and 7 below.

[0318] Stabilizing peptide-stabilizing peptide degron chimera

[0319] Provided herein are stable peptide-stable peptide degrader chimeras.These chimeras are composed of two stable peptides-a first stable peptide and a second stable peptide, wherein the first stable peptide is combined with a first protein, the first protein is a protein target to be degraded, and the second stable peptide is combined with a second protein, and the second protein is a degradation agent protein.Therefore, in some embodiments, the above-mentioned first stable peptide (for example, stapled, sutured) is connected to the above-mentioned second stable peptide (for example, stapled, sutured).The first and second stable peptides can be connected directly or indirectly.

[0320] In certain embodiments, the first protein is a protein target for degradation of the second protein, or a ligand or receptor of the second protein. In some embodiments, the first protein is an intracellular protein. In some embodiments, the first protein is an extracellular protein. In some embodiments, the first protein is a cell surface protein (e.g., a receptor). In some embodiments, the first protein is a disease-causing or disease-associated protein. In some embodiments, the first protein is a killer protein (e.g., BAX, BAK) or a protein that is harmful to cells or causes neurodegeneration (e.g., IgG, beta amyloid protein, tau, alpha-synuclein, TDP-43, HbS (hemoglobin-sickle cell), superoxide dismutase, Notch3, FUS, GFAP). In some embodiments, the first protein is a protein selected from the group consisting of: BCL2, BCLXL, MCL-1, BFL-1, BCL-w, BCL-B, EZH2, HDM2 / HDMX, KRAS / NRAS / HRAS, MYC, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion protein, MLL fusion protein, receptor tyrosine kinase, HOX homolog, JUN, Cyclin D, Cyclin E, BRAF, CRAF, CDK4, CDK2, HPV-E6 / E7, Aurora kinase, MITF, Wnt1, PD-1, BCR and CCR5. In some embodiments, the first protein is a bacterial protein. In some embodiments, the first protein is a viral protein. In some cases, the first protein is a protein aggregate (e.g., β-amyloid) that causes neurodegeneration.

[0321] In certain embodiments, the first stabilizing peptide has any one of the amino acid sequences shown below: SEQ ID NO.: 1-24 and 134 or variants thereof.

[0322] In certain embodiments, the first stabilizing peptide is coupled to the second stabilizing peptide via a linker. Non-limiting examples of linkers that can be used to connect the first and second stabilizing peptides to each other to form the chimeras described herein are described below. Figure 6 A subset is shown in .

[0323] In certain embodiments, the first stabilizing peptide is indirectly bound to the second stabilizing peptide.

[0324] In some embodiments, the second protein is a degrader protein, such as an E3 ubiquitin ligase or a substrate adaptor of an E3 ubiquitin ligase. In some embodiments, the second protein is an E3 ubiquitin ligase. Non-limiting examples of E3 ubiquitin ligases include VHL, COP1, and MDM2. In some embodiments, the second protein is selected from the group consisting of MDM2, MDMX, SKP2-CKS1, FBXW1, FBXW2, FBXW4, FBXW5, FBXW7, FBXW8, FBXW9, FBXW10, FBXW11, FBXW12, SPOP, VHL, ITCH, KEAP1, KLHL2, KLHL3, KLHL7, KLHL12, KLHL13, KLHL15, KLHL20, KLHL21, KLHL24, KLHL40, KLHL L42, COP1, TRAF7, RFWD3, DCAF1, DCAF2, DCAF3, DCAF4, DCAF5, DCAF6, DCAF7, DCAF8, DCAF9, DCAF10, DCAF11, DCAF12, DC AF13, DCAF14, DCAF15, DCAF16, DCAF17, DCAF19, SIAH1, TRPC4AC, DET1, WSB1, WSB2, HERC1, DDB2, CSA, CBL, CDC20 and FZR1. In certain embodiments, the second protein is a protein that binds to a protein selected from the group consisting of: MDM2, SKP2-CKS1, FBXW1, FBXW2, FBXW4, FBXW5, FBXW7, FBXW8, FBXW9, FBXW10, FBXW11, FBXW12, SPOP, VHL, ITCH, KEAP1, KLHL2, KLHL3, KLHL7, KLHL12, KLHL13, KLHL15, KLHL20, KLHL21, KLHL24, KLHL40, KLHL41, KLHL42, KLHL43, KLHL44, KLHL45, KLHL46, KLHL47, KLHL48, KLHL49, KLHL50, KLHL51, KLHL52, KLHL53, KLHL54, KLHL55 In some embodiments, the second protein binds to MDM2 or a protein complexed with MDM2, such as MDMX.

[0325] In certain embodiments, the second protein is a degrader protein, such as an E3 ubiquitin ligase or a substrate adapter of an E3 ubiquitin ligase. In certain embodiments, the second protein is an E3 ubiquitin ligase. In some embodiments, the second protein is bound to an E3 ligase (e.g., MDM2) or a protein complexed with an E3 ligase, such as MDMX bound to MDM2. In certain embodiments, the E3 ubiquitin ligase is a RING E3 ubiquitin ligase (e.g., Mdm2-MdmX, TRIM5α, c-CBL, cIAP, RNF4, BIRC7, IDOL, BRCA1-BARD1, RING1B-Bmi1, E4B, CHIP, Prp19). In certain embodiments, the E3 ubiquitin ligase is a HECT E3 ubiquitin ligase (e.g., Smurf1, Smurf2, Itch, E6AP). In certain embodiments, the E3 ubiquitin ligase is a RBR E3 ubiquitin ligase (e.g., Parkin, Parc, RNF144 (A / B), HOIP, HHARI). For example, non-limiting examples of E3 ubiquitin ligases are described in Morreale and Walden, Cell 165, 2016 DOI http: / dx.doi.org / 10.1016 / j.cell.2016.03.003.

[0326] In certain embodiments, the second stabilizing peptide has an amino acid sequence as shown in SEQ ID NO: 134 or a variant thereof. In certain embodiments, the second stabilizing peptide has an amino acid sequence as shown in SEQ ID NO: 6 or a variant thereof. In certain embodiments, the second stabilizing peptide has an amino acid sequence as shown in SEQ ID NO: 18 or a variant thereof.

[0327] In certain embodiments, the second stabilizing peptide is a stabilizing peptide described in U.S. Patents 8,889,632, 9,458,202, 9,505,804, 9,527,896, 9,957,299, 10,030,049, and 10,059,741, International Patent Application Publications WO 1998 / 001467 and WO2017 / 165617, and U.S. Patent Application Publication 2014 / 0018302A1, the entire contents of which are incorporated herein by reference.

[0328] In some cases, the first stabilizing peptide binds to the N-terminus of the second stabilizing peptide. In other cases, the first stabilizing peptide binds to the C-terminus of the second stabilizing peptide. In some cases, the first stabilizing peptide binds to the internal amino acid position of the second stabilizing peptide (i.e., any amino acid position in the stabilizing peptide except the N- or C-terminus, for example, 2, 3, 4, 5, 6, 7, 8, 9, etc.). In some cases, the second stabilizing peptide binds to the N-terminus of the first stabilizing peptide. In other cases, the second stabilizing peptide binds to the C-terminus of the first stabilizing peptide. In some cases, the second stabilizing peptide binds to the internal amino acid position of the first stabilizing peptide (i.e., any amino acid position in the stabilizing peptide except the N- or C-terminus, for example, 2, 3, 4, 5, 6, 7, 8, 9, etc.).

[0329] In certain embodiments, a stabilizing peptide-stabilizing peptide degron chimera comprises one or more (e.g., 2, 3, 4 or more) stabilizing peptides (which bind to one or more proteins to be degraded), and a stabilizing peptide degron that binds to a degrader protein. In certain embodiments, a stabilizing peptide-stabilizing peptide degron chimera comprises one or more (e.g., 2, 3, 4 or more) stabilizing peptide degron (which bind to one or more degrader proteins), and a stabilizing peptide that binds to a protein to be degraded. In certain embodiments, a stabilizing peptide-stabilizing peptide degron chimera comprises one or more (e.g., 2, 3, 4 or more) stabilizing peptides (which bind to one or more proteins to be degraded), and one or more (e.g., 2, 3, 4 or more) stabilizing peptide degron (which binds to one or more degrader proteins).

[0330] This disclosure covers Figure 21 Each chimeric construct listed in . The present disclosure covers Figure 21 Variants of each chimeric construct listed in .

[0331] In certain embodiments, the chimera is a chimera described in the Examples section below. For example, a non-limiting example of a stabilizing peptide-stabilizing peptide degron chimera is provided in Example 8 below.

[0332] Small molecule-stable peptide degron chimeras

[0333] Provided herein are small molecule-stable peptide degrader chimeras. These chimeras are composed of small molecules and stable peptides, wherein the small molecule is combined with a first protein, the first protein is a protein target to be degraded, and the stable peptide is combined with a second protein, and the second protein is a degradation agent protein. Therefore, in some embodiments, the small molecule is connected to the above-mentioned stable peptide (for example, stapling, suturing). Small molecules and stable peptides can be directly or indirectly connected.

[0334] In certain embodiments, the first protein is a protein target to be degraded by the second protein or a ligand or receptor of the second protein. In some embodiments, the first protein is an intracellular protein. In some embodiments, the first protein is an extracellular protein. In some embodiments, the first protein is a cell surface protein (e.g., a receptor). In some embodiments, the first protein is a disease-causing or disease-associated protein. In some embodiments, the first protein is a killer protein (e.g., BAX, BAK) or a protein that is harmful to cells or causes neurodegeneration (e.g., IgG, beta amyloid protein, tau, alpha-synuclein, TDP-43, HbS (hemoglobin-sickle cell), superoxide dismutase, Notch3, FUS, GFAP). In some embodiments, the first protein is a protein selected from the group consisting of: BCL2, BCLXL, MCL-1, BFL-1, BCL-w, BCL-B, EZH2, HDM2 / HDMX, KRAS / NRAS / HRAS, MYC, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion protein, MLL fusion protein, receptor tyrosine kinase, HOX homolog, JUN, Cyclin D, Cyclin E, BRAF, CRAF, CDK4, CDK2, HPV-E6 / E7, Aurora kinase, MITF, Wnt1, PD-1, BCR and CCR5. In some embodiments, the first protein is a bacterial protein. In some embodiments, the first protein is a viral protein. In some cases, the first protein is a protein aggregate (e.g., β-amyloid) that causes neurodegeneration.

[0335] In certain embodiments, a small molecule is any drug or compound that is capable of binding to a protein when forming part of a chimera without interfering with the ability of the stapled peptide of the chimera to interact with its target. Assays and methods for evaluating the interference of a drug or compound (in the case of its chimera) with the ability of the stapled peptide (of the chimera) to interact with its target (e.g., immunofluorescence and immunoreaction) are known in the art, for example, immunofluorescence and immunocoprecipitation. In certain embodiments, a small molecule is Figure 23 In certain embodiments, the small molecule is a kinase inhibitor. In certain embodiments, the small molecule is a histone deacetylase inhibitor.

[0336] In certain embodiments, the small molecule is connected to the stable peptide via a linker. Non-limiting examples of linkers that can be used to connect the small molecule and the stable peptide to each other to form the chimeras described herein are described below. Figure 6 A subset is shown in .

[0337] In certain embodiments, the small molecule is indirectly linked to the stabilizing peptide.

[0338] In some cases, the small molecule is attached to the N-terminus of the stabilizing peptide. In other cases, the small molecule is attached to the C-terminus of the stabilizing peptide. In some cases, the small molecule is attached to an internal amino acid position of the stabilizing peptide (i.e., any amino acid position in the stabilizing peptide except the N- or C-terminus, e.g., 2, 3, 4, 5, 6, 7, 8, 9, etc.).

[0339] In some embodiments, the second protein is a degrader protein, such as an E3 ubiquitin ligase or a substrate adaptor of an E3 ubiquitin ligase. In some embodiments, the second protein is an E3 ubiquitin ligase. Non-limiting examples of E3 ubiquitin ligases include VHL, COP1, and MDM2. In some embodiments, the second protein is selected from the group consisting of MDM2, MDMX, SKP2-CKS1, FBXW1, FBXW2, FBXW4, FBXW5, FBXW7, FBXW8, FBXW9, FBXW10, FBXW11, FBXW12, SPOP, VHL, ITCH, KEAP1, KLHL2, KLHL3, KLHL7, KLHL12, KLHL13, KLHL15, KLHL20, KLHL21, KLHL24, KLHL40, KLHL L42, COP1, TRAF7, RFWD3, DCAF1, DCAF2, DCAF3, DCAF4, DCAF5, DCAF6, DCAF7, DCAF8, DCAF9, DCAF10, DCAF11, DCAF12, DC AF13, DCAF14, DCAF15, DCAF16, DCAF17, DCAF19, SIAH1, TRPC4AC, DET1, WSB1, WSB2, HERC1, DDB2, CSA, CBL, CDC20 and FZR1. In certain embodiments, the second protein is a protein that binds to a protein selected from the group consisting of: MDM2, SKP2-CKS1, FBXW1, FBXW2, FBXW4, FBXW5, FBXW7, FBXW8, FBXW9, FBXW10, FBXW11, FBXW12, SPOP, VHL, ITCH, KEAP1, KLHL2, KLHL3, KLHL7, KLHL12, KLHL13, KLHL15, KLHL20, KLHL21, KLHL24, KLHL40, KLHL41, KLHL42, KLHL43, KLHL44, KLHL45, KLHL46, KLHL47, KLHL48, KLHL49, KLHL50, KLHL51, KLHL52, KLHL53, KLHL54, KLHL55 In some embodiments, the second protein binds to MDM2 or a protein that conforms to MDM2, such as MDMX.

[0340] In certain embodiments, the second protein is a degrader protein, such as an E3 ubiquitin ligase or a substrate adapter of an E3 ubiquitin ligase. In certain embodiments, the second protein is an E3 ubiquitin ligase. In some embodiments, the second protein is bound to an E3 ligase (e.g., MDM2) or a protein complexed with an E3 ligase, such as MDMX bound to MDM2. In certain embodiments, the E3 ubiquitin ligase is a RING E3 ubiquitin ligase (e.g., Mdm2-MdmX, TRIM5α, c-CBL, cIAP, RNF4, BIRC7, IDOL, BRCA1-BARD1, RING1B-Bmi1, E4B, CHIP, Prp19). In certain embodiments, the E3 ubiquitin ligase is a HECT E3 ubiquitin ligase (e.g., Smurf1, Smurf2, Itch, E6AP). In certain embodiments, the E3 ubiquitin ligase is a RBR E3 ubiquitin ligase (e.g., Parkin, Parc, RNF144 (A / B), HOIP, HHARI). For example, non-limiting examples of E3 ubiquitin ligases are described in Morreale and Walden, Cell 165, 2016 DOI http: / dx.doi.org / 10.1016 / j.cell.2016.03.003.

[0341] In some embodiments, the stabilizing peptide has the amino acid sequence shown below: SEQ ID NO: 134 or a variant thereof. In some embodiments, the second stabilizing peptide has the amino acid sequence shown below: SEQ ID NO: 6 or a variant thereof. In some embodiments, the second stabilizing peptide has the amino acid sequence shown below: SEQ ID NO: 18 or a variant thereof.

[0342] In certain embodiments, a small molecule-stable peptide degron chimera comprises one or more (e.g., 2, 3, 4 or more) stable peptide degron and a small molecule. In certain embodiments, a small molecule-stable peptide degron chimera comprises one or more (e.g., 2, 3, 4 or more) small molecules and a stable peptide degron. In certain embodiments, a small molecule-stable peptide degron chimera comprises one or more (e.g., 2, 3, 4 or more) stable peptide degron and one or more (e.g., 2, 3, 4 or more) small molecules.

[0343] This disclosure covers Figure 23 The present disclosure covers the chimeric constructs depicted in . Figure 23 Variants of the chimeric constructs depicted in .

[0344] In certain embodiments, the chimera is a chimera described in the Examples section below. For example, a non-limiting example of a small molecule-stable peptide degron chimera is provided in Example 9 below.

[0345] Linker

[0346] There is no particular limitation on the linkers that can be used in the above constructs. In some embodiments, the linker is an amino acid, such as aminopropionic acid, aminobutyric acid, aminovaleric acid, or aminocaproic acid. In some embodiments, the linker is an oligoethylene glycol, i.e., NH 2 -(CH 2 -CH 2 -O) x -CH 2 -CH 2 -COOH. In some embodiments, the joint is a peptide joint. In some embodiments, any single-stranded peptide comprising about 1 to 30 residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids) can be used as a joint. In other embodiments, the joint length is 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 144 or 10 to 150 amino acids. In some cases, the joint only comprises glycine and / or serine residues. Examples of such peptide linkers include: Gly, Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser (SEQ ID NO: 47); Ser Gly Gly Gly (SEQ ID NO: 48); Gly Gly Gly Gly Ser (SEQ ID NO: 49); Ser Gly Gly Gly Gly (SEQ ID NO: 50); Gly Gly Gly Gly Gly Ser (SEQ ID NO:51); Ser Gly Gly Gly Gly Gly (SEQ ID NO:52); Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO:53); Ser Gly Gly Gly Gly Gly Gly (SEQ ID NO:54); (Gly Gly Gly Gly Gly Ser) n (SEQ ID NO:49)n, wherein n is an integer greater than 1; and (Ser Gly Gly Gly Gly) n(SEQ ID NO:50)n, wherein n is an integer greater than 1. In some cases, the linker has the amino acid sequence of SEQ ID NO:4, except that the serine residue is substituted with another amino acid. In some cases, the linker has multiple copies (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) of the amino acid sequence of SEQ ID NO:4, except that the serine residue in each copy of the linker is substituted with another amino acid.

[0347] In other embodiments, the linker peptide is modified so that the amino acid sequence GSG (which appears at the junction of the traditional Gly / Ser linker peptide repeat sequence) is absent. For example, the peptide linker comprises an amino acid sequence selected from the following: (GGGXX) n GGGGS(SEQ ID NO:55) and GGGGS(XGGGS) n (SEQ ID NO: 56), wherein X is any amino acid that can be incorporated into the sequence without resulting in a polypeptide comprising the sequence GSG, and n is 0 to 4. In one embodiment, the sequence of the linker peptide is (GGGX 1 X 2 ) n GGGGS, and X 1 is P, and X 2 is S, and n is 0 to 4 (SEQ ID NO: 57). In another embodiment, the sequence of the linker peptide is (GGGX 1 X 2 ) n GGGGS, and X 1 is G, and X 2 is Q and n is 0 to 4 (SEQ ID NO: 58). In another embodiment, the sequence of the linker peptide is (GGGX 1 X 2 ) n GGGGS, and X 1 is G, and X 2 is A, and n is 0 to 4 (SEQ ID NO: 59). In another embodiment, the sequence of the linker peptide is GGGGS (XGGGS) n , and X is P and n is 0 to 4 (SEQ ID NO: 60). In one embodiment, the linker peptide of the present invention comprises the amino acid sequence (GGGGA) 2 GGGGS (SEQ ID NO: 61), or consisting thereof. In another embodiment, the linker peptide comprises the amino acid sequence (GGGGQ) 2 GGGGS (SEQ ID NO: 62), or consisting thereof. In another embodiment, the linker peptide comprises (GGGPS) 2GGGGS (SEQ ID NO: 63), or consisting thereof. In another embodiment, the linker peptide comprises GGGGS (PGGGS) 2 (SEQ ID NO:64), or consisting thereof.

[0348] In certain embodiments, the linker is a synthetic compound linker (chemical cross-linker). Examples of commercially available cross-linkers include N-hydroxysuccinimide (NHS), bis-succinimidyl suberate (DSS), bis-(sulfosuccinimidyl) suberate (BS3), dithiobis-(succinimidyl propionate) (DSP), dithiobis-(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis-(succinimidyl succinate) (EGS), ethylene glycol bis-(sulfosuccinimidyl succinate) (sulfoEGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis-[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES) and bis-[2-(sulfosuccinimidyloxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0349] In certain embodiments, the linker is Figure 6 Connectors shown.

[0350] Method for synthesizing stable peptide degrader chimera

[0351] Synthesis of stapled peptide-small molecule degron chimeras

[0352] Hydrocarbon-stapled peptides can be synthesized, purified, and quantified using previously reported methods (Bird et al., Methods Enzymol., 446:369-86 (2008); Bird et al., Curr. Protoc. Chem. Biol., 3(3):99-117 (2011) with the following modifications and added details: The peptide was synthesized using established methods (i.e., Fmoc-protected amino acids, HATU coupling agent) until the desired sequence was achieved. The peptide was then stapled using Grubbs catalyst (1st generation) and the N-terminus was deprotected using piperidine. A polyatom linker such as β-alanine or aminocaproic acid was incorporated. Thalidomide-COOH was then coupled using HCTU. The imide bond is particularly sensitive to nucleophiles, so piperidine or hydrazine was not used after incorporation. The polypeptide was then cleaved with TFA for 1 hour and purified by LCMS.

[0353] Synthesis of stapled peptide-peptide degron chimeras

[0354] The stapled peptide portion of the chimera can be synthesized as above and then coupled to a fully protected peptide degradation determinant. The fully protected degradation determinant peptide can be synthesized on a weakly acid cleavable resin (e.g., Sieber amide resin), and the final synthetic step is the reaction of glycolic anhydride with the peptide N-terminus. After 1% TFA cleavage, the protected peptide is precipitated in ether, dissolved in acetic acid / water, and then lyophilized. The fully protected degradation determinant peptide is then mixed with a coupling agent and a base and reacted with the stapled peptide N-terminus bound to the resin for 2 hours, followed by TFA cleavage and purification to obtain the stapled peptide-peptide degradation determinant.

[0355] Synthesis of stapled peptide-stapled peptide degron chimeras

[0356] The first stapled peptide portion can be synthesized using the method established above, and then a linker portion, such as β-alanine or aminocaproic acid, is introduced. The second half of the stapled peptide chimera can then be synthesized using the same protocol as the first stapled peptide portion, and then the entire chimera can be stapled using Grubbs catalyst (1st generation), followed by acetylation of the N-terminus. The chimera is then cleaved with TFA for 1 hour and purified by LCMS.

[0357] Synthesis of small molecule-stapled peptide degron chimeras

[0358] The fixed peptide portion of the chimera can be synthesized using the methods established above, followed by peptide stapling using Grubbs catalyst (1st generation), deprotection of the N-terminus with piperidine, and introduction of a polyatom linker such as β-alanine or aminocaproic acid. Coupling of small molecules to the stapled peptide can be performed as described above.

[0359] The properties and functional activities of the stabilized (eg, stapled) peptide degron chimeras of the invention can be determined, for example, using the methods described below.

[0360] Binding of stapled peptide degron chimeras to protein targets

[0361] Competitive fluorescence polarization assays are performed to monitor (1) the retention of binding affinity of the stapled peptide (or molecule) portion of the chimera for its protein target and (2) the ability of the degradation determinant component (whether molecule or peptide) to maintain affinity for binding to its protein target. Exemplary fluorescence polarization methods for stapled peptides and molecular degradation determinants include Pitter et al. Methods Enzymol 446: 387-408 (2008) and Nowak et al. Nat Chem Biol 14: 706-714 (2018). In a cell degradation assay using a GFP-tagged target protein substrate (e.g., GFP-BRD4), it is also used to determine the ability of the stapled peptide degradation determinant chimera to penetrate intact cells and compete with a positive control molecule degradation determinant chimera (e.g., dBET6) to inhibit induced degradation. Exemplary methods for such competitive cell degradation assays can be found in Nowak et al. Nat Chem Biol 14: 706-714 (2018).

[0362] Monitoring targeted ubiquitination of recombinant proteins by stapled peptide degron chimeras

[0363] In order to monitor the ubiquitination of protein targets in vitro, a commercial Mdm2 / HDM2 ubiquitin ligase kit (K-200B) can be used. In brief, chimeras (10 μM), recombinant full-length MDM2 (1 μM with a GST tag), E1 enzyme (UBE1, 50 nM), E2 enzyme (UBE2D3, 1 μM), ubiquitin (100 μM), ATP (1 mM) and recombinant target protein (100 nM) are combined in a 1.5 mL microtube into a reaction buffer. The mixture is incubated at 37 ° C for 6 hours. Subsequently, 20 μL of the reaction mixture is tested using standard protein blotting techniques to visualize the upward band shift caused by ubiquitination using antibodies against the target protein.

[0364] Monitoring native protein degradation in cells caused by stapled peptide degradation determinant chimeras

[0365] To measure intracellular protein degradation, cancer cells (e.g., SJSA-1, SJSA-X, U2OS) were cultured in a humidity-controlled CO atmosphere at 37°C. 2The cells were passaged in DMEM (Life Technologies, Grand Island, NY) medium (CM) in a balanced incubator containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Pen Strep). The day before treatment, the cells were passaged and seeded in six-well plates at a density of 100,000 cells / mL. After 24 hours, the cells were treated with stapled peptide degradation determinant chimeras (e.g., 10 μM) for 0, 2, 4, and 6 hours, then harvested and lysed. The cell lysates were then tested using standard Western blotting techniques, protein levels were assessed using antibodies against the target protein, and loading controls were performed using actin antibodies.

[0366] Monitoring the effects of target protein degradation induced by stapled peptide degradation determinant chimeras on cancer cell viability

[0367] For stapled peptide degradation determinant chimeras that retain binding to both protein targets, achieve cellular uptake, and can contact both targets within the cell to induce degradation of the targeted proteins, their cytotoxic effects on cancer cells are evaluated using established cell viability and apoptosis assays (including Cell Titer Glo) 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). Control studies of specificity of action are performed using, for example, point mutant peptides that cannot engage their protein targets and / or cell lines that do not express the target protein and / or the degrader protein of interest.

[0368] Therapeutic method

[0369] The chimeras disclosed herein can promote the degradation of disease-related proteins bound by stable peptides or small molecules. In some cases, the degraded protein is a killer protein, such as BAX or BAK (which is useful as a cell protector during stress such as stroke, neurodegenerative diseases and heart attack hypoxia). In some cases, the degraded protein is a protein that is harmful to cells, such as Ig in myeloma, amyloid in Alzheimer's disease, and other protein deposits that cause disease. In some cases, the protein to be degraded is a protein selected from the group consisting of: BCL2, / BCLX L, MCL-1, BFL-1, BCL-w, BCL-B, EZH2, HDM2 / HDMX, PUMA, SOSKRAS / NRAS / HRAS, MYC, b-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI, MLL fusion protein, receptor tyrosine kinase, HOX homolog, JUN, Cyclin D, Cyclin E, BRAF, CRAF, CDK4, CDK2, HPV-E6 / E7, Aurora kinase, MITF, Wnt1, PD-1, BCR and CCR5. In some cases, the protein to be degraded is a protein selected from the group consisting of amyloid beta (Alzheimer's disease), tau protein (Alzheimer's disease), alpha-synuclein (Alzheimer's disease), TDP-43 (frontotemporal lobar degeneration), superoxide dismutase (ALS), Notch3 (CADASIL), FUS (sarcoma, ALS), amyloid A, Ig heavy and light chains, and GFAP (Alexander disease).

[0370] The disclosure features methods of preventing and / or treating cancer, autoimmune diseases, or inflammatory diseases using any of the stabilized peptides or chimeras described herein.As used herein, the term "treat" means to alleviate, inhibit, or improve a disease or condition that a subject is suffering from.

[0371] The peptides or chimeras described herein can be used to treat human subjects with cancer. The peptides or chimeras described herein can also be used to treat people with melanoma, leukemia, lymphoma or other hematological malignancies or solid tumors. In some cases, the solid tumor is melanoma, breast cancer or lung cancer. In some embodiments, the peptides or chimeras described herein can be used to treat human subjects with autoimmune diseases or other inflammatory conditions characterized by cell excess diseases. In some cases, the autoimmune disease is autoimmune colitis, thyroiditis, arthritis, nephritis, dermatitis, vasculitis, systemic lupus erythematosus, diabetes or Sjögren's syndrome. In some cases, the inflammatory disease is asthma, psoriasis, inflammatory colitis, thyroiditis, arthritis, nephritis, dermatitis or vasculitis.

[0372] For example, where an endogenous protein (e.g., an oncoprotein such as MDM2) is to be modified to include a degron, any gene editing technology can be used (see, e.g., U.S. Patents 9,840,713; 9,840,702; 9,840,699; 9,834,791; 9,822,372; 9,816,080; 9,790,490; 9,783,490; 9,771,601; 9,758,775; 9,738,908; 9,616,090; 9,574,211, all of which are incorporated herein by reference in their entireties). The presence of the newly introduced degron should result in degradation of the protein.

[0373] Typically, the method includes selecting a subject and administering to the subject an effective amount of one or more peptides herein, e.g., in or as a pharmaceutical composition, and optionally repeated administration as needed to prevent or treat cancer such as melanoma or lymphoma, and can be administered orally, intravenously, or topically. The subject can be selected for treatment based on, for example, determining that the subject suffers from a cancer that expresses a protein targeted by the stapled peptide (e.g., MCL-1, BFL-1).

[0374] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, the age, weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptom, the patient's predisposition to the disease, condition or symptom, and the judgment of the treating physician.

[0375] The effective amount can be administered in one or more administrations, applications or dosages. The therapeutically effective amount (i.e., effective dose) of the therapeutic compound depends on the selected therapeutic compound. The composition can be administered once or more per day to once or more per week; including once every other day. The skilled person will appreciate that certain factors may affect the dose and time required to effectively treat the subject, including but not limited to the severity of the disease or condition, previous treatment, the overall health and / or age of the subject, and other diseases present. In addition, treatment of the subject with a therapeutically effective amount of the therapeutic compound described herein may include a single treatment or a series of treatments. For example, an effective amount may be administered at least once.

[0376] Pharmaceutical composition

[0377] Any stable peptide or chimera described herein can be formulated to be used as or in a pharmaceutical composition. These compositions can be formulated into preparations or adapted to be administered to a subject by 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 004 (available from fda.give / cder / dsm / DRG / drg00301.htm). For example, the composition can be formulated into or adapted to be administered by inhalation (e.g., oral and / or nasal inhalation (e.g., by a nebulizer or spray)), injection (e.g., intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular and / or subcutaneous); and / or for oral administration, transmucosal administration and / or topical administration (including topical (e.g., nasal) sprays and / or solutions).

[0378] In some cases, the pharmaceutical composition may include an effective amount of one or more stable peptides. As used herein, the terms "effective amount" and "therapeutically effective" refer to the amount or concentration of one or more compounds or pharmaceutical compositions described herein used over a period of time (including acute or chronic administration and periodic or continuous administration) that effectively induces a desired effect or physiological result (e.g., treatment of infection) within its scope of administration.

[0379] The pharmaceutical compositions of the present invention may comprise one or more peptides and any pharmaceutically acceptable carrier and / or vehicle. In some cases, the medicament may further comprise one or more additional therapeutic agents in an amount effective to achieve modulation of the disease or disease symptoms.

[0380] The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that can be administered to a patient together with the compound of the present invention without destroying its pharmacological activity and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the compound.

[0381] Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin; self-emulsifying drug delivery systems (SEDDS), such as d-α-tocopherol polyethylene glycol 1000 succinate; surfactants for pharmaceutical dosage forms, such as Tween or other similar polymer delivery matrices; serum proteins, such as human serum albumin; buffer substances, such as phosphates; glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin. Cyclodextrins such as α-, β- and γ-cyclodextrins can also be advantageously used to enhance the delivery of compounds of the formula described herein.

[0382] The pharmaceutical composition of the present invention may comprise any conventional nontoxic pharmaceutically acceptable carrier, adjuvant or support. In some cases, the pH of the preparation may be adjusted with a pharmaceutically acceptable acid, base or buffer to enhance the stability of the compound or its delivery form of the formulation. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0383] The pharmaceutical composition can be in the form of a solution or powder for inhalation and / or nasal administration. Such compositions can be prepared using suitable dispersants or wetting agents (e.g., Tween 80) and suspending agents according to techniques known in the art. Sterile injection preparations can also be sterile injection solutions or suspensions in nontoxic parenteral acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among the acceptable carriers and solvents that can be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives can be used to prepare injections, such as natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially its polyoxyethylated form. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, or carboxymethyl cellulose or similar dispersants, which are generally used to prepare pharmaceutically acceptable dosage forms, such as emulsions and / or suspensions. For the purposes of formulation, other commonly used surfactants such as Tweens or Span and / or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used.

[0384] The pharmaceutical composition can be orally administered in any oral acceptable dosage form, including but not limited to capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. For oral tablets, commonly used carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are also usually added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When oral aqueous suspensions and / or emulsions, the active ingredient can be suspended or dissolved in an oil phase and mixed with an emulsifier and / or suspending agent. If necessary, certain sweeteners and / or flavoring agents and / or coloring agents can be added.

[0385] Alternatively or additionally, the pharmaceutical composition can be administered by nasal spray or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0386] In some cases, one or more peptides disclosed herein can be conjugated to, for example, a carrier protein. Such a conjugated composition can be monovalent or multivalent. For example, a conjugated composition can include a peptide disclosed herein conjugated to a carrier protein. Alternatively, a conjugated composition can include two or more peptides disclosed herein conjugated to a carrier.

[0387] As used herein, when two entities are "conjugated" to each other, they are linked by direct or indirect covalent or non-covalent interactions. In certain embodiments, the association is covalent. In other embodiments, the association is non-covalent. Non-covalent interactions include hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, and the like. Indirect covalent interactions are when two entities are covalently linked, optionally through a linker group.

[0388] Carrier proteins may include any protein that increases or enhances the immunogenicity of a subject. Exemplary carrier proteins are described in the art (e.g., see Fattom et al., Infect. Immun., 58: 2309-2312, 1990; Devi et al., Proc. Natl. Acad. Sci. USA 88: 7175-7179, 1991; Li et al., Infect. Immun. 57: 3823-3827, 1989; Szu et al., Infect. Immun. 59: 4555-4561, 1991; Szu et al., J. Exp. Med. 166: 1510-1524, 1987; and Szu et al., Infect. Immun. 62: 4440-4444, 1994). The polymer carrier may be a natural or synthetic material comprising one or more primary and / or secondary amino groups, azido groups or carboxyl groups. The carrier may be water soluble.

[0389] Example

[0390] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can develop equivalent means or reactants without having to practice inventive capabilities and without departing from the scope of the invention.

[0391] Example 1: Synthesis of stapled peptide degron chimeras

[0392] A series of stapled peptide degron chimeras were generated, including: (i) stapled peptide-small molecule degron chimeras, (ii) stapled peptide-peptide degron chimeras, (iii) stapled peptide-stapled peptide degron chimeras, and (iv) small molecule-stapled peptide degron chimeras. Methods for generating each of these classes of stapled peptide degron chimeras are described below. Figures 26-29 The diversity of approaches to designing the stapled peptide portion of these chimeras is demonstrated. Exemplary components of the chimeras are shown in Figures 1-7 、15-16、21、23.

[0393] Synthesis of stapled peptide-small molecule degron chimeras

[0394] Hydrocarbon-stapled peptides were synthesized, purified, and quantified using previously reported methods (Bird et al., Methods Enzymol., 446:369-86 (2008); Bird et al., Curr. Protoc. Chem. Biol., 3(3):99-117 (2011) with the following modifications and added details: Peptides were synthesized using established methods (i.e., Fmoc-protected amino acids, HATU coupling agent) until the desired sequence was achieved. The peptides were then stapled using Grubbs catalyst (1st generation) and the N-terminus was deprotected using piperidine. Polyatom linkers such as β-alanine or aminocaproic acid (see also Figure 6 The thalidomide-COOH was then coupled using HCTU. The imide bond is particularly sensitive to nucleophiles; therefore, no piperidine or hydrazine was used after incorporation. The peptide was then cleaved with TFA for 1 hour and purified by LCMS.

[0395] Synthesis of stapled peptide-peptide degron chimeras

[0396] The stapled peptide portion of the stapled peptide-peptide degron chimera is synthesized as described above (i.e., as described for the stapled peptide-small molecule degron). The stapled peptide is then coupled to the fully protected peptide degron. The fully protected degron peptide is synthesized on a resin that can be cleaved by a weak acid, particularly a Sieber amide resin, and the final synthetic step is the reaction of glycolic anhydride with the N-terminus of the degron peptide. After cleavage with 1% TFA, the protected degron peptide is precipitated in ether, dissolved in acetic acid / water, and lyophilized. The fully protected degron peptide is then mixed with a coupling agent and a base and reacted with the resin-bound stapled peptide N-terminus for 2 hours, followed by TFA cleavage and purification to obtain the stapled peptide-peptide degron.

[0397] Synthesis of stapled peptide-stapled peptide degron chimeras

[0398] As described above (i.e., as described for stapled peptide-small molecule degron), the first stapled peptide of the stapled peptide-stapled peptide degron chimera is synthesized. A linker moiety, such as β-alanine or aminocaproic acid, is then introduced into the first stapled peptide. The second stapled peptide of the chimera is synthesized using the same protocol as the first stapled peptide of the chimera. Next, the entire chimera (i.e., two stapled peptides) is stapled using Grubbs catalyst (1st generation), and then the N-terminus is acetylated. The chimera is then cleaved with TFA for 1 hour and purified by LCMS.

[0399] Synthesis of small molecule-stapled peptide degron chimeras

[0400] The stapled peptide portion of the small molecule-stapled peptide degron chimera is synthesized as described above (i.e., as described for the stapled peptide-small molecule degron), and the peptide is then stapled using Grubbs catalyst (1st generation), the N-terminus is deprotected with piperidine, and a polyatom linker, such as β-alanine or aminocaproic acid, is introduced.

[0401] Small molecule-stapled peptide degron chimeras containing JQ1 as a small molecule were generated. Since the carboxyl group of JQ1 (L. Anders et al., Nat. Biotechnol. 32, 92–96 (2014)) can tolerate chemical substitution, we incubated the resin with JQ1 acid (11.3 mg, 0.0281 mmol, 1 eq) and N-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide trifluoroacetate (14.5 mg, 0.0281 mmol, 1 eq) dissolved in DMF (0.28 ml, 0.1 M) at room temperature. DIPEA (14.7 μL, 0.0843 mmol, 3 eq) and HATU (10.7 mg, 0.0281 mmol, 1 eq) were added and then reacted under nitrogen for 20 hours. The small molecule-stapled peptide degron chimera was then cleaved with TFA for 1 hour and purified by LCMS.

[0402] Example 2: Stapled peptide degron chimeras retain target protein binding affinity and enable cellular uptake to access their native targets within cells

[0403] An exemplary fluorescence polarization (FP) binding assay (see Nat Chem Biol. 2018 Jul; 14(7):706–714) was performed to demonstrate that a series of stapled peptide degron chimeras incorporating stapled peptide, linker, and thalidomide moieties could variably retain binding to cereblon as monitored by competitive FP ( Figure 8 ). Using a cellular assay involving expression of GFP-BRD4 (an exemplary target protein) and dBET6 (a small molecule proteolysis targeting chimera (“PROTAC”) that binds to Cereblon and BRD4 to induce BRD4 degradation), we further demonstrated that the stapled peptide-thalidomide chimera can enter cells to compete with dBET6 for Cereblon binding, thereby restoring GFP-BRD4 ( FIG. 9 ). These data indicate that the stapled peptide degron chimera can retain in vitro target protein interactions, can enter cells, and bind to natural protein degraders in cells.

[0404] Example 3: Targeted degradation of BCL-2 family proteins that resist apoptosis by stapled BIM BH3 peptide helix-thalidomide degradation determinant chimera

[0405] In the pro-apoptotic BIM BH3 domain and the incorporated degron (e.g. Lys-degron, Figures 2-4 ) after modeling the stapled peptide helix-degradant chimeric peptide ( Figure 1 ) was applied to A375P melanoma cells expressing anti-apoptotic BCL-2 family proteins such as MCL-1, which promotes cancer cell survival and chemoresistance. Treatment with 10 μM compound and monitoring of cellular MCL-1 levels by Western blotting of lysates revealed a time-dependent decrease in MCL-1 protein as early as the 2-hour time point ( Figure 10 Importantly, no decrease in actin levels was observed in the western blot control. These data suggest that targeting MCL-1 with the BIM BH3 helix derived from the degron moiety is able to reduce the levels of MCL-1 protein in cancer cells within hours of treatment.

[0406] Example 4: Targeted degradation of MDM2 oncoprotein by stapled p53 peptide helix-thalidomide degradation determinant chimera

[0407] The stapled peptide degron chimeras modeled after coupling the transactivation domain helix of p53 (ATSP-7041) to the thalidomide degron were applied to cultured cancer cells (SJSA-1, SJSA-X) and MDM2 protein levels were monitored by western blot and compared to cells treated with ATSP-7041 alone. The experiment was repeated and cell viability was measured by CellTiter Glo assay. The data showed that MDM2 levels were reduced in cells treated with the thalidomide degron coupled to ATSP-7041 compared to cells treated with ATSP-7041 alone ( Figure 11 In addition, each stapled peptide degron chimera impaired the viability of cancer cells in a dose-responsive manner ( Figure 12 The composition of the linker affects the presence of biological activity ( Figure 12 , left) or does not exist ( Figure 12 ,right).

[0408] Example 5: Cop1-mediated protein degradation

[0409] Primary degradation determinants are defined as peptide motifs containing specific sequence patterns that can be recognized by homologous ubiquitin E3 ligases. Primary degradation determinants are usually short and linear motifs within structurally disordered protein regions (Guharoy, M. et al., Nat Commun., 7: 10239, doi: 10.1038 / ncomms10239 (2016)). The primary degradation determinant sequence of the protein Trib1 recognized by the E3 ligase Cop1 is the amino acid sequence DQIVPEY (SEQ ID NO: 25). In the context of protein Trib1, the sequence DQIVPEY (SEQ ID NO: 25) confers Trib1 bound to Cop1, so that Trib1 acts as a substrate adapter, and proteins bound to Trib1 are targeted for degradation (Uljon, S. et al., Structure, doi: 10.1016 / j.str.2016.03.002 (2016)). The reported binding affinity of the sequence DQIVPEY (SEQ ID NO: 25) to Copl is 250±40 nM.

[0410] We have discovered that the sequence DQIVPEY (SEQ ID NO: 25) and its derivatives that retain binding affinity to Cop1 can be used as transplantable degron sequences to cause Cop1-mediated degradation of cellular proteins, resulting in therapeutic benefits as described below.

[0411] (1) Direct genetic modification: replacement of native protein residues in disordered regions with derivatives of the sequence DQIVPEY (SEQ ID NO: 25) results in degradation of the chimeric protein by Cop1. For example, replacement of the C-terminal sequence GFDVPD (SEQ ID NO: 26) of the p60 isoform of protein HDM2 with the Trib1-derived sequence DQIVPD (SEQ ID NO: 30) results in Cop1-mediated degradation of the mutant protein when exogenously expressed in human embryonic kidney 293T cells expressing Cop1. Figure 13 ). Co-immunoprecipitation studies in 293T cells showed that degradation occurred after incorporation of specific degron sequences ( Figure 13 ) and direct binding between Cop1 and the corresponding Myc-tagged MDM2 p60 mutant constructs, such as DQIVPD (SEQ ID NO: 30) ( Figure 14 ).

[0412] (2) Peptide ligand targeting: Conjugation of derivatives of the sequence DQIVPEY (SEQ ID NO: 25) to protein-targeted stapled peptides can target the binding partners of the stapled peptides for Cop1-mediated degradation. The design of these conjugates is similar to Figure 1The same as outlined in , except that the small molecule thalidomide is replaced by a derivative of the peptide sequence DQIVPEY (SEQ ID NO: 25), and conjugation is achieved via a peptide linker ( Figure 15 ).

[0413] Example 6: Targeted degradation of MDM2 oncoprotein by stapled p53 peptide helix-Trib degron chimera

[0414] The stapled peptide degron chimera modeled after conjugation of the transactivation domain helix of p53 (ATSP-7041) with a peptide degron modeled after the Trib sequence that binds Cop1 was applied to cultured cancer cells (SJSA-1, SJSA-X) and MDM2 protein levels were monitored by western blot and compared to cells treated with ATSP-7041 alone. The experiment was repeated and cell viability was measured by Cell Titer Glo assay. The data showed that MDM2 levels were lower in cells treated with Trib degron coupled to ATSP-7041 compared to cells treated with ATSP-7041 alone ( Figure 17 In addition, each stapled peptide degron chimera impaired the viability of cancer cells in a dose-responsive manner ( Figure 18 ).

[0415] Example 7: Targeted degradation of MDM2 oncoprotein by stapled p53 peptide helix-VHL degron chimera

[0416] The stapled peptide degron chimera modeled after coupling the transactivation domain helix of p53 (ATSP-7041) to a small molecule degron that binds VHL was applied to cultured cancer cells (SJSA-1, SJSA-X), and MDM2 protein levels were monitored by Western blot and compared to cells treated with ATSP-7041 alone. The experiment was repeated and cell viability was measured by Cell Titer Glo assay. The data showed that MDM2 levels were lower in cells treated with VHL degron coupled to ATSP-7041 compared to cells treated with ATSP-7041 alone ( Figure 19 In addition, each stapled peptide degron chimera impaired the viability of cancer cells in a dose-responsive manner ( Figure 20 ).

[0417] Example 8: Targeted degradation of MCL-1 oncoprotein by selectively stapled BH3 peptide helix-stapled p53 peptide degron chimera

[0418] Stapled peptide degron chimera modeled after selective targeting of the MCL-1 BH3 helix of MCL-1 ( Figure 21) was conjugated to a stapled peptide degron modeled after the transactivation domain helices of p53, with the goal of recruiting MDM2 to MCL-1 to induce non-canonical MDM2-mediated ubiquitination and degradation of MCL-1. Using an in vitro ubiquitination assay (described below), we observed that addition of the stapled peptide degron chimera to recombinant MCL-1, recombinant MDM2, E1 enzyme, E2 enzyme (UBE2D3), and ATP induced ubiquitination of MCL-1 ( Figure 22 ). These results indicate that only in the presence of the stapled peptide degron chimera is MDM2 successfully recruited to MCL-1, leading to the transfer of ubiquitin to MCL-1 via the ubiquitination mechanism.

[0419] Example 9: Targeted degradation of BRD4 oncoprotein by selective small molecule BRD4 inhibitor-stapled p53 peptide degron chimera

[0420] Small molecules that effectively target BRD4 are coupled to stapled peptide degrons modeled after the transactivation domain helices of p53 ( Figure 23 ), with the goal of recruiting MDM2 to BRD4 to induce non-canonical MDM2-mediated BRD4 ubiquitination and degradation. In a similar manner as described above (see Example 8) to evaluate the induced ubiquitination of recombinant MCL-1 protein, we used an in vitro assay to test whether our stapled peptide degradation determinant chimeras can recruit MDM2 to ubiquitinate recombinant BRD4 protein (e.g., portion: amino acids 342-460 or amino acids 49-170). The addition of stapled peptide degradation determinant chimeras induced an upshift of each recombinant BRD4 protein, indicating that MDM2 transfers ubiquitin to the target BRD4 protein ( Figure 24 To assess the effect on native BRD4 levels in cancer cells, we treated the U2OS cancer cell line with the stapled peptide degron chimera at a dose of 10 μM and observed a time-responsive degradation of native BRD4, as reflected by a gradual decrease in BRD4 protein levels over time (e.g., 0 to 6 hours) ( Figure 25 ). These data indicate that our stapled peptide degron chimeras can effectively repurpose MDM2 for BRD4 ubiquitination in vitro and induce native BRD4 degradation in cells.

[0421] Materials and methods

[0422] Monitoring ubiquitination of recombinant protein targets induced by stapled peptide degron chimeras

[0423] To monitor the ubiquitination of protein targets in vitro, a commercial Mdm2 / HDM2 ubiquitin ligase kit (K-200B) was used. Briefly, chimeras (10 μM), recombinant full-length MDM2 (with a GST tag, 1 μM), E1 enzyme (UBE1, 50 nM), E2 enzyme (UBE2D3, 1 μM), ubiquitin (100 μM), ATP (1 mM), and recombinant target protein (100 nM) were combined in a reaction buffer in a 1.5 mL microtube. The mixture was incubated at 37 ° C for 6 hours. Subsequently, 20 μL of the reaction mixture was tested using standard Western blotting techniques to visualize the upward band shift due to ubiquitination using antibodies against the target protein.

[0424] Monitoring the degradation of native proteins in cells induced by stapled peptide degron chimeras

[0425] To measure intracellular protein degradation, cancer cells (e.g., SJSA-1, SJSA-X, U2OS) were cultured in a humidity-controlled CO atmosphere at 37°C. 2 The cells were passaged in DMEM (Life Technologies, Grand Island, NY) medium (CM) in a balanced incubator containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Pen Strep). The day before treatment, the cells were passaged and seeded in six-well plates at a density of 100,000 cells / mL. After 24 hours, the cells were treated with stapled peptide degradation determinant chimeras (e.g., 10 μM) for 0, 2, 4, and 6 hours, then harvested and lysed. The cell lysates were then tested using standard Western blotting techniques, protein levels were assessed using antibodies against the target protein, and loading controls were performed using actin antibodies.

[0426] Other Implementations

[0427] Although the invention has been described in conjunction with the detailed description of the invention, 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 appended claims.

Claims

1. A chimera, comprising: a first part that binds to a second part; wherein the first part binds to a first protein to be targeted for degradation; and the second part binds to a second protein, wherein the second protein is a proteolytic agent.

2. A method of treating a disease or disorder driven by a pathological peptide or protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the chimera of claim 1.

3. A chimeric polypeptide, comprising a stapled peptide and a peptide that binds to a WD40 repeat protein, the WD40 repeat protein being a substrate adaptor of an E3 ubiquitin ligase, wherein the peptide comprises a modified form of a natural binding sequence or a natural binding consensus sequence of amino acids that bind to the WD40 repeat protein, and wherein the modified form comprises at least one amino acid substitution, at least one amino acid deletion, at least one amino acid insertion, or any combination thereof within the natural binding consensus sequence.

4. A modified protein of a first protein comprising a structurally disordered region, wherein the modified protein differs from the first protein in that the structurally disordered region comprises a peptide that binds to a WD40 repeat protein, the WD40 repeat protein being a substrate adaptor of an E3 ubiquitin ligase, and the peptide comprises a modified form of a natural binding sequence or a natural binding consensus sequence, and wherein the modified form comprises at least one amino acid substitution, at least one amino acid deletion, at least one amino acid insertion, or any combination thereof within the natural binding consensus sequence.

5. A method of treating a disease or disorder driven by a pathological peptide or protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the chimeric fusion polypeptide of claim 3.

6. A peptide-small molecule fusion comprising a protein-targeting stapled peptide and a thalidomide degrader moiety.

7. A method of treating a disease or disorder driven by a pathological peptide or protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the peptide-small molecule fusion of claim 6.

8. A peptide-small molecule fusion comprising a protein-targeting stapled peptide and a Von Hippel-Lindau (VHL) degrader moiety.

9. A method of treating a disease or disorder driven by a pathological peptide or protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the peptide-small molecule fusion of claim 8.

10. A peptide that binds to the constitutive photomorphogenesis 1 (Cop1) protein, wherein the peptide comprises a modified form of the amino acid sequence DQIVPEY (SEQ ID NO:25), and wherein the modified form comprises at least one amino acid substitution, at least one amino acid deletion, at least one amino acid insertion, or any combination thereof in SEQ ID NO:25, provided that if the modified form consists of a single amino acid substitution, the amino acid substitution is not A or R at any position from 1 to 7 in SEQ ID NO:25, or V at position 4 in SEQ ID NO:

25.

11. A chimeric fusion polypeptide comprising a protein-targeting stapled peptide and the peptide of claim 10.

12. A method of treating a disease or disorder driven by a pathological peptide or protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the chimeric fusion polypeptide of claim 11.

Images