Stabilized peptide-mediated targeted protein degradation

Abstract

To provide stabilized peptide-mediated targeted protein degradation.SOLUTION: This disclosure relates to small molecule chimeric compounds and stabilized peptide, termed stapled peptide-degron chimeras, that act as a combination of a protein targeting moiety (by the stapled peptide or molecular portion) and a protein degradation-inducing moiety (by another stapled peptide or molecular portion). The chimeras include a stapled peptide fused to either (i) a small molecule degron (e.g., a cereblon- or VHL-binding small molecule as the degron), (ii) a polypeptide sequence degron, (iii) a stapled peptide as the degron, or (iv) a small molecule as the protein targeting compound (with the stapled peptide serving as the degron). This disclosure also relates to methods of use thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 599,608, filed December 15, 2017, which is incorporated herein by reference in its entirety.

[0002] Technical Field The present disclosure relates to stabilized peptide and small molecule chimeric compounds, termed stapled peptide degron chimeras, which serve as a combination of a protein-targeting moiety (via a stapled peptide or molecule moiety) and a proteolysis-inducing moiety (via another stapled peptide or molecule moiety). The chimeras comprise a stapled peptide fused to either (i) a small molecule degron (e.g., a cereblon or VHL-binding small molecule as the degron), (ii) a polypeptide sequence degron, (iii) a stapled peptide as the degron, or (iv) a small molecule (including a stapled peptide that serves as a degron) as the protein-targeting compound, and the present disclosure relates to methods of their use. [Background technology]

[0003] background Degrons are parts of proteins that play a key role in their degradation. Degrons are usually short amino acid sequences that 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 the National Academy of Sciences, 93 (22):12142-12149 (1996)). In fact, some Proteins in the genome possess multiple degrons. Degrons have been identified in both prokaryotic and eukaryotic cells. Although several types of degrons exist, despite the fact that there is a high degree of variability within these groups, degrons are all similar in terms of their involvement in regulating the rate of protein degradation. Degradation can involve ubiquitin or can be ubiquitin-independent. Degrons that are ubiquitin-dependent contain specific sequences recognized by the cognate ubiquitin E3 ligase.

[0004] The ubiquitin proteasome pathway (UPP) is a major pathway that regulates key regulatory proteins and degrades misfolded or abnormal proteins. 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 Cullin4 (CUL4A), which then functions as a substrate receptor, ubiquitinating proteins recognized by CRBN and making them available for degradation by the proteasome. CRBN has also been identified to bind immunomodulatory drugs (IMiDs), such as thalidomide. Such binding is associated with the teratogenic mechanism and cytotoxicity of IMiDs, such as lenalidomide, used to treat multiple myeloma. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Cho et al., Genes & Development, 24 (5): 438-442 (2010) [Non-patent document 2] Fortmann et al., Journal of Molecular Biology, 427 (17): 2748-2756 (2015) [Non-patent document 3] Dohmen et al., Science, 263(5151):1273-1276 (1994) [Non-patent document 4] Varshavsky, Proceedings of the National Academy of Sciences, 93 (22):12142-12149 (1996) Summary of the Invention [Means for solving the problem]

[0006] Abstract The present disclosure relates to the synthesis and characterization of bifunctional stabilized peptide-small molecule (e.g., thalidomide degron) conjugates, stabilized peptide-peptide (e.g., primary degron sequence) conjugates, stabilized peptide-stabilized peptide (e.g., primary degron sequence) conjugates, and small molecule-stabilized peptide (e.g., primary degron sequence) conjugates (these conjugates are also referred to as "chimeras") that can be used to target any protein of interest. For example, these conjugates are useful for targeting proteins involved in or causing disease. The targeted proteins may be of viral, bacterial, animal, or human origin. In certain cases, the conjugates are useful for targeting disease-causing or disease-associated proteins. Such stabilized peptide conjugates are useful for treating diseases caused by such pathological proteins. The ability of stapled peptides to target large and typically non-druggable protein interaction surfaces, coupled with degron functionality, can expand the utility of degron technology beyond the scope of small molecules and enhance the biological activity of stapled peptides.

[0007] In a first aspect, the disclosure features peptide-small molecule fusions that include a protein-targeting staple peptide and a small molecule degron moiety (e.g., a thalidomide moiety or a von Hippel-Lindau (VHL) moiety).

[0008] In some embodiments, the small molecule degron (e.g., a thalidomide or VHL moiety) is conjugated to the N-terminus of the protein-targeting stapled peptide. In some cases, the small molecule degron (e.g., a thalidomide or VHL moiety) is conjugated to the C-terminus of the protein-targeting stapled peptide. In certain cases, the small molecule degron (e.g., a thalidomide or VHL moiety) is contained within an unnatural amino acid inserted into the peptide sequence between the N-terminus and C-terminus of the protein-targeting stapled peptide. In some cases, the stapled peptide binds to a disease-causing protein. In some cases, the stapled peptide binds to an intracellular protein. In some cases, the stapled peptide binds to an extracellular protein. In some cases, the stapled peptide binds to a cell surface protein (e.g., a receptor). In some cases, the stapled peptides bind to killer proteins (e.g., BAX, BAK) or proteins that damage cells or cause neurodegeneration (e.g., IgG, beta-amyloid, tau, alpha-synuclein, TDP-43, hemoglobin (sickle cell), superoxide dismutase, Notch3, FUS, GFAP). In some cases, the stapled peptides bind to BCL2, BCLX L , MCL-1, BFL-1, BCL-w, BCL-B, EZH2, HDM2 / HDMX, KRAS / NRAS / HRAS, MYC, b-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI, MLL fusions, 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. In some cases, the stapled peptide binds to a bacterial protein. In some cases, the stapled peptide binds to a viral protein. In certain cases, the thalidomide moiety comprises the structure shown below. [ka] In some cases, the thalidomide moiety, when conjugated at the N-terminus of the stabilizing peptide, comprises the structure shown below: [ka] In some cases, the thalidomide moiety, when conjugated at the C-terminus of the stabilizing peptide, comprises the structure shown below: [ka]

[0009] In some instances, the VHL moiety comprises the structure: [ka]

[0010] In some instances, the VHL moiety comprises the structure: [ka]

[0011] In a second aspect, the disclosure features a method of treating a disease or disorder caused by a pathological peptide or protein in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a peptide-small molecule fusion described herein.

[0012] In a third aspect, the disclosure features a peptide degron that binds to a WD40 repeat protein, where the WD40 repeat protein is a substrate adaptor for an E3 ubiquitin ligase. The peptide comprises a modified version of a native binding sequence or native binding consensus sequence for an amino acid sequence that binds to a WD40 repeat protein. The modified version 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 for an amino acid sequence that binds to a WD40 repeat protein. Exemplary peptides comprising a modified version of a native binding sequence or native binding consensus sequence for an amino acid sequence that binds to a WD40 repeat protein are provided as SEQ ID NOS: 26-30 and 106-118. In one illustrative example, the disclosure provides a peptide that binds to constitutive photomorphogenesis 1 (Cop1) protein. The peptide comprises a modified version 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. When the modified form consists of a single amino acid substitution, the amino acid substitution is not made to A or R at any one of positions 1 to 7 of SEQ ID NO: 25, nor to V at position 4 of SEQ ID NO: 25.

[0013] In some embodiments, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for at least one amino acid substitution. In some cases, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for at least one amino acid deletion. In some cases, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for at least one amino acid substitution and at least one amino acid deletion. In certain cases, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for one to six amino acid substitutions. In some cases, positions 4 (V) and / or 5 (P) of SEQ ID NO: 25 are not substituted. 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, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for one 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 1 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 example, the peptide has the amino acid sequence set forth in SEQ ID NO: 30.

[0014] In certain embodiments, the peptide is 4 to 10 amino acids in length.

[0015] 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.

[0016] In a fourth aspect, the present disclosure relates to a chimeric fusion polypeptide comprising a protein-targeting staple peptide and a Trib1 peptide degron or a variant thereof.

[0017] 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 protein that causes or is associated with a disease. In some embodiments, the stapled peptide binds to a killer protein (e.g., BAX, BAK) or a protein that damages cells or causes neurodegeneration (e.g., IgG, beta-amyloid, tau, alpha-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 fusions, MLL fusions, 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. In some embodiments, the stapled peptide binds to a bacterial protein. In some embodiments, the stapled peptide binds to a viral protein.

[0018] In a fifth aspect, the disclosure features a chimeric polypeptide including a staple peptide and a peptide that binds to a WD40 repeat protein, where the WD40 repeat protein is a substrate adaptor for an E3 ubiquitin ligase. The peptide comprises a native binding sequence or a modified version of a native binding consensus sequence for the amino acid sequence that binds to the WD40 repeat protein. The modified version 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 native binding consensus sequence for the amino acid sequence that binds to the WD40 repeat protein.

[0019] In some embodiments, the WD40 repeat protein that is 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.

[0020] In some embodiments, the naturally occurring binding sequence or naturally occurring binding consensus sequence is a sequence selected from the group consisting of SEQ ID NOs: 25, 31-46, and 65-105. In some cases, the naturally occurring binding consensus sequence is SEQ ID NO: 25. In other cases, the naturally occurring binding consensus sequence is SEQ ID NO: 46.

[0021] In some embodiments, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for at least one amino acid substitution. In some cases, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for at least one amino acid deletion. In some cases, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for at least one amino acid substitution and at least one amino acid deletion. In certain cases, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for one to six amino acid substitutions. In some cases, positions 4 (V) and / or 5 (P) of SEQ ID NO: 25 are not substituted. 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, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for one 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 1 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 example, the peptide has the amino acid sequence set forth in SEQ ID NO: 30.

[0022] 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.

[0023] 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.

[0024] 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 protein that causes or is associated with a disease. In some embodiments, the stapled peptide binds to a killer protein (e.g., BAX, BAK) or a protein that damages cells or causes neurodegeneration (e.g., IgG, beta-amyloid, tau, alpha-synuclein, TDP-43, HbS (hemoglobin sickle), 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, MLL fusion, 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 (e.g., beta-amyloid) that cause neurodegeneration.

[0025] In a sixth aspect, the disclosure features a variant of a first protein that includes a structurally irregular region. The variant protein differs from the first protein in that the structurally irregular region includes a peptide that binds to a WD40 repeat protein, a substrate adaptor for E3 ubiquitin ligase. The peptide includes a variant of a native binding consensus sequence, where the variant includes at least one amino acid substitution, deletion, insertion, or any combination thereof within the native binding consensus sequence.

[0026] In certain embodiments, WD40 repeat proteins that are substrate adaptors for E3 ubiquitin 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, CDC20, and FZR1. In some cases, the natural binding consensus sequence is a sequence selected from the group consisting of SEQ ID NOs: 25, 31-46, and 65-105. In some cases, the natural binding consensus sequence is SEQ ID NO: 25. In some cases, the natural binding consensus sequence is SEQ ID NO: 46.

[0027] In some embodiments, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for at least one amino acid substitution. In some cases, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for at least one amino acid deletion. In some cases, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for at least one amino acid substitution and at least one amino acid deletion. In certain cases, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for one to six amino acid substitutions. In some cases, positions 4 (V) and / or 5 (P) of SEQ ID NO: 25 are not substituted. 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, a peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for one 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 1 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 example, the peptide has the amino acid sequence set forth in SEQ ID NO: 30.

[0028] In certain embodiments, the peptide is 4 to 10 amino acids in length.

[0029] 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.

[0030] In a seventh aspect, the disclosure features a method of treating a disease or disorder caused by a pathological peptide or protein in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a chimeric fusion polypeptide described herein.

[0031] In an eighth aspect, the disclosure features a peptide degron selected from the group consisting of SEQ ID NOs: 106-118.

[0032] In some embodiments, the peptides are linked to a stabilizing peptide.

[0033] In a ninth aspect, the present disclosure provides a stabilized peptide-peptide degron chimera selected from the group consisting of SEQ ID NOs: 119 to 126.

[0034] In certain embodiments of all of the above aspects, the stabilized peptide-degron chimera is a chimera that binds to BCL2, BCLX L , BCL W , MCL-1, BFL-1, BAX, MDM2, or MDMX.

[0035] In a tenth aspect, the present disclosure provides a stabilizing peptide-stabilizing peptide combination comprising two stabilizing peptides, a first stabilizing peptide and a second stabilizing peptide, wherein the first stabilizing peptide binds to a first protein, which is a target protein to be degraded, and the second stabilizing peptide binds to a second protein, which is a degrader protein. In certain embodiments, a first stabilizing peptide binds to a disease-associated protein targeted for degradation, and a second stabilizing peptide binds to a degradation-inducing protein, such as an E3 ligase (e.g., MDM2).

[0036] 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 protein that causes or is associated with a disease. In some embodiments, the first protein is a killer protein (e.g., BAX, BAK) or a protein that damages 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, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion, MLL fusion, 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., beta-amyloid) that causes neurodegeneration.

[0037] In certain embodiments, the first stabilizing peptide has the amino acid sequence set forth 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 set forth in SEQ ID NO: 6, or a variant thereof. In certain embodiments, the second stabilizing peptide has the amino acid sequence set forth in SEQ ID NO: 18, or a variant thereof.

[0038] In certain embodiments, the second protein is a degradation-inducing protein, such as an E3 ubiquitin ligase, or a substrate adaptor for 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 that forms a complex with an E3 ligase, such as MDMX, which binds 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-Bmil, 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 an RBR E3 ubiquitin ligase (e.g., Parkin, Parc, RNF144(A / B), HOIP, HHARI). For non-limiting examples of E3 ubiquitin ligases, see, e.g., Morreale and Walden, Cell 165, 2016 DOI http: / dx.doi.org / 10.1016 / j.cell.2016.03.003.

[0039] In certain embodiments, the second stabilizing peptide portion of the chimera has the amino acid sequence set forth in SEQ ID NO: 134 or a variant thereof. In certain embodiments, the second stabilizing peptide has the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof. In certain embodiments, the second stabilizing peptide has the amino acid sequence set forth in SEQ ID NO: 18 or a variant thereof.

[0040] In an eleventh aspect, the present disclosure provides a small molecule-stabilizing peptide degron chimera comprising a small molecule and a stabilizing peptide, wherein the small molecule binds to a first protein, which is a target protein to be degraded, and the stabilizing peptide binds to a second protein, which is a degradation-inducing protein. In certain embodiments, the stabilizing peptide binds to and recruits the degradation-inducing protein, such as an E3 ligase (e.g., MDM2), or a degradation-inducing protein complex, such as the MDM2 / MDMX complex.

[0041] 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 protein that causes or is associated with a disease. In some embodiments, the first protein is a killer protein (e.g., BAX, BAK) or a protein that damages 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, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion, MLL fusion, 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., beta-amyloid).

[0042] In certain embodiments, the second protein is a degradation-inducing protein, such as an E3 ubiquitin ligase, or a substrate adaptor for 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 that forms a complex with an E3 ligase, such as MDMX, which binds 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-Bmil, 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 an RBR E3 ubiquitin ligase (e.g., Parkin, Parc, RNF144(A / B), HOIP, HHARI). For non-limiting examples of E3 ubiquitin ligases, see, e.g., Morreale and Walden, Cell 165, 2016 DOI http: / dx.doi.org / 10.1016 / j.cell.2016.03.003.

[0043] In a twelfth aspect, the present disclosure provides a chimera comprising a first moiety bound to a second moiety, wherein the first moiety binds to a first protein targeted for degradation and the second moiety binds to a second protein, wherein the second protein is a protein degradation inducer.

[0044] In certain embodiments, the first and second moieties are covalently bonded to one another. In certain embodiments, the first and second moieties are bonded to one another via a linker.

[0045] 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 protein that causes or is associated with a disease. In some embodiments, the first protein is a killer protein (e.g., BAX, BAK) or a protein that damages 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, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion, MLL fusion, 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., beta-amyloid).

[0046] In certain cases, the first portion comprises a first staple peptide that binds to a first protein targeted for degradation. In certain cases, the first portion comprises a small molecule that binds to the first protein targeted for degradation. In certain cases, the second portion comprises a second staple peptide that binds to a second protein, e.g., a proteolysis drug. In certain cases, the second portion comprises a small molecule that binds to a second protein, e.g., a proteolysis drug. In certain cases, the second portion comprises a peptide degron that binds to a proteolysis drug. In certain cases, the first portion comprises a first staple peptide that binds to a first protein, and the second portion comprises a second staple peptide that binds to a second protein. In certain cases, the first portion comprises a first staple peptide that binds to a first protein, and the second portion comprises a small molecule that binds to a second protein. In certain cases, the first portion comprises a first staple peptide that binds to a first protein, and the second portion comprises a peptide degron that binds to a proteolysis drug. In certain cases, the first portion comprises a small molecule that binds to a first protein, and the second portion comprises a stapled peptide that binds to a second protein.

[0047] In certain cases where the first portion is a stapled peptide, the stapled peptide does not include a Bcl-2 homology 3 (BH3) domain polypeptide. In certain cases where the first portion is a stapled peptide, the stapled peptide also does not include (a) a Bcl-2 homology 3 domain from MCL-1, (b) an MCL-1 stabilizing alpha helix of the BCL2 domain, or (c) an MCL-1 SAHBD.

[0048] In certain cases where the first moiety is a stapled peptide, the second moiety is attached to the N-terminus of the first moiety. In certain cases where the first moiety is a stapled peptide, the second moiety is attached to the C-terminus of the first moiety. In certain cases where the first moiety is a first stapled peptide, the second moiety is attached to an internal amino acid position of the first moiety.

[0049] In certain cases where the second portion is a stapled peptide, the first portion is attached to the N-terminus of the second portion. In certain cases where the second portion is a stapled peptide, the first portion is attached to the C-terminus of the second portion. In certain cases where the second portion is a stapled peptide, the first portion is attached to an internal amino acid position of the second portion.

[0050] In some embodiments, the protein degradation-inducing agent degrades a first protein that is targeted for degradation.

[0051] In a thirteenth aspect, the present disclosure provides a method of treating a disease or disorder caused by a pathological peptide or protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a chimera described herein.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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 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 are not intended to be limiting.

[0053] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0054] [Figure 1]Figure 1 provides peptide sequences and degron types / locations for a representative set of stapled peptide sequences. The amino acid sequences in the first column are assigned SEQ ID NOS: 1-24. # = N-terminal Ac or degron Ahx, as indicated; % = C-terminal Lys(ivdde) or Lys(degron), as indicated; X = S-pentenylalanine; 8 = R-octenylalanine; B = norleucine; * (within amino acid sequence) = cyclobutylalanine (ivdde = 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl). [Figure 2] FIG. 2 shows the carboxydegron thalidomide moiety coupled to a resin-bound primary amine to yield the stapled peptide degron shown in FIG. [Figure 3] FIG. 3 shows a C-terminal degron-containing moiety that is a side chain conjugated lysine linkage to a peptide. [Figure 4] FIG. 4 shows an N-terminal degron-containing moiety that is an aminohexanoic acid linkage to a peptide. [Figure 5] FIG. 5 shows the structure of diaminobutanoic acid ("DAB") used for coupling to acids or amines, as well as the chemical structures for the THAL and VHL ligands. [Figure 6] FIG. 6 shows the structures of various linkers (Gly, βAla, and linkers 1-8). [Figure 7]Figure 7 shows a series of stapled peptide degron chimeras whereby diaminobutanoic acid is incorporated into the stapled peptide and linked to the small molecule degron, and linkers of various compositions and lengths are introduced to separate the stapled peptide from the small molecule or peptide degron (THAL, TRIB, or VHL). The stapled peptide sequences illustrated in Figure 7 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), where % is indicated in the figure, 8 is (R)-2-(7-octenyl)alanine, and X is (S)-2-(4-pentenyl)alanine. [Figure 8] Figure 8 shows the ability of a stapled peptide degron chimera composed of a stapled peptide linked to a thalidomide degron to retain binding to recombinant cereblon, as monitored by a competitive fluorescence polarization assay. Lenalidomide served as a positive control for the 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 indicated in the figure. "N-terminal Ac" = N-terminal acetylation; Lys(ivDde) ​​= N-α-Fmoc-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine; "Lys-degron" = structure illustrated in Figure 3; "N-terminal degron Ahx" = structure illustrated in Figure 4. [Figure 9-1]Figures 9A-9I show that the stapled peptide degron chimeras can enter cells, compete with dBET6 to interact with cereblon, and inhibit induced GFP-BRD4 degradation. Sequences: #RRFFGIXLTNXLKTEEGN% (SEQ ID NO: 3); #FSSNRXKILXRTQILNQEWKQRRIQPV% (SEQ ID NO: 2); #NLWAAQRYGRELRXBSDXFVDSFKK% (SEQ ID NO: 10); #LSQEQLEHRERSLXTLRXIQRBLF% (SEQ ID NO: 5); and #NLWAAQRYGRELRXBDDXFVDSFKK% (SEQ ID NO: 9) (where X is (S)-2-(4-pentenyl)alanine, and # and % are as indicated in the figure). "N-terminal Ac" = N-terminal acetylation; Lys(ivDde) ​​= N-α-Fmoc-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine; "Lys-degron" = structure illustrated in Figure 3; "N-terminal degron Ahx" = structure illustrated in Figure 4. [Figure 9-2] Figures 9A-9I show that the stapled peptide degron chimeras can enter cells, compete with dBET6 to interact with cereblon, and inhibit induced GFP-BRD4 degradation. Sequences: #RRFFGIXLTNXLKTEEGN% (SEQ ID NO: 3); #FSSNRXKILXRTQILNQEWKQRRIQPV% (SEQ ID NO: 2); #NLWAAQRYGRELRXBSDXFVDSFKK% (SEQ ID NO: 10); #LSQEQLEHRERSLXTLRXIQRBLF% (SEQ ID NO: 5); and #NLWAAQRYGRELRXBDDXFVDSFKK% (SEQ ID NO: 9) (where X is (S)-2-(4-pentenyl)alanine, and # and % are as indicated in the figure). "N-terminal Ac" = N-terminal acetylation; Lys(ivDde) ​​= N-α-Fmoc-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine; "Lys-degron" = structure illustrated in Figure 3; "N-terminal degron Ahx" = structure illustrated in Figure 4. [Figure 10]Figure 10 shows that the BIM C-terminal degron induces degradation of MCL-1 when added to the A375P melanoma cell line at a concentration of 10 μM. The BIM C-terminal degron is #IWIAQELRXIGDXFNAYYARR% (SEQ ID NO: 134; # = N-terminal Ac; % = Lys-degron (see Figure 3 for the structure of the Lys-degron)). [Figure 11] Figure 11 shows that SJSA-1 cells treated with a panel of stapled peptide degron (1 μM) chimeras composed of MDM2 / MDMX-targeting stapled peptide ("ATSP") and thalidomide degron moieties demonstrate lower MDM2 levels in cancer cells compared to cells treated with ATSP-7041 alone, as assessed by anti-MDM2 Western 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 as depicted in Figure 6. [Figure 12] Figure 12 shows that treatment of SJSA-1 cells with a panel of stapled peptide degron chimeras composed of an MDM2 / MDMX-targeting stapled peptide ("ATSP"), different linkers, and a thalidomide degron results in a variably reduced cell viability of the cancer cells (left), with "5-L5" (LTF8EYWAQLX%AA (SEQ ID NO: 130) (where % is DAB / Linker 5 / THAL) exhibiting the most potent cytotoxic activity. Certain compositions with different linkers show no cytotoxicity (right). Linkers 1-5 are as illustrated in Figure 5. [Figure 13]Figure 13 shows how genetic modification of the MDM2 p60 isoform with a Trib1-derived sequence results in Cop1-mediated degradation using expression of a Myc-tagged MDM2 p60 chimeric construct in 293T cells. The native peptide sequence GFDVPD (SEQ ID NO: 26) in MDM2 is replaced by 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). Replacing the native sequence with the mutant sequence DQIVPD (SEQ ID NO: 30) results in destruction of the p60 chimeric protein by the Cop1 protein. Bottom panel: Cop1 loading control. [Figure 14] Figure 14 shows binding of Myc-tagged MDM2p60 mutant constructs to Cop1 as assessed by co-immunoprecipitation from 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). [Figure 15] Figure 15 provides SAH+Trib peptide sequences generated to assess the activity of targeted Trib-degron-mediated proteolysis. Stapled peptide chimeric sequences are assigned SEQ ID NOS: 119-126. [Figure 16] FIG. 16 shows the structure of a peptide degron modeled on the TRIB sequence and appropriate moieties incorporated therein for coupling to amines or acids. [Figure 17] Figure 17 shows that treatment of SJSA-1 or SJSA-X cells with a panel of stapled peptide degron chimeras (20 μM) composed of MDM2 / MDMX-targeting stapled peptides (e.g., ATSP-7041-like stapled p53 peptides) and a TRIB degron moiety resulted in a clear and variably reduced MDM2 levels in cancer cells compared to those treated with ATSP-7041 alone (1 μM), as assessed by anti-MDM2 Western analysis. Actin represents a loading control. Sequences: LTF8EYWAQ#XSAA (SEQ ID NO: 6) and LTF8%YWAQLXSAA (SEQ ID NO: 129). [Figure 18] Figure 18 shows that treatment of SJSA-1 or SJSA-X cells with a panel of stapled peptide degron chimeras composed of a stapled peptide targeting MDM2 / MDMX (e.g., an ATSP-7041-like stapled p53 peptide) and a TRIB degron variably reduces cell viability of the cancer cells. Sequence: LTF8%YWAQLXSAA (SEQ ID NO: 129). [Figure 19] Figure 19 shows that treatment of SJSA-1 or SJSA-X cells with a panel of stapled peptide degron chimeras (1 μM) composed of MDM2 / MDMX-targeting stapled peptides (e.g., ATSP-7041-like stapled p53 peptides) and VHL degron moieties resulted in a clear and variably reduced MDM2 levels in cancer cells compared to those treated with ATSP-7041 alone, as assessed by anti-MDM2 Western analysis. Actin represents a loading control. Sequences: LTF8EYWAQ#XSAA (SEQ ID NO: 6) and LTF8EYWAQLX%AA (SEQ ID NO: 130). [Figure 20] Figure 20 shows that treatment of SJSA-1 or SJSA-X cells with a panel of stapled peptide degron chimeras composed of stapled peptides targeting MDM2 / MDMX (e.g., ATSP-7041-like stapled p53 peptides) and a VHL degron variably reduces cell viability of the cancer cells. Sequences: %TF8EYWAQLXSAA (SEQ ID NO: 131) and LTF8EYWAQLX%AA (SEQ ID NO: 130). [Figure 21]Figure 21 shows the structure of an exemplary stapled peptide degron chimera containing one stapled peptide and another stapled peptide. One stapled peptide targets a protein of interest (e.g., a protein associated with a disease of interest), and the other stapled peptide binds to a degradation-inducing protein (e.g., stapled peptide ATSP-7041 (SEQ ID NO: 6, for binding to MDM2). Also shown is an example of a stapled peptide degron chimera containing two copies of the same stapled peptide, where the stapled peptide targets a protein of interest (e.g., a protein associated with a disease of interest) that is a degradation-inducing protein, such that upon binding of the stapled peptide degron chimera to the target protein, protein dimerization and autolysis can be triggered. In each illustrated example, the two stapled peptides are linked by a linker of variable length (e.g., see Figure 6 for exemplary linkers). Left column (top to bottom): SEQ ID NOs: 1-5, 132, 7-10, 133, and 12. Right column: SEQ ID NO: 134. [Figure 22] Figure 22 shows that incubation of the ubiquitination machinery, including E1, E2, and recombinant MDM2, with recombinant MCL-1 and a stapled peptide degron chimera that binds to MDM2 and MCL-1 induces ubiquitination of MCL-1 (amino acids 1-327) by MDM2. [Figure 23] Figure 23 shows the structure of the stapled peptide degron chimera. The stapled peptide (ATSP-7041 (LTF8EYWAQ#XSAA (SEQ ID NO: 6)) is incorporated to bind to and recruit the degradation-directed protein (MDM2), and a small molecule (JQ1) is included to bind to the disease-associated protein (BRD4). [Figure 24]Figure 24 shows that incubating the ubiquitination machinery, including E1, E2, and MDM2, with recombinant BRD4 species (e.g., amino acids 342-460; amino acids 49-170) and a stapled peptide degron chimera linked to MDM2 (e.g., stapled p53 peptide ATSP-7041) and BRD4 (e.g., small molecule JQ1) and with a linker composed of two beta-alanine amino acids, can induce ubiquitination of BRD4 at two distinct regions by MDM2. [Figure 25] Figure 25 shows that treatment of U2OS cells with a stapled peptide degron chimera incorporating a stapled peptide for binding to MDM2 and a small molecule for binding to BRD4, e.g., JQ1, with a linker composed of two beta-alanine amino acids as shown in Figure 23, results in time-dependent degradation of native BRD4. Actin represents a loading control. [Figure 26] The top panel of Figure 26 shows the chemical structures of exemplary non-naturally occurring amino acids used to generate different types of staples for insertion into peptides. The middle panel of Figure 26 shows peptides with staples of various lengths. The bottom panel of Figure 26 shows a staple walk along the peptide sequence. [Figure 27] FIG. 27 is a schematic diagram showing representative examples of different types of double and triple stapling strategies, as well as exemplary staple walks for generating stapled peptides. [Figure 28] FIG. 28 is a schematic diagram showing an exemplary staple walk using branched double-staple moieties of various lengths to generate stapled peptides. [Figure 29] FIG. 29 is a schematic diagram showing exemplary chemical modifications used to generate stapled peptides. DETAILED DESCRIPTION OF THE INVENTION

[0055] Detailed Description The present disclosure features stabilized peptide degron chimeras that act as protein degradation-inducing moieties by combining stabilized peptides targeting disease-associated proteins, e.g., as "degrons" or more generally as alternative small molecule degrons, with the cereblon-binding small molecule thalidomide or with polypeptide sequences containing the stabilized polypeptide sequence "degron." Stabilized peptide degron chimeras also include combining stabilized peptides that bind to and recruit degradation-inducing proteins with small molecules or peptides that are incorporated to target disease-associated proteins. By combining the ability of stabilized peptides to effectively target a wide range of intracellular proteins previously inaccessible to small molecules with small molecule or peptide degron moieties that can recruit degradation-inducing drug proteins that degrade the bound proteins, or by combining stabilized peptides that effectively bind to and recruit degradation-inducing proteins with small molecules or peptides that target disease-associated proteins, this novel class of stapled peptide degron chimeras expands the potential and breadth of biological activity of stapled peptides. The present disclosure also relates to methods for targeted degradation of endogenous proteins by using stapled peptide degron chimeras, which can be utilized in the treatment of disorders (e.g., proliferative disorders) caused by the presence of disease-associated proteins. The present application also provides methods for making the compounds of the present application and intermediates thereof.

[0056] Stabilizing Peptides Peptide helices are important mediators of key protein-protein interactions that regulate many important biological processes (e.g., apoptosis). However, when such helices are interpreted out of context within proteins and prepared in isolation, they may misfold and adopt a random coil conformation, leading to a dramatic decrease in biological activity and therefore reduced therapeutic potential. To circumvent this problem, conformationally stabilized peptides can be used. In some cases, conformationally stabilized peptides contain at least two modified amino acids joined by an internal (intramolecular) bridge (or staple). Stabilizing peptides as described herein include stapled peptides, stitched peptides, peptides containing multiple stitches, peptides containing multiple staples, or peptides containing a mix of staples and stitches, as well as peptides structurally strengthened by other chemical strategies (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. 2001;123:5382; Chin JW, et al., Int. Ed. 2001;40:3806; Chapman RN, et al., J. Am. Chem. Soc. 2001;123:5382; Chin JW, et al., Int. Ed. 2001;123:5382; Chin JW, et al., J. Am. Chem. Soc ... 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., See Org. Biomol. Chem., 2016, 14:8002-8013, all of which are incorporated herein by reference in their entireties.

[0057] In certain embodiments, the polypeptide can be stabilized by stapling a peptide (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 "stabilized" in that it maintains its natural secondary structure. For example, stapling allows a polypeptide that tends to have an α-helical secondary structure to maintain its natural α-helical conformation. This secondary structure may increase the resistance of the polypeptide to proteolytic cleavage and heat, and may also increase the binding affinity to the target, hydrophobicity, and cell permeability. Therefore, the stapled (crosslinked) polypeptides described herein have improved biological activity compared to the corresponding non-stapled (non-crosslinked) polypeptides.

[0058] "Peptide stapling" is a term coined from synthetic methodology in which two olefin-containing side chains (e.g., crosslinkable side chains) present on a polypeptide chain are covalently joined (e.g., "stapled together") to form a bridged ring using a ring-closing metathesis (RCM) reaction (e.g., Blackwell et al., J. Org. Chem., 66: 5291-5302, 2001; Angew et al., Chem. Int. Ed. 37:3281, 1994). The term "peptide stapling," as used herein, includes the joining of two (e.g., at least one pair) double-bond-containing, triple-bond-containing, or double- and triple-bond-containing side chains that may be present in a polypeptide chain using any number of reaction conditions and / or catalysts that facilitate such reaction and provide a single "staple" polypeptide. The term "multiple-staple" polypeptide refers to a polypeptide that contains more than one individual staple and may contain two, three, or more independent staples at various intervals. Furthermore, the term "peptide stitching," as used herein, refers to multiple, tandem "stapling" events in a single polypeptide chain, providing a "stitched" (e.g., tandem or multi-staple) polypeptide in which two staples are linked, for example, to a common residue. Peptide stitching is disclosed, for example, in WO2008 / 121767 and WO2010 / 068684, both of which are incorporated by reference in their entireties. In some cases, staples, as used herein, can retain or reduce unsaturated bonds.

[0059] In certain embodiments, polypeptides can be stabilized, for example, by hydrocarbon stapling. In certain cases, the stapled peptide contains at least two (e.g., 2, 3, 4, 5, 6) amino acid substitutions, the substituted amino acids are separated by 2, 3, or 6 amino acids, and the substituted amino acids are unnatural amino acids having olefinic side chains. There are many known unnatural or unnatural amino acids, any of which may be included in the stapled peptide. Some examples of unnatural amino acids include 4-hydroxyproline, desmosine, gamma-aminobutyric acid, beta-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-cyclopentanecarboxylic acid, 3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid, 4-amino-l-methyl-L-threonine ... Examples of amino acids include thylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioic acid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta-, and para-substituted phenylalanines (e.g., substituted with -C(=O)C6H5; -CF3; -CN; -halo; -NO2; CH3), disubstituted phenylalanines, substituted tyrosines (e.g., further substituted with -C=O)C6H5; -CF3; -CN; -halo; -NO2; CH3), and statins. Additionally, amino acids can be derivatized to include hydroxylated, phosphorylated, sulfonated, acylated, or glycosylated amino acid residues.

[0060] Hydrocarbon-stapled polypeptides contain one or more tethers (links) between two unnatural amino acids, which significantly reinforce the α-helical secondary structure of the polypeptide. Typically, the tether extends over a length of one or two helical turns (i.e., about 3.4 or about 7 amino acids). Thus, amino acids at positions 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, if a peptide has the sequence ...X1, X2, X3, X4, X5, X6, X7, X8, X9..., a cross-link between X1 and X4, or between X1 and X5, or between X1 and X8, is a useful hydrocarbon-stapled form of the peptide, as are cross-links between X2 and X5, or between X2 and X6, or between X2 and X9, etc. The use of multiple cross-links (e.g., 2, 3, 4, or more) is also contemplated. The use of multiple crosslinks is very effective in stabilizing and optimizing peptides, especially with increasing peptide length.Therefore, the present disclosure encompasses the incorporation of more than one crosslink into a polypeptide sequence to further stabilize the sequence or to promote the structural stabilization, proteolytic resistance, acid stability, thermal stability, cell permeability, and / or enhanced biological activity of longer polypeptide stretches.Additional descriptions regarding the production and use of carbohydrate-stapled polypeptides can be found, for example, in U.S. Patent Application Publication Nos. 2012 / 0172285, 2010 / 0286057, and 2005 / 0250680, the contents of all of which are incorporated herein by reference in their entirety.

[0061] In certain embodiments, when the staple is at the i and i+3 residues, R-propenylalanine and S-pentenylalanine; or R-pentenylalanine and S-pentenylalanine are substituted with amino acids at these positions. In certain embodiments, when the staple is at the i and i+4 residues, S-pentenylalanine is substituted with amino acids at these positions. In certain embodiments, when the staple is at the i and i+7 residues, S-pentenylalanine and R-octenylalanine are substituted with amino acids at these positions. In some cases, when a peptide is stitched, the amino acids of the peptide involved in the "stitch" are substituted with bis-pentenylglycine, S-pentenylalanine, and R-octenylalanine; or bis-pentenylglycine, S-octenylalanine, and R-octenylalanine.

[0062] The staple or stitch location can be varied by testing different staple locations in the staple walk.

[0063] Figure 26 (top) shows exemplary chemical structures of unnatural amino acids that can be used to generate various cross-linked compounds. Figure 26 (center) shows peptides with hydrocarbon bridges between residues i and i+3; between residues i and i+4; and between residues i and i+7. Figure 26 (bottom) shows staple walks along peptide sequences. Figure 27 shows various peptide sequences, including double and triple stapling strategies, as well as exemplary staple walks. Figure 28 shows exemplary staple walks using branched stitch segments of various lengths.

[0064] In one embodiment, the stabilized polypeptide has formula (I): [ka] (In the formula, Each R1 and R2 is independently H or C1-C 10alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; R3 is alkyl, alkenyl, or alkynyl; [R4-K-R4] n each of which is substituted with 0 to 6 R5; R4 is alkyl, alkenyl, or alkynyl; R5 is halo, alkyl, OR6, N(R6)2, SR6, SOR6, SO2R6, CO2R6, R6, a fluorescent moiety, or a radioisotope; K is O, S, SO, SO2, CO, CO2, CONR6, or [ka] and R6 is H, alkyl, or a therapeutic agent; n is an integer from 1 to 4; x is an integer from 2 to 10; each y is independently an integer from 0 to 100; z is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); and each Xaa is independently an amino acid).

[0065] The tether may be an alkyl, alkenyl, or alkynyl moiety (e.g., C5, C8, or C 11 Alkyl, C5, C8, or C 11 Alkenyl, or C5, C8, or C 11 The tethered amino acids may include alpha disubstituted (e.g., C1-C3 or methyl).

[0066] In some cases, x is 2, 3, or 6. In some cases, each y is independently an integer from 1 to 15 or 3 to 15. In some cases, R1 and R2 are each independently H or C1-C6 alkyl. In some cases, R1 and R2 are each independently C1-C3 alkyl. In some cases, at least one of R1 and R2 is methyl. For example, R1 and R2 can both be methyl. In some cases, R3 is alkyl (e.g., C8 alkyl) and x is 3. In some cases, R3 is C 11 In some cases, R is alkyl and x is 6. In some cases, R is alkenyl (e.g., C alkenyl) and x is 3. In some cases, x is 6 and R is C 11 In some cases, R3 is a straight chain alkyl, alkenyl, or alkynyl. In some cases, R3 is -CH2-CH2-CH2-CH=CH-CH2-CH2-CH2-.

[0067] In another embodiment, the two alpha, alpha disubstituted stereocenters are both in the R or S configuration (e.g., the i, i+4 bridges), or one stereocenter is R and the other is S (e.g., the i, i+7 bridges). Thus, Formula I [ka] For example, when x is 3, the C' and C" disubstituted stereocenters can both be of the R configuration or both of the S configuration. When x is 6, the C' disubstituted stereocenter is of the R configuration and the C" disubstituted stereocenter is of the S configuration. The R3 double bond can be of either the E or Z stereochemical configuration.

[0068] In some cases, R3 is [R4-K-R4] n and R4 is a straight chain alkyl, alkenyl, or alkynyl.

[0069] In some embodiments, the disclosure features internally crosslinked ("stapled" or "stitched") peptides in which the side chains of two amino acids separated by two, three, or six amino acids have been replaced with internal staples; the side chains of three amino acids have been replaced with internal stitches; the side chains of four amino acids have been replaced with two internal staples; or the side chains of five amino acids have been replaced with a combination of internal staples and internal stitches. 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.

[0070] In certain cases, the stabilizing peptide is a peptide of an intracellular protein. In certain cases, the stabilizing peptide is a peptide of a protein that causes or is associated with a disease. In certain cases, the stabilizing peptide is a peptide of a bacterial protein. In certain cases, the stabilizing peptide is a peptide of a human protein. In certain 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 fusions, MLL fusions, 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.

[0071] Non-limiting examples of stapled peptides include: [ka] [ka] (where 8 = R-octenylalanine; B = norleucine; # = cyclobutylalanine; X = S-pentenylalanine, and in some cases, X1 and X2 are the same (e.g., S-pentenylalanine).

[0072] In certain embodiments, the staple polypeptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 1-24 and 134. In certain embodiments, the present disclosure features stabilizing peptides that differ from the peptides disclosed above by having different staple / stitch locations. In certain embodiments, the present disclosure features stabilizing peptides that differ from the peptides disclosed above by having 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, 7) amino acid substitutions on the non-interacting faces of the alpha-helices of the peptides. In certain cases, the substitutions are conservative. In other cases, the substitutions are non-conservative. In certain embodiments, the present disclosure features stabilizing peptides that differ from the peptides disclosed above by having 1 to 5 (e.g., 1, 2, 3, 4, 5) amino acid substitutions on the interacting faces of the alpha-helices of the peptides. In certain cases, the substitutions are conservative. Exemplary types of variations / modifications to staple peptides are shown in Figure 29.

[0073] In certain embodiments, the stapled peptide is not a Bcl-2 homology 3 (BH3) domain polypeptide (e.g., not a BH3 domain from MCL-1, not a MCL-1 stabilizing alpha helix (SAHB) of the BCL2 domain, but rather an MCL-1SAHB). D But not.)

[0074] In certain embodiments, the stabilizing peptide (e.g., a stapled peptide) directly binds to and recruits a degradation-inducing protein, such as the ubiquitin E3 ligase MDM2. For example, the E3 ligase MDM2 can be strongly bound by a stapled p53 peptide known in the art, the entire contents of which are incorporated herein by reference. In certain cases, the peptide degron is a stabilizing or stapled peptide that directly binds to and recruits a complex comprising a degradation-inducing protein, such as a complex between MDMX and the ubiquitin E3 ligase MDM2. In this example, the stapled p53 peptide can strongly bind to MDMX and recruit the MDMX / MDM2 complex, allowing MDM2 to recruit the degradation-inducing protein.

[0075] In certain embodiments, the stabilization peptide directly or indirectly binds to a degradation-inducing protein, such as an E3 ubiquitin ligase or a substrate adaptor for the E3 ubiquitin ligase. In certain embodiments, the stabilization peptide directly or indirectly binds to an E3 ubiquitin ligase. In some embodiments, the stabilization peptide directly or indirectly binds to an E3 ligase (e.g., MDM2) or a protein that forms a complex with an E3 ligase, such as MDMX, which binds 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-Bmil, 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 an RBR E3 ubiquitin ligase (e.g., Parkin, Parc, RNF144(A / B), HOIP, HHARI). For non-limiting examples of E3 ubiquitin ligases, see, e.g., Morreale and Walden, Cell 165, 2016 DOI http: / dx.doi.org / 10.1016 / j.cell.2016.03.003.

[0076] Non-limiting examples of other stabilizing peptides that can be used in the chimeric fusions described herein are those described in U.S. Patent Nos. 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, No. 252; No. 9,505,816; No. 9,505,804; No. 9,505,801; No. 9,493,510; No. 9,464,125; No. 9,485,202; No. 9,45 No. 8,189; No. 9,416,162; No. 9,408,885; No. 9,346,868; No. 9,296,805; No. 9,227,995; No. 9,175,047; No. 9, Nos. 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. 20170247423; Nos. 20170240604; 20170212125; 20170165320; 20170066747; 20170015716; 20160376336; and 20160244494, the contents of all of which are incorporated by reference in their entireties (particularly the disclosure of stabilizing (e.g., stapled or stitched) peptides).

[0077] While hydrocarbon tethers are common, other tethers can also be used in the stabilized peptides described herein. For example, the tether can contain one or more ether, thioether, ester, amine, or amide, or triazole moieties. In some cases, naturally occurring amino acid side chains can be incorporated into the tether. For example, the tether can be coupled to functional groups such as the hydroxyl in serine, the thiol in cysteine, the primary amine in lysine, the acid in aspartic acid or glutamic acid, or the amide in asparagine or glutamine. Thus, rather than using a tether made by coupling two non-naturally occurring amino acids, it is possible to generate a tether using a naturally occurring amino acid. It is also possible to use a single non-naturally occurring amino acid together with a naturally occurring amino acid. Triazole-containing (e.g., 1,4 triazole or 1,5 triazole) bridges can be used (see, e.g., Kawamoto et al. 2012 Journal of Medicinal Chemistry 55:1137; WO2010 / 060112). Furthermore, different Other methods of performing this type of stapling are known in the art and can be used (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: JC Phelan et al.,, J. Am. Chem. Soc., 119:455-460 (1997); and bis-arylated stapling: A. See M. Spokoyny et al., J. Am. Chem. Soc., 135:5946-5949 (2013).

[0078] It is further contemplated that the length of the tether may vary: for example, if it is desired to impose a relatively high degree of constraint on the secondary alpha-helical structure, a short tether length may be used, whereas in some cases a lower constraint on the secondary alpha-helical structure may be desired, and thus a longer tether may be desired.

[0079] Furthermore, while the overall tether lengths of amino acids i to i+3, i to i+4, and i to i+7 are common to provide tethers primarily on one face of the alpha helix, tethers can be synthesized spanning any combination of amino acid numbers and can be used in combination to introduce multiple tethers.

[0080] In some cases, the hydrocarbon tethers (i.e., crosslinks) described herein can be further manipulated. In one example, the double bond of a hydrocarbon alkenyl tether (e.g., as synthesized using ruthenium-catalyzed ring-closing metathesis (RCM)) can be oxidized (e.g., via epoxidation, aminohydroxylation, or dihydroxylation) to provide one of the following compounds: [ka]

[0081] Either the epoxide moiety or one of the free hydroxyl moieties can be further functionalized. For example, the epoxide can be treated with a nucleophile to provide additional functionality that can be used, for example, to bind a therapeutic agent. Alternatively, such derivatization can be achieved by synthetic manipulation of the amino or carboxy terminus of the polypeptide or via an amino acid side chain. Other agents, such as agents that promote the entry of the polypeptide into cells, can be attached to the functionalized tether.

[0082] In some cases, alpha-disubstituted amino acids are used in polypeptides to improve the stability of alpha-helical secondary structures, however, alpha-disubstituted amino acids are not required, and the use of mono-alpha substituents (e.g., in tethered amino acids) is also contemplated.

[0083] The staple polypeptide may comprise a drug, a toxin, a polyethylene glycol derivative; a second polypeptide; a carbohydrate, etc. When a polymer or other agent is linked to the staple polypeptide, it may be desirable for the composition to be substantially homogeneous.

[0084] The addition of polyethylene glycol (PEG) molecules can improve the pharmacokinetic and pharmacodynamic properties of polypeptides. For example, PEGylation can decrease renal clearance and may result in more stable plasma concentrations. PEG is a water-soluble polymer. Formula:XO--(CH2CH2O) n --CH2CH2-Y wherein n is 2 to 10,000 and X is H or a terminal modification, e.g., C 1~4 and Y is alkyl; and Y can be represented as linked to the polypeptide as an amide, carbamate, or urea linkage to an amine group of the polypeptide (including, but not limited to, the epsilon amine of lysine, or the N-terminus). Y can also be a maleimide linkage to a thiol group (including, but not limited to, the thiol group of cysteine). Other methods for directly or indirectly linking PEG to a polypeptide are known to those skilled in the art. PEG can be linear or branched. Various forms of PEG, including various functionalized derivatives, are commercially available.

[0085] PEG can be used that has degradable linkage in the backbone.For example, PEG can be prepared with ester linkage that is subject to hydrolysis.The conjugate with degradable PEG linkage is described in WO99 / 34833, WO99 / 14259 and US6,348,558.

[0086] In certain embodiments, a polymeric polymer (e.g., PEG) is attached to the drug described herein via an intermediate linker. In certain embodiments, the linker is made up of 1 to 20 amino acids linked by peptide bonds, and the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those skilled in the art. In other embodiments, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Non-peptide linkers are also possible. For example, alkyl linkers, such as -NH(CH2) n C(O)- (where n=2-20) can be used. These alkyl linkers may be further substituted with any sterically unhindered group, such as lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. U.S. Patent No. 5,446,090 describes bifunctional PEG linkers and their use in forming conjugates with peptides at each PEG linker terminus.

[0087] In some embodiments, the stabilizing peptides may also be modified, for example, to further enhance cellular uptake or to increase in vivo stability. For example, acylation or PEGylation of the peptidomimetic macrocycle may enhance cellular uptake, increase bioavailability, increase circulation, alter pharmacokinetics, reduce immunogenicity, and / or reduce the required dosing frequency.

[0088] In some embodiments, the stapled peptides disclosed herein have an enhanced ability to penetrate cell membranes (eg, compared to non-stapled peptides).

[0089] Methods for synthesizing the stabilized peptides described herein are known in the art. Nevertheless, the following exemplary method may be used. It will be understood that various steps may be performed in an alternative sequence or order to obtain the desired compound. Synthetic chemical transformations and methodologies for protecting functional groups (protection and deprotection) useful in synthesizing the compounds described herein are known in the art, and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd 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 editions thereof.

[0090] Stabilizing peptides can be prepared by chemical synthesis methods well known to those skilled in the art. See, for example, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W.H. Freeman & Co., New York, NY, 1992, p. 77. Thus, peptides can be prepared by, for example, Applied Biosystems Synthesis can be performed using automated Merrifield techniques for solid phase synthesis using side-chain protected amino acids with α-NH2 protected with t-Boc or Fmoc chemistry on a Peptide Synthesizer Model 430A or 431.

[0091] One way to prepare the peptides described herein is to use solid-phase peptide synthesis (SPPS). The C-terminal amino acid is attached to a cross-linked polystyrene resin via an acid-labile bond using a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products. The N-terminus is protected with an acid-stable Fmoc group, but is base-removable. Any side chain functional groups are protected with base-stable, acid-labile groups.

[0092] Long peptides may be produced by joining individual synthetic peptides using native chemical ligation. Alternatively, long synthetic peptides can be synthesized using well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals containing detailed protocols. To construct a gene encoding a peptide of the present invention, the amino acid sequence is reverse-translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with optimal codons for the organism in which the gene will be expressed. A synthetic gene is then typically produced by synthesizing oligonucleotides encoding the peptide and any regulatory elements, as needed. The synthetic gene is inserted into a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods.

[0093] Peptides can be produced in a high-throughput combinatorial manner, for example, using a high-throughput multichannel combinatorial synthesizer available from Advanced Chemtech. Peptide bonds can be replaced with retro-inverso bonds (C(O)-NH); reduced amide bonds (NH-CH2); thiomethylene bonds (S-CH2 or CH2-S); oxomethylene bonds (O-CH2 or CH2-O); ethylene bonds (CH2-CH2); thioamide bonds (C(S)-NH); trans-olefin bonds (CH=CH); fluoro-substituted trans-olefin bonds (CF=CH); ketomethylene bonds (C(O)-CHR) or CHR-C(O) (wherein R is H or CH3); and fluoroketomethylene bonds (C(O)-CFR or CFR-C(O) (wherein R is H, F, or CH3). For example, to enhance the physiological stability of the peptide, peptide bonds can be replaced with:

[0094] Polypeptides can be further modified by acetylation, amidation, biotinylation, cinnamoylation, farnesylation, fluoresceination, formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyr, or Thr), stearoylation, succinylation, and sulfurylation. As noted above, peptides can be conjugated to, for example, polyethylene glycol (PEG); alkyl groups (e.g., C1-C20 straight-chain or branched alkyl groups); fatty acid radicals; and combinations thereof. α,α-disubstituted unnatural amino acids containing olefinic side chains of various lengths 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, the i-th staple linked to the i+7th staple is When using the second amino acid (helix 2 turn stabilized), either a) one S5 amino acid and one R8 amino acid are used, or b) one S8 amino acid and one R5 amino acid are used. R8 is synthesized using a similar route, except that the starting chiral auxiliary provides the R-alkyl-stereoisomer. Also, 8-iodooctene is used instead of 5-iodopentene. Inhibitors are synthesized on a solid support using solid-phase peptide synthesis (SPPS) on MBHA resin (see, e.g., WO2010 / 148335).

[0095] Fmoc-protected α-amino acids (other than the olefinic amino acids Fmoc-S5-OH, Fmoc-R8-OH, Fmoc-R8-OH, Fmoc-S8-OH, and Fmoc-R5-OH), 2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), and Rink Amide MBHA are commercially available, for example, from Novabiochem (San Diego, CA). Dimethylformamide (DMF), N-methyl-2-pyrrolidinone (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. Olefinic amino acid synthesis has been reported in the art (Williams et al., Org. Synth., 80:31, 2003).

[0096] Suitable methods for obtaining (e.g., synthesizing) the stapled, purified 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 Publication No. 12 / 525,123, filed March 18, 2010; and U.S. Patent No. 7,723,468, issued May 25, 2010, each of which is incorporated herein by reference in its entirety).

[0097] In some embodiments, the peptide is substantially free of or isolated from non-staple peptide contaminants. Methods for purifying peptides include, for example, peptide synthesis on a solid support. After cyclization, the solid support may be isolated and suspended in a solvent solution, 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 certain embodiments, a 50% / 50% DMSO / NMP solution is used. The solution may be incubated for 1, 6, 12, or 24 hours, after which the resin may be washed with, for example, dichloromethane or NMP. In one embodiment, the resin is washed with NMP. Shaking and bubbling of an inert gas into the solution may be performed.

[0098] The properties of stabilized (eg, stapled) polypeptides of the invention can be assayed, for example, using the methods described below.

[0099] Assay for determining α-helicity: Compounds are dissolved in aqueous solution (e.g., 25-50 μM in 5 mM potassium phosphate solution at pH 7 or distilled H2O). Circular dichroism (CD) spectra are measured 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; accumulations, 100 Hz). 10; response, 1 s; bandwidth, 1 nm; path length, 0.1 cm). The α-helical content of each peptide is calculated by dividing the residue-average ellipticity by the reported value for a model helical decapeptide (Yang et al., Methods Enzymol. 130:208 (1986)).

[0100] Assay for determining melting temperature (Tm): Crosslinked or unmodified template peptide is dissolved in distilled HO or other buffer or solvent (e.g., at a final concentration of 50 μM), and Tm is determined by measuring the change in ellipticity over a temperature range (e.g., 4 to 95°C) with a spectropolarimeter (e.g., Jasco J-710, Aviv) using standard parameters (e.g., wavelength 222 nm; step resolution, 0.5 nm; rate, 20 nm / sec; accumulation, 10; response, 1 sec; bandwidth, 1 nm; temperature ramp rate: 1°C / min; path length, 0.1 cm).

[0101] In vitro protease resistance assay The amide bonds of the peptide backbone are susceptible to hydrolysis by proteases, making the peptide compound vulnerable to rapid degradation in vivo. However, typically, when a peptide helix is ​​formed, the amide backbone becomes buried and / or twisted and / or shielded, thereby preventing or substantially slowing proteolytic cleavage. The peptidomimetic macrocycles of the present invention may be subjected to in vitro enzyme proteolysis (e.g., trypsin, chymotrypsin, pepsin) and evaluated for any changes in degradation rate compared with comparable uncrosslinked or alternative stapled polypeptides. For example, the peptidomimetic macrocycle and comparable uncrosslinked polypeptide are incubated with trypsin agarose, the reaction quenched at various time points by centrifugation, and then injected into an HPLC system, and the remaining substrate is quantified by UV absorption at 280 nm. Briefly, peptidomimetic macrocycles and peptidomimetic precursors (5 mcg) are incubated with trypsin agarose (Pierce) (S / E ~125) for 0, 10, 20, 90, and 180 min. The reaction is quenched at high speed by tabletop centrifugation, and the remaining substrate in the isolated supernatant is quantified by HPLC-based peak detection at 280 nm. The proteolytic reaction is first-order, and the rate constant k is determined from a plot of ln[S] versus time.

[0102] The peptidomimetic macrocycle and / or comparable uncrosslinked polypeptide may be incubated with fresh mouse, rat, and / or human serum (e.g., 1–2 mL) at 37°C for, e.g., 0, 1, 2, 4, 8, and 24 hours, respectively. Macrocycle samples at different concentrations may be prepared by serial dilution with serum. To determine the level of intact compound, the following procedure may be used: For example, extract the sample by transferring 100 μL of serum to a 2 mL centrifuge tube, followed by the addition of 10 μL of 50% formic acid and 500 μL of acetonitrile and centrifugation at 14,000 RPM and 4 ± 2°C for 10 minutes. The supernatant is then transferred to a fresh 2 mL tube and evaporated in a Turbovap under N2 at <10 psi and 37°C. The sample is reconstituted in 100 μL of 50:50 acetonitrile:water and subjected to LC-MS / MS analysis. Similar or analogous procedures for testing ex vivo stability are known and may be used to determine the stability of the macrocycle in serum.

[0103] In vivo protease resistance assay: The advantage of stapling key peptides is that in vitro protease resistance translates into significantly improved pharmacokinetics in vivo.

[0104] In vitro binding assays: Fluorescence polarization assays (FPA), for example, can be used to assess the binding and affinity of peptidomimetic macrocycles and peptidomimetic precursors to acceptor proteins. FPA techniques measure molecular orientation and mobility using polarized light and fluorescent tracers. When excited with polarized light, fluorescent tracers (e.g., FITC) bound to molecules with large apparent molecular weights (e.g., FITC-labeled peptides bound to large proteins) emit high levels of polarized fluorescence due to their slower rotation rate compared to fluorescent tracers bound to smaller molecules (e.g., FITC-labeled peptides free in solution).

[0105] cell analysis Cultured cells (e.g., cancer cells) are treated with the stapled peptide-degron chimera, and the levels of the targeted protein are monitored over time by Western analysis. A negative control protein not targeted by the chimeric peptide is also monitored to demonstrate the specificity of the targeted degradation. 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.

[0106] Peptide degron The present disclosure features peptide degrons that bind to proteins that are substrate adaptors for ubiquitin E3 ligases. In some cases, the degrons bind to WD-40 proteins, which are substrate adaptors for ubiquitin E3 ligases. The degrons bind to the substrate recognition domain of ubiquitin E3 ligases in the minor groove and are resistant to assimilation (i.e., conjugation of staple peptide sequences) at either 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, and KLHL51. These include HL42, 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. WD40 containing E3 ligases include the majority of E3 ligases, however, there are other types of E3 ligases and degrons that bind to proteins that are substrate adaptors for such E3 ligases and are encompassed by the present disclosure.

[0107] In certain cases, the peptide degron is based on the Trib1 protein sequence: DQIVPEY (SEQ ID NO: 25) or a variant thereof. [Table 1]

[0108] Degrons of the present disclosure include variants of SEQ ID NO:25, where the variants include 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. In some cases, variants of SEQ ID NO:25 have one or more (e.g., 1, 2, 3, 4, 5) substitutions. In certain cases, one or more (e.g., 1, 2, 3, 4, 5, 6, 7) of these substitutions are not for A, R at any of positions 1 to 6 of SEQ ID NO:25. In certain cases, these substitutions do not include a substitution of V at position 4 in SEQ ID NO:25 for I. 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, variants of SEQ ID NO:25 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, variants of SEQ ID NO:25 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, variants of SEQ ID NO:25 have one or more (e.g., 1, 2, 3) deletions and one or more (e.g., 1, 2, 3) insertions. In some cases, variants of SEQ ID NO:25 have 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1 amino acid substitutions within SEQ ID NO:25. In certain cases, positions 4 (V) and / or 5 (P) of SEQ ID NO: 25 are not substituted. 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 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, the variant of SEQ ID NO: 25 has a single deletion. The deletion may be at the C-terminus or N-terminus of SEQ ID NO: 25.

[0109] In some cases, peptide degrons bind to the F-box / WD repeat-containing protein 7 (FBXW7) protein. In one embodiment, the peptide degron has 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) a and X b are independently any amino acid). In some 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 differ from SEQ ID NO: 46 in having 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 a combination of any two or more thereof. In some cases, a variant of SEQ ID NO: 46 has one or more (e.g., 1, 2, 3, 4) substitutions. In some cases, a variant of SEQ ID NO: 46 has one or more (e.g., 1, 2, 3) deletions. In some cases, a variant of SEQ ID NO: 46 has one or more (e.g., 1, 2, 3) insertions. In some cases, a 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, a 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, a 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, a variant of SEQ ID NO: 46 has 1 to 5, 1 to 4, 1 to 3, 2, or 1 amino acid substitutions within SEQ ID NO: 46. In certain cases, position 2 (P) is not substituted. In certain cases, position 1 is pS and position 2 is P. In certain cases, position 1 is pS, position 2 is P, and position 5 is E. In certain cases, position 1 is pS, position 2 is P, and position 5 is pS. In certain cases, position 1 is pS, position 2 is P, and position 5 is pT. In one particular case, the first place is pT, the second place is P, and the fifth place is E. In one particular case, the first place is pT, the second place is P, and the fifth place is pS. In one particular case, the first place is pT, the second place is P, and the fifth place is pT.

[0110] In certain cases, peptide degrons are based on natural binding consensus sequences of peptides that bind to WD40 repeat proteins, which are substrate adaptors for E3 ubiquitin ligases. In some cases, peptide degrons are variants (e.g., substitution, deletion, or insertion variants) of natural binding consensus sequences of peptides that bind to WD40 repeat proteins, which are substrate adaptors for E3 ubiquitin ligases. Non-limiting examples of natural binding consensus sequences of peptides that bind to WD40 repeat proteins, which are substrate adaptors for E3 ubiquitin ligases, are shown below (sequences are assigned SEQ ID NOS: 65 to 92, from top to bottom): [Table 2-1] [Table 2-2]

[0111] †Motif patterns use the following nomenclature: "." specifies any amino acid type, "[X]" specifies the amino acid type(s) allowed at that position, "^X" at the beginning of the pattern specifies that the sequence starts with amino acid type X, "[^X]" indicates that the position can have amino acids other than type X, and numbers "X{x,y}" where x and y specify the minimum and maximum number of "X" amino acid type required at that position. The "$" symbol indicates the C-terminus of the protein chain. Positions of conserved residues within primary degrons known to be post-translationally modified (e.g., phosphorylation and proline hydroxylation) are shown in bold.

[0112] Any other peptide degrons known in the art can also be used in the present invention. For example, Meszaros et al., Sci. Signal., 10(470):eaak9982 (2017); Guharoy et al., Nature Communications, 7:10239, doi:10.1038 / ncomms10239 (2016); U.S. Patent Nos. 9,783,575; 9,297,017; and 9,1 15,184, all of which are incorporated herein by reference in their entireties.

[0113] In certain cases, the peptide degron has the amino acid sequence of the following peptides or variants thereof: FSDLWKLL (SEQ ID NO: 31)-E3 ligase: MDM2; SVEQTPKK (SEQ ID NO: 32)-E3 ligase: SKP2-CKS1; DSGIHS (SEQ ID NO: 32)-E3 ligase: β-TrCP1; LLPTPPLS (SEQ ID NO: 33)-E3 ligase: FBXW7; ASSSS (SEQ ID NO: 34)-E3 ligase: SPOP; LAPAAGDTIISLDF (SEQ ID NO: 35) - E3 ligase: VHL; PFLTPSPE (SEQ ID NO: 36)-E3 ligase: FBXW7; PPPY (SEQ ID NO: 37)-E3 ligase: ITCH; DEETGE (SEQ ID NO: 38)-E3 ligase: KEAP1; QDIDLGV (SEQ ID NO: 39) - E3 ligase: KEAP1; LLQPNNYQFC (SEQ ID NO: 40)-E3 ligase: CBL; DYR-E3 ligase: CBL; RAVENQYSFY (SEQ ID NO: 41)-E3 ligase: CBL; QKENS (SEQ ID NO: 42)-E3 ligase: CDH1; FDIYMD (SEQ ID NO: 43) - E3 ligase: CDC20 / CDH1; PRTALGDIG (SEQ ID NO: 44) - E3 ligase: CDC20 / CDH1; DKENG (SEQ ID NO: 45)-E3 ligase: PTTG1; HRKHLQEIP (SEQ ID NO: 93)-E3 ligase: APC / C; SKENV (SEQ ID NO: 94)-E3 ligase: APC / C; TRIR (SEQ ID NO: 95)-E3 ligase: APC / C; DQIVPEY (SEQ ID NO: 96) - E3 ligase: COP1; TSMTDFYHSKRRL (SEQ ID NO: 97) - E3 ligase: DCAF2; SPETGE (SEQ ID NO: 98)-E3 ligase: KEAP1; EPEEPEADQH (SEQ ID NO: 99) - E3 ligase: KLHL3; LAPYIPMDDDFQL (SEQ ID NO: 100) - E3 ligase: VHL; LTPPQS (SEQ ID NO: 101)-E3 ligase: FBXW7; SVEQTPRK (SEQ ID NO: 102) - E3 ligase: SKP2 / CKS1; DSGNYS (SEQ ID NO: 103)-E3 ligase: beta-TrCP1; KPAAVVAPI (SEQ ID NO: 104) - E3 ligase: Siah; or ADSST (SEQ ID NO: 105)-E3 ligase: SPOP

[0114] 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 a combination of any two or more thereof. Variants are selected that interact with an appropriate E3 ligase. In some cases, the selected peptide degron binds to the appropriate E3 ligase with a binding affinity of 1 nM to 300 nM. In some cases, the selected peptide degron binds to the appropriate E3 ligase with a binding affinity of 10 nM to 300 nM. In some cases, the selected peptide degron binds to the appropriate E3 ligase with a binding affinity of 50 nM to 300 nM. In some cases, the selected peptide degron binds to the appropriate E3 ligase with a binding affinity of 100 nM to 300 nM. In some cases, the selected peptide degron binds to a suitable E3 ligase with a binding affinity of 200 nM to 300 nM. In some cases, the selected peptide degron binds to a suitable E3 ligase with a binding affinity of 200 nM to 250 nM. In some cases, the selected peptide degron binds to a suitable E3 ligase with a binding affinity of 1 nM to 1000 nM. In some cases, the selected peptide degron binds to a suitable E3 ligase with a binding affinity of 200 nM to 1000 nM.

[0115] Non-limiting exemplary variants are shown below: 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); 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 for DCAF2 E3 ligase: TSMTDFYHSKRRI (SEQ ID NO:116), TSMTDFYHSKRKL (SEQ ID NO:117), TSMTDFYHSKRRS (SEQ ID NO:118).

[0116] In certain cases, the 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).

[0117] In certain cases, the peptide degron has the amino acid sequence of a peptide set forth in any one of SEQ ID NOs: 26 to 30. In some cases, the peptide degron has an amino acid sequence that is a variant of the peptide set forth in any one of SEQ ID NOs: 26 to 30. In certain cases, the peptide degron has the amino acid sequence of a peptide set forth in any one of SEQ ID NOs: 31 to 45. In some cases, the peptide degron has an amino acid sequence that is a variant of the peptide set forth in any one of SEQ ID NOs: 31 to 45. In certain cases, the peptide degron has the amino acid sequence of a peptide set forth in any one of SEQ ID NOs: 65 to 118. In some cases, the peptide degron has an amino acid sequence that is a variant of the peptide set forth in any one of SEQ ID NOs: 65 to 118. Variants include peptide degrons with 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 of these.

[0118] The peptide degrons described above bind to their appropriate substrate adaptors for ubiquitin E3 ligases (e.g., Cop1, FBXW7, FBXW8). In some cases, the peptide degrons bind to substrate adaptors for ubiquitin E3 ligases (e.g., Cop1, FBXW7, FBXW8) with a binding affinity of 1 nM to 300 nM. In some cases, the peptide degrons bind to substrate adaptors for ubiquitin E3 ligases (e.g., Cop1, FBXW7, FBXW8) with a binding affinity of 10 nM to 300 nM. In some cases, the peptide degrons bind to substrate adaptors for ubiquitin E3 ligases (e.g., Cop1, FBXW7, FBXW8) with a binding affinity of 50 nM to 300 nM. In some cases, the peptide degron binds to a substrate adaptor for a ubiquitin E3 ligase (e.g., Cop1, FBXW7, FBXW8) with a binding affinity of 100 nM to 300 nM. In some cases, the peptide degron binds to a substrate adaptor for a ubiquitin E3 ligase (e.g., Cop1, FBXW7, FBXW8) with a binding affinity of 200 nM to 300 nM. In some cases, the peptide degron binds to a substrate adaptor for a ubiquitin E3 ligase (e.g., Cop1, FBXW7, FBXW8) with a binding affinity of 200 nM to 250 nM. In some cases, the peptide degron binds to a substrate adaptor for a ubiquitin E3 ligase (e.g., Cop1, FBXW7, FBXW8) with a binding affinity of 1 nM to 1000 nM. In some cases, the peptide degron binds to a substrate adaptor for a ubiquitin E3 ligase (e.g., Cop1, FBXW7, FBXW8) with a binding affinity of 200 nM to 1000 nM.

[0119] The present disclosure also features a method for selecting a protein degron. Such a selected protein degron can be used in the chimeric constructs of the present disclosure. The method involves contacting a substrate adaptor for a ubiquitin E3 ligase with a naturally occurring variant of a peptide degron and selecting a degron that binds to the substrate adaptor with a desired affinity. For example, the method includes contacting a WD40 repeat protein, which is a substrate adaptor for an E3 ubiquitin ligase, with a variant of an amino acid sequence of a natural binding consensus sequence that binds to the WD40 repeat protein (e.g., Cop1, FBXW7, FBXW8), and selecting a peptide that binds to the WD40 repeat protein. In some cases, the selected peptide degron binds to the WD40 repeat protein with a binding affinity of 1 nM to 1000 nM. In some cases, the selected peptide degron binds to the WD40 repeat protein with a binding affinity of 10 nM to 300 nM. In some cases, the selected peptide degron binds to the WD40 repeat protein with a binding affinity of 50 nM to 300 nM. In some cases, the selected peptide degron binds to the WD40 repeat protein with a binding affinity of 100 nM to 300 nM. In some cases, the selected peptide degron binds to the WD40 repeat protein with a binding affinity of 200 nM to 300 nM. In some cases, the selected peptide degron binds to the WD40 repeat protein with a binding affinity of 200 nM to 250 nM. In some cases, the selected peptide degron binds to the WD40 repeat protein with a binding affinity of 1 nM to 1000 nM. In some cases, the selected peptide degron binds to the WD40 repeat protein with a binding affinity of 200 nM to 1000 nM. In some cases, the selected peptide degron binds to the WD40 repeat protein with a binding affinity of less than 1 nM (e.g., about 0.01 nM, about 0.05 nM, about 0.1 nM, about 0.5 nM). In some cases, the selected peptide degron binds to the WD40 repeat protein with a binding affinity of greater than 300 nM (e.g., about 350 nM, about 400 nM, about 500 nM, about 1000 nM).

[0120] In certain cases, proteins that are desired to be targeted for degradation are directly modified. The protein is examined for regions containing structurally disordered regions. A structurally disordered region is one that has a disorder score of less than or equal to 0.4, as calculated by IUPred:iupred.enzim.hu. The protein is modified in the structurally disordered region to include the above-mentioned peptide degron sequence or a variant thereof. In some cases, the structurally disordered region is at the N- or C-terminus of the protein. In some cases, the structurally disordered region is between the N-terminus and C-terminus of the protein. The peptide degron sequence can be inserted into the structurally disordered region. In other cases, the peptide degron sequence is substituted to replace an amino acid sequence in the structurally disordered region of the protein. Such substitutions can be made, for example, by CRISPR / Cas9 modification.

[0121] Small molecule degrons The present disclosure features small molecule degrons that can be utilized in the chimeras described herein. In one embodiment, the degron is based on thalidomide, which has the structure shown below. [ka]

[0122] In one particular case, when a small molecule degron is conjugated at the N-terminus of a stabilizing peptide, it has the structure shown below. [ka]

[0123] In one particular case, when a small molecule degron is conjugated at the C-terminus of a stabilizing peptide, it has the structure shown below. [ka]

[0124] In certain cases, the small molecule degrons used herein are based on ligands that bind to the von Hippel-Lindau ("VHL") protein and have the structure shown below (compatible with coupling to acid residues). [ka]

[0125] In certain cases, when the small molecule VHL degron is conjugated to an amine, the carboxylate analog shown below is used. [ka]

[0126] Any small molecule degron known in the art can be used in the chimera described herein.In some cases, the small molecule degron used herein is any degron described in U.S. Patent No. 9,694,084; U.S. Patent No. 9,750,816; U.S. Patent No. 9,770,512; U.S. Patent No. 9,821,068; U.S. Patent No. 9,783,575; U.S. Patent No. 9,765,019; U.S. Patent No. 9,632,089; and U.S. Patent No. 9,500,653, the contents of all of which are incorporated herein by reference in their entirety.

[0127] Stabilizing peptide and degron chimeras The present disclosure provides stabilized peptide (e.g., stapled, stitched) chimeras containing degrons (e.g., small molecule degrons, primary sequence degrons, and stabilizing (e.g., stapled) peptide degrons). Such chimeras, previously inaccessible to small molecules, can effectively target a wide range of proteins in conjunction with small molecules (e.g., cereblon-binding molecules) or other small molecule or peptide degron moieties capable of targeted degradation of binding proteins. Similarly, stabilized peptide degrons capable of binding and recruiting degradation-inducing proteins can be combined with small molecules that bind to a wide range of proteins and degrade disease-associated proteins. These novel classes of stapled peptide degron chimeras expand the potential and breadth of biological activity of stapled peptides. Chimeras containing more than one (e.g., two, three, four, or more) stabilized peptides and one degron (e.g., one small molecule degron, one primary sequence degron, or one stabilized (e.g., stapled) peptide degron) are also encompassed herein. Also encompassed herein are chimeras that include more than one (e.g., 2, 3, 4, or more) degrons (e.g., more than one small molecule degron, more than one primary sequence degron, or more than one stabilizing (e.g., stapled) peptide degron) and one stabilizing peptide. Also encompassed herein are chimeras that include 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 degron, more than one primary sequence degron, or more than one stabilizing (e.g., stapled) peptide degron).

[0128] In certain embodiments, the chimeric stapled peptide is not a Bcl-2 homology 3 (BH3) domain polypeptide (e.g., not a BH3 domain from MCL-1, not a MCL-1 stabilizing alpha helix (SAHB) of the BCL2 domain, but rather an MCL-1SAHB). D But not.)

[0129] Stabilized peptide-peptide degron chimera Provided herein are stabilized peptide-peptide degron chimeras. These chimeras are composed of a stabilized peptide and a peptide degron, where the stabilized peptide binds to a first protein, which is a protein targeted for degradation, and the peptide degron binds directly or indirectly to a second protein, which is a substrate adaptor for ubiquitin E3 ligase. Thus, in certain embodiments, the stabilized (e.g., staple, stitch) peptide is linked to the peptide degron. Exemplary chimeras are shown in Figures 7, 15, 17, and 18.

[0130] The stabilizing peptide may be linked to the degron by 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 the chimeras described herein are described below, and a subset is illustrated in Figure 6.

[0131] In certain embodiments, the stabilized peptide has an amino acid sequence set forth in any one of SEQ ID NOS: 1-24 and 134, or a variant thereof. In some embodiments, the peptide degron has an amino acid sequence set forth in any one of SEQ ID NOS: 25-46, 65-118, or a variant thereof. In certain cases, the peptide degron is attached to the N-terminus of the stabilized peptide. In other cases, the peptide degron is attached to the C-terminus of the stabilized peptide. In some cases, one or more degrons are attached to both the N-terminus and the C-terminus of the stabilized peptide. In some cases, the degron is attached to an internal amino acid position of the stabilized peptide (i.e., any amino acid position in the stabilized peptide other than the N- or C-terminus, e.g., positions 2, 3, 4, 5, 6, 7, 8, 9, etc.) in the stabilized peptide. In some cases where more than one degron (e.g., two or three) is attached to the stabilized peptide, one degron may be attached to a terminus of the stabilized peptide and one degron may be attached to an internal position of the stabilized peptide. In some cases where more than one (e.g., two or three) degrons are attached to a stabilized peptide, one degron may be attached to each end of the stabilized peptide. In some cases where more than one (e.g., two or three) degrons are attached to a stabilized peptide, more than one degron is attached to an internal position of the stabilized peptide. Figure 7 shows an exemplary chimera in which a degron is attached to a stabilized peptide at an internal amino acid position.

[0132] In certain cases, the stabilized peptide-peptide degron chimera has the amino acid sequence of one of SEQ ID NOs: 119-126.

[0133] In certain embodiments, a stabilized peptide-peptide degron chimera comprises one or more (e.g., 2, 3, 4, or more) stabilizing peptides and one peptide degron. In certain embodiments, a stabilized peptide-peptide degron chimera comprises one or more (e.g., 2, 3, 4, or more) peptide degrons and one stabilizing peptide. In certain embodiments, a stabilized 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 degrons.

[0134] Each of the chimeric constructs listed in Figures 7, 15, 17, and 18 are encompassed by the present disclosure. Variants of each of the chimeric constructs listed in Figures 7, 15, 17, and 18 are encompassed by the present disclosure.

[0135] In certain embodiments, the chimera is a chimera described in the Examples section below, e.g., see Example 6 below for a non-limiting example of a stabilized peptide-peptide degron chimera.

[0136] Stabilizing peptide-small molecule degron chimeras Stabilizing peptide-small molecule degron chimeras are provided herein. These chimeras are composed of a stabilizing peptide and a small molecule degron, where the stabilizing peptide binds to a first protein that is a target for degradation, and the small molecule degron binds to a second protein that is a degradation-inducing protein. Thus, in certain embodiments, the stabilizing (e.g., staple, stitch) peptide described above is linked to the small molecule degron described above. The stabilizing peptide may be linked to the degron by any linker of interest (e.g., a synthetic compound linker). Exemplary chimeras are shown in Figures 1, 7, 8, 9, 11, 12, 19, and 20.

[0137] In certain embodiments, the first protein is a target for degradation by a 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 protein that causes or is associated with a disease. In some embodiments, the first protein is a killer protein (e.g., BAX, BAK) or a protein that damages 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, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion, MLL fusion, 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., beta-amyloid) that causes neurodegeneration.

[0138] In certain embodiments, the stabilizing peptide has an amino acid sequence set forth in any one of SEQ ID NOs: 1-24, and 134, or a variant thereof.

[0139] In certain embodiments, the stabilizing peptide is linked to the small molecule degron via a linker. Non-limiting examples of linkers that can be used to link the stabilizing peptide and the small molecule to each other and form the chimeras described herein are described below, and a subset is illustrated in Figure 6.

[0140] In certain embodiments, the stabilizing peptide is indirectly attached to the small molecule peptide.

[0141] In certain cases, the small molecule degron is attached to the N-terminus of the stabilizing peptide. In other cases, the small molecule degron is attached to the C-terminus of the stabilizing peptide. In some cases, one or more degrons are attached to both the N-terminus and C-terminus of the stabilizing peptide. In some cases, the degron is attached to an 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., positions 2, 3, 4, 5, 6, 7, 8, 9, etc.). In some cases, more than one (e.g., two or three) degrons are attached to the stabilizing peptide. In some cases where more than one (e.g., two or three) degrons are attached to the stabilizing peptide, one degron may be attached to either end of the stabilizing peptide, and one degron may be attached to an internal position of the stabilizing peptide. In some cases where more than one (e.g., two or three) degrons are attached to the stabilizing peptide, one degron may be attached to each end of the stabilizing peptide. In some cases where more than one (eg, two or three) degrons are attached to a stabilizing peptide, each of the more than one degrons is attached to an internal position of the stabilizing peptide.

[0142] In certain embodiments, the second protein is a degradation-inducing protein, such as an E3 ubiquitin ligase or a substrate adaptor for an E3 ubiquitin ligase. In certain embodiments, the second protein is an E3 ubiquitin ligase. Non-limiting examples of E3 ubiquitin ligases include VHL, COP1, and MDM2. In certain embodiments, the second protein is 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, TR AF7, 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 certain embodiments, the second protein is 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 embodiments, the second protein binds to MDM2 or a protein that forms a complex with MDM2, such as MDMX.

[0143] In certain embodiments, the second protein is a degradation-inducing protein, such as an E3 ubiquitin ligase, or a substrate adaptor for 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 that forms a complex with an E3 ligase, such as MDMX, which binds 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-Bmil, 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 an RBR E3 ubiquitin ligase (e.g., Parkin, Parc, RNF144(A / B), HOIP, HHARI). For non-limiting examples of E3 ubiquitin ligases, see, e.g., Morreale and Walden, Cell 165, 2016 DOI http: / dx.doi.org / 10.1016 / j.cell.2016.03.003.

[0144] In certain embodiments, the small molecule degron is based on thalidomide (see, e.g., the structure above). In certain embodiments, the small molecule degron is based on a ligand that binds to von Hippel-Lindau protein (see, e.g., the structure above). 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. Patent Nos. 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 contents of all of which are incorporated herein by reference in their entireties.

[0145] Non-limiting examples of stapled peptide-small molecule degron chimeras are shown below: [ka] where 8 = R-octenylalanine; B = norleucine; # = cyclobutylalanine; X = S-pentenylalanine, and in some cases, X1 and X2 are the same (e.g., S-pentenylalanine), ^ = thalidomide-aminohexanoic, & = Lys-epsilon-amino-thalidomide, % = A1, A2, A3, A4, A5, A6, A7, A8, A9, A 10 , or A 11 , where A1 = DAB-thalidomide, A2 = DAB-Gly-Thal, A3 = DAB-βAla-Thal, A4 = DAB-linker 1-Thal, A5 = DAB-linker 2-Thal, A6 = DAB-linker 3-Thal, A7 = DAB-linker 4-Thal, A8 = DAB-linker 5-Thal, A9 = DAB-linker 6-Thal, A 10 =DAB-Linker 7-Thal, and A 11=DAB-Linker8-Thal; $=B1, B2, B3, B4, B5, or B6, where B1=DAB-TRIB carboxylate, B2=DAB-Gly-TRIB carboxylate, B3=DAB-βAla-TRIB carboxylate, B4=DAB-Linker3-TRIB carboxylate, B5=DAB-Linker5-TRIB carboxylate, and B6=DAB-Linker7-TRIB carboxylate; @=C1, C2, C3, C4, C5, or C6, where C1=DAB-VHL carboxylate, C2=DAB-Gly-VHL carboxylate, C3=DAB-βAla-VHL carboxylate, C4=DAB-Linker3-VHL carboxylate, C5=DAB-Linker5-VHL carboxylate, and C6=DAB-Linker7-VHL carboxylate, where Linker1-Linker8 are illustrated in Figure 6. In some embodiments, thalidomide-aminohexanoic acid (^) or Lys-epsilon-amino-thalidomide (&) is linked to the staple peptide via a linker.

[0146] In certain embodiments, a stabilized peptide-small molecule degron chimera comprises one or more (e.g., 2, 3, 4, or more) stabilizing peptides and one small molecule degron. In certain embodiments, a stabilized peptide-small molecule degron chimera comprises one or more (e.g., 2, 3, 4, or more) small molecule degrons and one stabilizing peptide. In certain embodiments, a stabilized 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 degrons.

[0147] Each of the chimeric constructs listed above and variants thereof are encompassed by the present disclosure. Each of the chimeric constructs listed in Figures 1, 7, 8, 9, 11, 12, 19, and 20 are encompassed by the present disclosure. Each of the variants of the chimeric constructs listed in Figures 1, 7, 8, 9, 11, 12, 19, and 20 are encompassed by the present disclosure.

[0148] In certain embodiments, the chimera is a chimera described in the Examples section below, see, e.g., Examples 2, 3, 4, and 7 below for non-limiting examples of stabilized peptide-small molecule degron chimeras.

[0149] Stabilizing peptide-stabilizing peptide degron chimera The present specification provides stabilizing peptide-stabilizing peptide degron chimeras.These chimeras are composed of two stabilizing peptides, a first stabilizing peptide and a second stabilizing peptide, where the first stabilizing peptide binds to the first protein, which is the target protein to be degraded, and the second stabilizing peptide binds to the second protein, which is the degradation-inducing protein.Therefore, in certain embodiments, the first stabilizing peptide (e.g., staple, stitch) is linked to the second stabilizing peptide (e.g., staple, stitch) described above.The first and second stabilizing peptides may be linked directly or indirectly.

[0150] In certain embodiments, the first protein is a target protein to be degraded by the second protein or the 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 protein that causes or is associated with a disease. In some embodiments, the first protein is a killer protein (e.g., BAX, BAK) or a protein that damages 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, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion, MLL fusion, 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., beta-amyloid) that causes neurodegeneration.

[0151] In certain embodiments, the first stabilizing peptide has an amino acid sequence set forth in any one of SEQ ID NOs: 1-24, and 134, or a variant thereof.

[0152] In certain embodiments, the first stabilizing peptide is linked to the second stabilizing peptide via a linker. Non-limiting examples of linkers that can be used to link the first and second stabilizing peptides to each other and form the chimeras described herein are described below, and a subset is illustrated in Figure 6.

[0153] In certain embodiments, the first stabilizing peptide is indirectly linked to the second stabilizing peptide.

[0154] In certain embodiments, the second protein is a degradation-inducing protein, such as an E3 ubiquitin ligase or a substrate adaptor for an E3 ubiquitin ligase. In certain embodiments, the second protein is an E3 ubiquitin ligase. Non-limiting examples of E3 ubiquitin ligases include VHL, COP1, and MDM2. In certain embodiments, the second protein is 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, 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 certain embodiments, the second protein is 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 embodiments, the second protein binds to MDM2 or a protein that forms a complex with MDM2, such as MDMX.

[0155] In certain embodiments, the second protein is a degradation-inducing protein, such as an E3 ubiquitin ligase, or a substrate adaptor for 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 that forms a complex with an E3 ligase, such as MDMX, which binds 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-Bmil, 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 an RBR E3 ubiquitin ligase (e.g., Parkin, Parc, RNF144(A / B), HOIP, HHARI). For non-limiting examples of E3 ubiquitin ligases, see, e.g., Morreale and Walden, Cell 165, 2016 DOI http: / dx.doi.org / 10.1016 / j.cell.2016.03.003.

[0156] In certain embodiments, the second stabilizing peptide has the amino acid sequence set forth in SEQ ID NO: 134 or a variant thereof. In certain embodiments, the second stabilizing peptide has the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof. In certain embodiments, the second stabilizing peptide has the amino acid sequence set forth in SEQ ID NO: 18 or a variant thereof.

[0157] In certain embodiments, the second stabilizing peptide is a stabilizing peptide described in U.S. Pat. Nos. 8,889,632, 9,458,202, 9,505,804, 9,527,896, 9,957,299, 10,030,049, and 10,059,741, WO1998 / 001467 and WO2017 / 165617, and U.S. Patent Application Publication No. 2014 / 0018302A1, each of which is incorporated by reference in its entirety.

[0158] In certain cases, the first stabilizing peptide is attached to the N-terminus of the second stabilizing peptide. In other cases, the first stabilizing peptide is attached to the C-terminus of the second stabilizing peptide. In some cases, the first stabilizing peptide is attached to an internal amino acid position of the second stabilizing peptide (i.e., any amino acid position in the stabilizing peptide other than the N- or C-terminus, e.g., positions 2, 3, 4, 5, 6, 7, 8, 9, etc.). In certain cases, the second stabilizing peptide is attached to the N-terminus of the first stabilizing peptide. In other cases, the second stabilizing peptide is attached to the C-terminus of the first stabilizing peptide. In some cases, the second stabilizing peptide is attached to an internal amino acid position of the first stabilizing peptide (i.e., any amino acid position in the stabilizing peptide other than the N- or C-terminus, e.g., positions 2, 3, 4, 5, 6, 7, 8, 9, etc.).

[0159] In certain embodiments, a stabilized peptide-stabilized peptide degron chimera comprises one or more (e.g., two, three, four, or more) stabilized peptides that bind to one or more proteins to be degraded and one stabilized peptide degron that binds to a degradation-inducing protein. In certain embodiments, a stabilized peptide-stabilized peptide degron chimera comprises one or more (e.g., two, three, four, or more) stabilized peptide degrons that bind to one or more degradation-inducing proteins and one stabilized peptide that binds to a protein to be degraded. In certain embodiments, a stabilized peptide-stabilized peptide degron chimera comprises one or more (e.g., two, three, four, or more) stabilized peptides that bind to one or more proteins to be degraded and one or more (e.g., two, three, four, or more) stabilizing peptide degrons that bind to one or more degradation-inducing proteins.

[0160] Each chimeric construct listed in Figure 21 is encompassed by the present disclosure. Variants of each chimeric construct listed in Figure 21 are encompassed by the present disclosure.

[0161] In certain embodiments, the chimera is a chimera described in the Examples section below. See, e.g., Example 8 below for a non-limiting example of a stabilizing peptide-stabilizing peptide degron chimera.

[0162] Small Molecule-Stabilizing Peptide Degron Chimera Provided herein is a small molecule-stabilizing peptide degron chimera.These chimeras are composed of a small molecule and a stabilizing peptide, wherein the small molecule binds to a first protein, which is the target protein to be degraded, and the stabilizing peptide binds to a second protein, which is the degradation-inducing protein.Therefore, in certain embodiments, the small molecule is linked to the above-mentioned stabilizing (for example, staple, stitch) peptide.The small molecule and the stabilizing peptide may be linked directly or indirectly.

[0163] In certain embodiments, the first protein is a target protein to be degraded by the second protein or the 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 protein that causes or is associated with a disease. In some embodiments, the first protein is a killer protein (e.g., BAX, BAK) or a protein that damages 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, β-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI fusion, MLL fusion, 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., beta-amyloid) that causes neurodegeneration.

[0164] In certain embodiments, the small molecule is any drug or chemical compound that, when forming part of the chimera, can bind to a protein without interfering with the ability of the chimeric staple peptide to interact with its target. Assays and methods, such as immunofluorescence and co-immunoprecipitation, for assessing the interference of a drug or chemical compound with the ability of a (chimeric) staple peptide to interact with its target (in the context of the chimera) are known in the art. In certain embodiments, the small molecule is a compound illustrated in Figure 23. In certain embodiments, the small molecule is a kinase inhibitor. In certain embodiments, the small molecule is a histone deacetylase inhibitor.

[0165] In certain embodiments, the small molecule is attached to the stabilizing peptide via a linker. Non-limiting examples of linkers that can be used to attach a small molecule and a stabilizing peptide to each other and form the chimeras described herein are described below, and a subset is illustrated in Figure 6.

[0166] In certain embodiments, the small molecule is indirectly attached to the stabilizing peptide.

[0167] In certain 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 other than the N- or C-terminus, e.g., positions 2, 3, 4, 5, 6, 7, 8, 9, etc.).

[0168] In certain embodiments, the second protein is a degradation-inducing protein, such as an E3 ubiquitin ligase or a substrate adaptor for an E3 ubiquitin ligase. In certain embodiments, the second protein is an E3 ubiquitin ligase. Non-limiting examples of E3 ubiquitin ligases include VHL, COP1, and MDM2. In certain embodiments, the second protein is 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, 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 certain embodiments, the second protein is 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 embodiments, the second protein binds to MDM2 or a protein that forms a complex with MDM2, such as MDMX.

[0169] In certain embodiments, the second protein is a degradation-inducing protein, such as an E3 ubiquitin ligase, or a substrate adaptor for 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 that forms a complex with an E3 ligase, such as MDMX, which binds 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-Bmil, 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 an RBR E3 ubiquitin ligase (e.g., Parkin, Parc, RNF144(A / B), HOIP, HHARI). For non-limiting examples of E3 ubiquitin ligases, see, e.g., Morreale and Walden, Cell 165, 2016 DOI http: / dx.doi.org / 10.1016 / j.cell.2016.03.003.

[0170] In certain embodiments, the stabilizing peptide has the amino acid sequence set forth in SEQ ID NO: 134 or a variant thereof. In certain embodiments, the second stabilizing peptide has the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof. In certain embodiments, the second stabilizing peptide has the amino acid sequence set forth in SEQ ID NO: 18 or a variant thereof.

[0171] In certain embodiments, a small molecule-stabilized peptide degron chimera comprises one or more (e.g., 2, 3, 4, or more) stabilizing peptide degrons and one small molecule. In certain embodiments, a small molecule-stabilized peptide degron chimera comprises one or more (e.g., 2, 3, 4, or more) small molecules and one stabilizing peptide degron. In certain embodiments, a small molecule-stabilized peptide degron chimera comprises one or more (e.g., 2, 3, 4, or more) stabilizing peptide degrons and one or more (e.g., 2, 3, 4, or more) small molecules.

[0172] The chimeric construct illustrated in Figure 23 is encompassed by the present disclosure. Variants of the chimeric construct illustrated in Figure 23 are encompassed by the present disclosure.

[0173] In certain embodiments, the chimera is a chimera described in the Examples section below, e.g., see Example 9 below for a non-limiting example of a small molecule-stabilized peptide degron chimera.

[0174] Linker There are no particular limitations regarding the linkers that can be used in the above-described constructs. In some embodiments, the linker is an amino acid, such as aminopropionic acid, aminobutanoic acid, aminopentanoic acid, or aminohexanoic acid. In some embodiments, the linker is an oligoethylene glycol, i.e., NH2-(CH2-CH2-O) xIn some embodiments, the linker is a peptide linker. In some embodiments, any arbitrary single-chain peptide containing 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 linker. In other embodiments, the linker 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 length. In certain cases, the linker contains only glycine and / or serine residues. Examples of such peptide linkers include Gly, Ser; GlySer; GlyGlySer; SerGlyGly; GlyGlyGlySer (SEQ ID NO: 47); SerGlyGlyGly (SEQ ID NO: 48); GlyGlyGlyGlySer (SEQ ID NO: 49); SerGlyGlyGlyGly (SEQ ID NO: 50); GlyGlyGlyGlyGlySer (SEQ ID NO: 51); SerGlyGlyGlyGlyGly (SEQ ID NO: 52); GlyGlyGlyGlyGlyGlySer (SEQ ID NO: 53); SerGlyGlyGlyGlyGlyGly (SEQ ID NO: 54); (GlyGlyGlyGlySer) n (SEQ ID NO: 49)n (wherein n is an integer of 1 or more); and (SerGlyGlyGlyGly) n (SEQ ID NO:50)n, where n is an integer of 1 or more. In some cases, the linker has the amino acid sequence of SEQ ID NO:4, except that the serine residue is replaced with another amino acid. In some cases, the linker has multiple copies (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 copies) of the amino acid sequence of SEQ ID NO:4, except that the serine residue in each copy of the linker is replaced with another amino acid.

[0175] In other embodiments, the linker peptide is modified so that the amino acid sequence GSG (which occurs at the junction of a traditional Gly / Ser linker peptide repeat) is absent. For example, the peptide linker may be modified to have the structure (GGGXX) n GGGGS (SEQ ID NO: 55) and GGGGS (XGGGS) n (SEQ ID NO: 56), where X is any amino acid that can be inserted into the sequence and does not result in a polypeptide containing the sequence GSG, and n is 0 to 4. In one embodiment, the sequence of the linker peptide is (GGGX1X2) n GGGGS (wherein X1 is P, X2 is S, and n is 0 to 4) (SEQ ID NO: 57). In another embodiment, the sequence of the linker peptide is (GGGX1X2) n GGGGS (wherein X1 is G, X2 is Q, and n is 0 to 4) (SEQ ID NO: 58). In another embodiment, the sequence of the linker peptide is (GGGX1X2) n GGGGS (wherein X1 is G, X2 is A, and n is 0 to 4) (SEQ ID NO: 59). In yet another embodiment, the sequence of the linker peptide is GGGGS(XGGGS) n (wherein X is P and n is 0 to 4) (SEQ ID NO: 60). In one embodiment, a linker peptide of the invention comprises or consists of the amino acid sequence (GGGGA)2GGGGS (SEQ ID NO: 61). In another embodiment, a linker peptide comprises or consists of the amino acid sequence (GGGGQ)2GGGGS (SEQ ID NO: 62). In yet another embodiment, a linker peptide comprises or consists of the amino acid sequence (GGGPS)2GGGGS (SEQ ID NO: 63). In a further embodiment, a linker peptide comprises or consists of the amino acid sequence GGGGS(PGGGS)2 (SEQ ID NO: 64).

[0176] In certain embodiments, the linker is a synthetic compound linker (chemical crosslinker). Examples of commercially available crosslinkers include N-hydroxysuccinimide (NHS), disuccinimidyl 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) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0177] In certain embodiments, the linker is the linker depicted in FIG.

[0178] Methods for synthesizing stabilized peptide degron chimeras Synthesis of stapled peptide-small molecule degron chimeras Carbohydrate-stapled peptides were synthesized as previously reported (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 additional details. The peptides can be synthesized using established methods (i.e., Fmoc-protected amino acids, HATU coupling reagent) until the desired sequence is complete. The peptides are then stapled using Grubbs' catalyst (first generation) and the N-terminus is deprotected using piperidine. A multiatom linker, such as beta-alanine or aminohexanoic acid, is then incorporated. Thalidomide-COOH is then coupled using HCTU. Because the imide bond is particularly sensitive to nucleophiles, piperidine or hydrazine is not used after incorporation. The peptides are then cleaved with TFA for 1 hour and purified by LCMS.

[0179] Synthesis of stapled peptide-peptide degron chimeras The staple peptide portion of the chimera may be synthesized as described above, followed by coupling of a fully protected peptide degron. The fully protected degron peptide can be synthesized on a weak acid-cleavable resin, such as Sieber amide resin, simultaneously with the final synthesis step, which is the reaction of glycolic anhydride with the peptide N-terminus. After cleavage with 1% TFA, the protected peptide is precipitated in ether, dissolved in acetic acid / water, and lyophilized. The fully protected degron peptide is then mixed with a coupling reagent and base and reacted with the resin-bound staple peptide N-terminus for 2 hours, followed by TFA cleavage and purification to yield the staple peptide-peptide degron.

[0180] Synthesis of stapled peptide-stapled peptide degron chimeras The first stapled peptide portion may be synthesized using established methods described above, followed by the incorporation of a linker moiety, such as beta-alanine or aminohexanoic acid. The second half of the stapled peptide chimera may then be synthesized using the same protocol as for the first stapled peptide portion, and the entire chimera may then be stapled using Grubbs' catalyst (first generation), followed by N-terminal acetylation. The chimera is then cleaved with TFA for 1 hour and purified by LCMS.

[0181] Synthesis of small molecule-stapled peptide degron chimeras The stapled peptide portion of the chimera may be synthesized using established methods described above, followed by stapling the peptide using Grubbs' catalyst (first generation), deprotecting the N-terminus with piperidine, and incorporating a multiatom linker, such as beta-alanine or aminohexanoic acid. Coupling of small molecules to the stapled peptide can be carried out as described above.

[0182] The properties and functional activity of the stabilized (eg, stapled) peptide degron chimeras of the invention can be assayed using, for example, the methods described below.

[0183] Binding of stapled peptide degron chimeras to protein targets A competitive fluorescence polarization assay is performed to monitor (1) the ability of the stapled peptide (or molecular) portion of the chimera to retain binding affinity for its protein target and (2) the ability of the degron component (whether molecular or peptide) to retain binding affinity for its protein target. Exemplary fluorescence polarization assays for stapled peptides and molecular degrons include those described by Pitter et al. Methods Enzymol 446: 387-408 (2008). and Nowak et al. Nat Chem Biol 14:706-714 (2018). In cellulo degradation assays using a targeted protein substrate (e.g., GFP-BRD4) can also be used to confirm the ability of the stapled peptide degron chimera to penetrate intact cells and inhibit induced degradation by competing with a positive control molecule degron chimera (e.g., dBET6). An exemplary method for such a competitive cellulolysis assay can be found in Nowak et al. Nat Chem Biol 14:706-714 (2018).

[0184] Monitoring ubiquitination of recombinant protein targets induced by stapled peptide degron chimeras To monitor in vitro ubiquitination of protein targets, a commercially available Mdm2 / HDM2 ubiquitin ligase kit (K-200B) can be used. Briefly, chimeric (10 μM), recombinant full-length MDM2 (GST-tagged, 1 μM), 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 in reaction buffer. The mixture is incubated at 37 °C for 6 h. Then, 20 μL of the reaction mixture is assayed using standard Western blot techniques, thereby visualizing the band shift resulting from ubiquitination using an antibody raised against the target protein.

[0185] Monitoring native protein degradation in cellulo induced by stapled peptide degron chimeras To assay for intracellular protein degradation, cancer cells (e.g., SJSA-1, SJSA-X, U2OS) are passaged in DMEM (Life Technologies, Grand Island, NY) culture medium (CM) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Pen Strep) in a humidity-controlled, CO2-equilibrated incubator at 37°C. The day before treatment, cells are passaged and seeded into 6-well plates at a density of 100,000 cells / mL. 24 hours later, cells are treated with staple peptide degron chimeras (e.g., 10 μM) for 0, 2, 4, and 6 hours, after which they are harvested and lysed. Cell lysates are then assayed using standard Western blot techniques with antibodies raised against the target protein to assess protein levels and actin antibodies against loading controls.

[0186] Monitoring the effects of targeted protein degradation induced by stapled peptide degron chimeras on cancer cell viability Stapled peptide degron chimeras that retain binding to both protein targets, achieve cellular uptake, access their dual targets in cellulo, and induce target protein degradation were developed to assess established cell viability and cell titer. Apoptosis assays, including Glo and caspase 3 / 7 activation assays, were performed as described (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)) and evaluated for their cytotoxic effects on cancer cells. For example, they may be unable to bind to their protein targets and / or cell lines that do not express the target protein and / or engage the degradation-inducing protein of interest. Specificity of action controls studies were performed using mutant peptides.

[0187] Treatment method The chimeras disclosed herein can promote the degradation of disease-related proteins to which the stabilizing peptide or small molecule binds. In certain cases, the degraded protein is a killer protein such as BAX or BAK (which is useful as a cytoprotective agent during stress, such as hypoxia in stroke, neurodegenerative diseases, and heart attacks). In certain cases, the degraded protein is a cell-damaging protein, such as Ig in myeloma, amyloid in Alzheimer's disease, or other protein deposits that lead to disease. In certain cases, the degraded protein is 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 fusions, 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. In certain cases, the protein being 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's disease).

[0188] The present disclosure features methods of using any of the stabilized peptides or chimeras described herein to prevent and / or treat cancer, autoimmune disease, or inflammatory disease. The term "treat" or "treating," as used herein, refers to reducing, inhibiting, or reversing the disease or condition from which a subject suffers.

[0189] The peptides or chimeras described herein may be useful for treating human subjects with cancer. The peptides or chimeras described herein may also be useful for treating human subjects with melanoma, leukemia, lymphoma, or other hematological malignancies or solid tumors. In certain cases, the solid tumor is melanoma, breast cancer, or lung cancer. In some embodiments, the peptides or chimeras described herein may be useful for treating human subjects with autoimmune diseases or other inflammatory conditions that exhibit characteristics of hypercellular diseases. In certain cases, the autoimmune disease is autoimmune colitis, thyroiditis, arthritis, nephritis, dermatitis, vasculitis, systemic lupus erythematosus, diabetes, or Sjogren's disease. In some cases, the inflammatory disease is asthma, psoriasis, inflammatory colitis, thyroiditis, arthritis, nephritis, dermatitis, or vasculitis.

[0190] When endogenous protein (for example, oncogenic protein such as MDM2) is modified to contain degron, any gene editing technology can be used (see, for example, United States Patent No. 9,840,713; United States Patent No. 9,840,702; United States Patent No. 9,840,699; United States Patent No. 9,834,791; United States Patent No. 9,822,372; United States Patent No. 9,816,080; United States Patent No. 9,790,490; United States Patent No. 9,783,490; United States Patent No. 9,771,601; United States Patent No. 9,758,775; United States Patent No. 9,738,908; United States Patent No. 9,616,090; United States Patent No. 9,574,211, all of which are incorporated herein by reference in their entirety).The existence of newly introduced degron should lead to protein degradation.

[0191] Generally, the methods involve 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, repeatedly as needed, to prevent or treat cancer, e.g., melanoma or lymphoma, and may be administered orally, intravenously, or topically. A subject may be selected for treatment based, for example, on a determination that the subject has a cancer that expresses a protein targeted by the staple peptide (e.g., MCL-1, BFL-1).

[0192] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the particular compound used, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, 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.

[0193] An effective amount may be administered in one or more administrations, applications, or dosages. The therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) will depend on the therapeutic compound selected. The composition can be administered from one or more times per day to one or more times per week, including once every other day. Those skilled in the art will understand that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatment, the subject's general health and / or age, and other diseases present, may influence the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a 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.

[0194] Pharmaceutical Composition Any one or more stabilized peptides or chimeras described herein can be formulated as or used in pharmaceutical compositions.Such compositions can be formulated or adapted for administration to subjects via any route, for example, any route approved by the Food and Drug Administration (FDA).Exemplary methods are described in the FDA CDER Data Standards Manual, 004th edition (available at fda.give / cder / dsm / DRG / drg00301.htm).For example, compositions can be administered by inhalation (for example, oral and / or nasal inhalation (for example, via nebulizer or spray)), injection (for example, intravenous, intraarterial, subdermal, intraperitoneal, intramuscular, and / or subcutaneous); and / or may be formulated or adapted for oral, transmucosal, and / or topical administration (including topical (e.g., nasal) sprays and / or solutions).

[0195] In some cases, the pharmaceutical composition may comprise an effective amount of one or more stabilized peptides. The terms "effective amount" and "effective for treatment," as used herein, refer to an amount or concentration of one or more compounds or pharmaceutical compositions described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) effective within the context of its administration (e.g., treatment of an infectious disease) to produce an intended effect or physiological outcome.

[0196] 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 symptoms of the disease.

[0197] The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that may be administered to a patient, together with a compound of the invention, which does not destroy its pharmacological activity and which is non-toxic when administered in dosages sufficient to deliver a therapeutic amount of the compound.

[0198] Pharmaceutically acceptable carriers, adjuvants and vehicles that may 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 used in pharmaceutical dosage forms, such as Tween® or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. Cyclodextrins, such as α-, β-, and γ-cyclodextrin, may also be advantageously used to enhance delivery of the compounds of the formulations described herein.

[0199] The pharmaceutical composition of the present invention can comprise any conventional non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle.In some cases, the pH of formulation can be adjusted with pharmaceutically acceptable acid, base or buffer to enhance the stability of formulated compound or its delivery form.The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0200] Pharmaceutical compositions may be in the form of solutions or powders for inhalation and / or nasal administration. Such compositions may be formulated according to techniques known in the art using suitable dispersants or wetting agents (e.g., Tween® 80) and suspending agents. Sterile injectable preparations may also be injectable sterile solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any sterile fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in injectable preparations, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated forms. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethylcellulose, or similar dispersing agents commonly used in the preparation of pharmaceutically acceptable dosage forms, e.g., emulsions and / or suspensions. Other commonly used surfactants, such as Tween® or Span, and / or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes.

[0201] Pharmaceutical compositions can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.For tablets for oral use, commonly used carriers include lactose and cornstarch.Lubricants, such as magnesium stearate, are also commonly added.For oral administration in capsule form, useful diluents include lactose and dried cornstarch.When aqueous suspensions and / or emulsions are orally administered, the active ingredient may be suspended or dissolved in an oily phase in combination with an emulsifier and / or suspending agent.If necessary, certain sweeteners and / or flavorings and / or coloring agents may be added.

[0202] Alternatively, or additionally, pharmaceutical compositions may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation, and may be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0203] In some cases, one or more peptides disclosed herein can be conjugated to, for example, carrier protein.Such conjugate composition can be monovalent or multivalent.For example, conjugate composition can comprise one peptide disclosed herein conjugated to carrier protein.Alternatively, conjugate composition can comprise two or more peptides disclosed herein conjugated to carrier.

[0204] As used herein, when two entities are "conjugated" to one another, 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 occur when two entities are covalently linked, optionally via a linker group.

[0205] The carrier protein may include any protein that increases or enhances immunogenicity in a subject. Exemplary carrier proteins are described in the art (e.g., 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 (See, e.g., Szu et al., J. Exp. Med. 166:1510-1524, 1987; and Szu et al., Infect. Immun. 62:4440-4444, 1994). The polymeric carrier may be a natural or synthetic material containing one or more primary and / or secondary amino, azide, or carboxyl groups. The carrier may be water-soluble. [Example]

[0206] The following examples are provided to further 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 may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

[0207] Example 1 Synthesis of stapled peptide degron chimeras. The present inventors have generated a series of classes of stapled peptide degron chimeras, including (i) stapled peptide-small molecule degron chimeras, (ii) stapled peptide-peptide degron chimeras, (iii) stapled peptide-staple peptide degron chimeras, and (iv) small molecule-staple peptide degron chimeras. Methods for producing each of these classes of stapled peptide degron chimeras are described below. Figures 26-29 demonstrate the versatility of approaches in designing the stapled peptide portion of these chimeras. Exemplary components of the chimeras are illustrated in Figures 1-7, 15-16, 21, and 23.

[0208] Synthesis of stapled peptide-small molecule degron chimeras Carbohydrate-stapled peptides were synthesized as previously reported (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 additional details: The peptides were synthesized using established methods (i.e., Fmoc-protected amino acids, HATU coupling reagent) until the desired sequence was completed. The peptides were then stapled using Grubbs' catalyst (first generation) and N-terminally deprotected using piperidine. A multiatom linker, such as beta-alanine or aminohexanoic acid, was incorporated (see also linkers in Figure 6). Thalidomide-COOH was then coupled using HCTU. The imide bond is particularly sensitive to nucleophiles; therefore, piperidine or hydrazine was not used after incorporation. The peptides were then cleaved with TFA for 1 h and purified by LCMS.

[0209] Synthesis of stapled peptide-peptide degron chimeras The staple peptide portion of the staple peptide-peptide degron chimera was synthesized as described above (i.e., as for the synthesis of staple peptide-small molecule degron). The staple peptide was then coupled to a fully protected peptide degron. The fully protected degron peptide was synthesized on a weak acid-cleavable resin, specifically, Sieber amide resin, simultaneously with the final synthesis step, which was the reaction of glycolic anhydride with the degron peptide N-terminus. After cleavage with 1% TFA, the protected degron peptide was precipitated in ether, dissolved in acetic acid / water, and lyophilized. The fully protected degron peptide was then mixed with coupling reagents and base and reacted with the resin-bound staple peptide N-terminus for 2 hours, followed by TFA cleavage and purification to yield the staple peptide-peptide degron.

[0210] Synthesis of stapled peptide-stapled peptide degron chimeras The first stapled peptide of the stapled peptide-staple peptide degron chimera was synthesized using the established method described above (i.e., as for the synthesis of the stapled peptide-small molecule degron). A linker moiety, such as beta-alanine or aminohexanoic acid, was then incorporated into the first stapled peptide. The second stapled peptide of the chimera was synthesized using the same protocol as the first stapled peptide. The entire chimera (i.e., both stapled peptides) was then stapled using Grubbs' catalyst (first generation), followed by N-terminal acetylation. The chimera was then cleaved with TFA for 1 hour and purified by LCMS.

[0211] Synthesis of small molecule-stapled peptide degron chimeras The stapled peptide portion of the small molecule-stapled peptide degron chimera was synthesized using established methods described above (i.e., as for the synthesis of stapled peptide-small molecule degrons), followed by stapled peptides using Grubbs' catalyst (first generation), N-terminal deprotection with piperidine, and incorporation of a multiatom linker, e.g., beta-alanine or aminohexanoic acid.

[0212] We generated a small molecule-stapled peptide degron chimera containing JQ1 as the small molecule. The carboxyl group of JQ1 (L. Anders et al., Nat. Biotechnol. 32, 92-96 (2014)) can tolerate chemical substitution, so the resin was loaded with JQ1-acid (11.3 mg, The resulting mixture was incubated with 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 2,6-dioxopiperidin-3-yl-1,3-dioxoisoindolin-4-yl)oxy)acetamide trifluoroacetate (14.5 mg, 0.0281 mmol, 1 eq) and DMF (0.28 ml, 0.1 M) at room temperature. DIPEA (14.7 microliters, 0.0843 mmol, 3 eq) and HATU (10.7 mg, 0.0281 mmol, 1 eq) were added, followed by reaction under nitrogen for 20 hours. The small molecule-staple peptide degron chimera was then cleaved with TFA for 1 hour and purified by LCMS.

[0213] Example 2 The stapled peptide degron chimera retains target protein binding affinity, achieves cellular uptake, and can access its natural target in cellulo. An exemplary fluorescence polarization (FP) binding assay (see Nat Chem Biol. 2018 Jul; 14(7): 706-714) was performed to identify the binding sites of the staple peptide, linker, and thalidomide moiety. We demonstrated that a series of stapled peptide degron chimeras incorporating thalidomide can 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 administration of dBET6, a small molecule proteolysis-targeting chimera ("PROTAC") that binds cereblon and BRD4 and induces BRD4 degradation, we further demonstrated that stapled peptide-thalidomide chimeras can enter cells and compete with dBET6 for cereblon binding, thereby restoring GFP-BRD4 (Figure 9). These data demonstrate that stapled peptide degron chimeras can retain target protein interactions in vitro, access cells, and bind natural proteolysis-inducing drugs in cellulo.

[0214] Example 3 Degradation of anti-apoptotic BCL-2 family proteins targeted by stapled BIM BH3 peptide helix-thalidomide degron chimeras. We applied a stapled peptide helix-degron chimeric peptide (Figure 1) modeled after the pro-apoptotic BIM BH3 domain and incorporating a degron (e.g., Lys-degron, Figures 2-4) to A375P melanoma cells expressing anti-apoptotic BCL-2 family proteins, such as MCL-1, which promotes cancer cell viability and chemotherapy resistance. Treatment with 10 μM compounds followed by Western blot monitoring of cellular MCL-1 levels demonstrated a time-dependent decrease in MCL-1 protein as early as 2 h (Figure 10). Importantly, Western blot control of actin protein levels showed no decrease. These data demonstrate that targeting MCL-1 with the BIM BH3 helix derivatized with a degron moiety reduced MCL-1 protein levels in cancer cells within the treatment timeframe.

[0215] Example 4 Degradation of the MDM2 oncoprotein targeted by a stapled p53 peptide helix-thalidomide degron chimera. A stapled peptide degron chimera modeled on the p53 transactivation domain helix (ATSP-7041) coupled to a thalidomide degron was applied to cultured cancer cells (SJSA-1, SJSA-X). MDM2 protein levels were monitored by Western blot and compared to those in cells treated with ATSP-7041 alone. The experiment was repeated, and cell viability was measured by Cell Titer Glo assay. The data demonstrate that MDM2 levels were reduced in cells treated with thalidomide degron coupled to ATSP-7041 compared to cells treated with ATSP-7041 alone (Figure 11). Furthermore, each stapled peptide degron chimera reduced cancer cell viability in a dose-responsive manner (Figure 12). The composition of the linker influenced the presence (Figure 12, left) or absence (Figure 12, right) of biological activity.

[0216] Example 5 Cop1-mediated protein degradation Primary degrons are defined as peptide motifs containing specific sequence patterns that can be recognized by the cognate ubiquitin E3 ligase. Primary degrons are usually short, linear motifs within structurally disordered protein regions (Guharoy, M. et al., Nat Commun., 7:10239, doi:10.1038 / ncomms10239 (2016)). Recognized by the E3 ligase Cop1. The primary degron sequence from the protein Trib1 is the amino acid sequence DQIVPEY (SEQ ID NO: 25). In the context of the protein Trib1, the sequence DQIVPEY (SEQ ID NO: 25) binds Trib1 to Cop1, allowing Trib1 to function as a substrate adaptor and targeting proteins bound to Trib1 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 Cop1 is 250±40 nM.

[0217] The inventors have found that the sequence DQIVPEY (SEQ ID NO: 25), and its derivatives, which retain binding affinity for Cop1, can be used as portable degron sequences to direct Cop1-mediated degradation of cellular proteins, resulting in therapeutic benefits as described below. (1) Direct genetic modification: Replacing native protein residues in structurally disordered regions with derivatives of the sequence DQIVPEY (SEQ ID NO: 25) can result in the chimeric protein being degraded by Cop1. For example, when exogenously expressed in Cop1-expressing human embryonic kidney 293T cells, replacing the C-terminal sequence GFDVPD (SEQ ID NO: 26) of the p60 isoform of the protein HDM2 with the Trib1-derived sequence DQIVPD (SEQ ID NO: 30) results in Cop1-mediated degradation of the mutant protein (Figure 13). Co-immunoprecipitation studies in 293T cells demonstrate a correlation between degradation upon integration of specific degron sequences (Figure 13) and direct binding between Cop1 and the corresponding Myc-tagged MDM2 p60 mutant construct (e.g., DQIVPD (SEQ ID NO: 30)) (Figure 14). (2) Peptide Ligand Targeting: Conjugation of a derivative of the sequence DQIVPEY (SEQ ID NO: 25) to a protein-targeting stapled peptide can target the stapled peptide's binding partner for Cop1-mediated degradation. The design of these conjugates is the same as that outlined in Figure 1, except that the small molecule thalidomide is replaced with a derivative of the peptide sequence DQIVPEY (SEQ ID NO: 25), and conjugation is achieved via a peptide linker (Figure 15).

[0218] Example 6 Degradation of MDM2 oncoprotein targeted by stapled p53 peptide helix-Trib degron chimera. Stapled peptide degron chimeras modeled on the transactivation domain helix of p53 (ATSP-7041) coupled to a peptide degron modeled on a sequence from Trib that binds to Cop1 were applied to cultured cancer cells (SJSA-1, SJSA-X), and MDM2 protein levels were monitored by Western blot and compared to those in cells treated with ATSP-7041 alone. The experiment was repeated, and cell viability was measured by Cell Titer Glo assay. The data demonstrate that MDM2 levels were reduced in cells treated with the Trib degron coupled to ATSP-7041 compared to cells treated with ATSP-7041 alone (Figure 17). Furthermore, each stapled peptide degron chimera reduced cancer cell viability in a dose-responsive manner (Figure 18).

[0219] Example 7 Degradation of MDM2 oncoprotein targeted by stapled p53 peptide helix-VHL degron chimera. Stapled peptide degron chimeras modeled on the p53 transactivation domain helix (ATSP-7041) coupled to a small molecule degron that binds VHL were applied to cultured cancer cells (SJSA-1, SJSA-X), and MDM2 protein levels were monitored by Western blot and compared to those in cells treated with ATSP-7041 alone. The experiment was repeated, and cell viability was measured by Cell Titer Glo assay. The data demonstrate that MDM2 levels were reduced in cells treated with the VHL degron coupled to ATSP-7041 compared to those in cells treated with ATSP-7041 alone (Figure 19). Furthermore, each stapled peptide degron chimera reduced cancer cell viability in a dose-responsive manner (Figure 20).

[0220] Example 8 Degradation of MCL-1 oncoprotein targeted by selective stapled BH3 peptide helix-stapled p53 peptide degron chimeras. We aimed to selectively target MCL-1 by coupling a stapled peptide degron chimera modeled on the MCL-1 BH3 helix (Figure 21) and a stapled peptide degron modeled on the p53 transactivation domain helix to MCL-1, thereby recruiting MDM2 to MCL-1 and inducing noncanonical MDM2-mediated ubiquitination and degradation. Using an in vitro ubiquitination assay (described below), we observed that adding the stapled peptide degron chimera to recombinant MCL-1, recombinant MDM2, E1 enzyme, E2 enzyme (UBE2D3), and ATP induced MCL-1 ubiquitination (Figure 22). These results indicate that MDM2 was successfully recruited to MCL-1 only in the presence of the stapled peptide degron chimera, indicating that ubiquitin was transferred to MCL-1 by the ubiquitination machinery.

[0221] Example 9 Selective small molecule BRD4 inhibitor-BRD4 oncoprotein degradation targeted by stapled p53 peptide degron chimeras. We coupled small molecules that potently target BRD4 to a stapled peptide degron modeled on the p53 transactivation domain helix (Figure 23) to recruit MDM2 to BRD4, induce non-canonical MDM2-mediated ubiquitination, and degrade BRD4. In a similar manner to that described above (see Example 8), we used an in vitro assay to assess the induced ubiquitination of recombinant MCL-1 protein and tested whether our stapled peptide degron chimeras could recruit MDM2 to ubiquitinated recombinant BRD4 protein (e.g., portions: amino acids 342–460 or amino acids 49–170). Addition of the stapled peptide degron chimeras induced an upshift of each recombinant BRD4 protein, indicating the transfer of ubiquitin to the target BRD4 protein by MDM2 (Figure 24). To assess the effect on native BRD4 levels in cancer cells, U2OS cancer cell lines were treated with our stapled peptide degron chimera at a 10 μM dose, and time-responsive degradation of native BRD4 was observed as reflected by a gradual decrease in BRD4 protein levels over time (e.g., from 0 to 6 hours) (Figure 25). These data demonstrate that our stapled peptide degron chimera can effectively repurpose MDM2 to ubiquitinate BRD4 in vitro and induce native BRD4 degradation in cellulo.

[0222] material and method Monitoring ubiquitination of recombinant protein targets induced by stapled peptide degron chimeras To monitor in vitro ubiquitination of protein targets, we used a commercially available MDM2 / HDM2 ubiquitin ligase kit (K-200B). Briefly, chimeric (10 μM), recombinant full-length MDM2 (GST-tagged, 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 reaction buffer in a 1.5 mL microtube. The mixture was incubated at 37 °C for 6 h. Subsequently, 20 μL of the reaction mixture was assayed using standard Western blot techniques, whereby an antibody raised against the target protein was used to visualize the band shift resulting from ubiquitination.

[0223] Monitoring native protein degradation in cellulo induced by stapled peptide degron chimeras. To assay for intracellular protein degradation, cancer cells (e.g., SJSA-1, SJSA-X, U2OS) were passaged in DMEM (Life Technologies, Grand Island, NY) culture medium (CM) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Pen Strep) in a humidity-controlled, CO2-equilibrated incubator at 37°C. The day before treatment, cells were passaged and seeded at a density of 100,000 cells / mL in 6-well plates. After 24 hours, cells were treated with staple peptide degron chimeras (e.g., 10 μM) for 0, 2, 4, and 6 hours, after which they were harvested and lysed. Cell lysates were then assayed using standard Western blot techniques with antibodies raised against the target protein to assess protein levels and actin antibodies for loading controls.

[0224] Other embodiments While the present invention has been described in conjunction with a detailed description thereof, the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and improvements are within the scope of the following claims. In certain embodiments, for example, the following items are provided: (Item 1) a chimera comprising a first portion linked to a second portion, the first moiety binds to a first protein targeted for degradation; The chimera, wherein the second portion binds to a second protein, and the second protein is a proteolytic agent. (Item 2) 2. The chimera of claim 1, wherein the first portion and the second portion are covalently linked to each other. (Item 3) Item 1, wherein the first portion and the second portion are linked to each other via a linker. (Item 4) 4. The chimera of any one of items 1 to 3, wherein the first protein is a disease-causing or disease-associated protein. (Item 5) 5. The chimera of any one of items 1 to 4, wherein the first protein targeted for degradation is a killer protein, a cell-damaging protein, a protein causing neurodegeneration, 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, MLL fusion, receptor tyrosine kinase, HOX homolog, JUN, cyclin D, cyclin E, BRAF, CRAF, CDK4, CDK2, HPV-E6 / E7, Aurora kinase, MITF, Wnt1, PD-1, BCR, CCR5, a bacterial protein, or a viral protein. (Item 6) 6. The chimera of any one of items 1 to 5, wherein the first portion comprises a first staple peptide that binds to a first protein targeted for degradation. (Item 7) 7. The chimera of item 6, wherein the first staple peptide does not comprise a Bcl-2 homology 3 (BH3) domain polypeptide. (Item 8) 7. The chimera of item 6, wherein the first staple peptide does not include (a) the Bcl-2 homology 3 domain from MCL-1, (b) the MCL-1 stabilizing alpha helix of the BCL2 domain, or (c) the MCL-1 SAHBD. (Item 9) 9. The chimera of any one of items 6 to 8, wherein the second portion is linked to the N-terminus of the first portion. (Item 10) 9. The chimera of any one of items 6 to 8, wherein the second portion is linked to the C-terminus of the first portion. (Item 11) 9. The chimera of any one of items 6 to 8, wherein the second portion is linked to an internal amino acid position of the first portion. (Item 12) 12. The chimera of any one of items 1 to 11, wherein the first portion comprises a small molecule that binds to the first protein targeted for degradation. (Item 13) 12. The chimera of any one of items 1 to 11, wherein the second portion comprises a peptide degron that binds to the proteolysis-inducing drug. (Item 14) 13. The chimera of any one of items 1 to 12, wherein the second portion comprises a second stapled peptide that binds to the proteolysis-inducing drug. (Item 15) 12. The chimera of any one of items 1 to 11, wherein the second portion comprises a small molecule that binds to the protein degradation-inducing drug. (Item 16) 9. The chimera of any one of items 1 to 8, wherein the second portion comprises a second staple peptide that binds to the proteolysis-inducing drug, and the first portion is attached to the N-terminus of the second portion. (Item 17) 9. The chimera of any one of items 1 to 8, wherein the second portion comprises a second staple peptide that binds to the proteolysis-inducing drug, and the first portion is attached to the C-terminus of the second portion. (Item 18) 9. The chimera of any one of items 1 to 8, wherein the second portion comprises a second staple peptide that binds to the proteolysis-inducing drug, and the first portion is attached to an internal amino acid position of the second portion. (Item 19) 19. The chimera of any one of items 1 to 18, wherein the protein degradation-inducing drug degrades the first protein targeted for degradation. (Item 20) 20. A method for treating a disease or disorder caused by a pathological peptide or protein in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a chimera according to any one of items 1 to 19. (Item 21) A chimeric polypeptide comprising a staple peptide and a peptide that binds to a WD40 repeat protein, which is a substrate adaptor for E3 ubiquitin ligase, wherein the chimeric polypeptide comprises a natural binding sequence or a modified version of a natural binding consensus sequence of an amino acid sequence that binds to the WD40 repeat protein, wherein the modified version 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. (Item 22) The WD40 repeat proteins, which are substrate adaptors for E3 ubiquitin 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, T 22. The chimeric polypeptide of item 21, selected from the group consisting of RAF7, 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. (Item 23) 22. The chimeric polypeptide according to item 21, wherein the natural binding consensus sequence is a sequence selected from the group consisting of SEQ ID NOs: 25, 31 to 46, and 65 to 105. (Item 24) 22. The chimeric polypeptide of item 21, wherein the native binding sequence is SEQ ID NO: 25. (Item 25) 22. The chimeric polypeptide of item 21, wherein the natural binding consensus sequence is SEQ ID NO: 46. (Item 26) 25. The chimeric polypeptide according to item 24, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except that the peptide has at least one amino acid substitution. (Item 27) 25. The chimeric polypeptide according to item 24, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except that the peptide has at least one amino acid deletion. (Item 28) 25. The chimeric polypeptide according to item 24, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except that the peptide has at least one amino acid substitution and at least one amino acid deletion. (Item 29) 25. The chimeric polypeptide according to item 24, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except that the peptide has 1 to 6 amino acid substitutions. (Item 30) 25. The chimeric polypeptide according to item 24, wherein positions 4 (V) and 5 (P) of SEQ ID NO: 25 are not substituted. (Item 31) 25. The chimeric polypeptide according to item 24, wherein one or more of positions 1 (D), 2 (Q), 3 (I), and 6 (E) of SEQ ID NO: 25 are substituted. (Item 32) 25. The chimeric polypeptide according to item 24, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except that it has one amino acid deletion. (Item 33) 25. The chimeric polypeptide according to item 24, wherein position 7 (Y) of the amino acid sequence set forth in SEQ ID NO: 25 is deleted. (Item 34) 25. The chimeric polypeptide according to item 24, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except that the peptide has 1 to 6 amino acid substitutions and at least 1 amino acid deletion. (Item 35) 25. The chimeric polypeptide of item 24, wherein the peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 to 30. (Item 36) 25. The chimeric polypeptide according to item 24, wherein the peptide has the amino acid sequence set forth in SEQ ID NO: 30. (Item 37) 37. The chimeric polypeptide of any one of items 24 to 36, wherein the peptide is 4 to 30 amino acids in length. (Item 38) The peptide is 1nM to 300nM; 10nM to 300nM; 100 nM to 300 nM; or 200nM to 300nM 38. The chimeric polypeptide of any one of items 24 and 25 to 37, which binds to Cop1 with a binding affinity of (Item 39) 40. The chimeric polypeptide of any one of claims 24 to 38, wherein the stapled peptide targets an intracellular protein, an extracellular protein, or a cell surface protein. 39. The chimeric polypeptide of any one of items 24 to 38, wherein the stapled peptide targets a protein that causes or is associated with a disease. (Item 41) 39. The chimeric polypeptide of any one of items 24 to 38, wherein the stapled peptide targets a killer protein (e.g., BAX, BAK) or a cell-damaging protein that causes neurodegeneration (e.g., IgG, beta-amyloid, tau, alpha-synuclein, TDP-43, HbS, superoxide dismutase, Notch3, FUS, GFAP). (Item 42) 29. The chimeric polypeptide of any one of paragraphs 24 to 28, wherein the stapled peptide targets 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 fusions, MLL fusions, 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. (Item 43) 29. The chimeric polypeptide of any one of items 24 to 28, wherein the staple peptide targets a bacterial protein. (Item 44) 39. The chimeric polypeptide of any one of items 24 to 38, wherein the staple peptide targets a viral protein. (Item 45) A modified protein of a first protein comprising a structurally irregular region, wherein the modified protein differs from the first protein in that the structurally irregular region comprises a peptide that binds to a WD40 repeat protein, which is a substrate adaptor for E3 ubiquitin ligase, and the peptide comprises a native binding sequence or a modified version of a native binding consensus sequence, wherein the modified version 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. (Item 46) The WD40 repeat protein, which is a substrate adaptor for 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, 46. ​​The modified protein of paragraph 45, selected from the group consisting of 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. (Item 47) 46. ​​The modified protein according to Item 45, wherein the native binding sequence or the native binding consensus sequence is a sequence selected from the group consisting of SEQ ID NOs: 25, 31 to 46, and 65 to 105. (Item 48) 46. ​​The engineered protein of claim 45, wherein the native binding sequence is SEQ ID NO: 25. (Item 49) 46. ​​The engineered protein of claim 45, wherein the natural binding consensus sequence is SEQ ID NO: 46. (Item 50) 49. The modified protein of item 48, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for having at least one amino acid substitution. (Item 51) 49. The modified protein of item 48, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except that the peptide has at least one amino acid deletion. (Item 52) 49. The modified protein of item 48, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except that the peptide has at least one amino acid substitution and at least one amino acid deletion. (Item 53) 49. The modified protein of item 48, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except that the peptide has one to six amino acid substitutions. (Item 54) 49. The modified protein of item 48, wherein positions 4 (V) and 5 (P) of SEQ ID NO: 25 are not substituted. (Item 55) 49. The modified protein of item 48, wherein one or more of positions 1 (D), 2 (Q), 3 (I), and 6 (E) of SEQ ID NO: 25 are substituted. (Item 56) 49. The modified protein of item 48, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 25, except for one amino acid deletion. (Item 57) 49. The modified protein according to Item 48, wherein position 7 (Y) of the amino acid sequence set forth in SEQ ID NO: 25 is deleted. (Item 58) 49. The peptide according to item 48, comprising the amino acid sequence set forth in SEQ ID NO: 25, except that it has 1 to 6 amino acid substitutions and at least 1 amino acid deletion. (Item 59) 49. The peptide according to item 48, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 to 30. (Item 60) 49. The peptide according to item 48, having the amino acid sequence set forth in SEQ ID NO: 30. (Item 61) 61. The peptide according to any one of items 45 to 60, which is 4 to 10 amino acids in length. (Item 62) 1nM to 300nM; 10nM to 300nM; 100 nM to 300 nM; or 200nM to 300nM 62. The peptide of any one of items 48 or 50 to 61, which binds to Cop1 with a binding affinity of (Item 63) 45. A method of treating a disease or disorder caused by a pathological peptide or protein in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a chimeric fusion polypeptide according to any one of items 21 to 44. (Item 64) A peptide-small molecule fusion comprising a protein-targeting staple peptide and a thalidomide degron moiety. (Item 65) 65. The peptide small molecule fusion of item 64, wherein the thalidomide moiety is conjugated to the N-terminus of the protein-targeting stapled peptide. (Item 66) 65. The peptide small molecule fusion of item 64, wherein the thalidomide moiety is conjugated to the C-terminus of the protein-targeting staple peptide. (Item 67) 65. The peptide small molecule fusion of item 64, wherein the thalidomide moiety is contained within an unnatural amino acid inserted into a peptide sequence between the N-terminus and C-terminus of the protein-targeting staple peptide. (Item 68) 68. The peptide-small molecule fusion of any one of items 64 to 67, wherein the stapled peptide targets a disease-causing protein. (Item 69) 70. The peptide-small molecule fusion of any one of claims 64 to 67, wherein the stapled peptide targets an intracellular protein, a receptor protein, or an extracellular protein. 68. The peptide-small molecule fusion of any one of paragraphs 64 to 67, wherein the stapled peptide targets a killer protein (e.g., BAX, BAK) or a cell-damaging protein that causes neurodegeneration (e.g., IgG, beta-amyloid, tau, alpha-synuclein, TDP-43, HbS, superoxide dismutase, Notch3, FUS, GFAP). (Item 71) 70. The peptide small molecule fusion of item 69, wherein the intracellular protein, receptor protein, or extracellular protein is selected from the group consisting of BCL2, BCLXL, MCL-1, BFL-1, BCL-w, BCL-B, EZH2, HDM2 / HDMX, KRAS / NRAS / HRAS, MYC, b-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI, MLL fusions, 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. (Item 72) 68. The peptide-small molecule fusion of any one of items 64 to 67, wherein the staple peptide targets a bacterial protein. (Item 73) 68. The peptide-small molecule fusion of any one of items 64 to 67, wherein the stapled peptide targets a viral protein. (Item 74) 74. The peptide small molecule fusion of any one of items 64 to 73, wherein the thalidomide portion comprises the structure shown below: [ka] (Item 75) 74. The peptide small molecule fusion of any one of items 64 and 67 to 73, wherein the thalidomide portion comprises the structure shown below: [ka] (Item 76) 74. The peptide small molecule fusion of any one of items 68 to 73, wherein the thalidomide portion comprises the structure shown below: [ka] (Item 77) 77. A method of treating a disease or disorder caused by a pathological peptide or protein in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a peptide small molecule fusion according to any one of items 64 to 76. (Item 78) A peptide-small molecule fusion comprising a protein-targeting stapled peptide and a von Hippel-Lindau (VHL) degron moiety. (Item 79) 79. The peptide-small molecule fusion of claim 78, wherein the VHL degron portion is conjugated to the N-terminus of the protein-targeting stapled peptide. (Item 80) 80. The peptide-small molecule fusion of item 79, wherein the VHL degron portion comprises the following structure: [ka] (Item 81) 79. The peptide-small molecule fusion of claim 78, wherein the VHL degron portion is conjugated to the C-terminus of the protein-targeting stapled peptide. (Item 82) 82. The peptide small molecule of item 81, wherein the VHL degron portion comprises the following structure: [ka] (Item 83) 79. The peptide small molecule fusion of claim 78, wherein the thalidomide moiety is contained within an unnatural amino acid inserted into a peptide sequence between the N-terminus and C-terminus of the protein-targeting staple peptide. (Item 84) 84. The peptide-small molecule fusion of any one of items 78 to 83, wherein the stapled peptide targets a disease-causing protein. (Item 85) 86. The peptide-small molecule fusion of any one of claims 78 to 83, wherein the stapled peptide targets an intracellular protein, a receptor protein, or an extracellular protein. 85. The peptide-small molecule fusion of any one of paragraphs 78 to 84, wherein the stapled peptide targets a killer protein (e.g., BAX, BAK) or a cell-damaging protein that causes neurodegeneration (e.g., IgG, beta-amyloid, tau, alpha-synuclein, TDP-43, HbS, superoxide dismutase, Notch3, FUS, GFAP). (Item 87) 86. The peptide small molecule fusion of item 85, wherein the intracellular protein, receptor protein, or extracellular protein is selected from the group consisting of BCL2, BCLXL, MCL-1, BFL-1, BCL-w, BCL-B, EZH2, HDM2 / HDMX, KRAS / NRAS / HRAS, MYC, b-catenin, PI3K, PTEN, TSC, AKT, BRCA1 / 2, EWS-FLI, MLL fusions, 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. (Item 88) 84. The peptide-small molecule fusion of any one of items 78 to 83, wherein the stapled peptide targets a bacterial protein. (Item 89) 84. The peptide-small molecule fusion of any one of items 78 to 83, wherein the stapled peptide targets a viral protein. (Item 90) 90. A method of treating a disease or disorder caused by a pathological peptide or protein in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a peptide small molecule fusion according to any one of items 78 to 89. (Item 91) A peptide that binds to constitutive photomorphogenesis 1 (Cop1) protein, comprising a modified version of the amino acid sequence DQIVPEY (SEQ ID NO: 25), wherein the modified version 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, but when the modified version consists of a single amino acid substitution, the amino acid substitution is not made to A or R at any one of positions 1 to 7 of SEQ ID NO: 25, nor to V at position 4 of SEQ ID NO: 25. (Item 92) 92. The peptide according to item 91, comprising the amino acid sequence set forth in SEQ ID NO: 25, except for having at least one amino acid substitution. (Item 93) 92. The peptide according to Item 91, comprising the amino acid sequence set forth in SEQ ID NO: 25, except that it has at least one amino acid deletion. (Item 94) 92. The peptide according to item 91, comprising 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. (Item 95) 92. The peptide according to item 91, comprising the amino acid sequence set forth in SEQ ID NO: 25, except that it has 1 to 6 amino acid substitutions. (Item 96) 92. The peptide according to item 91, wherein position 4 (V) and / or position 5 (P) of SEQ ID NO: 25 is not substituted. (Item 97) 92. The peptide according to item 91, wherein one or more of positions 1 (D), 2 (Q), 3 (I), and 6 (E) of SEQ ID NO: 25 are substituted. (Item 98) 92. The peptide according to item 91, comprising the amino acid sequence set forth in SEQ ID NO: 25, except that it has one amino acid deletion. (Item 99) 92. The peptide according to Item 91, wherein position 7 (Y) of the amino acid sequence set forth in SEQ ID NO: 25 is deleted. (Item 100) 92. The peptide according to item 91, comprising the amino acid sequence set forth in SEQ ID NO: 25, except that it has 1 to 6 amino acid substitutions and at least 1 amino acid deletion. (Item 101) 92. The peptide according to item 91, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 to 30. (Item 102) 92. The peptide according to item 91, having the amino acid sequence set forth in SEQ ID NO: 30. (Item 103) 103. The peptide according to any one of items 91 to 102, which is 4 to 10 amino acids in length. (Item 104) 103. The peptide of any one of items 91 to 102, which binds to Cop1 with a binding affinity of 1 nM to 300 nM. (Item 105) 103. The peptide of any one of items 91 to 102, which binds to Cop1 with a binding affinity of 10 nM to 300 nM. (Item 106) 103. The peptide of any one of items 91 to 102, which binds to Cop1 with a binding affinity of 100 nM to 300 nM. (Item 107) 103. The peptide of any one of items 91 to 102, which binds to Cop1 with a binding affinity of 200 nM to 300 nM. (Item 108) 108. A chimeric fusion polypeptide comprising a protein-targeting staple peptide and the peptide of any one of items 91 to 107. (Item 109) 109. The chimeric fusion polypeptide of claim 108, wherein the stapled peptide targets an intracellular protein or a cell surface receptor. (Item 110) 109. The chimeric fusion polypeptide of claim 108, wherein the stapled peptide targets a protein that causes or is associated with a disease. (Item 111) 109. The chimeric fusion polypeptide of paragraph 108, wherein the stapled peptide targets a killer protein (e.g., BAX, BAK) or a cell-damaging protein that causes neurodegeneration (e.g., IgG, beta-amyloid, tau, alpha-synuclein, TDP-43, HbS, superoxide dismutase, Notch3, FUS, GFAP). (Item 112) 113. The chimeric fusion polypeptide of claim 109, wherein the intracellular protein or cell surface receptor is 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 fusions, MLL fusions, 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. 109. The chimeric fusion polypeptide of claim 108, wherein the staple peptide targets a bacterial protein. (Item 114) 109. The chimeric fusion polypeptide of claim 108, wherein the staple peptide targets a viral protein. (Item 115) 115. A method of treating a disease or disorder caused by a pathological peptide or protein in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the chimeric fusion polypeptide of any one of items 108 to 114.

Claims

Claim 1: A chimera comprising a first portion linked to a second portion, the first portion comprises a small molecule that binds to BRD4; the second portion comprises a stapled peptide that binds to MDM2; the MDM2-binding stapled peptide comprises a sequence of LTF8EYWAQ#XSAA or LTF8EYWAQLXSAA (SEQ ID NO: 134), wherein 8 is R-octenylalanine, # is cyclobutylalanine, and X is S-pentenylalanine, but has 1 to 3 amino acid substitutions at positions other than 8 and X; The first portion and the second portion are: 【Chemistry 1】 are bonded to each other via a linker comprising the structure The chimera.

2. The chimera described in claim 1, wherein the 1 to 3 amino acid substitutions are selected from conservative amino acid substitutions and substitutions with alanine.

3. The chimera described in claim 1, wherein the 1 to 3 amino acid substitutions are substitutions with alanine.

4. The chimera according to claim 1, wherein the staple peptide that binds to MDM2 consists of a sequence having 1 to 3 amino acid substitutions at positions other than the 8 and the X in the sequence LTF8EYWAQ#XSAA or LTF8EYWAQLXSAA (SEQ ID NO: 134).

5. A chimera comprising a first portion bound to a second portion, the first portion comprises a small molecule that binds to BRD4; the second portion comprises a stapled peptide that binds to MDM2; the MDM2-binding stapled peptide comprises the sequence LTF8EYWAQ#XSAA or LTF8EYWAQLXSAA (SEQ ID NO: 134), wherein the 8 is R-octenylalanine, the # is cyclobutylalanine, and the X is S-pentenylalanine; The first portion and the second portion are: 【Chemistry 2】 are bonded to each other via a linker comprising the structure The chimera

6. The chimera described in claim 5, wherein the staple peptide that binds to MDM2 has the sequence LTF8EYWAQ#XSAA or LTF8EYWAQLXSAA (sequence number: 134).

7. A chimera described in any one of claims 1 to 6, wherein the first portion is bound to the N-terminus of the second portion.

8. A chimera described in any one of claims 1 to 6, wherein the first portion is bound to the C-terminus of the second portion.

9. A chimera described in any one of claims 1 to 6, wherein the first portion is bound to an internal amino acid position of the second portion.

10. A chimera described in any one of claims 1 to 9, wherein the small molecule is JQ1.

11. The method of claim 10, wherein the first moiety attached to the linker is: 【Transformation 3】 (Wherein, Linker is the linker.) The chimera of claim 10 having the structure:

12. A composition for use in treating a disease or disorder caused by a pathological peptide or protein in a subject in need of treatment, the composition comprising a chimera described in any one of claims 1 to 11.

13. The composition of claim 12, wherein the pathological peptide or protein binds to the chimera.

14. The composition described in claim 12 or 13, wherein the protein targeted for degradation is BRD4 and the disease or disorder is cancer.

15. A composition described in any one of claims 12 to 14, wherein the subject is a human.

16. A pharmaceutical composition comprising a chimera according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier and / or vehicle.

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