Bifunctional stapled polypeptides and uses thereof

• US 9,163,330 B2
• Filed: 07/13/2010
• Issued: 10/20/2015
• Est. Priority Date: 07/13/2009
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Verdine, Gregory L., Grossmann, Tom N., Moellering, Raymond E., Yeh, Tsung-Han Johannes, Oak, Youbean, Liang, Yue Rebecca Yue
• Primary Examiner(s): Skowronek, Karlheinz R
• Assistant Examiner(s): MADER, CATHERINE J
• Application Number:
  US13/383,881
• Publication Number:
  US 20120270800A1
• Time in Patent Office: 1,925 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: A61K 47/65 Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• CPC Class Codes: A61K 47/66 the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: C07K 1/107 by chemical modification of precursor peptides
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 1/113 without change of the primary structure
• CPC Class Codes: C07K 14/435 from animals; from humans
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/4747 Apoptosis related proteins
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/01 containing a localisation/targetting motif
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12Y 603/02019 Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding

Abstract

The invention relates to bifunctional stapled or stitched peptides comprising a targeting domain, a linker moiety, and an effector domain, that can be used to tether, or to bring into close proximity, at least two cellular entities (e.g., proteins). Certain aspects relate to bifunctional stapled or stitched peptides that bind to an effector biomolecule through the effector domain and bind to a target biomolecule through the targeting domain. Polypeptides and/or polypeptide complexes that are tethered by the bifunctional stapled or stitched peptides of the invention, where the effector polypeptide bound to the effector domain of the bifunctional stapled or stitched peptide modifies or alters the target polypeptide bound to the targeting domain of the bifunctional peptide. Uses of the inventive bifunctional stapled or stitched peptides including methods for treatment of disease (e.g., cancer, inflammatory diseases) are also provided.

Claims

1. A bifunctional peptide comprising:

a targeting domain;

a linker moiety; and

an effector domain;

wherein

the linker moiety links the targeting domain to the effector domain, and

both the targeting domain and the effector domain are independently stapled or stitched peptides.

2. The peptide of claim 1, wherein both the targeting domain and the effector domain are stapled.

3. The peptide of claim 1, wherein at least one of the targeting domain and the effector domain comprises a sub-domain of Formula (I):

4. The peptide of claim 3, wherein is a double bond in the sub-domain of Formula (I).

5. The peptide of claim 1, wherein at least one of the targeting domain and the effector domain comprises a sub-domain of Formula (II):

6. The peptide of claim 5, wherein is a double bond in the sub-domain of Formula (II).

7. The peptide of claim 1, wherein the linker moiety is an aminohexanoic acid monomer or a polymer of aminohexanoic acid.

8. The peptide of claim 1, wherein the linker moiety is an aminohexanoic acid monomer.

9. The peptide of claim 1, wherein the linker moiety is a polymer of aminohexanoic acid.

10. The peptide of claim 1, wherein the targeting domain binds β

-catenin, Myc, Ras, or hypoxia-inducible factor.

11. The peptide of claim 1, wherein the targeting domain is a ligand for β

-catenin.

12. The peptide of claim 11, wherein the targeting domain is a stapled Bcl9 or a derivative thereof, or a stapled Tcf-4 or a derivative thereof.

13. The peptide of claim 12, wherein the targeting domain is a stapled Bcl9 or a derivative thereof.

14. The peptide of claim 12, wherein the targeting domain is a stapled Tcf-4 or a derivative thereof.

15. The peptide of claim 1, wherein the targeting domain is a ligand for Myc.

16. The peptide of claim 15, wherein the targeting domain is a stapled Max or a derivative thereof, a stapled Mad or a derivative thereof, or a stapled Mxi or a derivative thereof.

17. The peptide of claim 1, wherein the targeting domain is a ligand for Ras or hypoxia-inducible factor.

18. The peptide of claim 1, wherein the effector domain modulates the activity of an enzyme.

19. The peptide of claim 18, wherein the enzyme is a ubiquitinating enzyme, a glycosylating enzyme, a histone deacetylase, a histone acetyl transferase, a phosphorylating enzyme, or a dephosphorylating enzyme.

20. The peptide of claim 1, wherein the effector domain is a ligand for a ubiquitinating enzyme.

21. The peptide of claim 20, wherein the ubiquitinating enzyme is an E3 ubiquitin ligase.

22. The peptide of claim 21, wherein the E3 ubiquitin ligase is hDM2, MDM2, ubiquitin protein ligase E3A, a RING finger domain, or an SCF E3 ligase complex.

23. The peptide of claim 22, wherein the E3 ubiquitin ligase is hDM2, and the ligand for hDM2 is a stapled p53.

24. The peptide of claim 1, wherein the effector domain is a ligand for a glycosylating enzyme, a histone deacetylase, a histone acetyl transferase, a kinase, or a phosphatase.

25. The peptide of claim 1, having the Formula (III):

26. The peptide of claim 25, wherein

27. The peptide of claim 25, wherein

28. The peptide of claim 25, wherein is a linker comprising 2, 3, or 4 aminohexanoic acid residues.

29. The peptide of claim 25, having the sequence DELISFKDEGEQE(β

Ala)₂ERDLS₅DVKS₅SLVN(Ahx)_nQSQQTFR₈NLWRLLS₅QN (SEQ ID NO;

     15) or DELISFKDEGEQE(β

Ala)₂ER₈DLADVKS₅SLVN(Ahx)_nQSQQTFR₈NLWRLLS₅QN (SEQ ID NO;

     16), wherein β

Ala is β

-alanine, Ahx is aminohexanoic acid, and n is 2, 3, or 4.

30. The peptide of claim 1, having the Formula (IV):

31. The peptide of claim 30, wherein

32. The peptide of claim 30, wherein

33. The peptide of claim 30, wherein is a linker comprising 2, 3, or 4 aminohexanoic acid residues.

34. The peptide of claim 30, having the sequence QSQQTFR₈NLWRLLS₅QN(Ahx)_nDELISFKDEGEQE(β

Ala)₂ERDLS₅DVKS₅SLVN (SEQ ID NO;

     13) or QSQQTFR₈NLWRLLS₅QN(Ahx)_nDELISFKDEGEQE(β

Ala)₂ER₈DLADVKS₅SLVN (SEQ ID NO;

     14), wherein β

Ala is β

-alanine, Ahx is aminohexanoic acid, and n is 2, 3, or 4.

35. The peptide of claim 1, having the Formula (V):

36. The peptide of claim 35, wherein

37. The peptide of claim 35, wherein is a linker comprising 2, 3, or 4 aminohexanoic acid residues.

38. The peptide of claim 35, wherein

39. The peptide of claim 35, having the sequence SQEQLR₈HRERSLS₅TLRDIQRMLF(Ahx)_nQSQQTFR₈NLWRLLS₅QN (SEQ ID NO:

4), SQEQLEHRERSLS₅TLRS₅IQRMLF(Ahx)_nQSQQTFR₈NLWRLLS₅QN (SEQ ID NO;

     5), or SQEQLEHRS₅RSLS₅TLRDIQRMLF(Ahx)_nQSQQTFR₈NLWRLLS₅QN (SEQ ID NO;

     6), wherein Ahx is aminohexanoic acid, and n is 2, 3, or 4.

40. The peptide of claim 1, having the Formula (VI):

41. The peptide of claim 40, having the sequence:

42. A composition comprising the peptide of claim 1, and an excipient.

43. A method of treating a cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of the peptide of claim 1, to the subject in need thereof;

wherein the cancer is selected from the group consisting of carcinoma, sarcoma, metastatic cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing'"'"'s tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm'"'"'s tumor, cervical cancer, testicular cancer, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, Kaposi'"'"'s sarcoma, and any combination thereof.

44. The peptide of claim 1, wherein the targeting domain is a ligand for Sin3.

45. The peptide of claim 44, wherein the ligand for Sin3 is a stapled Sin3 interacting domain (SID) of Mad1 or a derivative thereof.

46. The peptide of claim 45, wherein the stapled Sin3 interacting domain (SID) of Mad1 comprises the sequence:

47. The peptide of claim 45, wherein the stapled Sin3 interacting domain (SID) of Mad1 comprises the sequence:

48. The peptide of claim 1, wherein the effector domain is a stapled, truncated MLL.

49. The peptide of claim 1, wherein the effector domain is a stapled, truncated cMyb.

50. The peptide of claim 48, wherein the effector domain comprises the sequence:

51. The peptide of claim 49, wherein the effector domain comprises the sequence:

52. The peptide of claim 3, wherein R^a is hydrogen.

53. The peptide of claim 3, wherein R^b is hydrogen.

54. The peptide of claim 3, wherein R^b is alkyl.

55. The peptide of claim 3, wherein R^b is methyl.

56. The peptide of claim 1, having the sequence QSQQTFR8NLWRLLS₅QN(Ahx)₂NPES₅ILDS₅HVQRVMR (SEQ ID NO:

26), wherein Ahx is aminohexanoic acid.