G protein coupled receptor agonists and antagonists and methods of activating and inhibiting G protein coupled receptors using the same

• US 7,696,168 B2
• Filed: 09/20/2002
• Issued: 04/13/2010
• Est. Priority Date: 04/21/2000
• Status: Expired due to Fees

• Current Assignee: Tufts Medical Center Incorporated (Healthcare Realty Trust, Inc.)
• Original Assignee: Tufts Medical Center Incorporated (Healthcare Realty Trust, Inc.)
• Inventors: Covic, Lidija, Kuliopulos, Athan
• Primary Examiner(s): Allen; Marianne P
• Application Number:
  US10/251,703
• Publication Number:
  US 20030148449A1
• Time in Patent Office: 2,762 Days
• Field of Search: None
• US Class Current: 514/20.6
• 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: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/723 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• CPC Class Codes: G01N 33/567 utilising isolate of tissue or organ as binding agent

Abstract

The invention relates generally to G protein coupled receptors and in particular to agonists and antagonists of G protein receptors and methods of using the same.

Claims

1. A polypeptide comprising:

a) a first domain comprising a third intracellular loop (i3 loop), or a fragment thereof of a G protein coupled receptor (GPCR), and

b) a second domain, attached to the first domain, wherein said second domain is a naturally or non-naturally occurring cell-penetrating, membrane-tethering moiety, wherein said polypeptide is a GPCR agonist or antagonist and does not comprise an extracellular portion of the GPCR and said first domain is at least 3 contiguous amino acid residues.

2. The polypeptide of claim 1, wherein said first domain comprises at least 5 contiguous amino acid residues of the third intracellular loop.

3. The polypeptide of claim 1, wherein said first domain comprises at least 7 contiguous amino acid residues of the third intracellular loop.

4. The polypeptide of claim 1, wherein the first domain comprises 7 to 24 contiguous amino acid residues of the third intracellular loop.

5. The polypeptide of claim 1, wherein the cell-penetrating, membrane-tethering moiety is hydrophobic.

6. The polypeptide of claim 1, wherein the cell-penetrating, membrane-tethering moiety is non-peptidic.

7. The polypeptide of claim 6, wherein the moiety comprises a lipid.

8. The polypeptide of claim 6, wherein the moiety is selected from the group consisting of cholesterol, phospholipid, steroid, sphingosine, ceramide, octyl-glycine, 2-cyclohexylalanine, benzolylphenylalanine, and fatty acid.

9. The polypeptide of claim 8, wherein the fatty acid moiety is selected from the group consisting of a nonanoyl (C₉), capryl (C₁₀), undecanoyl (C₁₁), lauroyl (C₁₂), tridecanoyl (C₁₃), myristoyl (C₁₄), pentadecanoyl (C₁₅), palmitoyl (C₁₆), phytanoyl (methyl substituted C₁₆), heptadecanoyl (C₁₇), stearoyl (C₁₈), nonadecanoyl (C₁₉), arachidoyl (C₂₀), heniecosanoyl (C₂₁), behenoyl (C₂₂), trucisanoyl (C₂₃), and a lignoceroyl (C₂₄) group.

10. The polypeptide of claim 9, wherein the fatty acid moiety is the palmitoyl or myristoyl group.

11. The polypeptide of claim 1, wherein said fragment comprises one or more peptide bonds that have been replaced with a covalent bond which is not susceptible to cleavage by peptidases.

12. The polypeptide of claim 1, wherein said GPCR is selected from the group consisting of a luteinizing hormone receptor, a follicle stimulating hormone receptor, a thyroid stimulating hormone receptor, a calcitonin receptor, a glucagon receptor, a glucagon-like peptide 1 receptor (GLP-1), a metabotropic glutamate receptor, a parathyroid hormone receptor, a vasoactive intestinal peptide receptor, a secretin receptor, a growth hormone releasing factor (GRF) receptor, protease-activated receptors (PARs), cholecystokinin receptors, somatostatin receptors, melanocortin receptors, ADP receptors, adenosine receptors, thromboxane receptors, platelet activating factor receptor, adrenergic receptors, 5-HT receptors, chemokine receptors, neuropeptide receptors, opioid receptors, parathyroid hormone (PTH) receptor, and vasoactive intestinal peptide (VIP) receptor.

13. The polypeptide of claim 12, wherein the GPCR is selected from CCR5 or CXCR4.

14. The polypeptide of claim 1, wherein said GPCR is selected from the group consisting of a protease-activated receptor (PAR), a peptide receptor, a chemokine receptor, and a nucleotide receptor.

15. The polypeptide of claim 14, wherein said GPCR is selected from the group consisting of a PAR1, PAR2, PAR3, and a PAR4 receptor.

16. The polypeptide of claim 1, wherein said first domain comprises an i3 loop or fragment thereof of a protease-activated receptor and said second domain is a lipid.

17. The polypeptide of claim 1, wherein the second domain of the polypeptide further comprises 1-15 contiguous amino acid residues of a naturally-occurring transmembrane helix region of the GPCR immediately adjacent to the intracellular fragment.

18. The polypeptide of claim 1, wherein the second domain of said polypeptide further comprises at least 1-7 contiguous amino acid residues of a transmembrane helix region of the GPCR.

19. The polypeptide of claim 1, wherein the cell-penetrating, membrane-tethering moiety is attached to an N-terminal amino acid residue of said first domain.

20. The polypeptide of claim 1, wherein said first domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:

1-16, 19-23, 29, and 33-36.

21. The polypeptide of claim 20, wherein said first domain comprises a sequence selected from the group consisting of SEQ ID NO:

1-10 and 23.

22. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

1.

23. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

2.

24. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

3.

25. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

4.

26. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

5.

27. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

6.

28. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

7.

29. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

8.

30. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

9.

31. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

10.

32. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

11.

33. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

12.

34. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

13.

35. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

14.

36. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

15.

37. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

16.

38. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

19.

39. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

20.

40. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

21.

41. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

22.

42. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

23.

43. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

29.

44. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

33.

45. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

34.

46. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

35.

47. The polypeptide of claim 20, wherein said first domain comprises the amino acid sequence of SEQ ID NO:

36.

48. The polypeptide of claim 20, wherein said cell-penetrating, membrane-tethering moiety is a palmitoyl group.

49. The polypeptide of claim 20, wherein said cell-penetrating, membrane-tethering moiety is a myristoyl group.

50. The polypeptide of claim 1, wherein said cell-penetrating, membrane-tethering moiety comprises the amino acid sequence YCYVSII (SEQ ID NO:

40).

51. The polypeptide of claim 1, wherein the GPCR is a protease activated receptor (PAR).

52. The polypeptide of claim 1, wherein the GPCR is a thrombin receptor.

53. The polypeptide of claim 1, wherein the GPCR is a P2Y₁₂ receptor.

54. A pharmaceutical composition comprising a polypeptide comprising:

a) a first domain comprising a third intracellular loop (i3 loop) or a fragment thereof of a G protein coupled receptor (GPCR), and

b) a second domain, attached to said first domain, wherein said second domain is a naturally or non-naturally occurring cell-penetrating, membrane-tethering moiety,

wherein said polypeptide is a GPCR agonist or antagonist and does not comprise an extracellular portion of the GPCR and said first domain is at least 3 contiguous amino acid residues; and

a pharmaceutically acceptable carrier.

55. A polypeptide comprising:

a) a first domain comprising a third intracellular loop or a fragment thereof of a G protein coupled receptor, and

b) a second domain, attached to the first domain, wherein said second domain is a palmitoyl group,

wherein said polypeptide is a GPCR agonist or antagonist and does not comprise an extracellular portion of the GPCR and said first domain is at least 3 contiguous amino acid residues.