Phosphoinositide 3-kinase mediated inhibition of GPCRs

• US 20030182669A1
• Filed: 03/19/2002
• Published: 09/25/2003
• Est. Priority Date: 03/19/2002
• Status: Abandoned Application

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Caron, Marc G., Rockman, Howard A., Barak, Larry S., Laporte, Stephane A., Naga Prasad, Sathyamangla V.
• Application Number:
  US10/101,235
• Publication Number:
  US 20030182669A1
• Time in Patent Office: Days
• US Class Current: 800/18
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Abstract

The present invention relates to compounds that alter GPCR internalization and new methods for their identification. Compounds of this invention include modified phosphoinositide 3-kinase (PI3K), modified HEAT domain, modified β-adrenergic receptor kinase 1 (βARK1), as well as other peptides or small molecules that alter GPCR internalization. The present invention also includes the use of such compounds to treat GPCR-related diseases, such as cardiovascular disease, heart failure, asthma, nephrogenic diabetes insipidus, or hypertension.

Claims

1. A method of screening compound(s) for modulating G protein-coupled receptor (GPCR) internalization, comprising the steps of:

(a) providing a cell comprising molecules involved in GPCR internalization, wherein the molecules involved in GPCR internalization comprise β

-adrenergic receptor kinase 1 (β

ARK1), phosphoinositide 3-kinase (PI3K), GPCR, and arrestin, and wherein at least one of said molecules is detectably labeled;

(b) exposing the cell to the compound(s);

(c) identifying the location in the cell of the labeled molecule;

(d) comparing the location of the labeled molecule in the cell in the presence of the compound(s) to the location of the labeled molecule in the cell in the absence of the compound(s); and

(e) correlating a difference between (1) the location of the labeled molecule in the cell in the presence of the compound and (2) the location of the labeled molecule in the cell in the absence of the compound(s) to modulation of GPCR internalization.

2. The method of claim 1, wherein the GPCR is complexed with one or more other molecules.

3. The method of claim 2, wherein the GPCR is complexed with arrestin.

4. The method of claim 3, wherein the GPCR/arrestin complex is labeled.

5. The method of claim 4, wherein the GPCR is labeled.

6. The method of claim 4, wherein the arrestin is labeled.

7. The method of claim 1, wherein the labeled molecule is localized at the plasma membrane, endocytic vesicles, or endosomes.

8. The method of claim 1, wherein the compound alters PI3K catalytic activity.

9. The method of claim 1, wherein the compound alters formation of the PI3K/β

ARK1 complex.

10. The method of claim 1, wherein the compound inhibits GPCR internalization.

11. The method of claim 1, wherein the compound inhibits β

ARK1-mediated translocation of PI3K to the plasma membrane of the cell.

12. The method of claim 11, wherein the location of PI3K within the cell is identified.

13. The method of claim 11, wherein the β

ARK1 forms a complex with PI3K.

14. The method of claim 13, wherein the β

ARK1/PI3K is labeled.

15. The method of claim 14, wherein the β

ARK1 is labeled.

16. The method of claim 14, wherein the PI3K is labeled.

17. The method of claim 11, wherein PI3K is localized in the cytosol or the plasma membrane.

18. The method of claim 11, wherein the compound inhibits formation of a β

ARK1/PI3K complex.

19. The method of claim 17, wherein the β

ARK1/PI3K complex is labeled.

20. The method of claim 18, wherein the β

ARK1 is labeled.

21. The method of claim 18, wherein the PI3K is labeled.

22. The method of claim 1, wherein the cell further comprises adaptin.

23. The method of claim 22, wherein the adaptin is labeled.

47. A compound identified by the method of claim 1.

54. A method of preventing and/or treating a disease associated with GPCR activity in mammals, comprising providing to an animal a therapeutically effective amount of a compound according to claim 47 and a pharmaceutically acceptable carrier.

55. A method of preventing and/or treating a disease associated with PI3K activity in mammals, comprising providing to an animal a therapeutically effective amount of a compound according to claim 47 and a pharmaceutically acceptable carrier.

56. The method of claim 54, wherein the treated disease is a cardiovascular disease, heart failure, asthma, nephrogenic diabetes insipidus, or hypertension.

24. A modified PI3K, wherein GPCR desensitization is altered when said modified PI3K is expressed in a cell.

25. The modified PI3K of claim 24, wherein the modified PI3K comprises a Huntingtin Elongation Factor 3, A subunit of protein phosphatase 2A and TOR (HEAT) domain.

26. The modified PI3K of claim 24, wherein the modified PI3K lacks a HEAT domain.

27. The modified PI3K of claim 24, wherein the modified PI3K lacks catalytic activity.

28. The modified PI3K of claim 24, comprising a polypeptide with the amino acid sequence of SEQ ID NO:

2, 4, 6, 8, or 9.

29. The modified PI3K of claim 24, wherein the PI3K is a class IB PI3K.

30. The modified PI3K of claim 24, wherein the modified PI3K is a class IA PI3K.

31. The modified PI3K of claim 24, wherein the ability of a GPCR to bind adaptin is altered when said modified PI3K is expressed in a cell.

32. The modified PI3K of claim 24, wherein the modified PI3K is PI3Kγ

.

33. The modified PI3K of claim 24, wherein cellular phosphatidylinositol 3,4,5-triphosphate (Ptdlns (3,4,5) P₃) levels are altered when said modified PI3K is expressed in a cell.

34. The modified PI3K of claim 24, wherein the ability of wild-type PI3K to bind β

ARK1 is altered.

35. The modified PI3K of claim 25, wherein the modified PI3K has the ability to bind β

ARK1.

36. The modified PI3K of claim 26, wherein the modified PI3K lacks the ability to bind β

ARK1.

37. The modified PI3K of claim 24, wherein the modified PI3K is conjugated to a detectable molecule.

39. An isolated nucleic acid sequence encoding the modified PI3K of claim 24.

40. The isolated nucleic acid of claim 39, comprising a nucleic acid with the nucleic acid sequence of SEQ ID NO:

1.

41. An expression vector comprising the nucleic acid of claim 40 operably linked to an expression control sequence.

42. A host cell comprising the expression vector of claim 41.

43. A host cell comprising the nucleic acid of claim 40 integrated in its genome.

44. A non-human transgenic animal which expresses a modified PI3K of claim 24.

45. The animal of claim 44, wherein the animal is a mouse.

46. The animal of claim 44, wherein the non-human transgenic animal is a primate, a feline, a canine, a porcine, a bovine, a caprine, or an ovine.

57. A method of preventing and/or treating a disease associated with GPCR activity in mammals, comprising administering to a mammal an amount of the isolated nucleic acid of claim 39 sufficient to reduce or alleviate symptoms of said disease.

58. The method of claim 57, wherein the treated disease is a cardiovascular disease, heart failure, asthma, nephrogenic diabetes insipidus, or hypertension.

38. A modified β

ARK1 which lacks the ability to bind PI3K.

48. A kit for detecting a β

ARK1/PI3K complex in a biological sample comprising an antibody which recognizes and binds to the β

ARK1/PI3K complex and reagents which detect the antibody that binds to the β

ARK1/PI3K complex.

49. An isolated immunoglobulin which binds to a β

ARK1/PI3K complex.

50. The immunoglobulin of claim 49, wherein the immunoglobulin is a monoclonal antibody, a chimeric antibody, a human antibody, a bispecific antibody, a humanized antibody, a primatized antibody, or an antibody fragment.

51. The immunoglobulin of claim 49, wherein the antibody fragment is Fab, Fab′

, F(ab′

)2, F(v), and scFv.

52. A method of altering GPCR internalization, comprising providing an effective amount of LY294002.

53. A method of altering GPCR internalization, comprising providing an effective amount of wortmannin