Methods and compositions for identifying receptor effectors

• US 7,319,009 B2
• Filed: 10/22/2002
• Issued: 01/15/2008
• Est. Priority Date: 10/07/1998
• Status: Expired due to Fees

• Current Assignee: Cadus Technologies Incorporated
• Original Assignee: Cadus Technologies Incorporated
• Inventors: Paul, Jeremy, Klein, Christine A., Murphy, Andrew J.
• Primary Examiner(s): ULM, JOHN D
• Application Number:
  US10/277,607
• Publication Number:
  US 20030166143A1
• Time in Patent Office: 1,911 Days
• Field of Search: 435/7.21, 435/69.1, 435/254.2
• US Class Current: 435/7.1
• CPC Class Codes: C07K 14/723 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor

Abstract

The present invention makes available rapid, effective assays for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular receptor or ion channel. The subject assays enable rapid screening of large numbers of polypeptides in a library to identify those polypeptides which induce or antagonize receptor bioactivity. The subject assays are particularly amenable for identifying agonists and antagonists for orphan receptors. In particular the present invention makes available novel ligand agonists of human formyl peptide receptor like-1 (FPRL-1) receptors. These novel ligand agonists are used in the assays of the invention to identify modulators of FPRL-1 receptor.

Claims

1. A recombinant cell which comprises:

a heterologous formyl peptide receptor like-1 (FPRL-1) receptor expressed in the cell membrane of said cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal; and

a polypeptide, or analog thereof, that is a ligand agonist of said FPRL-1 receptor, wherein said ligand agonist is transported to a location allowing interaction with the extracellular region of said FPRL-1 receptor expressed in the cell membrane, and is expressed at a level sufficient for said ligand agonist to bind to and activate said FPRL-1 receptor, thereby causing a detectable signal to be generated.

2. The cell of claim 1, wherein said heterologous FPRL-1 receptor is coupled to a signal transduction pathway.

3. The cell of claim 1, wherein said heterologous FPRL-1 receptor acts as a surrogate for an endogenous cell receptor in a signal transduction pathway of the cell and wherein binding of said ligand agonist to said receptor activates the signal transduction activity of said receptor thereby generating a detectable signal.

4. The cell of claim 3 further comprising a reporter construct that is activated by the signal transduction pathway, wherein the detectable signal provided by said ligand agonist is mediated by the reporter construct.

5. The cell of claim 4, wherein said detectable signal is selected from the group consisting of a growth signal, intracellular calcium mobilization, an optical signal, second messenger production, changes in GTP hydrolysis, and phospholipid hydrolysis.

6. The cell of claim 1, wherein said ligand agonist has an EC₅₀ of 2×

10<sup>−

5</sup>M or less and comprises a polypeptide comprising from 3 to 80 amino acid residues.

7. The cell of claim 6, wherein said polypeptide comprises from 3 to 40 amino acid residues.

8. The cell of claim 7, wherein said ligand agonist comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, and analogs thereof.

9. The cell of claim 1, wherein said FPRL-1 receptor is associated with an indicator molecule which provides a detectable signal upon binding of said ligand agonist to said receptor.

10. The cell of claim 9, wherein said indicator molecule comprises GFP or a β

-arrestin-GFP conjugate.

11. The cell of claim 1 further comprising a heterologous test polypeptide, wherein the heterologous test polypeptide is transported to a location allowing interaction with the extracellular region of said FPRL-1 receptor expressed in the cell membrane;

and wherein the heterologous test polypeptide is expressed at a sufficient level such that modulation of the signal transduction activity of the receptor by the heterologous test polypeptide alters said detectable signal.

12. The cell of claim 11, wherein said heterologous test polypeptide includes a signal sequence that facilitates transport of the polypeptide to a location allowing interaction with the extracellular region of the receptor.

13. The cell of claim 1, wherein said heterologous FPRL-1 is human FPRL-1.

14. The cell of claims 1 or 3, wherein said cell is a eukaryotic cell.

15. The cell of claim 14, wherein said eukaryotic cell is a yeast cell, and wherein said heterologous FPRL-1 receptor acts as a surrogate for an endogenous yeast pheromone receptor in a pheromone response pathway of said yeast cell.

16. The cell of claim 15, which belongs to the species Saccharomyces cerevisiae.

17. A recombinant yeast cell which comprises:

a heterologous formyl peptide receptor like-1 (FPRL-1) receptor expressed in the cell membrane of said yeast cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal; and

a polypeptide, or analog thereof, that is a ligand agonist of said FPRL-1 receptor, wherein said ligand agonist is transported to a location allowing interaction with the extracellular region of said FPRL-1 receptor expressed in the cell membrane, and is expressed at a level sufficient for said ligand agonist to bind to and activate said FPRL-1 receptor, thereby causing a detectable signal to be generated.

18. The yeast cell of claim 17, wherein said heterologous FPRL-1 receptor is coupled to a signal transduction pathway.

19. The yeast cell of claim 17, wherein said heterologous FPRL-1 receptor acts as a surrogate for an endogenous yeast pheromone receptor in a pheromone response pathway of the cell and wherein binding of said ligand agonist to said receptor activates the signal transduction activity of said receptor thereby generating a detectable signal.

20. The yeast cell of claim 19 further comprising a reporter construct that is activated by the signal transduction pathway, wherein the detectable signal provided by said ligand agonist is mediated by the reporter construct.

21. The yeast cell of claim 20, wherein said reporter construct comprises a pheromone-responsive promoter operably linked to a selectable gene.

22. The yeast cell of claim 21, wherein said pheromone-responsive promoter is the FUS1 promoter.

23. The yeast cell of claim 21, wherein said selectable gene is selected from the group consisting of URA3, LYS2, HIS3, LEU2, TRP1, ADE1, ADE2, ADE3, ADE4, ADE5, ADE7, ADE8, ARG1, ARG3, ARG4, ARG5, ARG6, ARG8, HIS1, HIS4, HIS5 ILV1, ILV2, ILV5, THR1, THR4, TRP2, TRP3, TRP4, TRP5, LACZ, LEU1, LEU4, MET2, MET3, MET4, MET8, MET9, MET14, MET16, MET19, URA1, URA2, URA4, URA5, URA10, H0M3, H0M6, ASP3, CHO1, ARO2, ARO7, CYS3, OLE1, IN01, IN02, IN04, PRO1, and PRO3.

24. The yeast cell of claim 19 further comprising a heterologous test polypeptide, wherein the heterologous test polypeptide is transported to a location allowing interaction with the extracellular region of said FPRL-1 receptor expressed in the cell membrane;

and wherein the heterologous test polypeptide is expressed at a sufficient level such that modulation of the signal transduction activity of the receptor by the heterologous test polypeptide alters said detectable signal.

25. The yeast cell of claim 24, wherein said heterologous test polypeptide includes a signal sequence that facilitates transport of the polypeptide to a location allowing interaction with the extracellular region of the receptor.

26. The yeast cell of claim 24, wherein said signal sequence corresponds to a leader peptide of the Saccharomyces cerevisiae α

factor or a-factor.

27. The yeast cell of claim 19, which belongs to the species Saccharomyces cerevisiae, and wherein said yeast cell further comprises a mutation in at least one gene selected from the group consisting of STP22, VPS1, KRE1, CAV1, STE50, SGV1, PIK1, AFR1, FAR1, SST2, BAR1, STE2, STE3, STE14, MFa1, and MFa2.

28. The yeast cell of claim 19, which is a mutant strain of a yeast cell having a pheromone system pathway that is desensitized at slower rate than a wild type strain under the same conditions of continuous stimulation of the pheromone system pathway.

29. The yeast cell of claim 19, which belongs to the species Saccharomyces cerevisiae, and wherein said yeast cell has a ste14 mutation.

30. The yeast cell of claim 19, which belongs to the species Saccharomyces cerevisiae, and wherein said yeast cell has a ste2 or ste3 mutation.

31. The yeast cell of claim 19, which belongs to the species Saccharomyces cerevisiae.

32. The yeast cell of claim 17, wherein said ligand has an EC₅₀ of 2×

10<sup>−

5</sup>M or less and comprises a polypeptide comprising from 3 to 80 amino acid residues.

33. The yeast cell of claim 32, wherein said polypeptide comprises from 3 to 40 amino acid residues.

34. The yeast cell of claim 33, wherein said ligand agonist comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, and analogs thereof.

35. The yeast cell of claim 17, wherein said FPRL-1 receptor is associated with an indicator molecule which provides a detectable signal upon binding of said ligand agonist to said receptor.

36. The yeast cell of claim 35, wherein said indicator molecule comprises GFP or a β

-arrestin-GFP conjugate.

37. The yeast cell of claim 17, wherein said ligand agonist is associated with an indicator molecule which provides a detectable signal upon binding of said ligand agonist to said receptor.

38. The yeast cell of claim 17, wherein said FPRL-1 receptor is human FPRL-1.