Methods and compositions for identifying receptor effectors

• US 7,250,263 B2
• Filed: 01/05/2004
• Issued: 07/31/2007
• Est. Priority Date: 03/31/1993
• Status: Expired due to Fees

• Current Assignee: Cadus Technologies Incorporated
• Original Assignee: Cadus Technologies Incorporated
• Inventors: Trueheart, Joshua, Broach, James, Paul, Jeremy, Klein, Christine A., Fowlkes, Dana M., Murphy, Andrew J. M., Manfredi, John
• Primary Examiner(s): Ulm; John
• Application Number:
  US10/752,478
• Publication Number:
  US 20050059135A1
• Time in Patent Office: 1,303 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: C07K 14/395 from Saccharomyces
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 15/81 for yeasts

Abstract

The present invention makes available a rapid, effective assay for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular receptor or ion channel. The subject assay enables rapid screening of large numbers of polypeptides in a library to identifying those polypeptides which induce or antagonize receptor bioactivity. The subject assay is particularly amenable for identifying surrogate ligands for orphan receptors.

Claims

1. A mixture of recombinant yeast cells, each cell of which has a cell membrane and each cell of which comprises:

(a) a heterologous receptor selected from the group consisting of;

an FPRL-1 receptor, a C5a receptor and a thrombin receptor, which receptor is 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

(b) a heterologous polypeptide, wherein the heterologous polypeptide is transported to a location allowing interaction with the extracellular region of the heterologous receptor expressed in the cell membrane;

wherein collectively the mixture of cells expresses a library of said heterologous polypeptides, said library being expressible at a sufficient level such that modulation of the signal transduction activity of the heterologous receptor by a heterologous polypeptide within the library provides a detectable signal.

2. The mixture of recombinant yeast cells of claim 1, wherein each yeast cell further comprises a chimeric G protein subunit with which the heterologous receptor interacts.

3. The mixture of recombinant yeast cells of claim 1, which are a mutant strain of yeast cells 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.

4. The mixture of recombinant yeast cells of claim 1, wherein the yeast cells have a mutation in a gene selected from the group consisting of:

STE50, bar1, pik1, msg5, sig1, and aft1.

5. The mixture of recombinant yeast cells of claim 1, wherein the yeast cells have a ste2 or ste3 mutation.

6. The mixture of recombinant yeast cells of claim 1, wherein an endogenous pheromone gene is not functionally expressed in the yeast cells.

7. The mixture of recombinant yeast cells of claim 1, wherein an endogenous FAR1 gene is not functionally expressed in the yeast cells.

8. The mixture of recombinant yeast cells of claim 1, wherein an endogenous SST2 gene is not functionally expressed in the yeast cells.

9. The mixture of recombinant yeast cells of claim 2, which comprise a chimeric Gα

i2 subunit consisting of the first 41 amino acids of GPA1 fused to a region of human Gα

i2.

10. The mixture of recombinant yeast cells of claim 1, which further comprise a selectable marker that is activated by a pheromone system pathway of the yeast cells, thereby providing the detectable signal.

11. The mixture of recombinant yeast cells of claim 10, wherein the selectable marker comprises a pheromone-responsive promoter operably linked to a selectable gene.

12. The mixture of recombinant yeast cells of claim 11, wherein the pheromone-responsive promoter is the FUS1 promoter.

13. The mixture of recombinant yeast cells of claim 11, wherein the selectable gene is a HIS 3 gene.

14. The mixture of recombinant yeast cells of claim 1, wherein the heterologous polypeptide includes a signal sequence that facilitates transport of the polypeptide to a location allowing interaction with the extracellular region of the receptor.

15. The mixture of recombinant yeast cells of claim 14, wherein the signal sequence corresponds to a leader peptide of the Saccharomyces cerevisiae α

factor or a-factor.

16. The mixture of recombinant yeast cells of claim 1, which belong to the species Saccharomyces cerevisiae.

17. The mixture of recombinant yeast cells of claim 1, wherein the library of heterologous polypeptides includes more than 1000 different polypeptide sequences.

18. The mixture of recombinant yeast cells of claim 1, wherein said receptor is a C5a receptor.

19. The mixture of recombinant yeast cells of claim 1, wherein said heterologous receptor acts as a surrogate for an endogenous yeast pheromone receptor in a pheromone system pathway of said cell.

20. The mixture of recombinant yeast cells of claim 1, which further comprise a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the signal transduction activity of the receptor protein which thereby provides the detectable signal.

21. The mixture of recombinant yeast cells of claim 20, wherein said reporter gene encodes a gene product which confers a growth signal.

22. The mixture of recombinant yeast cells of claim 1, wherein said detectable signal is selected from the group consisting of:

color, fluorescence, luminescence, cell viability, relief of a cell nutritional requirement, cell growth, and drug resistance.

23. A mixture of recombinant yeast cells, each cell of which has a cell membrane and each cell of which comprises:

(a) a heterologous receptor selected from the group consisting of an FPRL-1 receptor, a C5a receptor, and a thrombin 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, said heterologous receptor acting as a surrogate for an endogenous yeast pheromone receptor in a pheromone system pathway of the yeast cell;

(b) a heterologous polypeptide, wherein the heterologous polypeptide is transported to a location allowing interaction with the extracellular region of the heterologous receptor expressed in the cell membrane; and

(c) a selectable marker that is activated by the pheromone system pathway;

wherein collectively the mixture of cells expresses a library of said heterologous polypeptides, said library being expressible at a sufficient level such that modulation of the signal transduction activity of the heterologous receptor by a heterologous polypeptide within the library provides a detectable signal mediated by the selectable marker; and

wherein said yeast cells have a mutation in at least one gene selected from the group consisting of FAR1, SST2, BAR1, SVG1, STE2, STE3, MFa1, MFa2, MFα

1 and MFα

2.

24. The mixture of recombinant yeast cells of claim 23, wherein the selectable marker comprises a pheromone-responsive promoter operably linked to a selectable gene.

25. The mixture of recombinant yeast cells of claim 23, wherein the heterologous polypeptide includes a signal sequence that facilitates transport of the polypeptide to a location allowing interaction with the extracellular region of the receptor.