System for detection of a functional interaction between a compound and a cellular signal transduction component

• US 20030211615A1
• Filed: 03/31/2003
• Published: 11/13/2003
• Est. Priority Date: 06/14/1999
• Status: Active Grant

• Current Assignee: Lambertus J. Oehlen
• Original Assignee: Lambertus J. Oehlen
• Inventors: Oehlen, Lambertus J.
• Granted Patent: US 7,090,991 B2
• Time in Patent Office: Days
• US Class Current: 436/4
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: G01N 2333/4719 G-proteins
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways

Abstract

The present invention makes available a rapid, reproducible, robust assay system for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular protein, e.g., a receptor. or ion channel. The subject assay enables rapid screening of large numbers of compounds to identify those which act as an agonist or antagonist to the bioactivity of the cellular protein. In this system, the first cell is treated with a compound, and functional interaction of this compound with a cellular receptor yields a secreted signal. A second cell, bearing a receptor for this secreted signal, makes use of an indicator gene in a signaling pathway coupled to this second receptor. The subject assays include methods of identifying compounds which specifically modulate, for example, heterologous receptors coupled to the pheromone response pathway in yeast. The subject assays are particularly amenable to the identification of specific agonists and antagonists of G protein-coupled receptors.

Claims

1. A method for identifying a test compound that modulates a heterologous receptor in a cell, said method comprising:

providing a first cell which comprises a heterologous receptor that is functionally integrated into a first signal transduction pathway of said first cell, wherein a signal molecule is produced by said first cell upon activation of said first signal transduction pathway;

providing a second cell which is responsive to production of said signal molecule by said first cell, such that a detectable signal is generated by said second cell in response to production of said signal molecule by said first cell; and

contacting said first cell with said test compound;

whereby modulation of said heterologous receptor by said test compound is indicated by generation of said detectable signal by said second cell.

2. The method of claim 1, further comprising contacting said signal molecule and said second cell.

3. The method of claim 2, wherein said second cell further comprises a receptor for said signal molecule, wherein said receptor is functionally integrated into a second signal transduction pathway of said second cell such that a detectable signal is generated upon activation of said second signal transduction pathway.

4. The method of claim 3, wherein said first and second cells are yeast cells.

5. The method of claim 4, wherein said first and second signal transduction pathways are yeast pheromone response pathways.

6. The method of claim 1, wherein said first and second cells are of opposite mating types.

7. The method of claim 4, wherein said first cell is a MATα

S. cerevisiae cell and said second cell is a MATa S. cerevisiae cell.

8. The method of claim 5, wherein said second cell further comprises a reporter gene, wherein activation of said second signal transduction pathway causes transcription of said reporter gene, said transcription providing said detectable signal.

9. The method of claim 8, wherein said reporter gene is operatively linked to a pheromone responsive promoter.

10. The method of claim 9, wherein said reporter gene is selected from the group consisting of lacZ, HIS3 and LEU2, and said pheromone responsive promoter is FUS1.

11. The method of claim 7, wherein said MATα

S. cerevisiae cell has the endogenous MFα

1 gene inactivated.

12. The method of claim 11, wherein said MFα

1 gene is inactivated by deletion.

13. The method of claim 11, wherein said MFα

1 gene is inactivated by mutation.

14. The method of claim 4, wherein said receptor in said second cell is STE2 or STE3.

15. The method of claim 7, wherein said signal molecule is a protein secreted by said first cell.

16. The method of claim 15, wherein said secreted protein is MFα

.

17. The method of claim 16, wherein said heterologous receptor is selected from the group consisting of melatonin receptor 1a, galanin receptor 1, neurotensin receptor, adenosine receptor 2a, somatostatin receptor 2, and, corticotropin releasing factor receptor 1.

18. The method of claim 17, wherein said heterologous receptor is melatonin receptor 1a.

19. The method of claim 4, wherein said heterologous receptor is a G protein-coupled receptor.

20. The method of claim 19, wherein said first cell further comprises a heterologous Gα

protein.

21. The method of claim 20, wherein said heterologous Gα

protein is a chimeric Gα

protein.

22. The method of claim 20, wherein said heterologous Gα

protein is a mutated Gα

protein.

23. The method of claim 19, wherein said heterologous G protein-coupled receptor functionally couples to the endogenous yeast GPA-1 protein subunit.

24. The method of claim 1, wherein said second cell further comprises a gene which produces a detectable protein.

25. The method of claim 24, wherein said gene is an endogenous gene at its natural location in said second cell.

26. The method of claim 25, wherein said natural location in said second cell is the natural location of said endogenous gene in the cellular genome, and wherein activation of said second signal transduction pathway causes said endogenous gene to produce said detectable protein, thereby generating a detectable signal.

27. The method of claim 1, wherein said heterologous receptor is expressed in the membrane of said first cell.

28. The method of claim 27, wherein said test compound comprises a heterologous test polypeptide expressed by said first cell, wherein said heterologous test polypeptide is transported to a location allowing interaction with the extracellular region of said heterologous receptor, and wherein said 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 generates said detectable signal.

29. The method of claim 28, 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.

30. The method of claim 15, further comprising contacting said second cell with said secreted protein, wherein said protein is secreted to the extracellular region of said first cell.

31. The method of claim 20, wherein said heterologous G_α

protein is a mammalian G_α

protein.

35. The recombinant cell of claim 1, wherein said signal transduction pathway is a yeast pheromone response pathway.

36. The recombinant cell of claim 1, wherein said signal molecule is a gene product.

37. The recombinant cell of claim 3, wherein said gene product is a natural product.

41. The method of claim 7, which further comprises:

providing a first cell, said first cell comprising a surrogate of a signal transduction pathway of said first cell, wherein a gene product is produced by said surrogate upon activation of said signal transduction pathway; and

.

providing a second cell, said second cell comprising means for generating a detectable signal, such that a detectable signal is generated by said second cell in response to stimulation of said second cell by said gene product.

42. The method of claim 8, wherein said signal transduction pathway is a yeast pheromone response pathway.

32. A kit for screening of test compounds that modulate a heterologous receptor in a cell, said kit comprising:

a first cell which comprises a heterologous receptor that is functionally integrated into a first signal transduction pathway of said first cell, wherein a signal molecule is produced by said first cell upon activation of said first signal transduction pathway; and

second cell which is responsive to production of said signal molecule by said first cell, such that a detectable signal is generated by said second cell in response to production of said signal molecule by said first cell.

33. The kit of claim 32, further comprising appropriate buffers and instructional materials for quantitating said detectable signal.

34. A recombinant cell comprising a surrogate of a signal transduction pathway of said cell, wherein a signal molecule is produced by said surrogate upon activation of said signal transduction pathway, and wherein production of said signal molecule is capable of detection by a second cell comprising means for generating a detectable signal, such that a detectable signal is generated by said second cell in response to stimulation of said second cell by said signal molecule.

38. A mixture of recombinant cells comprising a first recombinant cell and a second recombinant cell, wherein said first cell comprises a surrogate of a signal transduction pathway of said cell, such that a signal molecule is produced by said surrogate upon activation of said signal transduction pathway, and wherein said second cell comprises means for generating a detectable signal such that a detectable signal is generated by said second cell in response to stimulation of said second cell by said signal molecule.

39. A mixture of recombinant cells comprising a first recombinant cell and a second recombinant cell, wherein said first cell comprises a heterologous receptor that is functionally integrated into a signal transduction pathway of said first cell, and produces a signal molecule upon activation of said signal transduction pathway, and wherein said second cell comprises means for generating a detectable signal, and is responsive to the signal molecule produced by said first cell, such that a detectable signal is generated by said second cell in response to production of said signal molecule by said first cell.

40. A method for detecting a gene product produced by a cell, comprising:

causing a signal to be transmitted through a signal transduction pathway of a first cell, such that a gene product is produced by said first cell upon transmission of said signal through said signal transduction pathway; and

detecting said gene product by means of a second cell which is responsive to the gene product produced by said first cell, wherein said second cell generates a detectable signal in response to production of said gene product by said first cell.

43. A method for identifying a compound that modulates production of a gene product produced by a cell, said method comprising:

providing a first cell, said first cell comprising a surrogate of a signal transduction pathway of said first cell, wherein a gene product is produced by said surrogate upon activation of said signal transduction pathway;

providing a second cell, said second cell comprising means for generating a detectable signal and being responsive to said gene product, such that a detectable signal is generated by said second cell in response to stimulation of said second cell by said gene product; and

contacting said first cell with a test compound;

whereby said modulation is indicated by generation of said detectable signal by said second cell.