High throughput assays for inhibitors and activators of PAQR receptors
The subject invention provides methods of screening compounds or ligands that interact with human and/or non-human PAQR receptors or fungal osmotin receptors. These methods utilize a colorimetric assay to ascertain whether a compound binds to and activates a PAQR receptor or the osmotin receptor.
1. A method of identifying candidate ligands of “Progestin and AdipoQ Receptor” (PAQR) receptors, comprising:
a) providing a yeast cell expressing one or more PAQR receptor and a reporter molecule, said reporter molecule driven by a promoter containing a CCCTC binding motif providing a signal in response to activation of said one or more PAQR receptor;
b) contacting said yeast cell with a candidate ligand; and
c) detecting the activation of said one or more PAQR receptor by the development of a signal within the cell or in medium in which the cell is cultured, the detection of a signal indicating the activation of said one or more PAQR receptor.
2. The method according to claim 1 , wherein said one or more PAQR receptor is a human PAQR receptor selected from PAQR1, PAQR2, PAQR3, PAQR4, PAQR5, PAQR6, PAQR7, PAQR8, PAQR9, PAQR10 PAQR11 or various combinations thereof.
3. The method according to claim 2 , wherein said one or more human PAQR receptor or said one or more PAQR receptor is overexpressed in said yeast cell.
4. The method according to claim 1 , wherein said cell is cultured in medium that contains 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.10%, 0.05% or 0.025% galactose (w/v).
5. The method according to claim 4 , wherein said cell is cultured in medium that contains galactose at a concentration less than or equal to 0.10% galactose (w/v).
6. The method according to claim 4 , wherein said cell is cultured in medium that contains galactose at a concentration less than or equal to 0.05% galactose (w/v).
7. The method according to claim 1 , wherein said promoter is a truncated FET3 promoter that comprises nucleotides 102 to 603 of SEQ ID NO: 181.
8. The method according to claim 1 , wherein expression of said one or more PAQR receptor is driven by a galactose inducible promoter.
9. The method according to claim 8 , wherein said galactose inducible promoter is GAL1.
10. The method according to claim 1 , wherein said yeast cell is permeabilized prior to detecting the activation of PAQR receptors and β-galactosidase activity is measured.
11. The method according to claim 1 , wherein the activation of said PAQR receptor is detected by contacting intact yeast cells with a ferroxidase substrate and measuring ferroxidase activity.
12. The method according to claim 1 , wherein said reporter molecule is:
a) an enzyme;
b) a fluorescent protein; or
c) a protein providing antibiotic resistance.
13. The method according to claim 12 , wherein said enzyme is an alpha-D-glucosidase, a beta-D-glucosidase, an esterase, a lipase, an acid or alkaline phosphatase, a beta-D-galactosidase, an alpha-L-arabinofuranosidase, a leucine aminopeptidase, a chymotrypsin, an alpha-D-galactosidase, a beta-D-glucuronidase, an aminopeptidase, or a phosphohydralase.
14. The method according to 1 , wherein said promoter is FET3, ZRT1, OLE1, ZRC1 and ZPS1 or truncations thereof containing the CCCTC motif and capable of driving the expression of a gene operably linked thereto.
15. The method according to claim 1 , wherein said candidate ligand is an agonist of said receptor.
16. The method according to claim 1 , wherein the PAQR receptor is PAQR6 and/or PAQR9 and the candidate ligand is a progesterone receptor antagonist or agonist.
17. The method according to claim 2 , wherein said one or more PAQR receptor is human PAQR1, human PAQR2 or a combination thereof.
18. The method according to claim 17 , wherein said one or more PAQR receptor is human PAQR1.
19. The method according to claim 17 , wherein said one or more PAQR receptor is human PAQR2.
20. The method according to claim 2 , wherein said one or more PAQR receptor is a combination of human PAQR1 and human PAQR2.