Screening methods using G-protein coupled receptors and related compositions
The present invention provides screening methods for GPCRs based on the discovery that the affinity of a receptor agonist for a GPCR (such as the parathyroid hormone receptor) when not bound to a G-protein is correlated with the length of time over which the agonist is effective, independently of its pharmacokinetic properties. The invention also provides PTH- and PTHrP-derived polypeptides.
1. A method for identifying a candidate compound as a long-acting agonist of a secretin family receptor, said method comprising:
(a) contacting said secretin family receptor with said compound, wherein said secretin family receptor is in the RG form;
(b) measuring the affinity of said compound for the RG form of said secretin family receptor;
(c) contacting said secretin family receptor with said compound, wherein said secretin family receptor is in the R 0 form;
(d) measuring the affinity of said compound for the R 0 form of said secretin family receptor; and
(e) identifying said compound as a long-acting agonist of said secretin family receptor if said compound
(i) has an affinity for the RG form of said secretin family receptor that is at least 10% of the affinity of an endogenous agonist for the RG form of said secretin family receptor, and
(ii) has a greater affinity for the R 0 form of said secretin family receptor than the affinity of said endogenous agonist for the R 0 form of said secretin family receptor.
2. The method of claim 1 , further comprising the steps of:
(f) administering said candidate compound to an animal, and
(g) measuring at least one physiological response of said animal to said compound.
3. The method of claim 1 , wherein said receptor is a PTH/PTHrP receptor.
4. The method of claim 3 , wherein said PTH/PTHrP receptor is a human receptor.
5. The method of claim 3 , wherein said measuring step (b) is performed by measuring intracellular or blood calcium levels.
6. The method of claim 1 , wherein said measuring step (b) or step (d) is performed using a competition binding assay.
7. The method of claim 6 , wherein said competition binding assay uses a ligand that is specific for the RG form or specific for the R 0 form-of said secretin family receptor.
8. The method of claim 1 , wherein said measuring step (b) is performed using a delayed cAMP assay.
9. The method of claim 1 , wherein said R 0 form of said secretin family receptor is enriched using a nonhydrolizable nucleotide analog.
10. The method of claim 9 , wherein said nucleotide analog is GTPγS.
11. The method of claim 1 , wherein said RG form of said secretin family receptor is enriched using a dominant-negative G-protein.
12. The method of claim 1 , wherein said candidate compound comprises a peptide.
13. The method of claim 1 , wherein said candidate compound is from a chemical library or natural product library.
14. A method for identifying a candidate compound as a short-acting agonist of a secretin family receptor, said method comprising:
(a) contacting said secretin family receptor with said compound, wherein said secretin family receptor is in the RG form;
(b) measuring the affinity of said compound for the RG form of said secretin family receptor;
(c) contacting said secretin family receptor with said compound, wherein said secretin family receptor is in the R 0 form;
(d) measuring the affinity of said compound for the R 0 form of said secretin family receptor; and
(e) identifying said compound as a short-acting agonist of said secretin family receptor if said compound
(i) has an affinity for the RG form of said secretin family receptor that is at least 10% of the affinity of an endogenous agonist for the RG form of said GPCR, and
(ii) has a lower affinity for the R 0 form of said secretin family receptor than the affinity of said endogenous agonist for the R 0 form of said secretin family receptor.
15. The method of claim 14 , further comprising the steps of:
(f) administering said candidate compound to an animal, and
(g) measuring at least one physiological response of said animal to said compound.
16. The method of claim 14 , wherein said receptor is a PTH/PTHrP receptor.
17. The method of claim 16 , wherein said PTH/PTHrP receptor is a human receptor.
18. The method of claim 16 , wherein said measuring step (b) is performed by measuring intracellular or blood calcium levels.
19. The method of claim 14 , wherein said measuring step (b) or step (d) is performed using a competition binding assay.
20. The method of claim 19 , wherein said competition binding assay uses a ligand that is specific for the RG form or specific for the R 0 form of said secretin family receptor.
21. The method of claim 14 , wherein said measuring step (b) is performed using a delayed cAMP assay.
22. The method of claim 14 , wherein said R 0 form of said secretin family receptor is enriched using a nonhydrolizable nucleotide analog.
23. The method of claim 22 , wherein said nucleotide analog is GTPγS.
24. The method of claim 14 , wherein said RG form of said secretin family receptor is enriched using a dominant-negative G-protein.
25. The method of claim 14 , wherein said candidate compound comprises a peptide.
26. The method of claim 14 , wherein said candidate compound is from a chemical library or a natural product library.
27. The method of claim 1 , wherein said endogenous agonist is human PTH(1-34).
28. The method of claim 14 , wherein said endogenous agonist is human PTH(1-34).