Methods for detecting allosteric modulators of protein
The present invention discloses, inter alia, methods for labeling a target protein with an SHG-active probe for detection by second harmonic or sum-frequency generation in order to identify agents which bind to an allosteric site on the target protein thereby altering its structural conformation.
1. A method for identifying an agent that binds to an allosteric site on a G protein-coupled receptor (GPCR), the method comprising:
a) contacting the GPCR with a known ligand, wherein the known ligand is known to bind to the GPCR, wherein the known ligand is labeled with a second harmonic active moiety, and wherein the labeled ligand has a net orientation at an interface upon binding to the GPCR;
b) contacting the GPCR with the agent, wherein a detectable signal is generated by the second harmonic active label using a surface selective technique, and wherein the detectable signal indicates a conformational change in the structure of the GPCR produced when the agent binds to a site on the GPCR; and
c) measuring the presence or absence of the detectable signal after the target protein has been contacted with the agent.
2. The method of claim 1 , wherein the GPCR is located on the surface of a biological cell, a liposome, or a synthetic biological membrane.
3. The method of claim 1 , wherein the biological cell expresses the GPCR naturally.
4. The method of claim 1 , wherein the biological cell comprises a heterologous nucleic acid encoding the GPCR.
5. The method of claim 1 , wherein the GPCR is selected from the group consisting of alpha-1 adrenegic receptors (al-AR), urotensin (UT) receptors, 5-HT2 and 5-HT6 serotonin receptors, hypocretic (orexin) receptors, histamine HI receptors, bradykinin B1 and B2 receptors, bombesin BB2 receptors, P2Y purinergic receptors, acetycholine receptors, mGluR5 glutamate receptors, vasopressin V2 and VI receptors, angiotensin AGTR1 receptors, cholecystokinin CCKAR and CCKBR receptors, endothelin ENDRA receptors, ghrelin GHSR1a receptors, melatonin MTNR1 A receptors, neurotensin NTSR1 receptors, platelet-activating factor PTAFR receptors, prolactin releasing peptide receptor PRLHR receptors, G-coupled 5-HT2, 5-HT2A, 5-HT6, and 5-HT7 serotonin receptors, G-coupled GABA-B, histamine H3, and mGluR2/4 glutamate receptors.
6. A method for identifying an agent B that binds to an allosteric site on a target protein, the method comprising:
a) contacting the target protein with an agent A that binds to an active site of the target protein, wherein the agent A is labeled with a second harmonic-active moiety that has a net orientation at an interface, wherein a detectable signal is generated by the second harmonic-active moiety using a surface selective technique, and wherein the detectable signal indicates a conformational change in the structure of the target protein produced when an agent B binds to an allosteric site on the target protein; and
b) contacting the target protein with the agent B; and
c) measuring the presence or absence of the detectable signal after the target protein has been contacted with the agent B.
7. The method of claim 6 , wherein the second harmonic-active moiety is bound to the agent A by one or more sulfhydryl groups on the surface of the agent A.
8. The method of claim 7 , wherein said one or more sulfhydryl groups are native sulfhydryl groups.
9. The method of claim 7 , wherein said one or more sulfhydryl groups are engineered sulfhydryl groups.
10. The method of claim 6 , wherein the second harmonic-active moiety is PyMPO-maleimide.
11. The method of claim 6 , wherein the second harmonic-active moiety is bound to the agent A by one or more amine groups on the surface of the agent A.
12. The method of claim 11 , wherein said one or more amine groups are native amine groups.
13. The method of claim 11 , wherein said one or more amine groups are engineered amine groups.
14. The method of claim 11 , wherein the second harmonic-active moiety is PyMPO-succinimidyl ester.
15. The method of claim 6 , wherein the second harmonic-active moiety is an unnatural amino acid.
16. The method of claim 15 , wherein the unnatural amino acid is Aladan.
17. The method of claim 6 , wherein the target protein is a G protein-coupled receptor, a steroid hormone receptor, or a tyrosine kinase receptor.
18. The method of claim 17 , wherein the interface is selected from the group consisting of: a glass surface, a polyethylene glycol surface, a supported lipid bilayer surface, a lipid analog bilayer surface, a plastic surface, a metal surface, a latex surface, a rubber surface, a ceramic surface, a polymeric surface, a polypropylene surface, a polyvinylidene difluoride surface, a polyethylene surface.
19. The method of claim 18 , wherein the surface is derivatized with oligo-PEG molecules or lipids.
20. The method of claim 19 , wherein the oligo-PEG molecules or lipids are Ni-NTA-bearing oligo-PEG molecules or Ni-NTA-bearing lipids.
21. The method of claim 6 , wherein the surface is a supported lipid bilayer or a lipid analog bilayer.
22. The method of claim 21 , wherein the target protein comprises an affinity tag.
23. The method of claim 22 , wherein the agent A or the agent B is a small molecule chemical compound, an antibody, a non-antibody polypeptide, a carbohydrate, an inhibitory nucleic acid, or any combination thereof.