Methods for detecting allosteric modulators of protein

• US 8,932,822 B1
• Filed: 09/10/2014
• Issued: 01/13/2015
• Est. Priority Date: 04/25/2012
• Status: Active Grant

• Current Assignee: Quanta Therapeutics, Inc.
• Original Assignee: Biodesy, Inc.
• Inventors: Salafsky, Joshua S.
• Primary Examiner(s): Cheu, Jacob
• Application Number:
  US14/482,899
• Publication Number:
  US 20140377776A1
• Time in Patent Office: 125 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: G01N 2021/6463 Optics
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 2333/91205 Phosphotransferases in general
• CPC Class Codes: G01N 2333/91215 with a definite EC number (2.7.1.-)
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

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.

Claims

1. 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 or wherein agent A is naturally bound to the active site of the target protein when isolated;

b. contacting the target protein with the agent B, wherein the target protein is labeled with a second harmonic-active moiety having 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 the agent B binds to an allosteric site on the target protein; and

c. measuring the presence or absence of the detectable signal after the target protein has been contacted with the agent B.

2. The method of claim 1, wherein the second harmonic-active moiety is selected from the group consisting of PyMPO maleimide, PyMPO-NHS, PyMPO-succinimidyl ester, Badan, and Acrylodan.

3. The method of claim 1, wherein the second harmonic-active moiety is bound to the target protein by one or more sulfhydryl groups on the surface of the target protein.

4. The method of claim 3, wherein said one or more sulfhydryl groups are native sulfhydryl groups.

5. The method of claim 3, wherein said one or more sulfhydryl groups are engineered sulfhydryl groups.

6. The method of claim 5, wherein said one or more sulfhydryl groups are located on target protein amino acid residues known to contact one or more ligand.

7. The method of claim 2, wherein the second harmonic-active moiety is PyMPO-maleimide.

8. The method of claim 2, wherein the second harmonic-active moiety is bound to the target protein by one or more amine groups on the surface of the target protein.

9. The method of claim 8, wherein said one or more amine groups are native amine groups.

10. The method of claim 8, wherein said one or more amine groups are engineered amine groups.

11. The method of claim 10, wherein said one or more amine groups are located on target protein amino acid residues known to contact one or more one or more ligands.

12. The method of claim 11, wherein the second harmonic-active moiety is PyMPO-succinimidyl ester.

13. The method of claim 12, wherein the target protein is labeled in situ while bound to a surface.

14. The method of claim 1, wherein the second harmonic-active moiety is an unnatural amino acid.

15. The method of claim 14, wherein the unnatural amino acid is located in a region of the target protein known to contact one or more ligands.

16. The method of claim 5, wherein the unnatural amino acid is Aladan.

17. The method of claim 6 wherein the ligand is one or more of a protein, a nucleic acid, a phospholipid, a carbohydrate, or a co-factor.

18. The method of claim 17, wherein the ligand is a protein member of a kinase signaling cascade.

19. The method of claim 18, wherein the target protein is a G protein-coupled receptor, a steroid hormone receptor, or a tyrosine kinase receptor.

20. The method of claim 19, 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, and a polyethylene surface.

21. The method of claim 20, wherein the surface is derivatized with oligo-PEG molecules or lipids.

22. The method of claim 21, wherein the oligo-PEG molecules or lipids are Ni-NTA-bearing oligo-PEG molecules or Ni-NTA-bearing lipids.

23. The method of claim 20, wherein the surface is a supported lipid bilayer or a lipid analog bilayer.

24. The method of claim 23, wherein the target protein comprises an affinity tag.

25. The method of claim 24, wherein the conformational change in the structure of the target protein is detected in real time.

26. The method of claim 25, wherein the agent A is a small molecule chemical compound, an antibody, a non-antibody polypeptide, a carbohydrate, an inhibitory nucleic acid, or any combination thereof that specifically binds to the target protein active site.

27. The method of claim 26, wherein 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.

37. The method of claim 1, wherein the agent A is not naturally bound to the active site of the target protein when isolated.

38. The method of claim 37, wherein the agent A is a synthetic ligand.

39. The method of claim 37, wherein the agent B is a small molecule chemical compound.

40. The method of claim 1, wherein the target protein is further labeled with a second harmonic-active moiety at additional amino acid residues located at one or more different sites in the target protein.

41. The method of claim 40, wherein the presence of a detectable signal at a single location alone indicates that the agent B binds specifically to the target protein and induces a conformational change to the structure of the target protein at that location.

42. The method of claim 40, wherein the presence of identical detectable signals at different locations indicates that the agent B binds non-specifically to the target protein.

43. The method of claim 1, wherein the measuring in step (c) is performed by a detector, and wherein the detector records data of the target protein conformational change on a data storage medium that is accessible via a system for reading the storage medium, wherein the system for reading the storage medium comprises a computer.

44. The method of claim 1, wherein the target protein is a G protein-coupled receptor, a steroid hormone receptor or a protein kinase.

28. A method for identifying a site-specific conformational change in the structure of a target protein upon binding of an agent to the protein, the method comprising:

a. contacting the target protein with the agent in a first sample, wherein the target protein is labeled at a first amino acid residue with a second harmonic-active moiety having a net orientation at an interface, wherein a first detectable signal is generated by the second harmonic-active moiety using a surface selective technique, and wherein the first detectable signal indicates a conformational change in the structure of the target protein produced when the agent binds to a site on the target protein;

b. contacting the target protein with the agent in a second sample, wherein the target protein is labeled at a second amino acid residue with a second harmonic-active moiety having a net orientation at an interface, wherein the second amino acid residue is located in a region of the target protein that differs from the location of the first amino acid residue, wherein a second detectable signal is generated by the second harmonic-active moiety using a surface selective technique, and wherein the second detectable signal indicates a conformational change in the structure of the target protein produced when the agent binds to a different site on the target protein; and

c. comparing the first detectable signal with the second detectable signal, wherein a conformational change at the site of the first amino acid residue alone indicates that the agent induces a conformational change to the structure of the protein at the site of the first amino acid residue and wherein a conformational change at the site of the second amino acid residue alone indicates that the agent induces a conformational change to the structure of the protein at the site of the second amino acid residue.

29. The method of claim 28, wherein the method includes the initial step of contacting the target protein with an agent that binds to the active site of the target protein.

30. The method of claim 28, further comprising repeating steps (b) and (c), wherein one or more additional amino acid residues located at one or more different sites in the target protein are labeled with a second harmonic-active moiety having a net orientation at an interface, wherein one or more additional detectable signals are generated by the second harmonic-active moiety(ies) using a surface selective technique, and wherein the one or more additional detectable signals indicates a conformational change in the structure of the target protein produced when the agent binds to a site on the target protein.

31. The method of claim 30, further comprising determining whether the agent specifically or non-specifically binds to an allosteric site on the target protein, wherein the agent specifically binds to an allosteric site on the target protein if the agent induces a conformational change in the structure of the target protein at the site of one second harmonic-active moiety-labeled amino acid but does not induce a conformational change in the structure of the target protein at the site of one or more other second harmonic-active moiety-labeled amino acids, and wherein the agent non-specifically binds to an allosteric site on the target protein if the agent induces an identical conformational change in the structure of the target protein at the sites of all second harmonic-active moiety-labeled amino acids.

32. The method of claim 28, wherein the second harmonic-active moiety is bound to the target protein by one or more engineered amino acid residues.

33. The method of claim 28, wherein the target protein is labeled in situ while bound to a surface.

34. The method of claim 28, wherein the conformational change in the structure of the target protein is detected in real time.

35. The method of claim 28 wherein the agent is a small molecule chemical compound, an antibody, a non-antibody polypeptide, a carbohydrate, an inhibitory nucleic acid, or any combination thereof.

36. The method of claim 28, wherein the agent that binds to the site of the target protein is a synthetic ligand.