Methods for identifying allosteric sites

• US 20040014140A1
• Filed: 04/08/2003
• Published: 01/22/2004
• Est. Priority Date: 04/08/2002
• Status: Abandoned Application

• Current Assignee: Sunesis Pharmaceuticals, Inc. (Viracta Therapeutics, Inc.)
• Original Assignee: Sunesis Pharmaceuticals, Inc. (Viracta Therapeutics, Inc.)
• Inventors: OʼBrien, Thomas, Lam, Joni, Hardy, Jeanne, Hansen, Stig K., Erlanson, Daniel A.
• Application Number:
  US10/409,968
• Publication Number:
  US 20040014140A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/485 involving kinase
• 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 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins

Abstract

The present invention relates to exosites and methods for identifying exosites in protein targets. The present invention also relates to methods for identifying allosteric sites and identifying compounds that bind therein.

Claims

1. A method of identifying an allosteric site comprising:

a) providing a target comprising a first binding site, a second binding site, and a chemically reactive group at or near the second binding site;

b) contacting the target with a compound that is capable of forming a covalent bond with the chemically reactive group;

c) forming a covalent bond between the target and the compound thereby forming a target-compound conjugate;

d) determining whether the target-compound conjugate possesses a change in the primary binding site as compared with the target.

2. The method of claim 1 wherein the chemically reactive group is a thiol.

3. The method of claim 1 wherein the chemically reactive group is a masked thiol.

4. The method of claim 3 wherein the masked thiol is in the form of a disulfide.

5. The method of claim 2 or 3 wherein the covalent bond is a disulfide bond and the contacting step occurs in the presence of a reducing agent.

6. The method of claim 5 wherein the compound is a ligand candidate selected from the group consisting of:

7. The method of claim 1 wherein the change is a functional change in the activity of the target.

8. The method of claim 1 wherein the change is a structural change.

9. The method of claim 8 wherein the structural change is determined using x-ray crystallography.

10. The method of claim 8 wherein the structural change is determined using NMR.

11. The method of claim 8 wherein the structural change is determined using circular dichroism.

12. The method of claim 1 wherein the target is a protease.

13. The method of claim 1 wherein the target is a kinase.

14. The method of claim 1 wherein the target is a phosphatase.

15. A method of identifying an allosteric inhibitor comprising:

a) performing a first tethering experiment and

b) performing a second tethering experiment wherein both tethering experiments comprise;

i) providing a target comprising a first binding site, a second binding site, and a chemically reactive group at or near the second binding site;

ii) contacting the target with a compound that is capable of forming a covalent bond with the chemically reactive group;

iii) forming covalent bond between the target and the compound thereby forming a target-compound conjugate; and

iv) identifying the target-compound conjugate wherein the first tethering experiment is performed in the presence of a ligand that binds to the first binding site and the second tethering experiment is performed in the absence of the ligand that binds to the first binding site.

16. The method of claim 15 wherein the chemically reactive group is a thiol or a masked thiol.

17. The method of claim 16 wherein the covalent bond is a disulfide bond and the contacting step occurs in the presence of a reducing agent.

18. The method of claim 17 wherein the compound is a ligand candidate selected from the group consisting of:

19. A method of identifying an allosteric inhibitor comprising:

a) providing a target that is capable of allosteric regulation and a mutant thereof that is not capable of allosteric regulation;

b) contacting the target with a compound;

c) contacting the mutant with the compound; and

d) comparing the activity of the compound against the target with the activity of the compound against mutant.

20. An allosteric inhibitor of a caspase.