Methods for ligand discovery

• US 20020155505A1
• Filed: 04/10/2002
• Published: 10/24/2002
• Est. Priority Date: 06/26/1998
• Status: Active Grant

• Current Assignee: Sunesis Pharmaceuticals, Inc. (Viracta Therapeutics, Inc.)
• Original Assignee: Sunesis Pharmaceuticals, Inc. (Viracta Therapeutics, Inc.)
• Inventors: Wells, Jim, Erlanson, Dan, Braisted, Andrew C.
• Granted Patent: US 6,998,233 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C07D 207/46 with hetero atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 333/38 Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
• CPC Class Codes: C07D 333/70 attached in position 2
• CPC Class Codes: C07D 401/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C40B 20/08 Direct analysis of the library members per se by physical methods, e.g. spectroscopy
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/04 Libraries containing only organic compounds
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

The present invention provides novel methods for ligand discovery. The inventive methods rely on a process termed “tethering” where potential ligands are covalently bonded or “tethered” to a target and subsequently identified.

Claims

1. A method comprising

a) contacting a target that comprises a chemically reactive group at or near a site of interest with a compound that is capable of forming a covalent bond with the chemically reactive group;

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

,

c) identifying the target-compound conjugate by subjecting the target-compound conjugate to mass spectrometry.

2. A mass spectrometer comprising a target-compound conjugate.

3. A target-compound conjugate selected from the group consisting of

4. The target-compound conjugate of claim 3 wherein the target is a polypeptide.

5. The target-compound conjugate of claim 4 wherein the covalent bond between the —

S—

S—

group and the

6. The target-compound conjugate of claim 4 wherein the covalent bond between the —

S—

S—

group and the

7. The target-compound conjugate of claim 4 wherein the target is selected from the group consisting of enzymes, receptors, transcription factors, ligands for receptors, growth factors, cytokines, immunoglobulins, nuclear proteins, signal transduction components, and allosteric enzyme regulators.

8. A method comprising:

a) contacting a target protein that is capable of forming a disulfide bond with a ligand candidate that is also capable of forming a disulfide bond;

b) forming a disulfide bond between the target protein and the ligand candidate thereby forming a target-ligand conjugate; and

c) identifying the ligand present in the target protein-ligand conjugate.

9. The method as in claim 8 wherein the contacting step occurs in the presence of a reducing agent.

10. The method as in claim 8 wherein the identification occurs using mass spectrometry.

11. The method as in claim 8 wherein the identification occurs using a labeled probe.

12. The method as in claim 8 wherein the identification occurs using a functional assay.

13. The method as in claim 8 wherein the identification occurs using chromatography.

14. The method as in claim 8 wherein the identification occurs using surface plasmon resonance.

15. The method as in claim 8 wherein the ligand candidate is selected from the group comprising

16. The method of claim 8 wherein the target protein comprises an —

SH group from a cysteine that is part of the native amino acid sequence of said protein.

17. The method of claim 8 wherein the target protein has comprises an engineered —

SH group from a cysteine introduced into the amino acid sequence of said protein.

19. The library of claim 18 having at least 5 members.

20. The library of claim 18 having at least 100 members.

21. The library of claim 18 wherein each member library has a mass that differs from another member of the library by at least 5 atomic mass units.

22. The library of claim 18 wherein each member has a mass that differs from another member of the library by at least 10 atomic mass units.

24. The method of claim 23 wherein the identification of the second compound that binds to the target occurs in the presence of the first compound.

25. The method of claim 23 wherein R^DSSR¹ and R^ESSR¹ are each independently selected from the group consisting of

27. The method of claim 26 wherein the anchoring group is selected from a group consisting of a reactive electrophile, a reactive nucleophile, and a metal coordination site.

29. The method as in claim 28 wherein the reactive nucleophile on the target is a thiol or a masked thiol.

30. The method of claim 28 wherein the extender is of the formula:

31. The method of claim 28 wherein the extender is of the formula:

18. A library of compounds wherein each member comprises a moiety —

SSR¹ where R¹ is unsubstituted C₁-C₁₀ aliphatic, substituted C₁-C₁₀ aliphatic, unsubstituted aryl, and wherein each member has a different mass.

23. A method comprising:

a) identifying a first compound of the formula R^DSSR¹ that binds to a target protein;

b) identifying a second compound of the formula R^ESSR¹ that binds to a target protein; and

c) forming a conjugate compound comprising R^D and R^E wherein R^D and R^E are each independently C₁-C₂₀ unsubstituted aliphatic, C₁-C₂₀ substituted aliphatic, unsubstituted aryl, and substituted aryl; and

R¹ is unsubstituted C₁-C₁₀ aliphatic, substituted C₁-C₁₀ aliphatic, unsubstituted aryl.

26. A method comprising

a) providing a target having an anchoring group that is capable of forming a covalent bond or coordinating a metal at or near a site of interest;

b) contacting the target with an extender thereby forming a target-extender complex wherein the extender comprises a first functionality that forms either a covalent bond or coordinates a metal and a second functionality that is capable of forming a covalent bond;

c) contacting the target-extender complex with a candidate ligand that comprises a group that is capable of forming a covalent bond with the second functionality;

d) forming a covalent bond between the target-extender complex and the candidate ligand; and

,

e) identifying the candidate ligand present in the target-extender-ligand conjugate.

28. A method comprising:

a) providing a target having a reactive nucleophile at or near a site of interest;

b) contacting the target with an extender thereby forming a target-extender complex wherein the extender comprises a first functionality that reacts with the nucleophile in the target to form a covalent bond and a second functionality that is capable of forming a disulfide bond;

c) contacting the target-extender complex with a ligand candidate that is capable of forming a disulfide bond;

d) forming a disulfide bond between the target-extender complex and the ligand candidate thereby forming a target-extender-ligand conjugate; and

,

e) identifying the ligand candidate present in the target-extender-ligand conjugate.