Methods for ligand discovery

• US 6,998,233 B2
• Filed: 04/10/2002
• Issued: 02/14/2006
• Est. Priority Date: 06/26/1998
• Status: Expired due to Fees

• Current Assignee: Sunesis Pharmaceuticals, Inc. (Viracta Therapeutics, Inc.)
• Original Assignee: Sunesis Pharmaceuticals, Inc. (Viracta Therapeutics, Inc.)
• Inventors: Wells, Jim, Erlanson, Dan, Braisted, Andrew C.
• Primary Examiner(s): Wang, Andrew
• Assistant Examiner(s): Epperson, Jon D.
• Application Number:
  US10/121,216
• Publication Number:
  US 20020155505A1
• Time in Patent Office: 1,406 Days
• Field of Search: 435/6, 435/7.1
• US Class Current: 435/6.19
• 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) 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, wherein the extender is of the formula;

where R is unsubstituted C₁–

C₂₀ aliphatic, substituted C₁–

C₂₀ aliphatic, unsubstituted aryl, and substituted aryl;

R′

is H, —

SR¹ wherein R¹ is unsubstituted C₁–

C₁₀ aliphatic, substituted C₁–

C₁₀ aliphatic, unsubstituted aryl, and substituted aryl;

X is a leaving group, and the boxes in each formula represent a binding determinant;

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

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

,

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

2. The method of claim 1, wherein the candidate ligand present in the target-extender-ligand conjugate is identified by mass spectrometry.

3. The method of claim 1, wherein the candidate ligand present in the target-extender-ligand conjugate is identified using a labeled probe.

4. The method of claim 1, wherein the candidate ligand present in the target-extender-ligand conjugate is identified using a functional assay.

5. The method of claim 1, wherein the candidate ligand present in the target-extender-ligand conjugate is identified using chromatography.

6. The method of claim 1, wherein the candidate ligand present in the target-extender-ligand conjugate is identified using surface plasmon resonance.

7. 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, wherein the extender is of the formula;

where R is unsubstituted C₁–

C₂₀ aliphatic, substituted C₁–

C₂₀ aliphatic, unsubstituted aryl, and substituted aryl;

R′

is H, —

SR¹ wherein R¹ is unsubstituted C₁–

C₁₀ aliphatic, substituted C₁–

C₁₀ aliphatic, unsubstituted aryl, and substituted aryl;

X is a leaving group, and the box in the formula represents a binding determinant;

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

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

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

8. The method of claim 7, wherein the candidate ligand present in the target-extender-ligand conjugate is identified by mass spectrometry.

9. The method of claim 7, wherein the candidate ligand present in the target-extender-ligand conjugate is identified using a labeled probe.

10. The method of claim 7, wherein the candidate ligand present in the target-extender-ligand conjugate is identified using a functional assay.

11. The method of claim 7, wherein the candidate ligand present in the target-extender-ligand conjugate is identified using chromatography.

12. The method of claim 7, wherein the candidate ligand present in the target-extender-ligand conjugate is identified using surface plasmon resonance.