Methods of characterizing molecular interaction sites on proteins

• US 20030235862A1
• Filed: 04/04/2003
• Published: 12/25/2003
• Est. Priority Date: 04/05/2002
• Status: Abandoned Application

• Current Assignee: Sunesis Pharmaceuticals, Inc. (Viracta Therapeutics, Inc.)
• Original Assignee: Sunesis Pharmaceuticals, Inc. (Viracta Therapeutics, Inc.)
• Inventors: Fucini, Raymond, Cunningham, Brian, Arkin, Michelle, DeLano, Warren
• Application Number:
  US10/407,416
• Publication Number:
  US 20030235862A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/7155 for interleukins [IL]
• CPC Class Codes: C12N 2740/16222 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding

Abstract

The present invention is directed to methods of characterizing molecular interaction sites on a biological target molecule. The methods involve contacting a polypeptide with a library of ligand candidates each of which is capable of tethering to the polypeptide at a site of interest, and analyzing the physicochemical properties of the ligand candidates tethering to the polypeptide. The methods herein are useful for discovery of and development of small molecule drugs.

Claims

1. A method for characterizing a molecular interaction site on a protein, the method comprising:

a) contacting a polypeptide comprising a first chemically reactive group at a site of interest with a library of ligand candidates wherein each ligand candidate comprises a second chemically reactive group that is capable of forming a reversible covalent bond with the first chemically reactive group;

b) forming a reversible covalent bond between the first chemically reactive group and the second chemically reactive group of a plurality of the ligand candidates;

c) detecting the ligand candidates that formed the reversible covalent bond;

d) identifying a physicochemical property of at least one of the ligand candidates that formed the reversible covalent bond;

e) determining the number of ligand candidates that formed the reversible covalent bond having the physicochemical property; and

f) determining the number of ligand candidates in the library having the physicochemical property.

2. The method of claim 1 further comprising:

comparing the prevalence of ligand candidates with the physicochemical property that form the reversible covalent bond with the prevalence of ligand candidates with the physicochemical property in the library and measuring the statistical significance of the comparison.

3. The method of claim 2 further comprising:

characterizing the molecular interaction site by a significant enrichment of the prevalence of the physicochemical property among ligand candidates that formed the reversible covalent bond.

4. The method of claim 2 further comprising:

characterizing the molecular interaction site by a significant depletion of the prevalence of the physicochemical property among ligand candidates that formed the reversible covalent bond.

5. The method of claim 1 wherein the protein is individually contacted with members of the library of ligand candidates.

6. The method of claim 1 wherein the site of interest is an active site, an allosteric site, a small molecule binding site, or a protein-protein binding site.

9. The method of claim 1 wherein the physicochemical property of the ligand candidate is a molecular property selected from the group consisting of molecular weight, molar refractivity, LogP, polar surface area, total number of rotatable bonds, total number of aromatic atoms, total number of hydrogen bond donors and total number of hydrogen bond acceptors.

10. The method of claim 1 wherein the physicochemical property of the ligand candidate is the presence of a chemical feature selected from the group consisting of a molecular shape, an atomic skeleton and a peripheral functionality.

11. The method of claim 10 wherein the molecular shape is selected from the group consisting of:

12. The method of claim 10 wherein the atomic skeleton is selected from the group consisting of:

13. The method of claim 10 wherein the peripheral functionality is selected from the group consisting of:

14. The method of claim 10 wherein the peripheral functionality is selected from the group consisting of:

15. The method of claim 10 wherein the physicochemical property is the presence of a chemical feature selected from the group consisting of a nitro, an amine, an amide, a sulfonamide, a carboxylic acid, an alcohol, a phenol, a halogen, an aryl halide and an ether.

7. A method for characterizing a molecular interaction site on a protein, the method comprising:

a) contacting a polypeptide comprising a first chemically reactive group at a site of interest with a library of ligand candidates wherein each ligand candidate comprises a second chemically reactive group that is capable of forming a reversible covalent bond with the first chemically reactive group;

b) forming a reversible covalent bond between the first chemically reactive group and the second chemically reactive group of a plurality of the ligand candidates;

c) detecting the ligand candidates that formed the reversible covalent bond;

d) identifying a physicochemical property of at least one of the ligand candidates that formed the reversible covalent bond;

e) calculating the statistical significance of the incidence of ligand candidates having the physicochemical property in the plurality; and

f) repeating each of step a) through step e) for at least one positional variant of the polypeptide, the positional variant comprising an identical first chemically reactive group at a different sequence location.

8. The method of claim 7 wherein at least the formation of the reversible covalent bond is performed in the presence of a reducing agent.

16. A method for characterizing a molecular interaction site on a protein, the method comprising:

a) contacting a polypeptide comprising a thiol or a masked thiol at a site of interest with a library of ligand candidates wherein each ligand candidate comprises a disulfide group that is capable of engaging in disulfide exchange to form a disulfide linkage between the polypeptide and the ligand candidate;

b) forming the disulfide linkage between the polypeptide and a plurality of the ligand candidates;

c) detecting the ligand candidates that formed the disulfide linkage;

d) identifying a physicochemical property of at least one of the ligand candidates that formed the disulfide linkage; and

e) calculating the statistical significance of the incidence of ligand candidates having the physicochemical property in the plurality;

wherein the physicochemical property is selected from the group consisting of molecular weight, LogP, number of hydrogen bond donors, and number of hydrogen bond acceptors.