Methods for identifying compounds that modulate enzymatic activity

• US 20040005632A1
• Filed: 02/25/2003
• Published: 01/08/2004
• 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: Hansen, Stig, Erlanson, Daniel A., McDowell, Robert S.
• Granted Patent: US 7,214,487 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C07K 1/1072 by covalent attachment of residues or functional groups
• CPC Class Codes: G01N 2333/916 acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

The present invention relates to the use of “tethering” to identify compounds that modulate enzymatic activity.

Claims

1. A method comprising:

a) providing a target having a reactive nucleophile located outside of 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 a first covalent bond with the nucleophile and a second functionality that is capable of forming a second covalent bond;

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

d) forming a second covalent 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 reactive nucleophile is a thiol.

3. A method comprising:

a) providing a target having a first reactive nucleophile located inside of the site of interest and a second reactive nucleophile located outside of a site of interest;

b) contacting the target with an extender thereby forming a target-extender complex, the extender comprising a first functionality and a latent second functionality, a cleavable linker and a binding determinant wherein the first functionality forms a first covalent bond with the second reactive nucleophile and the binding determinant binds to the site of interest;

c) cleaving the extender at the cleavable linker thereby forming a modified target-extender complex by exposing the second functionality and releasing the binding determinant from the site of interest;

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

e) forming a second covalent bond between the modified target-extender complex and the candidate ligand thereby forming a target-extender-ligand conjugate; and

,

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

4. The method of claim 3 wherein the first and second reactive nucleophiles are each a thiol.

5. A method comprising:

a) providing a PTP having a reactive thiol located outside of the active site;

b) contacting the PTP with an extender thereby forming a PTP-extender complex wherein the extender comprises a first functionality that forms a covalent bond with the reactive thiol and a second functionality that is capable of forming a disulfide bond;

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

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

,

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

6. The method of claim 5 wherein the reactive thiol on the target is a naturally occurring —

SH from a cysteine that is part of the naturally occurring protein sequence.

7. The method of claim 5 wherein the reactive thiol on the target is from a cysteine where mutagenesis was used to replace a naturally occurring amino acid.

8. The method of claim 5 wherein the reactive thiol is masked as a disulfide.

9. The method of claim 5 wherein the covalent bond between the reactive thiol and the first functionality is an irreversible covalent bond.

10. The method of claim 5 wherein the covalent bond between the reactive thiol and the first functionality is a reversible covalent bond.

11. The method of claim 5 wherein the target-extender complex is contacted with a candidate ligand in the presence of a reducing agent.

12. The method of claim 11 wherein the reducing agent is selected from the group consisting of:

cysteine, cysteamine, dithiothreitol, dithioerythritol, glutathione, 2-mercaptoethanol, 3-mercaptoproprionic acid, a phosphine such as tris-(2-carboxyethyl-phosphine), and sodium borohydride.

13. The method of claim 5 wherein the extender is selected from the group consisting of

14. A method comprising:

a) providing a PTP having an active site, a cysteine located in the active site and a reactive thiol located outside of the active site;

b) contacting the PTP with an extender thereby forming a PTP-extender complex, the extender comprising a first functionality and a latent second functionality, a cleavable linker and a binding determinant comprising a phosphotyrosine or a phosphotyrosine mimetic wherein the first functionality forms a first covalent bond with the reactive thiol and the binding determinant binds to the active site;

c) cleaving the extender at the cleavable linker thereby forming a modified PTP-extender complex by exposing the second functionality and releasing the binding determinant from the active site;

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

e) forming a second covalent bond between the modified PTP-extender complex and the candidate ligand thereby forming a PTP-extender-ligand conjugate; and

,

f) identifying the candidate ligand present in the PTP-extender-ligand conjugate.

15. The method of claim 14 wherein the reactive thiol on the target is a naturally occurring —

SH from a cysteine that is part of the naturally occurring protein sequence.

16. The method of claim 14 wherein the reactive thiol on the target is from a cysteine where mutagenesis was used to replace a naturally occurring amino acid.

17. The method of claim 14 wherein the reactive thiol is masked as a disulfide.

18. The method of claim 14 wherein the covalent bond between the reactive thiol and the first functionality is an irreversible covalent bond.

19. The method of claim 14 wherein the covalent bond between the reactive thiol and the first functionality is a reversible covalent bond.

20. The method of claim 14 wherein the target-extender complex is contacted with a candidate ligand in the presence of a reducing agent.

21. The method of claim 20 wherein the reducing agent is selected from the group consisting of:

cysteine, cysteamine, dithiothreitol, dithioerythritol, glutathione, 2-mercaptoethanol, 3-mercaptoproprionic acid, a phosphine such as tris-(2-carboxyethyl-phosphine), and sodium borohydride.

22. The method of claim 14 wherein the extender is of the formula

23. The method of claim 22 wherein A is selected from the group consisting of: