Methods for identifying compounds that modulate enzymatic activities by employing covalently bonded target-extender complexes with ligand candidates

1. A method for identifying a candidate ligand comprising:

a) providing a PTP (protein tyrosine phosphatase) having a reactive thiol located outside of its 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.

2. The method of claim 1 wherein the reactive thiol on PTP is a naturally occurring -SH from a cysteine that is part of the naturally occurring protein sequence.

3. The method of claim 1 wherein the reactive thiol on the PTP is from a cysteine where mutagenesis was used to replace a naturally occurring amino acid.

4. The method of claim 1 wherein the reactive thiol is masked as a disulfide.

5. The method of claim 1 wherein the covalent bond between the reactive thiol and the first functionality is an irreversible covalent bond.

6. The method of claim 1 wherein the covalent bond between the reactive thiol and the first functionality is a reversible covalent bond.

7. The method of claim 1 wherein the PTP-extender complex is contacted with a candidate ligand in the presence of a reducing agent.

8. The method of claim 7 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-carboxyethylphosphine), and sodium borohydride.

9. The method of claim 1 wherein the extender is selected from the group consisting of

10. A method of identifying a candidate ligand comprising:

a) providing a PTP (protein tyrosine phosphatase) having an active site, a cysteine located in its 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 thereby 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.

11. The method of claim 10 wherein the reactive thiol on PTP is a naturally occurring —SH from a cysteine that is part of the naturally occurring protein sequence.

12. The method of claim 10 wherein the reactive thiol on the PTP is from a cysteine where mutagenesis was used to replace a naturally occurring amino acid.

13. The method of claim 10 wherein the reactive thiol is masked as a disulfide.

14. The method of claim 10 wherein the covalent bond between the reactive thiol and the first functionality is an irreversible covalent bond.

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

16. The method of claim 10 wherein PTP-extender complex is contacted with a candidate ligand in the presence of a reducing agent.

17. The method of claim 16 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-carboxyethylphosphine), and sodium borohydride.

18. The method of claim 10 wherein the extender is of the formula

where A is selected from the group consisting of:

n is 1–5; m is 2–5; and, X is a halide.

19. The method of claim 18 wherein A is selected from the group consisting of:

n and m are independently 2 or 3; and X is bromide.