Methods for identifying compounds that modulate enzymatic activity

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.

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.

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.