Methods for identifying compounds that modulate enzymatic activities by employing covalently bonded target-extender complexes with ligand candidates
First Claim
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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.
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Abstract
The present invention relates to methods for identifying compounds that modulate enzymatic activity by contacting covalently bonded PTP-extender complexes or modified covalently bonded PTP-extender complexes with ligand candidates.
8 Citations
19 Claims
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1. A method for identifying a candidate ligand comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method of identifying a candidate ligand comprising:
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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. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification