Covalent target engagement
AssigneePromega Corporation
InventorsMatthew ROBERS, Cesear CORONA, Jim VASTA, Inhong HWANG, Kelly TESKE
Life of this patent
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Abstract
Provided herein are systems and methods for conducting covalent target engagement assays. In particular, cysteine-reactive tracers are provided for covalent binding to labeled target proteins, and signals produced by the interaction or proximity of the tracers and labeled target proteins are used to detect/quantify binding of a cysteine-reactive test agent.
Claims32
A system comprising:
- (a) a target protein tethered to a first component of a luminescent reporter;
- and
- (b) a cysteine-reactive tracer comprising second component of the luminescent reporter tethered to a cysteine-reactive group.
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A method of quantifying the covalent binding of a cysteine-reactive test agent to a target protein, the method comprising:
- (a) contacting a target protein tethered to a first component of a luminescent reporter with the cysteine-reactive test agent;
- (b) contacting the target protein with a cysteine-reactive tracer comprising second component of the luminescent reporter tethered to a cysteine-reactive group,
- (c) detecting a luminescent signal from the luminescent reporter, wherein the amount of the luminescent signal emitted is inversely proportional to the amount of the cysteine-reactive test agent covalently bound to the target protein.
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A method of quantifying the covalent binding of a cysteine-reactive test agent to a target protein within a cell, the method comprising:
- (a) providing a cell comprising a target protein tethered to a first component of a luminescent reporter;
- (b) contacting the cell with the cysteine-reactive test agent, and allowing the cysteine-reactive test agent to enter the cell and interact with the target protein;
- (c) lysing the cell to produce a cell lysate;
- (d) contacting the cell lysate with a cysteine-reactive tracer comprising second component of the luminescent reporter tethered to a cysteine-reactive group, and allowing the cysteine-reactive tracer to interact with the target protein;
- (e) detecting a luminescent signal from the luminescent reporter, wherein the amount of the luminescent signal emitted is inversely proportional to the amount of the cysteine-reactive test agent covalently bound to the target protein.
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Dependencies are inferred from the claim text.