Ke Sherry Li, John G Quinn, Matthew J Saabye, Jesus F Salcido Guerrero, Jim Nonomiya, Qihui Lian, Wilson Phung, Yevgeniy Izrayelit, Benjamin T Walters, Amy Gustafson, Nicholas F Endres, Maureen H Beresini, Melinda M Mulvihill
Analytical chemistry 2022 Jan 18With recent advances and success in several drugs designed to treat acute and chronic diseases, targeted covalent inhibitors show a resurgence in drug discovery. As covalent inhibition is time-dependent, the preferred quantitative potency metric of irreversible inhibitors is the second-order rate constant kinact/Ki, rather than IC50. Here, we present the development of a mass spectrometry-based platform for rapid kinetic analysis of irreversible covalent inhibitors. Using a simple liquid handling robot for automated sample preparation and a solid-phase extraction-based RapidFire-MS system for rapid MS analysis, kinetic characterization of covalent inhibitors was performed in high throughput both by intact protein analysis and targeted multiple reaction monitoring (MRM). In addition, a bimolecular reaction model was applied to extract kinact/Ki in data fitting, providing tremendous flexibility in the experimental design to characterize covalent inhibitors with various properties. Using KRASG12C inhibitors as a test case, the platform was demonstrated to be effective for studying covalent inhibitors with a wide range of kinact/Ki values from single digit to 3 × 105 M-1 s-1.
Ke Sherry Li, John G Quinn, Matthew J Saabye, Jesus F Salcido Guerrero, Jim Nonomiya, Qihui Lian, Wilson Phung, Yevgeniy Izrayelit, Benjamin T Walters, Amy Gustafson, Nicholas F Endres, Maureen H Beresini, Melinda M Mulvihill. High-Throughput Kinetic Characterization of Irreversible Covalent Inhibitors of KRASG12C by Intact Protein MS and Targeted MRM. Analytical chemistry. 2022 Jan 18;94(2):1230-1239
PMID: 34990117
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