High-Throughput Kinetic Characterization of Irreversible Covalent Inhibitors of KRASG12C by Intact Protein MS and Targeted MRM
化学
共价键
组合化学
质谱法
色谱法
有机化学
作者
Ke Sherry Li,John Quinn,Matthew J Saabye,Jesus F Salcido Guerrero,Jim Nonomiya,Qihui Lian,Wilson Phung,Yevgeniy Izrayelit,Benjamin T. Walters,Amy Gustafson,Nicholas Endres,Maureen H. Beresini,Melinda M. Mulvihill
出处
期刊:Analytical Chemistry [American Chemical Society] 日期:2022-01-06卷期号:94 (2): 1230-1239被引量:12
With 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.