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High-Throughput Kinetic Characterization of Irreversible Covalent Inhibitors of KRAS by Intact Protein MS and Targeted MRM

Analytical Chemistry
January 10, 2022

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, the development of a
mass spectrometry-based platform for rapid kinetic analysis of irreversible covalent inhibitors is presented. 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/Kvalues from single digit to 3 × 105 M−1 s−1.

In this study, researchers at Genentech used Genedata Expressionist to batch process sample data and automatically annotate adducts based on the expected mass of the covalent modification.