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/Ki values 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.