High-Throughput Kinetic Characterization of Irreversible Covalent Inhibitors of KRAS^G12C by Intact Protein MS and Targeted MRM

Abstract

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 <i>k</i>_inact/<i>K</i>_i, 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 <i>k</i>_inact/<i>K</i>_i in data fitting, providing tremendous flexibility in the experimental design to characterize covalent inhibitors with various properties. Using KRAS^G12C inhibitors as a test case, the platform was demonstrated to be effective for studying covalent inhibitors with a wide range of <i>k</i>_inact/<i>K</i>_i values from single digit to 3 × 10⁵ M^-1 s^-1.