Abstract 5678: LC-HRMS based kinetic characterization platform for irreversible covalent inhibitor screening

Abstract Covalent inhibitors are recognized as indispensable parts in drug discovery and therapeutic researches. The irreversibility of covalent conjugation makes Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) a suitable tool for covalent inhibitor screening. LC-HRMS allows label-free, direct detection of the native protein and the covalent adduct in the sample, providing relative quantification of target engagement. Several methods have been developed for covalent screening using intact protein MS or peptide mapping, including binding site mapping, covalent binding rate determination and Kinact/Ki value measurement. In this study, the final protein concentration was 1.5 μM and the serial concentrations of positive control AMG510 were from 0.49 μM to 4.00 μM, and the total incubation time was 100 seconds. The intensity of the native protein and covalent adducts were collected on a QE Plus coupled with a C4 column. A relatively high binding rate was observed by intact mass analysis. And a sequence coverage of GDP-KRASG12C was above 75% using peptide mapping, the specific binding site was confirmed by MS2 data. Kinact/Ki value were fitted using more than six concentrations and five timepoints. This method was used for covalent inhibitors screening, binding site characterization and Kinact/Ki measurement. Citation Format: Cai Fu, Tong Wu, Li Li, Tj (Tiejun) Bing. LC-HRMS based kinetic characterization platform for irreversible covalent inhibitor screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5678.