ICE Bioscience Posters

Download

Covalent inhibitors are recognized as an 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 K_inact/K_i 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-KRAS^G12C was above 75% using peptide mapping, the specific binding site was confirmed by MS² data. K_inact/K_i value were fitted using more than six concentrations and five timepoints. This method was used for covalent inhibitors screening, binding site characterization and K_inact/K_i measurement.