Accelerating the Validation of Endogenous On-Target Engagement and In Cellulo Kinetic Assessment for Covalent Inhibitors of KRAS(G12C )in Early Drug Discovery

Covalent inhibition is a valuable modality in drug discovery because of its potential ability in decoupling pharmacokinetics from pharmacodynamics by prolonging the residence time of the drug on the target of interest. This increase in target occupancy is limited only by the rate of target turnover. However, a limitation in such studies is to translate the in vitro inhibition assessment to the appropriate in cellulo target engagement parameter by covalent probes. Estimation of such parameters is often impeded by the low-throughput nature of current probe-free approaches. In this study, an ultra-performance liquid chromatography-multiple reaction monitoring mass spectrometry platform was utilized to develop a targeted proteomics workflow that can evaluate cellular on-target engagement of covalent molecules in an increased throughput manner. This workflow enabled a throughput increase of 5-10 fold when compared to traditional nanoLC-based proteomics studies. To demonstrate the applicability of the method, KRAS(G12C )was used as a model system to investigate the interaction of an irreversible covalent small molecule, compound 25, both in vitro and in cellulo. Initial biochemical studies confirmed that the small molecule forms an adduct with the targeted cysteine on the protein, as assessed at the level of both intact protein and on the target peptide. In cellulo studies were carried out to quantify target engagement and allele selectivity assessment for the small molecule in the heterozygous NCI-H358 cell line for KRAS(G12C) with respect to the WT type protein. The workflow enabled evaluation of in vitro and in cellulo target engagement kinetics, providing mechanistic insights into the irreversible mode of inhibition. In summary, the method has the potential for target agnostic application in the assessment of on-target engagement of covalent probes compatible with the high-throughput requirements of early drug discovery.