Accelerating the validation of endogenous on-target engagement and assessment of in-cellulo kinetics for covalent inhibitors in early drug discovery

We have developed a complete workflow employing label-free mass-spectrometry to accelerate the characterization of covalent molecules from hit generation to lead-optimization. This workflow has fully integrated aspects of endogenous target engagement and kinetic parameter estimation to support structure-activity relationship (SAR), translate activity from invitro to the cell and to accurately determine the selectivity.

Covalent mechanism of inhibition is becoming increasingly popular in drug-discovery due to its potential ability in decoupling pharmacokinetics from pharmacodynamics by prolonging the residence time of the drug on the target of interest. This increases the therapeutic index which is limited only by the rate of target turnover. However, a key limitation in early covalent probe discovery is the translation of the in-vitro to appropriate in-cellulo target engagement parameters in a physiologically relevant cell models.

Using a classical model system, we interrogate the interaction of an irreversible covalent small-molecule both in-vitro and in-cellulo using an UPLC-MRM MS method with run time of 7.2min/sample only. Biochemically, we demonstrate the specificity of adduct formation with the cysteine of interest using mass shift both at the level of intact protein and on the peptide of interest. Further, we carry out in-cellulo studies to quantify target engagement as a function of compound concentration in NCI-H358 cell lines. Finally, we demonstrate the ability of the method to discriminate the selectivity of target engagement for the mutant variant vis-à-vis the WT in the heterozygous NCI-H358 cell line. Time and dose response of the inhibitor showing endogenous target engagement enabled the understanding and estimation of kinetics parameters such as kinact/KI.

The study demonstrates that under conditions where sample is not limiting and the adduct enrichment protocols are robust, use of UPLC rather than nanoLC decreases the turnaround time for the assessment of on-target engagement of covalent probes compatible with the high-throughput nature of early drug discovery.