High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2.

Target engagement metrics provide predictive value for in vivo efficacy of low-molecular-weight compounds. While direct observation of compound binding to its intended target builds the most confidence, the throughput of these measurements tends to be low. Indirect competition approaches that ask if an unlabeled compound can displace a tracer offer a higher throughput option for compound profiling. Most indirect target engagement assays employ reversible tracers; by contrast, programs focused on developing irreversible drugs can benefit from covalent tracers whose properties better match the mechanism of action of the test compounds. We demonstrate how covalent tracers could be employed in high-throughput assays to indirectly measure target occupancy of either endogenous or exogenously overexpressed MK2 and how these cellular assays could be adapted to monitor the kinetics of compound-target binding and bioavailability in the presence of human serum.