Rapid Evaluation of Small Molecule Cellular Target Engagement with a Luminescent Thermal Shift Assay

Determination
of target engagement for candidate drug molecules
in the native cellular environment is a significant challenge for
drug discovery programs. The cellular thermal shift assay (CETSA)
has emerged as a powerful tool for determining compound target engagement
through measurement of changes to a protein's thermal stability
upon
ligand binding. Here, we present a HiBiT thermal shift assay (BiTSA)
that deploys a quantitative peptide tag for determination of compound
target engagement in the native cellular environment using a high
throughput, plate-based luminescence readout. We demonstrate that
BiTSA can rapidly assess cellular target engagement of small molecule
ligands against their cognate targets and highlight two applications
of BiTSA for differentiating small molecules targeting mutant KRAS
and TP53.