High-Throughput Cellular Thermal Shift Assay Using Acoustic Transfer of Protein Lysates

Cellular thermal shift assay (CETSA) is a valuable method to confirm target engagement within a complex cellular environment, by detecting changes in a protein's thermal stability upon ligand binding. The classical CETSA method measures changes in the thermal stability of endogenous proteins using immunoblotting, which is low-throughput and laborious. Reverse-phase protein arrays (RPPAs) have been demonstrated as a detection modality for CETSA; however, the reported procedure requires manual processing steps that limit throughput and preclude screening applications. We developed a high-throughput CETSA using an acoustic RPPA (HT-CETSA-aRPPA) protocol that is compatible with 96- and 384-well microplates from start-to-finish, using low speed centrifugation to remove thermally destabilized proteins. The utility of HT-CETSA-aRPPA for guiding structure-activity relationship studies was demonstrated for inhibitors of lactate dehydrogenase A. Additionally, a collection of kinase inhibitors was screened to identify compounds that engage MEK1, a clinically relevant kinase target.

Automated summary

Cellular thermal shift assay (CETSA) is a valuable method for confirming target engagement in cellular environment. Traditional CETSA method measures changes in protein thermal stability using immunoblotting, which is low throughput and laborious. This study developed a high-throughput CETSA method that is compatible with 96- and 384-well microplates and removes thermally destabilized proteins using low speed centrifugation. It has been demonstrated to guide structure-activity relationship studies and compound screening.