posted on 2022-02-04, 15:05authored byAshley
E. Owens, Michael J. Iannotti, Tino W. Sanchez, Ty Voss, Abhijeet Kapoor, Matthew D. Hall, Juan J. Marugan, Sam Michael, Noel Southall, Mark J. Henderson
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.