Quantitative Interpretation of Intracellular Drug Binding and Kinetics Using the Cellular Thermal Shift Assay

Evidence
of physical interaction with the target protein is essential
in the development of chemical probes and drugs. The cellular thermal
shift assay (CETSA) allows evaluation of drug binding in live cells
but lacks a framework to support quantitative interpretations and
comparisons with functional data. We outline an experimental platform
for such analysis using human kinase p38α. Systematic variations
to the assay’s characteristic heat challenge demonstrate an
apparent loss of compound potency with an increase in duration or
temperature, in line with expectations from the literature for thermal
shift assays. Importantly, data for five structurally diverse inhibitors
can be quantitatively explained using a simple model of linked equilibria
and published binding parameters. The platform further distinguishes
between ligand mechanisms and allows for quantitative comparisons
of drug binding affinities and kinetics in live cells and lysates.
We believe this work has broad implications in the appropriate use
of the CETSA for target and compound validation.