A real-time cellular thermal shift assay (RT-CETSA) to monitor target engagement

Determining a molecule's mechanism of action is paramount during chemical probe development and drug discovery. The cellular thermal shift assay (CETSA) is a valuable tool to confirm target engagement in cells for a small molecule that demonstrates a pharmacological effect. CETSA directly detects biophysical interactions between ligands and protein targets, which can alter a protein's unfolding and aggregation properties in response to thermal challenge. In traditional CETSA experiments, each temperature requires an individual sample, which restricts throughput and requires substantial optimization. To capture the full aggregation profile of a protein from a single sample, we developed a prototype real-time CETSA (RT-CETSA) platform by coupling a real-time PCR instrument with a CCD camera to detect luminescence. A thermally stable Nanoluciferase variant (ThermLuc) was bioengineered that withstood unfolding at temperatures greater than 90 degrees Celsius and was compatible with monitoring target engagement events when fused to diverse targets. Utilizing well-characterized inhibitors of lactate dehydrogenase alpha, RT-CETSA showed significant correlation with enzymatic, biophysical, and other cell-based assays. A data analysis pipeline was developed to enhance the sensitivity of RT-CETSA to detect on-target binding. The RT-CETSA technology advances capabilities of the CETSA method and facilitates the identification of ligand-target engagement in cells, a critical step in assessing the mechanism of action of a small molecule. ### Competing Interest Statement T.W.S., M.H.R., A.E.O., B.B., S.M., A.S. and M.J.H. are inventors on a patent REAL-TIME CELLULAR THERMAL SHIFT ASSAY (RT-CETSA) FOR RESEARCH AND DRUG DISCOVERY (PCT/US21/45184). T.W.S., M.H.R., B.B. N.S., S.M. A.S. T.V. and M.J.H. are inventors on a related provisional patent METHODS AND SYSTEMS FOR ANALYZING TARGET ENGAGEMENT DATA FROM BIOLOGICAL ASSAYS (HHS E-022-2022-0-US-01).