biostudies-literatureUnknown17(9)Sanchez TWNational Center for Advancing Translational SciencesDetermining 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 ACS chemical biology2471-2482https://www.ebi.ac.uk/biostudies/studies/S-EPMC9486815biostudies-literatureReal-Time Cellular Thermal Shift Assay to Monitor Target Engagement.PMC9486815Baljinnyam BRonzetti MHMichael SGTalley DSanchez TWHenderson MJRai GMarugan JJLeyes Porello SEBalakrishnan KSimeonov AAntony MSouthall NOwens AEVoss TWallgren EfalseReal-Time Cellular Thermal Shift Assay 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 2022-01-01T00:00:00Z2022 Sep2025-04-20T00:39:51.395Z2025-04-20T00:39:51.395ZS-EPMC94868153604911910.1021/acschembio.2c00334