Real-Time Cellular Thermal Shift Assay to Monitor Target Engagement

Abstract

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 to withstand unfolding at temperatures greater than 90 °C 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. 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.

β-carboline derivative Z86 attenuates colorectal cancer cell proliferation and migration by directly targeting PI3K

“…Binding of chemicals to the target protein appears to improve the thermal stability and relative protease resistance [ 14 , 15 ]. CETSA (cellular thermal shift assay, CETSA) is widely used to verify the thermal stabilization improvement under heating conditions on binding of small molecules to target proteins [ 16 ]. We performed CETSA to further confirm the interaction between Z86 with PI3Kα.…”

A structural and mechanistic model for BSEP dysfunction in PFIC2 cholestatic disease

“…To examine the thermal stability of proteins in a cellular environment, we carried out CETSA experiments using a live cell HiBiT split luciferase detection (Promega, Madison, WI)) of nonaggregated BSEP and BSEP variants. Using a strategy like that described in the literature [38][39][40][41] (Figure 2), we developed plasmid constructs containing the BSEP protein, followed by a Cterminal GSGS linker and an 11-amino acid HiBiT tag. Briefly, plasmids containing the appropriate BSEP construct were transfected into HEK293 cells for 24 hours.…”

Next Generation Gold Drugs and Probes: Chemistry and Biomedical Applications

“…Cellular thermal shift assay (CETSA) 173 can be used as a profiling tool for gold-based drug discovery when coupled with proteome-wide MS. Current use of CETSA in the context of gold-derived complexes has been limited to validating identified targets by assessing thermal protein stability changes via Western blot. This is achieved by incubating cells with a desired Au agent or vehicle, followed by cell lysis and heat across a range of temperatures.…”