Real-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 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.