The Real-Time Cellular Thermal Shift Assay to Monitor Target Engagement

Authors

• P Massa
• E Chiang
• R Luke
• Y Chen
• N Cawley
• D Porter
• K Cheng

Abstract

Evaluating a small molecule's mechanism of action is crucial for drug discovery and the development of chemical probes. The Cellular Thermal Shift Assay (CETSA) provides a means for conducting direct target engagement studies by measuring protein thermal stability alterations upon ligand binding. However, conventional CETSA methods are low-throughput, requiring considerable optimization and sample allocation. We present a real-time CETSA (RT-CETSA) platform designed to overcome these shortcomings. This platform combines a bioengineered, thermally stable Nanoluciferase variant, known as ThermLuc, with a prototype real-time PCR instrument equipped with a CCD camera for luminescence detection. This configuration enables the generation of a comprehensive protein aggregation profile from a single sample. The novel Nanoluciferase variant, ThermLuc, designed for enhanced thermal stability, resists denaturation even at temperatures above 90 °C, demonstrating its compatibility with monitoring target engagement across a spectrum of targets. The effectiveness of the RT-CETSA was validated using well-studied inhibitors of lactate dehydrogenase alpha. This validation revealed a significant correlation with established enzymatic, biophysical, and cell-based assays. A dedicated data analysis pipeline accompanies RT-CETSA, heightening sensitivity in the detection of on-target binding. This innovative approach broadens the capabilities of CETSA, facilitating real-time, target-agnostic, and high-throughput evaluations of ligand-target interaction, a key component in determining a small molecule's mechanism of action.

Scientific Focus Area: Chemical Biology

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