Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay

TLDR

Therapeutic efficacy depends on drug binding to its target, yet measuring target engagement in cells is difficult because binding cannot be monitored intracellularly. The cellular thermal shift assay (CETSA) assesses drug binding by exploiting ligand‑induced thermal stabilization of target proteins. We developed CETSA to evaluate drug binding in cells and tissues, validated it on key clinical targets, and used it to monitor drug transport, activation, off‑target effects, resistance, and distribution, establishing it as a valuable tool for target‑engagement validation and optimization.

Abstract

The efficacy of therapeutics is dependent on a drug binding to its cognate target. Optimization of target engagement by drugs in cells is often challenging, because drug binding cannot be monitored inside cells. We have developed a method for evaluating drug binding to target proteins in cells and tissue samples. This cellular thermal shift assay (CETSA) is based on the biophysical principle of ligand-induced thermal stabilization of target proteins. Using this assay, we validated drug binding for a set of important clinical targets and monitored processes of drug transport and activation, off-target effects and drug resistance in cancer cell lines, as well as drug distribution in tissues. CETSA is likely to become a valuable tool for the validation and optimization of drug target engagement.