The cellular thermal shift assay : a novel strategy to study drug target engagement and resistance development in cancer therapy

The drug binding to its target molecule determines the efficacy of therapeutics. Therefore, it is important to study drug target engagement in cells. This is often difficult because drug binding cannot be directly measured inside cells. We have developed a technique called cellular thermal shift assay (CETSA) for studying drug binding to target proteins in cells and tissues. This assay is based on the biophysical principle of ligand-induced thermal stabilization of target proteins. In this thesis, I have used CETSA for various applications. The assay was employed to study drug binding in cancer cell lines for a set of important clinical targets and to monitor processes of drug transport and activation in cells. We showed that CETSA has the potential to monitor drug resistance development during cancer therapy using antifolate and fluoropyrimide drug resistant cell lines. CETSA enables to understand the effects of drug usage and find out the most effective drug in targeting proteins in cancer patients. Thermal precipitation assay (TPA) was developed in parallel to CETSA that uses the same principle to perform high throughput fragment screening at very low protein concentration. TPA was used for fragment screening of different clinically relevant targets. The CETSA method combined with quantitative mass spectrometry detection permits the monitoring of a wide range of drug targets and the downstream effectors. We have used this strategy to study the interactions of metabolites with the human proteome. The method was validated with known nucleotide-protein interactions and most importantly, novel interactions were discovered. These studies show that CETSA is likely to become a valuable tool for drug research and development.​Doctor of Philosophy (SBS