Thermal shift assays in drug discovery - Troubleshooting from biochemical to cellular applications

Abstract

Thermal shift assays (TSAs) have become valuable tools within drug discovery and development pipelines. These techniques detect direct physical interactions between small, drug-like molecules and their target proteins in biochemical and cellular settings. While there are several recent articles that outline TSA protocols and highlight their utility in drug discovery projects, this article aims to highlight common issues and solutions for challenges within cutting-edge TSAs. Herein, we have described three commonly used thermal stability assays, differential scanning fluorimetry (DSF), the protein thermal shift assay (PTSA) and the cellular thermal shift assay (CETSA), highlighting specific considerations for their use and implementation. We have also described frequently experienced challenges, including irregular melt curves, challenges with protein detection, and the impacts of buffer and test compounds on TSA performance. Overall, this troubleshooting guide serves to complement established methods and protocols to make TSAs more accessible and of greater use to the scientific community. We believe this will facilitate the use of thermal stability assays in biochemical (cell-free) and biological (cell-based) settings within drug discovery projects and support researchers seeking to develop the next-generation of therapeutic agents.

Keywords: Cellular thermal shift assay; Differential scanning fluorimetry; Drug discovery; Protein thermal shift assay; Target engagement; Thermal shift assay; Troubleshooting.