Applications of the Cellular Thermal Shift Assay to Drug Discovery in Natural Products: A Review

Natural products play a crucial role in drug discovery because of their structural diversity and biological activity. However, identifying their molecular targets remains a challenge. Traditional target identification approaches such as affinity-based protein profiling and activity-based protein profiling are limited by the need for chemical modification or reactive groups in natural products. The emergence of label-free techniques offers a powerful alternative for studying drug–target engagement in a physiological context. In particular, the cellular thermal shift assay (CETSA) exploits ligand-induced protein stabilization—a phenomenon where ligand binding enhances a protein’s thermal stability by reducing conformational flexibility—to assess drug binding without requiring chemical modifications. CETSA’s integration with advanced mass spectrometry and high-throughput platforms has dramatically expanded proteome coverage and sensitivity, enabling the simultaneous quantification of thousands of proteins and the identification of low-abundance targets in native cellular environments. This review highlights the application of key CETSA-based methods to target identification in natural products including Western blot-based CETSA, isothermal dose–response CETSA, mass spectrometry-based CETSA, and high-throughput CETSA. Case studies are presented that demonstrate their effectiveness in uncovering the mechanisms of action of different drugs. The current limitations of CETSA-based strategies are also explored, and future improvements to optimize their potential for drug discovery are discussed. Integrating CETSA with complementary approaches can enhance the target identification accuracy and efficiency for natural products and ultimately advance development of therapeutic applications.