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

Abstract

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.

Keywords: cellular thermal shift assay (CETSA); chemical biology; drug discovery; natural products; target identification.

Figure 1

Principle of the cellular thermal shift assay (CETSA). When exposed to increasing temperature, proteins treated with a vehicle (control) undergo thermal denaturation and aggregation, which reduces their solubility. In contrast, proteins bound to a natural product exhibit enhanced thermal stability, and they maintain their solubility at higher temperatures. The remaining soluble protein fraction is analyzed via a technique such as Western blotting, where an increased band intensity at higher temperatures indicates protein stabilization by the natural product. Rel. Intensity: relative intensity, Tm: melting temperature.

Figure 2

Workflow of MS-CETSA for target identification in natural products. Living cells or cell lysates are treated with either a vehicle or natural product and are subjected to a temperature gradient. The obtained soluble proteome is subjected to multiplexed mass spectrometry for proteome-wide analysis. Thermal stability curves are then generated, where a shift in the melting temperature (∆Tm) indicates drug–target binding and stabilization. MS analysis: Mass Spectrometry analysis, Rel. Intensity: relative intensity, Tm: melting temperature.

Figure 3

Workflow of the HT-CETSA reporter assay. Cells are transfected with a target protein fused to small reporter tags (e.g., ePL, 86b, HiBiT) and plated into a microtiter plate. After incubation with natural products and heat treatment, cells are lysed. Reporter activity is reconstituted by adding a large reporter fragment and substrate. Luminescence is then measured to assess protein stabilization using different complementation systems (ePL-EA complementation for β-galactosidase activity, 86b-11S and HiBiTLgBiT complementation for nanoluciferase activity).