Cellular Thermal Shift Assays and Validation of Intracellular Target Engagement

The ability to directly verify the interaction of small molecules with their putative protein targets, by biophysical methods in native cellular environments, is a critical step in elucidating and validating pharmacological concepts.  The CEllular Thermal Shift Assay (CETSA) was developed as a means to investigate ligand binding to target proteins in intact cells (Martinez Molina, et la, Science, 2013, 341, 84-87). In contrast to most other target engagement techniques no modification of the ligand or cloning work to include reporter groups on the target protein itself is required. Instead the methodology is based on ligand induced resistance of target proteins from thermal denaturation when the cells are heated, i.e. whereas ligand-stabilized protein remains in solution unbound protein denatures and aggregates.

In collaboration with Professor Pär Nordlund’s group, CBCS has demonstrated that CETSA is amenable to automation and screening, thus making it possible to apply large chemical libraries for direct screening purposes. As a result CBCS has now successfully developed several homogeneous AlphaScreen®-based assays to monitor target engagement and also performed a screening campaign in live, non-engineered cells expressing thymidylate synthase (TS). The screen successfully identified all drugs within the test library that are known to modulate TS, as well as novel compounds capable of binding and inhibiting this enzyme.

CBCS has established itself as a world-leading authority for developing modifications of this technology for high-throughput screening and target identifications purposes.  Investigators collaborating with CBCS have access to our full arsenal of expertise and technology allowing for powerful, conclusive scientific deduction of mollecular mechanisms of action.