Determination of the cellular target engagement by direct-to-biology Cellular Thermal Shift Assay (CETSA)

The direct-to-biology approach enables rapid, high-throughput evaluation of large compound libraries in biological assays, eliminating costly and time-consuming purification steps. This strategy has been widely used in the development of proteolysis-targeting chimeras (PROTACs) to facilitate rapid linker optimization and the identification of active degraders from thousands of crude candidate compounds. Similarly, some other direct-to-biology biophysical assays have been utilized for the optimization of small-molecule ligands. However, a direct-to-biology strategy for evaluating cellular target engagement has not yet been demonstrated. Here, we extend this approach to the cellular thermal shift assay (CETSA). By systematically comparing crude reaction mixtures of reported DCAF11 covalent ligands with their corresponding purified analogues, we demonstrate that unpurified compounds can be directly evaluated in CETSA to assess cellular target engagement.