Target-based drug screening typically relies on biochemical or affinity-based assays to identify compounds that modulate or bind to purified target proteins in vitro. However, additional cellular validation is essential to confirm genuine drug-target engagements. Integrating screening and validation within a single cellular assay could greatly expedite the drug discovery process. Herein, we developed a cellular ligand discovery method called CPSEA (cellular protein stability enhancement assay), which leverages the biophysical principle of ligand-induced stabilization of target proteins containing destabilizing mutations. Using CPSEA, we identified arteannuin B and colchicine as novel ligands for FKBP12 and KRASG₁₂S, respectively. Importantly, we introduced both experimental and computational strategies to identify destabilizing mutations, thereby broadening the applicability of CPSEA for target proteins with and without known stabilizing ligands. Overall, CPSEA represents a powerful cell-based screening strategy with significant potential in target-based drug discovery.

Target-based drug screening  /  Destabilizing mutations  /  KRAS  /  Protein stability-based screening  /  Cellular ligand discovery