High-throughput ligand discovery in living cells using the cellular protein stability enhancement assay

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 KRAS G12S , 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. We developed CPSEA, along with experimental and computational methods to identify destabilizing mutations and applied this strategy to discover novel ligands for FKBP12 and KRAS G12S in live cells.