A Time‐Resolved Förster Resonance Energy Transfer Platform for the Facile Quantification of Small Molecule Drugs in Cell Lysate

Many pharmacologically relevant drug targets are located inside cells, requiring small‐molecule therapeutics to penetrate the plasma membrane faster than they are metabolically inactivated or expelled by efflux pumps. Accurate measurement of intracellular concentrations is crucial for correlating biochemical potency with cellular efficacy and optimizing ligands. Despite the critical importance of this metric, current methods for quantifying intracellular levels of nonfluorescent small molecules are technically demanding and limited in throughput, significantly restricting the ability of medicinal chemists to readily and routinely measure cellular drug concentration. Here, we introduce a straightforward, high‐throughput time‐resolved Förster resonance energy transfer assay platform based on CoraFluor technology, a class of highly luminescent terbium complexes, to readily quantify the abundance of biologically active small molecules in whole cell lysates. Our approach conceptually extends existing homogeneous ligand displacement assays and does not require any additional reagents or equipment, enabling the comprehensive biochemical and cellular characterization of small‐molecule ligands.