A lysate-based TR-FRET approach for the facile quantification of cellular drug concentration

Many pharmacologically relevant drug targets are located inside cells. The development of therapeutically active small molecule modulators for intracellular targets requires that successful drug candidates can efficiently pass through the plasma membrane at higher rates than their metabolic inactivation or expulsion by drug efflux pumps. Such knowledge is invaluable for correlating biochemical and cellular activity, and for guiding ligand optimization. Despite the critical im-portance of this metric, current methodologies for measuring the intracellular concentration of non-fluorescent small molecules have many limitations, significantly restricting the ability of medicinal chemists to readily and routinely measure cellular drug concentration. We here introduce a straightforward, high-throughput TR-FRET assay platform based on CoraFluor technology to readily quantify the abundance of biologically active small molecules in whole cell lysates. Our approach builds on existing homogenous ligand displacement assays and does not require any additional reagents or equipment, enabling the comprehensive biochemical and cellular characterization of small molecule ligands.