Abstract
FK506-binding proteins (FKBPs) are promising targets for a variety of disorders and infectious diseases. High FKBP occupancy is thought to be necessary for ligands to effectively compete with the endogenous intracellular functions of FKBPs. Here, we report the development of NanoBRET assays for the most prominent cytosolic FKBPs, FKBP12, 12.6, 51 and 52. These assays allowed rapid profiling of FKBP ligands for target engagement and selectivity in living cells. These assays confirmed the selectivity of SAFit-type ligands for FKBP51 over FKBP52 but revealed a substantial offset for the intracellular activity of these ligands compared to bicyclic ligands or natural products. Our results stress the importance to control for intracellular FKBP occupancy and provide the assays to guide further FKBP ligand optimization.