Fluorescent Probes to Characterise FK506-Binding Proteins

FK506-binding proteins (FKBPs) convey the immunosuppressive action of FK506 and rapamycin and mediate the neuroprotective properties of these compounds, and participate in the regulation of calcium channels. In addition, the larger homologues FKBP51 and FKBP52 act as cochaperones for Hsp90 and regulate the transactivational activity of steroid hormone receptors. To further characterize these FKBPs, we have synthesized fluorescein-coupled rapamycin analogues. In fluorescence polarization assays one of these compounds retained high affinity to all tested proteins (K-d: 0.1-20 nM) and could be used for active-site titrations. To adapt the fluorescence polarization assay for high-throughput purposes, a simplified rapamycin derivative was synthesized and labelled with fluorescein. This probe showed moderate affinity for the FK1 domains of FKBP51 (177 nM) and FKBP52 (469 nM) and allowed a highly robust, optimized, miniaturized assay (Z' > 0.7) sufficient for high-throughput screening of large compound libraries.