Fluorescent Probes to Characterise FK506‐Binding Proteins

Abstract

Talented all-rounders: Fluorescence polarisation assays were developed for members of the FK506-binding protein family by using fluorescent rapamycin analogues (demonstrated in the figure). These tracers retain medium to high affinity to all tested proteins (FKBP12, -12.6, -13, -25, -51, -52). They can be used for active-site titrations, competition assays with unlabelled ligands and enable a robust, miniaturized assay adequate for high-throughput screening.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.

FKBPs and the Akt/mTOR pathway

“…These model organisms might therefore not suited to assess the contribution of the larger FKBPs. Intrigued by our initial finding that rapamycin can tightly bind to most members of the FKBP family, 14 we asked whether larger FKBP homologs contribute to the pharmacology of rapamycin in mammals. 15 We found that all tested human FKBPs (FKBP12, 12.6, 13, 25, 51, 52) could potently form rapamycin-induced ternary complexes with the FRB domain of mTOR and inhibit mTOR kinase activity.…”

Nanolayers for early diagnostics of proteins involved in degenerative amyloidosis

“…An efficient fluorescence quenching of FKBP12 was observed upon addition of the ligands in solution. The dissociation constants, K d , for FK506 and Rfx were calculated according to literature and recent studies in our group [7,8] The ligands were embedded in a planar scaffold using a biomimetic approach: that is to say preparing Langmuir-Blodgett films of phospholipids. The optimal phospholipid composition for each ligand has been established from the analysis of previous literature [9] combined with our own experimental work.…”

Large FK506-Binding Proteins Shape the Pharmacology of Rapamycin

“…Plasmids for the expression of His-tagged FKBPs in E. coli were prepared as described before (13). For expression in mammalian cell lines, cDNA of FKBPs was cloned into pcDNA3 (Invitrogen), and for expression in yeast cells, it was cloned into pYX223 (Invitrogen).…”

“…5) (13). FK1706, a nonimmunosuppressive analog of FK506 (38) that tightly binds to FKBP12, as well as to FKBP51 (K i ϭ 50 nM) and to FKBP52 (K i ϭ 20 nM) (13,38), likewise blocked the effects of rapamycin. Biricodar (39) and compound 44 (40) are two structurally different substances that bind to FKBP12 almost as tightly as FK506 and FK1706 but more strongly discriminate against the larger FKBP51 and FKBP52 proteins (Ͼ500-fold selectivity for FKBP12).…”

“…Rapamycin does not exclusively interact with FKBP12 but rather binds with high affinity to most members of the FK506-binding protein (FKBP) family (13), resulting in the inhibition of their peptidyl-prolyl cis-trans isomerase activity. For FKBP38, Bai and colleagues reported direct inhibitory effects on mTOR (14) but these results were challenged by others (15).…”