Increasing the efficiency of ligands for FK506-binding protein 51 by conformational control.

The design of efficient ligands remains a key challenge in drug discovery. In the quest for lead-like ligands for the FK506-binding protein 51 (FKBP51), we designed two new classes of bicyclic sulfonamides to probe the contribution of conformational energy in these ligands. The [4.3.1] scaffold had consistently higher affinity compared to the [3.3.1] or monocyclic scaffolds, which could be attributed to better preorganization of two key recognition motifs. Surprisingly, the binding of the rigid [4.3.1] scaffold was enthalpy-driven and entropically disfavored compared to the flexible analogues. Cocrystal structures at atomic resolution revealed that the sulfonamide nitrogen in the bicyclic scaffolds can accept an unusual hydrogen bond from Tyr(113) that mimics the putative FKBP transition state. This resulted in the first lead-like, functionally active ligand for FKBP51. Our work exemplifies how atom-efficient ligands can be achieved by careful conformational control even in very open and thus difficult binding sites such as FKBP51.

Evaluation of synthetic FK506 analogues as ligands for the FK506-binding proteins 51 and 52.

The FK506-binding proteins (FKBP) 51 and 52 are cochaperones that modulate the signal transduction of steroid hormone receptors. Both proteins have been implicated in prostate cancer. Furthermore, single nucleotide polymorphisms in the gene encoding FKBP51 have been associated with a variety of psychiatric disorders. Rapamycin and FK506 are two macrocyclic natural products that bind to these proteins indiscriminately but with nanomolar affinity. We here report the cocrystal structure of FKBP51 with a simplified α-ketoamide analogue derived from FK506 and the first structure-activity relationship analysis for FKBP51 and FKBP52 based on this compound. In particular, the tert-pentyl group of this ligand was systematically replaced by a cyclohexyl ring system, which more closely resembles the pyranose ring in the high-affinity ligands rapamycin and FK506. The interaction with FKBPs was found to be surprisingly tolerant to the stereochemistry of the attached cyclohexyl substituents. The molecular basis for this tolerance was elucidated by X-ray cocrystallography.

Determining a synthetic approach to pierisformaside C.

An efficient route detailing the construction of the central core of pierisformaside C, the first grayanane-type diterpene to possess three central double bonds, is reported.

Fluorescent probes to characterise FK506-binding proteins.

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.