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A series of C32-O-aralkyl ether derivatives of the FK-506 related macrolide ascomycin have been prepared based on an earlier reported C32-O-cinnamyl ether design. In the present study, the nature of the aryl tethering group was varied in an attempt to improve oral activity. An imidazol-2-yl-methyl tether was found to be superior among those investigated and has resulted in an ascomycin analog, L-733,725, with in vivo immunosuppressive activity comparable to FK-506 but with an improved therapeutic index.

The total synthesis and in vitro activities of a series of chemical inducers of dimerization (CIDs) is described. The use of small-molecule CIDs to control the dimerization of engineered FKBP12-containing fusion proteins has been demonstrated to have broad utility in biological research as well as potential medical applications in gene and cell therapies. The facility and flexibility of preparation make this new class of wholly synthetic compounds exceptionally versatile tools for the study of intracellular signaling events mediated by protein-protein interactions or protein localization. While some congeners possess potency comparable to or better than the first generation natural product-derived CID, FK1012, structure-activity relationships are complex and underscore the need for application-specific compound optimizations.

Background: The 12 kD FK506 binding protein FKBP12 is a cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin. In addition to its critical role in drug-induced T-cell immunosuppression, FKBP12 associates physiologically with ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors, regulating their ability to flux calcium. We investigated a role for FKBP12 in male reproductive physiology on the basis of our identification of extremely high levels of [3H]FK506 binding in male reproductive tissues., Materials and Methods: [3H]FK506 binding studies were performed to identify tissues enriched with FK506 binding sites. The abundant [3H]FK506 binding sites identified in the male reproductive tract were localized by [3H]FK506 autoradiography. FK506 affinity chromatography was employed to purify FKBP from epididymal fluid. Anti-FKBP12 Western analysis was used to confirm the identity of the purified FKBP. The binding of exogenous FKBP12 to sperm was evaluated by [32P]FKBP12 binding studies and [33P]FKBP12 autoradiography. The effect of recombinant FKBP12 on sperm motility was investigated using a Hamilton Thorne motility analyzer., Results: Male reproductive tissues contained high levels of [3H]FK506 binding. The localization of [3H]FK506 binding sites to the tubular epithelium of the caput epididymis and the lumen of the cauda and vas deferens suggested that FKBP is released in the male reproductive tract. FKBP12 was purified from epididymal plasma by FK506 affinity chromatography. Radiolabeled FKBP12 specifically bound to immature but not mature sperm. In sperm motility studies, FKBP12-treated caput sperm exhibited double the curvilinear velocity of untreated controls., Conclusions: High levels of FKBP12 are released in the male reproductive tract and specifically associate with maturing sperm. Recombinant FKBP12 enhances the curvilinear velocity of immature sperm, suggesting a role for FKBP12 in motility initiation. The highest concentrations of soluble FKBP12 in the male reproductive tract occur in the lumen of the vas deferens, a site of sperm storage and the conduit for ejaculated sperm. Preservation of mammalian sperm for reproductive technologies may be optimized by supplementing incubation or storage media with FKBP12.

Protection or regeneration of the dopaminergic (DA) system would be of significant therapeutic value for Parkinson's disease. Immunophilin ligands, such as FK506, can produce neurotrophic effects in vitro and in vivo, but their immunosuppressive effects make them unsuitable for neurological application. This study demonstrates that a novel, nonimmunosuppressive immunophilin ligand (V-10,367) increased the number of neurites extended by tyrosine hydroxylase positive (TH+) DA neurons in embryonic day 14 primary DA neuronal cultures. In contrast, the immunosuppressive immunophilin ligand FK506 increased the length of TH+ neurites. After oral administration in MPTP-treated mice, V-10,367 completely protected against MPTP-induced loss of striatal TH+ axonal density, while FK506 did not. These experiments demonstrate that nonimmunosuppressive immunophilin ligands specifically increase neurite branching in primary DA neuronal culture and possess neurotrophic actions in vivo with potential application to neurodegenerative disease.

Immuonosuppressive drugs FK506 and rapamycin block a number of signal transduction pathways in eukaryotic systems. The 12 kDa FK506 binding protein (FKBP12) mediates the action of both FK506 and rapamycin against their functional targets. In this report, we cloned, sequenced and characterized a gene encoding FKBP12 in Vicia faba (VfFKBP12). While VfFKBP12 is highly homologous to animal and yeast FKBP12, it does not mediate the action of FK506 and rapamycin. There are unique features in plant FKBP12 sequences that cause the variation in their function. One lies in the domain that is critical for interaction with calcineurin (CaN), the mammalian and yeast target of FKBP12-FK506 complex. Protein-protein interaction assays revealed a low-affinity and unstable VfFKBP12-FK506-CaN ternary complex. In the genetic assay, VfFKBP12 did not restore the sensitivity of yeast FKBP12 mutant to rapamycin or FK506, supporting that plant FKBP12-ligand complexes are unable to block the function of the drug target. Also unique to plant FKBP12 proteins, a pair of cysteines is spatially adjacent to potentially form disulfide linkage. Treatment of VfFKBP12 with reductant dithiothreitol (DTT) abolished the formation of VfFKBP12-FK506-CaN ternary complex. Site-directed mutagenesis to substitute one of the cysteines, Cys26, with Ser produced a similar effect as DTT treatment. These results indicate that an intramolecular disulfide bond is a novel structural feature required for the low-affinity interaction between plant FKBP12 and CaN. In conclusion, plant FKBP12 proteins have evolved structural changes that modify their protein-protein interacting domains and cause loss of function against the drug targets.

Several recent reports have described the rational redesign of small molecule-protein interfaces, generating modified ligands that interact only with suitably mutated proteins. These studies provide powerful reagents for specifically manipulating engineered proteins inside cells, as well as general insights into the factors underlying the binding affinity and specificity of small molecules in general. Progress this year includes the development of allele-specific inhibitors of Src-family tyrosine kinases and the crystal structure determination of a remodeled FKBP-ligand interface.

Ligand-protein docking simulations are employed to analyze the binding energy landscape of the pipecolinyl fragment that serves as a recognition core of the FK506 ligand in binding with the FKBP12 protein. This fragment acts as a molecular anchor that specifically binds within the protein active site in a unique binding mode, in harmony with the structure of the FK506-FKBP12 complex. Molecular anchors are characterized by a large stability gap, defined to be the free energy of a ligand bound in the native binding mode relative to the free energy of alternative binding modes. For ligands that share a common anchor fragment, a linear binding free energy relationship may be expected for hydrophobic substituents provided they do not abrogate the anchor binding mode. Changes in solvent-accessible surface area for these peripheral groups are used to rationalize the relative binding affinities of a series of FKBP12-ligand complexes which share the pipecolinyl anchor fragment. A series of benzene derivatives that bind to a mutant form of T4 lysozyme is also analyzed, and implications for structure-based drug design are discussed.