Elongation factor 2 (EF2) is an essential protein catalyzing ribosomal translocation during protein synthesis and is highly conserved in all eukaryotes. It is largely interchangeable in translation systems reconstituted from such divergent organisms as human, wheat, and fungi. We have identified the sordarins as selective inhibitors of fungal protein synthesis acting via a specific interaction with EF2 despite the high degree of amino acid sequence homology exhibited by EF2s from various eukaryotes. In vitro reconstitution assays using purified components from human, yeast, and plant cells demonstrate that sordarin sensitivity is dependent on fungal EF2. Genetic analysis of sordarin-resistant mutants of Saccharomyces cerevisiae shows that resistance to the inhibitor is linked to the genes EFT1 and EFT2 that encode EF2. Sordarin blocks ribosomal translocation by stabilizing the fungal EF2-ribosome complex in a manner similar to that of fusidic acid. The fungal specificity of the sordarins, along with a detailed understanding of its mechanism of action, make EF2 an attractive antifungal target. These findings are of particular significance due to the need for new antifungal agents.The elongation phase of translation in fungi requires the soluble elongation factors EF1␣, EF2, and EF3. EF1␣ and EF2 are members of the GTPase superfamily of proteins and are characterized by common structural motifs and their ability to alternate between conformational states in response to binding GDP or GTP. These proteins are required for translation in all eukaryotes, while EF3 is unique to fungi and essential for fungal protein synthesis (1). EF2 catalyzes the translocation of the ribosome along messenger RNA, presumably by stimulating a gross rearrangement of the ribosome that results in peptidyl-tRNA transfer and the movement of mRNA by one codon. The protein sequence of EF2 has been highly conserved throughout evolution, with Saccharomyces cerevisiae EF2 sharing 66% identity and 85% homology to human EF2. Despite this high degree of similarity, a class of tetracyclic diterpene glycoside natural products, the sordarins, has now been identified as selective inhibitors of EF2 function in fungal protein synthesis. Sordarin, produced by species of the fungal genus Sordaria, was described as an antifungal agent in 1970 (2, 3), but the mode of action of this family has not been examined until now. In this report, we show that sordarins specifically bind to the S. cerevisiae EF2-ribosome complex and block protein synthesis by inhibiting the release of EF2 from the posttranslocational ribosome. Our observations show that it is possible to inhibit fungal EF2 specifically, which may provide an opportunity for developing antifungal agents with a unique and selective mechanism of action. EXPERIMENTAL PROCEDURESSordarin was isolated essentially as described for Sordaria arenosa (2). Reticulocyte and wheat germ lysates were obtained from Promega.Assays-IC 50 values were determined from growth inhibition assays in which cells were inoculated a...

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Retinoid-related orphan receptor gamma (RORγ) directly controls the differentiation of Th17 cell and the production of interleukin-17, which plays an integral role in autoimmune diseases. To obtain insight into RORγ, we have determined the first crystal structure of a ternary complex containing RORγ ligand-binding domain (LBD) bound with a novel synthetic inhibitor and a repressor peptide, 22-mer peptide from silencing mediator of retinoic acid and thyroid hormone receptor (SMRT). Comparison of a binary complex of nonliganded (apo) RORγ-LBD with a nuclear receptor co-activator (NCoA-1) peptide has shown that our inhibitor displays a unique mechanism different from those caused by natural inhibitor, ursolic acid (UA). The compound unprecedentedly induces indirect disruption of a hydrogen bond between His479 on helix 11 (H11) and Tyr502 on H12, which is crucial for active conformation. This crystallographic study will allow us to develop novel synthetic compounds for autoimmune disease therapy.

Through a trans-lactonization reaction, galbonolide B (1) was converted to 3 with the chiral secondary alcohol at C13 exposed for derivatization. Two independent methods were employed to determine the absolute chirality at C13. Both of these methods established S chirality at C13. Since the relative stereochemistry of galbonolide B had been determined from the X-ray structure, the absolute stereochemistry of galbonolide B was therefore formally established to be structure 1, which contradicted earlier speculations in the literature. A total synthesis of galbonolide B has been completed. A highly selective method was developed for the assembly of the peculiar diene unit using Martin's sulfurane reagent for the dehydration of the preceding tertiary alcohol 20. The chiral center at C4 was installed by "contra-steric" enolate chemistry. A novel macro-Dieckmann cyclization was employed to generate the macrocycle. The desired configuration at C2 was obtained from the kinetic protonation of the corresponding enolate. Finally, a seldom used protecting group, 2,4,6-trimethylbenzylidene acetal, was employed for the glycol unit. It exhibited extremely facile hydrolysis under mildly acidic conditions without causing any decomposition of synthetic intermediates.

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