Elongation Factor 2 as a Novel Target for Selective Inhibition of Fungal Protein Synthesis

Abstract

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...

Mutations in Ribosomal Protein L10e Confer Resistance to the Fungal-specific Eukaryotic Elongation Factor 2 Inhibitor Sordarin

“…This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.the sordarin analog L-793,422 has been described previously (3,20). The sordarin-resistant strains sRb1, sRb2, sRb5, sRb13, and sRb14 were generated essentially as described previously (3), except that mutant strains that demonstrated sordarin resistance unlinked to EFT1 or EFT2 were selected for characterization in this study.…”

“…The sordarin-resistant strains sRb1, sRb2, sRb5, sRb13, and sRb14 were generated essentially as described previously (3), except that mutant strains that demonstrated sordarin resistance unlinked to EFT1 or EFT2 were selected for characterization in this study. The wild-type yeast strain YPH54 used for genetic analysis and the plasmid YCplac111 used for subcloning the S. cerevisiae L12e and L10e genes have both been described previously (3).…”

“…The ribosome, although not contributing significantly to the fungal specificity of sordarin, is a critical partner in forming the stabilized post-translocational complex (3). Detection of a complex between fungal eEF2 and a labeled sordarin analog is strongly dependent upon the presence of ribosomes.…”

“…Hydrolysis of GTP to GDP drives translocation and is associated with a presumed conformational change in eEF2. Sordarin (1) and its analogs are fungal-specific translation inhibitors (2, 3) that bind to the eEF2-ribosome-GDP complex in Saccharomyces cerevisiae, stabilizing the post-translocational GDP form in a manner similar to that of fusidic acid (3). However, in contrast to fusidic acid, which binds both EF-G and eEF2 and is a general translocation inhibitor, sordarin inhibits translation only in susceptible fungi, deriving its unique specificity from the source of eEF2 (3)(4)(5).…”

“…Sordarin (1) and its analogs are fungal-specific translation inhibitors (2, 3) that bind to the eEF2-ribosome-GDP complex in Saccharomyces cerevisiae, stabilizing the post-translocational GDP form in a manner similar to that of fusidic acid (3). However, in contrast to fusidic acid, which binds both EF-G and eEF2 and is a general translocation inhibitor, sordarin inhibits translation only in susceptible fungi, deriving its unique specificity from the source of eEF2 (3)(4)(5). The observation that eEF2 is the major determinant of sordarin specificity was confirmed by the identification of 15 unique sordarin-resistant alleles of EFT1 and EFT2 that encode eEF2 in S. cerevisiae.…”