A Novel Inhibitor of Translation Initiation Factor eIF5B in Saccharomyces cerevisiae

Abstract

The eukaryotic translation initiation factor eIF5B is a bacterial IF2 ortholog that plays an important role in ribosome joining and stabilization of the initiator tRNA on the AUG start codon during the initiation of translation. We identified the fluorophenyl oxazole derivative 2,2-dibromo-1-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)ethanone quinolinol as an inhibitor of fungal protein synthesis using an in vitro translation assay in a fungal system. Mutants resistant to this compound were isolated in Saccharomyces cerevisiae and were demonstrated to contain amino acid substitutions in eIF5B that conferred the resistance. These results suggest that eIF5B is a target of potential antifungal compound and that mutation of eIF5B can confer resistance. Subsequent identification of 16 other mutants revealed that primary mutations clustered mainly on domain 2 of eIF5B and secondarily mainly on domain 4. Domain 2 has been implicated in the interaction with the small ribosomal subunit during initiation of translation. The tested translation inhibitor could act by weakening the functional contact between eIF5B and the ribosome complex. This data provides the basis for the development of a new family of antifungals.

Keywords: Translation inhibitor; antifungal; eIF5B; target; translation initiation factor.

Fig. 1. In vitro translation activities in the presence of inhibitors.

Crude yeast S30 extracts were prepared from strain H1515. In vitro translation products from mRNA encoding the LUC protein were analyzed by luminescence. Data are expressed as the percent of LUC protein produced in extracts treated with 0.3–3.0 μg/ml of chemical. The results obtained from each experiment were expressed as mean and standard deviation SD) in triplicate. *** p < 0.001, ** p < 0.01, * p < 0.05, and n.s. stands for not significant.

Fig. 2. The amino acid residues changed in eIF5B identified in 1282NJ0069 resistant mutants.

Mapping of the PSY125 mutations on the primary structure of eIF5B. Position and nature of the amino acid change in the sequenced mutant alleles of eIF5B, and the amino acid residue found in the resistant mutant is shown as arrow below the line. Star indicates single residues changed in mutations representing the primary position showing resistance to the chemical.

Fig. 3. Overexpression of PSY76 mutant allele to wild type strain confers resistance to the inhibitor.

Overexpression of PSY76 mutant alleles in wild type strain restores growth on media containing inhibitor. PSY76 mutant and PSY125 mutant strains was transformed with the high–copy number plasmid or with vector carrying mutant allele each. Transformants were streaked on minimal SD medium supplemented only with the required nutrients and inhibitor, and then incubated 5 days at 30°C.

Fig. 4. Overexpression of PSY76 mutant allele to eIF5BΔ strain partially restores slow-growth phenotype.

PSY76 mutant and PSY125 mutant strains was transformed with the high–copy number plasmid or with vector carrying mutant allele to eIF5BΔ strain. Transformants were streaked on minimal SD medium supplemented only with the required nutrients, and then incubated 5 days at 30°C.