Pentamidine binds to tRNA through non-specific hydrophobic interactions and inhibits aminoacylation and translation.

The selective and potent inhibition of mitochondrial translation in Saccharomyces cerevisiae by pentamidine suggests a novel antimicrobial action for this drug. Electrophoresis mobility shift assay, T1 ribonuclease footprinting, hydroxyl radical footprinting and isothermal titration calorimetry collectively demonstrated that pentamidine non-specifically binds to two distinct classes of sites on tRNA. The binding was driven by favorable entropy changes indicative of a large hydrophobic interaction, suggesting that the aromatic rings of pentamidine are inserted into the stacked base pairs of tRNA helices. Pentamidine binding disrupts the tRNA secondary structure and masks the anticodon loop in the tertiary structure. Consistently, we showed that pentamidine specifically inhibits tRNA aminoacylation but not the cognate amino acid adenylation. Pentamidine inhibited protein translation in vitro with an EC(50) equivalent to that binds to tRNA and inhibits tRNA aminoacylation in vitro, but drastically higher than that inhibits translation in vivo, supporting the established notion that the antimicrobial activity of pentamidine is largely due to its selective accumulation by the pathogen rather than by the host cell. Therefore, interrupting tRNA aminoacylation by the entropy-driven non-specific binding is an important mechanism of pentamidine in inhibiting protein translation, providing new insights into the development of antimicrobial drugs.

Characterization of versilin-sensitive sites in self-sensitive producer and sensitive non-producer or unrelated organism.

Studies on self-sensitivity of producer mutant vs. sensitivity of non-producer parent and unrelated organism showed that versilin inhibited spore germination and sporulation in the self-sensitive producer mutant, non-producer parent Aspergillus versicolor N5 and the unrelated sensitive Trichophyton rubrum. Sporulation appeared to be more sensitive than spore germination. The inhibition of in vivo synthesis of protein was very marked, but inhibition of RNA and DNA was slight and moderate, respectively. Thus versilin was not specific in its action, but the principal sensitive site was protein synthesis, as further suggested by inhibition of polyU-directed in vitro synthesis of polyphenylalanine. The activation of leucine was unaffected, but the formation of leucyl-tRNA was severely inhibited in all three strains. The differences in sensitivities between the strains were the same, whether as whole cells or as cell-free extracts. Thus the nature of the sensitive site appeared to be identical in the self-sensitive producer and sensitive non-producer or unrelated organism.