Assays for the identification of inhibitors targeting specific translational steps

While bacterial protein synthesis is the target of about half of the known antibiotics, the great structural-functional complexity of the translational machinery still offers remarkable opportunities for identifying novel and specific inhibitors of unexploited targets. We designed a knowledge-based in vitro translation assay to identify inhibitors selectively targeting the bacterial or the yeast translational apparatus, preferentially blocking the early steps of protein synthesis. Using a natural-like, "universal" model mRNA and cell-free extracts prepared from Eschericha coli, Saccharomyces cerevisiae, and HeLa cells, we were able to translate, with comparable yields in the three systems, the immunogenic peptide encoded by this "universal" mRNA. The immuno-enzymatic quantification of the translated peptide in the presence of a potential inhibitor can identify a selective bacterial or fungal inhibitor inactive in the human system. When applied to the high-throughput screening (HTS) of a library of approximately 25,000 natural products, this assay led to the identification of two novel and specific inhibitors of bacterial translation.