Discovery and Analysis of Natural-Product Compounds Inhibiting Protein Synthesis in Pseudomonas aeruginosa

Abstract

ABSTRACT Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation (A/T) protein synthesis system composed of phenylalanyl-tRNA synthetases (PheRS), ribosomes, and ribosomal factors from Pseudomonas aeruginosa . This system has been used for high-throughput screening of a natural-compound library. Assays were developed for each component of the system to ascertain the specific target of inhibitory compounds. In high-throughput screens, 13 compounds were identified that inhibit protein synthesis with 50% inhibitory concentrations ranging from 0.3 to >80 μM. MICs were determined for the compounds against the growth of a panel of pathogenic organisms, including Enterococcus faecalis , Escherichia coli , Haemophilus influenzae , Moraxella catarrhalis , P. aeruginosa , Staphylococcus aureus , and Streptococcus pneumoniae . Three of the compounds were observed to have broad-spectrum activity and inhibited a hypersensitive strain of P. aeruginosa with MICs of 8 to 16 μg/ml. The molecular target of each of the three compounds was determined to be PheRS. One compound was found to be bacteriostatic, and one compound was bactericidal against both Gram-positive and Gram-negative pathogens. The third compound was observed to be bacteriostatic against Gram-positive and bactericidal against Gram-negative bacteria. All three compounds were competitive with the substrate ATP; however, one compound was competitive, one was uncompetitive, and one noncompetitive with the amino acid substrate. Macromolecular synthesis assays confirm the compounds inhibit protein synthesis. The compounds were shown to be more than 25,000-fold less active than the control staurosporine in cytotoxicity MTT testing in human cell lines.