title: High Throughput Screen Identifies Natural Product Inhibitor of Phenylalanyl-tRNA Synthetase from Pseudomonas aeruginosa and Streptococcus pneumoniae.
abstract: Pseudomonas aeruginosa and Streptococcus pneumoniae are causative agents in a wide range of infections. Genes encoding proteins corresponding to phenylalanyl-tRNA synthetase (PheRS) were cloned from both bacteria. The two forms of PheRS were kinetically evaluated and the K(m)'s for P. aeruginosa PheRS with its three substrates, phenylalanine, ATP and tRNA(Phe) were determined to be 48, 200, and 1.2 µM, respectively, while the K(m)'s for S. pneumoniae PheRS with respect to phenylalanine, ATP and tRNA(Phe) were 21, 225 and 0.94 µM, respectively. P. aeruginosa and S. pneumoniae PheRS were used to screen a natural compound library and a single compound was identified that inhibited the function of both enzymes. The compound inhibited P. aeruginosa and S. pneumoniae PheRS with IC50's of 2.3 and 4.9 µM, respectively. The compound had a K(I) of 0.83 and 0.98 µM against P. aeruginosa and S. pneumoniae PheRS, respectively. The minimum inhibitory concentration (MIC) of the compound was determined against a panel of Gram positive and negative bacteria including efflux pump mutants and hyper-sensitive strains. MICs against wild-type P. aeruginosa and S. pneumoniae cells in culture were determined to be 16 and 32 µg/ml, respectively. The mechanism of action of the compound was determined to be competitive with the amino acid, phenylalanine, and uncompetitive with ATP. There was no inhibition of cytoplasmic protein synthesis, however, partial inhibition of the human mitochondrial PheRS was observed.
authors.forename: Yanmei
authors.initials: Y
authors.lastname: Hu
authors.forename: Stephanie O
authors.initials: SO
authors.lastname: Palmer
authors.forename: Hector
authors.initials: H
authors.lastname: Munoz
authors.affiliation: Chemistry Department, SCIE. 3.320, The University of Texas-Pan American, 1201 W. University Drive, Edinburg, TX 78541, USA. bullardj@utpa.edu.
authors.forename: James M
authors.initials: JM
authors.lastname: Bullard
mesh_terms: D000595:Amino Acid Sequence; D001688:Biological Products / Q000032:analysis / Q000494:pharmacology*; D002478:Cells, Cultured; D057166:High-Throughput Screening Assays; D006801:Humans; D020128:Inhibitory Concentration 50; D008826:Microbial Sensitivity Tests; D008928:Mitochondria / Q000187:drug effects; D008969:Molecular Sequence Data; D009284:Naphthols / Q000494:pharmacology*; D010652:Phenylalanine-tRNA Ligase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics
chemical_list: C000603858:BPP 03B04; D001688:Biological Products; D009284:Naphthols; D011500:Protein Synthesis Inhibitors; D010652:Phenylalanine-tRNA Ligase