title: Identification and Characterization of a Chemical Compound that Inhibits Methionyl-tRNA Synthetase from Pseudomonas aeruginosa.
abstract: BACKGROUND
  Pseudomonas aeruginosa is an opportunistic pathogen problematic in causing nosocomial infections and is highly susceptible to development of resistance to multiple antibiotics. The gene encoding methionyl-tRNA synthetase (MetRS) from P. aeruginosa was cloned and the resulting protein characterized.
  
  
  METHODS
  MetRS was kinetically evaluated and the KM for its three substrates, methionine, ATP and tRNAMet were determined to be 35, 515, and 29 μM, respectively. P. aeruginosaMetRS was used to screen two chemical compound libraries containing 1690 individual compounds.
  
  
  RESULTS
  A natural product compound (BM01C11) was identified that inhibited the aminoacylation function. The compound inhibited P. aeruginosa MetRS with an IC50 of 70 μM. The minimum inhibitory concentration (MIC) of BM01C11 was determined against nine clinically relevant bacterial strains, including efflux pump mutants and hypersensitive strains of P. aeruginosa and E. coli. The MIC against the hypersensitive strain of P. aeruginosa was 16 μg/ml. However, the compound was not effective against the wild-type and efflux pump mutant strains, indicating that efflux may not be responsible for the lack of activity against the wild-type strains. When tested in human cell cultures, the cytotoxicity concentration (CC50) was observed to be 30 μg/ml. The compound did not compete with methionine or ATP for binding MetRS, indicating that the mechanism of action of the compound likely occurs outside the active site of aminoacylation.
  
  
  CONCLUSIONS
  An inhibitor of P. aeruginosa MetRS, BM01C11, was identified as a flavonoid compound named isopomiferin. Isopomiferin inhibited the enzymatic activity of MetRS and displayed broad spectrum antibacterial activity. These studies indicate that isopomiferin may be amenable to development as a therapeutic for bacterial infections.
authors.affiliation: Chemistry Department, The University of Texas-RGV, 1201 W. University Drive, Edinburg, TX 78541. United States.
authors.forename: Sara
authors.initials: S
authors.lastname: Robles
authors.affiliation: Chemistry Department, The University of Texas-RGV, 1201 W. University Drive, Edinburg, TX 78541. United States.
authors.forename: Yanmei
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Chemistry Department, The University of Texas-RGV, 1201 W. University Drive, Edinburg, TX 78541. United States.
authors.forename: Tahyra
authors.initials: T
authors.lastname: Resto
authors.affiliation: Chemistry Department, The University of Texas-RGV, 1201 W. University Drive, Edinburg, TX 78541. United States.
authors.forename: Frank
authors.initials: F
authors.lastname: Dean
authors.affiliation: Chemistry Department, The University of Texas-RGV, 1201 W. University Drive, Edinburg, TX 78541. United States.
authors.forename: James M
authors.initials: JM
authors.lastname: Bullard
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology*; D008718:Methionine-tRNA Ligase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification; D008826:Microbial Sensitivity Tests
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D007529:Isoflavones; C533098:isopomiferin; D008718:Methionine-tRNA Ligase
keywords: Protein synthesis; Pseudomonas.; antibiotics; drug discovery; in vitro screening; tRNA aminoacylation