RESUMEN
BACKGROUND: Pseudomonas aeruginosa is an opportunistic multi-drug resistance pathogen implicated as the causative agent in a high-percentage of nosocomial and community acquired bacterial infections. The gene encoding leucyl-tRNA synthetase (LeuRS) from P. aeruginosa was overexpressed in Escherichia coli and the resulting protein was characterized. METHODS: LeuRS was kinetically evaluated and the KM values for interactions with leucine, ATP and tRNA were 6.5, 330, and 3.0 µM, respectively. LeuRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen over 2000 synthetic and natural chemical compounds. RESULTS: The initial screen resulted in the identification of two inhibitory compounds, BT03C09 and BT03E07. IC50s against LeuRS observed for BT03C09 and BT03E07 were 23 and 15 µM, respectively. The minimum inhibitory concentrations (MIC) were determined against nine clinically relevant bacterial strains. In time-kill kinetic analysis, BT03C09 was observed to inhibit bacterial growth in a bacteriostatic manner, while BT03E07 acted as a bactericidal agent. Neither compound competed with leucine or ATP for binding LeuRS. Limited inhibition was observed in aminoacylation assays with the human mitochondrial form of LeuRS, however when tested in cultures of human cell line, BT03C09 was toxic at all concentration whereas BT03E07 only showed toxic effects at elevated concentrations. CONCLUSION: Two compounds were identified as inhibitors of LeuRS in a screen of over 2000 natural and synthetic compounds. After characterization one compound (BT03E07) exhibited broad spectrum antibacterial activity while maintaining low toxicity against human mitochondrial LeuRS as well as against human cell cultures.
Asunto(s)
Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Pseudomonas aeruginosa/efectos de los fármacos , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/aislamiento & purificación , Aminoacil-ARNt Sintetasas/metabolismo , Antibacterianos/química , Antibacterianos/uso terapéutico , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Pruebas de Enzimas , Ensayos Analíticos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad Microbiana , Infecciones Oportunistas/tratamiento farmacológico , Infecciones Oportunistas/microbiología , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Pruebas de Toxicidad AgudaRESUMEN
Pseudomonas aeruginosa, an opportunistic pathogen, is highly susceptible to developing resistance to multiple antibiotics. The gene encoding aspartyl-tRNA synthetase (AspRS) from P. aeruginosa was cloned and the resulting protein characterized. AspRS was kinetically evaluated, and the KM values for aspartic acid, ATP, and tRNA were 170, 495, and 0.5 µM, respectively. AspRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen 1690 chemical compounds, resulting in the identification of two inhibitory compounds, BT02A02 and BT02C05. The minimum inhibitory concentrations (MICs) were determined against nine clinically relevant bacterial strains, including efflux pump mutant and hypersensitive strains of P. aeruginosa. The compounds displayed broad-spectrum antibacterial activity and inhibited growth of the efflux and hypersensitive strains with MICs of 16 µg/mL. Growth of wild-type strains were unaffected, indicating that efflux was likely responsible for this lack of activity. BT02A02 did not inhibit growth of human cell cultures at any concentration. However, BT02C05 did inhibit human cell cultures with a cytotoxicity concentration (CC50) of 61.6 µg/mL. The compounds did not compete with either aspartic acid or ATP for binding AspRS, indicating that the mechanism of action of the compound occurs outside the active site of aminoacylation.