Development of Glutaminyl‐tRNA Synthetase from Pseudomonas aeruginosa as a Platform to Screen for Inhibitors of Protein Synthesis

Abstract

IntroductionPseudomonas aeruginosa is a common cause of nosocomial infections and the leading cause of mortality in patents with cystic fibrosis. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis.ResultsP. aeruginosa glutaminyl‐tRNA synthetase (GlnRS) was overexpressed in E. coli cells, and purified to homogeneity. GlnRS from P. aeruginosa is a discriminating synthetase with respect to the requirement for the presence of tRNAGln to produce a stable glutaminyl‐AMP intermediate. When compared with GlnRS from E. coli the amino acid sequence of GlnRS from P. aeruginosa are only 60% conserved. The kinetic parameters for interaction with tRNA were determined and the KM and Vmax were 1.0 μM and 0.23 μM min−1, respectively. The observed kcat was calculated to be 0.15 s−1 resulting in a kcat/KM value of 0.15 s−1μM−1. Scintillation proximity assay (SPA) technology was adapted to the aminoacylation assay and then used to screen for inhibitors of activity of P. aeruginosa GlnRS in a high throughput format. Using this assay, natural product (800 compounds) and synthetic compound (890 compounds) libraries were screened to detect compounds with the ability to inhibit function of the enzyme. Three compounds were identified which inhibit greater than 50% of enzymatic activity. Compounds BM02E04, BM04B05, and BM04H03 inhibit the activity of P. aeruginosa ArgRS with IC50 values of 1.7, 2.5, and 39.3 μM, respectively. All three of these compounds exhibited promising MICs against Gram+ bacteria and moderate activity against Gram‐ bacteria causing respiratory infections.ConclusionGlnRS identified in P. aeruginosa was cloned, expressed characterized and developed into a screening platform to identify compounds that have the potential for development as an antibacterial agent against pathogenic organisms.Support or Funding InformationResearch was funded by NIH grant 1SC3GM098173‐01A1. Partial student support was from a Departmental Grant from the Robert A. Welch Foundation (Grant No. BG‐0017) and from the NIH UTPA RISE program grant # 1R25GM100866‐01.