Identification of Inhibitors of Glutaminyl‐tRNA synthetase from Pseudomonas aeruginosa

Abstract

IntroductionAntibiotic resistance is a growing global threat and infections caused by resistant bacteria have become increasingly difficult to treat with most current antibiotics. Pseudomonas aeruginosa is a Gram‐negative opportunistic pathogen and is the leading cause of mortality in cystic fibrosis patients. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis. The glutaminyl‐tRNA synthetase (GlnRS) from P. aeruginosa was over‐expressed, enzymatically characterized and developed into a screening platform for the discovery of chemical compounds that inhibit protein synthesis.MethodsThe P. aeruginosa GlnRS was developed, using scintillation proximity assay (SPA) technology, as a platform to screen chemical compounds for detection of compounds that inhibited function. Using this assay, natural product (800 compounds) and synthetic compound (890 compounds) libraries were screened to detect compounds with inhibitory activity.ResultsFrom this screening campaign three compounds (BM02E04, BM04B05, and BM04H03) were identified and confirmed as inhibitors of the activity of P. aeruginosa GlnRS. BM02E04, BM04B05, and BM04H03 were observed to inhibit the activity of GlnRS with IC50 values of 1.7, 2.5, and 39.3 μM, respectively. The minimum inhibitory concentration (MICs) was determined against a panel of bacteria including; E. coli, E. coli tolC mutant, E. faecalis, H. influenzae, M. cattarrhalis, P. aeruginosa, P. aeruginosa PAO200 (efflux pump mutant), P. aeruginosa hypersensitive strain, S. aureus, and S. pneumoniae. There was no activity observed against the wild‐type strain of E. coli or P. aeruginosa, but broad spectrum activity was observed against other Gram‐negative and Gram‐positive bacteria. Time‐kill studies indicated a bacteriostatic mode of inhibition for each compound against cultures of S. aureus and M. catarrhalis. When tested in cultures of human embryonic kidney 293 (HEK‐293) cell lines using MTT cell proliferation assays, there was no toxicity observed by either BM02E04 or BM04B05 at any concentration tested up to 400 μg/ml.ConclusionP. aeruginosa GlnRS was cloned, over‐expressed, characterized and developed into a screening platform to identify compounds that have the potential for development as an antibacterial agent against pathogenic organisms. Three compounds were identified as having inhibitory activity against the aminoacylation activity of GlnRS. Two of the compounds were further characterized for inhibition of enzymatic activity, bacterial growth and toxic effects in human cell cultures.Support or Funding InformationNational Institutes of Health (grant number: 1SC3GM098173)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.