Identification of Chemical Compounds That Inhibit the Function of Glutamyl-tRNA Synthetase from Pseudomonas aeruginosa. (October 2015)

• Record Type: Journal Article
• Authors: Hu, Yanmei; Guerrero, Edgar; Keniry, Megan; Manrrique, Joel; Bullard, James M.
• Is Part Of: Journal of biomolecular screening. Volume 20:Number 9(2015)
• Page Start: 1160
• Page End: 1170
• Publication Date: 2015-10
• Subjects: glutamyl-tRNA synthetase -- high-throughput screening -- tRNA aminoacylation -- drug discovery -- antibiotics; Drugs -- Analysis -- Periodicals; Drugs -- Testing -- Periodicals; Biomolecules -- Analysis -- Periodicals; 572.36
• DOI: 10.1177/1087057115591120
• Languages: English
• ISSNs: 1087-0571

Abstract

Pseudomonas aeruginosa glutamyl-tRNA synthetase (GluRS) was overexpressed in Escherichia coli . Sequence analysis indicated that P. aeruginosa GluRS is a discriminating GluRS and, similar to other GluRS proteins, requires the presence of tRNA Glu to produce a glutamyl-AMP intermediate. Kinetic parameters for interaction with tRNA were determined and the k cat and KM were 0.8 s –1 and 0.68 µM, respectively, resulting in a k cat / KM of 1.18 s –1 µM –1 . A robust aminoacylation-based scintillation proximity assay (SPA) assay was developed and 800 natural products and 890 synthetic compounds were screened for inhibitory activity against P. aeruginosa GluRS. Fourteen compounds with inhibitory activity were identified. IC50 s were in the low micromolar range. The minimum inhibitory concentration (MIC) was determined for each of the compounds against a panel of pathogenic bacteria. Two compounds, BT_03F04 and BT_04B09, inhibited GluRS with IC50 s of 21.9 and 24.9 µM, respectively, and both exhibited promising MICs against Gram-positive bacteria. Time-kill studies indicated that one compound was bactericidal and one was bacteriostatic against Gram-positive bacteria. BT_03F04 was found to be noncompetitive with both ATP and glutamic acid, and BT_04B09 was competitive with glutamic acid but noncompetitive with ATP. The compounds were not observed to be toxic to mammalian cells in MTT assays.