Lysyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Identification of Inhibitory Compounds. (January 2020)

• Record Type: Journal Article
• Authors: Balboa, Samantha; Hu, Yanmei; Dean, Frank B.; Bullard, James M.
• Is Part Of: SLAS discovery. Volume 25:Number 1(2020)
• Page Start: 57
• Page End: 69
• Publication Date: 2020-01
• Subjects: lysyl-tRNA synthetase (LysRS) -- protein synthesis -- Pseudomonas aeruginosa -- aminoacyl-tRNA synthetase -- drug discovery -- antibiotics; Drugs -- Analysis -- Periodicals; Drugs -- Testing -- Periodicals; Biomolecules -- Analysis -- Periodicals; Biomolecules -- Analysis; Drugs -- Analysis; Drugs -- Testing; Drug Evaluation, Preclinical; Molecular Biology -- methods; Periodicals; Periodicals; 615.1
• DOI: 10.1177/2472555219873095
• Languages: English
• ISSNs: 2472-5552

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections and has highly developed systems for acquiring resistance against numerous antibiotics. The gene ( lys S) encoding P. aeruginosa lysyl-tRNA synthetase (LysRS) was cloned and overexpressed, and the resulting protein was purified to 98% homogeneity. LysRS was kinetically evaluated, and the K m values for the interaction with lysine, adenosine triphosphate (ATP), and tRNA Lys were determined to be 45.5, 627, and 3.3 µM, respectively. The k cat obs values were calculated to be 13, 22.8, and 0.35 s −1, resulting in k cat obs / K M values of 0.29, 0.036, and 0.11 s −1 µM −1, respectively. Using scintillation proximity assay technology, natural product and synthetic compound libraries were screened to identify inhibitors of function of the enzyme. Three compounds (BM01D09, BT06F11, and BT08F04) were identified with inhibitory activity against LysRS. The IC50 values were 17, 30, and 27 µM for each compound, respectively. The minimum inhibitory concentrations were determined against a panel of clinically important pathogens. All three compounds were observed to inhibit the growth of gram-positive organisms with a bacteriostatic mode of action. However, two compounds (BT06F11 and BT08F04) were bactericidal against cultures of gram-negative bacteria. When tested against human cell cultures, BT06F11 was not toxic at any concentration tested, and BM01D09 was toxic only at elevated levels. However, BT08F04 displayed a CC50 of 61 µg/mL. In studies of the mechanism of inhibition, BM01D09 inhibited LysRS activity by competing with ATP for binding, and BT08F04 was competitive with ATP and uncompetitive with the amino acid. BT06F11 inhibited LysRS activity by a mechanism other than substrate competition.