Identification and Characterization Chemical Compounds that Inhibit Lysyl‐tRNA Synthetase from Pseudomonas aeruginosa

Abstract

IntroductionPseudomonas aeruginosa is an opportunistic pathogen and a major cause of nosocomial infections. The enhanced ability of P. aeruginosa to acquire resistance to most antibiotics places it in a special class of pathogens, the so‐called “superbugs”. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis and are validated targets for development of antibacterial agents. The gene encoding P. aeruginosa lysyl‐tRNA synthetase (LysRS) was cloned and the resulting overexpressed protein was purified to 98% homogeneity.Methods and ResultsAminoacylation assays were used to measure kinetic parameters for interactions of LysRS with tRNA and ATP:PPi assays were used to determine the kinetic parameters for interactions with lysine and ATP. The KM values for interaction with lysine, ATP and tRNALys were determined to be 45.3, 627, and 3.3 μM, respectively. The kcatobs values were calculated to be 13.3, 22.8, and 0.35 sec−1. This resulted in kcatobs/KM values of 0.29, 0.036, 0.11 s−1μM−1. P. aeruginosa LysRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen natural product (800) and synthetic (890) compound libraries to identify compounds with the ability to inhibit the function of the enzyme. Three compounds (BM01D09, BT06F11, and BT08F04) were identified and inhibitory activity was confirmed. These compounds inhibited the ability of LysRS to aminoacylate tRNALys with IC50 values of 35.3, 66.9, and 36.4 μM, respectively. The minimum inhibitory concentration (MIC) was determined for each compound against a panel of ten pathogenic bacteria. Each compound exhibited broad spectrum activity. Time‐kill studies indicated a bacteriostatic mode of inhibition for each compound against cultures of S. aureus, however in cultures of M. catarrhalis, BT06F11 and BT08F04 was observed to be bactericidal. When tested in cultures of human embryonic kidney 293 (HEK‐293) cell lines using MTT cell proliferation assays, BT06F11 was not toxic at any concentration tested. BM01D09 was observed to only inhibit human cell growth at high concentrations (CC50 = 370 μM). However, BT08F04 was toxic at much lower concentrations (CC50 = 61 μM).ConclusionLysRS from P. aeruginosa was characterized and developed into a screening platform to identify potential anti‐bacterial compounds. Three compounds were identified as having inhibitory activity against the aminoacylation of LysRS. The compounds were then 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.