Characterization of the Tyrosyl‐S and Tyrosyl‐Z tRNA Synthetases from Pseudomonas aeruginosa and Discovery of Inhibitors

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IntroductionPseudomonas aeruginosa, a Gram‐negative opportunistic pathogen, is a leading cause of nosocomial infections. The ability of this bacteria to form resilient biofilms on implanted medical devices results in high mortality rates. Patients with cystic fibrosis (CF) are especially at risk as P. aeruginosa colonizes the lungs of these patients forming biofilms which leads to chronic infections and is a leading cause of death. Aminoacyl‐tRNA synthetases (aaRSs) catalyze the covalent attachment of amino acids to their cognate tRNAs. These enzymes are essential for protein synthesis in bacteria and serve as validated targets for the development of new anti‐infectives. Unlike in other bacteria, the P. aeruginosa genome contains two genes (tyrS and tyrZ) which encode two distinct TyrRS enzymes, which are very different at the amino acid level containing only 27% conserved amino acid residues.Methods and ResultsThe kinetic parameters for the interaction of these enzymes with their three substrates (tRNA, ATP, tyrosine) were determined using timed aminoacylation and ATP:PPi exchange assays. The KM for interaction with tRNA, ATP, and tyrosine were 1.5, 204, 172 μM and 2.1, 496, 29 μM for TyrRS‐S and TyrRS‐Z, respectively. The kcat values for interaction with tRNA, ATP, tyrosine were 0.19, 1.0, 3.8 s−1 and 1.9, 3.8, 3.1 s−1 for TyrRS‐S and TyrRS‐Z, respectively. Both forms of TyrRS were developed into screening platforms using scintillation proximity assay (SPA) technology. Using this assay, a synthetic compound library from ChemDiv, Inc. containing over 2000 distinct compounds was screened to detect compounds with the ability to inhibit the function of TyrRS. Four compounds (BCD37H06, BCD38C11, BCD49D09, and BCD54B04) were confirmed as hit compounds. All four compounds inhibited the activity of TyrRS‐S and one of the compounds inhibited TyrRS‐Z. The compounds exhibited IC50 values in the low μM range. These compounds were found to have moderate broad spectrum inhibitory activity (MICs) against a panel of ten pathogenic bacteria. The compounds were all shown to be bacteriostatic in time‐kill kinetic assays. In competition assays, none of the compounds competed with tyrosine for binding, however BCD38C11 and BCE49D09 were competitive with ATP for binding. None of the compounds inhibited growth of human cell cultures.ConclusionTyrRS‐S and TyrRS‐Z from P. aeruginosa were characterized and used for development of screening platforms to identify compounds that have the potential for development as antibacterial agents against drug resistant pathogenic organisms.Support or Funding InformationThe authors are grateful for the financial support provided by the National Institutes of Health (grant number: 1SC3GM098173).