A streamlined process for discovery and characterization of inhibitors against phenylalanyl-tRNA synthetase of Mycobacterium tuberculosis

Aminoacyl-tRNA synthetases (aaRSs) catalyze aminoacylation of tRNAs to produce aminoacyl-tRNAs for protein synthesis. Bacterial aaRSs have distinctive features, play an essential role in channeling amino acids into biomolecular assembly, and are vulnerable to inhibition by small molecules. The aaRSs continue to be targets for potential antibacterial drug development. The first step of aaRS reaction is the activation of amino acid by hydrolyzing ATP to form an acyladenylate intermediate with the concomitant release of pyrophosphate. None-radioactive assays usually measure the rate of ATP consumption or phosphate generation, offering advantages in high-throughput drug screening. These simple aaRS enzyme assays can be adapted to study the mode of inhibition of natural or synthetic aaRS inhibitors. Taking phenylalanyl-tRNA synthetase (PheRS) of Mycobacterium tuberculosis (Mtb) as an example, we describe a process for identification and characterization of Mtb PheRS inhibitor.