Global Health Drug Discovery Institute, Haidian, Beijing, China.
Global Health Drug Discovery Institute, Haidian, Beijing, China.
Methods Enzymol. 2023;679:275-293. doi: 10.1016/bs.mie.2022.07.032. Epub 2022 Aug 26.
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.
氨酰-tRNA 合成酶（aaRSs）催化 tRNA 的氨酰化，生成用于蛋白质合成的氨酰-tRNA。细菌 aaRSs 具有独特的特征，在将氨基酸导入生物分子组装中发挥着重要作用，并且容易受到小分子的抑制。aaRSs 仍然是潜在抗菌药物开发的目标。aaRS 反应的第一步是通过水解 ATP 激活氨基酸，形成带有焦磷酸的酰基腺苷酸中间体，同时释放焦磷酸。无放射性测定法通常测量 ATP 消耗或磷酸盐生成的速率，在高通量药物筛选中具有优势。这些简单的 aaRS 酶测定法可以适应研究天然或合成 aaRS 抑制剂的抑制模式。以结核分枝杆菌（Mtb）的苯丙氨酰-tRNA 合成酶（PheRS）为例，我们描述了一种鉴定和表征 Mtb PheRS 抑制剂的方法。
