Abstract：
Tuberculosis (TB) is the leading infectious disease caused by Mycobacterium tuberculosis and the second-most contagious killer after COVID-19. The emergence of drug-resistant TB has caused a great need to identify and develop new anti-TB drugs with novel targets. Indole propionic acid (IPA), a structural analog of tryptophan (Trp), is active against M. tuberculosis in vitro and in vivo. It has been verified that IPA exerts its antimicrobial effect by mimicking Trp as an allosteric inhibitor of TrpE, which is the first enzyme in the Trp synthesis pathway of M. tuberculosis. However, other Trp structural analogs, such as indolmycin, also target tryptophanyl-tRNA synthetase (TrpRS), which has two functions in bacteria: synthesis of tryptophanyl-AMP by catalyzing ATP + Trp and producing Trp-tRNATrp by transferring Trp to tRNATrp. So, we speculate that IPA may also target TrpRS. In this study, we found that IPA can dock into the Trp binding pocket of M. tuberculosis TrpRS (TrpRSMtb), which was further confirmed by isothermal titration calorimetry (ITC) assay. The biochemical analysis proved that TrpRS can catalyze the reaction between IPA and ATP to generate pyrophosphate (PPi) without Trp as a substrate. Overexpression of wild-type trpS in M. tuberculosis increased the MIC of IPA to 32-fold, and knock-down trpS in Mycolicibacterium smegmatis made it more sensitive to IPA. The supplementation of Trp in the medium abrogated the inhibition of M. tuberculosis by IPA. We demonstrated that IPA can interfere with the function of TrpRS by mimicking Trp, thereby impeding protein synthesis and exerting its anti-TB effect.
摘要：
结核病（TB）是由结核分枝杆菌引起的主要传染病，是仅次于COVID-19的第二大传染性杀手。耐药结核病的出现引起了对鉴定和开发具有新靶标的新抗结核药物的极大需求。吲哚丙酸(IPA),色氨酸（Trp）的结构类似物，在体外和体内对结核分枝杆菌具有活性。已经证实，IPA通过模拟Trp作为TrpE的变构抑制剂来发挥其抗菌作用，它是结核分枝杆菌Trp合成途径中的第一个酶。然而,其他Trp结构类似物，如吲哚霉素，还靶向色氨酸-tRNA合成酶（TrpRS），在细菌中具有两个功能：通过催化ATPTrp合成色氨酸-AMP，通过将Trp转移到tRNATrp来产生Trp-tRNATrp。所以,我们推测IPA也可能靶向TrpRS。在这项研究中,我们发现IPA可以对接到结核分枝杆菌TrpRS（TrpRSMtb）的Trp结合袋中，等温滴定量热法(ITC)进一步证实了这一点。生化分析表明，TrpRS可以催化IPA与ATP之间的反应生成焦磷酸盐（PPi），而无需Trp作为底物。野生型trpS在结核分枝杆菌中的过表达使IPA的MIC增加到32倍,在耻垢分枝杆菌中敲除trpS使其对IPA更加敏感。在培养基中补充Trp消除了IPA对结核分枝杆菌的抑制。我们证明了IPA可以通过模仿Trp来干扰TrpRS的功能，从而阻碍蛋白质合成并发挥其抗TB作用。
