Department of Biotechnology & Bioinformatics, School of Life Sciences, University of Hyderabad, Prof. C.R. Rao Road, Hyderabad 500046, India.
Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500039, India.
Int J Biol Macromol. 2023 May 15;237:124118. doi: 10.1016/j.ijbiomac.2023.124118. Epub 2023 Mar 23.
Aminoacyl-tRNA synthetases are crucial enzymes for cellular protein metabolism and have been considered as an attractive target for development of new antimicrobials. In the current study, seryl tRNA synthetase of Leishmania donovani (LdSerRS) and its mutants were purified and characterized through biochemical and structural methods. Purified LdSerRS was found to be enzymatically active and exhibited more alpha helices in secondary structure. The enzymatic activity of purified protein was observed as highest near physiological temperature and pH. Mutation in ATP binding residues (R295 and E297) demonstrated reduction in the affinity for cofactor with no significant deviation in secondary structure. In vitro inhibition studies with ureidosulfocoumarin derivatives helped to identify Comp 5l as a specific inhibitor for leishmanial SerRS that showed lesser potency towards purified HsSerRS. The identified compound presented competitive mode of inhibition for LdSerRS and also revealed druglikeness along with very low toxicity for human macrophages. Structural analysis of protein and ligand complex depicted the binding of Comp 5l into the cofactor binding site of LdSerRS with high affinity succeeded by validation employing molecular dynamics simulations. Altogether, our study presents a promising scaffold to explore small molecules to target the enzymatic activity of leishmanial SerRS to develop the specific therapeutics.
氨酰-tRNA 合成酶是细胞蛋白质代谢的关键酶，已被认为是开发新抗菌药物的有吸引力的靶标。在本研究中，通过生化和结构方法对杜氏利什曼原虫（LdSerRS）及其突变体的丝氨酰 tRNA 合成酶进行了纯化和表征。纯化的 LdSerRS 被发现具有酶活性，并表现出更多的二级结构中的α螺旋。纯化蛋白的酶活性在接近生理温度和 pH 值时最高。在 ATP 结合残基（R295 和 E297）发生突变的情况下，观察到与辅因子的亲和力降低，而二级结构没有明显偏差。脲基磺酰基香豆素衍生物的体外抑制研究有助于确定 Comp 5l 是一种针对利什曼原虫 SerRS 的特异性抑制剂，对纯化的 HsSerRS 的效力较低。鉴定出的化合物对 LdSerRS 表现出竞争性抑制模式，并且还显示出对人巨噬细胞的低毒性和类药性。蛋白质和配体复合物的结构分析表明，Comp 5l 以高亲和力结合到 LdSerRS 的辅因子结合位点，随后通过分子动力学模拟进行验证。总之，我们的研究提供了一个有前途的支架，以探索小分子来靶向利什曼原虫 SerRS 的酶活性，从而开发出特异性的治疗方法。
