College of Life Sciences and Biotechnology, The State Key Laboratory of Microbial Metabolism , Shanghai Jiao Tong University , 800 Dongchuan Road , Shanghai , Minhang District, China 200240.
Department of Bioinformatics and Biosciences , Capital University of Science and Technology , Islamabad , Pakistan 44000.
J Chem Inf Model. 2019 Jan 28;59(1):498-508. doi: 10.1021/acs.jcim.8b00525. Epub 2018 Dec 7.
In an effort to discover the mechanism of resistance offered by Mycobacterium tuberculosis (Mtb) toward the pyrazinamide (PZA) drug, an extensive molecular dynamics strategy was employed. PZA is a first-line prodrug that effectively cuts therapy time by 33% (from 9 to 6 months). Pyrazinamidase enzyme (PZase), encoded by the pncA gene, is responsible for the activation of prodrug PZA into pyrazinoic acid (POA). POA is toxic and potently inhibits the growth of latent Mtb even at low pH values. PZA resistance is caused by three genes pncA, rpsA, and panD. Among them, the pncA gene contributes 72-99% to the resistance. Hence, the present study focused on the novel mutations N11K, P69T, and D126N in the pncA gene. In the present study, the possible mechanism of these three mutations was studied through molecular dynamics simulation and docking techniques. Our in-depth analysis and results are in strong agreement with our experimental observation. The binding pocket analysis showed that mutations decrease the volume of the active site and hinder the correct orientation of PZA drug in the active site. Moreover, the Patchdock score was found to be low as compared to WT showing the disturbance of shape complementarity between PZase and PZA drug. These mutations were found to disturb the position of the Fe ion. Among the mutations, D126N allosterically disturbed the position of the Fe ion. MMGBSA analyses showed that these mutations decrease the binding affinity toward the PZA drug. In conclusion, mutations N11K, P69T, and D126N result in weak binding affinity with PZA and also cause significant structural deformations that lead to PZA resistance. This study provides useful information that mutations in other than active parts may also cause protein folding and ligand displacement effects, altering the biological functions.
为了探究结核分枝杆菌（Mtb）对吡嗪酰胺（PZA）药物产生耐药性的机制，我们采用了广泛的分子动力学策略。PZA 是一种一线前药，能将治疗时间从 9 个月缩短至 6 个月，有效减少 33%的治疗时间。吡嗪酰胺酶（PZase）由 pncA 基因编码，负责将前药 PZA 激活为吡嗪酸（POA）。POA 具有毒性，即使在低 pH 值下，也能强烈抑制潜伏性 Mtb 的生长。PZA 耐药性是由 pncA、rpsA 和 panD 三个基因引起的。其中，pncA 基因对耐药性的贡献为 72%-99%。因此，本研究主要关注 pncA 基因中的新型突变 N11K、P69T 和 D126N。在本研究中，通过分子动力学模拟和对接技术研究了这三种突变的可能机制。我们的深入分析和结果与实验观察结果高度一致。结合口袋分析表明，这些突变降低了活性位点的体积，阻碍了 PZA 药物在活性位点中的正确定向。此外，与 WT 相比，Patchdock 评分较低，表明 PZase 与 PZA 药物之间的形状互补性受到干扰。这些突变被发现干扰了 Fe 离子的位置。在这些突变中，D126N 变构地干扰了 Fe 离子的位置。MMGBSA 分析表明，这些突变降低了与 PZA 药物的结合亲和力。总之，N11K、P69T 和 D126N 突变与 PZA 的结合亲和力较弱，并且还导致显著的结构变形，导致 PZA 耐药性。本研究提供了有用的信息，即除了活性部位之外的突变也可能导致蛋白质折叠和配体位移效应，改变生物功能。
