Ligands includes in this study.

Drug-resistant tuberculosis is a pressing global health issue that requires the development of new drugs or the identification of new therapeutic targets. The ESX-3 secretion system is essential for the Mycobacterium tuberculosis growth and plays a role in iron/zinc homeostasis and virulence. The aim of this study was to evaluate the quaternary interface of EccD3, a component of the ESX-3 secretion system, and to evaluate the association of an eccD3 mutant with drug resistance. The molecular structures of EccD3 protein and other ESX-3 secretion system proteins of the M. tuberculosis were predicted based in homology with the Mycolicibacterium smegmatis tertiary protein structures. According to the in silico results, selamectin, avermectin, ivermectin, and moxidectin were selected as prospective drugs. Selamectin and moxidectin had favorable ΔG values for the EccB3 and EccD3 dimer interfaces, whereas the ESX-3 Protomer 1 interface had the best ΔG + with avermectin, ivermectin, and moxidectin. Furthermore, ivermectin susceptibility increased when the eccD3 gene was inhibited using CRISPRi in M. smegmatis. Blockage of EccD3 increased the ivermectin action, but the modest changes observed may be explained by the compensatory mechanisms or other ivermectin targets in absence of this Esx3 component. Further in vitro and preclinical studies are required to validate our findings.

耐药结核病（drug-resistant tuberculosis）是亟待解决的全球性公共卫生难题，亟需开发新型药物或确定全新治疗靶点。ESX-3分泌系统（ESX-3 secretion system）对结核分枝杆菌（Mycobacterium tuberculosis）的生长至关重要，同时参与铁/锌稳态调控与毒力发挥。本研究旨在解析ESX-3分泌系统组分EccD3的四级界面，并探究eccD3突变体与耐药性的关联。本研究基于耻垢分枝杆菌（Mycolicibacterium smegmatis）的蛋白质三级结构同源性，预测了结核分枝杆菌EccD3蛋白及其他ESX-3分泌系统蛋白的分子结构。基于计算机模拟实验（in silico）结果，本研究筛选出司拉克丁、阿维菌素、伊维菌素与莫西克汀作为潜在候选药物。司拉克丁与莫西克汀对EccB3与EccD3二聚体界面的吉布斯自由能变化（ΔG）值更为优异；而在ESX-3原聚体1界面中，阿维菌素、伊维菌素与莫西克汀的ΔG+值表现最佳。此外，在耻垢分枝杆菌中利用CRISPR干扰（CRISPR interference，CRISPRi）抑制eccD3基因后，菌株对伊维菌素的敏感性显著提升。阻断EccD3可增强伊维菌素的作用效果，但本研究观察到的变化幅度较为有限，这或许可归因于在缺失该Esx3组分的情况下，机体存在补偿机制或其他伊维菌素作用靶点。后续需开展更多体外实验与临床前研究，以验证本研究的发现。