结核分枝杆菌分枝菌酸合成途径脱水酶HadABC复合物与多种抗结核抑制剂的结构和功能研究

Tuberculosis (TB) is the most lethal and widely spread bacterial infectious disease over the world, mainly caused by the pathogenic Mycobacterium tuberculosis (Mtb). Seriously, the emergence of Multi-drug resistant TB (MDR-TB) and extensively drug resistant TB (XDR-TB) make currently used drugs ineffective to cure TB. Therefore, developing novel and effective anti-TB drugs following research of newly found drug targets has already been an urgent and important mission now. Mtb is hard to kill under host immune attack and survives from toxicant invasion because of the dense and hydrophobic mycolic acid layer of cell wall. Both of HadAB and HadBC were the dehydratases, which were the last and latest identified piece of the type II fatty acid synthesis (FAS-II) system, involved in the elongation of mycolic acid chain. The importance of mycolic acid for Mtb makes all the enzymes in this synthesis pathway potential drug targets including HadAB and HadBC. In fact, they were exact downstream enzymes of InhA, an approved target of currently first-line drug isoniazid. So far as we know, there are no crystal structures of HadAB and HadBC reported and the knowledge of catalytic mechanism is unclear as well. Thus, our research will fill in these gaps by deepening our understanding of it enzymatic catalysis from molecular perspective. Our research also aims to unveil inhibition mechanisms by studying the complex structures of HadABC with either flavonoid inhibitors or anti-TB drugs Isoxyl and Thiacetazone, which will lay sound structural basis and provide important clues for the anti-TB drug development.

结核病是目前世界上致死率最高、传播范围最广的细菌性疾病。结核分枝杆菌是引发结核病的病原菌，由于耐多药菌株（MDR-TB）和广泛耐药菌株（XDR-TB）的出现，使得现有药物的治疗几乎失效。因此，寻找新的药物靶点以及研究全新作用机制的强力抗结核药物已经成为迫在眉睫的重要任务。由致密的分枝菌酸层构成的细胞壁使得结核分枝杆菌可以逃避免疫攻击和有害物质入侵从而能够存活。脱氢酶HadAB和HadBC是II型分枝菌酸脂链延伸系统（FAS-II）中最晚被发现的两对复合物，也是一线药物异烟肼靶点InhA的下游蛋白。迄今为止，其三维结构和催化机制尚不清楚。本研究项目可以填补这方面的空白，从分子水平上推进其酶活机制的研究；而HadABC与黄酮类抑制剂和抗结核药物Isoxyl、Thiacetazone复合物结构研究将揭示其抗结核的抑制机制，对基于HadABC的抗结核药物开发提供重要线索和可靠的结构生物学基础。

结核病是目前世界上致死率最高、传播范围最广的细菌性疾病。结核分枝杆菌是引发结核病的病原菌，由于耐多药菌株(MDR-TB)和广泛耐药菌株(XDR-TB)的出现，使得现有药物的治疗几乎失效。因此，寻找新的药物靶点以及研究全新作用机制的强力抗结核药物已经成为迫在眉睫的重要任务。由致密的分枝菌酸层构成的细胞壁使得结核分枝杆菌可以逃避免疫攻击和有害物质入侵从而能够存活。脱氢酶HadAB和HadBC是II型分枝菌酸脂链延伸系统(FAS-II)中最晚被发现的两对复合物，也是一线药物异烟肼靶点InhA的下游蛋白。迄今为止，其三维结构和催化机制尚不清楚。.本项目以HadABC为研究对象，解析了结核分枝杆菌HadAB与HadBC两个复合物以及HadAB与butein、2’,4,4’-trihydroxychalcone和fisetin三个抗结核黄酮抑制剂复合物的晶体结构。HadAB结构与HadBC非常相似。结构分析表明，HadAB的两个亚基整体结构较为相似，都属于单热狗折叠，通过反向平行的方式结合形成异源二聚体。这两个亚基最大的不同在于热狗螺旋与中心折叠片的夹角，HadA的夹角更大，使得其有较大的空间与底物结合，而HadB上存在催化残基Asp36和His41，表明HadA是底物结合亚基，而HadB是催化亚基。HadAB异源二聚体的背面有一个明显的凹面，表面带正电荷，可以通过空间互补效应和电荷效应与表面带负电荷的结核分枝杆菌酰基载体蛋白（AcpM）相结合。HadAB之间存在一条底物结合通道，通道的下方有一个与之垂直的狭窄口袋。HadAB与抑制剂的复合物结构显示，三个黄酮小分子都结合在这个口袋中，抑制剂的头部向上突出，正好位于底物结合通道处，表明这些黄酮类抑制剂的抑制机理是在空间上阻挡了底物与酶的结合。已有研究证明二线抗结核药Thiacetazone (TAC)就是作用于HadB，但是作用机制一直不为人知。我们根据黄酮分子作用机制提出了一个TAC与HadAB相互作用的模型。该模型较合理地解释了TAC的MIC值比黄酮抑制剂小一个数量级的原因，也解释了把Cys61突变成Ser后，结核杆菌产生了对TAC的抗性。本研究从分子水平上探究了HadAB的催化机制，揭示了抗结核小分子的作用机理，对基于HadABC的抗结核药物开发提供了重要线索和可靠的结构生物学基础。