开发标的结核分枝杆菌二萜烯合成酶之抗生素：蛋白质晶体结构与高通量筛选

Tuberculosis caused by Mycobacterium tuberculosis (Mtb) infection is one of the most serious threats for human population which leads to more than one million deaths annually. Antibiotics-based treatments are effective but have been severely hampered by the emergence of drug-resistant variants. Rv3377c and Rv3378c are two virulence factors specifically expressed in pathogenic mycobacteria. Genetic manipulation experiments demonstrated that more than 80% reduction in bacterial replication in macrophages was resulted from deletion of either one of these genes. Therefore, Rv3377c and Rv3378c are considered novel targets of anti-mycobacteria agents. Rv3377c and Rv3378c have been functionally characterized as class II and class I diterpene synthases, respectively, which act sequentially to convert geranylgeranyl pyrophosphate (GGPP) into a group of unique diterpenoid compounds: tuberculosinol (TOH) and (±)-isotuberculosinols (iso-TOHs). These Mtb-specific diterpenoids can protect bacteria from degradation through arresting phagosome acidification and maturation. In order to investigate the molecular machinery of Rv3377c and Rv3378c as a basis for developing novel inhibitors, three dimensional structures of these proteins and complex structures with their ligands are prerequisite information. The numbers of diterpene synthase structures are very limited, and none of them is from bacteria. Currently, we have solved crystal structures of Rv3378c and in complex with its substrate, and illustrated the catalytic mechanism and potential strategy of drug designations accordingly. Here, we propose to solve the crystal structure and complex structures of Rv3377c with its substrates. Furthermore, a high-throughput screening utilizing Rv3377c-Rv3378c coupled reaction and chemical compound library will be carried out to select potent enzyme inhibitors. Next, the inhibitory effects, specific target, and toxic effects in mammalian cells of the most potent inhibitor(s) will be further verified. Finally, the complex structures of the selected inhibitors and Rv3377c/Rv3378c will also be analyzed. This proposal aims in revealing the molecular mechanism of novel Mtb virulence factors through studying the crystal structures, and providing fundamental information for designation of new anti-tuberculosis agents.

肺结核是重要的人类传染疾病,病原菌为结核分枝杆菌(Mtb),其抗药性的出现为治疗肺结核带来极大的困难.Rv3377c及Rv3378c分别为Mtb的第二型及第一型二萜烯合成酶,任一基因缺失导致Mtb在巨噬细胞内的复制降低80%以上,故Rv3377c/Rv3378c为抗结核菌药物的新型标靶.为了解Rv3377c及Rv3378c的分子作用机理并设计新型药物,本计划将解析其蛋白质晶体结构及底物的复合物结构,并使用化合物文库进行高通量筛选以获得Rv3377c/Rv3378c之抑制剂,进而解析其蛋白质复合体晶体结构以探讨其作用机理.二萜烯合成酶蛋白质结构报导非常少(目前仅有三个晶体结构,且序列与Rv3377c/Rv3378c完全不同),细菌的二萜烯合成酶的结构则完全没有被报导过.本计划从蛋白质结构分子作用机理的水平上研究Mtb的致病机理并针对特定标靶筛选新型抗生素,更将为二萜烯合成酶的研究打下重要基础

肺结核是重要的人类传染疾病,病原菌为结核分枝杆菌(Mtb),其抗药性的出现为治疗肺结核带来极大的困难.Rv3377c及Rv3378c分别为Mtb的第二型及第一型二萜烯合成酶,任一基因缺失导致Mtb在巨噬细胞内的复制降低80%以上,故Rv3377c/Rv3378c为抗结核菌药物的新型标靶.本项目研究了Rv3377c及Rv3378c的分子作用机理并设计新型药物,解析其蛋白质晶体结构及底物的复合物结构,通过高通量筛选以获得Rv3377c/Rv3378c之抑制剂,对于Mtb的致病机理并对特定标靶筛选新型抗生素,二萜烯合成酶的研究打下重要基础..金黄色葡萄球菌是人类的一种重要的病原菌，它可以引起许多的感染。MRSA至今感染几乎遍布全球，成为院内和社区感染的重要病原菌之一。Merochlorin A和Merochlorin B是新发现的MRSA的重要抗生素。Mcl22，异二酰基二磷酸合成酶（isosesquilavandulyl diphosphate synthase），可以催化一份子的DMAPP和GPP合成ILPP，而ILPP是合成Merochlorin A和Merochlorin B的重要前体。项目首次成功解析了不同结构特征的新型顺式“头-碰-中”萜类合成酶(Mcl22)和其底物及底物类似物的复合体结构，并揭示了催化机理，对重要萜类化合物的生物合成及merochlorin类抗生素的研发具有重要指导意义。