结核分枝杆菌十异戊二烯焦磷酸合成酶功能研究及抗结核药物开发

Pulmonary tuberculosis (TB) caused by human pathogen Mycobacterium tuberculosis (Mtb) is one of the most serious health threats for global populations, which leads to more than one million death annually. Although antibiotics-based regimens are available as routine TB treatments, the therapeutic efficacy has been severely hampered by the emergence of drug-resistant Mtb mutants. Therefore, searching for novel targets for antibiotics and developing next-generation anti-tuberculosis agents are important goals in the related research fields. Rv2361c, decaprenyl diphosphate synthase of Mtb, belongs to the cis-prenyltransferase superfamily. It catalyzes the chain elongation of farnesyl diphosphate (FPP) with seven molecules of isopentenyl diphosphate (IPP) to generate C50 decaprenyl diphosphate (DPP). Since DPP is the important intermediate of cell wall components and Rv2361c is indispensable for the bacteria growth, Rv2361c is an attractive target of anti-tuberculosis drugs. To investigate the molecular mechanism of Rv2361c, the crystal structure in complex with substrates is prerequisite information. In addition, several Rv2361c inhibitors with nanomolar potency have been obtained via preliminary in vitro screening assays. It would be of great interest to explore the anti-tuberculosis activity of these inhibitors. Accordingly, three goals are designated in the proposal: (1) resolving the complex structure of Rv2361c-FPP-IPP to demonstrate the enzymatic catalysis mechanism; (2) resolving the complex structures of Rv2361c and potent inhibitors to demonstrate the inhibitory mechanisms; (3) evaluating the anti-tuberculosis activity and synergistic effects of current drugs of Rv2361c in H37Ra strain. This proposal aims in revealing the molecular mechanism of an Mtb essential gene and testing the anti-tuberculosis efficacy of novel inhibitors. Furthermore, the Rv2361c mechanism study shall provide a hint of the chain-length determination rule of cis-prenyltransferase members, such as rubber synthase, and may shed light on the path of artificial synthesis of long-chain isoprenoid compounds.

肺结核是重要的人类传染疾病，其病原菌是结核分枝杆菌（Mtb），抗药性Mtb的出现已成为治疗肺结核疾病的重大挑战。Rv2361c是十异戊二烯焦磷酸合成酶，它以法呢基焦磷酸和异戊烯焦磷酸为底物，合成Mtb菌株中特有的顺式-五十碳单元的十异戊二烯焦磷酸(decaprenyl diphosphate，DPP)，由于DPP是Mtb细胞壁合成过程中的重要中间物，因此，Rv2361c是Mtb中不可或缺的基因。深入剖析Rv2361c的作用机理将能进一步了解Mtb细菌的关键生理步骤，这将是开发新型抗结核药物重要方向。本项目将解析Rv2361c与底物复合体的晶体结构，分析其催化机理，并利用功能试验分析链长决定因子；另一方面，我们将从化合物文库中筛选效力较高的抑制剂，解析Rv2361c与抑制剂的复合体结构，测试它们抑制细菌生长的能力，分析抑制剂作用位点以探讨药物作用机制，并作为后续改良抗结核药物的分子基础。

人类病原体引起的肺结核（TB）结核分枝杆菌（Mtb）是全球人口面临的最严重的健康威胁之一，每年导致一百万人死亡。Rv2361c是Mtb的萜烯合酶，属于顺式戊二烯基转移酶超家族。它通过7分子异戊烯基二磷酸酯（IPP）催化法呢基二磷酸酯（FPP）的链伸长，生成C50癸二烯基二磷酸酯（DPP），Rv2361c是抗结核药物的引人注目的靶标。本项目的研究中，我们解析了与复合体结合的Rv2361的晶体结构，并解释了其催化机理。我们还解析了在Mtb合成中很重要的富含丙氨酸和脯氨酸的蛋白质的晶体结构，以及其他功能类似于Rv2361c萜烯合酶的蛋白质晶体结构，薰衣草中的lavandulyl diphosphate Synthase（LPPS）, Streptomyces sp. CL190 中的cyclolavandulyl diphosphate from (CLDS)，以及Streptomyces sp. strain CNH-189中的 Isosesquilavandulyl diphosphate（Mcl22），通过结构解析，我们发现这些蛋白与Rv2361c具有相似的结构和功能。我们还通过本项目的支持，解析了吲哚生物碱合成中的重要stig-环化酶,来源于Clonostachys rosea的玉米赤霉烯酮水解酶以及PET水解酶的晶体结构，并解释了相应催化机理，完成项目任务。