结核分枝杆菌GlmU乙酰基转移酶抑制剂的作用机制研究及抑菌效果评价

Mycobacterium tuberculosis is a pathogen to cause tuberculosis. Due to the emergence of multi-drug-resistant strains and extensively drug-resistant strains, the existing anti-tuberculosis drugs are unable to cure tuberculosis effectively. Tuberculosis is serious threat to the human health. Therefore, it is urgent for us to develop new anti-tuberculosis drugs. The cell wall is an important structure for the survival of Mycobacterium tuberculosis. The bacteria will die once the cell wall is damaged. UDP-N-acetyl glucosamine is the glycosyl donor and precursor in the biosynthesis of cell wall polysaccharide. Bifunctional enzyme GlmU catalyzes the last two steps of reactions in the formation of UDP-N-acetyl glucosamine. The knockout of glmU gene caused mycobacterial lysis and death in our previous studies. Therefore, GlmU is an ideal target for new anti-tuberculosis drugs. We acquired five kinds of inhibitors for GlmU acetyl transferase by high throughput screening compound libraries from different sources. In this study we will investigate the action modes of five inhibitors on GlmU acetyl transferase and understand the interaction mechanism between the each inhibitor and GlmU enzyme by using molecular docking and site-specific mutagenesis methods. We will evaluate the inhibitory activities of GlmU inhibitors to growth of Mycobacterium tuberculosis. We also investigate the effects of five inhibitors on composition of polysaccharide in the cell wall and on the morphology and structure of Mycobacterium tuberculosis. The results from this study will provide a theoretical basis for the discovery of new anti-tuberculosis drugs.

结核分枝杆菌是引起结核病的病原体。由于多重耐药菌株和广泛耐药菌株的出现，现有的抗结核药物不能有效地治愈结核病，使人类健康受到严重的危害。因此，急需研发新型抗结核药物。细胞壁是维持结核分枝杆菌生存的重要结构，一旦细胞壁遭到破坏，细菌将会死亡。UDP-N-乙酰葡糖胺是细胞壁多糖合成的糖基供体和前体分子，双功能酶GlmU催化UDP-N-乙酰葡糖胺生物合成途径的最后两步反应。我们敲除glmU基因后，发现细菌细胞壁结构改变，细菌裂解、死亡。因此，GlmU是抗结核新药的理想靶标。我们通过高通量筛选得到5种不同分子结构的GlmU乙酰基转移酶抑制剂。本课题将确定5种抑制剂对GlmU乙酰基转移酶的抑制作用类型，利用分子对接及基因定点突变方法揭示抑制剂与GlmU酶相互作用的特点；评价抑制剂对结核分枝杆菌的抑菌效果并探讨抑制剂对细胞壁糖组分及细胞形态和结构的影响。本研究结果为发现新型抗结核药物提供理论依据。

催化结核分枝杆菌UDP-N-乙酰葡糖胺生物合成的双功能酶GlmU是抗结核新药作用靶点。本课题通过酶学实验确定了5 种抑制剂对结核分枝杆菌GlmU乙酰基转移酶的抑制类型；利用分子对接和氨基酸定点突变方法，明确了抑制剂CBBS、TPSA和DIC对GlmU乙酰基转移酶作用的分子机制；CBBS、TPSA和DIC均能靶向对菌体中的GlmU乙酰基转移酶，TPSA和DIC与现有抗结核药物联合使用能增强抑菌效果，尤其是DIC对多种MDR菌株呈现较好的抑菌效果；CBBS、TPSA、DIC和MTGATI-5均影响结核分枝杆菌细胞壁糖组分，但TPSA和DIC对细胞壁糖组分的影响更大。说明一旦抑制剂干扰了GlmU乙酰基转移酶活性，便阻断UDP-N-乙酰葡糖胺的生物合成，使肽聚糖和二糖衔接分子形成受阻，无法通过二糖衔接分子将聚阿拉伯半乳糖连接到受损的肽聚糖分子上，导致菌体的形态学和细胞壁结构发生改变，最终引起细菌死亡。TPSA对菌体的转录组和蛋白质组数据显示，TPSA能上调甲基转移酶的表达。这使我们能够理解为什么TPSA的IC50为5.30 M，而对H37Ra的MIC为25 g/ml。我们克隆了编码甲基转移酶的Rv0558和Rv0560c基因，构建了分枝杆菌表达载体pVV2-Rv0558和pVV2-Rv0560c以及H37Ra/pVV2-Rv0558及H37Ra/pVV2-Rv0560c菌株。通过酶学实验和细菌生长实验，发现Rv0558和Rv0560c能降低TPSA对GlmU乙酰基转移酶的抑制作用及对H37Ra生长的抑制作用，并降低对H37Ra/pVV2-Rv0558及H37Ra/pVV2-Rv0560c菌株的抑制作用。同时，我们利用LC-质谱技术鉴定出经Rv0560甲基化修饰的TPSA。.研究结果为进一步评价GlmU乙酰基转移酶抑制剂对临床耐药菌株的抑制效果及动物试验奠定基础。甲基转移酶对化合物的修饰作用将为抗结核新药研发及耐药机制研究提供新思路。