基于结核分枝杆菌莽草酸途径关键酶的新型抗结核药物靶标验证和深入研究

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (MTB), remains one of deadliest communicable diseases in the world. In 2013, an estimated 9.0 million people developed TB and 1.5 million died from the disease. With MDR-TB and XDR-TB infections being reported worldwide, drug resistance and dying from HIV-associated TB have been serious problems. In our previous study, an active anti-TB compound IMB-T130 targeting the tyrosyl-tRNA synthetase was identified. It showed better activity than rifampicin and isoniazid when tested with MDR-TB and XDR-TB, and there was no significant cross-resistance between IMB-T130 and first-line anti-TB drugs. By computer-aided molecular docking and inhibition test of enzyme activity, we found that IMB-T130 also inhibited the activity of 3-dehydroquinate synthase (DHQS), which is a key enzyme in shikimate pathway of MTB. Furthermore, the screening model of shikimate kinase (SK) in shikimate pathway was established and an anti-TB compound IMB-T168 inhibiting SK was obtained in prescreening. In this project, we plan to perform the following studies: (1) Combined screening with M. smegmatis and DHQS/SK screening models in order to obtain more leading compounds with better anti-TB activity. (2) Confirming the interaction between small molecular inhibitors and enzymes by molecular and genetic experiments . (3) Evaluating the synergistic antibacterial effect between the different inhibitors targeting DHQS and SK separately. This work will help us to further understand the shikimate pathway and provide new anti-TB drug targets in the future.

全球每年有900万人患上结核病，因结核病死亡人数约150万。近年来由于耐药结核的广泛传播，以及艾滋病相关结核病例的大量出现，使得新型抗结核药物的研发更加迫切。在前期工作中我们发现了抗结核先导物IMB-T130，对结核敏感株和耐药株均具有较好的活性。深入研究发现其对结核生存所必须的莽草酸途径3-脱氢奎尼酸合酶（DHQS）具有较强的抑制作用。同时我们用相同途径关键酶—莽草酸激酶（SK）抑制剂筛选模型筛选获得了抗结核先导物IMB-T168。本项目拟继续联用耻垢分枝杆菌表型筛选和以DHQS/SK为靶点的筛选模型，获得活性和成药性更好的小分子抑制剂，并在分子水平和遗传水平上考察小分子抑制剂与DHQS或SK的相互作用，验证莽草酸途径关键酶能否成为抗结核药物的有效靶标，考察联用两类小分子抑制剂双重阻断莽草酸途径的协同作用。本课题将促进对莽草酸途径成靶性的深入研究，为抗结核新药开发提供新的理论和实验依据。

近年来耐药结核的广泛传播和艾滋病相关结核病例的大量出现使得新型抗结核药物的研发更加迫切。莽草酸途径是结核分枝杆菌生长的必需途径，但在哺乳动物细胞中不存在，因此以该途径为靶标的抗结核先导化合物可能选择性较高。3-脱氢奎尼酸合酶（DHQS）和莽草酸激酶（SK）是该途径的关键酶，前期工作发现具有良好的抗结核活性的IMB-T130能够抑制DHQS活性。本研究开展了两个酶的抑制剂筛选，以考察DHQS和SK作为抗结核药物分子靶点的可行性。主要研究进展如下：IMB-T130对DHQS的IC50为2.703μM；SPR、圆二色谱及高分辨质谱分析发现化合物能够与DHQS蛋白结合，结合后蛋白二级结构明显变化，为非共价结合；分子对接发现7个结合相关的关键氨基酸残基，这些氨基酸突变蛋白的催化活性明显降低；IMB-T130对DHQS过表达菌株的MIC明显上调，证明其抗结核活性与DHQS的抑制相关。建立了SK激酶抑制剂的高通量测活方法，筛选得到的IMB-T5297对SK酶的IC50为1.745 μg/ml，为莽草酸竞争性抑制剂；SPR证明IMB-T5297能够与SK蛋白结合；分子对接显示其能够与蛋白活性中心的的关键氨基酸产生相互作用；Arg58突变对SK活性影响不大， IMB-T5297对Arg58突变蛋白的IC50明显高于原蛋白，表明Arg58可能是二者结合的关键位点；IMB-T5297在浓度为100 μg/mL时，对Hela细胞和HEK 293的生长无明显影响，提示细胞毒性小；对商业库ChemDiv和TargetMol进行结核杆菌SK蛋白小分子抑制剂的虚拟筛选，得到89个小分子化合物用于进一步的生物活性实测试；IMB-T168、IMB-T6337对SK激酶的抑制活性不强，但是它们对结核分枝杆菌标准株和耐药株的活性较好，两个化合物的进一步活性评价和可能的机制研究目前正在进行。总之，通过本项目的执行明确了IMB-T130和IMB-T5297的作用机制，初步探索了DHQS与SK激酶作为抗结核药物筛选的可行性，基于IMB-T130良好的抗结核活性和体内动力学活性，以及两个化合物IMB-T168与IMB-T6337良好的体外抗结核活性，为抗结核药物的研发提供了先导化合物。