新型抗结核先导物的筛选发现与分子药理学研究

发布时间：2019-06-28 09:02

【摘要】：结核病是由结核分枝杆菌引起的严重危害人类健康的传染性疾病。全世界约有三分之一的人感染结核分枝杆菌。在2014年结核病导致约150万人死亡。虽然近两年有新的抗结核药物上市,但已有耐药菌株出现的报道。因此仍然急需开发新机制的高效低毒的抗结核药物。氨酰-tRNA合成酶(AaRS)邑够将氨基酸和对应的tRNA合成氨酰-tRNA (aa-tRNA),在蛋白质生物合成中起着至关重要的作用。酪氨酰-tRNA合成酶(TyrRS)是该家族中的一员,阻断其功能就能抑制病原菌的生长繁殖。结核中的TyrRS蛋白与人体内的同源蛋白结构差异巨大,因此针对该靶点可能发现新的低毒高效的抗结核化合物。本研究中我们利用大肠杆菌原核表达系统,在体外表达纯化了具有良好催化活性的结核TyrRS蛋白。在此基础上,利用同位素方法建立了酶抑制剂筛选模型。同时使用结核分枝杆菌的近缘菌株耻垢分枝杆菌作为检定菌建立了表型筛选体系。联用两种方法对本实验室保存的10万个化合物进行了筛选,最终发现了一个同时具有酶抑制活性和抗结核活性的化合物IMB-T130。测定了该化合物对结核分枝杆菌的抑制活性,发现它对结核标准菌株H37Rv具有良好的抑制作用,MIC为0.08 μg／mL。特别的是它对临床分离的耐药菌株表现出良好的抑制活性,强于一线抗结核药物异烟肼和利福平。我们通过表面等离子共振(SPR)实验在分子水平证明了化合物和TyrRS蛋白之间存在相互作用。进一步构建了TyrRS蛋白过表达的耻垢分枝杆菌菌株,发现对化合物敏感性降低,在细胞水平验证了化合物的靶向抑制作用。通过计算机软件进行分子对接,我们分析了化合物与蛋白的相互作用机制。进一步对化合物细胞毒性,急性毒性和药代动力学性质进行了检测,结果显示化合物毒性较小,具有良好成药性。测定了化合物对于一系列革兰氏阳性、阴性菌株的抗菌谱,结果显示化合物对于结核分枝杆菌具有良好的选择性。考虑到化合物对TyrRS酶抑制活性和体外抗菌活性之间存在一定差异,我们推测该化合物可能还作用于其他重要的抗结核靶标。收集整理了已报道的结核分枝杆菌关键蛋白结构,通过计算机软件与IMB-T130进行分子对接,分析对接模型以寻找化合物新的作用靶标,我们推测莽草酸途径中的3-脱氢奎尼酸合酶(DHQS)可能是IMB-T130发挥抗菌作用的另一靶标。以此为基础,我们在体外表达纯化了结核DHQS蛋白,通过孔雀绿定磷法检测催化反应中生成的游离磷酸,建立了酶活性测定体系,在酶水平验证IMB-T130对该蛋白具有更强的抑制作用。SPR实验结果进一步证明了化合物和DHQS蛋白之间存在相互作用。通过高效液相技术对反应底物测定,同样证明阳性化合物确实阻断了催化反应的发生。以上实验结果初步证实DHQS蛋白可能是化合物IMB-T130抗结核机制中更为关键的靶标蛋白。本研究首次报道了化合物IMB-T130的抗结核分枝杆菌活性,它是第一个能够有效抑制结核DHQS蛋白活性且同时具有优秀抗结核作用的化合物。该化合物具有新的靶点与作用机制,与现有抗结核药物不存在交叉耐药,有可能发展成为新型抗结核先导化合物。
[Abstract]:Tuberculosis is an infectious disease that is seriously harmful to human health caused by Mycobacterium tuberculosis. About one third of the world's people are infected with Mycobacterium tuberculosis. Tuberculosis killed some 1.5 million people in 2014. Although new antituberculotic drugs have been listed in the last two years, there have been reports of drug-resistant strains. Therefore, it is still in urgent need of developing new mechanism's high-efficiency and low-toxic anti-tuberculosis drugs. The amino acid and the corresponding tRNA synthetic ammonia-tRNA (aa-tRNA) can play an important role in the biosynthesis of protein. TyrRS, a member of the family, can inhibit the growth and reproduction of pathogenic bacteria. The structural difference of the TyrRS protein in the tuberculosis and the homologous protein in the human body is great, so that the new low-toxicity and high-efficiency anti-tuberculosis compound can be found aiming at the target point. In this study we used the E. coli prokaryotic expression system to express and purify the TyrRS protein with good catalytic activity in vitro. On this basis, the enzyme inhibitor screening model was established by the isotope method. And a phenotype screening system is established by using the mycobacteria of the near-edge strain of the mycobacterium tuberculosis as a test bacterium. One hundred and ten thousand compounds stored in the laboratory were screened in combination with two methods, and a compound IMB-T130 with both an enzyme-inhibitory activity and an anti-tuberculosis activity was found. The inhibitory activity of the compound on the Mycobacterium tuberculosis was determined, and it was found to have a good inhibitory effect on the tuberculosis standard strain H37Rv, and the MIC was 0.08. mu.g/ mL. In particular, it has good inhibitory activity on the clinical isolated drug-resistant strains, and is stronger than that of the first-line anti-tuberculosis drugs and the rifampin. We demonstrated the interaction between the compound and the TyrRS protein at the molecular level by surface plasmon resonance (SPR) experiments. The strain of the Mycobacterium smegmatis that has been overexpressed by the TyrRS protein is further constructed, and the sensitivity of the compound is reduced, and the targeted inhibition effect of the compound is verified at the cell level. The interaction between the compound and the protein is analyzed by means of computer software for molecular docking. The toxicity, the acute toxicity and the pharmacokinetic properties of the compound are further detected, and the result shows that the compound has small toxicity and good drug property. The antibacterial spectrum of the compound for a series of Gram-positive and negative strains was determined, and the results showed that the compound has good selectivity for Mycobacterium tuberculosis. Considering that the compound has a certain difference in the inhibition activity of TyrRS and the in vitro antibacterial activity, we assume that the compound may also act on other important anti-tuberculosis targets. The key protein structure of the reported Mycobacterium tuberculosis is collected and arranged, the molecular docking is carried out by the computer software and the IMB-T130, and the butt-joint model is analyzed to find a new action target of the compound. We have speculated that the 3-dehydroquinic acid synthase (DHQS) in the shikimic acid pathway may be another target for IMB-T130 to play an antibacterial role. On the basis of this, we expressed the purified tuberculosis DHQS protein in vitro, and the free phosphoric acid produced in the catalytic reaction was detected by the malachite green method. The enzyme activity determination system was established, and the inhibition of IMB-T130 on the protein was demonstrated at the enzyme level. The SPR experimental results further demonstrated the interaction between the compound and the DHQS protein. The reaction substrate was determined by high-performance liquid-phase technology, and it was also proved that the positive compound did block the occurrence of the catalytic reaction. The above experimental results confirm that the DHQS protein may be a more critical target protein in the anti-tuberculosis mechanism of the compound IMB-T130. In this study, the activity of the compound IMB-T130 is reported for the first time, and it is the first compound capable of effectively inhibiting the activity of the tuberculosis DHQS protein and having excellent anti-tuberculosis effect. The compound has novel target and action mechanism, does not have cross resistance with the existing anti-tuberculosis drugs, and can be developed into a novel anti-tuberculosis lead compound.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R966

【相似文献】