新型抗结核药靶TPP及先导化合物的研究
发布时间：2019-02-26 19:58
【摘要】：结核分枝杆菌是结核病的病原菌,结核病是当今人类的第一杀手,严重危害人类健康。二十世纪中期抗结核药物的发现及使用,结核病曾一度得到控制,但是近十几年来耐药结核杆菌的广泛出现,使得结核病这一全球性的疾病卷土重来,给人类带来严重的威胁与挑战,因此研发新型抗结核药物迫在眉睫。 结核分枝杆菌细胞壁内的某些成份独特,细胞壁合成途径中的因子可以成为抗结核药物设计的合适靶标。研究表明海藻糖磷酸磷酸酶(Trehalose phosphate phosphatase, TPP)与结核分枝杆菌的生长紧密相关,并且TPP在耐异烟肼结核分枝杆菌中呈现上调表达。 本研究选取结核分枝杆菌海藻糖磷酸磷酸酶作为药物靶标,开展了基于虚拟筛选抗结核先导化合物的发现和晶体学研究的工作。 1)运用分子生物学技术克隆了TPP编码基因Rv3372,然后重组构建到原核表达载体上,在大肠杆菌中进行表达并纯化了TPP蛋白,通过生化分析重组蛋白具有海藻糖磷酸磷酸酶活性； 2)采用同源模建方法获得了TPP蛋白的三维结构,并进行活性位点的分析； 3)通过基因定点突变研究,发现突变蛋白几乎丧失海藻糖磷酸磷酸酶的活性,验证了活性位点的正确性； 4)基于TPP蛋白结构的虚拟筛选方法从LeadQuest化合物数据库中获得67个小分子化合物,通过抗结核活性筛选后进一步进行基于配体的筛选研究,得到41个小分子化合物,最后通过对活性先导化合物进行改造,有机合成获得21个小分子化合物； 5)将小分子化合物在体外分别对结核分枝杆菌H37Ra菌株、H37Rv菌株和临床耐药分离菌株进行抑制作用评价试验,发现有5个小分子化合物对H37Ra菌株的MIC低于0.39μ g/ml,其中有1个小分子化合物对H37Rv菌株和临床耐药菌株的MIC达到0.14μ g/m1； 6)通过分析11个小分子化合物对TPP蛋白酶活性的抑制作用,结果表明有4个化合物在一定程度上能够有效地降低酶活性,其中有3个小分子化合物能够有效地抑制结核杆菌的生长； 7)对重组TPP蛋白进行大量表达和蛋白纯化方法的摸索,最终确立了TPP蛋白的纯化思路和方法； 8)用符合晶体生长要求的TPP蛋白样品进行晶体培养试验,进行大量的生长条件筛选和优化以促使TPP蛋白分子能够规律有序地堆积成晶体,并对获得的晶体进行了X-射线衍射分析。 本论文研究工作中获得的先导化合物为研发新型抗结核药物打下了坚实的基础。
[Abstract]:Mycobacterium tuberculosis is the pathogen of tuberculosis. Tuberculosis is the first killer of human and seriously endangers human health. The discovery and use of anti-tuberculosis drugs in the middle of the twentieth century once brought tuberculosis under control, but the widespread emergence of drug-resistant TB bacteria in recent years has brought back tuberculosis, a global disease. Because of the serious threat and challenge to human being, it is urgent to develop new anti-tuberculosis drugs. Some components in the cell wall of Mycobacterium tuberculosis are unique. The factors in the cell wall synthesis pathway can be used as suitable targets for anti-tuberculosis drug design. The results showed that trehalose phosphatase (Trehalose phosphate phosphatase, TPP) was closely related to the growth of Mycobacterium tuberculosis and the expression of TPP was up-regulated in isoniazid-resistant Mycobacterium tuberculosis. In this study, Mycobacterium tuberculosis trehalose phosphatase was selected as a drug target, and the discovery and crystallographic study of antituberculous lead compounds based on virtual screening were carried out. The main results are as follows: 1) the TPP encoding gene Rv3372, was cloned by molecular biological technique and then recombined into prokaryotic expression vector. The TPP protein was expressed and purified in E. coli. The recombinant protein had trehalose phosphatase activity by biochemical analysis. 2) the three-dimensional structure of TPP protein was obtained by homologous modeling, and the active sites were analyzed. 3) We found that the mutant protein almost lost the activity of trehalose phosphatase by site-directed mutation study, which verified the correctness of the active site. 4) A virtual screening method based on the structure of TPP protein was used to obtain 67 small molecular compounds from the LeadQuest compound database. After screening for anti-tuberculosis activity, 41 small molecular compounds were obtained by further screening based on ligands. At last, 21 small molecular compounds were synthesized by modifying the active lead compounds. 5) the inhibitory effects of small molecular compounds on Mycobacterium tuberculosis H37Ra strain, H37Rv strain and clinical drug resistant isolate were evaluated in vitro. It was found that the MIC of five small molecular compounds to H37Ra was less than 0.39 渭 g / ml. Among them, the MIC of one small molecule compound to H37Rv strain and clinical drug resistant strain was 0.14 渭 g / ml; 6) by analyzing the inhibitory effect of 11 small molecular compounds on the activity of TPP protease, the results showed that 4 compounds could decrease the enzyme activity to a certain extent. Three small molecular compounds could effectively inhibit the growth of Mycobacterium tuberculosis. 7) A large number of expression and purification methods of recombinant TPP protein were carried out, and the idea and method of purification of TPP protein were finally established. 8) the crystal culture experiments were carried out with TPP protein samples which meet the requirements of crystal growth, and a large number of growth conditions were screened and optimized in order to promote the regular and orderly accumulation of TPP protein molecules to form crystals. The obtained crystals were analyzed by X-ray diffraction. The lead compounds obtained in this paper lay a solid foundation for the research and development of new anti-tuberculosis drugs.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R52
本文编号：2431102
[Abstract]:Mycobacterium tuberculosis is the pathogen of tuberculosis. Tuberculosis is the first killer of human and seriously endangers human health. The discovery and use of anti-tuberculosis drugs in the middle of the twentieth century once brought tuberculosis under control, but the widespread emergence of drug-resistant TB bacteria in recent years has brought back tuberculosis, a global disease. Because of the serious threat and challenge to human being, it is urgent to develop new anti-tuberculosis drugs. Some components in the cell wall of Mycobacterium tuberculosis are unique. The factors in the cell wall synthesis pathway can be used as suitable targets for anti-tuberculosis drug design. The results showed that trehalose phosphatase (Trehalose phosphate phosphatase, TPP) was closely related to the growth of Mycobacterium tuberculosis and the expression of TPP was up-regulated in isoniazid-resistant Mycobacterium tuberculosis. In this study, Mycobacterium tuberculosis trehalose phosphatase was selected as a drug target, and the discovery and crystallographic study of antituberculous lead compounds based on virtual screening were carried out. The main results are as follows: 1) the TPP encoding gene Rv3372, was cloned by molecular biological technique and then recombined into prokaryotic expression vector. The TPP protein was expressed and purified in E. coli. The recombinant protein had trehalose phosphatase activity by biochemical analysis. 2) the three-dimensional structure of TPP protein was obtained by homologous modeling, and the active sites were analyzed. 3) We found that the mutant protein almost lost the activity of trehalose phosphatase by site-directed mutation study, which verified the correctness of the active site. 4) A virtual screening method based on the structure of TPP protein was used to obtain 67 small molecular compounds from the LeadQuest compound database. After screening for anti-tuberculosis activity, 41 small molecular compounds were obtained by further screening based on ligands. At last, 21 small molecular compounds were synthesized by modifying the active lead compounds. 5) the inhibitory effects of small molecular compounds on Mycobacterium tuberculosis H37Ra strain, H37Rv strain and clinical drug resistant isolate were evaluated in vitro. It was found that the MIC of five small molecular compounds to H37Ra was less than 0.39 渭 g / ml. Among them, the MIC of one small molecule compound to H37Rv strain and clinical drug resistant strain was 0.14 渭 g / ml; 6) by analyzing the inhibitory effect of 11 small molecular compounds on the activity of TPP protease, the results showed that 4 compounds could decrease the enzyme activity to a certain extent. Three small molecular compounds could effectively inhibit the growth of Mycobacterium tuberculosis. 7) A large number of expression and purification methods of recombinant TPP protein were carried out, and the idea and method of purification of TPP protein were finally established. 8) the crystal culture experiments were carried out with TPP protein samples which meet the requirements of crystal growth, and a large number of growth conditions were screened and optimized in order to promote the regular and orderly accumulation of TPP protein molecules to form crystals. The obtained crystals were analyzed by X-ray diffraction. The lead compounds obtained in this paper lay a solid foundation for the research and development of new anti-tuberculosis drugs.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R52
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相关期刊论文 前3条
1 李洪林,沈建华,罗小民,沈旭,朱维良,王希诚,陈凯先,蒋华良;虚拟筛选与新药发现[J];生命科学;2005年02期
2 乐军,刘丽蓉,谢建平,雷建强,梁莉,王洪海;结核分枝杆菌异烟肼耐药相关分泌蛋白海藻糖磷酸磷酸酶的鉴定[J];中华结核和呼吸杂志;2004年10期
3 乐军,刘丽蓉,谢建平,刘蓓,梁莉,王洪海;异烟肼耐药和敏感结核分枝杆菌的比较蛋白质组学研究[J];中华微生物学和免疫学杂志;2004年04期
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