新型抗結(jié)核藥靶TPP及先導(dǎo)化合物的研究
發(fā)布時間：2019-02-26 19:58
【摘要】：結(jié)核分枝桿菌是結(jié)核病的病原菌,結(jié)核病是當(dāng)今人類的第一殺手,嚴重危害人類健康。二十世紀中期抗結(jié)核藥物的發(fā)現(xiàn)及使用,結(jié)核病曾一度得到控制,但是近十幾年來耐藥結(jié)核桿菌的廣泛出現(xiàn),使得結(jié)核病這一全球性的疾病卷土重來,給人類帶來嚴重的威脅與挑戰(zhàn),因此研發(fā)新型抗結(jié)核藥物迫在眉睫。 結(jié)核分枝桿菌細胞壁內(nèi)的某些成份獨特,細胞壁合成途徑中的因子可以成為抗結(jié)核藥物設(shè)計的合適靶標。研究表明海藻糖磷酸磷酸酶(Trehalose phosphate phosphatase, TPP)與結(jié)核分枝桿菌的生長緊密相關(guān),并且TPP在耐異煙肼結(jié)核分枝桿菌中呈現(xiàn)上調(diào)表達。 本研究選取結(jié)核分枝桿菌海藻糖磷酸磷酸酶作為藥物靶標,開展了基于虛擬篩選抗結(jié)核先導(dǎo)化合物的發(fā)現(xiàn)和晶體學(xué)研究的工作。 1)運用分子生物學(xué)技術(shù)克隆了TPP編碼基因Rv3372,然后重組構(gòu)建到原核表達載體上,在大腸桿菌中進行表達并純化了TPP蛋白,通過生化分析重組蛋白具有海藻糖磷酸磷酸酶活性； 2)采用同源模建方法獲得了TPP蛋白的三維結(jié)構(gòu),并進行活性位點的分析； 3)通過基因定點突變研究,發(fā)現(xiàn)突變蛋白幾乎喪失海藻糖磷酸磷酸酶的活性,驗證了活性位點的正確性； 4)基于TPP蛋白結(jié)構(gòu)的虛擬篩選方法從LeadQuest化合物數(shù)據(jù)庫中獲得67個小分子化合物,通過抗結(jié)核活性篩選后進一步進行基于配體的篩選研究,得到41個小分子化合物,最后通過對活性先導(dǎo)化合物進行改造,有機合成獲得21個小分子化合物； 5)將小分子化合物在體外分別對結(jié)核分枝桿菌H37Ra菌株、H37Rv菌株和臨床耐藥分離菌株進行抑制作用評價試驗,發(fā)現(xiàn)有5個小分子化合物對H37Ra菌株的MIC低于0.39μ g/ml,其中有1個小分子化合物對H37Rv菌株和臨床耐藥菌株的MIC達到0.14μ g/m1； 6)通過分析11個小分子化合物對TPP蛋白酶活性的抑制作用,結(jié)果表明有4個化合物在一定程度上能夠有效地降低酶活性,其中有3個小分子化合物能夠有效地抑制結(jié)核桿菌的生長； 7)對重組TPP蛋白進行大量表達和蛋白純化方法的摸索,最終確立了TPP蛋白的純化思路和方法； 8)用符合晶體生長要求的TPP蛋白樣品進行晶體培養(yǎng)試驗,進行大量的生長條件篩選和優(yōu)化以促使TPP蛋白分子能夠規(guī)律有序地堆積成晶體,并對獲得的晶體進行了X-射線衍射分析。 本論文研究工作中獲得的先導(dǎo)化合物為研發(fā)新型抗結(jié)核藥物打下了堅實的基礎(chǔ)。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R52
本文編號：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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R52
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