基于生物信息学的抗结核药物靶点的筛选与验证

【作者】 赖煦卉；

【导师】 王洪海；

【作者基本信息】 复旦大学 ， 微生物学， 2008， 博士

【摘要】 结核病是全球死亡率和发病率最高的传染性疾病之一,结核分枝杆菌(简称结核杆菌)是引起结核病的病原菌。世界卫生组织2008年的调查报告表明,2006年全球新增920万结核病人,有170万人死于结核病。结核杆菌持续感染及耐药结核杆菌的出现和蔓延,是目前结核病研究面临的两大难题,开发新型抗结核药物迫在眉睫。生物信息学是现代药物开发中必不可少的工具。1998年结核杆菌基因组测序的完成开启了新型抗结核药物研究的新时代。本论文首先利用生物信息学工具分析结核杆菌基因组中功能未知的基因,在此分析过程中,发现了两个新型蛋白质结构域(domain),DIM和MSTF。分析同源蛋白发现包含DIM domain的蛋白可能涉及细菌的致病性、信号转导或小分子物质运输;而MSTF domain可能在一些与细胞壁相关的过程中起作用。同时选取与结核杆菌持续感染及细胞壁合成、代谢相关的332个基因,利用生物信息学分析,最后筛选出与人及小鼠蛋白序列一致性低、在多种致病分枝杆菌中高度保守且可被用来进行计算机辅助药物设计的编码基因66个,为本课题组深入开展新型抗结核药物靶点的研究奠定基础。结核杆菌的致病性和耐药性与其厚且结构复杂的细胞壁有重要的联系。基于上一步生物信息学潜在药靶筛选的结果,本文选取与细胞壁合成相关基因galU/Rv0993和glmU/Rv1018c作为研究目标进行相关的功能研究。结核杆菌galU基因编码UDP葡萄糖焦磷酸化酶(简称MtGalU),用于合成UDP葡萄糖(UDP-Glc);glmU基因编码的蛋白同时具有葡糖胺-1-磷酸盐(GlcN-1-P)乙酰转移酶和UDP-N-乙酰葡糖胺(UDP-N-GlcNAc)焦磷酸化酶活性(简称MtGlmU),用于合成UDP-N-GlcNAc。在本研究中,我们首先分别克隆了Rv0993和Rv1018c基因,并表达、纯化获得MtGalU和MtGlmU重组蛋白,质谱分析分子量表明两个重组蛋白为所设计的目的蛋白。对MtGalU和MtGlmU重组蛋白比活力、最适反应条件和酶动力学参数的测定表明所获得的两个重组蛋白分别具有相应的活性。研究发现,具有活性的重组MtGalU蛋白为不均一的单聚体和多聚体混合物,而具有活性的重组MtGlmU蛋白为均一、稳定的六聚体。利用两个重组蛋白的多克隆抗体进行亚细胞定位研究,发现MtGalU主要定位于细胞壁,MtGlmU主要定位于细胞质中。同时利用同源模建方法模拟了MtGalU和MtGlmU的蛋白三维结构,为后期小分子化合物的虚拟筛选奠定基础。基于潜在药靶筛选结果,本文还选取与结核杆菌持续感染相关的异柠檬酸裂解酶(ICL)做为潜在靶点,进行了抗结核先导化合物的筛选。首先克隆了异柠檬酸裂解酶的编码基因icl/Rv0467,然后在大肠杆菌中表达并纯化了ICL蛋白,生化验证重组蛋白具有异柠檬酸裂解酶活性。基于ICL蛋白的晶体结构,从Specs数据库中虚拟筛选得到能与ICL活性位点结合的小分子化合物80种,酶活性检测表明其中67种化合物在200μg/ml的浓度下对ICL活性有抑制作用。通过体外抑菌实验,筛选到能有效抑制模拟持续感染状态(限制碳源培养)的结核杆菌H37Ra株和临床耐多药结核杆菌的先导化合物2种;其中1种化合物FD20具有较低的细胞毒性且对细胞内细菌有明显的抑制作用。本论文首先利用生物信息学分析从结核杆菌蛋白质组中发现了两个新型蛋白质domain,DIM和MSTF,对两个新型domain的分析为我们了解微生物未知蛋白的功能提供启示。同时利用生物信息学分析筛选到与持续感染及细胞壁合成、代谢相关的可作为潜在药靶的基因66个,为本课题组抗结核药靶研究工作提供参考。论文中对MtGalU和MtGlmU蛋白相关功能的研究工作为下一步以MtGalU和MtGlmU做为抗结核杆菌药物靶点筛选抑制剂奠定了基础。本论文针对ICL所筛选得到的小分子化合物FD20,是非常具有成药潜力的抗结核先导化合物。更多还原

【Abstract】 Tuberculosis,caused by Mycobacterium tuberculosis.is one of the leading infectious diseases worldwide.Globally,9.2 million new cases and 1.7 million deaths from TB occurred in 2006.The persistent infection of M.tuberculosis and the emergence of drug-resistant strains present two major obstacles in gaining control over tuberculosis worldwide.It is urgent to develop new anti-tuberculosis drugs.Modern drug development is largely target-driven.Bioinformatics has certainly come to stay and is now ubiquitous with drug discovery.In present work,we firstly analyzed the unknown function genes in the tuberculosis H37Rv genome and identified two novel protein domains,DIM and MSTF.The functions of these novel domains were discussed based on the homology sequences.To facilitate novel drug target identification, we started with 332genes which are involved in persistent infection and cell wall synthesis and metabolism of M.tuberculosis.Using bioinformatics methods,we finally identified 66 genes which fill the basic discipline of potential drug target.The thick and complex cell envelope has been implicated in many aspects of the pathogenicity and drug resistance of M.tuberculosis.Based on the drug target screening result,we cloned galU/Rv0993 and glmU/Rv1018c which involved in the cell wall synthesis.M.tuberculosis galU/Rv0993 which coded UDP-glucose pyrophosphorylase(MtGalU) was used to synthesis important sugar donor UDP-glucose.M.tuberculosis glmU/Rv1018c coded a bifunctional enzyme which inhabits both GleN-1-P acetyltransferase and UDP-N-GlcNAc pyrophosphorylase activities(MtGlmU).MtGlmU was used to synthesis another essential sugar donor UDP-N-GlcNAc.In current work, we purified and characterized the recombinant MtGalU and MtGlmU proteins. MtGalU exists as a mixture of monomer and different oligomers and MtGlmU exists as a stable hexamer.The specific activity,enzyme kinetic properties of MtGalU and MtGlmU were determined.The subcellular localization study shows that MtGalU located on cell wall and MtGlmU located in cell cytoplasm.The 3D structures of MtGalU and MtGlmU were modeled and the models could be further explored for in silico docking studies with suitable inhibitors.Ic1/Rv0467 which is related with persistence infection of M. tuberculosis was subjected to further characterization.We cloned the icl/Rv0467 gene and purified the recombinant functional isocitrate lyase (ICL) protein.The crystal structure of M.tuberculosis ICL was determined formerly and eighty compounds that "hits" the target ICL were screened in silico from the compound library Specs.The enzymatic inhibition effects of these compounds were confirmed by enzymatic activity assay. By testing the inhibition effect of the compounds on the growth of bacteria in vitro,we finally got two compounds which inhibit the proliferation of analogue persistent M.tuberculosis H37Ra(cultured in the defined minimal medium with acetate as only carbon source) and M.tuberculosis multi-drug resistant stains.One compound,FD20,has low cytotoxicity and can inhibit the proliferation of M.tuberculosisH37Ra in human macrophage THP-1.In summary,two novel protein domains,DIM and MSTF,were identified in M.tuberculosis proteome by bioinformatics analysis in this work. Combined with bioinformatics analysis,we identified 66 potential anti-tuberculosis drug target genes,three of which were subjected to further biochemical analysis.Finally,the compound FD20 targeted against ICL represents an attractive anti-tuberculosis lead compound.更多还原