基于HIV-1相关蛋白的分子模拟和药物设计研究

发布时间：2018-02-14 18:56

本文关键词： 分子模拟结合自由能虚拟筛选整合酶艾滋病　出处：《成都学院》2017年硕士论文　论文类型：学位论文

【摘要】：HIV-1是全球流行的传染病——艾滋病的病原体,严重威胁着人类的健康,针对HIV-1相关蛋白的药物设计成为抗艾滋病药物研究的热点领域。HIV-1相关的蛋白主要包括逆转录酶、整合酶和蛋白酶,它们分别在病毒的生命周期中发挥着不同的功能,因此,研究药物与三个酶的作用机制对于后续抗艾滋病的药物设计具有重要意义。本论文主要采用计算机辅助药物设计方法对三个蛋白进行了系列研究,内容包括4个方面:沙奎那韦与HIV-1蛋白酶的分子识别;HIV-1逆转录酶的功能运动性分析;HIV-1整合酶抑制剂的筛选平台及分子识别;HIV-1整合酶在大肠杆菌中的表达和纯化。(1)通过分子动力学模拟研究了SQV与蛋白酶的分子识别机理,并计算了二者形成的氢键以及重要氨基酸对识别的贡献。计算得到的B因子与实验值具有显著的相关性表明模拟过程是可靠的;SQV与Asp25'和Asp25之间稳定的氢键是结合的重要作用力;Gly49'、Gly27'、Pro81和Asp29'对于SQV-蛋白酶识别的能量贡献较大。(2)采用粗粒化模型分析了逆转录酶各个功能区域的运动性,并采用分子对接预测了逆转录酶与NAD化合物的复合物模型。结果显示,手指区与RNase H区的开合运动是逆转录酶最主要的功能性运动,对于发挥逆转录功能具有重要的意义;分子对接表明反式双键是NAD化合物与RT结合的优势构象。(3)通过序列比对、虚拟筛选和结合自由能计算研究PFV IN-DNA体系作为IN抑制剂筛选平台的潜力,并通过分子动力学模拟及构象分析研究了NRD化合物与IN的分子识别。结果表明,40.8%的序列相似度,26.43的筛选效率和结合自由能计算值与实验值的显著相关性都证明了PFV IN-DNA作为虚拟筛选平台的可靠性;DTG的结构可以分为亲水性和疏水性部分,其中2个Mg2+、3个水分子以及周围的DDE保守氨基酸与亲水性区域结合,而病毒DNA中的碱基以及Tyr212、Pro214等氨基酸则与疏水部分结合;构象运动性分析表明,导致IN-DNA的运动性降低可能是IN抑制剂的抑制机理之一。(4)将含有F185K/C280S双突变的pET28a-IN重组质粒导入大肠杆菌细胞内并表达,并用亲和层析及SDS-PAGE法纯化蛋白。结果显示,通过亲和层析得在细胞破碎上清液中得到较多的整合酶蛋白质,表明所采用的实验方法是可靠的。
[Abstract]:HIV-1 is the pathogen of AIDS, which is a global infectious disease. It is a serious threat to human health. Drug design for HIV-1 related proteins has become a hot topic in the research of anti-AIDS drugs. HIV-1 related proteins mainly include reverse transcriptase. Integrase and protease, which play different roles in the virus's life cycle, therefore, It is important to study the action mechanism of drugs and three enzymes for the subsequent anti-AIDS drug design. In this paper, a series of studies on the three proteins were carried out by using the method of computer-aided drug design. The content includes four aspects: molecular recognition of sarquinavir and HIV-1 protease; functional mobility analysis of HIV-1 reverse transcriptase; screening platform for HIV-1 integrase inhibitor; expression and purification of HIV-1 integrase in Escherichia coli. The molecular recognition mechanism of SQV and protease was studied by molecular dynamics simulation. The hydrogen bond formed by them and the contribution of important amino acids to recognition are also calculated. The calculated B factor is significantly correlated with the experimental value, which indicates that the simulation process is reliable and the stable hydrogen bond between SQV and Asp25 'and Asp25 is bound. The important action force of Gly49A, Gly27P27 Pro81 and Asp29', was used to analyze the motility of various functional regions of reverse transcriptase by using coarse granulation model, which contributed significantly to the energy contribution of SQV- protease recognition. Molecular docking was used to predict the complex model of reverse transcriptase and NAD compounds. The results showed that the opening and closing movement between finger region and RNase H region was the most important functional movement of reverse transcriptase, which had important significance for exerting reverse transcriptase function. Molecular docking indicates that trans-double bond is the dominant conformation for the binding of NAD compounds to RT.) by sequence alignment, virtual screening and calculation of binding free energy, the potential of PFV IN-DNA system as an IN inhibitor screening platform is studied. Molecular dynamics simulation and conformation analysis were used to study the molecular recognition of NRD compounds and IN. The results showed that the screening efficiency of 40.8% sequence similarity and the significant correlation between the calculated value of binding free energy and the experimental value were proved. The structure of PFV IN-DNA as a virtual screening platform can be divided into hydrophilic and hydrophobic parts. Two Mg2, three water molecules and the surrounding DDE conserved amino acids bind to the hydrophilic region, while the bases in the viral DNA and the amino acids Tyr212OPro214 are partially bound to the hydrophobic region. The reduction of IN-DNA motility may be one of the inhibitory mechanisms of IN inhibitors. (4) the recombinant pET28a-IN plasmid containing double mutation of F185K / C280S was introduced into Escherichia coli cells and expressed. The protein was purified by affinity chromatography and SDS-PAGE. Through affinity chromatography, more integrase proteins were obtained in the supernatant of cell fragmentation, which indicated that the experimental method was reliable.
【学位授予单位】：成都学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

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