【作者】 张涛；

【导师】 李亦学； 魏冬青；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2011， 博士

【摘要】 细胞色素P450酶家族是人体中重要的一类单氧化酶,在体内发挥着许多功能,特别在药物代谢过程中具有重要作用,是人体内主要的药物代谢酶。同时此类酶的另一特性是在基因水平上具有广泛的遗传多样性,可以引起不同个体之间药物代谢能力的明显差异。因此对于细胞色素P450酶介导的药物代谢过程、不同细胞色素P450酶单体与化合物之间的代谢关系、基因多态性对于细胞色素P450酶的功能影响以及不同细胞色素P450酶突变体的活性改变机制等一直是人们对于细胞色素P450酶研究的热点。本文主要针对细胞色素P450酶的非同义单核苷酸多态性对于酶功能活性的影响及细胞色素P450酶与不同化合物之间的代谢关系进行四方面相对独立的研究,下面对进行的研究内容和所得到的结论进行简要的描述。1、通过收集和阅读相关文献,构建了侧重于细胞色素P450酶的非同义单核苷酸多态性(nsSNP)影响酶活性变化的数据库CYP-nsSNP。该数据库不仅对已测定的相关实验数据进行了有效的整理,同时也可以为理论研究氨基酸在维持细胞色素P450酶活性中的作用提供了可靠的数据资源;2、利用分子动力学模拟和分子对接以及其它基于蛋白结构的方法对于蛋白表面具有重要作用的氨基酸突变F186L如何影响CYP1A2结构和功能进行了系统研究。从蛋白结构和蛋白运动的角度上分析了该表面氨基酸突变对于酶活性的作用,在原子水平上对于此表面氨基酸突变导致酶活性改变给予了合理的解释,并且首次在细胞色素P450酶研究中将蛋白运动和底物通道相结合进行分析。同时提出了与别构效应调节相类似,表面氨基酸突变可以借助影响蛋白运动和改变蛋白构象的方式来调节蛋白活性;3、利用机器学习方法从化合物结构出发对于不同细胞色素P450酶底物代谢的选择性进行研究。将遗传算法和人工神经网络方法以及多标签K邻近分类法联合使用,分别构建出可以对细胞色素P450酶底物进行分类的单标签多类别和多标签多类别分类模型。与其它细胞色素P450酶底物的分类模型相比,我们的模型涵盖了更多种类的细胞色素P450酶,更加符合体内细胞色素P450酶所介导的药物代谢;4、将基于蛋白结构和小分子结构的两类方法相结合,构建出针对CYP1A2酶活性中心的结构模型。通过该模型我们可以获得组成CYP1A2酶活性中心的重要氨基酸残基在底物结合过程中所发挥的作用,并且对于药物代谢位点预测、药物设计以及有目的酶功能改造提供了详实可靠的信息。更多还原

【Abstract】 The cytochrome P450 superfamily (CYP) is a group of heme-containing monooxygenases which perform many necessary functions in the human body. As the major drug-metabolizing enzymes, CYP enzymes are responsible for the metabolism of a large number of drugs. In addition, CYP enzymes are subject to genetic polymorphism resulting in the inter-individual difference in drug-metabolizing ability. Thus, the main focus of recent CYP research includes the CYP-mediated drug metabolism, the substrate specificity of different CYP isoenzymes, the influences of different genetic polymorphisms on function of CYP enzymes and the possible mechanism for changing enzymatic activity of CYP due to mutation of amino acid. Here we carried out four independent experiments focused on the effect of non-synonymous single nucleotide polymorphism (nsSNP) on the enzymatic activity of CYP and the substrate selectivity of different CYP isoenzymes. These studies and their conclusions are briefly described below.1. A database named CYP-nsSNP was constructed through collecting the relevant data from the published references. The CYP-nsSNP is used to organize the available data regarding effect of nsSNPs on function of CYP proteins and to provide the reliable information for theoretical investigation of the role of amino acids in CYP function.2. The effect of a surface mutation F186L on CYP1A2 structure and function was explored using the molecular dynamics simulation and molecular docking as well as other structure-based methods. In this study, the long-range effect of surface mutation was comprehensively investigated from the point of view of protein structure and motions. Additionally, the method of protein motion combined with substrate channel was firstly applied to reveal the relationship between structure and function of CYP protein. Based on results from this study, we can provide a rational explanation for the decrease in enzymatic activity caused by mutation. In addition, we also propose a new mechanism for alosteric regulation of enzyme induced by mutation through changing the access channels and protein conformations.3. The substrate selectivity of different CYP isoenzymes was investigated using the machine learning methods based on the structure of CYP substrates. The genetic algorithm was combined with the artificial neural network and multi-label K nearest neighbour method to construct the single-label multi-class and multi-label multi-class classification models. Compared with the traditional models, two models reflect the more realistic CYP-mediated drugs metabolism in the human body. After appropriate evaluation, both models exhibited more than 80% of predictive accuracy.4. Integrating the information about both protein and compounds, we constructed a structural model specific to CYP1A2 active site using the comparative molecular field analysis. Based on such a model, we can investigate the detailed role of residues lining the CYP1A2 active site in substrate binding. Therefore, such analysis is useful for the rational drug design and enzyme modification by providing the detailed and reliable information about protein-ligand interactions.更多还原