以苯丙氨酸为碳端的ACE抑制肽3D-QSAR、活性验证及分子作用机制研究

发布时间：2018-03-16 21:15

本文选题：苯丙氨酸　切入点：血管紧张素转化酶抑制肽　出处：《广东药科大学》2017年硕士论文　论文类型：学位论文

【摘要】：血管紧张素转化酶(ACE)在血压调节中的关键性作用,使其成为治疗高血压疾病的理想靶点。ACE抑制肽凭借其安全、易吸收等优点受到了广泛关注。本论文采用以苯丙氨酸为碳端的ACE抑制二、三肽进行三维定量构效关系(3D-QSAR)研究并建立相关模型,根据所建模型筛选并合成具有良好ACE抑制活性的三肽并进行体外活性验证,同时采用分子动力学模拟和二维核磁共振实验相结合的方法,研究新型ACE抑制肽的分子作用机制,并给出微观解释。本文的主要内容由以下四部分组成:第一部分,收集已报道的53种ACE抑制二、三肽进行3D-QSAR CoMFA/CoMSIA以及分子对接研究,建立了具有良好预测能力的CoMFA/CoMSIA模型,并获得抑制肽分子与ACE受体之间可能的结合模式。同时根据已建立的模型筛选并合成了四种三肽Gly-Glu-Phe(GEF),Val-Glu-Phe(VEF),Val-Arg-Phe(VRF)以及Val-Lys-Phe(VKF)进行体外活性实验,对模型进行进一步的验证。第二部分,采用改良的紫外分光光度法对第一部分中筛选并合成的四种三肽GEF,VEF,VRF以及VKF进行体外活性实验验证。其中,GEF的CoMFA/CoMSIA模型预测值均与实验值较为接近,误差分别为4.49%和7.14%;VEF的Co MSIA模型预测值与实验值的相对误差最小,仅为1.08%;VRF的CoMFA模型预测值与实验值之间的相对误差最大,为9.87%,但是两者仍然属于同一个数量级。上述四种三肽的CoMFA/CoMSIA模型预测值均接近与实验活性值,实验结果进一步证明了所建模型具有良好的预测能力及准确性。第三部分,采用分子动力学模拟与2D 1H-1H-NOESY图谱相结合的方法,研究生物活性水溶液GEF-H2O体系和小肽变性条件下的GEF-DMSO体系中GEF分子的构象变化以及分子间的弱相互作用。结果表明GEF分子在水溶液中主要以伸展构象存在,折叠构象和伸展构象之间可以快速相互转换,而在DMSO溶液中主要以折叠构象存在,较少存在伸展构象。2D 1H-1H-NOESY图谱的得到的结果与分子动力学模拟结论一致,理论和实验的结果得到很好的吻合。第四部分,本章采用分子动力学研究了GEF分子分别与ACE的C-结构域(C-ACE)、N-结构域(N-ACE)之间相互作用的机制。研究发现GEF分子在C-ACE活性区域的构象较在N-ACE活性区域的构象更舒展,C-ACE的Lys475氨基酸残基对配体与受体之间的结合影响最大,而N-ACE中影响最大的是Lys489氨基酸残基。通过配位作用分析发现,GEF分子与Zn离子之间的相互作用是维持配体-受体复合物稳定的主要因素。本论文运用理论与实验相结合的方法建立了一套筛选高活性ACE抑制肽及揭示分子作用机制的研究体系。这一体系包括:建立ACE抑制肽的数学模型;筛选并合成具有较高抑制活性的肽;体外活性实验验证模型的准确性;分子动力学模拟从微观角度解释活性分子结构与活性之间的关系。此方法对研究和开发新型ACE抑制肽具有一定的理论指导意义。
[Abstract]:The key role of angiotensin converting enzyme (ACE) in the regulation of blood pressure makes it an ideal target for the treatment of hypertension. The advantages of easy absorption have attracted much attention. In this paper, the three-dimensional quantitative structure-activity relationship (3D-QSAR) was studied by using phenylalanine (phenylalanine) as carbon terminal ACE to inhibit dipeptide and tripeptide, and the relevant models were established. According to the established model, tripeptides with good ACE inhibitory activity were selected, synthesized and verified in vitro. At the same time, the molecular mechanism of the novel ACE inhibitory peptides was studied by means of molecular dynamics simulation and two-dimensional nuclear magnetic resonance (NMR). The main contents of this paper are as follows: in the first part, we collect 53 kinds of ACE inhibitory dipeptide and tripeptide to study 3D-QSAR CoMFA/CoMSIA and molecular docking, and establish a CoMFA/CoMSIA model with good prediction ability. The possible binding patterns between inhibitory peptide molecules and ACE receptors were obtained. Four tripeptide Gly-Glu-Phegnon Val-Glu-Phegnon VRFs and Val-Lys-Phe-Phe-VKFs were screened and synthesized according to the established model. Modified UV spectrophotometry was used to verify the in vitro activity of four tripeptide GEFFV VRF and VKF, which were selected and synthesized in the first part. The predicted values of CoMFA/CoMSIA model were all close to the experimental values. The relative error between the predicted value and experimental value of Co MSIA model with errors of 4.49% and 7.14 respectively is the smallest, and the relative error between the predicted value of CoMFA model and the experimental value of 1.08VRF is the largest. The predicted values of the CoMFA/CoMSIA model of the above four tripeptides are all close to the experimental activity values. The experimental results further prove that the model has good predictive ability and accuracy. The method of combining molecular dynamics simulation with 2D 1H-1H-NOESY map was used. The conformational changes and weak intermolecular interactions of GEF molecules in bioactive aqueous solution GEF-H2O system and GEF-DMSO system with small peptide denaturation were studied. The results show that GEF molecules mainly exist in extensional conformation in aqueous solution. The folding conformation and extensional conformation can be converted rapidly, while in DMSO solution, the folding conformation mainly exists in the form of folded conformation, and the results obtained from the extensional conformation .2D 1H-1H-NOESY are in agreement with the results obtained by molecular dynamics simulation. The theoretical and experimental results are in good agreement. Part 4th, In this chapter, molecular dynamics was used to study the mechanism of interaction between GEF molecule and C-ACE-N-ACE-domain of ACE. It was found that the conformation of GEF molecule in C-ACE active region was more extensive than that in N-ACE active region. The amino acid residues of Lys475 have the greatest influence on the binding between ligand and receptor. In N-ACE, the amino acid residues of Lys489 were the most influential. The interaction between Lys489 molecules and Zn ions was found to be the main factor to maintain the stability of ligand receptor complexes. In this paper, the theoretical and experimental phase was used. A set of research systems for screening highly active ACE inhibitory peptides and revealing the molecular mechanism of action were established by the combined method. The system includes: establishing a mathematical model of ACE inhibitory peptides; The peptides with high inhibitory activity were screened and synthesized, and the accuracy of the model was verified by in vitro activity experiment. Molecular dynamics simulation explains the relationship between active molecular structure and activity from the microcosmic point of view. This method has a certain theoretical significance for the research and development of novel ACE inhibitory peptides.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R945

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