9-取代-2-氨基-6-胍基嘌呤类精子顶体酶抑制剂的设计、合成及活性评价

顶体酶是存在于精子顶体内的一种胰蛋白酶样的丝氨酸水解酶,是目前男性抗生育药物设计的潜在靶点之一.在前期对精子顶体酶同源模建的基础上,根据活性腔结构和活性位点性质,以KF950为先导化合物,设计合成了一类胍基鸟嘌呤化合物,采用核磁共振氢谱、质谱、红外光谱和元素分析等手段对其结构进行了表征,其中中间体5m的结构经X射线单晶衍射分析确证.以N-甲苯磺酰-L-赖氨酸-氯甲基酮(TLCK)为阳性对照,分别测定了目标化合物对精子顶体酶的体外抑制活性.结果表明,化合物6a～6z的酶抑制活性均强于TLCK,其中化合物6z抑制活性与KF950相当.     

人顶体酶活性腔性质及与抑制剂的结合模式

顶体酶是目前抗生育药物的一个潜在靶点. 在前期同源模建人顶体酶三维结构复合物的基础上, 采用多重拷贝同时搜寻(MCSS)等方法对人顶体酶活性腔进行分析. 结果显示, 活性腔的P1,P2和G 3个区域均具有较大极性, 且P1对抑制剂结合尤为重要. 另外, G和P1边缘及P2底部还具有一定疏水性, 且其中的部分重要残基还能与配体形成氢键作用和静电作用. MCSS计算结果确定的关键配体结合位点与人顶体酶复合物结构和定点突变实验结果相吻合. 在此基础上用分子对接方法将6个人顶体酶代表性抑制剂对接入活性腔, 阐明其结合模式, 确定与配体结合相关的关键残基.     

猪伪狂犬病毒胸苷激酶的三维结构模建与配体设计

以伪狂犬病毒(PRV)Fa为材料, 克隆测序胸苷激酶(TK)基因, 采用同源模建方法构建胸苷激酶的三维结构模型, 并经Ramachandran图和Profile_3D图验证了模型的可靠性. 采用InsightⅡ/Binding site, Delphi和Affinity方法定位了胸苷激酶的活性位点Site 1, 在此基础上设计出胸苷激酶抑制小分子N-苯基-N'-甲基脲, 通过柔性分子对接法阐明了胸苷激酶抑制剂与靶酶活性位点的相互作用模式, 发现模式中特异性的氢键相互作用可能是对靶酶产生抑制活性的重要分子基础. 研究结果为合理设计PRV胸苷激酶抑制剂, 探索新的治疗及预防伪狂犬病方案奠定了基础.     

嘧啶衍生物类PI3K/mTOR双重抑制剂的3D-QSAR及分子对接研究

PI3K/Akt/mTOR信号通路在癌细胞的生长和增殖中异常激活,对PI3K和mTOR位点的抑制可有效阻断信号通路的传导,是药物设计的理想靶点.本文选择38个嘧啶类小分子抑制剂进行3D-QSAR和分子对接研究.采用比较分子力场分析(CoMFA)和比较分子相似性指数分析(CoMSIA)方法,建立了三维定量构效关系模型,结果表明,该模型具有良好的稳定性和预测能力,可运用分子对接研究小分子抑制剂与PI3K和mTOR蛋白受体的作用模式.通过对QSAR模型的三维等势图以及受体与配体的相互作用模式分析后,优化出10个小分子化合物并预测其活性,发现一些小分子化合物活性提高,这为PI3K/mTOR双重抑制剂的设计筛选提供了借鉴.     

PTP1B和1,2-萘醌类抑制剂的分子动力学模拟

采用分子动力学和分子力学相结合的方法 ,研究了一类 1,2 萘醌类抑制剂与酪氨酸蛋白磷酸酯酶PTP1B之间的相互作用模式 .计算得到的抑制剂和靶酶之间的相互作用模式显示范德华相互作用、疏水相互作用以及氢键作用是主要的作用模式 .计算结果还表明抑制剂和PTP1B的相互作用能ΔE越低 ,抑制剂活性越高 .通过计算各种能量对ΔE的贡献 ,以及对复合物结构参数的分析 ,发现抑制剂和受体之间疏水相互作用是造成抑制剂活性差别的主要原因 .这为设计其他非酸类抑制剂提供了信息     

酮醇酸还原异构酶(KARI)与其抑制剂间相互作用的分子模拟研究

采用MCCE, Autodock及密度泛函方法对酮醇酸还原异构酶(KARI)与其抑制剂间相互作用进行了研究. 计算结果表明, KARI活性位点中的Mg^2＋在活性位点残基的离子化状态、活性位点的静电性质以及与抑制剂结合等方面起重要的作用; 同时, 抑制剂在结合方式、前线轨道布居及静电势等方面与酶促反应中间体(HOIV)具有一定程度的相似性; 可电离的羧基是当前发现的靶向KARI抑制剂一个重要的结构特征, 进一步推广可认为潜在的抑制剂应该拥有可电离成负电荷的功能团. 在药物设计中考虑到以上结论, 将有利于发现和改造靶向KARI的抑制剂.     

拟南芥TIR1与生长素IAA相互作用的分子对接及分子动力学研究

基于最新得到的拟南芥运输抑制剂响应蛋白质1(TIR1)与吲哚乙酸(IAA)复合物的晶体结构, 使用分子对接方法和分子动力学方法对TIR1与生长素IAA相互作用的方式进行了研究. 分子对接结果表明, 通过逐级考察辅酶InsP₆和中心水分子的影响, 发现辅酶InsP₆和中心水分子对生长素IAA正确结合到活性位点有重要作用. 分子动力学结果表明, 复合物体系在整个模拟过程中较为稳定, 2个水分子相继作为中心水分子与生长素IAA形成了稳定的氢键作用, IAA与活性位点处残基的相互作用与晶体结构相比略有差异.     

PTPlB和1，2-萘醌类抑制剂的分子动力学模拟

采用分子动力学和分子力学相结合的方法，研究了一类1，2-萘醌类抑制剂与酪氨酸蛋白磷酸酯酶PTP1B之间的相互作用模式．计算得到的抑制剂和靶酶之间的相互作用模式显示范德华相互作用、疏水相互作用以及氢键作用是主要的作用模式．计算结果还表明抑制剂和PTP1B的相互作用能△E越低，抑制剂活性越高．通过计算各种能量对△E的贡献，以及对复合物结构参数的分析，发现抑制剂和受体之间疏水相互作用是造成抑制剂活性差别的主要原因．这为设计其他非酸类抑制剂提供了信息．     

基于Topomer CoMFA和Surflex-dock的GSK-3β抑制剂的3D-QSAR与作用模式研究

GSK-3β的过度表达可导致人脑神经细胞内Tau蛋白的过磷酸化,从而介导阿尔兹海默病(Alzheimer’s disease,AD)的发生.本文旨在研究GSK-3β的马来酰胺类抑制剂的三维定量构效关系(3D-QSAR)及新抑制剂分子与GSK-3β的作用机制.采用基于R基团搜索技术的Topomer CoMFA建立了49个马来酰胺类GSK-3β抑制剂的3D-QSAR模型,并用包括25个样本的测试集验证模型的外部预测能力.所得优化模型的拟合、交互验证以及外部验证的复相关系数分别为0.928,0.790和0.725.采用Topomer search在ZINC分子数据库中进行虚拟搜索,设计了28个可能具有更高活性的新抑制剂.借助Surflex-dock分子对接研究了新抑制剂与GSK-3β作用模式与机制.结果显示,新抑制剂与GSK-3β的Asp133,Tyr134,Val135和Pro136等位点作用显著.     

2'-羟基-2,4-二溴二苯醚与人血清白蛋白作用机制的光谱研究与计算模拟

采用分子对接、分子动力学模拟和光谱法研究了2'-羟基-2,4-二溴二苯醚(2'-OH-BDE-7)与人血清白蛋白(HSA)的相互作用.同步荧光光谱研究表明,2'-OH-BDE-7诱导HSA的构象产生变化,并与分子动力学模拟的结果相吻合.荧光光谱表明,2'-OH-BDE-7能引起HSA荧光猝灭,且其作用机制为静态猝灭和非辐射能量转移.热力学分析得出ΔG0,ΔH0和ΔS0,表明氢键和范德华力在HSA-2'-OH-BDE-7的体系中起着重要作用.竞争实验和分子对接结果表明2'-OH-BDE-7主要结合在HSA的位点Ⅰ.将计算模拟和光谱实验研究相结合,为研究小分子和蛋白质相互作用机制提供更多的信息.     

Acrosin structure-based design,synthesis and biological activities of 7-azaindol derivatives as new acrosin inhibitors

A series of 7-azaindol derivatives were designed based on the homologous 3D model of human acrosin.These compounds were synthesized and evaluated for their human acrosin inhibitory activities in vitro.Compounds 7a,7i,7j,7k and 7n showed highly inhibitory activity against human acrosin.The three-dimensional structure-activity relationship was investigated through a CoMFA model,which provided valuable information to further study of potential human acrosin inhibitors.     

The discovery of novel auxin transport inhibitors by molecular modeling and three-dimensional pattern analysis

Summary Molecular modeling techniques and three-dimensional (3D) pattern analysis have been used to investigate the chemical and steric properties of compounds that inhibit transport of the plant hormone auxin. These compounds bind to a specific site on the plant plasma membrane characterized by its affinity for the herbicide N-1-naphthylphthalamic acid (NPA). A 3D model was derived from critical features of a set of ligands for the NPA receptor, a suggested binding conformation is proposed, and implications for the topographical features of the NPA receptor are discussed. This model, along with 3D structural analysis techniques, was then used to search the Abbott corporate database of chemical structures. Of the 467 compounds that satisfied the criteria of the model, 77 representative molecules were evaluated for their ability to compete for the binding of [³H]NPA to corn microsomal membranes. Nineteen showed activity that ranged from 16 to 85% of the maximum NPA binding. Four of the most active of these, representing chemical classes not included in the original compound set, were also found to inhibit polar auxin transport through corn coleoptile sections. Thus, this study demonstrates that 3D analysis techniques can identify active, novel ligands for biochemical target sites with concomitant physiological activity.     

Prediction of enzyme binding: human thrombin inhibition study by quantum chemical and artificial intelligence methods based on X-ray structures

Thrombin is a serine protease which plays important roles in the human body, the key one being the control of thrombus formation. The inhibition of thrombin has become a target for new antithrombotics. The aim of our work was to (i) construct a model which would enable us to predict Ki values for the binding of an inhibitor into the active site of thrombin based on a database of known X-ray structures of inhibitor-enzyme complexes and (ii) to identify the structural and electrostatic characteristics of inhibitor molecules crucially important to their effective binding. To retain as much of the 3D structural information of the bound inhibitor as possible, we implemented the quantum mechanical/molecular mechanical (QM/MM) procedure for calculating the molecular electrostatic potential (MEP) at the van der Waals surfaces of atoms in the protein's active site. The inhibitor was treated quantum mechanically, while the rest of the complex was treated by classical means. The obtained MEP values served as inputs into the counter-propagation artificial neural network (CP-ANN), and a genetic algorithm was subsequently used to search for the combination of atoms that predominantly influences the binding. The constructed CP-ANN model yielded Ki values predictions with a correlation coefficient of 0.96, with Ki values extended over 7 orders of magnitude. Our approach also shows the relative importance of the various amino acid residues present in the active site of the enzyme for inhibitor binding. The list of residues selected by our automatic procedure is in good correlation with the current consensus regarding the importance of certain crucial residues in thrombin's active site.     

An Approach for Visualization of the Active Site of Enzymes with Unknown Three-Dimensional Structures

Abstract

A new approach for virtual characterization of the active site structure of enzymes with unknown three-dimensional (3D) structure has been proposed. It includes analysis of data on enzyme interaction with reversible competitive inhibitors, their 3D structures and moulding of the substrate-binding region. The superposition of ligands in biologically active conformations allows to determine the shape and dimension of the active site cavity accommodating these compounds. Monoamine oxidase A (MAO-A), a “typical” enzyme with unknown spatial organisation, was used to test this method. The correctness of such approach was validated by the analysis of HIV protease interaction with its inhibitors using 3D structures of their complexes. Mould of the substrate/inhibitor binding site can be used for the visualization of this binding site and for searching new ligands in molecular databases.     

CoMFA and CoMSIA 3D-quantitative structure-activity relationship model on benzodiazepine derivatives, inhibitors of phosphodiesterase IV

Recently, we reported structurally novel PDE4 inhibitors based on 1,4-benzodiazepine derivatives. The main interest in developing bezodiazepine-based PDE4 inhibitors is in their lack of adverse effects of emesis with respect to rolipram-like compounds. A large effort has thus been made toward the structural optimization of this series. In the absence of structural information on the inhibitor binding mode into the PDE4 active site, 2D-QSAR (H-QSAR) and two 3D-QSAR (CoMFA and CoMSIA) methods were applied to improve our understanding of the molecular mechanism controlling the PDE4 affinity of the benzodiazepine derivatives. As expected, the CoMSIA 3D contour maps have provided more information on the benzodiazepine interaction mode with the PDE4 active site whereas CoMFA has built the best tool for activity prediction. The 2D pharmacophoric model derived from CoMSIA fields is consistent with the crystal structure of the PDE4 active site reported recently. The combination of the 2D and 3D-QSAR models was used not only to predict new compounds from the structural optimization process, but also to screen a large library of bezodiazepine derivatives.     

Ligand- and protein-based modeling studies of the inhibitors of human cytochrome P450 2D6 and a virtual screening for potential inhibitors from the Chinese herbal medicine, Scutellaria baicalensis (Huangqin,Baikal Skullcap)

We have previously examined the binding patterns of various substrates to human cytochrome P450 2D6 (CYP2D6) using a series of molecular modeling methods. In this study, we further explored the binding modes of various types of inhibitors to CYP2D6 using a combination of ligand- and protein-based modeling approaches. Firstly, we developed and validated a pharmacophore model for CYP2D6 inhibitors, which consisted of two hydrophobic features and one hydrogen bond acceptor feature. Secondly, we constructed and validated a quantitative structure-activity relationship (QSAR) model for CYP2D6 inhibitors which gave a poor to moderate prediction accuracy. Thirdly, a panel of CYP2D6 inhibitors were subject to molecular docking into the active site of wild-type and mutated CYP2D6 enzyme. We demonstrated that 8 residues in the active site (Leu213, Glu216, Ser217, Gln244, Asp301, Ser304, Ala305, and Phe483) played an important role in the binding to the inhibitors via hydrogen bond formation and/or π-π stacking interaction. Apparent changes in the binding modes of the inhibitors have been observed with Phe120Ile, Glu216Asp, Asp301Glu mutations in CYP2D6. Finally, we screened for potential binders/inhibitors from the Chinese herbal medicine Scutellaria baicalensis (Huangqin, Baikal Skullcap) using the established pharmacophore model for CYP2D6 inhibitors and molecular docking approach. Overall, 18 out of 40 compounds from S. baicalensis were mapped to the pharmacophore model of CYP2D6 inhibitors and most herbal compounds from S. baicalensis could be docked into the active site of CYP2D6. Our study has provided insights into the molecular mechanisms of interaction of synthetic and herbal compounds with human CYP2D6 and further benchmarking studies are needed to validate our modeling and virtual screening results.     

Discovery of novel human acrosin inhibitors by virtual screening

Human acrosin is an attractive target for the discovery of male contraceptive drugs. For the first time, structure-based drug design was applied to discover structurally diverse human acrosin inhibitors. A parallel virtual screening strategy in combination with pharmacophore-based and docking-based techniques was used to screen the SPECS database. From 16 compounds selected by virtual screening, a total of 10 compounds were found to be human acrosin inhibitors. Compound 2 was found to be the most potent hit (IC₅₀ = 14 μM) and its binding mode was investigated by molecular dynamics simulations. The hit interacted with human acrosin mainly through hydrophobic and hydrogen-bonding interactions, which provided a good starting structure for further optimization studies.     

A ligand-based comparative molecular field analysis (CoMFA) and homology model based molecular docking studies on 3′, 4′-dihydroxyflavones as rat 5-lipoxygenase inhibitors: Design of new inhibitors

In this study, ligand based comparative molecular field analysis (CoMFA) with five principal components was performed on class of 3′, 4′-dihydroxyflavone derivatives for potent rat 5-LOX inhibitors. The percentage contributions in building of CoMFA model were 91.36% for steric field and 8.6% for electrostatic field. R² values for training and test sets were found to be 0.9320 and 0.8259, respectively. In case of LOO, LTO and LMO cross validation test, q² values were 0.6587, 0.6479 and 0.5547, respectively. These results indicate that the model has high statistical reliability and good predictive power. The extracted contour maps were used to identify the important regions where the modification was necessary to design a new molecule with improved activity. The study has developed a homology model for rat 5-LOX and recognized the key residues at the binding site. Docking of most active molecule to the binding site of 5-LOX confirmed the stability and rationality of CoMFA model. Based on molecular docking results and CoMFA contour plots, new inhibitors with higher activity with respect to the most active compound in data set were designed.     

Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

In order to enhance the glutathione peroxidase(GPX) catalytic activity of the selenium-containing single-chain variable fragments(Se-scFv), a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional(3D) model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b(+), then the scFv protein was expressed in soluble form in Escherichia coli BL21(DE3) and purified by Ni2+-immobilized metal affinity chromatography(IMAC). The serine residue of scFv in the active site was converted into selenocysteine(Sec) with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mecha- nism.