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题名: 组织蛋白酶B、K和基质金属蛋白酶9抑制剂的对接与构效关系研究
作者: 潘蓄林
学位类别: 博士
答辩日期: 2007-06-05
授予单位: 中国科学院昆明植物研究所
授予地点: 昆明植物研究所
导师: 谭宁华
关键词: 分子对接 ; 3D-QSAR ; 组织蛋白酶B和K ; 基质金属蛋白酶9 ; 抑制剂
学位专业: 植物学
中文摘要: 本论文共包括六章:第一章围绕计算机辅助药物设计的基本方法及三种重要的蛋白酶靶点即组织蛋白酶B和K、 基质蛋白酶9进行简单介绍;第二章介绍用量子化学和分子对接方法研究本课题组从植物中分离筛选发 现的三个组织蛋白B新天然抑制剂双黄酮类化合物的作用机理,并进行了组织蛋白酶B的虚拟筛选和活性验 证;第三章和第四章用分子对接或数据库叠合和3D-QSAR方法研究组织蛋白K的合成类抑制剂醛类、酮酰胺 类化合物与组织蛋白酶K的构效关系;第五章用量子化学及多种分子对接方法研究基质金属蛋白酶9与七个黄酮类抑 制剂的作用机理及构效关系;第六章针对计算辅助药物设计一些研究进展进行了综述。主要论文工作如下: \textbf{第二章 组织蛋白酶B与穗花杉双黄酮类抑制剂的作用模式与其虚拟筛选及活性验证:}组织蛋白酶B(cathepsin B, CatB)是和肿瘤相关的药物靶点,寻找新型的CatB抑制剂具有重要的价值。前期我们从墨西哥落羽杉中分离得到 的三个穗花杉双黄酮类化合物,经测试具有抑制CatB的活性,并且和大多数合成类抑制剂不同,此类抑制剂是可逆性抑 制剂。通过密度函数和FlexX的结果证明此类抑制剂作用于CatB的活性空腔,和Cys29形成疏水相互作用;而其它的 不可逆抑制剂则与Cys29上的S原子形成共价相互作用。并且穗花杉双黄酮7$^{\prime\prime}$位羟基上的氢原子被甲基 取代后会增加其抑制活性,而4$^{\prime\prime\prime}$位羟基上的氢原子被甲基取代则由于空间效应而不利于抑制 活性的提高。为了寻找更多的CatB抑制剂,与中国科学院上海药物研究所合作进行了以CatB为靶点的虚拟筛选, 从具有24万个化合物的SPECS数据库中筛选出98个具有活性的化合物,通过进一步实验验证,发现26个化合物具 有抑制CatB的活性,其中有3个化合物的IC$_{50}$值小于1$\mu$M,阳性率为26.53\%。上述研究为进一步寻找 和改造双黄酮类抑制剂提供理论指导,在此基础上进行的虚拟筛选,能快速地发现新的抑制剂,为寻找其它 组织蛋白酶抑制剂提供了思路。 \textbf{第三章和第四章 组织蛋白酶K醛类和酮酰胺类抑制剂的3D-QSAR研究:}组织蛋白酶K(cathepsin K, CatK)是与骨质疏松密切相关的药物靶点,目前已有几个抑制剂进入临床前研究,所以寻找新型的CatK抑制剂具有重要意义。 应用分子对接或数据库叠合及3D-QSAR方法即CoMFA和CoMSIA分别研究了59个醛类抑制剂和74个酮酰胺类抑制剂与CatK的构 效关系,模型的交叉验证系数q$^2_{cv}$值都达到0.7以上,并且对检验集中化合物的活性预测都比较准确,表明所建立 的三维构效关系模型是可靠的。醛类和酮酰胺类化合物主要作用在CatK的S1、S2和S3位点,这几个活性位点组成一个深而 窄的空腔,所以对醛类和酮酰胺类化合物的空间结构有比较高的要求。S1位点是一个窄而深的空腔,计算结果表明在此位 置适当增加取代基的体积能增加其抑制活性,如醛类化合物的P1位上比较有利的取代基为正丁基;S2位点是一个浅而宽 的口袋,醛类化合物的P2位点取代基比较好的是四员环,而酮酰胺类由于体积较大,P2取代基已经暴露在活性位点外。 本工作所建立的三维构效关系模型不仅很好地解释了实验事实,而且为进一步合成和改造这两类抑制剂提供了理论基础。 \textbf{第五章 基质金属蛋白酶9与7个黄酮类抑制剂的作用机理:}基质金属蛋白酶9(matrix metalloproteinase 9, MMP9)是一类与多种疾病,特别是肿瘤相关的药物靶点,己有一些合成类抑制剂如氧肟酸类化合物 进入临床前研究,所以寻找新型的MMP9抑制剂具有重要的价值。选择了文献报道的七个芹菜素类黄酮化合物, 这几个黄酮类化合物环上的羟基数目和取代基位置不同,相应抑制MMP9的活性也不同。应用密度函数 和多种分子对接方法即Glide、MVD2006、FlexX、Gold3.0和Moe2004对这七个黄酮分子对MMP9的抑制机 理进行了研究。计算结果表明,黄酮类化合物环上的取代羟基数目越多,活性越高是因为羟基数目的增 多会增加整个分子的亲水性,而MMP9的活性位点有大量的亲水区域,所以有利于二者的相互作用; B环上3$^\prime$和4$^\prime$同时被羟基取代会增加其抑制活性是因为它能和MMP9活性位点上的Zn离 子形成较强的”二齿“螯合作用;C环上3位的羟基能增强整个分子的供电子效应和共轭效应,但3位羟基 的位置刚好处于MMP9的疏水区域,所以3位的羟基对该类黄酮分子的抑制活性贡献较小。这些计算结果不仅很好 地解释了实验事实,并且为我们进一步研究此类抑制剂提供了理论基础。
英文摘要: This thesis included six chapters, which reported studies on inhibitors of cathepsin B (CatB), cathepsin K (CatK) and matrix metalloproteinase 9 (MMP9) using molecular docking and QSAR methods. The first chapter gave simple introduction of computer-aided drug design methods and three proteases (CatB, CatK and MMP9). The second chapter elucidated the mechanism between three amentoflavone analogues isolated by us and cathepsin B using density functional theory (DFT) and molecular docking methods, virtual screen and IC$_{50}$ test. The third and fourth chapters built 3D-QSAR models of aldehyde and ketoamide compounds, which are two classes of cathepsin K inhibitors, employing molecular docking, database alignment, CoMFA and CoMSIA methods. The fifth chapter explained the inhibitory mechanism of seven flavones (apigenin analogues) on MMP9 using DFT and five molecular docking methods. The sixth chapter made a mini-review on computer-aided drug design methods. Main works as follows: \textbf{Chapter 2 Interaction mode of amentoflavone analogues, virtual screen and experimental test targeted on human cathepsin B.} It is valuable to find novel inhibitors of CatB because CatB related to cancer is one of important drug targets. Three amentoflavone analogues isolated from \emph{Taxodium mucronatum} showed inhibitory activity on CatB in our previously works. Calculated results by DFT and FlexX demonstrated that these inhibitors bind to active sites and have a hydrophobic interaction with Cys25 of CatB, which are different from most synthetic inhibitors that have a covalent interaction with Cys25; 7$^{\prime\prime}$-OCH$_3$ increases inhibitory activity as the result of hydrophobic effect and 4$^{\prime\prime\prime}$-OCH$_3$ decreases inhibitory activity because of steric effect. Virtual screen on SPECS library with 240,000 compounds targeted CatB was finished under the help of Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and 98 active compounds were found and selected to further verify activity by experiment. Results indicated that 26 compounds showed inhibitory activity on CatB, in which IC$_{50}$ values of three inhibitors are less than 1$\mu$M. The hit rate is equal to 26.53\%. These results will help us to find novel biflavones and other inhibitors of CatB and give some important clues to study on inhibitors of cathepsins. \textbf{Chapter 3 and 4 3D-QSAR models of aldehyde and ketoamide inhibitors of cathepsin K. } Cathepsin K is one of important drug targets related to bone disease and some inhibitors of CatK have been going into clinical trail. CoMFA and CoMSIA models of 59 aldehyde and 74 ketoamide compounds were respectively built based on molecular docking and database alignment results, and gave a reliable prediction of inhibitory activity, which have high r$^2$ values (r$^2$$>$0.7) and reasonable predicted values of test sets. Aldehyde and ketoamide compounds mainly bind to S1, S2 and S3 active sites of CatK, which is a narrow and steep cleft. 3D-QSAR model demonstrated that n-butane group is the favorable group on P1 and four-member cyclic groups on P2, and the P2 groups of ketoamides are out of active sites. These 3D-QSAR models may not only predict inhibitory activity of cathepsin K well, but also can guide synthesis and modification of new aldehyde and ketoamide inhibitors. \textbf{Chapter 5 Inhibitory mechanism between seven flavonoids and matrix metalloproteinase 9.} MMP9 is one of important drug targets involved many diseases especially cancer, and there are some hydroxamate inhibitors of MMP9 have been going into clinical trial. It is very important to find novel inhibitors of MMP9. Docking and QSAR of seven flavones (apigenin analogues) were optimized by DFT and five molecular docking (Glide, MVD2006, FlexX, Gold3.0 and Moe2004) methods. Calculated results indicated that number of hydroxyl affects the inhibitory activity of flavones because the more hydroxyl would lead to the higher hydrophilic ability, and the active sites of MMP9 are full of hydrophilic amino acid residues. 3$^\prime$ and 4$^\prime$ hydroxyl substitutions would enhance inhibitory activity because they can chelate the active-site zinc(\uppercase \expandafter {\romannumeral 2}) ion. 3 hydroxyl substitution has a little effects because the hydroxyl locates in the hydrophobic area of MMP9. Our calculated results may not only be identical to experimental results, but also can give theoretical bases for further reseaches on these inhibitors.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/180
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Recommended Citation:
组织蛋白酶B、K和基质金属蛋白酶9抑制剂的对接与构效关系研究.潘蓄林[d].中国科学院昆明植物研究所,2007.20-25
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