【作者】 张小轶；

【导师】 王存新；

【作者基本信息】 北京工业大学 ， 生物医学工程， 2009， 博士

【摘要】 艾滋病是由I型人类免疫缺陷病毒(Human immunodeficiency virus I,HIV-1)感染引发全身免疫系统严重损害,使人体对威胁生命的各种病原体丧失抵抗能力,最后导致死亡的严重疾病。目前,艾滋病仍是不治之症。整合酶(Integrase,IN)介导病毒cDNA与宿主细胞DNA的整合过程,在HIV-1生活周期中起必不可少的作用,且人体内没有IN的功能类似物,这使得IN抑制剂对人体正常细胞的毒性作用较小。近来,IN已成为发展抗艾滋病药物的最具吸引力的靶标,以IN为靶点开发新药及进行抑制剂改造成为研究热点。此外,现有的抗艾滋病药物大都产生了耐药性,这是成功进行高效抗逆转录病毒疗法(Highly active antiretroviral therapy,HAART)及新药设计的重大障碍。进一步从结构与功能的角度阐明耐药机制,特别是弄清最有前景的二酮酸(Diketoacids,DKAs)类IN抑制剂引起的耐药性机理,将有助于更合理地使用和设计针对IN的抑制剂。本论文的工作主要包括三部分:第一部分,是基于DKAs类IN抑制剂的药效团模型构建及针对IN蛋白的药效团模型构建;第二部分,是基于DKAs类IN抑制剂的三维定量构效关系(Quantitative Structure-Activity Relationships,QSAR)研究;第三部分,是多种IN耐药突变体的共有耐药机理研究。一.基于DKAs类IN抑制剂及IN蛋白的药效团模型构建药效团模型的构建可以找出化合物中对活性具有重要作用的药效团元素特征,利用建立好的模型进行数据库搜索,可能发现结构新颖的活性小分子。目前已获得几类DKAs小分子抑制剂的结构,且IN的晶体结构已被解析,本论文从配体及受体两个角度出发,进行DKAs类IN抑制剂的药效团模型构建,目前国际上还未见类似的工作报道。以作用于HIV-1 IN的DKAs类抑制剂构建药效团模型,尝试将抑制剂与药效团叠合后的构象和抑制剂与IN的对接构象进行叠合,得到药效团模型与分子对接构象中IN残基的相对位置,并基于抑制剂的药效团模型特征与周围IN氨基酸残基位置的匹配情况进行药效团特征的修改。所得最优药效团由1个疏水特征、3个氢键特征对和1个氢键供体特征组成。该药效团的命中物质量(Goodness of hit,GH)为0.56,产出率达63.6%,假阳性率为0.41%。结果表明,该药效团模型产出率较高,假阳性率较低,具有较好的置信度。另外,基于5类DKAs与IN的合理结合模式,建立了基于IN受体的药效团模型。分析了药效特征元素与周围蛋白残基环境的吻合程度。将基于配体的药效团与基于受体的药效团相互验证,确定了DKAs与IN相互作用中关键的相互作用残基及关键的药效特征元素。所得药效团模型及关键特征元素可用于数据库搜索,以发现新的具有DKAs药效团特征的活性化合物,也可为先导化合物的改造提供帮助。二.基于DKAs类IN抑制剂的QSAR研究通过研究药物小分子的化学结构和生物活性之间的关系,获得三维定量构效关系模型,所得模型可用来预测药物小分子的作用方式及改造后的化合物活性,并可指导新药设计和先导化合物的优化等。虽然国内外已有一些关于IN抑制剂的结构改造及构效关系研究发表,然而,迄今为止,尚未见到专门针对DKAs类IN抑制剂进行的构效关系研究。DKAs是目前最有前景的IN抑制剂,进行DKAs抑制剂的构效关系研究,会对更好的理解DKAs抑制剂与IN的相互作用以及如何改造DKAs类抑制剂提供帮助。论文将药效团模型、分子对接及三维定量构效关系研究相结合,进行了DKAs类IN抑制剂的构效关系研究。首先,构建了基于DKAs抑制剂的药效团模型。用所得药效团模型进行基于药效团模型的分子叠合,同时,将公共骨架叠合法作为对比,以寻找适合于体系的最佳模型。最佳模型由比较相似因子分析方法(Comparative molecular similarity indices analysis,CoMSIA)所得,即CoMSIA model 2。该模型具有最佳交叉验证回归系数(q2 = 0.665)以及非交叉回归系数(r2 = 0.955),并对测试集分子作出的较好的预测(r2 = 0.559)。为了评价所得最优QSAR模型的合理性,将IN与DKAs抑制剂小分子的对接结果与QSAR模型叠合,考察该模型所建议的取代基团是否与IN蛋白的周围残基环境相吻合。在此基础上对DKAs分子结构提出了合理的改造意见。三.多种IN耐药突变体的共有耐药机理研究现有的抗艾滋病药物大都产生了耐药性,HIV产生耐药突变是成功进行HARRT及新药设计的重大障碍。为了使已有的以及新开发的抑制剂在抗病毒治疗中最大限度的发挥其药效,进一步从结构与功能的角度,对HIV-1在这些抑制剂存在下产生的耐药性、特别是针对IN的耐药性机制进行研究是新的热点。已有关于IN突变体的耐药机理研究多来自对一个IN耐药突变体与野生型IN的比较,但多种耐药突变体IN共有的耐药机理尚未见报道。为了解DKAs引起的多种耐药株共有的耐药机理,论文选择三种针对S-1360耐药的HIV-1突变株,用分子对接和分子动力学模拟阐明IN对DKAs抑制剂耐药的机理。结果表明,在突变体中,由于T66残基突变成I66,阻止了抑制剂S-1360进一步进入结合口袋,因此在突变体中,S-1360与IN的结合位置靠近功能loop 3区却远离与病毒DNA结合的关键残基K156和K159,不能阻止病毒DNA与IN的结合;另外,发现对IN活性起重要作用的loop 3区和helix 1区的二级结构长度比野生型复合物中增长,且在野生型复合物中,抑制剂通过与helix 1区的E152及sheet 1区的D64形成氢键抑制了该区域的柔性,而在突变复合物中,抑制剂不与IN这两个区域的残基形成任何氢键作用,因此不能抑制该区域的柔性,使该区域表现出较高柔性。抑制剂结合位置的变化及loop 3区和helix 1区的构象变化是导致耐药性产生的主要原因。多种耐药突变体共有的耐药机理将对基于突变体复合物进行的3D药效团构建,对提高药物抑制IN耐药突变体的活性以及研发新药具有很大帮助。更多还原

【Abstract】 Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV). This disease results in heavily damage of human immune system, then makes people lost the resist to all kinds of pathogens, and finally lead to death. Nowadays, AIDS remains an irremediable disease. An essential step in HIV replication is the integration of the viral cDNA into host chromosomal DNA by integrase (IN), IN has no sequence homologue in the human host, therefore, it is considered as a pivotal and validated drug target. Recently, new drug development and inhibitor modification targeting IN become the focus of the medical research. Besides, drug resistance against most of the anti-HIV drugs has appeared. The drug resistant mutations in HIV are major impediment to successful highly active antiretroviral therapy (HAART) and new drug design. Understanding the mechanism of HIV drug resistance in terms of structure and function will be useful for the rational inhibitor modification and drug design targeting IN, whereas, the mechanism of drug resistance caused by diketoacids (DKAs), the most potent IN inhibitors is still unknown.A pharmacophore model could be used for database searching and new structural entities revealing. There are several classes of DKAs structures have been reported, simultaneously, the catalytic domain structure of IN with 5-CITEP determined by X-ray crystallography is available. Hence, both ligand based and receptor based pharmacophore models are developed in this study, no similar studies have been reported. Firstly, we developed a three-dimensional pharmacophore model for HIV IN from DKAs inhibitors, inhibitor conformations mapped into the pharmacophore model were superimposed with their docking conformations. Corresponding positions between the pharmacophore model and IN residues were thus obtained. The pharmacophore model was refined according to whether the pharmacophore features were compatible with residues around them. An optimal pharmacophore model was generated and consisted of 1 hydrophobic feature, 3 hydrogen pair features and 1 hydrogen-bond donor feature. This pharmacophore model based on DKAs had high reliability with a goodness of hit (GH) score of 0.56, a high percentage yield of actives (Y) of 63.6.1% and a lower false positive rate (FP) of 0.41%. The pharmacophore model had high reliability. Additionally, a receptor-based pharmacophore model was generated according to the binding mode between IN and 6 classes of DKAs. We attempted to refine the obtained pharmacophore model according to the corresponding residues of IN around pharmacophore features. Finally, the key interactions and the common pharmacophore features were obtained by comparing the pharmacophore models based on ligands and acceptor. The final pharmacophore model can contribute to the discovery and design of new IN inhibitors.A QSAR (Quantitative structure-activity relationship) analysis has been done to gain the relationship between structure and biological activity of drug molecules. The model obtained from QSAR analysis can be used to predict the binding mode and the biological activity of new inhibitors, and the model can also be used to guide the new drug design and lead compound optimization. There are some studies on QSAR analysis of IN inhibitors, whereas, we found no QSAR model of IN DKAs inhibitors was built. DKAs are the most potential IN inhibitors, QSAR studies on DKAs will benefit the understanding of interaction between DKAs and IN, and will benefit the modification of IN inhibitors. In this paper, QSAR analysis combined with pharmacophore model construction and molecular docking have been done against IN DKAs inhibitors. Firstly, a pharmacophore model was built. The structure alignment for all the inhibitors were performed based on the pharmacophore; for a comparison, the common substructure-based alignment was performed in the structure alignment of inhibitors. As a result, a optimal CoMSIA model gains a conventional correlation coefficient r2 of 0.955 and a leave-one-out cross-validated coefficient q2 of 0.665, and the model also can predict the activities of the test DKAs well (r2 = 0.559). In order to validate the rationality of the optimal CoMSIA model, the CoMSIA contours were mapped into the binding mode of IN and DKAs, and the rationality of the substitutions suggested by QSAR model were investigated according to the IN residues around these substitutions. Reasonably modification rules were suggested based on this QSAR study.Drug resistance against most of the anti-HIV drugs has appeared. The drug resistant mutations in HIV are major impediment to successful highly active antiretroviral therapy (HAART) and new drug design. In order to make drugs take full effects, understanding the mechanism of HIV drug resistance in terms of structure and function became a attractive issue. Previous studies on drug resistant IN mutants are all from comparing 1 drug-resistant IN strain with wild-type IN, whether the mechanism mutually existed for multiple drug-resistant strains remains unclear. In order to understand the drug resistance mechanism mutually existed for multiple drug-resistant strains caused by the most potent IN inhibitors of DKAs, 3 S-1360-resistant HIV strains were selected and molecular docking and molecular dynamics simulations were performed to illustrate the mechanism of drug resistance against DKAs inhibitors. The results showed that: after Thr66 was mutated to Ile66 in the three mutants, the long side chain of Ile66 occupied the center of IN active pocket, then prevented the inhibitor S-1360 from moving into depth of the active pocket, thus, the binding site of S-1360 is close to loop 3 region whereas far from the residues K156 and K159 which are crucial for virus DNA binding; Additionally, it was found from secondary structural analyses that the length of loop 3 and helix 1 regions are longer in the three mutant complexes in comparison to the wild type IN complex. Also, there are hydrogen bonds between S-1360 and E152 in helix 1 region, the hydrogen bond depressed the flexibility of the region, whereas, in the three mutant complexes, there is no hydrogen bond between S-1360 and the residues in helix 1 and loop 3 regions of IN, therefore, the regions show higher flexibility in the three mutant complexes. All above results contribute to drug resistance. Altogether, the drug resistance lies in the different binding sites of the inhibitor and the conformation changes of loop 3 and helix 1 regions. The mechanism mutually existed for multiple drug-resistant strains will be useful for improving the inhibition potency of DKA inhibitors against drug-resistant IN, and rational inhibitor modification and design targeting IN.更多还原