抗HIV-1 整合酶入核抑制剂筛选方法的建立及药物筛选
文献类型：学位论文
| 作者 | 刘亚娟 |
| 学位类别 | 硕士 |
| 答辩日期 | 2012-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 郑永唐 |
| 关键词 | 人类免疫缺陷病毒1 型 整合酶 晶状体上皮源性生长因子 核输入 |
| 其他题名 | Establishment of methods for screening nuclear import Inhibitors of HIV-1 integrase (IN) and drug candidates screening |
| 学位专业 | 生物化学与分子生物学专业 |
| 中文摘要 | 人类免疫缺陷病毒1型（HIV-1）整合酶（Integrase，IN）在病毒复制的多个步骤中均发挥着重要的作用。在细胞质，整合酶进行cDNA的3′-加工，然后携带cDNA以整合前复合物(PIC)的形式进入细胞核进行整合作用。整合酶进入细胞核是发生整合作用的前提条件，这是一个复杂的过程，有许多病毒蛋白和宿主细胞蛋白参与。特异性抑制非分裂细胞整合酶的核输入无疑将抑制HIV-1的感染，以此为靶点开发新型抗HIV-1整合酶药物具有很大前景。 本论文中，将野生型HIV-1整合酶片段克隆到表达载体pEGFP-C1，构建pEGFP-C1-IN质粒。pEGFP-C1-IN和pEGFP-C1分别转染293T细胞，24小时后用莱卡DMI4000B荧光显微镜观察，EGFP-IN融合蛋白绿色荧光主要积累在细胞核；而转染pEGFP-C1的细胞，EGFP绿色荧光均匀地分布于整个细胞。据报道苯甲酸衍生物D77对IN-LEDGF/p75的相互作用具有很强的抑制作用，影响IN的细胞核分布导致病毒不能正常复制。转染5h后加入D77，24小时后倒置荧光显微镜下观察，发现绿色荧光蛋白主要集中在细胞质，细胞核几乎无荧光。本文以D77为阳性对照药，运用建立的方法进行抗HIV-1整合酶入核抑制剂的筛选。 首先我们对化合物进行了体外抗HIV-1活性及对C8166细胞毒性的检测，然后挑选活性较好的化合物进行抗HIV-1 IN入核抑制剂的筛选。筛选结果显示某些化合物与阳性对照D77作用类似，绿色荧光蛋白分散的分布于细胞质，细胞核几乎观察不到荧光。为了进一步鉴定这些化合物的作用靶点，在HIV-1感染细胞后的不同时间点加药即分时加药，则药物的抑制作用将持续到靶点。分时加药结果显示，这些化合物在病毒感染7小时前均是抑制作用，7小时后抑制作用减弱。HIV-1感染细胞后一般4~6小时完成逆转录，然后cDNA与IN以PIC的形式向细胞核运输。以此来看，这些化合物可能作用于细胞核运输。分子对接分析发现化合物与IN-LEDGF/p75复合物的疏水区域相互作用。据报道 整合酶A链氨基酸残基Q168,E170,T174和B链氨基酸残基T125,W131与LEDGF/p75 I365，D366，F406，V408残基发生相互作用。本文分子对接结果显示化合物与整合酶A链T174形成氢键，另外与A链Q168,E170,T174和B链W131形成疏水键。显然，化合物破坏了IN-LEDGF/p75的作用，从而抑制了整合酶的入核。 通过以上实验，我们推测这些化合物可能与LEDGF/p75竞争性结合于整合酶，破坏了整合酶的正常入核作用。了解到化合物的可能机制，我们又进一步检测了其抗病毒活性。化合物对NVP耐药株HIV-1A17、AZT耐药株HIV-1AO18、ddI,ddC耐药株HIV-174V及HIV-2ROD、HIV-2CBL-20均有一定的抑制作用。 |
| 英文摘要 | HIV-1 integrase (IN) plays a key role in several steps of HIV-1 replication. IN carry out 3’-processing in cytoplasm and then translocate to the nucleus for integration in the formation of pre-integration complex (PIC) with the viral DNA. Translocation of viral IN into nucleus is a critical precondition of integration during the life cycle of HIV-1. This step is complicated and involve in a number of virion-derived and cellular proteins. Specifically inhibiting IN nuclear import would undoubtedly block HIV-1 infection in non-dividing cells, so this critical step of HIV-1 replication is of great prospect as a drug target. In this study, wild-type HIV-1IN was cloned into pEGFP-C1 expression vector to form pEGFP-C1-IN plasmid. pEGFP-C-IN and pEGFP-C1 plasmids were transfected into 293T cells respectively and the fusion proteins EGFP-IN mainly accumulated in the nucleus but EGFP was evenly full of the whole cells by Leica DMI4000B microscopy twenty four hours after transfection. It has reported that D77, one benzoic acid derivative, showed strong inhibition activity against IN-LEDGF/p75 interaction and affected the HIV-1 IN nuclear distribution resulting in inefficient replication. D77 was added at 5h post-transfection and cells were observed 24h later and EGFP-IN appeared diffusely distributed in cytoplasm and no fluorescence could be observed in nucleus. In this work, we screened for inhibitors of HIV-1 IN nuclear import using this method and D77 as positive control. Firstly, the anti-HIV activities and cytotoxicity of compounds were evaluated simultaneously in vitro. For the better candidates, the activities against IN nuclear import was detected and the results showed that some of them like D77 made the EGFP-IN diffusely distribution in cytoplasm and no fluorescence in nucleus. To further investigate the target of action of these compounds on IN translocation , a time-of-drug addition assay was carried out to target identification of potent antiviral compounds. Viral replication was inhibited up to the time point corresponding to the stage of HIV-1 replication targeted by compounds. The results of a time-of-drug addition assay showed that the compounds inhibited the syncytia formation before seven hours. Following reverse transcription completion after 4~6 h of HIV-1 infection, IN and cDNA will transport to the nucleus in the formation of PIC. According this, these compounds maybe target nuclear import of HIV-1 IN. Then a study of molecular docking suggested that these compounds bound to the hydrophobic pocket of IN catalytic core domain (CCD) in complex to the IN-binding domain (IBD) of LEDGF/p75. It has reported that residues Q168, E170, and T174 in chain A of IN, T125 and W131 in chain B of IN as well as I365, D366, F406 and V408 in LEDGF/p75 were responsible for their binding. Our docking studies found that these compounds could formed H-bond with T174 and hydrophobic contacts with Q168 in chain A, E170, T174 in chain A and W131 in chain B, respectively. Obviously, these compounds affected the interaction IN and LEDGF/p75 and resulted in the inhibition of IN nuclear translocation. According to above results, we speculate that these compounds maybe compete binding IN with LEDGF/p75 and inhibit the nuclear import of the HIV-1 IN. Further studies on anti-HIV activities exhibited the inhibition of drug-resistant viral strains (HIV-1A17, HIV-174V and HIV-1AO18) and HIV-2 (HIV-2ROD and HIV-2CBL-20). |
| 公开日期 | 2012-08-01 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6997]  |
| 专题 | 昆明动物研究所_分子免疫药理学 |
| 推荐引用方式 GB/T 7714 | 刘亚娟. 抗HIV-1 整合酶入核抑制剂筛选方法的建立及药物筛选[D]. 北京. 中国科学院研究生院. 2012. |
入库方式： OAI收割
来源：昆明动物研究所
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