| 题名 | 二种抗HIV-1 药物筛选模型的建立及β-咔啉类化合物抗HIV 活性研究 |
| 作者 | 王云华 |
| 学位类别 | 博士 |
| 答辩日期 | 2007-06 |
| 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 | HIV 蛋白酶 核衣壳蛋白 抑制剂 筛选 β-咔啉 flazinamide dehydroxymethylflazinamide 点柄乳牛肝菌 |
| 其他题名 | Establishment of two methods for anti-HIV drugs screening and anti-HIV effects of β-carboline compounds |
| 学位专业 | 动物学 |
| 中文摘要 | 艾滋病的流行严重威胁着人类健康和全球经济的发展，在某些国家已经造成严重的经济问题和社会问题。由于尚无安全有效的艾滋病疫苗问世，药物治疗仍是目前防治艾滋病的主要途径。二十多种抗HIV药物的临床使用以及HAART疗法的应用，艾滋病患者的死亡率呈一定的下降趋势。然而，目前使用的抗HIV药物能够抑制患者体内病毒的复制，但不能完全清除病毒。耐药病毒株的出现和流行更降低了药物治疗的成功率。为此需要不断研究开发作用于新靶点或具有不同作用机制的药物。 药物筛选是药物开发中的重要环节，其关键在于建立适合的药物筛选方法。传统药物筛选费时费力，近年来逐渐被高通量药物筛选方法所代替。本研究中，我们在大肠杆菌中表达并纯化了HIV-1蛋白酶，获得了纯度和活性较高的蛋白酶。利用荧光标记的蛋白酶底物在体外检测化合物对蛋白酶活性的影响，建立了适于高通量筛选蛋白酶抑制剂的体外筛选方法。通过对大量样本的筛选，发现一些具有蛋白酶抑制活性的化合物和粗提物。本研究还表达纯化了HIV-1核衣壳蛋白NCp7。利用锌离子特异性的荧光染料，建立了相应的高通量筛选方法。该方法能够筛选出通过逐出NCp7结合的锌离子而抑制该蛋白功能的化合物。这两种体外筛选方法的建立大大提高了药物筛选的效率，降低了工作强度，为进一步的研究打下了基础。 在药物开发过程中，常常通过对已知有效的化合物进行结构修饰以提高药物活性并降低细胞毒性，改善药物疗效。在先前的药物筛选中，我们发现从高等真菌中提取的β-咔啉类化合物flazin, 具有一定的抗HIV活性。通过对flazin的结构修饰，我们发现了治疗指数更高的化合物。本研究对其中两个，dehydroxymethylflazinamide和flazinamide，进行了更深入的抗HIV活性研究。dehydroxymethylflazinamide和flazinamide抑制HIV-1IIIB感染诱导的合胞体形成的EC50分别为0.31 μM 和 0.38 μM。 与flazin(EC50为2.37 μM）相比较，抗HIV活性提高了约6-7倍。这两个化合物对C8166细胞的半致死浓度(CC50)分别为27.34μM和118.64μM。Dehydroxymethylflazinamide的细胞毒性与flazin (CC50为28.71μM）相似，而flazinamide的细胞毒性降低了约4倍左右。与flazin相比，通过结构修饰，Dehydroxymethylflazinamide治疗指数从12.1提高到88.19，而flazinamide治疗指数从12.1提高到312.2。研究还发现，这两个化合物对临床分离株HIV-1KM018以及实验株HIV-2ROD、HIV-2CBL-20也有良好的抑制效果。 我们对dehydroxymethylflazinamide和flazinamide的作用机制也进行了初步探讨。二者均能有效抑制HIV-1IIIB, HIV-2ROD and HIV-2CBL-20病毒的细胞间传播，而不能抑制HIV-1IIB慢性感染的H9细胞中病毒复制，说明该化合物可能主要作用于病毒生活周期的早期阶段；进一步的研究表明，dehydroxymethylflazinamide对HIV-1IIIB进入阶段有很强的抑制活性，说明进入阶段是该化合物的主要作用靶点；而flazinamide对直接杀病毒、病毒吸附及进入均没有显著的抑制效果，该化合物的作用机制还需进一步研究。对酶靶点作用研究表明，两个化合物对HIV-1逆转录酶仅有微弱抑制活性说明该酶可能不是作用靶点；dehydroxymethylflazinamide对HIV-1蛋白酶有抑制活性但半效浓度（EC50）较高,说明该酶可能是化合物的次要靶点。而flazinamide在体外与重组的整合酶有较强的结合活性，暗示该化合物可能对整合酶有一定的抑制作用。以上结果还说明，虽然两个化合物具有类似的结构，但它们可能是通过完全不同的机制来抑制HIV的复制。 |
| 英文摘要 | The replication of HIV-1 in infected patients can be reduced considerably by treatment with combinations of drugs with multiple viral drugs. But none of currently available drug or combinations could eradicate HIV-1 from patients completely. The long-term clinical effectiveness of approved anti-HIV drugs has been hampered by the ascendance of drug-resistant mutants in response to antiretroviral therapies. The rates of success of HAART are predicated to decrease gradually with the increase in the emergence of drug resistant strains. Therefore, it is essential to develop drugs targeting alternative steps of the viral replication cycle. For many decades, the discovery of drugs has depended on screening. Traditional screening was labor intensive and expensive. More and more high-throughput methods were developed and enabled to screening large pool of compounds. In this research, we expressed and purified HIV-1 protease. A cell-free screening method based on FRET was established. Another HIV-1 viral protein, NCp7, was also expressed and purified in this research. A method to test zinc ejection activity of compounds by TSQ zinc ion indicator was developed. Both methods are amenable to high-throughput screening. A large pool of samples, either natural products or synthesized compounds, were examined and some of them are found to inhibit protease or NCp7 in vitro. Structural modification to improve activity of an identified lead molecular is a cost effective way for drug discovery. A β-carboline compound, flazin isolated from Svillus granvlatvs has been shown weak anti-HIV-1 activity. Based on the structure of flazin, dehydroxymethylflazinamide (1-(2’-furyl)-β-carboline-3- formamide) and flazinamide [1-(5’-Hydromethyl-2’-furyl)-β-carboline-3- carboxamide] was synthesized and their anti-HIV activities were evaluated in the present study. The cytotoxicity of dehydroxymethyl- flazinamide was similar with that of flazin and cytotoxicity of flazinamide was about 4.1-fold lower. Dehydroxymethylflazinamide and flazinamide potently reduced syncytium formation induced by HIV-1IIIB with EC50 values of 0.31 μM and 0.38 μM, about 7-fold lower than that of flazin. Dehydroxymethylflazinamide and flazinamide also inhibited HIV-2ROD and HIV-2CBL-20 infection. Dehydroxymethylflazinamide and flazinamide reduced p24 antigen expression in HIV-1IIIB acute infected C8166 and in clinical isolated strain HIV-1KM018 infected PBMC. Neither suppressed HIV-1 replication in chronically infected H9 cells. Both of them blocked the fusion between normal cells and HIV-1 or HIV-2 chronically infected cells. They did not show virucide or attachment inhibitory activities. Dehydroxymethylflazinamide inhibited HIV-1 entry with EC50 value of 1.08 μM. Dehydroxymethylflazinamide and flazinamide weakly inhibited activities of recombinant HIV-1 reverse transcriptase at higher concentrations. Dehydroxymethylflazinamide reduced protease activity and flazinamide bound to intergrase in vitro. The structural modification of flazin markedly enhanced the antiviral activity. TI value of dehydroxymethyl- flazinamide increased from 12.1 to 88.19 and flazinamide to 312.2. In despite of structural similarity, they may possess distinct action mechanisms. Dehydroxymethylflazinamide might target entry step and flazinamide might interfere other steps in the early stage of HIV life cycle. |
| 语种 | 中文 |
| 公开日期 | 2010-10-14 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6109]  |
| 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 | 王云华. 二种抗HIV-1 药物筛选模型的建立及β-咔啉类化合物抗HIV 活性研究[D]. 北京. 中国科学院研究生院. 2007. |
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