低聚凤梨参岩藻糖化硫酸软骨素FuCS-1 和N-取代苯磺酰基-3-乙酰基吲哚的体外抗HIV 活性及机制研究
文献类型：学位论文
| 作者 | 黄宁 |
| 学位类别 | 博士 |
| 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 | HIV 抗HIV活性 硫酸软骨素 进入抑制剂 构效关系 |
| 其他题名 | The Anti-HIV Effects and Mechanism of Depolymerized Fucosylated Chondroitin Sulfate derivative from Sea Cucumber Thelenata anana and N-arylsulfonyl-3-acetylindoles |
| 学位专业 | 细胞生物学 |
| 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。30多种抗HIV药物的临床使用以及HAART疗法的应用，使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，并且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新作用机制的药物。 硫酸软骨素是一类硫酸化的糖胺聚糖(glycosaminoglycan)，它存在于诸多动物组织内，在许多海洋生物中含量丰富，有研究显示其具有抗凝血、抗肿瘤、抗病毒等多种生物活性，其中包括对HIV的抑制作用，在实验中我们首先从海洋生物凤梨参中分离得到的岩藻糖化硫酸软骨素Thenenatan，然后对其进行了结构修饰得到了岩藻糖化硫酸软骨素FuCS-1。我们采用MTT方法测定了多糖硫酸软骨素FuCS-1对人T淋巴细胞系细胞C8166、H9、Hela、Jurkat、K562、MT-4；原代培养细胞正常人PBMC以及慢性感染的H9/HIV-1ⅢB细胞的体外细胞毒性，实验结果显示在最高浓度25mg/mL时都未观察到其细胞毒性。我们应用3种病毒株（实验株，耐药株，临床分离株）对其进行了抗HIV活性的系统评价。结果发现：FuCS-1对急性感染的C8166细胞中的实验株HIV-1IIIB和HIV-1RF病毒复制有极强的抑制作用，半数有效剂量浓度EC50分别为0.73μg/mL和1.14μg/mL，SI值分别>32426.57和 >21929.82； FuCS-1对临床病毒株HIV-1KM018 和 HIV-1TC-2也有很好的抑制作用，其EC50值分别为23.75和31.86μg/mL，选择指数SI分别为>1052.63和>784.68。该化合物对多种耐药病毒株都有较好的抑制作用，对非核苷类逆转录酶耐药株HIV-1A17, 蛋白酶耐药株HIV-1RF/V82F/184V 的EC50值分别为1.09、0.95μg/ml；对T-20敏感株(pNL4-3gp41(36G)N42S)和T-20耐药株 (pNL4-3gp41(36G)V38E,N42S, pNL4-3gp41(36G)V38A,N42D, pNL4-3gp41(36G)V38A,N42T)在C8166细胞中的复制也有同样的抑制效果，EC50值分别为0.79、0.94，0.76、1.13μg/mL，而对照药物T-20却只对敏感株有一定的抑制作用EC50为226.16μg/mL，对其耐药株在最高浓度1000ng/mL时都未显示出有效的抑制作用；FuCS-1对HIV-2也有较好的抑制作用，对HIV-2CBL-20和HIV-2R0D EC50分别为71.76μg/mL和97.63μg/mL。这些实验结果显示硫酸软骨素FuCS-1在体外有较好的抗HIV活性，不仅能够抑制实验室适应病毒株，而且能够抑制多种耐药株，临床分离病毒株和HIV-2在细胞内的复制，因此我们对其进行了抗HIV的作用机制研究，结果显示FuCS-1对HIV-1蛋白酶没有抑制活性，但对逆转录酶有较弱的抑制作用。我们用分时给药，融合阻断，进入抑制等实验方法证实了岩藻糖化硫酸软骨素FuCS-1主要作用于病毒复制的早期阶段，进一步的实验结果显示FuCS-1有可能是通过与gp120蛋白结合而干扰了gp120的功能，从而使病毒不能进入靶细胞。 吲哚（indole）是具有许多生物活性的天然物质。吲哚类化合物具有抗炎、抗肿瘤、抗菌、抗病毒、抗HIV等多种生理活性。以吲哚环为基本结构通过对其进行结构修饰得到的许多吲哚类化合物是开发活性新物质的重要方向之一。我们对21个吲哚类化合物进行抗HIV活性筛选，得到了几个活性较好的化合物，尤其是化合物4n N-对乙基硝基苯磺酰基-3-乙酰基-6-甲基吲哚，EC50为0.38μg/mL，SI值为>526.31。因此我们对其进行深入的抗HIV活性研究。我们首先检测了化合物4n N-对乙基硝基苯磺酰基-3-乙酰基-6-甲基吲哚对不同的细胞系和人外周血单个核细胞（PBMC）毒性作用，结果显示化合物的细胞毒性较低。在细胞水平上，通过观察致感染细胞病变和HIV-1 p24抗原表达抑制实验（ELISA方法），采用3类多株HIV病毒株（实验株、临床分离株、耐药株）和3类多种细胞（人T淋巴细胞传代株、HIV-1慢性感染人T淋巴细胞株、人外周血单个核细胞）对化合物进行体外抗HIV活性进行系统评价，实验结果表明，化合物对不同来源的HIV-1病毒株都显示出很好抗HIV活性。同时，我们也研究了该化合物抗HIV-2活性，发现该化合物也能抑制HIV-2在C8166细胞中复制。在作用机制和靶点研究上，我们检测了化合物不同时间给药对HIV的抑制作用，对感染与未感染细胞之间融合的抑制及对HIV-1慢性感染H9细胞（H9/HIV-1IIIB）中病毒复制的阻断作用。结果显示，N-对乙基硝基苯磺酰基-3-乙酰基-6-甲基吲哚在0-12小时给药后都可以抑制HIV的复制但它对感染与未感染细胞的融合没有抑制作用（EC50为 >200μg/mL），而对慢性感染H9细胞中病毒的复制有一定的抑制作用（EC50为24.26μg/mL）。提示化合物有可能作用于HIV DNA整合后的HIV复制的后期。其后对HIV-1逆转录酶活性的抑制，HIV整合酶入核抑制以及HIV蛋白酶活性抑制情况进行了分析，发现N-对乙基硝基苯磺酰基-3-乙酰基-6-甲基吲哚对HIV-1逆转录酶（RT）有一定的抑制作用，对整合酶入核没有抑制作用，而对蛋白酶有较好的抑制作用。 我们对21个N-苯磺酰基-3-乙酰基吲哚类化合物进行了初步的构效关系研究，发现N-苯磺酰基-3-乙酰基吲哚类化合物的吲哚环上引入吸电子基团（如硝基、氰基）后化合物的抗 HIV-1 活性明显低于吲哚环上是给电子基团的化合物如N-苯磺酰基-3-乙酰基-6-甲基吲哚类化合物（4h、4r 与4j 比较，4g、4q 与 4k 比较，4i、4p 与 4m 比较）。这些构效关系的研究将为将来抗HIV吲哚类化合物的设计指引新的方向。 |
| 英文摘要 | The replication of HIV-1 in infected patients can be reduced considerably by treatment with combinations of drugs with multiple anti-viral drugs. But none of currently these 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. Chondroitin sulfate is a sulfated glycosaminoglycan (glycosaminoglycans, GAG), which exists in many animal tissues and is abundant in many marine organisms, a number of studies show that the compound can anti-clotting, anti-tumor, anti-virusa, including the inhibition of HIV. FuCS-1 was synthesized based on the structure of thenenatan, a compound purified from Thelenata ananas. The cytotoxicities of FuCS-1 on C8166、H9、Hela、Jurkat、K562、MT-4、PBMC and H9/HIV-1ⅢB were assessed by MTT and the results manifested that FuCS-1 don’t show cytotoxicity to cultured cells even 25mg/mL. Anti-HIV activities of FuCS-1 were evaluated in the present study. The activities of FuCS-1 against 3 groups of human immunodeficiency virus (HIV) stains were determined in vitro. These were laboratory-derived virus (HIV-1IIIB and HIV-1RF), drug-resistant virus NNRTIs-resisant strain (HIV-1A17), PIs-resisant strain (HIV-1RF/V82F/I84V), T-20 sensitive strain (pNL4-3gp41(36G)N42S) and T-20 resistant strains pNL4-3gp41(36G)V38E,N42S, pNL4-3gp41(36G)V38A,N42D, pNL4-3gp41(36G)V38A,N42T) , pNL4-3gp41(V38E/N42S) and low-passage clinical isolated virus (HIV-1KM018 and HIV-1TC-2). In p24 antigen inhibition test, the EC50s of FuCS-1 against HIV-1IIIB and HIV-1RF infection in C8166 cells were 0.73 and 1.14μg/mL, respectively, and with selective index of >32426.57and >21929.82. FuCS-1 inhibit HIV-1KM018 and HIV-1TC-2 infection in PBMC with EC50 23.75 and 31.86μg/mL , SI >1052.63 and 784.68 respectively; FuCS-1 can against many kinds of HIV drug-resistant virus including NNRTIs-resisant strain (HIV-1A17), PIs-resisant strain (HIV-1RF/V82F/I84V) with EC50s1.09, 0.95μg/ml, respectively, T-20 sensitive strain (pNL4-3gp41(36G)N42S) and T-20 resistant strains pNL4-3gp41(36G)V38E,N42S, pNL4-3gp41(36G)V38A,N42D, pNL4-3gp41(36G)V38A,N42T) with EC50s 0.79, 0.94, 0.76, 1.13μg/mL, respectively, while the control drug T-20 only can inhibt the sensitive strain replication with EC50 226.16μg/mL but can’t inhibt the resisant strains at the highest concentration 1000ng/mL. FuCS-1 also can against HIV-2 CBL-20 and HIV-2R0D with EC50s 71.76μg/mL and 97.63μg/mL, respectively. All of these results indicatied that FuCS-1 has good anti-HIV activities in vitro. It not only can against laboratory-derived virus, but also can inhibit drug-resistant virus, clinical isolated virus and HIV-2. So we examine FuCS-1’s the mechanism of anti-HIV. The results show can’t inhibit HIV-1 protentase but can inhibit activities of recombinant HIV-1 reverse transcriptase weakly. FuCS-1 effectively inhibited HIV-1 replication at the early stages, blocked the fusion between normal cells and HIV-1 chronically infected cells and inhibit HIV virus enter thethe target cells. Further studies indicatied that FuCS-1 can bind to the recombinant HIV-1 gp120 protein potently. The interaction between FuCS-1 and gp120 interfered gp120 functions，so it can effectively inhibit HIV enter the the target cells. Indole is a natural substance with a number of biologically activities. Indole compounds can anti-inflammatory, antitumor, antibacterial, antiviral, anti-HIV and so on. So one of important direction of development new active substances is modificating the indoles. Twelone N-arylsulfonyl-3-acetylindoles (4a-4u) anti-HIV-1 activites were evaluated in the present study. The verified that there are several compounds inhibited HIV-1IIIB induced cytopathic effects (syncytia assay) in C8166 cell. Especially N-p-ethyl-benzenesulfonyl-3-acetyl-6-methylindole had the highest anti-HIV-1 activity with EC50 values of syncytium formation inhibition is 0.38 μg/mL and SI values is >543.78. The N-p-ethyl-benzenesulfonyl-3-acetyl-6-methylindole with the best activity deserved to be further study. The potential cell toxicity of N-p-ethyl-benzenesulfonyl-3-acetyl-6-methylindole to human peripheral blood mononuclear cells (PBMCs) and other cell lines was evaluated. The results showed that it was not significantly cytotoxic to these cells. In the cellular level, by observing cytopathic effects assay and ELISA method for HIV p24 antigen assay, compound was tested a panel of different HIV-1 strains, including laboratory-derived viruses (HIV-1IIIB and HIV-1RF), clinical isolated viruses (HIV-1KM018, HIV-1TC-1, HIV-1TC-2 and HIV-1wan) and drug-resistant viruses (HIV-1A17, HIV-1 RF/V82F/I84Vand pNL4-3gp41(36G)V38E,N42S), the compound also showed good anti-HIV-1 effect on these different HIV-1 strains, which exhibited potent and broad-spectrum anti-HIV-1 activities, as well as on the HIV-2 CBL-20. The mechanism of action in inhibiting HIV-1 by N-p-ethyl-benzenesulfonyl-3-acetyl-6-methylindolewas tested through a time-of-addition experiment, chronically infected H9 and uninfected C8166 cell fusion inhibition assay. The compound can inhibit HIV in 0-12 hour after compound interfering but did not interfere with virus fusion to target cells. This compound exerted its active mechanism through blocking the production of virus from chronically infected cells containing integrated proviral DNA.These results suggest its key mechinsm of N-p-ethyl-benzenesulfonyl-3-acetyl-6-methylindole is inhibition HIV-1 replication at the early stages. Furthermore, Mechanistic studies revealed that it can inhibit HIV-1 reverse transcriptase and HIV protease, but N-p-ethyl-benzenesulfonyl-3-acetyl-6-methylindole can’t inhibit HIV-1 integrase nuclear translocation. The structure-activity relationship (SAR) of these 21 compounds is also surveyed. It was found that antivities of the compounds with the electron- drawinggroups substituted on indole ring are not better than the compounds with electron-withdrawing groups, such as N-benzenesulfonyl-3-acetyl-6-methyl indoles (e.g., 4h and 4r vs 4j, 4g and 4q vs 4k, 4i and 4p vs 4j). The SAR research will pave the way for the design of indole derivatives as anti-HIV-1 drugs in the future. |
| 语种 | 中文 |
| 公开日期 | 2012-09-25 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7121]  |
| 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 | 黄宁. 低聚凤梨参岩藻糖化硫酸软骨素FuCS-1 和N-取代苯磺酰基-3-乙酰基吲哚的体外抗HIV 活性及机制研究[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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