| 题名 | 化合物DB-02和SJP-L-5体外抗HIV-1活性及耐药性机制研究 |
| 作者 | 张兴杰 |
| 学位类别 | 博士 |
| 答辩日期 | 2013-11 |
| 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 | DB-02 S-DABO S-DACO 耐药性 HIV-1 SJP-L-5 木脂素 非核苷类 逆转录酶抑制剂 五味子科 |
| 其他题名 | Anti-HIV-1 activity and the resistant mechanisms of compounds DB-02 and SJP-L-5 |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 高效抗逆转录病毒治疗（Highly Active Antiretroviral Therapy，HAART，也简称“鸡尾酒”疗法）是控制人免疫缺陷病毒（Human Immunodeficiency Virus，HIV）感染的重要手段，其中，非核苷类逆转录酶抑制剂（Nonnucleoside Reverse Transcriptase Inhibitors，NNRTIs）是重要的组分之一。然而，针对NNRTI的耐药株的快速出现，使得HAART的疗效大大降低，甚至可能导致治疗失败。为了应对这一挑战，研发新的具有高耐药屏障的药物显得迫在眉睫。 植物来源的天然产物是一个药用化合物的巨大宝库。在传统中药里，五味子（五味子科植物）常用于治疗慢性咳嗽、呼吸困难、遗精、遗尿、尿频和长期腹泻等病症。木脂素是五味子科植物的中的主要活性成分，其它成分还包括有机酸、糖苷、三萜类化合物等。 我们已经报道了一类新的化合物——硫-二氢芳基/烷基硫-环己甲基嘧啶酮，（dihydro-aryl/alkylsulfanyl-cyclohexylmethyl-oxopyrimidines，S-DACOs），这一类化合物具有较强的抗HIV活性。其中，6-环已甲基-5-乙基-2-（2-氧代-2-苯乙基硫）嘧啶-4（3H）-酮（6-（cyclohexylmethyl）-5-ethyl-2-（（2-oxo-2-phenylethyl）thio）pyrimidin-4（3H）-one，DB-02，中文简称“德宝”）是活性最好的化合物之一。体外抗HIV-1活性研究和耐药性研究表明DB-02对于细胞系细胞和原代PBMC细胞都具有很小的毒性（CC50>1 mM）。它对不同的病毒实验株、不同亚型嗜性的病毒临床株都有很好的抑制活性，EC50的范围在2.40和41.8 nM之间。基因型耐药实验和定点突变实验证实，V106A是该化合物的最主要的耐药突变位点。分子对接实验表明DB-02定位于逆转录酶的疏水口袋，与Lys101、Val106、Leu234、His235有较强的相互作用。DB-02与4类已批准上市的抗HIV药物联合使用时，不会表现出拮抗效应。所有的研究结果均提示DB-02是一个潜在的NNRTI，其毒性更低、活性更好。我们的研究结果还为进一步优化S-DACO类化合物提供了帮助，这将有利于研发耐药屏障更高、毒性更低的新的化合物。 我们前期的工作证实从红花五味子和小花五味子中分离到的木脂素具有抗HIV活性。在优化木脂素结构的过程中，我们研发了一系列新的具有抑制HIV复制能力的化合物。其中，SJP-L-5表现出显著的抗病毒能力。在本研究中，我们发现SJP-L-5可以有效抑制HIV-1实验株和临床株的复制，EC50的范围在0.4~16.2 μM。同时，SJP-L-5对于细胞系细胞和原代PBMC的毒性非常小，CC50均大于608 μM。SJP-L-5对NNRTI耐药株HIV-1A17敏感性降低，产生耐药现象，提示它可能是一个RT抑制剂；然而，它却不能抑制体外RT的RNA依赖的DNA聚合酶活性。这一矛盾的结果暗示，SJP-L-5可能通过一种特殊的机制抑制逆转录酶的活性。随后，我们发现SJP-L-5可以抑制体外RT的DNA依赖的DNA聚合酶活性，但不能抑制其RNaseH的活性。总之，SJP-L-5是一个新的较为特殊的逆转录酶抑制剂，它的抗病毒机制仍需要继续深入地探索。 |
| 英文摘要 | Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are the major components of highly active antiretroviral therapy (HAART) used to control human immunodeficiency virus (HIV) infections. However, the rapid emergence of resistant viruses of NNRTIs has decreased the efficiency of HAART and lead to failure of the therapy. To overcome this difficulty, it is necessary to develop new compounds with a high genetic barrier. Natural products from herbal plants are a great reservoir of medicinal compounds. In traditional Chinese medicine, Wuweizi (Schisandraceae plant) is commonly used to cure chronic cough and dyspnea, spermatorrhea, enuresis, frequent urination, protracted diarrhea and so on. Lignans are the major and characteristic constituents of Schisandraceae plants, also included are organic acids, glycosides and triterpenes. We have reported a new class of molecules, dihydro-aryl/alkylsulfanyl-cyclohexylmethyl-oxopyrimidines (S-DACOs), as novel anti-HIV-1 compounds. 6-(cyclohexylmethyl)-5-ethyl-2-((2-oxo-2-phenylethyl)thio) pyrimidin-4(3H)-one (DB-02) was one of the best compounds with high antiviral activity. In vitro anti-HIV-1 activity and resistance profile studies have suggested that DB-02 has very low cytotoxicity (CC50>1mM) to cell lines and peripheral blood mononuclear cells (PBMCs). It displays potent anti-HIV-1 activity against laboratory adapted strains and primary isolated strains including different subtypes and tropism strains (EC50s range from 2.40 to 41.8 nM). Studies on genotypic resistance profiles and site-directed mutagenesis revealed that V106A was the major mutation of RT for the compound. Molecular docking analysis showed that DB-02 located in the hydrophobic pocket with interactions of Lys101, Val106, Leu234, His235. DB-02 also showed non-antagonistic effects to four approved antiretroviral drugs. All studies indicated that DB-02 would be a potential NNRTI with low cytotoxicity and improved activity. Our results here could also give a clue to optimize S-DACOs compounds to obtain higher genetic barrier and lower cytotoxicity. Our previous study has demonstrated that lignans from Schisandra rebriflora and Schisandra micrantha show anti-HIV activities. During optimization of lignans, we developed a series of novel compounds that inhibit HIV replication in vitro. Among these compounds, SJP-L-5 showed obvious antiviral activity than others. In this study, we found that SJP-L-5 blocked HIV-1 lab strains and clinical isolates replication with EC50 varying from 0.4~16.2 μM, while it also exhibited low cytotoxic to cell lines and primary PBMCs with CC50 higher than 608 μM. SJP-L-5 showed low sensitivity to HIV-1A17, which is an NNRTI resistant strain, indicating that it might be an RT inhibitor. However, it did not inhibit RT RNA dependent DNA polymerase activity in vitro. This contradiction suggests that SJP-L-5 may have a special mechanism of action against RT. We next found that SJP-L-5 could inhibit RT DNA dependent DNA polymerase activity, but not its RNaseH activity. In conclusion, SJP-L-5 is a novel and special RT inhibitor based on lignan structure and its antiviral mechanism needs to be further investigated. |
| 语种 | 中文 |
| 公开日期 | 2014-02-11 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7798]  |
| 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 | 张兴杰. 化合物DB-02和SJP-L-5体外抗HIV-1活性及耐药性机制研究[D]. 北京. 中国科学院研究生院. 2013. |
| 个性服务 |
| 查看访问统计 |
| 相关权益政策 |
| 暂无数据 |
| 收藏/分享 |
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。
修改评论