棉酚氨基酸衍生物抗I型人类免疫缺陷病毒活性与机制的研究

发布时间：2018-06-11 18:57

本文选题：人类免疫缺陷病毒 + 获得性免疫缺陷综合症　；参考：《武汉大学》2013年博士论文

【摘要】：在与人类免疫缺陷病毒斗争的这三十年中,人类为预防和治疗艾滋病所作的努力主要有三个方面,抗HIV药物和疫苗的研发以及AIDS的基因治疗。尽管人们对HIV-1疫苗的研发投入了很多,但是HIV-1序列的多样性和相对不易接近到Env的保守结构域大大降低了诱发保护性抗体的可能,再加上我们对控制HIV-1复制的免疫反应了解甚少,导致至今还没有诞生一个有效的疫苗。虽然已经有几十例报道的处于临床或临床前试验的基因治疗案例,但是基因疗法的疗效、安全性特别是经济性仍需提高。迄今为止,32种被FDA批准的用于治疗HIV-1感染的药物中,大多数是逆转录酶和蛋白酶抑制剂,尽管它们在改善和延长HIV-1感染者的生命方面取得了不错的效果,但是这些药物的毒性以及服药后耐药毒株的产生极大的限制了它们的使用。因此,发展新型的口服利用率高且价格低廉的小分子抗病毒药物对于艾滋病患者特别是发展中国家的患者是迫切需要的。棉酚是一种从棉籽中提取出来的含醛基的多酚类物质,具有抗生育、抗病毒、抗癌、诱导凋亡和调节免疫等多种生物活性。为了提高它的抗HIV-1活性、降低其细胞毒性和消除男性抗生育活性,我们对棉酚进行了一系列的修饰和改造,以期将它发展成新的抗HIV-1药物。在对棉酚衍生物的筛选中我们发现,氨基酸修饰后的棉酚特别是中性脂肪族氨基酸的棉酚衍生物抗HIV-1的活性最高,我们认为这与该族氨基酸侧链R基的疏水性有关,因为后续的机制研究指出棉酚氨基酸衍生物的作用靶点也是疏水性的。为了进一步的提高其抗病毒活性并降低细胞毒性,我们对棉酚进行了拆分,并屏蔽了赋予它毒性的醛基,结果发现左旋棉酚的氨基酸衍生物比修饰前的治疗指数提高了100多倍。接着,我们还对棉酚和这几种衍生物的杀精活性进行了对比。以前人们普遍认为棉酚和它们的衍生物是通过作用在逆转录酶上来抑制HIV-1复制的,但是我们通过时间递加实验、HIV-1介导的细胞融合实验以及VSV-G假病毒实验分析表明丙氨酸的左旋棉酚衍生物((-)G-Ala)是一种HIV-1的进入抑制剂。深入的机制研究表明(-)G-Ala既不阻断gp120蛋白与CD4受体的结合,又不影响病毒与辅助受体的结合。夹心ELISA实验、N-PAGE实验和CD实验表明(-)G-Ala能有效地阻止gp41核心六聚体的形成。更进一步的实验表明(-)G-Ala不但能结合到HIV-5-Helix蛋白上,还能阻断PIE7结合到gp41内部的三螺旋束表面的疏水口袋中。以上这些结果说明(-)G-Ala可能结合到gp41内部的三螺旋束表面的疏水口袋中,通过阻断gp41融合活性的核心六聚体形成来抑制HIV-1介导的病毒与细胞间的融合进而阻止病毒的侵入。
[Abstract]:In the three decades of fighting HIV, human efforts to prevent and treat AIDS are mainly in three areas: the development of anti-HIV drugs and vaccines, and the genetic therapy of AIDS. Although there has been a lot of investment in the development of HIV-1 vaccine, the diversity of HIV-1 sequences and the relative inaccessibility of the conserved domain of Env have greatly reduced the possibility of inducing protective antibodies. Coupled with our little knowledge of the immune response to control HIV-1 replication, no effective vaccine has yet been produced. Although there have been dozens of reported cases of gene therapy in clinical or preclinical trials, the efficacy, safety and especially economic efficiency of gene therapy need to be improved. To date, most of the 32 drugs approved by the FDA for the treatment of HIV-1 infections are reverse transcriptase and protease inhibitors, although they have achieved good results in improving and prolonging the lives of HIV-1 infected people. But the toxicity of these drugs and the production of drug-resistant strains greatly limit their use. Therefore, the development of new small molecular antiviral drugs with high oral utilization and low cost is urgently needed for AIDS patients, especially in developing countries. Gossypol is an aldehyde-containing polyphenols extracted from cottonseed. It has many biological activities, such as anti-fertility, anti-virus, anti-cancer, inducing apoptosis and regulating immunity. In order to improve its anti-HIV-1 activity, reduce its cytotoxicity and eliminate male antifertility activity, we have modified and modified gossypol to develop into a new anti-HIV-1 drug. The amino acid modified gossypol, especially the gossypol derivative of neutral aliphatic amino acid, had the highest activity against HIV-1, which we think is related to the hydrophobicity of the R group of the amino acid side chain. It is indicated that the targets of gossypol amino acid derivatives are hydrophobic. In order to further improve the antiviral activity and reduce the cytotoxicity of gossypol, we separated gossypol and shielded its toxic aldehydes. The results showed that the amino acid derivatives of L-gossypol were more than 100 times higher than those before modification. Then we compared the spermicidal activity of gossypol and these derivatives, which were previously widely believed to inhibit HIV-1 replication by acting on reverse transcriptase. However, our analysis of HIV-1 mediated cell fusion and VSV-G pseudovirus assay showed that the L-gossypol derivative of alanine was an entry inhibitor of HIV-1. Further studies have shown that G-Ala does not block the binding of gp120 protein to CD4 receptor, nor does it affect the binding of virus to coreceptor. The results of sandwich Elisa, N-PAGE and CD showed that G-Ala could effectively prevent the formation of gp41 core hexamer. Further experiments show that G-Ala can not only bind to HIV-5-Helix protein, but also block the binding of PIE7 to the hydrophobic pocket of the surface of three-helix beam in gp41. These results suggest that G-Ala may bind to the hydrophobic pocket of the triple helix bundle surface of gp41 and inhibit the HIV-1 mediated fusion between the virus and cells by blocking the formation of the core hetamer of the fusion activity of gp41 and thus preventing the invasion of the virus.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.91

【相似文献】