【作者】 夏承来；

【导师】 姜世勃； 刘叔文；

【作者基本信息】 南方医科大学 ， 抗病毒药物药理学， 2010， 博士

【摘要】 研究背景艾滋病(AIDS)是由人免疫缺陷病毒(HIV)感染引起的,目前在全球已呈爆发性流行趋势。据2009年UNAIDS/WHO的报告,全球共有3340万HIV感染者,其中270万为新发感染,而每年有200万人死于AIDS相关疾病。我国现有艾滋病病毒感染者约70万人,其中艾滋病病人8万例。在云南、河南、新疆和我们广东等省份,HIV感染已出现高流行趋势。根据世界卫生组织统计,目前我国艾滋病病毒感染者占总人口的比例虽然很低,但感染人数在亚洲位居第二位,在全球居第十四位。如果防治措施不力,将对我国的人口健康和经济社会发展产生巨大的影响。艾滋病防治最好的手段应是疫苗。但二十多年来,还没有一个HIV疫苗的临床方案被证明为有效。在今后相当长的一段时期内,有效的HIV疫苗恐难有重大突破。因此,发展安全有效的抗艾滋病药物,依然是当前艾滋病预防和治疗的重点。迄今为止,已有三大类28种抗HIV药物被美国FDA批准用于临床,分别是逆转录酶抑制剂,蛋白酶抑制剂和HIV进入抑制剂。前两类药物易诱导HIV耐药性突变,而且对人体有较大的毒副作用,导致越来越多的艾滋病患者无法持续地接受抗HIV药物的治疗。而HIV进入抑制剂,尽管目前仅有T-20和Maraviroc两种,但它们对耐受前两类抗HIV药物的病毒依然有效,并可组成更多的“鸡尾酒”方案,用于艾滋病的治疗。因此,开发新的阻止病毒进入的第三类抗HIV药物已经成为目前抗艾滋病药物研究的热点。高通量药物筛选是当前新药发现的手段之一。自从20世纪90年代Jiang等发现第一个来自HIV-1gp41C-末端重复序列的HIV-1进入抑制剂以来,本实验室曾建立了一系列筛选HIV-1进入抑制剂的方法,并且采用这些方法发现了一系列靶向HIV-1gp41的融合抑制剂和靶向HIV-1gp120的粘附抑制剂。但是,由于HIV-1病毒具有传染性强、致病性高等特点,对该病毒只限于在生物安全级别三级(P3)实验室内进行,不便于广泛深入的开展研究,所以建立适用于普通实验室筛选HIV-1进入抑制剂高通量药物筛选的方法,对于开发抗HIV-1新药显得尤为重要。肽类药物T-20分子量大(超过4000 Da),容易被内源性蛋白酶降解,不能口服,只能注射,而且用量大(每次100mg,2次／天),合成步骤多,成本高,药品费用太昂贵(约2万美元／人／年)。因此,寻找小分子化合物代替T-20是HIV进入抑制剂研究的方向。我们课题组此前的研究表明,T-20的作用机制和C34并不相同,它不能与N-多肽形成六螺旋束结构,也不能阻止C34与N-多肽形成六螺旋束结构,它可能具有多个功能区。但是它具体的作用机制并不清楚。通过全序列多肽扫描,我们曾发现衍生至X4病毒株HIV-1MN gp120的辅助受体结合部位和gp41跨膜区段的15-mer多肽,能消除T-20的抑制活性,表明T-20抗HIV的作用位点还包括gp120上的CXCR4辅助受体结合部位。那T-20与gp120的CXCR4结合位点相互作用的关键序列是什么?T-20是否能结合到gp120-CD4复合物上的CCR5辅助受体结合位点?深入研究T-20的新作用机制,将可能为HIV进入抑制剂的研究提供新的药物作用靶点。为此,本课题根据我们在HIV-1病毒进入抑制剂研究领域积累的经验,以HIV-1假病毒作为研究对象,为确认HIV-I进入抑制剂新的药物作用靶点以及建立相应的高通量筛选方法,寻找替代多肽药物T-20的小分子化合物奠定研究基础。(1)建立一种非感染性的细胞融合的方法：根据本实验室积累的建立非感染性高通量筛选方法的经验,分别用HL2／3细胞和Hela-CD4-LTR/β-gal作为效应物和靶细胞,通过对β-半乳糖甘酶染色来判断细胞融合的效果。由于本方法具有非感染性,再结合基于细胞融合的非感染性的CHO-WT方法,可以在普通实验室用来快速筛选HIV-1融合抑制剂和Tat蛋白抑制剂。(2)天然来源的抗HIV-1药物筛选：利用假病毒体系和针对包膜蛋白亚基gp41的ELISA方法,结合分子对接,拟对来源于马尾树树皮的化合物蜡果杨梅酸B、日本蛇菰地上地下部分的葡萄糖类化合物和白桦酯酸衍生物等活性化合物抗HIV-1的作用机理。(3)T-20与gp120的关系的研究：拟合成覆盖409-431序列5个8-mer多肽(每个多肽相隔3个氨基酸残基),寻找消除T-20抑制活性的关键序列以及与T-20相互作用的关键序列。并且建立HIV-1假病毒感染U87. CD4. CXCR4细胞的活性检测方法,并筛选小分子化合物库,寻找能有效抑制HIV-1 env.进入靶细胞的活性分子,为开发病毒进入抑制剂类抗HIV-1药物提供新的思路。目的：1、根据HIV-1进入和复制的两个关键步骤,即病毒包膜蛋白介导的病毒-细胞膜融合和Tat-LTR的相互作用,拟建立一种非感染性的细胞融合的方法来同时快速筛选HIV-1融合抑制剂和Tat-LTR抑制剂。2、建立HIV-1假病毒细胞筛选模型,以及HIV-1假病毒感染U87.CD4. CXCR4细胞的活性检测方法,并筛选小分子化合物库,寻找能有效抑制HIV-1 env进入靶细胞的活性分子,研究活性小分子化合物的具体作用机制。3、深入研究T-20的具体作用机制,寻找HIV进入抑制剂新的药物作用靶点。方法：1、分别用HL2／3细胞和Hela-CD4-LTR/β-gal作为效应物和靶细胞。当HL2／3细胞和Hela-CD4-LTR/β-gal细胞结合后,HL2／3中的Tat蛋白可以启动B-半乳糖甘酶长末端的LTR的表达,进一步导致了β-半乳糖甘酶的表达。因此可以通过对B-半乳糖甘酶染色来判断细胞融合的效果。2、建立细胞水平的HIV-1假病毒活性检测方法：将包膜蛋白质粒(pHXB2或VSV-G)与pNL4,3, Luc. R-E(带有荧光素酶报告基因的HIV-1基因)共转染至293T细胞后,可分别获得HIV-1 env假病毒和VSV-G假病毒。3、抗HIV-1活性筛选：应用U87. CD4. CXCR4细胞作为筛选模型,利用假病毒体系和针对包膜蛋白亚基gp41的ELISA方法,结合分子对接,研究来源于马尾树树皮的化合物蜡果杨梅酸B、日本蛇菰地上地下部分的葡萄糖类化合物和白桦酯酸衍生物等活性化合物抗HIV-1的作用机理。4、合成覆盖409-431序列的5个8-mer多肽(每个多肽相隔3个氨基酸残基),寻找消除T-20抑制活性的关键序列以及与T-20相互作用的关键序列。构建相应的X4假病毒,检测T-20抑制这些X4假病毒与靶细胞融合的活性。结果：1、HIV进入抑制剂高通量筛选方法的建立1) HeLa-CD4-LTR-β-gal细胞与HL2／3细胞共孵育后导致了β-半乳糖甘酶的表达。HIV-1融合抑制剂(T-20、C34和ADS-J1)可以抑制表达了HIV-1包膜蛋白的HL2／3细胞和表达了β-半乳糖甘酶的HeLa-CD4-LTR-β-gal细胞的融合。两种Tat蛋白抑制剂姜黄素和氯丙嗪也可以抑制HL2／3细胞和HeLa-CD4-LTR-β-gal细胞的融合。2) CHO-WT和MT-2细胞共孵育后导致了大量融合泡的产生。HIV-1融合抑制剂(T-20、C34和ADS-J1)可以抑制稳定表达HIV-1包膜蛋白gp160的CHO/WT细胞株和含有表达CD4受体分子和CXCR4辅助受体的MT-2细胞之间融合泡的产生。两种Tat蛋白抑制剂姜黄素和氯丙嗪并不能抑制细胞之间融合泡的产生。2、天然来源的抗HIV-1化合物筛选1)从马尾树树皮中提取蜡果杨梅酸B (myriceric acid B)能抑制HIV-1 env假病毒感染,其半数抑制浓度(IC50)值为是8.3±0.2μg·ml-1。其抑制活性与其抑制gp41六螺旋束结构形成的机制有关,并且能靶向gp41上N-螺旋三聚体的靶穴位置。2)来源于日本蛇菰地上地下部分的咖啡酰葡萄糖类化合物,1,2,6-三-0-咖啡酰基-β-D-吡喃葡萄糖和1,3-双-0-咖啡酰基-4-0-没食子酰-β-D-吡喃葡萄糖具有较好抑制病毒的作用。它们的半数抑制浓度(IC50)值分别是5.5±0.2μg·ml-1和5.3±0.1μg·ml-1。这2种化合物的抑制活性与其抑制gp41六螺旋束结构形成的机制有关。它们均能靶向gp41上N-螺旋三聚体的靶穴位置。3)白桦酯酸衍生物LY7和LY14能够抑制HIV env假病毒,它们的半数抑制浓度(IC60)值分别为7.96±1.54μg·ml-1和5.67±0.40μg·ml-1。3、T-20作用机制的研究1)衍生于gp120的系列多肽(MN-6312,MN-6313,SW-1012,SW-1013)能够逆转T-20对HIV env的抑制作用。2)衍生于gp120的多肽SW-1011对U87.CD4.CXCR4具有细胞毒性,CC50值为5.92±1.53μM3)SW-1010与T-20不能直接结合。Biotin-6312并不能和衍生gp41的系列多肽作用。4)衍生于gp120的系列多肽(MN-6312,MN-6313,SW-1010,SW-1012)能够促进HIV env pseudovirus感染,但是它们对VSV-G pseudovirus均无促进作用。结论：1.由于实验中采用的细胞融合体系都是非感染性的,可以在普通实验室应用。2.蜡果杨梅酸B是作用于gp41的HIV-1进入抑制剂,可作为先导化合物来研发抗HIV-1新药。3.1,2,6-三-0-咖啡酰基-β-D-吡喃葡萄糖和1,3-双-0-咖啡酰基-4-0-没食子酰-β-D-吡喃葡萄糖可以较好的抑制HIV-1 env假病毒感染靶细胞,可作为先导化合物来研发抗HIV-1新药。4.白桦酯酸衍生物LY7和LY14能够抑制HIV env假病毒,可作为先导化合物来研发抗HIV-1新药。5.T-20不能和gp120直接结合,衍生于gp120的小肽逆转T-20的作用是由于它具有很强的促进病毒感染的能力。更多还原

【Abstract】 BACKGROUNDAcquired Immune Deficiency Syndrome (AIDS) was caused by human immunodeficiency virus (HIV-1). According to the UNAIDS/WHO report in 2009, an estimated 33.4 million people were living with HIV infection. It is also estimated that 2.7 million people became newly infected with HIV, and 2.0 million people died of AIDS-related illnesses in 2007. Nowadays, there are about 0.7 million HIV-1 infected people in China. The HIV-1 infection becomes a growing popular trend in many of provinces in our country, such as Yun-Nan, He-Nan, Xin-Jiang and Guang-Dong. From the statistical data in World Health Organization, the ratio of HIV-1 infection people to total people in our country is very low, but the real number of HIV-1 infected people is the second in Asia and the fourteenth in the world. This phenomenon became a serious social question in our life style.It is well known that developing vaccine is the best HIV-1 prevention strategy in all methods. But there is no effective HIV-1 vaccine in clinical treatment. Therefore, it is an important means to develop effective and safe drugs in treating HIV/AIDS. To this day, there are 28 kinds of anti-HIV drugs approved by FDA. These drugs were divided into three classes, such as reverse transcriptase inhibitors, protease inhibitors and HIV-1 entry inhibitors. But reverse transcriptase inhibitors and protease inhibitors could develop the anti-HIV-1 drug resistance mutations easily and showed strong adverse effect to human bodies. For these reasons more and more people could not tolerate these two group drugs. However, HIV-1 entry inhibitors, T-20 and Maraviroc, showed their anti-HIV activities to resistant-virus caused by reverse transcriptase inhibitors and protease inhibitors. It is a hot field to develop new anti-HIV drugs in these days.High-throughput drug screening is a hot strategy in current new-drug finding field. Since 1990s, Dr. Jiang found the first HIV-1 entry inhibitor which was derived from HIV-1 gp41 CHR. Our laboratory established a series of methods to screening HIV-1 entry inhibitors and found many active compounds targeting to gp41 or gp120.HIV-1 can cause serious infection, therefore, the researches on real virus could be only limited in P3 laboratory. So it is of great significance to identify the screening methods applying for common biological laboratory.The molecular weight of Fuzeon (also known as T-20 or enfuvirtide) is more 4000 dalton. It is easy degradated by endogenous protease. It can not be taken orally and can only be injected. It was too expensive for HIV-1 patients. So there is a hot research to find new small molecular drugs.In our previous studies, we found that even though it overlaps C34 sequence with 24 amino acids, T-20 has a different mechanism of action from C34. The interaction of T-20 with viral NHR region alone may not prevent the formation of the fusion active gp41 core. T-20-mediated anti-HIV activity can be significantly abrogated by peptides derived from the membrane-spanning domain in gp41 and coreceptor binding site in gp120. But the key sequence of interaction between T-20 and gp120 CXCR4 binding site is not known. Also it is not known whether T-20 can interact with the gp120-CD4 coreceptor binding site. Further elucidation of the mechanism of action of T-20 will provide new target(s) for development of novel HIV entry inhibitors. According to our laboratory research experience in HIV entry inhibitors, I focused on three projects in the present study. (1) Identify a method for for screening of HIV-1 fusion inhibitors and Tat-LTR interaction blockers by sequential application ofβ-galactosidase-and syncytium-based cell fusion assays. (2) Screening native source anti-HIV-1 compounds. (3) Identify the mechasim of T-20. For the first project, I intended to do following works:Using HL2/3 cells and Hela-CD4-LTR/β-gal cells as effector or target cells, respectively. Theβ-galactosidase staining kit was used to test the expression ofβ-galactosidase. At the same time, CHO/WT cells and MT-2 cells were selected as another pair of effector-target cells. Through the two systems, I can screen the HIV-1 entry inhibitor and Tat-LTR blocker rapidity. For the second project, I intended to do the following works:Screening the anti-HIV-1 activities and the mechanism of action of myriceric acid B and C isolated from Rhoiptelea chiliantha Diels et Hand.-Mazz, caffeoyl glucose compounds from Balanophora japonica Makino and Betulinic acid-derivated compounds. For the third project, I intended to do the following works:Synthesizing the serial peptides which overlap gp120 sequence from residue 409 to residue 431. Finding the key motif which could eliminate the activity of T-20 caused by gp120 co-receptor binding site.OBJECTIVES:a) Viral envelope glycoprotein (Env)-mediated membrane fusion and Tat-LTR interaction are two critical steps of HIV-1 entry and replication. In this experiment we want to develop a novel strategy to screen anti-HIV-1 agents that block HIV-1 fusion and Tat-LTR interaction by sequential use ofβ-galactosidase(gal)-and syncytium-based cell fusion assays.b) Establish the HIV-1 Env pseudovirus cell model to screen small molecular compounds. Try to clarify the detail mechanism of small molecular compounds.c) To identify the detail anti-HIV-1 mechanism of action of T-20.METHODS:a) HL2/3 Cells and Hela-CD4-LTR/β-gal cells were act as effector or target cells, respectively. Theβ-gal expressing cells were revealed by using aβ-gal staining set.b) Establish the system of testing HIV-1 pseudovirus in cell level:The plasmids encoding envelope proteins of HIV-1 (pHXB2) and VSV (pVSV-G) were cotransfected 293T cells with pNL4-3.Luc.R-E-to produce HIV-1 Env pseudovirus and VSV-G pseudovirus, respectively, which were used for testing the antiviral activities of these compounds. U87.CD4.CXCR4 cells were act as target cells; ELIS A and molecular docking were used to study the mechanism of action of the active compounds, such as Myriceric acid B derivatived from Rhoiptelea chiliantha Diels et Hand.-Mazz, caffeoyl glucose compound derived from Balanophora japonica Makino and Betulinic acid-derivated compounds.c) Synthesize serial peptides which overlap gp120 sequence from residue 409 to residue 431. Try to find the key motif which could eliminate the activity of T-20 caused by gp120 co-receptor binding site and the sequence of T-20-gp120 interaction. Construct HIV-1 Env pseudovirus to test the activity of T-20.RESULTS:a) Coculture of HeLa-CD4-LTR-B-gal cells with HL2/3 cells resulting in expression ofβ-galactosidase. To develop a B-gal-based cell-cell fusion assay, we selected HeLa-CD4-LTR-β-gal cells as the target cells and HL2/3 cells as the effector cells. HL2/3 cells also express HIV-1 Env and Tat proteins. After fusion of HeLa-CD4-LTR-B-gal cells with HL2/3 cells, the LTR-linkedβ-gal gene could be activated by the Tat protein in HL2/3 cells, resulting in expression of B-galactosidase in the fused cells which become visible after B-gal staining. A number of syncytia were revealed in the coculture of CHO-WT and MT-2 cells, while no syncytium was seen when CHO-WT and MT-2 cells were cultured separately. All of the three HIV-1 fusion inhibitors, T-20, C34 and ADS-Jl could inhibited, in dose-dependent manner, both B-gal-based cell fusion between HeLa-CD4-LTR-B-gal cells and HL2/3 cells and syncytium-based cell fusion between CHO-WT and MT-2 cells. However, two inhibitors of Tat-LTR interaction, curcumin and chlorpromazine, could only effectively inhibit B-gal-based cell fusion between HeLa-CD4-LTR-B-gal cells. Their IC50 values were 31.7 and 54.9μM, respectively.b) Myriceric acid B could significantly inhibit the infection of HIV-1 Env pseudovirus with an IC50 of 8.3±0.2μg·ml-1. The carbonoxyl group at C-28 position and the hydroxyl group at the C-3 position of myriceric acid B are important for its anti-HIV-1 activity. Like other HIV-1 entry inhibitors targeting gp41 (e.g., ADS-J1 and NB-64), myriceric acid B could also block the gp41 six-helix bundle formation. Molecular docking analysis suggests that myriceric acid B may bind to the hydrophobic cavity of the gp41 N-trimeric coiled coil. TCGP and DCGGP could inhibit HIV-1 Env pseudovirus infection. The IC50 values of these two compounds were 5.5±0.2μg·ml-1 and 5.3±0.1μg·ml-1 respectively. These two compounds could also block the gp41 six-helix bundle formation. They might bind to the hydrophobic cavity of the gp41 N-trimeric coiled-coil. Betulinic acid-derivated compounds, LY7 and LY14, can inhibit the HIV env pseudovirus entry. The value of IC50 is 7.96±1.54μg·ml-1 and 5.67±0.40μg·ml-1, respectively.c) MN-6312, MN-6313, SW-1012 and SW-1013, which which derived from gp120, can block the activity of T-20. Biotin-6312 can not interact with gp41-derived peptides. MN-6312, MN-6313, SW-1010 and SW-1012, which derived from gp120, are able to increase the HIV-1 Env pseudovirus infectivity on target cells. But they can not enhance the infection abilities of VSV-G pseudovirus.CONCLUSIONS:a) The cells used in both cell-cell assays are non-infectious and can be used in a general biology laboratory. This approach has great potential for rapid identification of HIV-1 fusion inhibitors and/or Tat-LTR interaction blockers.b) Myriceric acid B, TCGP, DCGGP, Betulinic acid-derivated compounds, LY7 and LY14, showed good activities of anti-HIV pseudovirus. They are potent HIV-1 entry inhibitors and can serve as a lead compound for developing novel anti-HIV-1 drug.c) T-20 can not interact with gp120. The peptides derived from gp 120 are able to enhance HIV-1 Env pseudovirus infection to target cells.更多还原