天然来源化合物抗HIV的活性筛选和ADS-J1抑制HIV进入的作用机制研究
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摘要
研究背景
艾滋病是由人类免疫缺陷病毒(human immunodeficiency virus, HIV)感染引起的,严重威胁人类健康的传染性疾病。据UNAIDS统计,2008年全球感染HIV的人数有3340多万,而08年新增的HIV感染者数量为270万,因艾滋病死亡的人数高达200万。我国自1985年首次发现HIV感染者,截止2009年10月底,估计有74万人发生了感染,且2009年新增的感染者数量为4.8万。在非洲的有些国家,HIV的感染率达总人口30%以上。因此,预防和治疗艾滋病,已不仅仅是挽救个人生命的问题,而是关系到民族存亡的大事。由于有效的艾滋病疫苗在短时期内还难以面世,开发高效、安全和廉价的抗HIV药物依然是挽救艾滋病人生命的关键举措,也是艾滋病防治的当务之急。目前应用于临床的抗HIV药物中逆转录酶抑制剂、蛋白酶抑制剂有26种、进入抑制剂和整合酶抑制剂才3种,但前两类药物容易引起病毒耐药现象和严重的毒副作用。因此,为了治疗感染了耐药病毒的艾滋病患者,组成更多有效的“鸡尾酒”治疗方案,必须大力开发后两类新作用机制的抗艾滋病药物。其中HIV进入抑制剂,由于能在病毒感染的早期进行阻断,更为有效地预防和治疗HIV感染,而倍受重视。
已被批准的HIV进入抑制剂有两种,分别是靶向HIV跨膜蛋白gp41的多肽药物T-20(也叫融合抑制剂)和靶向HIV辅助受体CCR5的受体拮抗剂Maraviroc。Maraviroc只能作用于利用CCR5作为辅助受体的R5病毒,对以CXCR4为辅助受体的X4病毒无效,而T-20则对两类病毒均具有抑制作用。然而,T-20是多肽药物,不能口服,容易被体内的蛋白酶降解,而且价格昂贵不是广大的发展中国家HIV感染者所能承受的,因此开发出具有类似作用机制的非肽类HIV进入抑制剂迫在眉睫。
本课题根据实验室在相关领域积累的经验,与相关的研究组合作对大量的有机合成小分子和天然产物提取成分进行抑制HIV进入的体外活性筛选,并进一步的开发完善新的实验方法,研究公认的HIV-1抑制剂ADS-J1对gp41产生抑制作用的具体位点。
第一章天然来源化合物抗HIV的活性筛选
目的：
通过对茶黄素衍生物单体进行结构改造,改变原样品颜色深,水溶性差等缺点,考察改造后的化合物体外抑制HIV-1包膜介导的细胞融合活性和相关的生物学活性,确定改造后成分的可利用度及进一步改造的方向,为该类化合物作为HIV进入抑制剂的作用机制研究提供依据。对番石榴叶提取成分进行体外的免疫,分生活性检测,筛选出能在HIV-1进入阶段发挥作用的成分,并初步探讨其作用机理,填补该类成分在HIV-1进入抑制研究方面的空白。
方法：
1.茶黄素类衍生物的结构改造和番石榴叶成分提取。茶黄素衍生物是文献中研究最多的从红茶中提取的TF1 (theaflavin), TF2A (theaflavin-3-monogallate), TF3 (theaflavin-3,3'-digallate),分别对它们的苯并酚酮七元环中的1”,2”,3”,7”4个双键进行还原得到对应的化合物TF1-4H,TF2A-4H, TF3-4H。具体操作是在Pt/C的催化下,通入H2还原,然后过滤除掉催化剂,对还原产物用反相柱层析分离纯化得到相应的化合物。参照相关文献,番石榴叶经两次乙醇提取的浸膏用SA-1型大孔树脂吸附,然后用80%(V/V)的乙醇溶液洗脱,得到总皂苷的粗品。番石榴叶的乙醇提取物用FL-1型大孔树脂吸附,然后用0.2%NaOH溶液洗脱,得到总黄酮的粗品。番石榴叶水浸泡液回流提取得到挥发油成分。
2.以上样品先用抗体特异性的检测抗原的酶联免疫吸附实验(Enzyme-linked immunosorbent assay, ELISA)进行第一次筛选,有效的通过浓度梯度实验考察量效关系,计算抑制率并确定半数抑制浓度(Half of the inhibiting concentration, IC50)范围。进一步的用可直观的从六螺旋束(six-helical bundle,6-HB)条带变化中反映化合物抑制强弱的天然非变性聚丙烯酰胺凝胶电泳(Native polyacrylamide gel electrophoresis, N-PAGE)确证活性。
3.另外用HIV-1包膜介导的细胞-细胞融合实验考察改造后的茶黄素类化合物和番石榴叶的有效成分细胞水平的抑制活性。根据融合时的细胞状态,对样品进行四甲基偶氮唑盐微量酶反应比色法(MTT)实验检测相应的细胞毒性。
结果：
1.分离纯化的茶黄素衍生物结构改造后纯化的产物由香港浸会大学提供,本研究主要鉴定结构改造后单体的活性大小。番石榴叶提取成分由南方医科大学中医药学院提供,黄酮粗品中总黄酮量为55.22%,皂苷粗品中总皂苷量为55.3%。
2.由于茶黄素化合物本身的不稳定性,多次反复冻融后有部分化合物活性下降或消失,且受样品量的限制,该部分有的实验结果不完善。首先利用抗体特异性的ELISA对茶黄素系列化合物进行第一次筛选,发现25μg/mL的终浓度时,化合物都有很高的抑制率。进一步的梯度稀释时,虽然没有做出TFl的IC50’范围,但在终浓度11.08μM时,抑制率为60%,而还原产物TF1-4H的IC50为0.63±0.27μM,另一组TF2A的IC50=2.60±0.56μM,还原产物TF2A-4H的IC50=1.13±0.27μM,第三组TF3的IC50=0.88±0.34 gM,还原产物TF3-4H的IC50=1.74±0.55 gM。与文献值相比,本研究中IC50值略低,但都在微摩尔水平,且<5μM的范围内。N-PAGE实验的条带显示六个单体随着浓度的降低,6-HB的颜色越来越深,表现出一定的量效依赖关系。用ELISA实验对番石榴叶的三个成分进行初筛时,发现以25μg/mL终浓度初筛,番石榴叶提取物总黄酮和总皂苷对6-HB的抑制率都在80%左右,挥发油成分的抑制活性较低,统计分析显示前两组与相同浓度的阳性对照TF间无差异,而挥发油成分与TF相比差异显著,所以后续实验中去掉该组。在浓度梯度实验中,番石榴叶总黄酮和总皂苷对6-HB的抑制也表现出明显的量效关系,IC50分别为3.9±1.3μg/mL,9.06±0.17μg/mL。进一步的N-PAGE实验结果确证了二者对gp41的作用,结果与ELISA一致，200μg/mL时与相同浓度的阳性药物TF的效果相近,通过对应浓度的条带,清楚的看到样品量与抑制作用之间成正比关系。
3.细胞融合结果显示,改造后TF1-4H,TF2A-4H,TF3-4H抑制细胞融合的活性比改造前的TF1,TF2A,TF3稍有增强,都在相应的微摩尔水平。TF1的IC50为14.63±1.40μM,TFl-4H的IC50为11.144±1.02μM。第二组TF2A的IC50为10.30±0.36μM,TF2A-4H的IC50为6.44±0.51μM。第三组TF3的IC50为8.14±0.21μM,TF3-4H的IC50为6.06±0.26μM。由于样品量较少,没有进一步的考察细胞毒性。细胞融合实验中番石榴叶总黄酮和总皂苷都表现出一定的抑制活性,总皂苷的IC50为7.33±0.40μg/mL,总黄酮的IC50为5.3±1.3μg/mL。MTT实验显示总黄酮产生的细胞毒性很小,半数致死浓度(concentration of 50% cytotoxicity, CC50)都在100μg/mL以上,总皂苷对CHO-WT和MT-2的CC50分别为46.86±1.27μg/mL和62.97±1.22μg/mL,统计学结果显示总皂苷在7.82μg/mL以下浓度时CHO-WT的生长与对照组之间没有显著性差异(P＞D.05),在25μg/mL以下MT-2的生长与对照组之间没有显著性差异(P>D.05)。
结论：
1.茶多酚衍生物是文献报道能对HIV感染的多个环节都有抑制效果的化合物,茶黄素衍生物低浓度时没有表现出抑制CD4,趋化因子受体与gp120的相互作用却表现出抑制gp41核心结构6-HB形成的活性,且观察发现化合物中含有没食子酰基的比不含的活性好,含的个数越多活性越好,推测其活性可能是通过化合物自身带的微弱的负电荷与靶点(如N末端574位的赖氨酸)的正电荷形成离子键实现的。茶多酚自身的多酚结构使它具有上述活性,但是正因为含有多个酚羟基使化合物整体易氧化,不稳定,且与相并的酚酮七元环一起形成一个大共轭体系使得化合物颜色较深,水溶性也不好。我们的研究通过对三个衍生物的酚酮七元环的双键进行还原,在不破坏酚羟基的前提下尽量减少生色基团,并对改造后的产物进行体外活性检测。还原后化合物的颜色变浅,水溶性也增加了。ELISA, N-PAGE实验的结果都显示改造后化合物表现出良好的抑制六螺旋束形成的活性。细胞融合实验显示茶黄素衍生物表现出与文献报道值相似的活性,结构改造后的三个化合物活性比改造前还好,推测可能是因为改造后化合物水溶性提高,稳定性增强,且空间构象也发生了一些变化,相同条件下到达靶位的有效浓度提高,更加有利于化合物与靶点的结合。这些结果既为进一步的结构改造提供了方向又在化合物的作用机制研究方面提供了有力的证据。
2.番石榴叶提取物总黄酮和总皂苷虽然是混合成分,但主要成分黄酮和皂苷含量都在55%以上。二者都能在很大范围内安全有效的抑制HIV-1包膜介导的细胞融合,这说明二者可以在HIV的进入阶段起到抑制作用,且细胞毒性实验证明在有效范围内样品对细胞的生长没有抑制作用。而靶向gp41的抗体免疫实验ELISA表明二个样品都可以在微克每毫升的水平抑制gp41核心构象6-HB的形成,该结果还被N-PAGE实验证实,这说明二者在进入过程中的抑制作用可能是通过抑制了该过程中很重要的环节六股螺旋体的结构形成来实现的。
第二章ADS-J1抑制HIV进入的作用机制研究
目的：
通过建立和完善酸性非变性聚丙烯酰胺凝胶电泳(Acid native polyacrylamide gel electrophoresis AN-PAGE),研究和探讨ADS-J1与gp41相互作用的机制。
方法：
建立并完善AN-PAGE,在已有的ELISA, N-PAGE等结果的支持下更清楚直接的反映ADS-J1(4-羟基-5-((E)-(4-(5-羟基-6-((E)-2-甲氧基-5-磺苯基)二氮烯基)-7-磺基萘-2-苯基氨基)-6-苯胺-1,3,5-三嗪-2-苯基氨基)-5-甲氧基-2-甲基苯)二氮烯基)萘-2,7-二磺酸在HIV-1gp41上的作用位点,为其机制研究提供直接可靠的证据。
结果：
采用AN-PAGE能显示来源于gp41 CHR (C-terminal heptad repeat), NHR (N-terminal heptad repeat)不同区段的多肽条带,同时也能显示6-HB,以及与不同浓度的ADS-J1作用后的6-HB条带。结果表明,ADS-J1能梯度抑制C34与N36形成的6-HB条带,从0到100μM,6-HB的条带颜色逐渐变淡,与单独的C34共同孵育,C34条带没有变化,与单独的包含574位赖氨酸的N36共同孵育后,N36条带消失,与NHR上不包含574位点的其他多肽片段单独孵育,多肽条带都没有变化,与574位位点突变后的多肽片段孵育,对条带也没影响。
结论：
AN-PAGE的结果表明,从含有20000个化合物的化合物库中筛选出来的偶氮化合物ADS-J1对HIVgp41的作用是通过与gp41核心结构中的NHR同源三聚体螺旋结构表面的疏水深穴的第574位赖氨酸相互作用来实现的,可能是由于ADS-J1自身的磺酸基团与赖氨酸的正电荷之间形成了离子键,从而阻碍了gp41CHR上632位带负电荷的天冬氨酸与赖氨酸的相互作用,破坏了分子内部盐桥的形成,也破坏了六股α螺旋束的稳定性,从而发挥HIV进入抑制的功效。本实验中所用AN-PAGE方法采用pH值3.4的缓冲液体系,并反转了电极,对比N-PAGE中所用的pH值8.3的缓冲液体系,前者能使pI值在3.4-8.3之间的多肽片段都跑出条带,满足更多的实验需要,为靶向HIV-1包膜的病毒进入抑制剂的研究提供了一个方便有效的方法。
Background:
AIDS, caused by human immunodeficiency virus (HIV), threatens human health severely. According to the survey made by UNAIDS, the number of people infecting HIV was more than 33.4 million in 2008. Among them,2.7 million were newly infected in 2008. It was estimated that about 740,000 people were infected HIV till the end of October in 2009 dating from 1985 when the first HIV infected was reported in China. Some countries in Africa, HIV infection rates are more than 30% of the total population. Therefore, the prevention and treatment of AIDS is not only saving lives of individuals, but also the nation. Due to the difficulty of developing an effective AIDS vaccine in a short time, it's still very important to develop efficient, safe and cheap anti-HIV drugs. Currently, there are 26 reverse transcriptase inhibitors and protease inhibitors but only 3 entry inhibitors and integrase inhibitors used in clinic. However, the first two categories of drugs prone to induce viral resistance and serious side effects. Therefore, we should vigorously develop the latter two kinds of anti-HIV drugs to form more "cocktail" therapy protocols for patients infected with drug-resistant HIV strains. The HIV entry inhibitors would be more effective in the treatment and prevention of HIV infection since they block HIV replication at the early stages of infection.
Two HIV entry inhibitors have been approved, one targeted HIV transmembrane protein gp41 peptide named T-20 and the other, Maraviroc, targeted HIV co-receptor CCR5. The Maraviroc can only act on the R5 viruses which use CCR5 as the coreceptor but is no inhibition to X4 virus taking CXCR4 as a coreceptor. The T-20 is effective to both R5 and X4 virus. However, as a peptide, T-20 cannot act by oral administration and is easy to be degradated by protease in vivo. Furthermore, it's too expensive for people in developing county to afford for a long time. Therefore, it's urgently and necessary to develop small compounds with similar mechanism of action as T-20.
Based on laboratory experience in related fields, we screened a synthetic compound library and several natural products offered by other studying groups, to see whether these compounds have in vitro anti-HIV activities.. We also establish a new experimental method to investigate the interaction between ADS-J1 and gp41 to find out the binding sites of ADS-J1, a small molecular HIV entry inhibitor.
Part I Screening theaflavin derivates and Psidium guajava leaf extracts against HIV-1 entry
Aim:
To identify the inhibitory activity of theaflavin derivatives against HIV envelope glycoprotein induced cell-cell fusion. The mechanism of action of the compounds as HIV entry inhibitor was also investigated. The extracts from Psidium guajava leaf were tested on the formation of HIV gp41 six-helical bundle by immune and molecular biology testing.
Method:
1. Theaflavin derivatives, including TF1 (theaflavi), TF2A (theaflavin-3- monogallate), TF3 (theaflavin-3,3'-digallate), are extracted from black tea. We showed these compounds can inhibit HIV entry by targeting HIV gp41. However, these compounds are not stable and have deep colour, which are the shortcomings for developing them as microbicides to prevent HIV sexual transmission. Therefore, these theaflavin compounds were modified by adding H atoms to the double bonds of benzo annulen. The modified compounds were purified using reversed-phase column chromatography. The TSGL (total saponin of Psidium guajava) was purified and concentrated by loading the alcohol extractum of Psidium guajava leaf on SA-1 macroporous resin and then washed out with 80% (v/v) alcohol. Similarly, the TFGL (total flavone of Psidium guajava) was purified by FL-1 macroporous resin and washed out by 0.2% NaOH. The essential oil was gained by extracting the aqueous solution of Psidium guajava leaf for serval hours and collected the remainder at a high temperature.
2. The activities of the samples on blocking HIV-1 gp41 six-helical bundles (6-HB) forming were analyzed by ELISA and N-PAGE. All of the samples were firstly detected by antigen-specific antibody enzyme-linked immunosorbent assay. Sample with good activity would experience a second screening of gradient experimental to calculate the IC50. N-PAGE assay was used to identify the biologic activity by comparing the intensity of bands at the site of 6-HB.
3. Cell-cell fusion assay mediated by HIV-1 env was used to study the inhibitory activity at cellular level of theaflavins compound and active ingredients of guava leaves. According to the cell stated in the fusion, the cytotoxicity of samples were tested on CHO-WT and MT-2 by MTT.
Results:
1. All the purification and characterization of theaflavin derivates were carried out by the provider. We mainly focused on the biologic ability screening. Extractum of Psidium guajava leaf was offered by School of Traditional Chinese Medicine in Southern Medical University. The content of TSGL purified with SA-1 macropore resin was 55.3% and the TFGL with FL-1 was 55.22%.
2. The activities of theaflavin derivates against 6-HB formation tested by ELISA. Compound TF1 showed inhibition of 60% at 11.08μM while TF1-4H showed 50% inhibition at 0.63±0.27μM. In the second pair, the IC50 of TF2A is 2.60±0.56μM and TF2A-4H is 1.13±0.27μM. In the third pair, IC50 of TF3 is 0.88±0.34μM and TF3-4H is 1.74±0.55μM. The results of N-PAGE are consistent with that of ELISA. The intensity of bands at 6-HB became darker when the concentration of compounds getting lower. Our ELISA results of TF1, TF2A, TF3 are at the same level as reported in literature, ranging from 0 to 5 micromole. Results of Psidium guajava leaf by ELISA showed good activity against 6-HB formation, with an inhibition more than 80% at 25μg/mL, except essential oil. The IC50 of TFGL is 3.9±1.3μg/mL and the IC50 of TSGL is 9.06±0.17μg/mL. The results were confirmed by N-PAGE.
3. Results of cell-cell fusion for theaflavin derivates showed that the modified compounds had a smaller IC50 comparing with those before modifying. The IC50 of TF1 is 14.63±1.40μM while that of TF1-4H is 11.14±1.02μM. The IC50 of TF2A is 10.30±0.36μM while that of TF2A-4H is 6.44±0.51μM. Similarly, the IC50 of TF3 is 8.14±0.21μM and TF3-4H is 6.06±0.26μM. Both TFGL and TSGL showed inhibition activity against cell-cell fusion mediated by HIV-1 env. The IC50 of TFGL is 5.3±1.3μg/mL and that of TSGL is 7.33±0.40μg/mL. Cytotoxicity of TFGL to two cells are low, with CC50>100μg/mL. The CC50 of TSGL to CHO-WT is 46.86±1.27μg/mL and to MT-2 is 62.97±1.22μg/mL. Statistic analysis shows that there are no distinct difference on cell growth between compounds group and cell control group when the compounds group at concentration<7.82μg/mL (P>0.05), the same as MT-2 with compounds concentration<25μg/mL (P>0.05).
Conclusions:
1. Theaflavin derivates are reported to inhibit HIV-1 replication with multiple mechanisms. Theaflavin derivates at low concentration can't block the interaction between gp120 and CD4 molecules, nor interact with CXCR4 or CCR5, but can significantly inhibit the gp41 conformation changing by blocking the formation of six-helical bundles on gp41. The more the compounds contain galloyls, the better the inhibition activity is. It's presumed that the active polyphenols contain weak negative charges and these compounds may interact with the target proteins via ionic interactions. The structure of polyphenol gives the compounds weak negative charges together with the reducibility, instability, darker color and bad water solubility which make it difficult to use extensively. Our study planned to modify the structure to light color, enhance its stability and water solubility but not change the biologic ability especially on anti-HIV. Results of ELISA, N-PAGE and cell fusion assay show that they can still prevent HIV infecting at the same level as before modification. The results imply that we can continue in the direction and also it provides the proofs that the mechanism of action of theaflavin derivates is blocking HIV entry into target cells.
2. Though both the TFGL and the TSGL are admixtures, the main content of flavones and saponin are both up to 55%. It's reported that mount of polyphenol in the TFGL is no more than 1%. The admixture can inhibit the formation of syncytium at micromole level and has little cytotoxicity, indicating that they could act as an lead HIV entry inhibitors. Results of ELISA and N-PAGE showed they might act through preventing the formation of gp41 core six-helical bundle.
PartⅡThe study of mechanism of ADS-J1 on HIV-1 by acid-native polypropylene amide gel electrophoresis
Aim:
To investigate the mechanism of HIV-1 inhibitor ADS-J1 on HIV gp41, offer proof for the designing and developing of new small molecular compounds against HIV-1.
Method:
Establishing and improving the acid-native polypropylene amide gel electrophoresis to find out the action site of ADS-J1, (7-[6-phenylamino-4-[4-[(3,5-disulfo-8-hyd-roxyna phthyl)azo]-2-methoxy-5-Methyl phenylamino]-1,3,5-triazine-2-yl]-4-hydroxy-3-[(2-methoxy-5-sulfophenyl)azo]-2-naphthalene sulfonic acid) on gp41.
Results:
Acid-native PAGE can show bands of peptides from different fraction of gp41. Results of AN-PAGE implied that ADS-J1 can inhibit the interaction of N36 and C34, with the band of 6-HB get lighter while the concentration of ADS-J1 varied from 0 to 100μM. ADS-J1 did not change the band of only C34, but made the band disappeared when incubated with only N36 ADS-J1 did not change the band of peptides from different fraction of HIV gp41 NHR without cavity region, neither the band of mutated N36 on the residue at 574th position. These results showed that ADS-J1 can bind to the cavity region on gp41, and the residue at 574th position (K574) is the most important residue for the activity of ADS-J1 against gp416-HB formation.
Conclusion:
Result of AN-PAGE indicated that the ADS-J1 inhibit HIV via ionic interactions. Computer-aided molecule docking analysis in the present study indicates that the negative charged groups in acid groups can interact with a positive charge residue K574 located in the gp41 NHR cavity region that participates in the formation of hydrophobic pocket, thus blocking the salt bridge formation between K574 and D632, a negatively charged residue in the pocket binding region of the gp41 CHR. In the experiment AN-PAGE, a running buffer with pH 3.4 was used and a reversed electrode. AN-PAGE can show bands of peptides with the pⅠranged from 3.4-8.8 compare to the traditional N-PAGE. The method will make the study of HIV entry inhibitor easier.
艾滋病是由人类免疫缺陷病毒(human immunodeficiency virus, HIV)感染引起的,严重威胁人类健康的传染性疾病。据UNAIDS统计,2008年全球感染HIV的人数有3340多万,而08年新增的HIV感染者数量为270万,因艾滋病死亡的人数高达200万。我国自1985年首次发现HIV感染者,截止2009年10月底,估计有74万人发生了感染,且2009年新增的感染者数量为4.8万。在非洲的有些国家,HIV的感染率达总人口30%以上。因此,预防和治疗艾滋病,已不仅仅是挽救个人生命的问题,而是关系到民族存亡的大事。由于有效的艾滋病疫苗在短时期内还难以面世,开发高效、安全和廉价的抗HIV药物依然是挽救艾滋病人生命的关键举措,也是艾滋病防治的当务之急。目前应用于临床的抗HIV药物中逆转录酶抑制剂、蛋白酶抑制剂有26种、进入抑制剂和整合酶抑制剂才3种,但前两类药物容易引起病毒耐药现象和严重的毒副作用。因此,为了治疗感染了耐药病毒的艾滋病患者,组成更多有效的“鸡尾酒”治疗方案,必须大力开发后两类新作用机制的抗艾滋病药物。其中HIV进入抑制剂,由于能在病毒感染的早期进行阻断,更为有效地预防和治疗HIV感染,而倍受重视。
已被批准的HIV进入抑制剂有两种,分别是靶向HIV跨膜蛋白gp41的多肽药物T-20(也叫融合抑制剂)和靶向HIV辅助受体CCR5的受体拮抗剂Maraviroc。Maraviroc只能作用于利用CCR5作为辅助受体的R5病毒,对以CXCR4为辅助受体的X4病毒无效,而T-20则对两类病毒均具有抑制作用。然而,T-20是多肽药物,不能口服,容易被体内的蛋白酶降解,而且价格昂贵不是广大的发展中国家HIV感染者所能承受的,因此开发出具有类似作用机制的非肽类HIV进入抑制剂迫在眉睫。
本课题根据实验室在相关领域积累的经验,与相关的研究组合作对大量的有机合成小分子和天然产物提取成分进行抑制HIV进入的体外活性筛选,并进一步的开发完善新的实验方法,研究公认的HIV-1抑制剂ADS-J1对gp41产生抑制作用的具体位点。
第一章天然来源化合物抗HIV的活性筛选
目的：
通过对茶黄素衍生物单体进行结构改造,改变原样品颜色深,水溶性差等缺点,考察改造后的化合物体外抑制HIV-1包膜介导的细胞融合活性和相关的生物学活性,确定改造后成分的可利用度及进一步改造的方向,为该类化合物作为HIV进入抑制剂的作用机制研究提供依据。对番石榴叶提取成分进行体外的免疫,分生活性检测,筛选出能在HIV-1进入阶段发挥作用的成分,并初步探讨其作用机理,填补该类成分在HIV-1进入抑制研究方面的空白。
方法：
1.茶黄素类衍生物的结构改造和番石榴叶成分提取。茶黄素衍生物是文献中研究最多的从红茶中提取的TF1 (theaflavin), TF2A (theaflavin-3-monogallate), TF3 (theaflavin-3,3'-digallate),分别对它们的苯并酚酮七元环中的1”,2”,3”,7”4个双键进行还原得到对应的化合物TF1-4H,TF2A-4H, TF3-4H。具体操作是在Pt/C的催化下,通入H2还原,然后过滤除掉催化剂,对还原产物用反相柱层析分离纯化得到相应的化合物。参照相关文献,番石榴叶经两次乙醇提取的浸膏用SA-1型大孔树脂吸附,然后用80%(V/V)的乙醇溶液洗脱,得到总皂苷的粗品。番石榴叶的乙醇提取物用FL-1型大孔树脂吸附,然后用0.2%NaOH溶液洗脱,得到总黄酮的粗品。番石榴叶水浸泡液回流提取得到挥发油成分。
2.以上样品先用抗体特异性的检测抗原的酶联免疫吸附实验(Enzyme-linked immunosorbent assay, ELISA)进行第一次筛选,有效的通过浓度梯度实验考察量效关系,计算抑制率并确定半数抑制浓度(Half of the inhibiting concentration, IC50)范围。进一步的用可直观的从六螺旋束(six-helical bundle,6-HB)条带变化中反映化合物抑制强弱的天然非变性聚丙烯酰胺凝胶电泳(Native polyacrylamide gel electrophoresis, N-PAGE)确证活性。
3.另外用HIV-1包膜介导的细胞-细胞融合实验考察改造后的茶黄素类化合物和番石榴叶的有效成分细胞水平的抑制活性。根据融合时的细胞状态,对样品进行四甲基偶氮唑盐微量酶反应比色法(MTT)实验检测相应的细胞毒性。
结果：
1.分离纯化的茶黄素衍生物结构改造后纯化的产物由香港浸会大学提供,本研究主要鉴定结构改造后单体的活性大小。番石榴叶提取成分由南方医科大学中医药学院提供,黄酮粗品中总黄酮量为55.22%,皂苷粗品中总皂苷量为55.3%。
2.由于茶黄素化合物本身的不稳定性,多次反复冻融后有部分化合物活性下降或消失,且受样品量的限制,该部分有的实验结果不完善。首先利用抗体特异性的ELISA对茶黄素系列化合物进行第一次筛选,发现25μg/mL的终浓度时,化合物都有很高的抑制率。进一步的梯度稀释时,虽然没有做出TFl的IC50’范围,但在终浓度11.08μM时,抑制率为60%,而还原产物TF1-4H的IC50为0.63±0.27μM,另一组TF2A的IC50=2.60±0.56μM,还原产物TF2A-4H的IC50=1.13±0.27μM,第三组TF3的IC50=0.88±0.34 gM,还原产物TF3-4H的IC50=1.74±0.55 gM。与文献值相比,本研究中IC50值略低,但都在微摩尔水平,且<5μM的范围内。N-PAGE实验的条带显示六个单体随着浓度的降低,6-HB的颜色越来越深,表现出一定的量效依赖关系。用ELISA实验对番石榴叶的三个成分进行初筛时,发现以25μg/mL终浓度初筛,番石榴叶提取物总黄酮和总皂苷对6-HB的抑制率都在80%左右,挥发油成分的抑制活性较低,统计分析显示前两组与相同浓度的阳性对照TF间无差异,而挥发油成分与TF相比差异显著,所以后续实验中去掉该组。在浓度梯度实验中,番石榴叶总黄酮和总皂苷对6-HB的抑制也表现出明显的量效关系,IC50分别为3.9±1.3μg/mL,9.06±0.17μg/mL。进一步的N-PAGE实验结果确证了二者对gp41的作用,结果与ELISA一致，200μg/mL时与相同浓度的阳性药物TF的效果相近,通过对应浓度的条带,清楚的看到样品量与抑制作用之间成正比关系。
3.细胞融合结果显示,改造后TF1-4H,TF2A-4H,TF3-4H抑制细胞融合的活性比改造前的TF1,TF2A,TF3稍有增强,都在相应的微摩尔水平。TF1的IC50为14.63±1.40μM,TFl-4H的IC50为11.144±1.02μM。第二组TF2A的IC50为10.30±0.36μM,TF2A-4H的IC50为6.44±0.51μM。第三组TF3的IC50为8.14±0.21μM,TF3-4H的IC50为6.06±0.26μM。由于样品量较少,没有进一步的考察细胞毒性。细胞融合实验中番石榴叶总黄酮和总皂苷都表现出一定的抑制活性,总皂苷的IC50为7.33±0.40μg/mL,总黄酮的IC50为5.3±1.3μg/mL。MTT实验显示总黄酮产生的细胞毒性很小,半数致死浓度(concentration of 50% cytotoxicity, CC50)都在100μg/mL以上,总皂苷对CHO-WT和MT-2的CC50分别为46.86±1.27μg/mL和62.97±1.22μg/mL,统计学结果显示总皂苷在7.82μg/mL以下浓度时CHO-WT的生长与对照组之间没有显著性差异(P＞D.05),在25μg/mL以下MT-2的生长与对照组之间没有显著性差异(P>D.05)。
结论：
1.茶多酚衍生物是文献报道能对HIV感染的多个环节都有抑制效果的化合物,茶黄素衍生物低浓度时没有表现出抑制CD4,趋化因子受体与gp120的相互作用却表现出抑制gp41核心结构6-HB形成的活性,且观察发现化合物中含有没食子酰基的比不含的活性好,含的个数越多活性越好,推测其活性可能是通过化合物自身带的微弱的负电荷与靶点(如N末端574位的赖氨酸)的正电荷形成离子键实现的。茶多酚自身的多酚结构使它具有上述活性,但是正因为含有多个酚羟基使化合物整体易氧化,不稳定,且与相并的酚酮七元环一起形成一个大共轭体系使得化合物颜色较深,水溶性也不好。我们的研究通过对三个衍生物的酚酮七元环的双键进行还原,在不破坏酚羟基的前提下尽量减少生色基团,并对改造后的产物进行体外活性检测。还原后化合物的颜色变浅,水溶性也增加了。ELISA, N-PAGE实验的结果都显示改造后化合物表现出良好的抑制六螺旋束形成的活性。细胞融合实验显示茶黄素衍生物表现出与文献报道值相似的活性,结构改造后的三个化合物活性比改造前还好,推测可能是因为改造后化合物水溶性提高,稳定性增强,且空间构象也发生了一些变化,相同条件下到达靶位的有效浓度提高,更加有利于化合物与靶点的结合。这些结果既为进一步的结构改造提供了方向又在化合物的作用机制研究方面提供了有力的证据。
2.番石榴叶提取物总黄酮和总皂苷虽然是混合成分,但主要成分黄酮和皂苷含量都在55%以上。二者都能在很大范围内安全有效的抑制HIV-1包膜介导的细胞融合,这说明二者可以在HIV的进入阶段起到抑制作用,且细胞毒性实验证明在有效范围内样品对细胞的生长没有抑制作用。而靶向gp41的抗体免疫实验ELISA表明二个样品都可以在微克每毫升的水平抑制gp41核心构象6-HB的形成,该结果还被N-PAGE实验证实,这说明二者在进入过程中的抑制作用可能是通过抑制了该过程中很重要的环节六股螺旋体的结构形成来实现的。
第二章ADS-J1抑制HIV进入的作用机制研究
目的：
通过建立和完善酸性非变性聚丙烯酰胺凝胶电泳(Acid native polyacrylamide gel electrophoresis AN-PAGE),研究和探讨ADS-J1与gp41相互作用的机制。
方法：
建立并完善AN-PAGE,在已有的ELISA, N-PAGE等结果的支持下更清楚直接的反映ADS-J1(4-羟基-5-((E)-(4-(5-羟基-6-((E)-2-甲氧基-5-磺苯基)二氮烯基)-7-磺基萘-2-苯基氨基)-6-苯胺-1,3,5-三嗪-2-苯基氨基)-5-甲氧基-2-甲基苯)二氮烯基)萘-2,7-二磺酸在HIV-1gp41上的作用位点,为其机制研究提供直接可靠的证据。
结果：
采用AN-PAGE能显示来源于gp41 CHR (C-terminal heptad repeat), NHR (N-terminal heptad repeat)不同区段的多肽条带,同时也能显示6-HB,以及与不同浓度的ADS-J1作用后的6-HB条带。结果表明,ADS-J1能梯度抑制C34与N36形成的6-HB条带,从0到100μM,6-HB的条带颜色逐渐变淡,与单独的C34共同孵育,C34条带没有变化,与单独的包含574位赖氨酸的N36共同孵育后,N36条带消失,与NHR上不包含574位点的其他多肽片段单独孵育,多肽条带都没有变化,与574位位点突变后的多肽片段孵育,对条带也没影响。
结论：
AN-PAGE的结果表明,从含有20000个化合物的化合物库中筛选出来的偶氮化合物ADS-J1对HIVgp41的作用是通过与gp41核心结构中的NHR同源三聚体螺旋结构表面的疏水深穴的第574位赖氨酸相互作用来实现的,可能是由于ADS-J1自身的磺酸基团与赖氨酸的正电荷之间形成了离子键,从而阻碍了gp41CHR上632位带负电荷的天冬氨酸与赖氨酸的相互作用,破坏了分子内部盐桥的形成,也破坏了六股α螺旋束的稳定性,从而发挥HIV进入抑制的功效。本实验中所用AN-PAGE方法采用pH值3.4的缓冲液体系,并反转了电极,对比N-PAGE中所用的pH值8.3的缓冲液体系,前者能使pI值在3.4-8.3之间的多肽片段都跑出条带,满足更多的实验需要,为靶向HIV-1包膜的病毒进入抑制剂的研究提供了一个方便有效的方法。
Background:
AIDS, caused by human immunodeficiency virus (HIV), threatens human health severely. According to the survey made by UNAIDS, the number of people infecting HIV was more than 33.4 million in 2008. Among them,2.7 million were newly infected in 2008. It was estimated that about 740,000 people were infected HIV till the end of October in 2009 dating from 1985 when the first HIV infected was reported in China. Some countries in Africa, HIV infection rates are more than 30% of the total population. Therefore, the prevention and treatment of AIDS is not only saving lives of individuals, but also the nation. Due to the difficulty of developing an effective AIDS vaccine in a short time, it's still very important to develop efficient, safe and cheap anti-HIV drugs. Currently, there are 26 reverse transcriptase inhibitors and protease inhibitors but only 3 entry inhibitors and integrase inhibitors used in clinic. However, the first two categories of drugs prone to induce viral resistance and serious side effects. Therefore, we should vigorously develop the latter two kinds of anti-HIV drugs to form more "cocktail" therapy protocols for patients infected with drug-resistant HIV strains. The HIV entry inhibitors would be more effective in the treatment and prevention of HIV infection since they block HIV replication at the early stages of infection.
Two HIV entry inhibitors have been approved, one targeted HIV transmembrane protein gp41 peptide named T-20 and the other, Maraviroc, targeted HIV co-receptor CCR5. The Maraviroc can only act on the R5 viruses which use CCR5 as the coreceptor but is no inhibition to X4 virus taking CXCR4 as a coreceptor. The T-20 is effective to both R5 and X4 virus. However, as a peptide, T-20 cannot act by oral administration and is easy to be degradated by protease in vivo. Furthermore, it's too expensive for people in developing county to afford for a long time. Therefore, it's urgently and necessary to develop small compounds with similar mechanism of action as T-20.
Based on laboratory experience in related fields, we screened a synthetic compound library and several natural products offered by other studying groups, to see whether these compounds have in vitro anti-HIV activities.. We also establish a new experimental method to investigate the interaction between ADS-J1 and gp41 to find out the binding sites of ADS-J1, a small molecular HIV entry inhibitor.
Part I Screening theaflavin derivates and Psidium guajava leaf extracts against HIV-1 entry
Aim:
To identify the inhibitory activity of theaflavin derivatives against HIV envelope glycoprotein induced cell-cell fusion. The mechanism of action of the compounds as HIV entry inhibitor was also investigated. The extracts from Psidium guajava leaf were tested on the formation of HIV gp41 six-helical bundle by immune and molecular biology testing.
Method:
1. Theaflavin derivatives, including TF1 (theaflavi), TF2A (theaflavin-3- monogallate), TF3 (theaflavin-3,3'-digallate), are extracted from black tea. We showed these compounds can inhibit HIV entry by targeting HIV gp41. However, these compounds are not stable and have deep colour, which are the shortcomings for developing them as microbicides to prevent HIV sexual transmission. Therefore, these theaflavin compounds were modified by adding H atoms to the double bonds of benzo annulen. The modified compounds were purified using reversed-phase column chromatography. The TSGL (total saponin of Psidium guajava) was purified and concentrated by loading the alcohol extractum of Psidium guajava leaf on SA-1 macroporous resin and then washed out with 80% (v/v) alcohol. Similarly, the TFGL (total flavone of Psidium guajava) was purified by FL-1 macroporous resin and washed out by 0.2% NaOH. The essential oil was gained by extracting the aqueous solution of Psidium guajava leaf for serval hours and collected the remainder at a high temperature.
2. The activities of the samples on blocking HIV-1 gp41 six-helical bundles (6-HB) forming were analyzed by ELISA and N-PAGE. All of the samples were firstly detected by antigen-specific antibody enzyme-linked immunosorbent assay. Sample with good activity would experience a second screening of gradient experimental to calculate the IC50. N-PAGE assay was used to identify the biologic activity by comparing the intensity of bands at the site of 6-HB.
3. Cell-cell fusion assay mediated by HIV-1 env was used to study the inhibitory activity at cellular level of theaflavins compound and active ingredients of guava leaves. According to the cell stated in the fusion, the cytotoxicity of samples were tested on CHO-WT and MT-2 by MTT.
Results:
1. All the purification and characterization of theaflavin derivates were carried out by the provider. We mainly focused on the biologic ability screening. Extractum of Psidium guajava leaf was offered by School of Traditional Chinese Medicine in Southern Medical University. The content of TSGL purified with SA-1 macropore resin was 55.3% and the TFGL with FL-1 was 55.22%.
2. The activities of theaflavin derivates against 6-HB formation tested by ELISA. Compound TF1 showed inhibition of 60% at 11.08μM while TF1-4H showed 50% inhibition at 0.63±0.27μM. In the second pair, the IC50 of TF2A is 2.60±0.56μM and TF2A-4H is 1.13±0.27μM. In the third pair, IC50 of TF3 is 0.88±0.34μM and TF3-4H is 1.74±0.55μM. The results of N-PAGE are consistent with that of ELISA. The intensity of bands at 6-HB became darker when the concentration of compounds getting lower. Our ELISA results of TF1, TF2A, TF3 are at the same level as reported in literature, ranging from 0 to 5 micromole. Results of Psidium guajava leaf by ELISA showed good activity against 6-HB formation, with an inhibition more than 80% at 25μg/mL, except essential oil. The IC50 of TFGL is 3.9±1.3μg/mL and the IC50 of TSGL is 9.06±0.17μg/mL. The results were confirmed by N-PAGE.
3. Results of cell-cell fusion for theaflavin derivates showed that the modified compounds had a smaller IC50 comparing with those before modifying. The IC50 of TF1 is 14.63±1.40μM while that of TF1-4H is 11.14±1.02μM. The IC50 of TF2A is 10.30±0.36μM while that of TF2A-4H is 6.44±0.51μM. Similarly, the IC50 of TF3 is 8.14±0.21μM and TF3-4H is 6.06±0.26μM. Both TFGL and TSGL showed inhibition activity against cell-cell fusion mediated by HIV-1 env. The IC50 of TFGL is 5.3±1.3μg/mL and that of TSGL is 7.33±0.40μg/mL. Cytotoxicity of TFGL to two cells are low, with CC50>100μg/mL. The CC50 of TSGL to CHO-WT is 46.86±1.27μg/mL and to MT-2 is 62.97±1.22μg/mL. Statistic analysis shows that there are no distinct difference on cell growth between compounds group and cell control group when the compounds group at concentration<7.82μg/mL (P>0.05), the same as MT-2 with compounds concentration<25μg/mL (P>0.05).
Conclusions:
1. Theaflavin derivates are reported to inhibit HIV-1 replication with multiple mechanisms. Theaflavin derivates at low concentration can't block the interaction between gp120 and CD4 molecules, nor interact with CXCR4 or CCR5, but can significantly inhibit the gp41 conformation changing by blocking the formation of six-helical bundles on gp41. The more the compounds contain galloyls, the better the inhibition activity is. It's presumed that the active polyphenols contain weak negative charges and these compounds may interact with the target proteins via ionic interactions. The structure of polyphenol gives the compounds weak negative charges together with the reducibility, instability, darker color and bad water solubility which make it difficult to use extensively. Our study planned to modify the structure to light color, enhance its stability and water solubility but not change the biologic ability especially on anti-HIV. Results of ELISA, N-PAGE and cell fusion assay show that they can still prevent HIV infecting at the same level as before modification. The results imply that we can continue in the direction and also it provides the proofs that the mechanism of action of theaflavin derivates is blocking HIV entry into target cells.
2. Though both the TFGL and the TSGL are admixtures, the main content of flavones and saponin are both up to 55%. It's reported that mount of polyphenol in the TFGL is no more than 1%. The admixture can inhibit the formation of syncytium at micromole level and has little cytotoxicity, indicating that they could act as an lead HIV entry inhibitors. Results of ELISA and N-PAGE showed they might act through preventing the formation of gp41 core six-helical bundle.
PartⅡThe study of mechanism of ADS-J1 on HIV-1 by acid-native polypropylene amide gel electrophoresis
Aim:
To investigate the mechanism of HIV-1 inhibitor ADS-J1 on HIV gp41, offer proof for the designing and developing of new small molecular compounds against HIV-1.
Method:
Establishing and improving the acid-native polypropylene amide gel electrophoresis to find out the action site of ADS-J1, (7-[6-phenylamino-4-[4-[(3,5-disulfo-8-hyd-roxyna phthyl)azo]-2-methoxy-5-Methyl phenylamino]-1,3,5-triazine-2-yl]-4-hydroxy-3-[(2-methoxy-5-sulfophenyl)azo]-2-naphthalene sulfonic acid) on gp41.
Results:
Acid-native PAGE can show bands of peptides from different fraction of gp41. Results of AN-PAGE implied that ADS-J1 can inhibit the interaction of N36 and C34, with the band of 6-HB get lighter while the concentration of ADS-J1 varied from 0 to 100μM. ADS-J1 did not change the band of only C34, but made the band disappeared when incubated with only N36 ADS-J1 did not change the band of peptides from different fraction of HIV gp41 NHR without cavity region, neither the band of mutated N36 on the residue at 574th position. These results showed that ADS-J1 can bind to the cavity region on gp41, and the residue at 574th position (K574) is the most important residue for the activity of ADS-J1 against gp416-HB formation.
Conclusion:
Result of AN-PAGE indicated that the ADS-J1 inhibit HIV via ionic interactions. Computer-aided molecule docking analysis in the present study indicates that the negative charged groups in acid groups can interact with a positive charge residue K574 located in the gp41 NHR cavity region that participates in the formation of hydrophobic pocket, thus blocking the salt bridge formation between K574 and D632, a negatively charged residue in the pocket binding region of the gp41 CHR. In the experiment AN-PAGE, a running buffer with pH 3.4 was used and a reversed electrode. AN-PAGE can show bands of peptides with the pⅠranged from 3.4-8.8 compare to the traditional N-PAGE. The method will make the study of HIV entry inhibitor easier.
引文
[1]Root MJ, Steger HK. HIV-1 gp41 as a target for viral entry inhibition[J].Curr Pharm Des,2004,10(15):1805-25.
[2]Jiang S, Zhao Q, Debnath AK. Peptide and non-peptide HIV fusion inhibitors[J].Curr Pharm Des,2002,8(8):563-80.
[3]夏承来,姜世勃,刘叔文.HIV包膜蛋白的结构及其相应的病毒进入抑制剂[J].中国药理学通报,2009，25(8)：991-994.
[4]Lai W, Huang L, Ho P, et al. Betulinic acid derivatives that target gp120 and inhibit multiple genetic subtypes of human immunodeficiency virus type 1[J].Antimicrob Agents Chemother,2008,52(1):128-36.
[5]De Clercq E. Current lead natural products for the chemotherapy of human immunodeficiency virus (HIV) infection[J].Med Res Rev,2000,20(5):323-49.
[6]Debnath AK, Radigan L, Jiang S. Structure-based identification of small molecule antiviral compounds targeted to the gp41 core structure of the human immunodeficiency virus type 1[J].J Med Chem,1999,42(17):3203-09.
[7]Jiang S, Lu H, Liu S, et al. N-substituted pyrrole derivatives as novel human immunodeficiency virus type 1 entry inhibitors that interfere with the gp41 six-helix bundle formation and block virus fusion[J].Antimicrob Agents Chemother,2004,48(11):4349-59.
[8]Liu K, Lu H, Hou L, et al. Design, synthesis, and biological evaluation of N-carboxyphenylpyrrole derivatives as potent HIV fusion inhibitors targeting gp41[J].J Med Chem,2008,51(24):7843-54.
[9]Asres K, Seyoum A, Veeresham C, et al. Naturally derived anti-HIV agents[J]. Phytother Res,2005,19(7):557-81.
[10]Inder Pal Singh, Sandip B. Bharate and K. K. Bhutani, et al. Anti-HIV natural products[J].Current Science,2005,89(2):269-290.
[11]赵声兰,陈朝银,董其江,等.皂荚皂苷的提取及其抗HIV、抗解脲支原体和抗菌作用的研究[J].陕西中医,2007，28(7)：923-925.
[12]张楠,张杰,葛海涛.薯蓣皂苷及其水解物治疗高脂血症及冠心病研究进展[J].上海中医药杂志,2009,23(9)：79-81.
[13]张均田.人参皂菅Rgl的促智作用机制——对神经可塑性和神经发生的影响[J].药学学报,2005,40(5)：385-388.
[14]张赞彬,李彩侠,吴亚卿.黄酮类化合物的研究进展[J].食品与机械,2005,21(5)：70-73.
[15]Ono K, Nakane H, Fukushima M, et al. Inhibition of reverse transcriptase activity by a flavonoid compound 5,6,7-trihydroxyflavone[J]. Biochem Biophys Res Commun,1989,160(3):982-87.
[16]刘景云,吴伯平.中草药防治艾滋病作用的探索[J].中华中医药杂志,1991,6(2)：52-54.
[17]Kawai K, Tsuno NH, Kitayama J, et al. Epigallocatechin gallate, the main component of tea polyphenol, binds to CD4 and interferes with gp120 binding[J].J Allergy Clin Immunol,2003,112(5):951-57.
[18]Fassina G, Buffa A, Benelli R, et al. Polyphenolic antioxidant (-)-epigallocatechin-3-gallate from green tea as a candidate anti-HIV agent [J].Aids, 2002,16(6):939-41.
[19]Yamaguchi K, Honda M, Ikigai H, et al.Inhibitory effects of (-)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (HIV-1)[J].Antiviral Res,2002,53(1):19-34.
[20]Liu S, Lu H, Zhao Q, et al.Theaflavin derivatives in black tea and catechin derivatives in green tea inhibit HIV-1 entry by targeting gp41[J].Biochim Biophys Acta,2005,1723(1-3):270-81.
[21]Chiang LC, Ng LT, Liu LT, et al. Cytotoxicity and anti-hepatitis B virus activities of saikosaponins from Bupleurum species[J].Planta Med,2003,69(8): 705-09.
[22]Desai SH, Chouksey A, Poll J, et al. A pilot study of equal doses of 10% IGIV given intravenously or subcutaneously[J].J Allergy Clin Immunol,2009,124(4): 854-56.
[23]李珉珉,李琳,姜世勃,等.基于细胞-细胞融合的HIV进入抑制剂非感染性筛选方法的研究[J].暨南大学学报(医学版),2007,28(6)：576-580.
[24]Liu S, Zhao Q, Jiang S. Determination of the HIV-1 gp41 fusogenic core conformation modeled by synthetic peptides:applicable for identification of HIV-1 fusion inhibitors[J].Peptides,2003,24(9):1303-13.
[25]Liu S, Boyer-Chatenet L, Lu H, et al. Rapid and automated fluorescence-linked immunosorbent assay for high-throughput screening of HIV-1 fusion inhibitors targeting gp41[J].J Biomol Screen,2003,8(6):685-93.
[26]Gutierrez RM, Mitchell S, Solis RV. Psidium guajava:a review of its traditional uses, phytochemistry and pharmacology[J].J Ethnopharmacol,2008, 117(1):1-27.
[27]薄华本,邵红伟,胡凌波,等.淋巴细胞活性检测方法研究[J].南方医科大学学报,2008,28(3)：409-410.
[28]Stone A, Jiang S. Microbicides:stopping HIV at the gate[J].Lancet,2006, 368(9534):431-33.
[29]黄建林,张展.GC-MS分析番石榴叶乙醇提取物的化学成分[J].中山大学学报(自然科学版),2004,43(6)：117-120.
[30]Chan DC, Fass D, Berger JM, et al. Core structure of gp41 from the HIV envelope glycoprotein[J].Cell,1997,89(2):263-73.
[31]Chan DC, Kim PS. HIV entry and its inhibition[J].Cell,1998,93(5):681-84.
[32]Armand-Ugon M, Clotet-Codina I, Tintori C, et al. The anti-HIV activity of ADS-J1 targets the HIV-1 gp120[J].Virology,2005,343(1):141-49.
[33]Sackett K, Wexler-Cohen Y, Shai Y. Characterization of the HIV N-terminal fusion peptide-containing region in context of key gp41 fusion conformations [J].J Biol Chem,2006,281(31):21755-62.
[34]Jiang S, Debnath AK. A salt bridge between an N-terminal coiled coil of gp41 and an antiviral agent targeted to the gp41 core is important for anti-HIV-1 activity[J].Biochem Biophys Res Commun,2000,270(1):153-57.
[35]He Y, Liu S, Jing W, et al. Conserved residue Lys574 in the cavity of HIV-1 Gp41 coiled-coil domain is critical for six-helix bundle stability and virus entry[J].J Biol Chem,2007,282(35):25631-39.
[36]He Y, Liu S, Li J, et al. Conserved salt bridge between the N-and C-terminal heptad repeat regions of the human immunodeficiency virus type 1 gp41 core structure is critical for virus entry and inhibition[J].J Virol,2008,82(22): 11129-39.
[37]Lin PF, Blair W, Wang T, et al. A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding[J].Proc Natl Acad Sci U S A,2003,100(19):11013-18.
[38]Wang H, Qi Z, Guo A, et al. ADS-J1 inhibits human immunodeficiency virus type 1 entry by interacting with the gp41 pocket region and blocking fusion-active gp41 core formation[J].Antimicrob Agents Chemother,2009,53(12): 4987-98.
[39]Liu S, Xiao G, Chen Y, et al. Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus:implications for virus fusogenic mechanism and identification of fusion inhibitors[J].Lancet,2004, 363(9413):938-47.
[2]Jiang S, Zhao Q, Debnath AK. Peptide and non-peptide HIV fusion inhibitors[J].Curr Pharm Des,2002,8(8):563-80.
[3]夏承来,姜世勃,刘叔文.HIV包膜蛋白的结构及其相应的病毒进入抑制剂[J].中国药理学通报,2009，25(8)：991-994.
[4]Lai W, Huang L, Ho P, et al. Betulinic acid derivatives that target gp120 and inhibit multiple genetic subtypes of human immunodeficiency virus type 1[J].Antimicrob Agents Chemother,2008,52(1):128-36.
[5]De Clercq E. Current lead natural products for the chemotherapy of human immunodeficiency virus (HIV) infection[J].Med Res Rev,2000,20(5):323-49.
[6]Debnath AK, Radigan L, Jiang S. Structure-based identification of small molecule antiviral compounds targeted to the gp41 core structure of the human immunodeficiency virus type 1[J].J Med Chem,1999,42(17):3203-09.
[7]Jiang S, Lu H, Liu S, et al. N-substituted pyrrole derivatives as novel human immunodeficiency virus type 1 entry inhibitors that interfere with the gp41 six-helix bundle formation and block virus fusion[J].Antimicrob Agents Chemother,2004,48(11):4349-59.
[8]Liu K, Lu H, Hou L, et al. Design, synthesis, and biological evaluation of N-carboxyphenylpyrrole derivatives as potent HIV fusion inhibitors targeting gp41[J].J Med Chem,2008,51(24):7843-54.
[9]Asres K, Seyoum A, Veeresham C, et al. Naturally derived anti-HIV agents[J]. Phytother Res,2005,19(7):557-81.
[10]Inder Pal Singh, Sandip B. Bharate and K. K. Bhutani, et al. Anti-HIV natural products[J].Current Science,2005,89(2):269-290.
[11]赵声兰,陈朝银,董其江,等.皂荚皂苷的提取及其抗HIV、抗解脲支原体和抗菌作用的研究[J].陕西中医,2007，28(7)：923-925.
[12]张楠,张杰,葛海涛.薯蓣皂苷及其水解物治疗高脂血症及冠心病研究进展[J].上海中医药杂志,2009,23(9)：79-81.
[13]张均田.人参皂菅Rgl的促智作用机制——对神经可塑性和神经发生的影响[J].药学学报,2005,40(5)：385-388.
[14]张赞彬,李彩侠,吴亚卿.黄酮类化合物的研究进展[J].食品与机械,2005,21(5)：70-73.
[15]Ono K, Nakane H, Fukushima M, et al. Inhibition of reverse transcriptase activity by a flavonoid compound 5,6,7-trihydroxyflavone[J]. Biochem Biophys Res Commun,1989,160(3):982-87.
[16]刘景云,吴伯平.中草药防治艾滋病作用的探索[J].中华中医药杂志,1991,6(2)：52-54.
[17]Kawai K, Tsuno NH, Kitayama J, et al. Epigallocatechin gallate, the main component of tea polyphenol, binds to CD4 and interferes with gp120 binding[J].J Allergy Clin Immunol,2003,112(5):951-57.
[18]Fassina G, Buffa A, Benelli R, et al. Polyphenolic antioxidant (-)-epigallocatechin-3-gallate from green tea as a candidate anti-HIV agent [J].Aids, 2002,16(6):939-41.
[19]Yamaguchi K, Honda M, Ikigai H, et al.Inhibitory effects of (-)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (HIV-1)[J].Antiviral Res,2002,53(1):19-34.
[20]Liu S, Lu H, Zhao Q, et al.Theaflavin derivatives in black tea and catechin derivatives in green tea inhibit HIV-1 entry by targeting gp41[J].Biochim Biophys Acta,2005,1723(1-3):270-81.
[21]Chiang LC, Ng LT, Liu LT, et al. Cytotoxicity and anti-hepatitis B virus activities of saikosaponins from Bupleurum species[J].Planta Med,2003,69(8): 705-09.
[22]Desai SH, Chouksey A, Poll J, et al. A pilot study of equal doses of 10% IGIV given intravenously or subcutaneously[J].J Allergy Clin Immunol,2009,124(4): 854-56.
[23]李珉珉,李琳,姜世勃,等.基于细胞-细胞融合的HIV进入抑制剂非感染性筛选方法的研究[J].暨南大学学报(医学版),2007,28(6)：576-580.
[24]Liu S, Zhao Q, Jiang S. Determination of the HIV-1 gp41 fusogenic core conformation modeled by synthetic peptides:applicable for identification of HIV-1 fusion inhibitors[J].Peptides,2003,24(9):1303-13.
[25]Liu S, Boyer-Chatenet L, Lu H, et al. Rapid and automated fluorescence-linked immunosorbent assay for high-throughput screening of HIV-1 fusion inhibitors targeting gp41[J].J Biomol Screen,2003,8(6):685-93.
[26]Gutierrez RM, Mitchell S, Solis RV. Psidium guajava:a review of its traditional uses, phytochemistry and pharmacology[J].J Ethnopharmacol,2008, 117(1):1-27.
[27]薄华本,邵红伟,胡凌波,等.淋巴细胞活性检测方法研究[J].南方医科大学学报,2008,28(3)：409-410.
[28]Stone A, Jiang S. Microbicides:stopping HIV at the gate[J].Lancet,2006, 368(9534):431-33.
[29]黄建林,张展.GC-MS分析番石榴叶乙醇提取物的化学成分[J].中山大学学报(自然科学版),2004,43(6)：117-120.
[30]Chan DC, Fass D, Berger JM, et al. Core structure of gp41 from the HIV envelope glycoprotein[J].Cell,1997,89(2):263-73.
[31]Chan DC, Kim PS. HIV entry and its inhibition[J].Cell,1998,93(5):681-84.
[32]Armand-Ugon M, Clotet-Codina I, Tintori C, et al. The anti-HIV activity of ADS-J1 targets the HIV-1 gp120[J].Virology,2005,343(1):141-49.
[33]Sackett K, Wexler-Cohen Y, Shai Y. Characterization of the HIV N-terminal fusion peptide-containing region in context of key gp41 fusion conformations [J].J Biol Chem,2006,281(31):21755-62.
[34]Jiang S, Debnath AK. A salt bridge between an N-terminal coiled coil of gp41 and an antiviral agent targeted to the gp41 core is important for anti-HIV-1 activity[J].Biochem Biophys Res Commun,2000,270(1):153-57.
[35]He Y, Liu S, Jing W, et al. Conserved residue Lys574 in the cavity of HIV-1 Gp41 coiled-coil domain is critical for six-helix bundle stability and virus entry[J].J Biol Chem,2007,282(35):25631-39.
[36]He Y, Liu S, Li J, et al. Conserved salt bridge between the N-and C-terminal heptad repeat regions of the human immunodeficiency virus type 1 gp41 core structure is critical for virus entry and inhibition[J].J Virol,2008,82(22): 11129-39.
[37]Lin PF, Blair W, Wang T, et al. A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding[J].Proc Natl Acad Sci U S A,2003,100(19):11013-18.
[38]Wang H, Qi Z, Guo A, et al. ADS-J1 inhibits human immunodeficiency virus type 1 entry by interacting with the gp41 pocket region and blocking fusion-active gp41 core formation[J].Antimicrob Agents Chemother,2009,53(12): 4987-98.
[39]Liu S, Xiao G, Chen Y, et al. Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus:implications for virus fusogenic mechanism and identification of fusion inhibitors[J].Lancet,2004, 363(9413):938-47.