HIV假病毒的构建及抗HIV药物SPR、ELRSE筛选方法的建立
摘要
随着人类对艾滋病致病机制的深入研究,越来越多新的抗HIV药物进入临床试验阶段,但大量抗HIV药物的使用也导致了耐药性问题的出现。因此,建立高通量快速的抗HIV药物初步筛选平台对于研究新的抗HIV药物非常重要。针对传统抗HIV药物细胞水平筛选模型对实验室条件要求高,并且具有较高危险性等缺点,本论文通过构建HIV假病毒和应用体外药物靶向筛选方法来研究抗HIV药物的初步筛选,以建立快速方便且危险性较低抗HIV药物筛选和评价系统,用于新的抗HIV药物的开发。主要的研究内容如下：
(1)构建HIV假病毒,为进一步建立基于HIV假病毒的抗HIV药物筛选细胞模型提供基础。实验过程中成功构建出了具有感染活性的HIV假病毒。但由于其感染活性较低,尚不能应用于抗HIV药物的筛选。需要进一步对假病毒构建的实验过程进行优化,以提高假病毒的感染活性,
(2)利用SPR技术,建立抗HIV药物的筛选和分析方法,获得不同样品对HIV包膜蛋白rgp120 MN结合能力和平衡解离常数等数据,得出三个多糖样品中Za24-70-5-1002#与rgp120 MN的结合作用非常明显。动力学／亲和分析结果显示,Za24-70-5-1002#与rgp120 MN之间平衡解离常数KD(M)为1.172E-13M。两者之间具有很高的亲和力,因此Za24-70-5-10023可能具有较高的抗HIV活性,有必要对其药理作用进行进一步的研究。Za48-70-×100 J与rgp120 MN之间也具有比较好的亲和性,它们之间的平衡解离常数KD(M)为5.752E-6M。
(3)应用ELISA法建立抗HIV药物初步筛选方法,以细胞表面受体CD4作为研究靶位,研究多糖样品对gp120与CD4结合的抑制作用。通过实验测得13个不同样品对gp120与CD4结合的抑制效果,计算出不同样品对gp120与CD4结合抑制的IC5o值,得到不同样品的IC5o值在1.150μg/mL之间。为进一步的药物筛选和分析提供依据。
With increasingly understanding of mechanism of HIV infection, more and more new anti-HIV agents were developed and used in clinical experiments. However, the abuse of anti-HIV drugs was causing serious drug-resistance problem, so it is very important to develop new high-throughput screening systems for drug discovery. For the traditional anti-HIV drugs screening system based on cell model has high requirements on laboratory conditions and high risk of exposure to HIV, this dissertation study on anti-HIV drugs screening by constructing pseudovirus and using drug targeting screening in vitro, which are used for establishing high-thoughput and less dangerous anti-HIV drug evaluation systems. The main results can be listed as follows:
(1) The pseudotyped Human Immunodefieiency Virus-1(HIV-1) was constructed successfully and may lay foundations for establishing a pseudovirous-based system for anti-HIV drugs screening. Pseudovirous with infection activity have been successfully constructed. But the infection activities are low, so the pseudovirous can not be used in anti-HIV drug screening yet. Further research is needed to optimize the experimental conditions for improving the infection activity of pseudovirous.
(2) Establishing screening and evaluation system by surface plasmon resonance analysis is an effective way to develop new anti-HIV drugs. Three polysaccharide samples were been analyzed by this system. The equilibrium dissociation constant of interaction between rgp120 MN and Za24-70-5-1002# is 1.172E-13M by kinetics/affinity analysis through Biacore X100 evaluation software. The result shows that a strong interaction is exist between the two molecules. Therefore, this sample may have high anti-HIV activity. Otherwise, the equilibrium dissociation constant of interaction between rgp120 MN and Za48-70-×100 J is 5.752E-6M.
(3) Establishing screening system by ELISA can be also a good method to discover new anti-HIV drugs. Using the cell surface receptor CD4 as a research target, the inhibitory effects of the binding between gp120 and CD4 by polysaccharide were analyzed. The inhibitory effects of 13 different samples were detected by ELISA. The IC50 values of all samples were calculated out and they are 1-150μg/mL. Some samples may have anti-HIV activity. And it can be proved and analyzed in further experiments.
(1)构建HIV假病毒,为进一步建立基于HIV假病毒的抗HIV药物筛选细胞模型提供基础。实验过程中成功构建出了具有感染活性的HIV假病毒。但由于其感染活性较低,尚不能应用于抗HIV药物的筛选。需要进一步对假病毒构建的实验过程进行优化,以提高假病毒的感染活性,
(2)利用SPR技术,建立抗HIV药物的筛选和分析方法,获得不同样品对HIV包膜蛋白rgp120 MN结合能力和平衡解离常数等数据,得出三个多糖样品中Za24-70-5-1002#与rgp120 MN的结合作用非常明显。动力学／亲和分析结果显示,Za24-70-5-1002#与rgp120 MN之间平衡解离常数KD(M)为1.172E-13M。两者之间具有很高的亲和力,因此Za24-70-5-10023可能具有较高的抗HIV活性,有必要对其药理作用进行进一步的研究。Za48-70-×100 J与rgp120 MN之间也具有比较好的亲和性,它们之间的平衡解离常数KD(M)为5.752E-6M。
(3)应用ELISA法建立抗HIV药物初步筛选方法,以细胞表面受体CD4作为研究靶位,研究多糖样品对gp120与CD4结合的抑制作用。通过实验测得13个不同样品对gp120与CD4结合的抑制效果,计算出不同样品对gp120与CD4结合抑制的IC5o值,得到不同样品的IC5o值在1.150μg/mL之间。为进一步的药物筛选和分析提供依据。
With increasingly understanding of mechanism of HIV infection, more and more new anti-HIV agents were developed and used in clinical experiments. However, the abuse of anti-HIV drugs was causing serious drug-resistance problem, so it is very important to develop new high-throughput screening systems for drug discovery. For the traditional anti-HIV drugs screening system based on cell model has high requirements on laboratory conditions and high risk of exposure to HIV, this dissertation study on anti-HIV drugs screening by constructing pseudovirus and using drug targeting screening in vitro, which are used for establishing high-thoughput and less dangerous anti-HIV drug evaluation systems. The main results can be listed as follows:
(1) The pseudotyped Human Immunodefieiency Virus-1(HIV-1) was constructed successfully and may lay foundations for establishing a pseudovirous-based system for anti-HIV drugs screening. Pseudovirous with infection activity have been successfully constructed. But the infection activities are low, so the pseudovirous can not be used in anti-HIV drug screening yet. Further research is needed to optimize the experimental conditions for improving the infection activity of pseudovirous.
(2) Establishing screening and evaluation system by surface plasmon resonance analysis is an effective way to develop new anti-HIV drugs. Three polysaccharide samples were been analyzed by this system. The equilibrium dissociation constant of interaction between rgp120 MN and Za24-70-5-1002# is 1.172E-13M by kinetics/affinity analysis through Biacore X100 evaluation software. The result shows that a strong interaction is exist between the two molecules. Therefore, this sample may have high anti-HIV activity. Otherwise, the equilibrium dissociation constant of interaction between rgp120 MN and Za48-70-×100 J is 5.752E-6M.
(3) Establishing screening system by ELISA can be also a good method to discover new anti-HIV drugs. Using the cell surface receptor CD4 as a research target, the inhibitory effects of the binding between gp120 and CD4 by polysaccharide were analyzed. The inhibitory effects of 13 different samples were detected by ELISA. The IC50 values of all samples were calculated out and they are 1-150μg/mL. Some samples may have anti-HIV activity. And it can be proved and analyzed in further experiments.
引文
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[37]Takeuchi Y, Simpson G, Vile RG et al. Retroviral pseudotypes produced by rescue of a Moloney murine leukemia virus vector by C-type, but not D-type, retroviruses[J]. Virology,1992,186(2):792-794.
[38]苗文泉,李敬云.抗HIV药物的筛选评价方法[J].国外医学药学分册,2007,34(3)：170-173.
[39]Kageyama S, Kurokawa M, Shiraki K. Extract of prunella vulgaris spikes inhibits HIV replication at reverse transcription in vitro and can be absorbed from intestine in vivo[J]. Antivir Chem Chemother,2000,11 (2):157-164.
[40]Westby M, Nakayama GR, Butler SL, et al. Cell-based and biochemical screening approaches for the discovery of novel HIV-1 inhibitors [J]. Antiviral Res, 2005,67(3):121-140.
[41]Garcia-Perez J, Sanchez-Palomino S, Perez-Olmeda M, et al. A new strategy based on recombinant viroses as a tool for assessing drug susceptibility of human immunodeficiency virus type 1[J]J Med Virol,2007,79(2):127-137.
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[2]黄文林.分子病毒学.北京.人民卫生出版社,2002.
[3]Turner BG, Summers MF. Structural biology of HIV [J]. J Mol Biol.1999 285(1):1-32
[4]Frankel AD,Young JA.HIV-1:fifteen proteins and an RNA.Annu Rev Biochem 1998;67:1-25.
[5]Hager-Braun C, Tomer KB. Characterization of the tertiary structure of soluble CD4 bound to glycosylated full-length HIVgp120 by chemical modification of arginine residues and mass spectrometric analysis[J]. Biochemistry,2002, 41(6):1759-66
[6]Greenberg M, Cammack N, Salgo M, et al. HIV fusion and its inhibition in antiretroviral therapy[J]. Rev Med Virol,2004,14(5):321-37
[7]Weiss RA. Gulliver's travels in HIVland [J]. Nature,2001,410(6831):963-7
[8]Pata JD, Stirtan WG, Goldstein SW, et al. Structure of HIV-1 reverse transcriptase bound to an inhibitor active against mutant reverse transcriptases resistant to other nonnucleoside inhibitors[J]. Proc Natl Acad Sci,2004,101(29):10548-53
[9]Weiss RA. Gulliver's travels in HIVland [J]. Nature,2001,410(6831):963-7
[10]Rizzuto CD, Wyatt R, Hernandez-Ramos N, et al. A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding[J]. Science,1998 280(5371):1949-53
[11]Cochrane A. Controlling HIV-1 Rev function[J]. Curr Drug Targets Immune Endocr Metabol Disord,2004,4(4):287-95
[12]Bukrinskaya AG. HIV-1 assembly and maturation[J]. Arch Virol, 2004,149(6):1067-82.
[13]Spence RA,Kati WM,Anderson KS,et al.Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors.Science 1995;267(5200):988-93.
[14]Shnaper S, Sackett K, Gallo SA, et al. The C2and the N2termi2nal regions of glycop rotein 41 ectodomain fuse membranes en2 riched and not enriched with cholesterol, respectively[J]. J B iolChem,2004,279 (18):18526-18534.
[15]Morse C, Maldarelli F. Enfuvirtide antiviral activity desp ite rebound viremia and resistance mutations:fitness tampering or acase of persistent braking on entering? [J]. J Infect D is,2007,195 (3):318-321.
[16]Riccio R, Kinnel RB, Bifulco G, et al. Kakelokelose, a sulfated mannose polysaccharide with anti-HIV activity from the Pacific tunicate Didemnum molle[J]. Tetrahedron Letters,1996,37(12):1979-1982
[17]丁保金,金丽琴,吕建新.多糖的生物活性研究进展[J].中国药学杂志,2004,39(8)： 561-565
[18]Zhao HB, Lin SQ, Liu JH, et al. Polysaccharide extract isolated from ganoderma lucidum protects rat cerebral cortical neurons from hypoxia/reoxygenation injury [J]. J Pharm Sci,2004,95(2):294-298
[19]Talarico LB, Zibetti RGM, Faria PCS, et al. Anti-herpes simplex virus activity of sulfated galactans from the red seaweeds Gymnogongrus griffithsiae and Cryptonemia crenulata[J]. Int J Biol Macromol,2004,34(2):63-71
[20]Briggs JA, Wilk T, Fuller SD. Do lipid rafts mediate virus assembly and pseudotyping[J]. J Gen Virol,2003,84(pt4):757-768
[21]Sanders DA. No false start for novel pseudotyped vectors[J]. Curr Opin Biotechnol,2002,13(5):437-442.
[22]Cohen J. Did Merck's failed HIV vaccine cause harm?[J]. Science,2007, 318(5853):1048-1049.
[23]Cohen J. Promising AIDS vaccine's failure leaves field reeling[J]. Science,2007, 318(5847):28-29.
[24]Heyndrickx L, Vermoesen T, Vereecken K, et al. Antiviral compounds show enhanced activity in HIV-1 single cycle pseudovirus assays as compared to classical PBMC assays[J]. J Virol Methods,2008,148(1-2):166-173.
[25]种辉辉,许四宏,王佑春.评价HIV-1抗病毒药物的重组假病毒法的建立及其初步应用[J].药物分析杂志,2008,28(6):924-927.
[26]Adelson ME, Pacchia AL, Kaul M, et al. Toward the development of a virus-cell-based assay for the discovery of novel compounds against human immunodeficiency virus type 1[J]. Antimicrob Agents Chemother,2003,47(2): 501-508.
[27]Garcia JM, Gao A, He PL, et al. High-throughput screening using pseudotyped lentiviral particles:A strategy for the identification of HIV-1 inhibitors in a cell-based assay[J]. Antiviral Res,2009,81(3):239-247.
[28]Wei X, Decker JM, Liu H, et al. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy[J]. Antimicrob Agents Chemother,2002.46(6):1896-1905.
[29]Wei X, Decker JM, Wang S, et al. Antibody neutralization and escape by HIV-1. Nature,2003,422(6929):307-312.
[30]He J, Choe S, Walker R, et al. Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity[J]. J Virol,1995,69(11):6705-6711.
[31]仇超,彭虹,黄相刚等.携带绿色荧光蛋白基因的单轮感染活性HIV假病毒的建立及其活性检测[J].中华微生物学和免疫学杂志.2006,26(5):394-398.
[32]曹颖莉,郭颖.应用假病毒技术研究HIV-1复制抑制剂[J].药学学报,2008,43(3):253-258.
[33]Mastromarino P, Conti C, Goldoni P, et al. Characterization of membrane components of the erythrocyte involved in vesicular stomatitis virus attachment and fusion at acidic pH[J]. J. Gen. Virol,1987,68(pt9):2359-2369.
[34]Petropoulos CJ, Parkin NT, Limoli KL, et al. A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1[J]. Antimicrob Agents Chemother,2000,44(4):920-928.
[35]Chan E, Heilek-Snyder G, Cammack N, et al. Development of a Moloney murine leukemia virus-based pseudotype anti-HIV assay suitable for accurate and rapid evaluation of HIV entry inhibitors[J]. J Biomol Screen,2006,11(6):652-663.
[36]Lee MK, Seo JK, Kim HK, et al.A vector system for introducing foreign HIV-1 env genes and pseudotyping of MuLV particles with the recombinant HIV-1 envelope proteins for anti-HIV-1 assay [J]. Antiviral Res,2002,53(2):99-111.
[37]Takeuchi Y, Simpson G, Vile RG et al. Retroviral pseudotypes produced by rescue of a Moloney murine leukemia virus vector by C-type, but not D-type, retroviruses[J]. Virology,1992,186(2):792-794.
[38]苗文泉,李敬云.抗HIV药物的筛选评价方法[J].国外医学药学分册,2007,34(3)：170-173.
[39]Kageyama S, Kurokawa M, Shiraki K. Extract of prunella vulgaris spikes inhibits HIV replication at reverse transcription in vitro and can be absorbed from intestine in vivo[J]. Antivir Chem Chemother,2000,11 (2):157-164.
[40]Westby M, Nakayama GR, Butler SL, et al. Cell-based and biochemical screening approaches for the discovery of novel HIV-1 inhibitors [J]. Antiviral Res, 2005,67(3):121-140.
[41]Garcia-Perez J, Sanchez-Palomino S, Perez-Olmeda M, et al. A new strategy based on recombinant viroses as a tool for assessing drug susceptibility of human immunodeficiency virus type 1[J]J Med Virol,2007,79(2):127-137.
[42]Stuyver L,Wyseur A, Rombout A, et al. Line probe assay for rapid detection of drug-selected mutations in the human immunodeficiency virus type 1 reverse transcriptase gene[J]. Antimicrob Agents Chemother,1997,41(2):284-291.
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