从FDA批准药物库中筛选影响乙型肝炎病毒核衣壳形成的药物的初步研究
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摘要
目的从FDA已批准的药物库中,筛选可以干扰乙肝病毒核衣壳形成的药物。方法利用细胞模型SRluc-HBc6,通过分段荧光素酶互补实验来指示二聚体的形成,筛选靶向HBV核心蛋白二聚体形成的药物;对初筛出来的候选化合物,用颗粒凝胶电泳分析法鉴定其对HBV核衣壳形成的影响;用Southern blot方法鉴定候选药物对HBV DNA复制的影响。结果利用SRluc-HBc6细胞模型筛选了FDA批准的1 434种药物,第1轮筛选鉴定到5种药物:苯丁酸氮芥(Chlorambucil)、克拉屈滨(Cladribine)、亚甲蓝(Methylene Blue)、索拉菲尼(Sorafenib)、瑞格菲尼(Regorafenib);进一步测试发现,苯丁酸氮芥可以减少HBV核心蛋白和核衣壳的形成,抑制HepAD38细胞中HBV DNA的复制。结论苯丁酸氮芥通过减少HBV核心二聚体和衣壳的形成来抑制HBV DNA的复制。
Objective To identify the candidates targeting capsid formation of HBV by screening a FDA approved drug library. Methods SRluc-HBc6 cells, which indicates the formation of HBc dimers by the split luciferase complementation(SLC), were used as the 1 st round screening model. Compounds identified by the 1 st round screening were tested for their ability to inhibit the formation of capsid by using particle gel assay. Southern blotting was employed to detect the anti-HBV activity of these compounds on the replication of HBV DNA. Results According to the Rluc activities of SRluc-HBc6 cells, 5 candidates were screened out of 1 434 drugs in the first round screening. They are chlorambucil, cladribine, methylene blue, sorafenib, and regorafenib. Further tests showed that chlorambucil inhibited the replication of HBV DNA in HepAD38 cells by reducing the formation of HBV core dimers and capsids. Conclusion Chlorambucil inhibit HBV DNA replication in vitro by reducing HBV core dimers and the formation of capsid.
Objective To identify the candidates targeting capsid formation of HBV by screening a FDA approved drug library. Methods SRluc-HBc6 cells, which indicates the formation of HBc dimers by the split luciferase complementation(SLC), were used as the 1 st round screening model. Compounds identified by the 1 st round screening were tested for their ability to inhibit the formation of capsid by using particle gel assay. Southern blotting was employed to detect the anti-HBV activity of these compounds on the replication of HBV DNA. Results According to the Rluc activities of SRluc-HBc6 cells, 5 candidates were screened out of 1 434 drugs in the first round screening. They are chlorambucil, cladribine, methylene blue, sorafenib, and regorafenib. Further tests showed that chlorambucil inhibited the replication of HBV DNA in HepAD38 cells by reducing the formation of HBV core dimers and capsids. Conclusion Chlorambucil inhibit HBV DNA replication in vitro by reducing HBV core dimers and the formation of capsid.
引文
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[3] PERRILLO R.Benefits and risks of interferon therapy for hepatitis B[J].Hepatology,2009,49(5 Suppl):S103-S111.DOI:10.1002/hep.22956.
[4] ZOULIM F,LEBOSSé F,LEVRERO M.Current treatments for chronic hepatitis B virus infections[J].Curr Opin Virol,2016,18:109-116.DOI:10.1016/j.coviro.2016.06.004.
[5] LEVRERO M,TESTONI B,ZOULIM F.HBV cure:why,how,when?[J].Curr Opin Virol,2016,18:135-143.DOI:10.1016/j.coviro.2016.06.003.
[6] ZLOTNICK A,VENKATAKRISHNAN B,TAN Z N,et al.Core protein:A pleiotropic keystone in the HBV lifecycle[J].Antiviral Res,2015,121:82-93.DOI:10.1016/j.antiviral.2015.06.020.
[7] WU G Y,LIU B,ZHANG Y J,et al.Preclinical characterization of GLS4,an inhibitor of hepatitis B virus core particle assembly[J].Antimicrob Agents Chemother,2013,57(11):5344-5354.DOI:10.1128/AAC.01091-13.
[8] LAM A M,REN S P,ESPIRITU C,et al.Hepatitis B virus capsid assembly modulators,but not nucleoside analogs,inhibit the production of extracellular pregenomic RNA and spliced RNA variants[J].Antimicrob Agents Chemother,2017,61(8):e00680-e00617.DOI:10.1128/AAC.00680-17.
[9] DAWOOD A,BASIT S A,JAYARAJ M,et al.Drugs in development for hepatitis B[J].Drugs,2017,77(12):1263-1280.
[10] KATO N,JONES J.The split luciferase complementation assay[J].Methods Mol Biol,2010:359-376.DOI:10.1007/978-1-60761-765-5_24.
[11] AZAD T,TASHAKOR A,HOSSEINKHANI S.Split-luciferase complementary assay:applications,recent developments,and future perspectives[J].Anal Bioanal Chem,2014,406(23):5541-5560.DOI:10.1007/s00216-014-7980-8.
[12] LADNER S K,OTTO M J,BARKER C S,et al.Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells:A novel system for screening potential inhibitors of HBV replication[J].Antimicrob Agents Chemother,1997,41(8):1715-1720.
[13] WEI X F,GAN C Y,CUI J,et al.Identification of compounds targeting hepatitis B virus core protein dimerization through a split luciferase complementation assay[J].Antimicrob Agents Chemother,2018,62(12):e01302-e01318.DOI:10.1128/AAC.01302-18.
[14] GOEDE V,EICHHORST B,FISCHER K,et al.Past,present and future role of chlorambucil in the treatment of chronic lymphocytic leukemia[J].Leuk Lymphoma,2015,56(6):1585-1592.DOI:10.3109/10428194.2014.963077.
[15] YOGARAJAH M,TEFFERI A.Leukemic transformation in myeloproliferative neoplasms:A literature review on risk,characteristics,and outcome[J].Mayo Clin Proc,2017,92(7):1118-1128.DOI:10.1016/j.mayocp.2017.05.010.
[16] RAI K R,JAIN P.Chronic lymphocytic leukemia (CLL)—Then and now[J].Am J Hematol,2016,91(3):330-340.DOI:10.1002/ajh.24282.
[17] JAIN N,O’BRIEN S.Initial treatment of CLL:integrating biology and functional status[J].Blood,2015,126(4):463-470.DOI:10.1182/blood-2015-04-585067.