筛选附加型DNA的抑制剂，鉴定出dicumarol作为乙型肝炎病毒抑

StephenW

PLoS One. 2019 Feb 19;14(2):e0212233. doi: 10.1371/journal.pone.0212233. eCollection 2019.
Screening for inhibitor of episomal DNA identified dicumarol as a hepatitis B virus inhibitor.
Takeuchi F1,2, Ikeda S1,2, Tsukamoto Y2,3, Iwasawa Y1,2, Qihao C1,2, Otakaki Y1,2, Ryota O2,4, Yao WL1,2, Narita R2,5, Makoto H1,2, Watashi K6,7,8, Wakita T6, Takeuchi K9, Chayama K10, Kogure A2, Kato H1,2,3, Fujita T1,2.
Author information

1
    Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
2
    Department of Virus Research, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.
3
    Institute of Cardiovascular Immunology, University Hospital Bonn, Bonn, Germany.
4
    Department of Immunology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Hokkaido, Japan.
5
    Centre for Structural Biology, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
6
    Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
7
    Department of Applied Biological Science, Tokyo University of Science, Noda, Japan.
8
    CREST, Japan Science and Technology Agency (JST), Saitama, Japan.
9
    Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan.
10
    Liver Research Project Center, Hiroshima University, Hiroshima, Japan.

Abstract

Currently, there is no available therapy to eradicate hepatitis B virus (HBV) in chronically infected individuals. This is due to the difficulty in eliminating viral covalently closed circular (ccc) DNA, which is central to the gene expression and replication of HBV. We developed an assay system for nuclear circular DNA using an integration-deficient lentiviral vector. This vector produced non-integrated circular DNA in nuclei of infected cells. We engineered this vector to encode firefly luciferase to monitor the lentiviral episome DNA. We screened 3,840 chemicals by this assay for luciferase-reducing activity and identified dicumarol, which is known to have anticoagulation activity. We confirmed that dicumarol reduced lentiviral episome DNA. Furthermore, dicumarol inhibited HBV replication in cell culture using NTCP-expressing HepG2 and primary human hepatocytes. Dicumarol reduced intracellular HBV RNA, DNA, supernatant HBV antigens and DNA. We also found that dicumarol reduced the cccDNA level in HBV infected cells, but did not affect HBV adsorption/entry. This is a novel assay system for screening inhibitors targeting nuclear cccDNA and is useful for finding new antiviral substances for HBV.

PMID:
    30779774
DOI:
    10.1371/journal.pone.0212233

StephenW

PLoS One。 2019年2月19日; 14（2）：e0212233。 doi：10.1371 / journal.pone.0212233。 eCollection 2019。
筛选附加型DNA的抑制剂，鉴定出dicumarol作为乙型肝炎病毒抑制剂。
Takeuchi F1,2，Ikeda S1,2，Tsukamoto Y2,3，Iwasawa Y1,2，Qihao C1,2，Otakaki Y1,2，Ryota O2,4，Yao WL1,2，Narita R2,5，Makoto H1,2， Watashi K6,7,8，Wakita T6，Takeuchi K9，Chayama K10，Kogure A2，Kato H1,2,3，Fujita T1,2。
作者信息

1
    日本京都京都大学生物学研究科。
2
    日本京都京都大学边境生活与医学科学研究所病毒研究室。
3
    德国波恩大学医院心血管免疫学研究所。
4
    日本北海道北海道大学医学部和医学研究科免疫学系。
五
    丹麦奥胡斯奥胡斯大学分子生物学和遗传学系结构生物学中心。
6
    日本东京国立传染病研究所病毒学系II。
7
    日本野田大学东京理科大学应用生物科学系。
8
    CREST，日本科学技术厅（JST），日本埼玉县。
9
    日本东京国立先进工业科学技术研究所（AIST）药物发现分子谱分析研究中心。
10
    广岛大学肝脏研究项目中心，日本广岛。

抽象

目前，在慢性感染的个体中没有可用的根除乙型肝炎病毒（HBV）的疗法。这是由于难以消除病毒共价闭合环状（ccc）DNA，这是HBV基因表达和复制的核心。我们使用整合缺陷的慢病毒载体开发了核环状DNA的测定系统。该载体在感染细胞的细胞核中产生非整合的环状DNA。我们设计了这种载体来编码萤火虫荧光素酶来监测慢病毒附加体DNA。我们通过该测定筛选了3,840种化学物质用于荧光素酶还原活性，并鉴定了已知具有抗凝血活性的杀螨醇。我们证实dicumarol减少了慢病毒附加体DNA。此外，dicumarol使用表达NTCP的HepG2和原代人肝细胞抑制细胞培养物中的HBV复制。 Dicumarol减少细胞内HBV RNA，DNA，上清液HBV抗原和DNA。我们还发现，dicumarol降低了HBV感染细胞的cccDNA水平，但不影响HBV的吸附/进入。这是一种用于筛选靶向核cccDNA的抑制剂的新型测定系统，可用于寻找新的HBV抗病毒物质。

结论：
    30779774
DOI：
    10.1371 / journal.pone.0212233