Anti-viral mechanism of isosteviol derivative against hepatitis B virus

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Anti-viral mechanism of isosteviol derivative against hepatitis B virus

禮明）, and Shwu-Jiuan Lin（林淑娟）*

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Research Center for Biodiversity and Graduate Institute of Ecology and Evolutionary Biology, China Medical University, Taichung 404, Taiwan

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Department of Pharmaceutical Sciences, School of Pharmacy, Taipei Medical University, Taipei 110, Taiwan

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Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan

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Division of Medicinal Chemistry,National Research Institute of Chinese Medicine, Taipei 112, Taiwan

An isosteviol derived compound, ent-16-oxobeyeran-19-N-methylureido (NC-8), was synthesized and its anti-hepatitis B virus (HBV) ability was investigated in human hepatoma cell lines. Treatment with NC-8 specifically inhibited viral gene expression and reduced the encapsidated viral DNA intermediates in Huh7 cells that express replicating HBV. To further assess the NC-8 inhibitory mode, four promoter regions of viral genes were isolated and the promoter-reporter assay was conducted. Results revealed that treatment with NC-8 potently attenuated all of the viral promoter activity in HBV-expressing Huh7 cells, but not in HBV non-expressing cells. Further examination of the roles of this anti-viral action in cellular signaling pathways showed that NC-8 inhibited the activity of NF-B element containing promoter but enhanced the activities of AP-1 and ISRE containing promoters in HBV-expressing cells. Furthermore, treatment with NC-8 significantly eliminated NF-B and TLR2 protein levels in a dose-dependent manner in HBV transfected Huh7 cells, but not in non-transfected cells. In electrophoretic mobility shift assay (EMSA), the binding activity of NF-B to DNA element was enhanced in HBV transfected nuclear extracts of Huh7 cells, but treatment of NC-8 significantly reduced the DNA binding of NF-B. Taken together, this study suggests that NC-8 mediates the antiviral effect by disturbing the replication and gene expression of HBV, as well as inhibiting the host TLR2/NF-B signaling pathway.

Keywords: Isosteviol derivative; Hepatitis B virus; Human hepatoma cells; NF-B; Toll-like receptor 2