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Identification of Antiviral Agents Targeting Hepatitis B Virus Promoter from Extracts of Indonesian Marine Organisms by a Novel Cell-Based Screening Assay.

Yamashita A, Fujimoto Y, Tamaki M, Setiawan A, Tanaka T, Okuyama-Dobashi K, Kasai H, Watashi K, Wakita T, Toyama M, Baba M, de Voogd NJ, Maekawa S, Enomoto N, Tanaka J, Moriishi K - Mar Drugs (2015)

Bottom Line: Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy)-phenol (compound 1) and 3,4,5-tribromo-2-(2,4-dibromophenoxy)-phenol (compound 2), which are classified as polybrominated diphenyl ethers (PBDEs), were identified as anti-HBV agents in the extracts.Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner.The EC50 values of compounds 1 and 2 were 0.23 and 0.80 µM, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Division of Medical Sciences, Graduate School of Interdisciplinary Research, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan. atsuyay@yamanashi.ac.jp.

ABSTRACT
The current treatments of chronic hepatitis B (CHB) face a limited choice of vaccine, antibody and antiviral agents. The development of additional antiviral agents is still needed for improvement of CHB therapy. In this study, we established a screening system in order to identify compounds inhibiting the core promoter activity of hepatitis B virus (HBV). We prepared 80 extracts of marine organisms from the coral reefs of Indonesia and screened them by using this system. Eventually, two extracts showed high inhibitory activity (>95%) and low cytotoxicity (66% to 77%). Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy)-phenol (compound 1) and 3,4,5-tribromo-2-(2,4-dibromophenoxy)-phenol (compound 2), which are classified as polybrominated diphenyl ethers (PBDEs), were identified as anti-HBV agents in the extracts. Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner. The EC50 values of compounds 1 and 2 were 0.23 and 0.80 µM, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively. These results suggest that our cell-based HBV core promoter assay system is useful to determine anti-HBV compounds, and that two PBDE compounds are expected to be candidates of lead compounds for the development of anti-HBV drugs.

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Development of Hepatitis B virus (HBV) core promoter reporter system. (A) Schematic representation of the firefly luciferase reporter plasmid pGL4.18 CURS_BC_AeUS and pGL4.18 BC_AeUS; (B) HBV core promoter activity in three cell lines. Each plasmid described above was transfected with phRG-TK into hepatic (Huh7) and non-hepatic (HeLa and HT-1080) cells. Luciferase activity was measured at 48 h post-transfection as described in the Experimental Section. Firefly luciferase activity was normalized with Renilla luciferase activity. Luciferase activity was expressed as a fold induction compared with the value of cells transfected with pGL4.18 [luc2P/Neo] empty control vector (control). The data shown in this panel are representative of three independent experiments. Error bars indicate standard deviation.
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marinedrugs-13-06759-f001: Development of Hepatitis B virus (HBV) core promoter reporter system. (A) Schematic representation of the firefly luciferase reporter plasmid pGL4.18 CURS_BC_AeUS and pGL4.18 BC_AeUS; (B) HBV core promoter activity in three cell lines. Each plasmid described above was transfected with phRG-TK into hepatic (Huh7) and non-hepatic (HeLa and HT-1080) cells. Luciferase activity was measured at 48 h post-transfection as described in the Experimental Section. Firefly luciferase activity was normalized with Renilla luciferase activity. Luciferase activity was expressed as a fold induction compared with the value of cells transfected with pGL4.18 [luc2P/Neo] empty control vector (control). The data shown in this panel are representative of three independent experiments. Error bars indicate standard deviation.

Mentions: The core promoter consists of CURS and basal core promoter (BCP) (Figure 1A) and is responsible for transcription of 3.5 kb mRNA, pgRNA [4]. CURS negatively and positively regulates the promoter activity [4]. The region composed of both CURS and BCP or BCP only was cloned into pGL4.18 [luc2P/Neo] plasmid (Figure 1A). The resulting plasmids were designated as pGL4.18 CURS_BC_AeUS (CURS BCP) or pGL4.18 BC_AeUS (BCP) in this study (Figure 1A). The plasmid pGL4.18 CURS_BC_AeUS or pGL4.18 BC_AeUS was transfected with phRG-TK into human hepatoma cell line Huh7, human cervical cancer cell line HeLa, and human fibrosarcoma cell line HT-1080. The resulting cells were harvested 48 h post-transfection and suspended in lysis buffer in order to estimate luciferase activity. Previous findings suggested that HBV core promoter (CURS and BCP) is more active in hepatoma cell lines than other cell lines [6,20,21,22,23]. In this study, Huh7 cell line exhibited the highest luciferase activity under the control of CURS BCP or BCP among tested cell lines (Figure 1B). Moreover, the Huh7 cells transfected with pGL4.18 CURS_BC_AeUS exhibited 5-time higher luciferase activity than the cells transfected with pGL4.18 BC_AeUS (Figure 1B). These results suggest its potential for establishment of a cell-based screening assay based on HBV promoter activity. The plasmid pGL4.18 CURS_BC_AeUS was introduced into Huh7 cells again for establishment of a stable cell line. The transfected cells were incubated in the presence of G418 until colony formation. The Huh7 cell line exhibiting the highest luciferase activity was selected by colony isolation, and designated as Huh7 GL4.18 CURS_BC_AeUS.


Identification of Antiviral Agents Targeting Hepatitis B Virus Promoter from Extracts of Indonesian Marine Organisms by a Novel Cell-Based Screening Assay.

Yamashita A, Fujimoto Y, Tamaki M, Setiawan A, Tanaka T, Okuyama-Dobashi K, Kasai H, Watashi K, Wakita T, Toyama M, Baba M, de Voogd NJ, Maekawa S, Enomoto N, Tanaka J, Moriishi K - Mar Drugs (2015)

Development of Hepatitis B virus (HBV) core promoter reporter system. (A) Schematic representation of the firefly luciferase reporter plasmid pGL4.18 CURS_BC_AeUS and pGL4.18 BC_AeUS; (B) HBV core promoter activity in three cell lines. Each plasmid described above was transfected with phRG-TK into hepatic (Huh7) and non-hepatic (HeLa and HT-1080) cells. Luciferase activity was measured at 48 h post-transfection as described in the Experimental Section. Firefly luciferase activity was normalized with Renilla luciferase activity. Luciferase activity was expressed as a fold induction compared with the value of cells transfected with pGL4.18 [luc2P/Neo] empty control vector (control). The data shown in this panel are representative of three independent experiments. Error bars indicate standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663552&req=5

marinedrugs-13-06759-f001: Development of Hepatitis B virus (HBV) core promoter reporter system. (A) Schematic representation of the firefly luciferase reporter plasmid pGL4.18 CURS_BC_AeUS and pGL4.18 BC_AeUS; (B) HBV core promoter activity in three cell lines. Each plasmid described above was transfected with phRG-TK into hepatic (Huh7) and non-hepatic (HeLa and HT-1080) cells. Luciferase activity was measured at 48 h post-transfection as described in the Experimental Section. Firefly luciferase activity was normalized with Renilla luciferase activity. Luciferase activity was expressed as a fold induction compared with the value of cells transfected with pGL4.18 [luc2P/Neo] empty control vector (control). The data shown in this panel are representative of three independent experiments. Error bars indicate standard deviation.
Mentions: The core promoter consists of CURS and basal core promoter (BCP) (Figure 1A) and is responsible for transcription of 3.5 kb mRNA, pgRNA [4]. CURS negatively and positively regulates the promoter activity [4]. The region composed of both CURS and BCP or BCP only was cloned into pGL4.18 [luc2P/Neo] plasmid (Figure 1A). The resulting plasmids were designated as pGL4.18 CURS_BC_AeUS (CURS BCP) or pGL4.18 BC_AeUS (BCP) in this study (Figure 1A). The plasmid pGL4.18 CURS_BC_AeUS or pGL4.18 BC_AeUS was transfected with phRG-TK into human hepatoma cell line Huh7, human cervical cancer cell line HeLa, and human fibrosarcoma cell line HT-1080. The resulting cells were harvested 48 h post-transfection and suspended in lysis buffer in order to estimate luciferase activity. Previous findings suggested that HBV core promoter (CURS and BCP) is more active in hepatoma cell lines than other cell lines [6,20,21,22,23]. In this study, Huh7 cell line exhibited the highest luciferase activity under the control of CURS BCP or BCP among tested cell lines (Figure 1B). Moreover, the Huh7 cells transfected with pGL4.18 CURS_BC_AeUS exhibited 5-time higher luciferase activity than the cells transfected with pGL4.18 BC_AeUS (Figure 1B). These results suggest its potential for establishment of a cell-based screening assay based on HBV promoter activity. The plasmid pGL4.18 CURS_BC_AeUS was introduced into Huh7 cells again for establishment of a stable cell line. The transfected cells were incubated in the presence of G418 until colony formation. The Huh7 cell line exhibiting the highest luciferase activity was selected by colony isolation, and designated as Huh7 GL4.18 CURS_BC_AeUS.

Bottom Line: Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy)-phenol (compound 1) and 3,4,5-tribromo-2-(2,4-dibromophenoxy)-phenol (compound 2), which are classified as polybrominated diphenyl ethers (PBDEs), were identified as anti-HBV agents in the extracts.Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner.The EC50 values of compounds 1 and 2 were 0.23 and 0.80 µM, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Division of Medical Sciences, Graduate School of Interdisciplinary Research, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan. atsuyay@yamanashi.ac.jp.

ABSTRACT
The current treatments of chronic hepatitis B (CHB) face a limited choice of vaccine, antibody and antiviral agents. The development of additional antiviral agents is still needed for improvement of CHB therapy. In this study, we established a screening system in order to identify compounds inhibiting the core promoter activity of hepatitis B virus (HBV). We prepared 80 extracts of marine organisms from the coral reefs of Indonesia and screened them by using this system. Eventually, two extracts showed high inhibitory activity (>95%) and low cytotoxicity (66% to 77%). Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy)-phenol (compound 1) and 3,4,5-tribromo-2-(2,4-dibromophenoxy)-phenol (compound 2), which are classified as polybrominated diphenyl ethers (PBDEs), were identified as anti-HBV agents in the extracts. Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner. The EC50 values of compounds 1 and 2 were 0.23 and 0.80 µM, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively. These results suggest that our cell-based HBV core promoter assay system is useful to determine anti-HBV compounds, and that two PBDE compounds are expected to be candidates of lead compounds for the development of anti-HBV drugs.

Show MeSH
Related in: MedlinePlus