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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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Validation of cell based HBV core promoter reporter assay. Huh7 GL4.18 CURS_BC_AeUS cells (positive control) and Huh7 GL4.18 cells (negative control) were harvested at 72 h. The luciferase activity was determined as described in the Experimental Section. The Z′ factor and coefficient of variation (CV) value was calculated as described in the Experimental Section.
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marinedrugs-13-06759-f002: Validation of cell based HBV core promoter reporter assay. Huh7 GL4.18 CURS_BC_AeUS cells (positive control) and Huh7 GL4.18 cells (negative control) were harvested at 72 h. The luciferase activity was determined as described in the Experimental Section. The Z′ factor and coefficient of variation (CV) value was calculated as described in the Experimental Section.

Mentions: We calculated the Z′ factor in order to evaluate the Huh7 G4.18 CURS_BC_AeUS cell line for high-throughput screening. The Z′ factor is a useful tool for measurement of the quality or suitability of high throughput screening, and the value spanning from 0.5 to 1.0 exhibits an appropriate assay [24,25]. In this study, the value of Z′ factor was 0.79 (n = 48) using Huh7 GL4.18 CURS_BC_AeUS cells (Figure 2). The coefficient of variation (CV), which represents unevenness of the screening system, should be less than 10% for a correct assay [24]. The CV value of our system was 7.0% (Figure 2).


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)

Validation of cell based HBV core promoter reporter assay. Huh7 GL4.18 CURS_BC_AeUS cells (positive control) and Huh7 GL4.18 cells (negative control) were harvested at 72 h. The luciferase activity was determined as described in the Experimental Section. The Z′ factor and coefficient of variation (CV) value was calculated as described in the Experimental Section.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06759-f002: Validation of cell based HBV core promoter reporter assay. Huh7 GL4.18 CURS_BC_AeUS cells (positive control) and Huh7 GL4.18 cells (negative control) were harvested at 72 h. The luciferase activity was determined as described in the Experimental Section. The Z′ factor and coefficient of variation (CV) value was calculated as described in the Experimental Section.
Mentions: We calculated the Z′ factor in order to evaluate the Huh7 G4.18 CURS_BC_AeUS cell line for high-throughput screening. The Z′ factor is a useful tool for measurement of the quality or suitability of high throughput screening, and the value spanning from 0.5 to 1.0 exhibits an appropriate assay [24,25]. In this study, the value of Z′ factor was 0.79 (n = 48) using Huh7 GL4.18 CURS_BC_AeUS cells (Figure 2). The coefficient of variation (CV), which represents unevenness of the screening system, should be less than 10% for a correct assay [24]. The CV value of our system was 7.0% (Figure 2).

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