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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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Effect of PBDEs on HBV production. HepG2.2.15.7 cells were incubated with various concentrations of compound 1 or 2. Supernatant HBV DNA and cytotoxicity were estimated by real-time qPCR and MTS assay, respectively, as described in the Experimental section. The data were representative of three independent experiments. Error bars indicate standard deviation.
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marinedrugs-13-06759-f005: Effect of PBDEs on HBV production. HepG2.2.15.7 cells were incubated with various concentrations of compound 1 or 2. Supernatant HBV DNA and cytotoxicity were estimated by real-time qPCR and MTS assay, respectively, as described in the Experimental section. The data were representative of three independent experiments. Error bars indicate standard deviation.

Mentions: We next addressed the effects of compounds 1 and 2 on HBV production and cell viability. HBV-producing cultured cells, HepG2.2.15.7, were incubated in culture medium containing various concentrations of compound 1 or 2. Entecavir was used as the positive control for anti-HBV activities of compound 1 and 2. The amount of supernatant HBV DNA and cell viability were measured by using real-time PCR and MTS assay, respectively. Treatment with compound 1 or 2 impaired production of HBV DNA in a dose-dependent manner (Figure 5). The IC50 and CC50 values of compound 1 were 0.23 µM and 4.19 µM, respectively, while the EC50 and CC50 values of compound 2 were 0.80 µM and 10.26 µM, respectively. Thus, the selectivity indexes of compounds 1 and 2 were 18.2 and 12.8, respectively (Table 2). These results suggest that compounds 1 and 2 possess anti-HBV activity. However, IC50 values of compound 1 and 2 were higher than that of entecavir, while compounds 1 and 2 were more toxic than entecavir (Table 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)

Effect of PBDEs on HBV production. HepG2.2.15.7 cells were incubated with various concentrations of compound 1 or 2. Supernatant HBV DNA and cytotoxicity were estimated by real-time qPCR and MTS assay, respectively, as described in the Experimental section. The data were representative of three independent experiments. Error bars indicate standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06759-f005: Effect of PBDEs on HBV production. HepG2.2.15.7 cells were incubated with various concentrations of compound 1 or 2. Supernatant HBV DNA and cytotoxicity were estimated by real-time qPCR and MTS assay, respectively, as described in the Experimental section. The data were representative of three independent experiments. Error bars indicate standard deviation.
Mentions: We next addressed the effects of compounds 1 and 2 on HBV production and cell viability. HBV-producing cultured cells, HepG2.2.15.7, were incubated in culture medium containing various concentrations of compound 1 or 2. Entecavir was used as the positive control for anti-HBV activities of compound 1 and 2. The amount of supernatant HBV DNA and cell viability were measured by using real-time PCR and MTS assay, respectively. Treatment with compound 1 or 2 impaired production of HBV DNA in a dose-dependent manner (Figure 5). The IC50 and CC50 values of compound 1 were 0.23 µM and 4.19 µM, respectively, while the EC50 and CC50 values of compound 2 were 0.80 µM and 10.26 µM, respectively. Thus, the selectivity indexes of compounds 1 and 2 were 18.2 and 12.8, respectively (Table 2). These results suggest that compounds 1 and 2 possess anti-HBV activity. However, IC50 values of compound 1 and 2 were higher than that of entecavir, while compounds 1 and 2 were more toxic than entecavir (Table 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