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

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.

Human induced-pluripotent stem cell-derived hepatocyte-like cells as an in vitro model of human hepatitis B virus infection

“…The HBV inoculum used in this experiment was genotype D, and was derived from the culture supernatant of HepG2.2.15.7 cells30, which are a HepG2.2.15 subclone producing a high titer of HBV. HBV pregenome (pg) RNA and total HBV RNAs were detected in iPS-HLCs following inoculation (Fig.…”

“…HepG2.2.15.7 cells30, which are a HepG2.2.15 clone producing a higher level of HBV, were also cultured in DMEM/F-12-Glutamax (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml insulin, and 10 mM HEPES. All cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2.…”

Marine natural products

“…Two sponge-derived polybrominated biphenyls 689,690 isolated from Indonesian Dysidea species were found to possess hepatitis antiviral activity with selectivity indices of 12.8-18.2. 691 Transcriptomic analysis of HepG2 hepatocarcinoma cells treated with both Crambe crambe metabolites crambescin C1 (ref. 692) and A1 (ref.…”

Four Cytotoxic Spongian Diterpenes from the Sponge <i>Dysidea</i> cf. <i>arenaria</i>

“…Examples include dysidaminones, which show nuclear factorkappa B (NF-κB) inhibitory activity 3) ; dysifragilones, which are nitric oxide (NO) inhibitors 4) ; dysidinoids, which possess anti-Methicillin-resistant Staphylococcus aureus (MRSA) activity 5) ; polybrominated diphenyl ethers (PBDEs), which have antiviral activity against hepatitis B virus 6) ; etc. 2,[7][8][9][10][11][12] In the course of our studies of novel bioactive molecules from coral reef organisms, 13) we have reported a series of cytotoxic spongian diterpenes from the soft white encrusting sponge D. cf.…”