Table 1.
Citotoxicity of the compounds.
Fig 1.
Five ε-Pol binding inhibitors selected by pull-down assay-based screening.
(A) Scheme of the detection of ε-Pol binding by pull-down assays. The lysate of HEK-293T cells expressing 3×FLAG-Pol was incubated with ε-biotin, and pulled down using streptavidin sepharose. Precipitated 3×FLAG-Pol was detected by a Western blot analysis using an anti-FLAG antibody. (B) A Western blot analysis for Pol pulled-down by the indicated RNAs (ε RNA with biotin, ε RNA without biotin, and control RNA with biotin). (C and D) A Western blot analysis for Pol pulled-down by 10 pmol ε-biotin in the presence of the indicated compounds. Lamivudine was used as a negative control. Arrows: bands detected at a position of the estimated mass of full-length 3×FLAG-Pol.
Fig 2.
Rosmarinic acid and quercetin are specific inhibitors of ε-Pol binding.
(A) Seventy-one-base ssDNA-biotin was pulled-down in the presence of the indicated compounds, and detected by EtBr staining. Arrow: 71-base ssDNA. (B) dsRNA-RIG-I EMSA in the presence of the indicated compounds. *: monomeric dsRNA, **: dsRNA-RIG-I complex. (C) A Western blot analysis for ISG20 D94G pulled-down by ε-biotin in the presence of the indicated compounds. Arrow: 3×ISG20 D94G.
Fig 3.
Rosmarinic acid inhibits HBV DNA production in cell lines stably expressing HBV.
Hep38.7-Tet cells were treated with rosmarinic acid at the indicated concentrations, 100 nM lamivudine, or 400 ng/ml tetracycline (A and B), or with 30 μM rosmarinic acid and/or 10 nM lamivudine, 100 nM Lam, or 400 ng/ml tetracycline (C and D). Five days after the induction of HBV expression, extracellular HBV DNA was quantified by qPCR (A and C), and cell viability was measured using WST-1 reagent (B and D). Data are from one representative of at least two independent experiments; the means and S.D. of triplicate experiments are shown (*: p < 0.05, **: p < 0.01, ***: p < 0.001).
Fig 4.
Rosmarinic acid suppresses HBV replication in HBV-infected primary human hepatocytes.
(A-C) PXB-cells were infected with HBV, and treated with 30 μM rosmarinic acid or 500 nM lamivudine. Seven to 12 days post-infection, extracellular HBV DNA was quantified by qPCR, intracellular HBV 3.5 kb RNA was quantified by RT-qPCR, and SHBs were measured by ELISA. (D-F) PXB-cells were infected with HBV, and were treated with 30 μM rosmarinic acid and/or 20 nM lamivudine. Extracellular HBV DNA, intracellular HBV 3.5-kb RNA, and SHBs were measured as in (A-C). Data are from one representative of at least two independent experiments; the means and S.D. of duplicate or triplicate experiments are shown (* p < 0.05, ** p < 0.01). N.D.: not detected, n.s.: not significant.
Fig 5.
Rosmarinic acid derivatives also inhibit ε-Pol binding.
(A) Western blot analysis for Pol pulled-down by ε-biotin in the presence of the indicated compounds. Arrows: 3×FLAG-Pol. (B) ssDNA-biotin was pulled-down in the presence of the indicated compounds, and detected by EtBr staining. Arrow: 71-base ssDNA. (C) dsRNA-RIG-I EMSA in the presence of the indicated compounds (100 μM). *: monomeric dsRNA, **: the dsRNA-RIG-I complex. (D) Hep38.7-Tet cells were treated with 10 μM rosmarinic acid, compounds 12c, 13c, 2, and 21a, 100 nM lamivudine, or 400 ng/ml tetracycline. Extracellular HBV DNA and cell viability were measured as in (Fig 3A and 3B). Data are from one representative of at least two independent experiments; the means and S.D. of triplicate experiments are shown (* p < 0.05, ** p < 0.01, *** p < 0.001). (E) A Western blot analysis for Pol pulled-down by ε-biotin in the presence of the indicated compounds. Arrows: 3×FLAG-Pol. Spliced images are indicated with a dividing line.
Table 2.
Rosmarinic acid derivatives.
Fig 6.
Chemical structures of compounds with ε-Pol inhibitory activity (A), and without ε-Pol inhibitory activity (B).