Yamashita A et al. (2012), Inhibition of hepatitis C virus replication and...

ECB-ART-42463

Mar Drugs 2012 Apr 01;104:744-761. doi: 10.3390/md10040744.

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Inhibition of hepatitis C virus replication and viral helicase by ethyl acetate extract of the marine feather star Alloeocomatella polycladia.

Yamashita A , Salam KA , Furuta A , Matsuda Y , Fujita O , Tani H , Fujita Y , Fujimoto Y , Ikeda M , Kato N , Sakamoto N , Maekawa S , Enomoto N , Nakakoshi M , Tsubuki M , Sekiguchi Y , Tsuneda S , Akimitsu N , Noda N , Tanaka J , Moriishi K .

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Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC(50) of 11.7 µg/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC(50) value of 23 to 44 µg/mL, which is similar to the IC(50) value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C.

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Genes referenced: LOC100893907 LOC115919910 LOC579132 LOC581395 LOC587333 LOC587482 LOC587800 LOC590297 polr3a

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	Figure 1. Schematic representation of the PET assay system for unwinding activity of HCV NS3 helicase. The fluorescent dye (BODIPY FL) is attached to the cytosine at the 5'-end of the fluorescent strand and quenched by the guanine base at the 3'-end of the complementary strand via photoinduced electron transfer. When the helicase unwinds the double-strand RNA substrate, the fluorescence of the dye emits bright light upon the release of the dye from the guanine base. The capture strand, which is complementary to the complementary strand, prevents the reannealing of the unwound duplex.
	Figure 2. Alloeocomatella polycladia belongs to a class of feather star (Echinodermata, Crinoidea). The ethyl acetate fraction prepared from the marine organism was designated SG1-23-1 in this study.
	Figure 3. Effect of SG1-23-1 on the unwinding activity of NS3 helicase. (A) NS3 helicase activity was measured by PET assay. The reactions were carried out in the absence or presence of SG1-23-1. Helicase activity in the absence of SG1-23-1 was defined as 100% helicase activity. Each value represents the mean of three independent reactions. Error bars indicate standard deviation. The data represent three independent experiments. (B) The unwinding activity of NS3 helicase was measured by RNA unwinding assay using radioisotope-labeled RNA. The heat-denatured single-strand RNA (26-mer) and the partial duplex RNA substrate were applied to lanes 1 and 2, respectively. The duplex RNA was reacted with NS3 (300 nM) in the presence of SG1-23-1 (lanes 4 to 9, 16 to 500 µg/mL). The resulting samples were subjected to native polyacrylamide gel electrophoresis.
	Figure 4. Effect of SG1-23-1 on ATPase and RNA-binding activities of NS3 helicase.(A) The reaction mixtures were incubated with [γ-32P] ATP as described in Materials and Methods. The reaction mixtures were subjected to thin-layer chromatography. The start positions and migrated positions of ATP and free phosphoric acid are indicated as “Origin”, “32P-ATP”, and “32P-Pi”, respectively, on the left side of this figure. The data represent three independent experiments. (B) Gel mobility shift assay for RNA-binding activity of NS3 helicase. The reaction was carried out at the indicated concentration of SG1-23-1. The reaction mixture was subjected to gel mobility shift assay. The data represent three independent experiments.
	Figure 5. Effect of SG1-23-1 on viral replication in replicon cell lines. (A–D) Huh7 Lunet/Con1 LUN Sb #26 (A), Huh7 rep Feo (B), Huh7#94/ORN3-5B#24 (C), and OR6 (D) cell lines were incubated in medium containing various concentrations of SG1-23-1. Luciferase and cytotoxicity assays were carried out as described in Materials and Methods. Error bars indicate standard deviation. The data represent three independent experiments. (E) Protein extract was prepared from Huh7 Lunet/Con1 LUN Sb #26 cells treated for 72 h with an indicated concentration of SG1-23-1 and then was subjected to Western blotting using antibodies to NS3, NS5A, and beta-actin. (F) Huh7 cell line transfected with pEF Fluc IN vector or pEF Rluc IN was established in the presence of G418. Both cell lines were incubated without (control) and with 50 μg/mL SG1-23-1. Firefly or Renilla luciferase activity was measured 72 h post-treatment. Luciferase activity was normalized with protein concentration. Error bars indicate standard deviation. The data represent three independent experiments. (G) Schematic structure of the plasmid, pEFRluc EMCV IRES Feo. The bicistronic gene is transcribed under the control of elongation factor 1α (EF1α) promoter. The upstream cistron encoding Renilla luciferase (RLuc) is translated by a cap-dependent mechanism. The downstream cistron encodes the fusion protein (Feo), which consists of the firefly luciferase (Fluc) and neomycin phosphotransferase (Neor), and is translated under the control of the EMCV IRES. (H) Huh7 cell line transfected with pEF Rluc EMCV IRES Feo was established in the presence of G418. The cells were incubated for 72 h without (control) and with 50 µg/mL of SG1-23-1. Firefly or Renilla luciferase activity was measured by the method described in Materials and Methods and was normalized by the protein concentration. F/R: Relative ratio of Firefly luciferase activity to Renilla luciferase activity. F/R is presented as a percentage of the control condition. Error bars indicate standard deviation. The data represent three independent experiments.
	Figure 6. Effect of SG1-23-1 on interferon signaling pathway. Huh7 Lunet/Con1 LUN Sb #26 cells were treated without (lane 1) or with 1, 10, or 100 U/mL IFNa-2b (lanes 2–4), and 50 µg/mL SG1-23-1 (lane 5) for 48 h. The mRNAs of MxA, 2',5'-OAS, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control were detected by reverse-transcription polymerase chain reaction (RT-PCR). Error bars indicate standard deviation. The data represent three independent experiments.

References [+] :

Ahmed-Belkacem, Silibinin and related compounds are direct inhibitors of hepatitis C virus RNA-dependent RNA polymerase. 2010, Pubmed