Inhibition of Hepatitis C Virus NS3 Helicase by Manoalide

The hepatitis C virus (HCV) causes one of the most prevalent chronic infectious diseases in the world, hepatitis C, which ultimately develops into liver cancer through cirrhosis. The NS3 protein of HCV possesses nucleoside triphosphatase (NTPase) and RNA helicase activities. As both activities are essential for viral replication, NS3 is proposed as an ideal target for antiviral drug development. In this study, we identified manoalide (<b>1</b>) from marine sponge extracts as an RNA helicase inhibitor using a high-throughput screening photoinduced electron transfer (PET) system that we previously developed. Compound <b>1</b> inhibits the RNA helicase and ATPase activities of NS3 in a dose-dependent manner, with IC₅₀ values of 15 and 70 μM, respectively. Biochemical kinetic analysis demonstrated that <b>1</b> does not affect the apparent <i>K</i>_m value (0.31 mM) of NS3 ATPase activity, suggesting that <b>1</b> acts as a noncompetitive inhibitor. The binding of NS3 to single-stranded RNA was inhibited by <b>1</b>. Manoalide (<b>1</b>) also has the ability to inhibit the ATPase activity of human DHX36/RHAU, a putative RNA helicase. Taken together, we conclude that <b>1</b> inhibits the ATPase, RNA binding, and helicase activities of NS3 by targeting the helicase core domain conserved in both HCV NS3 and DHX36/RHAU

Inhibitory Effects of Caffeic Acid Phenethyl Ester Derivatives on Replication of Hepatitis C Virus

<div><p>Caffeic acid phenethyl ester (CAPE) has been reported as a multifunctional compound. In this report, we tested the effect of CAPE and its derivatives on hepatitis C virus (HCV) replication in order to develop an effective anti-HCV compound. CAPE and CAPE derivatives exhibited anti-HCV activity against an HCV replicon cell line of genotype 1b with EC₅₀ values in a range from 1.0 to 109.6 µM. Analyses of chemical structure and antiviral activity suggested that the length of the n-alkyl side chain and catechol moiety are responsible for the anti-HCV activity of these compounds. Caffeic acid n-octyl ester exhibited the highest anti-HCV activity among the tested derivatives with an EC₅₀ value of 1.0 µM and an SI value of 63.1 by using the replicon cell line derived from genotype 1b strain Con1. Treatment with caffeic acid n-octyl ester inhibited HCV replication of genotype 2a at a similar level to that of genotype 1b irrespectively of interferon signaling. Caffeic acid n-octyl ester could synergistically enhance the anti-HCV activities of interferon-alpha 2b, daclatasvir, and VX-222, but neither telaprevir nor danoprevir. These results suggest that caffeic acid n-octyl ester is a potential candidate for novel anti-HCV chemotherapy drugs.</p></div

Effect of caffeic acid esters 7and 8 on HCV replication.

<p>Chemical structures of both compounds are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082299#pone.0082299.s003" target="_blank">Figure S3</a></p><p>a: Fifty percent effective concentration based on the inhibition of HCV replication.</p><p>b: Fifty percent cytotoxicity concentration based on the reduction in cell viability.</p><p>c: Selectivity index (CC₅₀/EC₅₀).</p><p>d: Determined with ChemDraw software (Chem Bio Office Ultra, 2008).</p

Effect of CAPE derivatives on the interferon-signaling pathway.

<p>(A) Plasmids pISRE-TA-Luc and phRG-TK were co-transfect into Huh7 OK1 cells. The transfected cells were cultured with 1, 10, 100, or 1000 U/mL of interferon-alpha 2b, and compounds <b>1</b>, <b>6</b> and <b>10</b>. Treatment with DMSO corresponds to ‘0’. After 48 h of treatment, luciferase activities were measured, and the value were normalized against <i>Renilla</i> luciferase activities. Error bars indicate standard deviation. The data represent three independent experiments. (B) Huh7 replicon cell line of genotype 1b was treated with 1, 10, 100, or 1000 U/mL of interferon-alpha 2b, and compounds <b>1</b>, <b>6</b> and <b>10</b> for 48 h. Treatment with DMSO corresponds to the control. The mRNAs of Mx1, MxA, IFIT4, ISG15, OAS1, OAS2, OAS3, and GAPDH as an internal control were detected by RT-PCR.</p

Effect of CAPE on viral replication in the replicon cell line of genotype 1b.

<p>(A) Molecular structure of CAPE. (B) Huh7/Rep-Feo cells were incubated for 72 h in a medium containing various concentrations of CAPE. Luciferase and cytotoxicity assays were carried out by the method described in Materials and Methods. Error bars indicate standard deviation. The data represent results from three independent experiments. (C) Protein extract was prepared from Huh7/Rep-Feo cells treated for 72 h with the indicated concentration of CAPE and it was then was subjected to Western blotting using antibodies to NS3 and beta-actin.</p

Correlation between the inhibitory effect on HCV replication <i>and C</i>log <i>P</i> of CAPE analogues.

<p>Values of <i>x</i>-axis indicate EC₅₀ values of CAPE analogues, while values of <i>y</i>-axis show <i>C</i>log <i>P</i> values. (A) Correlation between the inhibitory effect on HCV replication and <i>C</i>log <i>P</i> of CAPE analogues (Compound 7–11). (B) Correlation between the inhibitory effect on HCV replication and <i>C</i>log <i>P</i> of CAPE analogues (Compound 10 and 13–16).</p

Anti-HCV activity of compound 10 in replicon cell lines of genotypes 1b and 2a.

<p>a: Fifty percent effective concentration based on the inhibition of HCV replication.</p><p>b: Fifty percent cytotoxicity concentration based on the inhibition of HCV replication.</p><p>c: Selectivity Index (CC₅₀/EC₅₀).</p

Effect of octyl esters 10 and 13–16 on HCV replication.

<p>The basic structure and side moieties are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082299#pone.0082299.s004" target="_blank">Figure S4</a>.</p><p>a: Fifty percent effective concentration based on the inhibition of HCV replication.</p><p>b: Fifty percent cytotoxicity concentration based on the reduction in cell viability.</p><p>c: Selectivity index (CC₅₀/EC₅₀).</p><p>d: Determined with ChemDraw software (Chem Bio Office Ultra, 2008).</p

Effect of compound 10 on the viral replication in the replicon cell line and HCVcc.

<p>(A) Molecular structure of compound <b>10</b>. (B) Huh7/Rep-Feo cells were incubated for 72 h in a medium containing various concentrations of compound <b>10</b>. Luciferase and cytotoxicity assays were carried out by the method described in Materials and Methods. Error bars indicate standard deviation. The data represent three independent experiments. (C) Protein extract was prepared from Huh7/Rep-Feo cells treated for 72 h with the indicated concentration of compound <b>10</b> and it was then subjected to Western blotting using antibodies to NS3 and beta-actin. (D) Huh7 cell line was transfected with pEF Fluc IN encoding firefly luciferase or pEF Rluc IN encoding <i>Renilla</i> luciferase. Both transfected cell lines were incubated with DMSO (Control) or 40 µg/ml compound <b>10</b>. Firefly or <i>Renilla</i> luciferase activity was measured 72 h post-treatment. Luciferase activity was normalized with protein concentration. Error bars indicate standard deviation. The data were represented from three independent experiments. (E) Schematic structure of RNA transcribed from the plasmids was shown (Top). The bicistronic gene is transcribed under the control of elongation factor 1α (EF1α) promoter. The upstream cistron encoding <i>Renilla</i> 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 (Neo^r), and is translated under the control of the EMCV or HCV IRES. Huh7 cell line was transfected with each plasmid and incubated for 72 h post-treatment with DMSO (control) or 40 µg/ml of compound <b>10</b>. Firefly and <i>Renilla</i> luciferase activities were measured. Relative ratio of Firefly luciferase activity to <i>Renilla</i> luciferase activity was represented as percentage of the control condition. Error bars indicate standard deviation. The data were represented from three independent experiments. (F) Huh7 OK1 cell line was infected with HCVcc derived from JFH-1 strain and then treated with several concentrations of compound <b>10</b> at 24 h post-infection. The resulting cells were harvested 72 h post-infection. The viral RNA of supernatant was purified and estimated by the method described in Materials and Methods. Error bars indicate standard deviation. The data represent three independent experiments. Treatment with DMSO corresponds to ‘0’.</p

Effect of caffeic acid esters 7, 9–14, including 1, on HCV replication.

<p>The basic structure and side moieties are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082299#pone.0082299.s002" target="_blank">Figure S2</a>.</p><p>a: Fifty percent effective concentration based on the inhibition of HCV replication.</p><p>b: Fifty percent cytotoxicity concentration based on the reduction in cell viability.</p><p>c: Selectivity index (CC₅₀/EC₅₀).</p><p>d: Determined with ChemDraw software (Chem Bio Office Ultra, 2008).</p