Inhibitory effects of caffeic acid phenethyl ester derivatives on replication of hepatitis C virus

Abstract

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.

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

(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.

Figure 2. Correlation between the inhibitory effect on HCV replication and Clog P of CAPE analogues.

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

Figure 3. Effect of compound 10 on the viral replication in the replicon cell line and HCVcc.

(A) Molecular structure of compound 10. (B) Huh7/Rep-Feo cells were incubated for 72 h in a medium containing various concentrations of compound 10. 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 10 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 Renilla luciferase. Both transfected cell lines were incubated with DMSO (Control) or 40 µg/ml compound 10. 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 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 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 (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 10. Firefly and Renilla luciferase activities were measured. Relative ratio of Firefly luciferase activity to Renilla 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 10 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’.

Figure 4. Effect of CAPE derivatives on the interferon-signaling pathway.

(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 1, 6 and 10. Treatment with DMSO corresponds to ‘0’. After 48 h of treatment, luciferase activities were measured, and the value were normalized against Renilla 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 1, 6 and 10 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.

Figure 5. Synergistic effect analyses for the combination of compound 10 with IFN-α 2b, daclatasvir, VX-222, telaprevir, or danoprevir.

The Huh7 cell line, including the subgenomic replicon RNA of genotype 1b strain Con1, was treated for 72h with combinations of compound 10 and IFN-α 2b, daclatasvir, VX-222, telaprevir, or danoprevir. Luciferase assay were carried out as described in Materials and Methods. (A) The calculated EC₉₀ values for combination were plotted as the fractional concentration (FC) of compound 10 and one of IFN-α 2b, daclatasvir, VX-222, telaprevir, and danoprevir on the x and y axes, respectively. Synergy, antagonism and additivity are indicated in a representative graph as a right end of lower graphs and are described in Materials and Methods. (B) Combination indexes of compound 10 with IFN-α 2b, daclatasvir, VX-222, telaprevir, or danoprevir at the EC50, EC75, and EC90 values were measured at various drug rations. Synergy, antagonism and additivity are indicated in a representative graph as a right end of lower graphs and are described in Materials and Methods.