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. 2007 May 22;46(20):6050-9.
doi: 10.1021/bi700181j. Epub 2007 Apr 25.

Biochemical mechanism of hepatitis C virus inhibition by the broad-spectrum antiviral arbidol

Eve-Isabelle Pécheur  1 , Dimitri Lavillette, Fanny Alcaras, Jennifer Molle, Yury S Boriskin, Michael Roberts, François-Loïc Cosset, Stephen J Polyak
Affiliations

Biochemical mechanism of hepatitis C virus inhibition by the broad-spectrum antiviral arbidol

Eve-Isabelle Pécheur et al. Biochemistry. .

Abstract

Hepatitis C affects approximately 3% of the world population, yet its current treatment options are limited to interferon-ribavirin drug regimens which achieve a 50-70% cure rate depending on the hepatitis C virus (HCV) genotype. Besides extensive screening for HCV-specific compounds, some well-established medicinal drugs have recently demonstrated an anti-HCV effect in HCV replicon cells. One of these drugs is arbidol (ARB), a Russian-made broad-spectrum antiviral agent, which we have previously shown to inhibit acute and chronic HCV infection. Here we show that ARB inhibits the cell entry of HCV pseudoparticles of genotypes 1a, 1b, and 2a in a dose-dependent fashion. ARB also displayed a dose-dependent inhibition of HCV membrane fusion, as assayed by using HCV pseudoparticles (HCVpp) and fluorescent liposomes. ARB inhibition of HCVpp fusion was found to be more effective on genotype 1a than on genotypes 1b and 2a. In vitro biochemical studies revealed association of ARB with membranelike environments such as detergents and with lipid membranes. This association was particularly prominent at acidic pH which is optimal for HCV-mediated fusion. Our results suggest that the affinity of ARB for lipid membranes could account for its anti-HCV actions, together with a differential level of interaction with key motifs in HCV glycoproteins of different genotypes.

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Figures

Figure 1
Figure 1. Structures of arbidol (A), indole (B) and tryptophan (C)
(D), scheme of the protonation of substituted indole, and its effect on the 3-position [from (22)].
Figure 2
Figure 2. Arbidol effect on HCVpp infectivity
Huh-7 cells were seeded at 4.104 cells per well in 24-well plates the day before infection (day -1). At day 0, HCVpp (as a 100 × concentrated fraction; see Experimental Procedures) were added to cells, and incubated for 3h at 37°C, in the absence or presence of arbidol (see Experimental Procedures for other details). Panel A, ARB was added at indicated concentrations during the course of infection. Panel B, ARB was first preincubated at indicated concentrations with concentrated pseudoparticles for 3h, and infection was then performed in the presence of the same concentration of ARB. Panel C and D, ARB was preincubated at indicated concentrations with Huh-7 cells for 3h before infection, and HCVpp infection was performed in the presence (panel C) or absence (panel D) of ARB. HCV pseudoparticles tested were harboring the E1 and E2 glycoproteins of genotypes 1a (AF009606), 1b (AY734975 for 1b-I, and AF333324 for 1b-II), and 2a (AB047639). HApp and RD114pp are shown as positive and negative controls, respectively. Infectivity is expressed as the percentage of control, i.e. pseudoparticle infection of Huh-7 cells not treated with ARB, taken as 100%. Results are the mean ± standard deviation of 4 separate experiments.
Figure 3
Figure 3. Arbidol inhibits HCVpp-mediated lipid mixing
Lipid mixing curves of HCVpp genotypes 1a (A), 1b (B), and 2a (C), in the absence or presence of ARB, with R18-labeled liposomes (representative of 4 separate experiments). Pseudoparticles (40 μl) were added to R18-labeled PC:chol liposomes (15 μM final lipid concentration), in PBS pH 7.4 at 37°C, with or without indicated concentrations of ARB. After a 2-min equilibration, lipid mixing was initiated by decreasing the pH to 5.0 (time 0), and recorded as R18 fluorescence dequenching as a function of time. Panel D, influence of ARB on Ca2+-induced fusion of PS vesicles. Liposomes consisting of PS:R18 (96:4) were mixed with non-labeled PS vesicles (molar ratio 1:4) in 5 mM Hepes/100 mM NaCl pH 7.4 at 37°C, at a final lipid concentration of 100 μM. Fusion was induced by rapid injection of Ca2+ into the medium (2 mM final), and the kinetics of lipid dilution was continuously monitored. ARB was incubated with labeled and non-labeled PS vesicles for 1 min in buffer at pH 7.4 or at pH 5.0 (by adding diluted HCl), then Ca2+ was added at 2 mM final to initiate fusion.
Figure 4
Figure 4. Fluorescence properties of arbidol
All measurements were recorded at 37°C, with excitation (exc) and emission (em) slits set to 8 and 4 nm respectively. Relative orientations of exc. and em. polarizers were 90° and 0°, respectively. ARB absorbance (dotted curve) was recorded at pH 7.4, and fluorescence emission spectra were recorded at pH 7.4 (thin solid line) or pH 5.0 (thick solid line). Fluorescence was monitored using a 113 μM (60 μg/ml) ARB solution, with λexc = 255 nm.
Figure 5
Figure 5. Arbidol interaction with detergent micelles
Fluorescence conditions were similar as those in Fig.4. ARB (18.8 μM, 10 μg/ml) was added to PBS buffer containing either 100 mM SDS, or 100 μM α-lysoPC, or 100 mM DM, or no detergent, (A) at pH 7.4 or (B) at pH 5.0.
Figure 6
Figure 6. Binding of arbidol to SDS (A), α-lysoPC (B) and DM (C) at pH 5.0
Fluorescence conditions were similar as those in Fig.4. Increasing concentrations of detergent were added to a cuvette containing ARB (18.8 μM) in PBS buffer acidified at pH 5.0 and equilibrated at 37°C. Emission spectra for each concentration were recorded after a 5-min equilibration. The fluorescence intensities obtained after each detergent addition, corrected using blank values (detergent alone in buffer), were plotted as a percentage of the initial value, as a function of final detergent concentration.
Figure 7
Figure 7. Binding of arbidol to DM micelles : effect of pH
DM (final concentration 5 mM) was added to a 18.8 μM (10 μg/ml) ARB solution in PBS pH 7.4. Fluorescence conditions were similar as those in Fig.4. pH was decreased by addition of diluted HCl to the cuvette, and (A) emission spectra were recorded at pH 7.4 (a), 6.5 (b), 5.5 (c) or 4.5 (d), after a 5-min equilibration at 37°C. (B) plot of the fluorescence obtained at 350 nm from panel A as a function of pH, and expressed as a percentage of the initial value at pH 7.4.
Figure 8
Figure 8. Influence of liposomes on ARB fluorescence
(A) Neutral pH. PC:chol (70:30 molar ratio) liposomes were added at 12.5 μM (a), 25 μM (b), 62.5 μM (c) or 100 μM (d), to a 18.8 μM (10 μg/ml) ARB solution in PBS pH 7.4 (dotted curve). Emission spectra were recorded after 5 min of incubation, and corrected for blank (buffer) and liposome scattering effects. (B) Acidic pH. ARB solution (10 μg/ml) in PBS buffer pH 7.4 was acidified with HCl to pH 5.0 final reading (dotted curve), and PC:chol liposomes were subsequently added [final lipid concentration 12.5 μM (a), 25 μM (b) or 62.5 μM (c)].
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