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

Арбидол - статья о эффекте арбидола при гепатите С. Сделана совместно с французами и американцами

Eve-Isabelle Pécheur,§* Dimitri Lavillette,§† Fanny Alcaras,§◊ Jennifer Molle,§◊ Yury S. Boriskin,¶ Michael Roberts,‡ François-Loïc Cosset,§† and Stephen J. Polyak#

IFR128 Biosciences Lyon Gerland, Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS-Université Claude Bernard Lyon I, 7 passage du Vercors, 69367 Lyon Cedex 07, France
† Université de Lyon, (UCBL 1), IFR128 Biosciences Lyon Gerland; INSERM, U758, Lyon; Ecole Normale Supérieure de Lyon, F-69007, France

Institute of Virology, Medical Academy of Sciences, Moscow, Russia

Global Phasing Ltd, Sheraton House, Castle Park, Cambridge CB3 0AX, UK

Virology Division, Department of Laboratory Medicine, School of Medicine, University of Washington, 325 9th avenue, Seattle, Washington 98104-2499, USA
* Corresponding author, IBCP, UMR 5086 CNRS-UCBL, 7 passage du Vercors, 69367 Lyon Cedex 07, France. Phone: 33-4-72-72-26-44; Fax: 33-4-72-72-26-04; E-mail: e.pecheur@ibcp.fr
◊These authors contributed equally to this work.
Small right arrow pointing to: The publisher‘s final edited version of this article is available at Biochemistry.


          o Abstract
          o Experimental procedures
          o Results
          o Discussion
          o References

Abstract

Hepatitis C affects about 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 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 more effective on genotype 1a than on genotypes 1b and 2a. In vitro biochemical studies revealed ARB association with membrane-like 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.

   

 
The hepatitis C virus (HCV) infects an estimated 3% or 170 million of the world‘s population, and hepatitis C is now the most frequent indication for liver transplantation. Current treatment options are limited to pegylated recombinant interferon alpha (IFN-α) in combination with ribavirin. However viremia eradication is variably achieved depending on the genotype, with only 50% of virus eradication in genotype 1-infected patients. This is clearly a problem in North America, Europe and Japan, where genotype 1 is the most prevalent genotype. HCV therefore appears resistant to IFN antiviral therapy, and this is likely to be due to some factors of viral origin (1).
Historically, the development of new anti-HCV drugs has been hampered due to the lack of cell culture and small animal models that are required for pre-clinical evaluations of antiviral compounds. The generation of an HCV replicon system (2) has afforded massive anti-viral drug screening efforts (3). Distinct from specific anti-HCV compounds that target key viral functions, are a group of broad-spectrum medicinal drugs that were originally designed for other treatments or targeted toward other viruses. Using the replicon system, these compounds have been shown to possess potent antiviral activity toward HCV (4, 5). The advantage of this group of antivirals is that they have already met the pharmacological criteria for medicinal drugs and are already approved for clinical use in some countries.
One of these compounds, the Russian-made arbidol [ARB; 1H-Indole-3-carboxylic acid, 6-bromo-4-((dimethylamino)methyl)-5-hydroxy-1-methyl-2-((phenylthio)methyl)-, ethyl ester, monohydrochloride; CAS Registry Number 131707-23-8] (Fig. 1Figure 1) was originally described as an anti-influenza drug with purported immunostimulant properties (6). ARB has been licensed for several years in Russia for use as prophylaxis and treatment for influenza A and B infections. It allegedly exerts its effect by activation of macrophage phagocytic activity, and may also stimulate aspects of cellular and humoral immunity. ARB inhibits influenza virus-induced membrane fusion, and may have the capacity to induce serum interferon (7). More recent studies extended its inhibitory activity to other human viruses such as the respiratory syncytial virus, parainfluenza virus 3, rhinovirus 14 (8) and hepatitis B virus (9). Bird viruses such as the avian coronavirus and the H5/N1 influenza A virus were shown to be sensitive to ARB as well (10, 11). We recently demonstrated that ARB inhibits both acute and chronic HCV infection in vitro, and HCV replication using the HCV replicon system (12). In the current study we further characterized ARB‘s mechanism of action. We present a detailed biochemical characterization of ARB interactions with membranes, and examin how ARB can inhibit HCV entry by using HCV pseudoparticles (HCVpp). We found that ARB inhibited the infection of cells with HCVpp of different genotypes, through the inhibition of HCVpp-mediated membrane fusion. This inhibition was particularly prominent at acidic pH that is optimal for ARB association with membranes.
Figure 1
Figure 1
   
Figure 1
Structures of arbidol (A), indole (B) and tryptophan (C)

    *  Other Sections▼
          o Abstract
          o Experimental procedures
          o Results
          o Discussion
          o References

Experimental procedures
Chemicals
Sodium dodecyl sulfate (SDS), n-dodecyl-β-D-maltoside (DM), L-α-palmitoyl-lysophosphatidylcholine (α-lysoPC), phosphatidylcholine from egg yolk (PC, 99% pure), cholesterol (chol, 99% pure) and Triton X-100 were purchased from Sigma. Phosphatidylserine (PS) from porcine brain was from Avanti Polar Lipids (Alabaster, AL). Octadecyl rhodamine B chloride (R18) was from Molecular Probes. Arbidol [ARB, 1H-Indole-3-carboxylic acid, 6-bromo-4-((dimethylamino)methyl)-5-hydroxy-1-methyl-2-((phenylthio)methyl)-, ethyl ester, monohydrochloride; Fig.1AFigure 1] was a kind gift from A.M. Schuster and I.A. Leneva. ARB was first dissolved in absolute ethanol, and then completed to final concentration (100 mM) in sterile twice-distilled water. The final concentration of ethanol in contact with membranes varied between 0.011 and 1.1 ‰.
Vector constructs, preparation of pseudoparticles and infection assay
The CMV-Gag-Pol murine leukemia virus (MLV) packaging construct, encoding the MLV gag and pol genes, and the MLV-GFP plasmid, encoding a MLV-based transfer vector containing a CMV-GFP internal transcriptional unit, were described previously (13). The phCMV-HA (14) and NA expression vectors encode the hemagglutinin and neuraminidase of fowl plague virus, respectively. The phCMV-E1E2-HCV (13) encodes both HCV E1 and E2 glycoproteins of genotype 1a, strain H77 (AF009606), of genotype 1b (AY734975 clone UKN1b12.6, and AF333324), or of genotype 2a, isolate JFH-1 (AB047639). The phCMV-RD expression vector encodes the feline endogenous oncoretrovirus RD114 glycoprotein (15). Pseudoparticles bearing either the E1 and E2 envelope glycoproteins of HCV (HCVpp), or the hemagglutinin of the influenza virus (HApp), or the RD114 envelope protein (RD114) were then generated and purified as previously described (13, 16). Infection of Huh7 cells by concentrated pseudoparticles was performed in the absence or presence of increasing concentrations of ARB (between 0.1 and 6 μg/ml), added to cell culture medium as the ethanol/water stock solution

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