Mechanism of Inhibition of Enveloped Virus Membrane Fusion by the Antiviral Drug Arbidol

The broad-spectrum antiviral arbidol (Arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell membrane. To better understand Arb mechanism of action, we investigated its interactions with phospholipids and membrane peptides. We demonstrate that Arb associates with phospholipids in the micromolar range. NMR reveals that Arb interacts with the polar head-group of phospholipid at the membrane interface. Fluorescence studies of interactions between Arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that Arb interacts with tryptophan in the micromolar range. Interestingly, apparent binding affinities between lipids and tryptophan residues are comparable with those of Arb IC50 of the hepatitis C virus (HCV) membrane fusion. Since tryptophan residues of membrane proteins are known to bind preferentially at the membrane interface, these data suggest that Arb could increase the strength of virus glycoprotein's interactions with the membrane, due to a dual binding mode involving aromatic residues and phospholipids. The resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the fusion process. Our findings pave the way towards the design of new drugs exhibiting Arb-like interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection

Differential In Vitro Effects of Intravenous versus Oral Formulations of Silibinin on the HCV Life Cycle and Inflammation

Silymarin prevents liver disease in many experimental rodent models, and is the most popular botanical medicine consumed by patients with hepatitis C. Silibinin is a major component of silymarin, consisting of the flavonolignans silybin A and silybin B, which are insoluble in aqueous solution. A chemically modified and soluble version of silibinin, SIL, has been shown to potently reduce hepatitis C virus (HCV) RNA levels in vivo when administered intravenously. Silymarin and silibinin inhibit HCV infection in cell culture by targeting multiple steps in the virus lifecycle. We tested the hepatoprotective profiles of SIL and silibinin in assays that measure antiviral and anti-inflammatory functions. Both mixtures inhibited fusion of HCV pseudoparticles (HCVpp) with fluorescent liposomes in a dose-dependent fashion. SIL inhibited 5 clinical genotype 1b isolates of NS5B RNA dependent RNA polymerase (RdRp) activity better than silibinin, with IC50 values of 40–85 µM. The enhanced activity of SIL may have been in part due to inhibition of NS5B binding to RNA templates. However, inhibition of the RdRps by both mixtures plateaued at 43–73%, suggesting that the products are poor overall inhibitors of RdRp. Silibinin did not inhibit HCV replication in subgenomic genotype 1b or 2a replicon cell lines, but it did inhibit JFH-1 infection. In contrast, SIL inhibited 1b but not 2a subgenomic replicons and also inhibited JFH-1 infection. Both mixtures inhibited production of progeny virus particles. Silibinin but not SIL inhibited NF-κB- and IFN-B-dependent transcription in Huh7 cells. However, both mixtures inhibited T cell proliferation to similar degrees. These data underscore the differences and similarities between the intravenous and oral formulations of silibinin, which could influence the clinical effects of this mixture on patients with chronic liver diseases

Characteristics of arbidol-resistant mutants of influenza virus: Implications for the mechanism of anti-influenza action of arbidol

The antiviral drug arbidol (ARB), which is licensed in Russia for use against influenza, is known to inhibit early membrane fusion events in influenza A and B virus replication. To investigate in more detail the target and mechanism of ARB action we generated and studied the characteristics of ARB-resistant influenza virus mutants. Observations of the ARB susceptibility of reassortants between A/Singapore/1/57(H2N2)and A/chicken/Germany/27(H7N7, "Weybridge" strain) and of mutants of the latter virus identified the virus haemagglutinin (HA) as the major determinant of ARB sensitivity. ARB-resistant mutants, selected from the most sensitive reassortant, possessed single amino acid substitutions in the HA2 subunit which caused an increase in the pH of fusion and the associated conformational change in HA. ARB was shown to stabilize the HA by causing a 0.2 pH unit reduction in the pH of the transition to the low pH form, which was specifically abrogated by the resistance mutations. Some of the resistance mutations, which reduce acid stability and would disrupt ARB-HA interactions, are located in the vicinity of a potential ARB binding site identified using the docking programme Gold. Together, the results of these investigations indicate that ARB falls within a class of inhibitor which interacts with HA to stabilize it against the low pH transition to its fusogenic state and consequently inhibit HA-mediated membrane fusion during influenza virus infection. (C) 2008 Elsevier B.V. All rights reserved.</p