Silibinin and Related Compounds Are Direct Inhibitors of Hepatitis C Virus RNA-Dependent RNA Polymerase

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http://www.gastrojournal.org/article/PIIS0016508509021052/abstract?rss=yesGASTRO\

ENTEROLOGYVolume 138, Issue 3, Pages 1112-1122 (March 2010) Silibinin and

Related Compounds Are Direct Inhibitors of Hepatitis C Virus RNA-Dependent RNA

PolymeraseAbdelhakim Ahmed–BelkacemâŽ, Nazim AhnouâŽ, Laetitia BarbotteâŽ,

Czeslaw Wychowski‡, Coralie PallierâŽÂ§, Rozenn BrilletâŽ, Ralf–Torsten

Pohl∥, Jean–Michel PawlotskyâŽÂ¶Received 20 February 2009; accepted 30

November 2009. published online 07 December 2009.Background & AimsSilymarin is a

mixture of flavonolignans extracted from the milk thistle. Silymarin contains

several molecules, including silibinin A, silibinin B, isosilibinin A,

isosilibinin B, silicristin, and silidianin. Intravenous infusion of silibinin

induces dose-dependent reduction of hepatitis C virus (HCV) RNA levels. The aim

of this study was to test the principal isomers contained in silymarin

preparations for their ability to inhibit HCV enzymatic functions and

replication in different models.MethodsThe inhibitory activity of silymarin

components was tested in HCV RNA-dependent RNA polymerase and NS3/4A protease

enzyme assays. Their ability to inhibit replication of an HCV genotype 1b

replicon model and the JFH1 infectious HCV model in cell culture was also

studied.ResultsSilibinin A, silibinin B, their water-soluble dihydrogen

succinate forms and Legalon SIL, a commercially available intravenous

preparation of silibinin, inhibited HCV RNA-dependent RNA polymerase function,

with inhibitory concentrations 50% of the order of 75−100 μM. Silibinin A and

silibinin B also inhibited HCV genotype 1b replicon replication and HCV genotype

2a strain JFH1 replication in cell culture. None of these compounds inhibited

HCV protease function.ConclusionsSilibinin A and silibinin B, as well as Legalon

SIL, inhibit HCV replicon and JFH1 replication in cell culture. This effect is

at least partly explained by the ability of these compounds to inhibit HCV

RNA-dependent RNA polymerase activity. Our results provide a basis for the

optimization and subsequent development of members of the Flavonoid family as

specific HCV antivirals.Keywords: Hepatitis C Virus, Silibinin, Polymerase

Inhibitor, Enzyme AssayAbbreviations used in this paper: DMSO, dimethyl

sulfoxide, EC50, effective concentration 50%, HCV, hepatitis C virus, HCVcc,

infectious HCV particles, IC50, inhibitory concentration 50%, IFN, interferon,

NS3/4A, nonstructural 3/4A, RdRp, RNA-dependent RNA polymerase, NS5B,

nonstructural 5B⎠Research Team “Pathophysiology and Therapy of Chronic

Viral Hepatitis,†INSERM U955, Créteil, France‡ Institut de Biologie de

Lille (CNRS UMR8161), Université de Lille I and II and Institut Pasteur de

Lille, Lille, France§ Department of Virology, Hôpital de Bicêtre, Le

Kremlin-Bicêtre, France∥ Rottapharm/Madaus, Cologne, Germany¶ National

Reference Center for Viral Hepatitis B, C and Delta, Department of Virology,

Hôpital Henri Mondor, Université Paris 12, Créteil, FranceReprint requests

Address requests for reprints to: Jean-Michel Pawlotsky, MD, PhD, Department of

Virology, Hôpital Henri Mondor, 51 Avenue du Maréchal de Lattre de Tassigny,

94010 Créteil, France. ax: +33-1-4981-4831 Conflicts of interest These authors

disclose the following: Dr Pawlotsky is an advisor for Rottapharm/Madaus. Dr

Pohl is an employee of Rottapharm/Madaus. The remaining authors disclose no

conflicts. Funding This work is part of the activity of the VIRGIL European

Network of Excellence on Antiviral Drug Resistance supported by a grant

(LSHM-CT-2004-503359) from the Priority 1 “Life Sciences, Genomics and

Biotechnology for Health†programme in the 6th Framework Programme of the

European Union. A. A. is a recipient of a postdoctoral fellowship, and N. A. is

under contract with the Agence Nationale de Recherche sur le SIDA et les

Hépatites Virales (ANRS). L. B. is a recipient of a pre-doctoral fellowship

from the Ministère de l'Enseignement Supérieur et de la Recherche. The

purified silymarin compounds and their dihydrogen succinates have been provided

by Madaus GmbH (Rottapharm Group), Cologne, Germany.PII:

S0016-5085(09)02105-2doi:10.1053/j.gastro.2009.11.053© 2010 AGA Institute.

Published by Elsevier Inc. All rights reserved.