Direct Inhibitors of Hepatitis C Virus RNA-Dependent RNA Polymerase

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

Reported by Jules Levin
AASLD Oct 31-Nov 3 2009, Boston, MA

A. Ahmed-Belkacem1; N. Ahnou1; L. Barbotte1; C. Wychowski2; R. Brillet1; R. Pohl3; J. Pawlotsky1

1. Henri Mondor Hospital, University of Paris 12, Creteil, France.

2. Institut de Biologie de Lille, Lille, France.

3. Madaus-Rottapharm, Cologne, Germany.




Only approximately 50% of patients with HCV genotype 1 infection eradicate infection upon pegIFN-ribavirin therapy. Current HCV drug discovery efforts focus on developing molecules that specifically inhibit HCV enzymes, such as the RNA-dependent RNA polymerase (RdRp) or the NS3/4A protease. Silymarin is a mixture of flavonolignans extracted from the milk thistle, which contains several molecules including silibinin A, silibinin B, isosilibinin A, isosilibinin B, silichristin, and silidianin. Intravenous infusion of Legalon SIL®, a commercially available preparation of silibinin, induces dose-dependent reduction of HCV RNA levels. Our aim was to test the isomers contained in silymarin preparations for their ability to inhibit HCV enzymatic functions and replication in different models.

METHODS: The inhibitory activity of silymarin components was tested in HCV RdRp and NS3/4A protease enzyme assays. Their ability to inhibit replication of an HCV genotype 1b replicon and the JFH1 infectious HCV model in cell culture was also studied. The effect of amino acid substitutions known to confer HCV resistance to RdRp inhibitors was tested.

RESULTS: Silibinin 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% (IC50s) of the order of 75-100 micromolar. Silibinin A and silibinin B also inhibited HCV genotype 1b replicon replication with effective concentrations 50% (EC50s) of the micromolar order, and HCV genotype 2a strain JFH1 replication in cell culture with EC50s approximately one log above those observed in the replicon system. None of the tested silymarin components showed any inhibitory activity in the NS3/4A protease assay, up to a concentration of 200 µM. No cytotoxic effect was observed at inhibitory concentrations in two different human cell lines (Huh7 and HEK 293). Amino acid substitutions known to confer resistance to RdRp inhibitors, including 2?-methyl nucleoside analogues (S282T) and non-nucleoside inhibitors (P495L, M423T, H95Q, and C316Y, located in thumb 1, thumb 2, palm 1 and palm 2 RdRp domains, respectively) did not confer resistance to silibinin in the RdRp enzyme assay.

CONCLUSIONS: Silibinin A and silibinin B, as well as Legalon SIL®, inhibit HCV replication in cell culture. This effect is at least partly explained by the ability of these compounds to directly inhibit HCV RdRp activity. These results provide a basis for the optimization and subsequent development of members of the Flavonoid family as specific HCV antivirals.