AAC Accepts, published online ahead of print on 13 October 2008

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Antimicrob. Agents Chemother. doi:10.1128/AAC.00669-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

1,5-Benzodiazepines, A Novel Class Of Hepatitis C Virus Polymerase Non-nucleoside Inhibitors

Origène Nyanguile^*, Frederik Pauwels, Walter Van den Broeck, Carlo W. Boutton, Ludo Quirynen, Tania Ivens, Liesbet van der Helm, Geneviève Vandercruyssen, Wendy Mostmans, Frédéric Delouvroy, Pascale Dehertogh, Maxwell D. Cummings, Jean-Francois Bonfanti, Kenneth A. Simmen, and Pierre Raboisson

HCV Research, Tibotec, Mechelen, Belgium; Johnson & Johnson Pharmaceutical and Research Development, Val de Reuil, France

^* To whom correspondence should be addressed. Email: onyangui{at}its.jnj.com.

The exogenous control of hepatitis C virus (HCV) replication can be mediated through inhibition of the RNA dependent RNA polymerase (RdRp) activity of NS5B. Small-molecule inhibitors of NS5B include nucleoside and non-nucleoside analogs. Here, we report the discovery of a novel class of HCV polymerase non-nucleoside inhibitors, 1,5 - benzodiazepines (1,5-BZDs), identified by high-throughput screening of a library of small-molecules. A fluorescence quenching assay and X-ray crystallography revealed that 1,5-BZD 4a binds stereospecifically to NS5B next to the catalytic site. Mutants known to confer resistance against chemotypes that bind at this site were detrimental to inhibition of 1,5-BZD 7a in replicons. Using a panel of enzyme isolates that covers genotypes 1 to 6, we show that compound 4a inhibits genotype 1 only. In mechanistic studies, 4a was found to inhibit the RNA dependent RNA polymerase (RdRp) activity of NS5B non-competitively with GTP, and to inhibit the formation of the first phosphodiester bond during the polymerization cycle. The specificity for the HCV viral target was evaluated by profiling the 1,5-BZDs against other viral and human polymerases, as well as benzodiazepine receptors.