Interdomain Communication in Hepatitis C Virus Polymerase Abolished by Small Molecule Inhibitors Bound to a Novel Allosteric Site

Stefania Di Marco,Cinzia Volpari,Licia Tomei,Sergio Altamura,Steven Harper,Frank Narjes,Uwe Koch,Michael Rowley,Raffaele De Francesco,Giovanni Migliaccio,Andrea Carfí

doi:10.1074/jbc.m505423200

Abstract

The hepatitis C virus (HCV) polymerase is required for replication of the viral genome and is a key target for therapeutic intervention against HCV. We have determined the crystal structures of the HCV polymerase complexed with two indole-based allosteric inhibitors at 2.3- and 2.4-Angstroms resolution. The structures show that these inhibitors bind to a site on the surface of the thumb domain. A cyclohexyl and phenyl ring substituents, bridged by an indole moiety, fill two closely spaced pockets, whereas a carboxylate substituent forms a salt bridge with an exposed arginine side chain. Interestingly, in the apoenzyme, the inhibitor binding site is occupied by a small alpha-helix at the tip of the N-terminal loop that connects the fingers and thumb domains. Thus, these molecules inhibit the enzyme by preventing formation of intramolecular contacts between these two domains and consequently precluding their coordinated movements during RNA synthesis. Our structures identify a novel mechanism by which a new class of allosteric inhibitors inhibits the HCV polymerase and open the way to the development of novel antiviral agents against this clinically relevant human pathogen.

Highlights

The hepatitis C virus (HCV)1 is a small positive-strand RNA virus responsible for a considerable proportion of acute and chronic hepatitis in humans [1, 2]
The hepatitis C virus (HCV) polymerase is required for replication of the viral genome and is a key target for therapeutic intervention against HCV
Inhibition of HCV Polymerase—The key pharmacophore elements of the allosteric inhibitors used in this study include a central scaffold of a 6,5-fused ring system, which proved to be optimal [27], an acid, a cyclohexyl and an aryl substituent (Table I)

Summary

Introduction

The hepatitis C virus (HCV) is a small positive-strand RNA virus responsible for a considerable proportion of acute and chronic hepatitis in humans [1, 2]. Crystal structures of NS5B bound to a short RNA template or to NTPs have been solved [20, 21] In all these structures, ligand binding occurred without rearrangements in the enzyme domains. The structure of the NS5B-GTP complex showed that, in addition to the active site, GTP can bind to an exposed site on the thumb domain in close proximity to the tip of the fingertip ⌳1 loop [21] This external GTP site, 30 Å away from the polymerase catalytic center, was proposed to exert a regulatory activity by modulating the interactions between the fingers and thumb domains during RNA synthesis. In the opened conformation the tip of the fingertip ⌳1 loop, which is ␣-helical in the closed conformation of the polymerase, moved away from the thumb domain and adopted a ␤-hairpin-like structure

Methods

Results

Conclusion