title: Mechanism of action and antiviral activity of benzimidazole-based allosteric inhibitors of the hepatitis C virus RNA-dependent RNA polymerase.
abstract: The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis of the mechanism of action of selected benzimidazole inhibitors. Kinetic data and binding experiments indicated that these compounds act as allosteric inhibitors that block the activity of the polymerase prior to the elongation step. Escape mutations that confer resistance to these compounds map to proline 495, a residue located on the surface of the polymerase thumb domain and away from the active site. Substitution of this residue is sufficient to make the HCV enzyme and replicons resistant to the inhibitors. Interestingly, proline 495 lies in a recently identified noncatalytic GTP-binding site, thus validating it as a potential allosteric site that can be targeted by small-molecule inhibitors of HCV polymerase.
authors.affiliation: Istituto di Ricerche di Biologia Molecolare "P. Angeletti," 00040 Pomezia-Rome, Italy. Licia_Tomei@Merck.com
authors.forename: Licia
authors.initials: L
authors.lastname: Tomei
authors.forename: Sergio
authors.initials: S
authors.lastname: Altamura
authors.forename: Linda
authors.initials: L
authors.lastname: Bartholomew
authors.forename: Antonino
authors.initials: A
authors.lastname: Biroccio
authors.forename: Alessandra
authors.initials: A
authors.lastname: Ceccacci
authors.forename: Laura
authors.initials: L
authors.lastname: Pacini
authors.forename: Frank
authors.initials: F
authors.lastname: Narjes
authors.forename: Nadia
authors.initials: N
authors.lastname: Gennari
authors.forename: Monica
authors.initials: M
authors.lastname: Bisbocci
authors.forename: Ilario
authors.initials: I
authors.lastname: Incitti
authors.forename: Laura
authors.initials: L
authors.lastname: Orsatti
authors.forename: Steven
authors.initials: S
authors.lastname: Harper
authors.forename: Ian
authors.initials: I
authors.lastname: Stansfield
authors.forename: Michael
authors.initials: M
authors.lastname: Rowley
authors.forename: Raffaele
authors.initials: R
authors.lastname: De Francesco
authors.forename: Giovanni
authors.initials: G
authors.lastname: Migliaccio
mesh_terms: D000495:Allosteric Site / Q000187:drug effects*; D000998:Antiviral Agents / Q000494:pharmacology*; D001562:Benzimidazoles / Q000494:pharmacology*; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D012367:RNA, Viral / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; D001562:Benzimidazoles; D004791:Enzyme Inhibitors; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C031000:benzimidazole; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase