Binding Site Characterization and Resistance to a Class of Non-nucleoside Inhibitors of the Hepatitis C Virus NS5B Polymerase

Abstract

The virally encoded NS5B RNA-dependent RNA polymerase has emerged as a prime target in the search for specific HCV antivirals. A series of benzimidazole 5-carboxamide compounds inhibit the cellular RNA replication of a HCV subgenomic replicon and we have advanced our understanding of this class of inhibitors through a combination of complementary approaches that include biochemical cross-linking experiments with a photoreactive analogue followed by mass spectrometry analysis of the enzyme. A novel binding site has been localized for these inhibitors at the junction of the thumb domain and the N-terminal finger loop. Furthermore, the isolation and characterization of resistant replicon mutants that co-localize to this region distinguished this class of compounds from other non-nucleoside NS5B inhibitors that bind to distinct allosteric sites. Resistant mutations that emerged with the benzim- More than 2% of the world population are chronically infected with hepatitis C virus (HCV), 2 a flavivirus that is the etiological agent of non-A non-B hepatitis (1, 2). A large proportion of patients fail to achieve a sustained response to current therapies consisting of a combination of pegylated interferon and ribavirin. The discovery and development of specific anti-HCV chemotherapies aims to address this unmet clinical need and has focused on inhibitors of virally encoded functions. HCV encodes a linear polyprotein of ϳ3010 amino acids that is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural (NS) proteins (for review, see Ref.3). One of the non-structural proteins, NS5B, catalyzes the RNA-dependent RNA polymerization of a negative strand intermediate and the subsequent generation of multiple copies of the plus strand viral genome; this enzyme has emerged as a principal target for chemotherapeutic inhibition of HCV replication (4).The three-dimensional structure of the NS5B polymerase reveals an organization comparable with other nucleic acid polymerases with the familiar features of fingers, palm, and thumb domains that are organized in a "right-hand" motif (5-7). A distinct feature of the HCV polymerase (and closely related RNA-dependent RNA polymerase) active site cavity is the protrusion of a unique ␤-hairpin from the thumb subdomain that apparently plays a role in the initiation of de novo RNA synthesis as demonstrated by both structural and biochemical studies (8 -11). Another additional feature of the HCV polymerase is two loops that bridge the fingers and thumb subdomain and result in an encircled active site. This feature is now known to be shared by other RNA-dependent RNA polymerase from rhinovirus, bacteriophage 6, rabbit hemorrhagic disease virus, bovine viral diarrhea virus, Norwalk virus,. Interestingly, the interface between the HCV polymerase N-terminal 1 loop and the thumb subdomain is the location of a GTP binding site (8), although its precise biological role is unsolved.A number of different HCV polymerase inhibitors have emerged that can be broadly di...

Dynamics of Hepatitis C Virus (HCV) RNA-dependent RNA Polymerase NS5B in Complex with RNA

“…14, a, d, and g, respectively. Pro 495 is part of thumb site 1 and provides critical contacts to compound C. Substitution of this residue with alanine is associated with resistance to this class of NNIs (11). We thus explored next the interaction dynamics of P495A with R A 20 and compared the results to those acquired with WT NS5B (Fig.…”

“…Substitutions of basic amino acids that are implicated in RNA binding (10,11) with glutamic acid resulted in reductions or loss of enzymatic activity (40). Here we focused on residues that constitute the template entrance (Lys-98, Lys-100), and the center of the RNA binding channel (Arg-394).…”

“…Thumb pocket 1 non-nucleoside inhibitors (NNIs) or finger loop NNIs bind to a region of the thumb that is otherwise occupied by the flexible ⌬1 finger loop, which seems to impede productive RNA binding (Fig. 1b) (11)(12)(13)(14).…”

“…Here, we used a prototype inhibitor, i.e. compound C (11,41), to study whether the mechanism of action involves changes in the dynamic interaction between NS5B and its ssRNA template.…”

Association of hepatitis C virus NS5B variants with resistance to new antiviral drugs among untreated patients

“…The region of the viral genome that codes for NS5B has been described as an important target in therapy with IFN and rBV (Kukolj et al 2005, Lu et al 2007, Lutchman et al 2007). The mechanisms that lead to viral resistance after IFN/ rBV treatment is still a matter of debate (Horiike et al 1999, Hadziyannis et al 2004, Kanda et al 2004, Hamano et al 2005, Hofmann et al 2007, Chung et al 2008, Kuntzen et al 2008, Kwong et al 2008, Nakamura et al 2008, Cannon et al 2009, Gaudieri et al 2009, rydberg et al 2009).…”