Non-nucleoside Inhibitors Binding to Hepatitis C Virus NS5B Polymerase Reveal a Novel Mechanism of Inhibition

Biswal, B.K.; Wang, Meitian; Cherney, M.M.; Chan, Laval; Yannopoulos, Constantin G.; Bilimoria, Darius; Bédard, Jean; James, Michael N.G.

doi:10.1016/j.jmb.2006.05.074

Cited by 75 publications

(58 citation statements)

References 53 publications

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“…Our present results are consistent with those of our earlier study and allow us to tentatively extend this conclusion to include the 1a enzyme. This is distinct from what has been observed for the NNI-1 and -2 inhibitors, where inhibitor binding involves significant conformational adjustment of NS5B (4,5,10).…”

Section: Differences In 15-bzd Inhibition Against Subtypes 1a and 1bcontrasting

confidence: 83%

1a/1b Subtype Profiling of Nonnucleoside Polymerase Inhibitors of Hepatitis C Virus

Nyanguile¹,

Devogelaere²,

Vijgen³

et al. 2010

J Virol

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The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor-and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.

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Section: Differences In 15-bzd Inhibition Against Subtypes 1a and 1bcontrasting

confidence: 83%

1a/1b Subtype Profiling of Nonnucleoside Polymerase Inhibitors of Hepatitis C Virus

Nyanguile¹,

Devogelaere²,

Vijgen³

et al. 2010

J Virol

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“…Type 2 inhibitors are non-nucleoside inhibitors (NNI) which bind to allosteric pockets of protein to block enzymatic activities; the mechanism of action of NNI includes structural alteration of polymerase to an inactive conformation, blocking the conformational switch from polymerase initiation to elongation, or impeding the processivity of polymerase elongation (11). A broad range of chemical classes have been identified as NNI, including inhibitors of HIV (9,35) and HCV (3,5,11,25).…”

mentioning

confidence: 99%

Inhibition of Dengue Virus Polymerase by Blocking of the RNA Tunnel

Niyomrattanakit

Chen

Dong

et al. 2010

J Virol

108

116

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Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G) 20 ]. Chemical modification of the initial "hit" improved the compound potency to an IC 50 (that is, a concentration that inhibits 50% RdRp activity) of 0.7 M. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC 50 of 5 M). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC 50 s of >100 M against hepatitis C virus RdRp and human DNA polymerase ␣ and ␤. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.

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“…The RdRp of other RNA viruses have been major targets for antiviral compounds. For instance, NS5A is the RdRp in hepatitis C virus (HCV) and a major target for nonnucleoside inhibitors (NNI) (Biswal et al, 2005(Biswal et al, , 2006. The binding sites for thiophene-based NNIs are located in the 'thumb' domain of NS5B, in close proximity to the allosteric GTP binding site and approximately 35 Å from the active site.…”

Section: Future Prospectsmentioning

confidence: 99%

The search for a structural basis for therapeutic intervention against the SARS coronavirus

2007

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The 2003 outbreak of severe acute respiratory syndrome (SARS), caused by a previously unknown coronavirus called SARS-CoV, had profound social and economic impacts worldwide. Since then, structure-function studies of SARSCoV proteins have provided a wealth of information that increases our understanding of the underlying mechanisms of SARS. While no effective therapy is currently available, considerable efforts have been made to develop vaccines and drugs to prevent SARS-CoV infection. In this review, some of the notable achievements made by SARS structural biology projects worldwide are examined and strategies for therapeutic intervention are discussed based on available SARS-CoV protein structures. To date, 12 structures have been determined by X-ray crystallography or NMR from the 28 proteins encoded by SARS-CoV. One key protein, the SARS-CoV main protease (M pro ), has been the focus of considerable structure-based drug discovery efforts. This article highlights the importance of structural biology and shows that structures for drug design can be rapidly determined in the event of an emerging infectious disease.

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Non-nucleoside Inhibitors Binding to Hepatitis C Virus NS5B Polymerase Reveal a Novel Mechanism of Inhibition

Cited by 75 publications

References 53 publications

1a/1b Subtype Profiling of Nonnucleoside Polymerase Inhibitors of Hepatitis C Virus

1a/1b Subtype Profiling of Nonnucleoside Polymerase Inhibitors of Hepatitis C Virus

Inhibition of Dengue Virus Polymerase by Blocking of the RNA Tunnel

The search for a structural basis for therapeutic intervention against the SARS coronavirus

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