Novel Nonnucleoside Inhibitor of Hepatitis C Virus RNA-Dependent RNA Polymerase

Howe, Anita Y. M.; Bloom, Johnathan; Baldick, Carl J.; Benetatos, Christopher A.; Cheng, Huiming; Christensen, Joel S.; Chunduru, Srinivas K.; Coburn, Glen A.; Feld, Boris; Gopalsamy, Ariamala; Gorczyca, William P.; Herrmann, Steve; Johann, S V; Jiang, Xian; Kimberland, Michelle L.; Krisnamurthy, Girija; Olson, Matthew W.; Orlowski, Mark; Swanberg, Steve; Thompson, Ian; Thorn, Megan; Vecchio, Alfred Del; Young, Dorothy C.; Zeijl, Marja van; Ellingboe, John W.; Upeslacis, Janis; Collett, Marc S.; Mansour, Tarek S.; O’Connell, John F.

doi:10.1128/aac.48.12.4813-4821.2004

Cited by 51 publications

(48 citation statements)

References 45 publications

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“…The cells were treated multiple times over a 16-day period in medium containing 2% FCS without G418 and 30 M HCV-570 dissolved in dimethyl sulfoxide (DMSO; final concentration of DMSO in the tissue culture medium, 0.5% [vol/vol]). HCV-570 is a racemic mixture that contains about 50% of the R enantiomer, HCV-371 (20). As a control, clone A cells were passaged in parallel in the same medium with 0.5% (vol/vol) DMSO and no compound.…”

Section: Methodsmentioning

confidence: 99%

“…The susceptibilities of the replicon-containing cells to compounds were evaluated as described previously (20). Briefly, cells were treated with increasing concentrations of compounds in a medium containing 2% FCS and no G418 for 3 days at 37°C in 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

“…The inhibitory activity of this racemate appears to reside in the R enantiomer (HCV-371) (20); no activity was found in the S enantiomer. The chiral structure-dependent inhibitory activity of HCV-371 is also consistent with the results of the cocrystal structure (R. Chopra, S. Sukits, and K. Svenson, unpublished data).…”

mentioning

confidence: 99%

“…Recent research efforts have led to the discovery of many inhibitors that specifically target HCV protease and polymerase (18,47). Among the HCV polymerase inhibitors, both nucleoside and nonnucleoside analogues have been reported (9,14,20,28,40,42,48). The nonnucleoside analogues, in general, bind to two regions of NS5B: near the catalytic site in the palm domain (33,45) and near the interface with the C-terminal extension of the polymerase at the base thumb domain (3,28,48).…”

mentioning

confidence: 99%

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Molecular Mechanism of a Thumb Domain Hepatitis C Virus Nonnucleoside RNA-Dependent RNA Polymerase Inhibitor

Howe¹,

Cheng²,

Thompson³

et al. 2006

Antimicrob Agents Chemother

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A new pyranoindole class of small-molecule inhibitors was studied to understand viral resistance and elucidate the mechanism of inhibition in hepatitis C virus (HCV) replication. HCV replicon variants less susceptible to inhibition by the pyranoindoles were selected in Huh-7 hepatoma cells. Variant replicons contained clusters of mutations in the NS5B polymerase gene corresponding to the drug-binding pocket on the surface of the thumb domain identified by X-ray crystallography. An additional cluster of mutations present in part of a unique ␤-hairpin loop was also identified. The mutations were characterized by using recombinant replicon variants engineered with the corresponding amino acid substitutions. A single mutation (L419M or M423V), located at the pyranoindole-binding site, resulted in an 8-to 10-fold more resistant replicon, while a combination mutant (T19P, M71V, A338V, M423V, A442T) showed a 17-fold increase in drug resistance. The results of a competition experiment with purified NS5B enzyme with GTP showed that the inhibitory activity of the pyranoindole inhibitor was not affected by GTP at concentrations up to 250 M. Following de novo initiation, the presence of a pyranoindole inhibitor resulted in the accumulation of a five-nucleotide oligomer, with a concomitant decrease in higher-molecular-weight products. The results of these studies have confirmed that pyranoindoles target the NS5B polymerase through interactions at the thumb domain. This inhibition is independent of GTP concentrations and is likely mediated by an allosteric blockade introduced by the inhibitor during the transition to RNA elongation after the formation of an initiation complex.A wealth of literature has documented that RNA viruses or viruses that use RNA replicative intermediates have high degrees of genetic variability (5, 15). The high mutation rates found in these organisms are largely due to the lack of proofreading mechanisms and/or a high recombination frequency during cellular DNA replication. A DNA-dependent DNA polymerase complex allows an error rate of 10 Ϫ9 mutations/ nucleotide, whereas the error rates for an RNA-dependent DNA polymerase (reverse transcriptase) and RNA-dependent RNA polymerase (RdRp), such as those in human immunodeficiency virus (HIV) and hepatitis C virus (HCV), respectively, are on the order of 10 Ϫ4 to 10 Ϫ5 mutations/nucleotide (35, 37). The robust viral replication that leads to a large population size during viral infection in these RNA viruses and retroviruses contributes to the generation of quasispecies in infected patients (7,12,13).During chemotherapy, the high rates of viral replication and the high frequency of mutation lead to the rapid generation of drug-resistant mutants. In the case of HIV, numerous mutations have been identified in patients treated with protease inhibitors as well as nucleoside and nonnucleoside reverse transcriptase inhibitors. The emergence of resistant viruses becomes one of the biggest challenges in developing effective antiviral therapies to eradicate these ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Molecular Mechanism of a Thumb Domain Hepatitis C Virus Nonnucleoside RNA-Dependent RNA Polymerase Inhibitor

Howe¹,

Cheng²,

Thompson³

et al. 2006

Antimicrob Agents Chemother

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“…The principle of the tryptophan fluorescence quenching assay has been described to determine compound binding to HCV NS5B and NS3 proteins (31)(32)(33)(34). In the present study, a new plate-based fluorescence quenching binding assay was developed to assess whether NNI binding affinity correlates with the potency of inhibition of NS5B polymerase.…”

Section: Correlation Of Binding Affinity and Inhibition Potency Of Nomentioning

confidence: 99%

Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus

Hang

Yang

Harris

et al. 2009

Journal of Biological Chemistry

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Hepatitis C virus (HCV)4 constitutes a global health problem. Current therapies are unable to effectively eliminate viral infection in a significant number of patients. The RNA-dependent RNA polymerase (RdRp) of HCV NS5B is an attractive target for the development of orally bioavailable small molecule inhibitors (1, 2). The structure of the NS5B apoenzyme and the NS5B-RNA complex reveals the characteristic right hand architecture of polymerase enzymes, comprising three distinct domains (palm, thumb, and finger) encircling the enzyme active site located in the palm domain (3-6). The structural and biochemical characterization of HCV NS5B polymerase can provide a basis for drug design efforts, and the elucidation of the mechanism of inhibition can guide the optimization of inhibitor efficiency against wild-type and resistant mutants.Among the extensively investigated non-nucleosides documented to inhibit the RdRp activity of HCV NS5B, derivatives of various benzofuran and benzothiadiazine have been reported to bind to allosteric binding sites in the palm domain of NS5B (7,8). The palm domain, whose geometry is conserved in virtually all DNA and RNA polymerases, contains catalytic aspartic acids responsible for the nucleotidyl transfer reaction. The benzofuran compound HCV-796 has been shown to have significant antiviral effects in patients chronically infected with HCV (9, 10). In addition, two series of compounds based on the thiophene and benzimidazole scaffolds have been reported to inhibit NS5B by binding to two different binding pockets in the thumb domain of NS5B (11,12). The thumb domain is connected to the palm domain by a ␤-hairpin termed the primer grip motif. The C-terminal region of the thumb protrudes toward the active site (3). The thumb binding inhibitors have been proposed to inhibit the RdRp activity of NS5B, perhaps by interfering with template/primer interaction and conformational dynamics of the protein (13,14).Despite the elucidation of a number of NNIs that bind to the thumb and palm binding sites, the mechanism by which NNIs cause inhibition of RNA synthesis is unclear. Also, our understanding of the kinetics of NNI interaction with NS5B, the role of NNI binding and kinetics for inhibition, and the inhibitor efficacy on NS5B-resistant mutations remains incomplete. The four representative palm-and thumb-binding NNIs selected in this study have been reported to effectively inhibit replication of subgenomic replicons with low toxicity. Noncompetitive inhibition of NS5B polymerase activity with respect to NTPs has been reported (2, 15, 16). Based on co-crystallization studies with NS5B, it has been proposed that allosteric inhibitors may lock the NS5B protein in an inactive formation by binding tightly to the protein (16,17). It is important to understand how the binding affinity relates to inhibition potency and resistance to HCV inhibition. Because the intrinsic potency of slowly binding compounds can be underestimated in the short time □ S The on-line version of this article (available at htt...

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