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<br>[1]. McCown MF, Rajyaguru S, Kular S, Cammack N, Nájera I. GT-1a or GT-1b subtype-specific resistance profiles for hepatitis C virus inhibitors telaprevir and HCV-796. Antimicrob Agents Chemother. 2009 May;53(5):2129-32.
Abstract
In vitro, telaprevir selects subtype-specific resistance pathways for hepatitis C virus GT-1a and GT-1b, as described to have occurred in patients. In GT-1a, the HCV-796 resistance mutation C316Y has low replication capacity (7%) that can be compensated for by the emergence of the mutation L392F or M414T, resulting in an increase in replication levels of > or = 10-fold.
<br>[2]. Flint M, Mullen S, Deatly AM, Chen W, Miller LZ, Ralston R, Broom C, Emini EA, Howe AY. Selection and characterization of hepatitis C virus replicons dually resistant to the polymerase and protease inhibitors HCV-796 and boceprevir (SCH 503034). Antimicrob Agents Chemother. 2009 Feb;53(2):401-11.
Abstract
HCV-796 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase, and boceprevir is an inhibitor of the NS3 serine protease. The emergence of replicon variants resistant to the combination of HCV-796 and boceprevir was evaluated. Combining the inhibitors greatly reduced the frequency with which resistant colonies arose; however, some resistant replicon cells could be isolated by the use of low inhibitor concentrations. These replicons were approximately 1,000-fold less susceptible to HCV-796 and 9-fold less susceptible to boceprevir. They also exhibited resistance to anthranilate nonnucleoside inhibitors of NS5B but were fully sensitive to inhibitors of different mechanisms: a pyranoindole, Hsp90 inhibitors, an NS5B nucleoside inhibitor, and pegylated interferon (Peg-IFN). The replicon was cleared from the combination-resistant cells by extended treatment with Peg-IFN. Mutations known to confer resistance to HCV-796 (NS5B C316Y) and boceprevir (NS3 V170A) were present in the combination-resistant replicons. These changes could be selected together and coexist in the same genome. The replicon bearing both changes exhibited reduced sensitivity to inhibition by HCV-796 and boceprevir but had a reduced replicative capacity.
<br>[3]. Howe AY, Cheng H, Johann S, Mullen S, Chunduru SK, Young DC, Bard J, Chopra R, Krishnamurthy G, Mansour T, O/’Connell J. Molecular mechanism of hepatitis C virus replicon variants with reduced susceptibility to a benzofuran inhibitor, HCV-796. Antimicrob Agents Chemother. 2008 Sep;52(9):3327-38.
Abstract
HCV-796 selectively inhibits hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In hepatoma cells containing a genotype 1b HCV replicon, HCV-796 reduced HCV RNA levels by 3 to 4 log(10) HCV copies/mug total RNA (the concentration of the compound that inhibited 50% of the HCV RNA level was 9 nM). Cells bearing replicon variants with reduced susceptibility to HCV-796 were generated in the presence of HCV-796, followed by G418 selection. Sequence analysis of the NS5B gene derived from the replicon variants revealed several amino acid changes within 5 A of the drug-binding pocket. Specifically, mutations were observed at Leu314, Cys316, Ile363, Ser365, and Met414 of NS5B, which directly interact with HCV-796. The impacts of the amino acid substitutions on viral fitness and drug susceptibility were examined in recombinant replicons and NS5B enzymes with the single-amino-acid mutations. The replicon variants were 10- to 1,000-fold less efficient in forming colonies in cells than the wild-type replicon; the S365L variant failed to establish a stable cell line. Other variants (L314F, I363V, and M414V) had four- to ninefold-lower steady-state HCV RNA levels. Reduced binding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effects of these resistance mutations were structurally rationalized using X-ray crystallography data. While different levels of resistance to HCV-796 were observed in the replicon and enzyme variants, these variants retained their susceptibilities to pegylated interferon, ribavirin, and other HCV-specific inhibitors. The combined virological, biochemical, biophysical, and structural approaches revealed the mechanism of resistance in the variants selected by the potent polymerase inhibitor HCV-796.
<br>[4]. Kim ND, Chun H, Park SJ, Yang JW, Kim JW, Ahn SK. Discovery of novel HCV polymerase inhibitors using pharmacophore-based virtual screening. Bioorg Med Chem Lett. 2011 Jun 1;21(11):3329-34.
Abstract
We report the use of pharmacophore-based virtual screening as an efficient tool for the discovery of novel HCV polymerase inhibitors. A three-dimensional pharmacophore model for the HCV-796 binding site, NNI site IV inhibitor, to the enzyme was built by means of the structure-based focusing module in Cerius2 program. Using these models as a query for virtual screening, we produced a successful example of using pharmacophore-based virtual screening to identify novel compounds with HCV replicon assay through inhibition of HCV polymerization. Among the hit compounds, compounds 1 and 2 showed 56% and 48% inhibition of NS5B polymerization activity at 20 μM, respectively. In addition, compound 1 also exhibited replicon activity with EC(50) value of 2.16 μM. Following up the initial hit, we obtained derivatives of compound 1 and evaluated polymerization inhibition activity and HCV replicon assay. These results provide information necessary for the development of more potent NS5B inhibitors.
<br>[5]. Reich S, Golbik RP, Geissler R, Lilie H, Behrens SE. Mechanisms of activity and inhibition of the hepatitis C virus RNA-dependent RNA polymerase. J Biol Chem. 2010 Apr 30;285(18):13685-93.
Abstract
The RNA-dependent RNA polymerase NS5B is a key enzyme of the replication of hepatitis C virus (HCV) and a major therapeutic target. Applying a novel continuous assay with highly purified protein and a fluorescent RNA-template we provide for the first time a comprehensive mechanistic description of the enzymatic reaction. Using fluorescence spectroscopy, the kinetics of NS5B was confirmed to consist of two half-reactions, namely substrate binding and turnover. Determining the binding constants of the substrates and the rate constants of individual reaction steps, NS5B was shown to bind the template single-stranded RNA with high affinity (nanomolar range) and in a stepwise process that reflects the substrate positioning. As demonstrated by CD, NTP(s) binding caused a tertiary structural change of the enzyme into an active conformation. The second half-reaction was dissected into a sequential polymerization and a subsequent, rate-limiting product release reaction. Taking advantage of these tools, we analyzed the mechanism of action of the NS5B inhibitor HCV-796, which was shown to interfere with the formation of double-stranded RNA by blocking the second half-reaction.
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