Background
PSI-6130 is a nucleoside inhibitor of the NS5B RNA polymerase of hepatitis C virus (HCV; Ki = 4.3 ?M).1 It inhibits HCV genotype 1b (GT-1b) Con1 and GT-1a H77 viral replication in Huh7 replicon cells using a luciferase-based assay (EC50s = 0.51 and 0.30 ?M, respectively). PSI-6130 also inhibits replication of HCV GT-1b and GT-1a replicons from clinical isolates, with EC50 values ranging from 0.60 to 1.41 ?M, and 0.20 to 0.43 ?M, respectively, in the same assay. It has little or no activity against West Nile Virus (WNV), Dengue type 2 virus (DV), human immunodeficiency virus (HIV), or hepatitis B virus (HBV; EC90s = 46.3, >100, >100, and >10 ?M, respectively).2
1.Murakami, E., Bao, H., Ramesh, M., et al.Mechanism of activation of β-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine and inhibition of hepatitis C virus NS5B RNA polymeraseAntimicrob. Agents Chemother.51(2)503-509(2007)
2.Stuyver, L.J., McBrayer, T.R., Tharnish, P.M., et al.Inhibition of hepatitis C replicon RNA synthesis by β-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine: A specific inhibitor of hepatitis C virus replicationAntivir. Chem. Chemother.17(2)79-87(2006)
Protocol
Kinase experiment: | The inhibition potency of compounds with respect to the RdRp activity of recombinant NS5B570-BK, NS5B570-Con1, and NS5B570-H77 proteins is determined by measuring the incorporation of radiolabeled NMP into acid-insoluble RNA products by use of a complement strand of internal ribosomal entry site (cIRES) RNA template. Briefly, 50% inhibitory concentration (IC50) determinations are carried out using 200 nM in vitro-transcribed cIRES RNA template, 1 μCi of tritiated UTP (42 Ci/mmol), 500 μM ATP, 500 μM GTP, 1 μM CTP, 1× TMDN buffer (40 mM Tris-HCl [pH 8.0], 4 mM MgCl2, 4 mM dithiothreitol, 40 mM NaCl), and 200 nM enzyme. The inhibition potency of compounds with respect to the RdRp activity of NS5B570-S282T-Con1 is determined under GT-1b assay conditions as. NS5B570-BK and NS5B570-Con1 enzymes are used as controls. The final reaction volume is 50 μL under all assay conditions. All reactions contain a final 10% dimethyl sulfoxide. Km and Ki values are measured. |
参考文献: [1]. Ali S, et al. Selected replicon variants with low-level in vitro resistance to the hepatitis C virus NS5B polymerase inhibitor PSI-6130 lack cross-resistance with R1479. Antimicrob Agents Chemother. 2008 Dec;52(12):4356-69.
[2]. Ma H, et al. Characterization of the metabolic activation of hepatitis C virus nucleoside inhibitor beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) and identification of a novel active 5'-triphosphate species. J Biol Chem. 2007 Oct 12;282(41):29812-20. Epub 2007 Aug 13. |
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