R-1479

Kinase experiment:

The membrane-associated, native HCV replicase complex is isolated from 2209-23 HCV replicon cells and a derived cell line carrying HCV replicon RNA with a S282T mutation in the NS5B coding sequence. The in vitro replicase assay contain 10 μL of cytoplasmic membrane fraction, 50 mM HEPES (pH 7.5), 10 mM KCl, 10 mM dithiothreitol, 5 mM MgCl2, 20 μg/mL actinomycin D, 1 mM ATP, 1 mM GTP, 1 mM UTP, 30 μCi of [α-33P]CTP (3000 Ci/mmol, 10 mCi/mL), 1 unit/μL SUPERase•In, 10 mM creatine phosphate, and 200 μg/mL creatine phosphokinase in a final volume of 25 μL. Inhibition by nucleotide analogs is determined[1].

Cell experiment:

The effect of R-1479 on the incorporation of tritiated thymidine into cellular DNA is measured using the [3H]thymidine incorporation scintillation proximity assay system. MTT and WST-1 assay systems are used to measure cell viability. The ATP bioluminescence assay kit HSII is used to measure intracellular ATP levels[1].

References:

[1]. Klumpp K, et al. The novel nucleoside analog R1479 (4'-azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture. J Biol Chem. 2006 Feb 17;281(7):3793-9.
[2]. Smith DB, et al. Design, synthesis, and antiviral properties of 4'-substituted ribonucleosides as inhibitors of hepatitis C virus replication: the discovery of R1479. Bioorg Med Chem Lett. 2007 May 1;17(9):2570-6.