| Assay Method Information | |
| | Radiolabeled Nucleotide Incorporation Assay |
| Description: | To measure inhibition of the enzymatic activity of the HCV NS5B RNA-dependent RNA polymerase by the nucleoside triphosphate compounds of the present invention, a radiolabeled nucleotide incorporation assay was used. This assay is a modified version of the assay described in International Publication No. WO2002/057287. Briefly, 50 uL reactions containing 20 mM HEPES (pH 7.3); 7.5 mM DTT; 20 units/ml RNasIN; 1 uM each of ATP, GTP, UTP and CTP; 20 uCi/mL [33P]-CTP; 10 mM MgCl; 60 mM NaCl; 100 ug/ml BSA; 0.021 uM DCoH heteropolymer RNA template; and 5 nM NS5B (1b-BKDelta55) enzyme are incubated at room temperature for 1 hour. The assay is then terminated by the addition of 500 mM EDTA (50 uL). The reaction mixture is transferred to a Millipore DE81 filter plate and the incorporation of labeled CTP is determined using Packard TopCount. Compound IC50 values can then be calculated from experiments with 10 serial 3-fold dilutions of the inhibitor in duplicate. |
| Affinity data for this assay | |
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