Lomibuvir (VCH-222) is a selective, non-nucleoside allosteric inhibitor of HCV NS5B polymerase (RdRp) with a Kd of 17 nM. Lomibuvir inhibits the 1b/Con1 HCV subgenomic replicon with an EC 50 of 5.2 nM. Lomibuvir preferentially inhibits elongative RNA synthesis rather than de novo -initiated RNA synthesis [1].

HCV NS5B 1a:0.94-1.2 μM

Anti-NS5B activity assay: The inhibitory effect of VX-222 on HCV NS5B activity is measured by evaluating the amount of radiolabeled UTP incorporated by the C-terminal ?21 truncated version of enzyme in a newly synthesized RNA using a homopolymeric RNA template / primer namely poly rA / oligo dT. Quantitative detection of incorporated radioactivity is done using a liquid scintillation counter. The in vitro kinetics of inhibition of HCV NS5B from genotype 1b strain BK by VX-222 are determined using the C-terminal ?21 truncated version of NS5B. VX-222 (1 to 1.5 μM) is tested in the presence of 10 to 75 μM nonradioactive UTP mixed with 0.89 to 6.70 μCi of [α-33P]-labeled UTP. RNA-dependent-RNA polymerase reactions are allowed to proceed for 18 min at 22 °C.

Huh7.5 cells harboring HCV RNA replicons are trypsinized and plated into 48-well plates at a concentration of 4 × 104 cells/well. The next day the medium is changed and VX-222 is added in 200 μL of complete medium. After 48 hours, total RNA is extracted and viral RNAs are quanti?ed by real-time reverse transcription-PCR (RT-PCR). The effective drug concentrations that reduced HCV RNA replicon levels by 50% (EC50) are calculated by nonlinear regression analysis with log curve ?tting.(Only for Reference)