In vitro activity: the antiviral activity of simeprevir (TMC435350) in Huh7-Luc cells is dose dependent, and the EC50 and EC90 values determined for TMC435350 are 8 nM and 24 nM, respectively. Inhibition of TMC435350 on NS3/4A protease is time dependent, and the overall Kis are estimated to be 0.5 nM for genotype 1a and 0.4 nM for genotype 1b, respectively. TMC435350 is a potent inhibitor of HCV NS3/4A protease (Ki=0.36 nM) and viral replication (replicon EC50=7.8 nM).

Kinase Assay: the in vitro inhibitory activity of simeprevir against NS3/4A is determined using a fluorescence resonance energy transfer cleavage assay with the RetS1 peptide substrate, derived from the genotype 1a NS4A-4B junction, and bacterially expressed full-length NS3 protease domain, supplemented with an NS4A peptide. Briefly, NS3/4A is preincubated in the presence of TMC435350 for 10 min, and then the RetS1 substrate is added and fluorescence is continuously measured for 20 min (excitation, 355 nm; emission, 500 nm). Cleavage of the substrate is expressed as a percentage of the cleavage seen with the vehicle control.

Cell Assay: Huh7-Luc cells are seeded at a density of 2,500 cells/well in a 384-well plate in Dulbecco's modified Eagle's medium plus 10% fetal calf serum and incubated with a range of concentrations of serially diluted simeprevir (TMC435350), in a final DMSO concentration of 0.5% in the absence of G418. After 72 h of incubation, Steady Lite reagent is added in a 1:1 ratio to the medium, and luciferase signal is measured using a ViewLux reader.