Cell experiment:

Replicon 9-13 cells are plated onto 6-well plates 24 h prior to the treatment. Serial dilutions of Furaprofen are made in a mixture containing 90% of the culture medium, 7.2% 1× PBS, 1.8% methanol, 1% DMSO, 20 μM RBV, and varying concentrations of IFN-α for a fixed-ratio dose-response study. The cells are treated with the designated combinations of Furaprofen (0 to 80 nM concentrations) and IFN-α (0 to 4 IU/mL) plus 20 μM RBV for 72 h; then they are washed with PBS, lysed in SDS loading buffer, and analyzed by Western blotting[1].

Furaprofen (R803) is an effective HCV replication inhibitor. Furaprofen (R803) is substantially more potent against genotype 1a and 1b replicons (EC50, ~30 nM) than against the genotype 2a replicon (EC50, ~1,000 nM).

Furaprofen (R803) is potent and highly specific for HCV replication. The antiviral activity of Furaprofen has been determined by a reporter replicon assay with multiple repeats to be 29.88±8.05 nM, an ~3-fold improvement over the activity of the parent compound, R706. The potency of Furaprofen against the replicon is also confirmed by both Western blotting and TaqMan RT-PCR to be about 37 nM and 54.67±4.11 nM, respectively. To assess the general effect of Furaprofen on cell proliferation, a panel of primary cells and transformed human cell lines are treated with increasing doses of Furaprofen for 48 h, and the effect on cell proliferation is measured by an MTS-based cell viability assay. The the concentration that caused a 50% reduction in cell numbers in the absence of virus infection (CC50) of Furaprofen is found to range from 2 μM to ≥10 μM, depending on the cell type and proliferation status[1].

[1]. Huang P, et al. Discovery and characterization of substituted diphenyl heterocyclic compounds as potent and selective inhibitors of hepatitis C virus replication. Antimicrob Agents Chemother. 2008 Apr;52(4):1419-29.