Telaprevir

Antiviral agents included two active site protease inhibitors telaprevir (Selleckchem, Houston, TX, USA) and boceprevir (ChemScene, Monmouth Junction, NJ, USA), NS5A inhibitor daclatasvir (Selleckchem), two nucleotide NS5B polymerase inhibitors, sofosbuvir (Advanced Chemblocks, Burlingame, CA, USA) and 2′-C-methylcytidine (US Biological, Salem, MA, USA), host-targeting cyclophillin inhibitor cyclosporin A (Sigma-Aldrich, St Louis, MO, USA) and entry-inhibitor (S)-chlorcyclizine (NCATS, Bethesda, MD, USA).
The HCVcc-Luc infectious virus consisted of a full-length J6/JFH-1 HCV with insertion of a luciferase reporter gene at the 3′ end of the p7 gene [16 (link)].
Huh7.5.1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Serum Source International, Charlotte, NC, USA), 100 IU/ml penicillin, and 100 μg/ml streptomycin in 5% CO₂, at 37°C. A stably expressing Con1b replicon cell line with luciferase reporter under the direction of 5′-NTR for quantification described previously [21 (link),22 (link)] was grown in the same condition as Huh7.5.1 cells with the addition of 500 μg/ml G-418.

HCV-Luc infection and ATPlite assays were carried out in 96-well plates in the presences of the compound of interest titrated in vertical and the known antiviral drug titrated in horizontal.^{13 (link),22} The known antiviral drugs include ribavirin (Sigma-Aldrich), sofosbuvir (Advanced Chemblocks), telaprevir (Selleckchem), daclatasvir (Selleckchem), cyclosporin A (Sigma-Aldrich), and boceprevir (ChemScene). Two independent mathematical models, the Bliss independence model and the Loewe additivity model, were used to predict the theoretical additive, synergistic, or antagonistic effects. By the Bliss independence model, log volumes of synergistic or antagonistic effect were calculated with the MacSynergy program. By the Loewe additivity model, combination indices were calculated at or near the EC₅₀ values of the compound and the antiviral drug when tested alone with CalcuSyn program (Biosoft).