Cell experiment:

Huh-luc/neo cells are seeded in 96-well plates at a density of 8000 cells per well in a final volume of 200 μL of Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. One day after seeding, ACH-806 is serially diluted in 100% dimethyl sulfoxide (DMSO) and added to cells at a 1:200 dilution, achieving a final concentration of 0.5% DMSO in a total volume of 200 μL. Cells are further incubated for 3 days (96 h post-seeding), and the inhibition of HCV replicon replication is quantified by measurement of luciferase activity using a commercial kit[1].

ACH-806 is an NS4A antagonist which can inhibit Hepatitis C Virus (HCV) replication with an EC50 of 14 nM.

ACH-806 is an NS4A antagonist which can inhibit Hepatitis C Virus (HCV) replication with an EC50 of 14 nM. ACH-806 treatment results in significant reductions of both NS3 and NS4A in the transfected cells. This finding is reminiscent of ACH-806-treated replicon cells in which the amounts of NS3 and NS4A are also both decreased. The total amount of NS3 in the ACH-806-treated sample is reduced by ~6-fold (100/16) and causes a reduction of NS4A-bound NS3 ~29-fold (261/9). The levels of labeled NS3 and NS4A immunoprecipitated by anti-NS3 antibody are apparently reduced after the treatment of ACH-806. ACH-806 also induces significant decreases of NS3 and NS4A and promotes p14 formation in the parental replicon cells but not in the ACH-806-resistant replicon cells[1].

[1]. Yang W, et al. ACH-806, an NS4A antagonist, inhibits hepatitis C virus replication by altering the composition of viral replication complexes. Antimicrob Agents Chemother. 2013 Jul;57(7):3168-77.