Hepatitis C virus (HCV) is the major cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer with over 170 million infected people worldwide. There is not any vaccine available for HCV treatment. NS3/4A serine protease is essential for viral replication, which shows a promising drug target for developing direct-acting anti-HCV agents. In this study, 150,000 small molecule compounds were extracted from chemical library and screened against NS3/4A protease by structure-based virtual screening (PyRx sofware). Ten compounds with the highest binding affinity were selected and used to evaluate their inhibitory activity by enzyme assay. The NS3/4A protease used for inhibitory kinetic analyses was the recombinant core enzyme, NS3-GSGS-NS4A. Among 10 selected compounds, compound 1 showed the highest inhibitory activity against the NS3/4A protease with IC50 and Ki values of 2.17 μM and 0.92 μM, respectively. Compound 1 was found to be a competitive inhibitor supposed to bind directly to the protease active site. In comparison with the control, the inhibitory activity of compound 1 was two-fold higher than that of hexapeptide, DEMEEC. Our newly identified compound 1 showed a promising anti-HCV agent for further drug development. These data suggested that virtual screening by means of molecular docking may an alternative way in drug discovery and development, which could save time, reduce chemical usage and find candidates efficiently.
