Potent cationic inhibitors of West Nile Virus NS2B/NS3 protease with serum stability, cell permeability and antiviral activity

West Nile virus (WNV) has spread rapidly around the globe, efficiently crossing species from migrating birds into humans and other mammals. The viral protease NS2B-NS3 is important for WNV replication and recognizes dibasic substrate sequences common to other flaviviral proteases but different from most mammalian proteases. Potent inhibitors of WNV protease with antiviral activity have been elusive to date. We report the smallest and most potent inhibitors known for this enzyme, cationic tripeptides with nonpeptidic caps at the N-terminus and aldehyde at the C-terminus. One of these, compound 3 (K(i) = 9 nM) is stable in serum (>90% intact after 3 h, 37 degrees C), cell permeable, and shows antiviral activity (IC(50) 1.6 PM) without cytotoxicity (IC(50) >400 mu M), thereby validating the approach of inhibiting WNV protease to suppress WNV replication.