Identification and Biochemical Characterization of Small-Molecule Inhibitors of West Nile Virus Serine Protease by a High-Throughput Screen

West Nile virus (WNV) and the four serotypes of dengue virus (DENV1 to DENV4) have recently emerged as significant human pathogens that cause millions of infections each year and result in considerable morbidity and mortality (16, 26). WNV was introduced into the Western Hemisphere during an outbreak in the United States in 1999. In the following years, WNV has spread throughout much of North America and has become a major public health concern (reviewed in reference 7). Most WNV infections are asymptomatic; however, about 20% of cases are associated with mild flu-like symptoms. A small fraction of these cases progresses to more-severe clinical manifestations, including encephalitis and/or flaccid paralysis. Currently, there are no approved vaccines or antiviral therapeutics available for WNV-infected humans. The WNV genome consists of approximately 11 kb of RNA of positive polarity, which encodes a single polyprotein that is processed co- and posttranslationally by the host signal peptidase and the viral serine protease into at least 10 proteins. The three structural proteins, capsid (C), prM, and envelope (E), arise from the N terminus of the polyprotein, and the seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) arise from the C-terminal portion during processing at the endoplasmic reticulum of the host cell (7, 23). The active form of the viral serine protease consists of a complex of two proteins, NS2B and NS3. NS3 is a multifunctional protein. The amino-terminal domain contains the serine protease catalytic triad, consisting of amino acid residues H51, D75, and S135 (5). This domain interacts with NS2B, a required cofactor, to form the active serine protease (3, 8, 9, 14, 15, 33, 35). Fine mapping of the minimal domain of NS3 has revealed that the amino-terminal 167 residues are sufficient for cis-cleavage at the NS2B-NS3 junction (22). The two-component NS2B/NS3 viral serine protease activity plays a key role in flaviviral polyprotein processing. This is an obligatory step prior to viral RNA replication, thus identifying the viral serine protease as an excellent therapeutic target. The protease cleavage sites in the polyprotein have a pair of basic amino acids (R and K) at the P2 and P1 (occasionally there is a Q at P2) positions, followed by a short-chain amino acid (G, S, or A) at the P1′ position (30). NS2B is an endoplasmic reticulum integral membrane protein (12). It consists of a conserved hydrophilic domain (NS2BH) and three hydrophobic domains; the former is essential both for interaction with the NS3 protease domain (NS3-pro) and for protease activity (9, 12, 14). In a previous study using peptide substrates, we reported that the interaction of DENV2 NS2BH with NS3-pro increased the kcat/Km of NS3-pro from ∼3.3 × 103-fold to 7.6 × 103-fold (34). The crystal structures of the NS2BH cofactor bound to NS3-pro of WNV and DENV2 were reported recently, the former in the presence of a substrate-based inhibitor peptide covalently linked to the active site (1, 13). These structures revealed the identities of the amino acid residues involved in substrate recognition and provided a structural basis for the activation of NS3-pro by NS2BH. They also provided a rational explanation for the mutational effects of the WNV NS2BH cofactor as well as for its role in the active protease (10). The goal of this study was to identify small-molecule inhibitors of the WNV protease. We employed previously described in vitro protease assays (27, 34) adapted to a high-throughput format. Further detailed biochemical and kinetic analyses of representative compounds led to the identification of two lead compounds (compounds A and B) that inhibited the WNV NS2BH/NS3-pro in vitro. Compound B was also found to inhibit WNV RNA replication in cultured cells when the replicon RNA was delivered by infection with WNV particles bearing replicon RNA (28). The kinetic data revealed that compound B functioned as a competitive inhibitor. This conclusion is supported by molecular modeling, which shows that there is only one plausible binding site for the compound within the NS3-pro domain in the vicinity of the substrate binding pockets.