Structure-based virtual screening leads to the discovery of a 4,5-dihydropyrazole derivative as the first dual inhibitor of West Nile virus NS3 helicase and protease.

West Nile Virus (WNV) non-structural protein 3 (NS3) is a multifunctional enzyme critical for viral replication and represents a highly well-conserved antiviral target. NS3 consists of an N-terminal serine protease (NS3pro), which relies on the NS2B cofactor for polyprotein processing, and a C-terminal superfamily 2 helicase (NS3hel) responsible for ATP-dependent RNA unwinding and associated NTPase/RTPase activities. Increasing evidence shows functional crosstalk between these domains, supporting the dual target inhibition as a promising antiviral approach. This study describes a structure-based virtual screening (SBVS) campaign using an in-house library (EMAC-DB) targeting both NS3hel and NS3pro. Following an integrated CADD workflow, we identified a new series of 4,5-dihydropyrazole derivatives as dual-site NS3 inhibitors. In vitro experiments confirmed that these compounds inhibit both NS3hel and NS3pro enzymatic activities at low micromolar concentrations. Notably, compound 5e emerged as a key candidate, demonstrating the highest potency and significantly reducing WNV replication in HuH-7 cells, with an EC50 of 0.42 ± 0.1 μM. These findings suggest that two mechanisms contribute to viral replication inhibition. Further investigations revealed that NS2B-NS3pro inhibition occurred through a non-competitive mechanism, whereas NS3hel inhibition was non-competitive with respect to ATP and competitive with respect to dsDNA, supporting the hypothesis that the compound interacts with the RNA-binding cleft of NS3. Overall, this work provides a structural and mechanistic framework for the optimization of 4,5-dihydropyrazole-based dual NS3 inhibitors, highlighting the importance of a dual-acting inhibitory strategy to potentially reduce the development of resistance.