Computational drug repurposing identifies dual-site inhibitors of the Kyasanur Forest disease virus NS3 helicase targeting its ATPase and RNA-binding domains

The tick-borne Flavivirus, Kyasanur Forest Disease Virus (KFDV), remains an emerging threat in India owing to its recurrent outbreaks, limited surveillance, and the absence of effective antiviral therapeutics. The highly conserved viral NS3 helicase, a critical enzyme responsible for viral genome replication through RNA unwinding, represents a promising target for antivirals. In light of this, our study presents a comprehensive structure-based drug repurposing computational analysis to identify a potential dual-site inhibitor targeting both the catalytic ATP-binding site (ABS) and RNA-binding site (RBS) of the KFDV NS3hel enzyme. A ligand library of 9,716 DrugBank compounds was screened for highly interacting molecules using the hierarchical docking (HTVS, SP, and XP) technique in the Schrödinger Maestro suite. The selected complexes were further evaluated by 300 ns molecular dynamics simulations and advanced post-simulation studies, such as binding free energy (MM/GBSA) and their energy landscape plots. Among the screened compounds, the prodrug Taribavirin exhibited stable binding interactions and favourable energetics across both the ABS and RBS pockets. Although not the strongest binder at either site, it can maintain dual-site interactions, suggesting its potential as a dual-site helicase inhibitor. Overall, this study provides the first in silico evidence of dual-site inhibition of the KFDV NS3hel, with Taribavirin identified as a promising candidate for further in vitro and in vivo validation studies.