Structural elucidation of Langat virus helicase unveils dual-target inhibition for broad-spectrum anti-flaviviruses strategy.

Introduction: Flaviviruses, such as dengue, Zika, and Langat virus (LGTV), pose significant global health threats, highlighting the urgent need for broad-spectrum antiviral therapies. This study focuses on the NS3 helicase of LGTV, a key enzyme in viral replication, aiming to elucidate its structure and identify high-potency inhibitors to facilitate rational drug design. Methods: The study employed an integrated approach: 1) Structural Biology: Determining the crystal structure of apo LGTV helicase. 2) Comparative Analysis: Aligning the structure with Zika (ZIKV) and dengue (DENV) virus helicases to assess conformational flexibility. 3) Virtual Screening: Screening 11,027 compounds to identify high-affinity inhibitors. 4) Molecular Modeling: Validating binding modes and stability via molecular docking and dynamics simulations. 5) Experimental Validation: Assessing pan-flavivirus affinity of lead compounds using Isothermal Titration Calorimetry (ITC). Results and discussion: The cloverleaf-shaped architecture of LGTV helicase was resolved, revealing conserved ATP- and RNA-binding sites. LGTV exhibited an intermediate conformational flexibility upon ATP binding compared to ZIKV and DENV helicases. Virtual screening identified six high-affinity hits, notably the repurposed drug Zafirlukast. Zafirlukast demonstrated a dual-targeting mechanism, engaging both the ATPase pocket and RNA-binding cleft. Molecular dynamics confirmed stable binding, and ITC validated its broad-spectrum affinity across flaviviruses. The study establishes the LGTV helicase as a robust model for antiviral development. Zafirlukast emerges as a promising prototype for dual-target inhibitors, capable of simultaneously obstructing ATP hydrolysis and RNA unwinding. These findings provide a strong foundation for designing novel broad-spectrum therapeutics against neurotropic flaviviruses.