Structural Elucidation of Langat Virus Helicase Unveils Dual-Target Inhibition for Broad-Spectrum Anti-Flaviviruses Strategy

Ruixue Li¹Zhen Han²Xiao He¹Rongrong Zhong³Chen Chen^1*

• ¹Tianjin Medical University, Tianjin, China
• ²Tianjin University, Tianjin, China
• ³Tianjin Medical University General Hospital, Tianjin, China

Flaviviruses, such as dengue, Zika, and Langat virus, pose significant global health threats, underscoring the clinical need for broad-spectrum antiviral therapies. It is imperative to conduct broader inhibitor panel studies on the crucial proteins that could serve as potential targets for therapeutics. The NS3 helicase of Langat virus (LGTV), a tick-borne flavivirus, plays a critical role in viral RNA replication and represents a promising antiviral target. Here, we resolved the apo LGTV helicase crystal structure and characterized its conserved ATP-and RNA-binding sites. The cloverleaf-shaped architecture comprises three domains, with a positively charged RNA-binding cleft and an ATPase pocket at the domain 1/2 interface. Comparative analysis with ZIKV and DENV helicases revealed LGTV's intermediate conformational flexibility in motif I upon ATP binding, suggesting species-specific dynamics. Virtual screening of 11,027 compounds identified six high-affinity inhibitors targeting these functional sites. Notably, the repurposed leukotriene antagonist Zafirlukast exhibited dual-target binding, engaging both ATP-and RNA-binding pockets via conserved residues by molecular docking. Molecular dynamics simulations confirmed stable complex formation, with Zafirlukast showing superior stabilization compared to other candidates like Bananins. Isothermal titration calorimetry (ITC) validated pan-flavivirus affinity, highlighting its broad-spectrum potential. Structural conservation of interacting residues across flaviviruses supports a dual-inhibition mechanism—blocking ATP hydrolysis and RNA unwinding—to circumvent resistance. These findings establish LGTV helicase as a model for rational drug design and position Zafirlukast as a prototype for dual-target inhibitors against neurotropic flaviviruses.