From virtual screening to animal models: chlorhexidine and indinavir as promising anti-Zika drug candidates.

Introduction: Zika virus (ZIKV) infection is associated with severe neurological complications, but no clinically approved antiviral therapies exist, leaving management reliant on symptomatic support. The essential NS2B/NS3 protease represents a promising drug target for ZIKV. Methods: We performed structure-based virtual screening of 5,980 FDA-approved compounds from the ZINC database against the ZIKV NS2B/NS3 protease. Molecular docking identified 10 high-affinity candidates (LibDock score >150), which were subsequently evaluated for cytotoxicity and antiviral activity in Vero cells. The most promising compounds were further validated using immunofluorescence and Western blot assays. Their in vivo efficacy was assessed in a lethal AG6 mouse model. Results: Chlorhexidine and indinavir exhibited potent anti-ZIKV activity in vitro, with EC50 values of 16.41 µM and 12.8 µM, respectively, and favorable selectivity indices (CC50: 57.56 µM and 38.96 µM). Both compounds demonstrated a dose-dependent inhibition of ZIKV replication (5-40 µM) at the protein level. In the AG6 mouse model, treatment with either compound (50 mg/kg/day) significantly prolonged survival (p<0.001), delayed disease-associated weight loss, and reduced viral loads in key tissues compared to untreated controls. Discussion: Our integrated computational and experimental approach identifies chlorhexidine and indinavir as promising repurposed anti-ZIKV agents. While toxicity concerns require further investigation, these findings provide a validated foundation for the development of therapeutics against ZIKV infection.