Darunavir and Fosamprenavir Inhibit Zika Virus Replication via Dual Targeting of the Envelope Protein and NS2B-NS3 Protease

Zika virus (ZIKV) remains a significant global health concern, underscoring the need for effective Antiviral agents. In this study, we evaluated the Antiviral activity and mechanisms of the HIV Protease Inhibitors darunavir (DRV), fosamprenavir (FPV), and amprenavir (APV) against ZIKV. DRV and FPV, but not APV, exhibited potent Antiviral activity in BHK-21 and TE-671 cells, as demonstrated by reduced cytopathic effects and decreased viral protein expression. Both compounds inhibited ZIKV infectivity and viral yield with submicromolar EC₅₀ values. Mechanistic analyses using time-of-addition and temperature-shift assays revealed that DRV and FPV act at multiple stages of the viral life cycle, including attachment, entry, and post-entry processes. Molecular docking and mutagenesis studies identified the β-octyl glucoside (β-OG) binding pocket within domain II of the ZIKV envelope (E) protein as a critical target, with Lys209 and Asp278 serving as key interaction residues. Disruption of these residues significantly reduced compound efficacy, confirming their functional importance in viral attachment inhibition. In addition, both DRV and FPV directly inhibited ZIKV NS2B-NS3 Protease activity, with NS2B Asp83 identified as a key determinant for drug binding. In contrast, neither compound significantly affected NS5 RNA-dependent RNA polymerase activity. In a suckling mouse model, both DRV and FPV reduced viral loads in brain tissues in a dose-dependent manner, with DRV showing superior efficacy at lower doses. Collectively, these findings demonstrate that DRV and FPV exert potent anti-ZIKV activity through dual targeting of viral entry and Protease function, highlighting their potential as repurposed therapeutics for ZIKV Infection.