Neferine inhibits Zika virus replication by targeting viral protease

Zika virus (ZIKV) belonging to the Flaviviridae family causes critical neurological abnormalities, including congenital microcephaly and Guillain-Barré syndrome. Despite being a major public health concern, effective therapeutic interventions against ZIKV remain unavailable. NS2B-NS3 (ZIKVpro), the viral serine protease critical for polyprotein processing as well as viral replication, is a potential target for developing antiviral agents PURPOSE: This study aims to develop ZIKV inhibitors targeting the ZIKVpro from natural products.

Surface plasmon resonance (SPR) based assay were used for drug screening from a natural product library. qRT-PCR, Western blot and plaque assay was used for the evaluation of their antiviral activity and studies of their mechanisms of action. Hydrogen-deuterium exchange mass spectrometry (HDXMS) analysis and Molecular dynamics (MD) simulation were used for confirmation of action site.

We demonstrate that neferine, a bisbenzylisoquinoline alkaloid derived from the green embryo of the mature seeds of Nelumbo nucifera Gaertn (Plumula Nelumbinis), exhibits potent inhibitory effects against ZIKV replication in vitro and in vivo. Mechanistic analyses revealed that neferine directly binds to ZIKVpro, interacting with catalytic residues Tyr161 and Ser135, thereby suppressing protease activity in enzymatic assays and impairing polyprotein processing in infected cells. Notably, neferine also demonstrated broad-spectrum antiviral activity against Dengue virus, suggesting its potential as a pan-Flavivirus inhibitor.

Our results highlight neferine's therapeutic potential for combating ZIKV and related Flavivirus infections, providing a robust foundation for further drug development.