Anti‐Zika Virus Activity of Azvudine via Inhibition of RNA Replication by Its Active Triphosphate Form

ABSTRACT The outbreak of the Zika virus (ZIKV) poses a significant threat to global public health. Currently, there are no approved antiviral drugs specifically targeting ZIKV. This highlights the urgent need for the development of effective therapeutic agents. Azvudine (FNC), an antiviral drug approved in China for treating HIV/AIDS and later granted conditional approval for COVID‐19, is a promising candidate for drug repurposing. In this study, we found FNC inhibited ZIKV replication in a cell‐based replicon system we established. This inhibition was confirmed by significant reductions in ZIKV RNA and NS5 protein levels, as demonstrated by RT‐qPCR, Western blot, and immunofluorescence analyses. Moreover, FNC effectively inhibited the ZIKV (SMGC‐1) strain with an EC 50 of 6.62 μM, comparable to that of the known ZIKV inhibitor NITD008. LC‐MS/MS analysis revealed efficient intracellular uptake of FNC and its conversion to the active triphosphate metabolite FNC‐TP. We further showed that FNC‐TP, but not FNC, acted as a nucleoside analog to directly inhibit the RNA‐dependent RNA polymerase (RdRp), preventing RNA chain elongation with an IC 50 of 4.93 μM. These findings provided the first evidence of FNC's anti‐ZIKV activity, revealing that its convention to FNC‐TP is the key mechanism for inhibiting viral RNA synthesis and highlighting its potential as a promising anti‐ZIKV agent.