Identification of a small compound that specifically inhibits Zika virus in vitro and in vivo by targeting the NS2B-NS3 protease

Zika virus (ZIKV) has rapid become a global threat, but no ZIKV-specific vaccines or drugs are currently available. In this study, inhibitors of ZIKV NS2B-NS3 protease were screened from a library containing 4,452 compound fragments. One of the compounds, 6-bromo-1,2-naphthalenedione, exhibited high specific inhibition against ZIKV NS2B-NS3 protease, but had no inhibitory effects against other viral proteases. A microscale thermophoresis (MST) assay confirmed that the compound bound to ZIKV NS2B-NS3 protein with a binding constant (Kd) of 12.26 μM. Indirect immunofluorescence assays, Western blots, and plaque assays indicated that the compound inhibited virus replication in cells. Virus titer was reduced by more than 75% when the compound was present at 1 μM. A time-of-addition assay showed that inhibition occurred at the virus replication stage, but not at the adsorption or invasion stages. The half cytotoxicity concentration (CC50) of the compound on HeLa, Vero, and BHK-21 cells were 445.44 μM, 123.87 μM, and 123.64 μM, respectively. In vivo tests using infected AG129 mice demonstrated that treatment with the compound reduced mortality by up to 60%. Mice treated with the compound showed a reduction in histopathological lesions in brain, testis, and ovary. Viral RNA, IL-1β, and IL-6 mRNA levels decreased significantly in these tissues. In summary, this study has identified a small compound with high and specific inhibitory effects on ZIKV. The compound can be used as a therapeutic agent and is also an ideal starting point for drug optimization. •A library containing 4452 compound fragments was screened to obtain inhibitors of Zika virus NS2B-NS3 protease.•One compound fragment was identified to have high inhibition to the activity of Zika virus NS2B-NS3 protease.•The compound fragment was proven to have high inhibitory effect to the virus in cells.•The compound fragment has therapeutic effect on mice infected with the Zika virus.•The compound screened in this study has very low molecular weight and has great space for modification and optimization.