Mateus Serafim, Thales Kronenberger, Rafael Rocha, Rafaela Ferreira, Vinícius Maltarollo, Bruno Mota and Erna Kroon

Approximately three billion people live in regions at risk of infections by flaviviruses. Dengue virus (DENV), Zika virus (ZIKV) and Yellow fever virus (YFV) presents outbreaks and severe complications. Currently, there are no antivirals available to treat these diseases. We screened and evaluated the potential antiviral activity of small molecules against these viruses, targeting the viral protease NS2B-NS3 (NS3PRO). We used a combination of HQSAR models and structural molecular modelling, based on structures of peptidomimetic DENV-3 NS3PRO inhibitors and molecular docking studies to screen for new compounds. Binding sites of DENV-3 and ZIKV NS3PRO were assessed to build a pharmacophoric model for virtual screening. Hits were selected after molecular dynamics simulations, with predictions of toxicity and biological activity. Biological activities were evaluated by the MTT assay. Antiviral activity was evaluated by plaque reduction, pre-treatment and virucide activity assays. Enzymatic inhibition assays against ZIKV NS3PRO were carried out. An optimal HQSAR model (q2 = 0.67; r2 = 0.87) was selected. A virtual screening of ~7,600,000 compounds was conducted (pharmacophore, docking and molecular dynamics), identifying eight potential inhibitors to the NS3PRO, with favorable biological activity (5/8) and toxicity (8/8) predictions. Five were active against ZIKV, YFV, DENV-2 or DENV-3 (EC50 from 4.21 ± 0.14 to 37.51 ± 0.8 µM, with selective indexes from 1.42 to 3.74), with one being active against all viruses. In plaque reduction assays, two substances reduced about 1.0 to 1.5 log10 of the viral titer of ZIKV, YFV and DENV-2. One also reduced about 1.0 log10 of YFV titer in pre-treatment assays. We have identified five compounds with antiviral activity, with one showing a potential panflavivirus activity. Preliminary ZIKV NS3PRO inhibition assays showed three active compounds with IC50 values between 28 and 69 µM.

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