Avaliação da atividade antiviral da ouabaína contra o Zika vírus em modelos in vitro, in vivo e in silico

Abstract

Ouabain, a cardiotonic steroid known as Na+/K+-ATPase inhibitor, has been described as an immunomodulatory substance by our group. In addition, the antiviral activity of ouabain has been reported in several studies. Zika virus (ZIKV) is an emerging arbovirus associated with neurological disorders. Currently, there are no vaccines or antivirals available to treat the disease caused by ZIKV. Then, this work aimed to evaluate the anti-ZIKV activity of ouabain in vitro, in silico and in vivo. Initially, Vero cells were used to determine the non-toxic concentrations of ouabain and then, the antiviral activity was evaluated in this cell type. Ouabain presented a dose-dependent inhibitory effect against ZIKV, mainly when added post infection. The reduction of infectious virus was accompanied by a decrease in ZIKV RNA levels, suggesting that the mechanism of ZIKV inhibition by ouabain occurred at the replication step. To evaluate the activity of ouabain in a more clinical approach in relation to ZIKV infection, an in vitro model of human neural progenitor stem cells (NS/PCs) and an animal model of Congenital Zika Virus Syndrome (SCZ) were performed. In NS/PCs, ouabain reduced ZIKV infection in this cell type and blocked the impairment of neurogenesis triggered by the virus. Additionally, in an animal model of SCZ, ouabain protected the fetus from microcephaly triggered by the infection. This is might related to the ability of ouabain to inhibit the virus in yolk sac microglial progenitors. In addition to antiviral activity, ouabain was able to reduce the pro-inflammatory cytokine IL-1β in the placenta, demonstrating its anti-inflammatory potential in this model. Finally, we performed a molecular docking procedure followed by molecular dynamics simulations to predict the interaction between ouabain and the main drug targets in ZIKV proteome (NS3 Helicase, NS5 Mtase and NS5 RdRp). Our in silico data demonstrated a conformational stability and favorable binding free energy of ouabain in the biding sites of the NS5-RdRp and NS3-helicase proteins, suggesting inhibition of these viral proteins by this steroid.Together, these data demonstrate the antiviral action of ouabain in ZIKV infection, revealing this substance as a potential compound for the treatment of infection caused by this virus. In this way, this study contributes to highlighting the effectiveness of cardiotonic steroids as promising antiviral agents, in addition to generating more understanding about the biological functions of ouabain.