During development of multicellular organisms, cells respond to extracellular cues through nonlinear signal transduction cascades whose principal components have been identified. Nevertheless, the molecular mechanisms underlying specificity of cellular responses remain poorly understood. Spatial distribution of signaling proteins may contribute to signaling specificity. Here, we tested this hypothesis by investigating the role of the Rab5 effector Appl1, an endosomal protein that interacts with transmembrane receptors and Akt. We show that in zebrafish, Appl1 regulates Akt activity and substrate specificity, controlling GSK-3beta but not TSC2. Consistent with this pattern, Appl1 is selectively required for cell survival, most critically in highly expressing tissues. Remarkably, Appl1 function requires its endosomal localization. Indeed, Akt and GSK-3beta, but not TSC2, dynamically associate with Appl1 endosomes upon growth factor stimulation. We propose that partitioning of Akt and selected effectors onto endosomal compartments represents a key mechanism contributing to the specificity of signal transduction in vertebrate development.

Zika virus (ZiKV) has been extensively studied since it was linked to congenital malformations, and recent research has revealed that astrocytes are targets of ZiKV. However, the consequences of ZiKV infection, especially to this cell type, remain largely unknown, particularly considering integrative studies aiming to understand the crosstalk among key cellular mechanisms and fates involved in the neurotoxicity of the virus. Here, the consequences of ZiKV infection in ipSc-derived astrocytes are presented. our results show RoS imbalance, mitochondrial defects and DnA breakage, which have been previously linked to neurological disorders. We have also detected glial reactivity, also present in mice and in post-mortem brains from infected neonates from the northeast of Brazil. Given the role of glia in the developing brain, these findings may help to explain the observed effects in congenital Zika syndrome related to neuronal loss and motor deficit.

The infection by the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes major public health concern and economic burden. Although clinically approved drugs have been repurposed to treat individuals with 2019 Coronavirus disease (COVID-19), the lack of safety studies and limited efficiency as well jeopardize clinical benefits. Daclatasvir and sofosbuvir (SFV) are clinically approved direct-acting antivirals (DAA) against hepatitis C virus (HCV), with satisfactory safety profile. In the HCV replicative cycle, daclatasvir and SFV target the viral enzymes NS5A and NS5B, respectively. NS5A is endowed with pleotropic activities, which overlap with several proteins from SARS-CoV-2. HCV NS5B and SARS-CoV-2 nsp12 are RNA polymerases that share homology in the nucleotide uptake channel. These characteristics of the HCV and SARS-CoV-2 motivated us to further study the activity of daclatasvir and SFV against the new coronavirus. Daclatasvir consistently inhibited the production of infectious SARS-CoV-2 virus particles in Vero cells, in the hepatoma cell line HuH-7 and in type II pneumocytes (Calu-3), with potencies of 0.8, 0.6 and 1.1 μM, respectively. Daclatasvir targeted early events during SARS-CoV-2 replication cycle and prevented the induction of IL-6 and TNF-α, inflammatory mediators associated with the cytokine storm typical of SARS-CoV-2 infection. Sofosbuvir, although inactive in Vero cells, displayed EC50 values of 6.2 and 9.5 μM in HuH-7 and Calu-3 cells, respectively. Our data point to additional antiviral candidates, in especial daclatasvir, among drugs overlooked for COVID-19, that could immediately enter clinical trials.

Background Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity. Methods SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir’s dose and schedule to maximize the probability of success for COVID-19. Results Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 μM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. Conclusions Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.

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