A diarylamine derived from anthranilic acid inhibits ZIKV replication

Abstract

Zika virus (ZIKV) is a mosquito-transmitted Flavivirus, originally identified in Uganda in 1947 and recently associated with a large outbreak in South America. Despite extensive efforts there are currently no approved antiviral compounds for treatment of ZIKV infection. Here we describe the antiviral activity of diarylamines derived from anthranilic acid (FAMs) against ZIKV. A synthetic FAM (E3) demonstrated anti-ZIKV potential by reducing viral replication up to 86%. We analyzed the possible mechanisms of action of FAM E3 by evaluating the intercalation of this compound into the viral dsRNA and its interaction with the RNA polymerase of bacteriophage SP6. However, FAM E3 did not act by these mechanisms. In silico results predicted that FAM E3 might bind to the ZIKV NS3 helicase suggesting that this protein could be one possible target of this compound. To test this, the thermal stability and the ATPase activity of the ZIKV NS3 helicase domain (NS3^Hel) were investigated in vitro and we demonstrated that FAM E3 could indeed bind to and stabilize NS3^Hel.

Figure 1

Inhibitory activity of FAM E3 on ZIKV replication. Schematic representation of ZIKV-Nanoluc that continuously expresses the Nanoluciferase reporter (a). Chemical structure of FAM E3 (b). Dose response assay: ZIKV-Nanoluc infected cells (MOI 0.1) were treated with FAM E3 at concentrations ranging from 1 to 10 µM and virus replication efficiency was evaluated 72 h.p.i. Simultaneously, Vero cells were equally treated with FAM E3 and cells viability was measured 72 h later (c). Effective and cytotoxic concentration of 50%: Vero cells were treated with increasing concentrations of FAM E3 ranging from 0.10 to 200 µM. ZIKV replication was measured by luciferase assay (indicated by ■) and cellular viability measured using an MTT assay (indicated by ●) (d). Vero cells were infected with ZIKV^BR and treated with FAM E3 at 3 µM and virus replication was accessed 72 h.p.i. The intracellular virus was titrated by analysing focus- forming units per milliliters (Ffu/mL), DMSO and OLX were used as negative and positive controls, respectively (e). Huh-7 or 293 T cell lines were infected with ZIKV-Nanoluc and treated with FAM E3 (3 µM) or DMSO (0.1%) for 72 h (f). Mean values of three independent experiments each measured in quadruplicate including the standard deviation are shown. P < 0.05 was considered significant compared to DMSO control.

Figure 2

Effects of FAM E3 on the different stages of the ZIKV replicative cycle. Vero cells were infected with ZIKV-Nanoluc at a MOI = 0.5 and simultaneously treated with FAM E3 for 2 h; cells were washed to remove the virus and replaced with fresh media. ZIKV replication was measured by Nanoluc activity at 72 h.p.i (a). Vero cells were treated with FAM E3 for 2 h. Then, cells were extensively washed and infected with ZIKV-Nanoluc at a MOI = 0.5 for 2 h. The inoculum was removed and the cells were washed and replaced with fresh media. ZIKV replication was measured by Nanoluc activity at 72 h.p.i. (b). Vero cells were infected with ZIKV-Nanoluc at a MOI = 0.5 for 2 h. The virus was removed, cells were washed and added of fresh media containing FAM E3. ZIKV replication was measured by Nanoluc activity at 72 h.p.i (c). For all assays, non-infected Vero cells were equally treated with FAM E3 and cell viability was measured 72 h later using MTT assay. DMSO was used as negative control and OLX as positive control for infectivity inhibition. Mean values of three independent experiments each measured in quadruplicate including the standard deviation are shown. P < 0.05 was considered significant.

Figure 3

Analysis of FAM E3 intercalation into the viral dsRNA and its interaction with the activity of phage SP6 RNA polymerase. Fifteen nanomoles of dsRNA were incubated with the FAM E3 or intercalating controls (DMSO) or (DOX) for 45 minutes at room temperature. The reaction products were subjected to 1% agarose electrophoresis gel containing Ethidium Bromide followed by densitometry analysis (a). FAM E3 and 5 µg of purified pCCI-SP6-ZIKV amplicon was used for in vitro transcription using SP6 RNA polymerase at the presence or absence of FAM E3. Reaction products were analysed by agarose gel electrophoresis followed by densitometry analysis (b). Results of a representative of three independent reproducible experiments are shown.

Figure 4

FAM E3 interference with the cell lipid metabolism of the host cells. Vero cells were infected with ZIKV at MOI = 0.1 and treated with FAM E3 3 µM or DMSO 0.1% or OLX controls for 72 h. Naïve Vero cells were treated with DMSO were used as non-infected cells control. After treatment, cells were fixed and nuclei, lipid droplets (LDs) and ZIKV NS3 were labeled using DAPI (blue), BODIPY 493/503 (green) and ZIKV anti-NS3 antibody (red), respectively. Scale bar 100 nm.

Figure 5

Predicted intermolecular interactions between FAM E3 and the RNA binding site of ZIKV NS3^Hel. 3D structure of the RNA binding site of ZIKV NS3^Hel docked with FAM E3, highlighting the main interactions between FAM E3 and amino acid residues, through hydrogen bonds (dotted black lines) and hydrophobic interactions (transparent green surface) (a). 2D representation of the protein-ligand interactions (b).

Figure 6

Predicted intermolecular interactions between FAM E3 and the ATP binding site of ZIKV NS3^Hel. 3D structure of the ATP binding site of ZIKV NS3^Hel docked with FAM E3, highlighting the main interactions between FAM E3 and amino acid residues, through hydrogen bonds (dotted black lines) and hydrophobic interactions (transparent green surface) (a). 2D representation of the protein-ligand interactions (b).

Figure 7

FAM E3 activity on NS3^Hel. Thermal denaturation curves of NS3^Hel and Boltzmann fitting in the presence of DMSO (Control) or FAM E3. Tm of NS3^Hel obtained after Boltzmann fitting is represented on the table (a). MicroScale Thermophoretic analysis of the interaction between FAM E3 and NS3^Hel. Data from four experiments were normalized to the fraction of bound ligand and averaged. Kd constant was obtained after a sigmoidal shape fitting with the Hill function (black continuous line) (b). NTPase activity of ZIKV NS3^Hel in the presence or absence of FAM E3 and using ATP as substrate (c).