Acridones as promising drug candidates against Oropouche virus

Abstract

Oropouche virus (OROV) is an emerging vector-borne arbovirus found in South America that causes Oropouche fever, a febrile infection similar to dengue fever. It has a high epidemic potential, causing illness in over 500,000 cases diagnosed since the virus was first discovered in 1955. Currently, the prevention of human viral infection depends on vaccination, but availability for many viruses is limited, and they are classified as neglected viruses. At present, there are no vaccines or antiviral treatments available. An alternative approach to limiting the spread of the virus is to selectively disrupt viral replication mechanisms. Here, we demonstrate the inhibitory effect of acridones, which efficiently inhibited viral replication by 99.9 % in vitro. To evaluate possible mechanisms of action, we conducted tests with dsRNA, an intermediate in virus replication, as well as MD simulations, docking, and binding free energy analysis. The results showed a strong interaction between FAC21 and the OROV endonuclease, which possibly limits the interaction of viral RNA with other proteins. Therefore, our results suggest a dual mechanism of antiviral action, possibly caused by ds-RNA intercalation. In summary, our findings demonstrate that a new generation of antiviral drugs could be developed based on the selective optimization of molecules.

Keywords: Acridone; Antiviral; Drug discovery; Endonuclease; Oropouche.

Fig. 1

Chemical structure of the acridones FAC21 and FAC22.

Fig. 2

Acridones have low cytotoxicity, and their effect on viral yield is concentration-dependent. (A) Cytotoxicity assay of the tested compounds in Vero cells. The cytotoxicity of acridones based on the dose-response was determined using MTT. The 50 % cytotoxic concentration (CC₅₀) was calculated for each compound using nonlinear regression analysis of GraphPad Prism software (version 8.0.1). (B/C) OROV production was measured in the presence of several dilutions of the tested compound in Vero cells, with an initial inoculum of MOI 0,1 under simultaneous treatment (bars chart and left axis). The diamond shape (♦) and the right axis represents percentages of viral inhibition under viral control. Error bars represent standard deviations. Values are the mean ± standard error obtained from three independent experiments.The dashed line indicates the detection limit for the viral titers assay. Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett's test (*, p< 0.05).

Fig. 3

Antiviral activity of acridones in Oropouche Virus Progeny. Culture supernatants were harvested at the indicated time points. OROV production was measured in the presence of 125 μM dilution of the FAC21 or FAC22 in cells infected with MOI 0.1 by plaque forming assay. DMSO represents diluent control. Values are the mean ± standard error obtained from three independent experiments. The numbers above the bars indicate the percentage of virus inhibition (under the viral control). The dashed line indicates the detection limit for the viral titers assay. Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett's test (*, p< 0.05).

Fig. 4

Interaction of FAC21 and FAC22 with viral dsRNA. Thirty nanomoles of JFH-1 HCV dsRNA were incubated with FAC21 and 22 at 18.5 µM or the controls (DMSO 0.1 % and doxorubicin at 100 µM) for 45 min at room temperature. The reaction products were subjected to 1 % agarose 1X TAE electrophoresis gel containing Ethidium Bromide, followed by densitometry analysis on ImageJ.JS version 1.53j.

Fig. 5

Inhibition test, IC50 value determination and inhibition mode of the acridones FAC21 and FAC22 against OROV endonuclease. The enzyme activity was determined using the FAM-TCT CTA GCA GTG GCG CC-TAM DNA substrate, and the fluorescence intensities after cleavage were measured at 495 nm (excitation) and 520 nm (emission). (A) Primary inhibition test of FAC21 and FAC22 (50 µM) against the OROV endonuclease activity. EDTA (50 µM) was used as a known inhibitor control. (B) Normalized activity and inhibition of the endonuclease protein under the effect of FAC21. FAC21 was tested using a concentration range of 0–90 µM. (C) Dose–response curve for the IC50 value of FAC21 was determined by nonlinear regression. The normalized response [%] of OROV Endonuclease is plotted against the Log of the FAC21 concentration. The determined IC50 value is presented insert. (D) Inhibition mode of FAC21. The experiment was performed using different final concentrations of the inhibitor (0, 0.5, 2 and 5 µM) and titration of the substrate.The data were analyzed using a Lineweaver-Burk plot; therefore, the reciprocal of velocity (1/V) vs the reciprocal of the substrate concentration (1/[S]) was compared. Data shown are the mean ± standard deviation (SD) from three independent measurements (n = 3).

Fig. 6

MD simulations analysis of the N-terminal Endonuclease of OROV. (A) Cα RMSD evolution by 200 ns trajectory of three replicates (1 - purple, 2 - red, 3 - blue) and mean (black line). (B) RMSF analysis of three replicates using the same color scheme used in (A). It shows the average fluctuation per residue along the MD trajectory. (C) Ribbon representation of the three replicas coloured according to the RMSF values (with low and high fluctuation in blue and red, respectively). The Mn²⁺ binding site is shown in a red circle.

Fig. 7

MD of complexes that presented the best Vina score values. The figure is organised by row: Per-residue decomposition analysis from MM/PBSA calculation; the best ΔG_binding binding mode and 2D interactions between ligand and protein residues. Ligand binding mode that presents the lower AG_binding residues less than 5 Å away from the FAC21(in gray stick): (A) replica 1, (B) replica 2, and (C) replica 3. Residues are numbered according to the protein sequence, and Mn²⁺ is 181. The residues marked with * in the middle panel appeared with ΔG_binding lower than −1 kcal/mol in per-residue MM/PBSA analysis. The Mn²⁺ is represented by a green ball in the middle panel figures.