Antiviral activity of myricetin glycosylated compounds isolated from Marcetia taxifolia against chikungunya virus

Abstract

The chikungunya virus (CHIKV) has produced epidemic outbreaks of significant public health impact. The clinical symptoms of this disease are fever, polyarthralgia, and skin rash, generally self-limiting, although patients may develop a chronic disabling condition or suffer lethal complications. Unfortunately, there is no specific treatment or vaccine available. Thus, the search for effective therapies to control CHIKV infection is an urgent need. This study evaluated the antiviral activity of flavonoids isolated from Marcetia taxifolia by in vitro and in silico analysis. Cytotoxicity of compounds was determined by MTT assay and viral load was assessed in cell substrates supernatants by plaque-forming and RT-qPCR assays. Selected molecules were analyzed by molecular docking assays. Myricetin 3-rhamnoside (MR) and myricetin 3-(6-rhamnosylgalactoside) (MRG) were tested for antiviral assays and analyzed by the TCID50 method and RT-qPCR. MR exhibited dose-dependent antiviral activity, reducing viral titer at concentrations of 150-18.8 μg/mL by at least 1-log. Similarly, MRG showed a significant decrease in viral titer at concentrations of 37.5, 9.4, and 2.3 μg/mL. RT-qPCR analysis also displayed a substantial reduction of CHIKV RNA for both flavonoids. Furthermore, molecular docking of the selected flavonoids proposed the nsP3 macrodomain as a possible target of action. Our study reveals that MR and MRG could be considered promising anti-CHIKV therapeutic agents. Molecular modeling studies showed MR and MRG ligands with a high affinity for the N-terminal region of the nsP3 macrodomain, postulating them as a potential target of action for the CHIKV control.

Keywords: Marcetia taxifolia; antiviral activity; chikungunya; cytotoxicity; molecular modeling; myricetin.

Table 1. Cytotoxic activity of Marcetia taxifolia flavonoids on BHK-21 cell line

Table 2. Favorable and unfavorable 3GPG-MR and 3GPG-MRG, protein-ligand interactions, distance in angstroms, total binding E kcal/mol from docking

Figure 1. Susceptibility of Vero and BHK-21 cell lines to CHIKV infection. Vero and BHK-21 cell lines were inoculated with CHIKV at MOI of 10^-4. Culture supernatants and infected cells were collected at 10, 22, 34, 46, 63, 70, and 106 hours. (A) Plaques were counted in plates previously seeded with cells incubated with the collected medium for five days. (B) RNA from infected cells was extracted, and the nsP1 gene was amplified by RT-qPCR. Data are presented as the mean and standard deviation of the mean (SEM). Statistical differences were analyzed using a student t-test *p<0.05, **p<0.005.

Figure 2. Antiviral activity of MR and MRG against CHIKV. BHK-21 cells infected with CHIKV were exposed to serial dilutions starting from 150 to 1.17 µg/mL for MR (A) and 75 to 0.58 µg/mL for MRG (B) for 72 hours. Viral titer was obtained after five days of cell incubation by the Reed and Muench method. Viral titer data are presented as log10 titer mean ± standard deviation of the mean (SEM). Statistical differences were analyzed using a student t-test (*p<0.05). nsP1 viral RNA copies from infected cells incubated with MR (C) or MRG (D) were amplified by RT-qPCR. Data were transformed using a base 10 logarithm and analyzed using a student t-test (*p<0.02, **p<0.05, ***p<0.001, ****p<0.0001). Using the dose-response algorithm, the Ct obtained was normalized to determine the effective concentration 50 (EC50) for MR (E) or MRG (F).

Figure 3. MR and MRG docked with nsP3 protein, 2D and 3D representation. A) MR-nsP3 complex, affinity energy of -9.1 kcal/mol. B) MRG-nsP3 complex, affinity energy of -8.5 kcal/mol. Molecular docking assays were carried out with AutoDock, 2D figures with PoseView of ProteinsPlus, and 3D figures with UCSF Chimera. Left superior figure presents H-bonds, right superior figure presents hydrophobicity, left inferior figure presents solvent accessibility and right inferior figure presents 2D interaction diagram.

Figure 4. Molecular dynamics of MR and MRG-nsP3 complexes. A) RMSD Root Mean Square Deviation. B) H-bonds between ligand and protein. C) RMSF Root Mean Square Fluctuation of nsP3 protein. D) Radius of gyration. For additional information see Supplementary Figure 1 and 2.