In silico and in vitro studies of potential inhibitors against Dengue viral protein NS5 Methyl Transferase from Ginseng and Notoginseng

Abstract

Background and aim: Dengue is a potentially deadly tropical infectious disease transmitted by mosquito vector Aedes aegypti with no antiviral drug available to date. Dengue NS5 protein is crucial for viral replication and is the most conserved among all four Dengue serotypes, making it an attractive drug target. Both Ginseng and Notoginseng extracts and isolates have been shown to be effective against various viral infections yet against Dengue Virus is understudied. We aim to identify potential inhibitors against Dengue NS5 Methyl transferase from small molecular compounds found in Ginseng and Notoginseng.

Experimental procedure: A molecular docking model of Dengue NS5 Methyl transferase (MTase) domain was tested with decoys and then used to screen 91 small molecular compounds found in Ginseng and Notoginseng followed by Molecular dynamics simulations and the per-residue free energy decompositions based on molecular mechanics/Poisson-Boltzmann (generalised Born) surface area (MM/PB(GB)SA) calculations of the hit. ADME predictions and drug-likeness analyses were discussed to evaluate the viability of the hit as a drug candidate. To confirm our findings, in vitro studies of antiviral activities against RNA and a E protein synthesis and cell toxicity were carried out.

Results and conclusion: The virtual screening resulted in Isoquercitrin as a single hit. Further analyses of the Isoquercitrin-MTase complex show that Isoquercitrin can reside within both of the NS5 Methyl Transferase active sites; the AdoMet binding site and the RNA capping site. The Isoquercitrin is safe for consumption and accessible on multikilogram scale. In vitro studies showed that Isoquercitrin can inhibit Dengue virus by reducing viral RNA and viral protein synthesis with low toxicity to cells (CC₅₀ > 20 μM). Our work provides evidence that Isoquercitrin can serve as an inhibitor of Dengue NS5 protein at the Methyl Transferase domain, further supporting its role as an anti-DENV agent.

Keywords: Antiviral; Dengue; Isoquercitrin; Molecular docking; Molecular dynamics simulations; NS5 methyl transferase.

Graphical abstract

Fig. 1

a) Superimposed 3D structures of NS5 MTase with ligands at AdoMet binding site (PDB ID: 5E9Q (SAM, cyan), 1R6A (SAH, pink), 4R8S (SFG, green), 3P8Z (36A, yellow), 5ULP (kb1, orange) and RVP (pink) at RNA capping site, b) Structures of AdoMet binding site ligands used as references in this study.

Fig. 2

Docking scores of the five ligands and the decoys against the DENV NS5 MTase.

Fig. 3

Top-scoring pose of Isoquercitrin in AdoMet binding site, overlaid with the protein DENV NS5 MTase (PDB ID:4r8s) a) Hydrogen bond interactions (purple arrows) in 2D representation, b) Hydrogen bonds (yellow lines) lengths (Å) in 3D representation.

Fig. 4

a) RMSD of the complex's C_α (Å) over 500 ns starting with ligand at AdoMet binding site (shown as blue mesh in 4e) in three replicates: AdoMet#1 (black), AdoMet#2 (red) and AdoMet#3 (green), b) RMSD of the complex's C_α (Å) over 500 ns starting with ligand at RNA capping site (shown as white mesh in 4e) in three replicates: RNA#1 (blue), RNA#2 (pink) and RNA#3 (orange), c) RMSF of the residue's C_α (Å) starting with ligand at AdoMet binding site in three replicates: AdoMet#1 (black), AdoMet#2 (red) and AdoMet#3 (green), d) RMSF of the residue's C_α (Å) starting with ligand at RNA capping site in three replicates: RNA#1 (blue), RNA#2 (pink) and RNA#3 (orange) and e) X-ray structure of MTase (grey ribbon, PDB ID: 4r8s) superimposed with snapshots at 500 ns of all six MD simulations (AdoMet#1–2 in cyan ribbons, AdoMet#3 and RNA#1–3 in yellow ribbons). High RMSF regions are represented in pink surfaces.

Fig. 5

a) RMSD of Isoquercitrin (Å) over 500 ns, starting with ligand at AdoMet binding site, in three replicates: first (AdoMet#1 in black), second (AdoMet#2 in red) and third (AdoMet#3 in green), b) Superimposed structures of Isoquercitrin showing the AdoMet binding site and the RNA capping site: staring from docked pose (relaxed) in light grey and after 500 ns simulations in three replicates: first (AdoMet#1 in cyan), second (AdoMet#2 in green) and third (AdoMet#3 in yellow) c) RMSD of Isoquercitrin (Å) over 500 ns, starting with ligand at RNA capping site, in three replicates: first (RNA#1 in blue), second (RNA#2 in pink) and third (RNA#3 in orange), and d) Superimposed structures of Isoquercitrin showing the AdoMet binding site and the RNA capping site: a starting pose (snapshot at 500 ns of AdoMet#3) shown in white and snapshots after 500 ns simulations of the three replicates: first (RNA#1 in yellow), second (RNA#2 in purle) and third (RNA#3 in grey).

Fig. 6

Antiviral activity of isoquercitrin against DENV2 in vitro. Vero cells were infected with DENV 2 at MOI 0.2, and then treated with 20, 10, 5 and 2.5 μM of isoquercitrin or with 0.5%DMSO (virus control). At 48h post infection cells were determine the RNA expression level and protein level of DENV2 virus. The experiments were performed independently in triplicate. Error bar showed mean ± SD.