In silico and in vitro inhibition of host-based viral entry targets and cytokine storm in COVID-19 by ginsenoside compound K

Abstract

SARS-CoV-2 is a novel coronavirus that emerged as an epidemic, causing a respiratory disease with multiple severe symptoms and deadly consequences. ACE-2 and TMPRSS2 play crucial and synergistic roles in the membrane fusion and viral entry of SARS-CoV-2 (COVID-19). The spike (S) protein of SARS-CoV-2 binds to the ACE-2 receptor for viral entry, while TMPRSS2 proteolytically cleaves the S protein into S1 and S2 subunits, promoting membrane fusion. Therefore, ACE-2 and TMPRSS2 are potential drug targets for treating COVID-19, and their inhibition is a promising strategy for treatment and prevention. This study proposes that ginsenoside compound K (G-CK), a triterpenoid saponin abundant in Panax Ginseng, a dietary and medicinal herb highly consumed in Korea and China, effectively binds to and inhibits ACE-2 and TMPRSS2 expression. We initially conducted an in-silico evaluation where G-CK showed a high affinity for the binding sites of the two target proteins of SARS-CoV-2. Additionally, we evaluated the stability of G-CK using molecular dynamics (MD) simulations for 100 ns, followed by MM-PBSA calculations. The MD simulations and free energy calculations revealed that G-CK has stable and favorable energies, leading to strong binding with the targets. Furthermore, G-CK suppressed ACE2 and TMPRSS2 mRNA expression in A549, Caco-2, and MCF7 cells at a concentration of 12.5 μg/mL and in LPS-induced RAW 264.7 cells at a concentration of 6.5 μg/mL, without significant cytotoxicity.ACE2 and TMPRSS2 expression were significantly lower in A549 and RAW 264.7 cells following G-CK treatment. These findings suggest that G-CK may evolve as a promising therapeutic against COVID-19.

Keywords: A549; ACE-2; CaCo-2; Compound K; Ginsenoside; MCF7; Molecular docking; Molecular dynamics simulation; Raw 264.7; TMPRSS2.

Graphical abstract

Fig. 1

Illustration of the mechanism of action of G-CK against the main drug targets (ACE2 and TMPRSS2) of COVID-19.

Fig. 2

The allosteric sites of angiotensin-converting enzyme 2. (a) Cartoon presentation of the human angiotensin-converting enzyme 2(3SCJ). Different black shapes highlight potential allosteric areas. (b) A yellow square highlights the orthosteric site of ACE-2. Essential amino acid residues are H345, H505, and R273. (c) Allosteric site 1 (red) and amino acid residues (blue) of ACE-2 participating in hydrogen bonding (H-bond) with the receptor-binding domain (RBD) of SARS-CoV-2 (d) Visual summary of all the possible binding sites of TMPRSS2. Adapted from Refs. [71,73].

Fig. 3

(A) 2D & 3D docking interactions of G-CK with ACE2 (PDB ID: 6M0J). (B) Docking interactions of G-CK (blue) and control drug inhibitors (Dexamethasone (Orange), NAG(Green), Remdesivir (magenta), Indinavir (cyan), Chloroquine (yellow), and Camostat (red)) with ACE2.

Fig. 4

(A) 2D & 3D docking interactions of G-CK with TMPRSS2 (PDB ID: 7MEQ). (B) Docking interactions of G-CK (blue) and control drug inhibitors (Dexamethasone (Orange), Nafamostat (Green), Remdesivir (magenta), Indinavir (cyan), Chloroquine (yellow), and Camostat (red)) with TMPRSS2.

Fig. 5

3D docking interactions of G-CK with (A) ACE2 (B) TMPRSS2.

Fig. 6

(A) RMSD of G-CK and Dexamethasone in complex with SARS-Cov-2 ACE2 as a function of MD simulation time. (B) RMSD of G-CK in complex with SARS-Cov-2 TMPRSS2 as a function of MD simulation time.

Fig. 7

(A) RMSF of G-CK and Dexamethasone in complex with SARS-Cov-2 ACE2 as a function of MD simulation time. (B) RMSF of G-CK in complex with SARS-Cov-2 ACE2 as a function of MD simulation time.

Fig. 8

(A) The radius of gyration plots of molecular dynamics (MD) simulation of ACE2 receptor- G-CK and Dexamethasone complexes. (B) The radius of gyration plots of molecular dynamics (MD) simulation of ACE2 receptor G-CK and Dexamethasone complexes.

Fig. 9

(A) Line plots of Ligand-protein H bonds for ACE2 with G-CK and Dexamethasone. (B) Line plots of Ligand-protein H bonds for ACE2 with G-CK and Dexamethasone.

Fig. 10

ADMET properties of G-CK and the control drugs (A) G-CK, (B) Dexamethasone, (C) NAG, (D) Nafamostat, (E) Remdesivir, (F) Indinavir, (G) Chloroquine, and (H) Camostat. Abbreviations: MW: Molecular weight; nRig: number of rigid bonds; fChar: formal charge; nHet: number of heteroatoms; MaxRing: number of atoms in the biggest ring; nRing number of rings; nRot: number of rotatable bonds; TPSA: topological polar surface area; nHD: number of hydrogen bond donors; nHA: number of hydrogen bond acceptor; LogD: logP at physiological pH 7.4; logS: log of the aqueous solubility; and LogP: log of the octanol/water partition coefficient.

Fig. 11

In vitro, cell cytotoxicity evaluation for G-CK (A) RAW 264.7, (B) A549 cells, (C) Caco-2, and (D) MCF-7 cells, cells. Every value is expressed as the mean ± standard error of three independent experiments. ***p < 0.001 compared with control.

Fig. 12

Effects of G-CK on mRNA expression levels of Anti-covid related genes in (A–B) A549 cells, (C–D) Caco-2 cells, and (E–F) MCF-7 cells.

Fig. 13

Effects of G-CK on mRNA expression levels of Anti-covid (A–B) and Cytokine storm (C–G) related genes on Raw 264.7 cells compared with LPS.