Bio-Guided Isolation of SARS-CoV-2 Main Protease Inhibitors from Medicinal Plants: In Vitro Assay and Molecular Dynamics

Abstract

Since the emergence of the pandemic of the coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the discovery of antiviral phytoconstituents from medicinal plants against SARS-CoV-2 has been comprehensively researched. In this study, thirty-three plants belonging to seventeen different families used traditionally in Saudi Arabia were tested in vitro for their ability to inhibit the SARS-CoV-2 main protease (M^PRO). Major constituents of the bio-active extracts were isolated and tested for their inhibition potential against this enzyme; in addition, their antiviral activity against the SARS-CoV-2 Egyptian strain was assessed. Further, the thermodynamic stability of the best active compounds was studied through focused comparative insights for the active metabolites regarding ligand-target binding characteristics at the molecular level. Additionally, the obtained computational findings provided useful directions for future drug optimization and development. The results revealed that Psiadia punctulata, Aframomum melegueta, and Nigella sativa extracts showed a high percentage of inhibition of 66.4, 58.7, and 31.5%, against SARS-CoV-2 M^PRO, respectively. The major isolated constituents of these plants were identified as gardenins A and B (from P. punctulata), 6-gingerol and 6-paradol (from A. melegueta), and thymoquinone (from N. sativa). These compounds are the first to be tested invitro against SARS-CoV-2 M^PRO. Among the isolated compounds, only thymoquinone (THY), gardenin A (GDA), 6-gingerol (GNG), and 6-paradol (PAD) inhibited the SARS-CoV-2 M^PRO enzyme with inhibition percentages of 63.21, 73.80, 65.2, and 71.8%, respectively. In vitro assessment of SARS-CoV-2 (hCoV-19/Egypt/NRC-03/2020 (accession number on GSAID: EPI_ISL_430820) revealed a strong-to-low antiviral activity of the isolated compounds. THY showed relatively high cytotoxicity and was anti-SARS-CoV-2, while PAD demonstrated a cytotoxic effect on the tested VERO cells with a selectivity index of CC₅₀/IC₅₀ = 1.33 and CC₅₀/IC₅₀ = 0.6, respectively. Moreover, GNG had moderate activity at non-cytotoxic concentrations in vitro with a selectivity index of CC₅₀/IC₅₀ = 101.3/43.45 = 2.3. Meanwhile, GDA showed weak activity with a selectivity index of CC₅₀/IC₅₀ = 246.5/83.77 = 2.9. The thermodynamic stability of top-active compounds revealed preferential stability and SARS-CoV-2 M^PRO binding affinity for PAD through molecular-docking-coupled molecular dynamics simulation. The obtained results suggest the treating potential of these plants and/or their active metabolites for COVID-19. However, further in-vivo and clinical investigations are required to establish the potential preventive and treatment effectiveness of these plants and/or their bio-active compounds in COVID-19.

Keywords: 6-gingerol; 6-paradol; SARS-CoV-2 Egyptian strain; SARS-CoV-2 main protease; coronavirus; gardenin A; thymoquinone.

Figure 1

Plant extracts with significant inhibitory activity against the viral protease (SARS-CoV-2 M^PRO) GC376; positive control, *** significantly different at p < 0.0001, ** significantly different at p < 0.001.

Figure 2

Inhibition of M^PRO protease enzyme activity by isolated compounds.

Figure 2

Inhibition of M^PRO protease enzyme activity by isolated compounds.

Figure 3

Dose-inhibition curves for bioactive compounds, where IC50 values were calculated using nonlinear regression analysis of GraphPad Prism software (version 5.01) by plotting log inhibitor versus normalized response.

Figure 4

Ligand/M^PRO binding modes and interactions. (A) Surface rendition of SARS-CoV-2 M^PRO, with an overlay of docked isolated compounds (yellow lines) and potent reference (blue or orange sticks for X77 or GC376, respectively). The protein is colored in dark and light gray colors for protomer A and B, respectively, while the target binding subsites are shown in red, magenta, green, and cyan for S1′, S1, S2, and S3 subsites, respectively; (B) Docked binding modes of investigated compounds (sticks), where residues (lines) only located within 5Å radius of bound ligands are displayed, labeled by sequence numbers, and colored based on respective subsite location. Polar interactions (hydrogen bonds) are shown as dashed black lines.

Figure 4

Ligand/M^PRO binding modes and interactions. (A) Surface rendition of SARS-CoV-2 M^PRO, with an overlay of docked isolated compounds (yellow lines) and potent reference (blue or orange sticks for X77 or GC376, respectively). The protein is colored in dark and light gray colors for protomer A and B, respectively, while the target binding subsites are shown in red, magenta, green, and cyan for S1′, S1, S2, and S3 subsites, respectively; (B) Docked binding modes of investigated compounds (sticks), where residues (lines) only located within 5Å radius of bound ligands are displayed, labeled by sequence numbers, and colored based on respective subsite location. Polar interactions (hydrogen bonds) are shown as dashed black lines.

Figure 5

Stability analysis of the ligand-M^PRO complex across the 100 ns explicit molecular dynamics simulation runs. The generated RMSD trajectories (Å) are represented across the simulation timeframes (ns).

Figure 6

Conformational analysis of the simulated ligand-M^PRO complexes at the start and end of the 100 ns explicit molecular dynamics simulation runs. (A) PAD; (B) GNG; (C) GDA; (D) THY; (E) X77; (F) GC376. Overlaid snapshots at 0 ns and 100 ns are presented in green and red colors, respectively; the target proteins (cartoon) and ligands (sticks) are colored corresponding to the extracted frame.

Figure 6

Conformational analysis of the simulated ligand-M^PRO complexes at the start and end of the 100 ns explicit molecular dynamics simulation runs. (A) PAD; (B) GNG; (C) GDA; (D) THY; (E) X77; (F) GC376. Overlaid snapshots at 0 ns and 100 ns are presented in green and red colors, respectively; the target proteins (cartoon) and ligands (sticks) are colored corresponding to the extracted frame.