Antiviral drug discovery by targeting the SARS-CoV-2 polyprotein processing by inhibition of the main protease

Abstract

The spread of SARS-CoV-2, the causative agent for COVID-19, has led to a global and deadly pandemic. To date, few drugs have been approved for treating SARS-CoV-2 infections. In this study, a structure-based approach was adopted using the SARS-CoV-2 main protease (M^pro) and a carefully selected dataset of 37,060 compounds comprising M^pro and antiviral protein-specific libraries. The compounds passed two-step docking filtration, starting with standard precision (SP) followed by extra precision (XP) runs. Fourteen compounds with the highest XP docking scores were examined by 20 ns molecular dynamics simulations (MDs). Based on backbone route mean square deviations (RMSD) and molecular mechanics/generalized Born surface area (MM/GBSA) binding energy, four drugs were selected for comprehensive MDs analysis at 100 ns. Results indicated that birinapant, atazanavir, and ritonavir potently bound and stabilized SARS-CoV-2 M^pro structure. Binding energies higher than -102 kcal/mol, RMSD values <0.22 nm, formation of several hydrogen bonds with M^pro, favourable electrostatic contributions, and low radii of gyration were among the estimated factors contributing to the strength of the binding of these three compounds with M^pro. The top two compounds, atazanavir and birinapant, were tested for their ability to prevent SARS-CoV-2 plaque formation. At 10 µM of birinapant concentration, antiviral tests against SARS-CoV-2 demonstrated a 37% reduction of virus multiplication. Antiviral assays demonstrated that birinapant has high anti-SARS-CoV-2 activity in the low micromolar range, with an IC50 value of 18 ± 3.6 µM. Therefore, birinapant is a candidate for further investigation to determine whether it is a feasible therapy option.

Keywords: COVID-19; Drug discovery; Main protease; Molecular modeling; SARS-CoV-2.

Figure 1. The docking site and ligands interactions with M^pro.

(A) The docking site of M^pro following XP docking protocol. (B) Surface representation (blue) of birinapant in the active site of M^pro. (C) The binding site of residues of atazanavir. (D) The binding site residues of birinapant. (E) The ligand interactions of atazanavir. (F) The ligand interactions of birinapant. Hydrogen bonds are shown in purple arrows, hydrophobic interactions in grey circles.

Figure 2. (A–C) RMSD plot of the top fourteen compounds after MDS for 20 ns.

Lopinavir was used for reference. Apo structure is M^pro without any ligands.

Figure 3. RMSD plot of the top four compounds, alpha-mangostin, atazanavir, birinapant and lopinavir, after MDs for 100 ns.

Figure 4. RMSF plot of the top four compounds after MDs for 100 ns.

Figure 5. Radius of gyration of the top four compounds after MDs for 100 ns.

Figure 6. The hydrogen bond length of the top four compounds after MDs for 100 ns.

Figure 7. Effect of birinapant and atazanavir on the replication of SARS-CoV-2.

Vero E6 cells were infected with 0.1 MOI SARS-CoV-2 in 6-well plate and then treated with DMSO (0.1%), birinapant (10 µM) or atazanavir (10 µM) at 3 h after virus infection (n = 3). Supernatants of virus-infected cell cultures were collected at 48 h after virus infection. Virus replication in the supernatants was quantified by plaque formation assay. ^∗∗p < 0.01.