Structure-Based Virtual Screening and Biochemical Validation to Discover a Potential Inhibitor of the SARS-CoV-2 Main Protease

Abstract

The recent pandemic caused by SARS-CoV-2 has led the world to a standstill, causing a medical and economic crisis worldwide. This crisis has triggered an urgent need to discover a possible treatment strategy against this novel virus using already-approved drugs. The main protease (Mpro) of this virus plays a critical role in cleaving the translated polypeptides that makes it a potential drug target against COVID-19. Taking advantage of the recently discovered three-dimensional structure of Mpro, we screened approved drugs from the Drug Bank to find a possible inhibitor against Mpro using computational methods and further validating them with biochemical studies. The docking and molecular dynamics study revealed that DB04983 (denufosol) showed the best glide docking score, -11.884 kcal/mol, and MM-PBSA binding free energy, -10.96 kcal/mol. Cobicistat, cangrelor (previous computational studies in our lab), and denufosol (current study) were tested for the in vitro inhibitory effects on Mpro. The IC₅₀ values of these drugs were ∼6.7 μM, 0.9 mM, and 1.3 mM, respectively, while the values of dissociation constants calculated using surface plasmon resonance were ∼2.1 μM, 0.7 mM, and 1.4 mM, respectively. We found that cobicistat is the most efficient inhibitor of Mpro both in silico and in vitro. In conclusion, cobicistat, which is already an FDA-approved drug being used against HIV, may serve as a good inhibitor against the main protease of SARS-CoV-2 that, in turn, can help in combating COVID-19, and these results can also form the basis for the rational structure-based drug design against COVID-19.

Figure 1

2D ligand interaction diagram of SARS-CoV-2 Mpro with (A) NADPH (DB02338), (B) oxo-nicotinamide adenine dinucleotide phosphate (DB01753), (C) 6-(adenosine tetraphosphate methyl)-7,8-dihydropterin (DB04158), and (D) denufosol (DB04983).

Figure 2

RMSD fluctuations of the protein backbone (light blue), DB02338 (dark blue), DB01753 (orange), DB04158 (gray), and DB04983 (yellow) during 200 ns molecular dynamics simulation runs on Mpro. This plot indicates that there were no significant fluctuations during the entire course of simulations.

Figure 3

Plot showing the change in radius of gyration (Rg) of the protein backbone (light blue), DB02338 (dark blue), DB01753 (orange), DB04158 (gray), and DB04983 (yellow) during 200 ns molecular dynamics simulation runs on Mpro.

Figure 4

Plot showing the number of stable hydrogen bonds as a function of run length for all the ligands: DB02338 (dark blue), DB01753 (orange), DB04158 (gray), and DB04983 (yellow) during 200 ns molecular dynamics simulation runs on Mpro.

Figure 5

Variations in the bond distances between donors and acceptors as a function of time.

Figure 6

SPR sensograms showing the mode of binding of (A) cobicistat, (B) cangrelor, and (C) denufosol to the immobilized Mpro on the CM-5 chip. Cobicistat was found to show the best K_D value of 2.1 μM followed by 0.7 mM for cangrelor and 1.44 mM for denufosol.

Figure 7

Inhibitory activity profile of the tested compounds (A) cobicistat, (B) cangrelor, and (C) denufosol. The corresponding IC₅₀ values are also indicated.

Figure 8

Three-dimensional individual snapshots of (A) cobicistat (pink), (B) cangrelor (cyan), and (C) denufosol (black) in the active site of Mpro. Subsite residues S1 (green), S1′, (yellow), S2 (blue), and S4 (magenta) are also indicated.

Figure 9

Three-dimensional representation of all the three FDA-approved drugs cobicistat (pink), cangrelor (cyan), and denufosol (black) in the active site of Mpro. The key amino acid residues forming S1 (green), S1′ (yellow), S2 (blue), and S4 (magenta) are also indicated.