Chlorhexidine and SARS-CoV-2 main protease: Molecular docking study

Abstract

As the beginning of the COVID-19 pandemic, numerous attempts have been made to identify specific antiviral mouth rinses which may help reduce the salivary viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). Although the results from in vivo well-controlled clinical studies are yet pending, many contemporary antimicrobial mouth rinses have been explored for potential antiviral properties with respect to SARS-CoV-2. The most widely used compounds such as povidone-iodine, chlorhexidine, hydrogen peroxide, and essential oils have been known to have antiviral activity by targeting the outer lipid membrane or by denaturing the capsid proteins of enveloped virus SARS-CoV. Until now, there has been scanty scientific evidence on the molecular basis of interaction of the gold standard antimicrobial mouth rinse as an underlying mechanism of its anti-SARS-CoV-2 effect. The current communication reports the findings of our in silico docking study pertaining to understand the interactions of chlorhexidine with the most well-studied target of the SARS-CoV main protease.

INTRODUCTION

Chlorhexidine, as an antimicrobial compound, has a long, time-tested track record from almost six decades and is a standalone gold standard for use in the oral cavity. The high rate of transmission and high mortality rate due to severe acute respiratory syndrome coronavirus (SARS-CoV19) require an urgent need of identifying potent drug targets, antiviral drug molecules, and strategies to control the spread of the notorious virus. Genome studies of mature miRNAs of SARS-CoV-2 predicted the ability of the virus to target the human genes involved in epidermal growth factor receptor signaling, apoptosis signaling, vascular endothelial growth factor signaling, and fibroblast growth factor receptor signaling pathways, which play a major role in lung diseases and other disorders.[1] The studies on coronaviruses reported virus spike protein, RNA-dependent RNA polymerase (RdRp), main protease (M^pro), and the papain-like protease (PL^pro) as potent drug targets for the family of SARS-CoV and the Middle East respiratory syndrome (MERS).[2,3] The M^pro has been widely used as a drug target for SARS-CoV-2, SARS-CoV, and MERS-CoV.[4] The crystal structures of the SARS-CoV-2 M^pro bound with various inhibitors such as calpain inhibitors II, XII, and other inhibitors are available in the Protein Data Bank (PDB) database. Therefore, to find out the interaction of chlorhexidine with coronavirus, the COVID-19 M^pro has been used in our in silico study.

MATERIALS AND METHODS

Protein–ligand docking

The crystal structure of the receptor protein, COVID-19 main protease (M^pro) bound with inhibitor was obtained from the PDB (PDB ID: 6w63). Based on the already bound inhibitor in the crystal structure, the binding site on the receptor molecule was identified by Discovery Studio Visualizer version 2020 (BIOVIA, Dassault Systèmes, Discovery Studio Visualizer), which is also confirmed using DoGSiteScorer.[5,6] Receptor structure was prepared for docking by removing the already-bound heteroatoms, addition of hydrogens, and addition of charges. The three-dimensional structure of chlorhexidine used as our ligand was downloaded from the PubChem (CID9552079) in structure data file format. Protein–ligand docking was performed in AutoDock tools (Scripps Research, La Jolla, California, USA).

AutoDock tools

The ligand was prepared for docking by adding any missing hydrogen atoms; charges were added and converted to PDB, partial charge (Q), and atom type (T) (PDBQT) format. The grid box for docking was manually adjusted based upon the already predicted active site and centered on coordinates – X: −17.370, Y: 18.944, and Z: −26.343, and dimensions – X: 15 Å, Y: 15 Å, and Z: 15 Å, thereby covering the entire possible active site residues. Docking was performed, and the results were exported to a Digital Line Graph (dlg) text file for ranking of docked conformations obtained according to the binding affinity, clustering analysis, and root-mean-square deviation (RMSD) analysis. The docked conformations with receptor complex were saved as PDB files and visualized in Discovery Studio Visualizer to know the interactions of chlorhexidine with the residues of the COVID-19 M^pro binding pocket. The residues of the binding site interacting with the ligand were also confirmed by the PoseView tool (ZBH Centre for Bioinformatics, University of Hamburg, Hamburg, Germany).[7]

RESULTS

After performing initial docking of chlorhexidine with COVID 19 Mpro in AutoDock 10, multiple docked poses/conformations were obtained. The first conformation of the ligand docked with Mpro, corresponding to the best binding energy (−10.10 kcal/mol) was selected as a base conformation to explore the binding residues [Table 1]. The RMSD value for this was zero (lower than 2 Å), which validates the accuracy of performance of docking/scoring functions.[8]

Table 1.

Ranking of the binding mode of ligand (conformations) based on binding energies (kcal mol^-1) and corresponding root-mean-square deviation (Å) values

Conformation (mode)	Rank	Sub rank	Binding energy	Cluster RMSD
1	1	1	−10.10	0.00
2	1	2	−8.66	1.67
3	2	1	−9.45	0.00
4	3	1	−9.22	0.00
5	4	1	−8.66	0.00
6	5	1	−7.72	0.00
7	5	2	−7.56	1.88
8	6	1	−7.69	0.00
9	7	1	−7.40	0.00
10	8	1	−7.39	0.00

RMSD - Root-mean-square deviation

The analysis of interactions between COVID-19 M^pro and the best binding poses of the chlorhexidine was performed in three different tools, i.e., AutoDock tools, PoseView, and Discovery Studio [Figure 1]. Binding residues predicted from these tools are recorded in [Table 2].

Figure 1.

Best docked conformation of chlorhexidine molecule (sticks format) bound with COVID-19 M^pro in the binding site. (a) M^pro in ribbon display format obtained in Discovery Studio Visualizer and (b) binding site of M^pro in molecular surface display format where H-bond with chlorhexidine (in green) is shown in red-colored wireframe circle obtained in AutoDock tools. M^pro: Main protease

Table 2.

Binding residues predicted using AutoDock tools, PoseView, and Discovery Studio

Docked conformation	Binding energy (kcal/mol)	Tool	Predicted binding residues
1	−10.10	AutoDock tools	Cys44, Tyr54, Asn142, Cys145, Leu141, His163, Met165, Glu166*, Pro168, Asp187, Arg188, Gln189, Thr190, Gln192
		PoseView	Glu166*, Arg188, Pro168, Gln189
		Discovery Studio	His41, Cys44, Pro52, Tyr54, Met49, Leu141, Asn142, Ser144, Cys145, His163, Met165, Glu166*, Leu167, Pro168, Gly170, Asp187, Arg188, Gln189, Ala191, Thr190*

^*The residue forming H-bond is marked with bold and asterisk symbol. Cys - Cysteine; Tyr - Tyrosine; Leu - Leucine; His - Histidine; Met - Methionine; Glu - Glutamate; Pro - Proline; Asp - Aspartate; Arg - Arginine; Thr - Threonine; Ser - Serine; Gly - Glycine; Ala - Alanine; Gln - Glutamine; Asn - Asparagine

DISCUSSION

The interaction results obtained from Discovery Studio Visualizer provided greater insight for interactions between receptor and bound ligand [Figure 2c]. Binding interaction studies have shown that the chlorhexidine molecule binds to M^pro through at least one hydrogen (H)-bond with glutamate (Glu) 166 residue [Figure 2], and the other possible residue forming H-bond is threonine (Thr) 190. Glu166 was involved in forming hydrogen bonds with an H-bond distance of 1.93 or 1.85 [Figure 2c] at two different points. Chlorhexidine also binds to M^pro through 8 van der Waals interactions, with the residues methionine (Met) 49, tyrosine 54, leucine (Leu) 141, serine 144, Leu167, glycine 170, aspartate 187, and alanine 191. In addition, two π-sulfur interactions with cysteine (Cys) 145 and Met165, two π-π T-shaped bond interactions with histidine (His) 41 and arginine 188, and four alkyl bond interactions involving residues Cys44, proline (Pro) 52, His163, and Pro168 were obtained between M^pro and chlorhexidine molecule. The docking interactions provide an insight into the possible molecular interactions underlying the anti-SARS-CoV activity of the chlorhexidine molecule, reported in our previous work.[9] In another recent computational investigation, chlorhexidine exhibited has the strongest binding affinity to the M^pro as compared to some flavonoid compounds, and similar residues for interaction as observed in our study have also been reported.[10] These key binding interactions provide strong evidence of effective inhibitor activity of chlorhexidine against the COVID-19 and related coronaviruses.

Figure 2.

Interaction map of binding residues of receptor and ligand molecule in (a) AutoDock tools; (b) PoseView; and (c) Discovery Studio Visualizer. Pi - inorganic phosphate; Ala - Alanine; Pro - Proline; His - Histidine; Cys - Cysteine; Leu - Leucine; Ser - Serine; Asn - Asparagine; Gly - Glycine; Glu - Glutamic acid; Arg - Arginine; Met - Methionine; Asp - Aspartic acid; Tyr - Tyrosine; Gln - Glutamine; Thr - Threonine

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.