Dental workers in front-line of COVID-19: an in silico evaluation targeting their prevention

Abstract SARS-CoV-2 has high human-human transmission rate. The aerosols and saliva droplets are the main contamination source. Thus, it is crucial to point out that dental practitioners become a high-risk group of contagion by SARS-CoV-2. Based on this, protocols have been recommended to avoid cross-contamination during dental care; however, appropriate evidence has not yet been established. Objective Our study sought to make a screening, by in silico analysis, of the potential of mouth rinses used in dental practices to prevent the dental workers' contamination by SARS-CoV-2. Methodology Multiple sequence comparisons and construction of the phylogenetic tree were conducted using the FASTA code. Therefore, molecular docking investigation between SARS-CoV-2 proteins (Main Protease, Spike Glycoprotein, Non-structure Protein, and Papain-like Protease) and molecules used in dental practices (chlorhexidine digluconate, hydrogen peroxide, cetylpyridinium chloride, povidone-iodine, gallic acid, β-cyclodextrin, catechin, and quercetin) was performed using AutoDock Vina. Moreover, 2D interactions of the complex protein-ligand structure were analyzed by Ligplot+. Results The obtained results showed a remarkable affinity between SARS-CoV-2 proteins and all tested compounds. The chlorhexidine digluconate, catechin, and quercetin presented a higher affinity with SARS-CoV-2. Conclusions The overall results allowed us to suggest that chlorhexidine is the most suitable active compound in reducing the SARS-CoV-2 salivary load due to its better binding energy. However, in vivo studies should be conducted to confirm their clinical use.


Introduction
The coronavirus disease 2019 (COVID-19) outbreak has drawn attention worldwide since its first identified case in Wuhan -China. 1 This infectious disease, caused by the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2), has spread globally and infected millions of people, leading thousands of individuals to death. 2 The SARS-CoV-2 has high human-human transmissibility, and the saliva plays an essential role in it. Through the saliva droplets/aerosols inhalation or ingestion from infected people, health people may fall ill. 3,4 Based on this rationale, asymptomatic patients also are considered a transmission vector, since they are freely carrying out their activities and endangering the population's health. 5 Salivary glands and saliva are the main reservoirs to SARS-CoV-2 due to the high-affinity with the host-cell receptor angiotensin-converting enzyme II (ACE2), also found in salivary glands. 6,7 Then, dental practitioners can be considered a high-risk group of contagion by SARS-CoV-2 due to their exposure to aerosols-generated procedures during dental care. 8,9 Moreover, a lack of evidence regarding what to do as a pre-procedural protocol to avoid SARS-CoV-2 crossinfection is worrying. It should be resolved to offer more protection to dental workers.
Regarding this issue, some protocols have been recommended worldwide to avoid SARS-CoV-2 dissemination during dental care. Based on this, mouth rinses with hydrogen peroxide, povidone-iodine, or chlorhexidine are used before any dental procedure. 10 However, once these suggestions have inadequate scientific support, 11 appropriate evidence must be produced to understand the possible mechanism of action of these compounds. Additionally, clinical studies involving drug testing require time and involve risks for both research groups and researchers. 12 In this context, in silico analyses play a fundamental role in simulating molecular processes to support validation studies between molecular and cellular processes. 13 Among in silico analyses, the docking studies can be emphasized, which evaluate proteinligand complexes through a series of algorithms to generate scoring functions. Thus, these analyses can predict the biological effects of chemical compounds due to their ability to interact with proteins responsible for the virulence present in the surface. 14 Therefore, the fastest scenario to test existing drugs on the SARS-CoV-2 proteins (such as Main or Papain-like proteases, Spike glycoprotein, and non-structured proteins) is the in silico analysis, which is a robust approach to provide remarkable results. Thus, it could propose initial therapeutic strategies to prevent SARS-CoV-2 contamination by dental workers. 15 Then, our study aimed at providing preliminary data, using computational tools, of the therapeutic potential of mouth rinses, widely used in dentistry practices, to prevent the contamination of dental workers by SARS-CoV-2 during dental procedures.
The absence of interaction between the SARS-CoV-2 proteins and mouth rinses' compounds was considered the null hypothesis.

Methodology
Retrieval of proteins sequences The ligand' rotatable bonds were defined using AutoDock, and the structures were saved as pdbqt files for the use in the docking studies.

SARS-CoV-2 proteins preparation
The AutoDock (RRID: SCR_012746) was used to delete repeated chains, heteroatoms, and water molecules, add polar hydrogens atoms, and add the charge atoms to the protein structure. Gasteiger charges were computed, and the structure was saved as a pdbqt file for the docking studies.

Molecular docking procedure
The 3D grids were created by the Autogrid algorithm to generate the grid parameter files (Autogrid, RRID: SCR_015982). Each grid map was set to the center of the Chain A. Docking parameters were set according to the protein (Table 1), and all analyses were conducted with "exhaustiveness = 8".

Molecular docking
The affinity between selected compounds and SARS-CoV-2 proteins was observed in our study (Table 2).
Nonetheless, chlorhexidine digluconate, catechin, and quercetin showed higher binding energy than others. and Non-structure Protein (PDB: 6WIQ). The specific binding sites to each compound are shown in Figure   3, in which we could observe that some compounds have the same binding site.

SARS-CoV-2 Proteins
Center Size Interactions of the complex protein-ligand structure The results of LigPlot+ analyses showed the interaction of chlorhexidine, catechin, and quercetin with M pro or Non-structure Protein (Figure 4, Table   3). Importantly, these compounds shared the same binding pocket in the M pro (PDB: 6Y84). The spike glycoprotein was also studied in our work. The ability to bind to the ACE 2 makes the spike glycoprotein a crucial factor of pathogenicity. 26,27 Thus, according to Walls, et al. 28 (2020), this group is an essential target to neutralize SARS-CoV-2. Based on this rationale, the scientific reports describe that the spike glycoprotein in an opened state (PDB: 6VYB) would be associated with the most pathogenic coronaviruses. In contrast, the closed state is

Conclusions
Finally, our findings suggest that chlorhexidine is the most active compound in reducing the SARS-CoV-2 salivary load due to its better binding energy. It can be considered to be used as a mouthwash before dentistry procedures, reducing the SARS-CoV-2 contamination risk of dental workers. However, in vivo studies should be conducted to confirm their clinical use.

Conflict of interest
The authors have no actual or potential conflicts of interest.