In silico phytochemicals analysis as inhibitors of the SARS-COV-2 main protease

Background: The world population's full immunization with vaccines against SARS-CoV-2 is still challenging. Therefore, more research must be needed to ﬁ nd an active antiviral drug against the virus, including new mutated strains. Results: Therefore, this research analyzes 35 natural compounds isolated from various plants against SARS-CoV-2 main protease (Mpro) using an in silico strategy. According to the results, it was possible to identify promising molecules using a molecular docking strategy. Furthermore, the results showed that the interaction of these molecules with protease-speci ﬁ c residues, including (2S)-Eriodictyol 7-O-(6''-O-galloyl)-beta-D-glucopyranoside (Trp207, Ser284, and Glu288), Hypericin (Glu166, Arg188, and Thr190), Calceolarioside B (Gly143, Ser144, Cys145, Glu166, Arg188, and Gln192), Epicatechin (Ser144, His163, and Leu167) and Myricitrin (Thr190) with ∆ G was -8.5, -9.6, -8.5, -9.3 and -9.3 kcal/mol, respectively. In addition, analyzing all compounds for their ADME properties shows that compounds present an excellent pharmacokinetic pro ﬁ le. Conclusion: In conclusion, the results of this study indicated that these major natural compounds can be considered potential inhibitors of Mpro and should be further explored in vitro and in vivo in accordance with our data.


Molecular docking studies
Molecular docking was performed via the Chimera tool, using the AutodockVina tool to predict the protein-ligand interaction described elsewhere [16]. Evolution at max steps 300 and neighbor distance faster 1.00 [16]. The method for docking calculation consists of using the MMFF94 parameter as a Geometry Optimization Method, where a force fi eld aims to minimize the energy of the ligand.
The solvation parameters are set by AutoDockVina concurrently with the docking simulations [17]. The docking validation was following the instructions presented elsewhere [18].   [20]. The protein was minimized using SwissProt PDBviewer 4.1 as described elsewhere [21].

Background
Although many countries have reviewed restrictive measures and have relaxed the use of masks [1], some countries do not have a fully immunized population. This scenario could highlight the middle-or low-income countries with few resources to acquire the developed immunizers and lack donations from richer countries [2,3]. In addition, in some countries, the population is waiting for newer vaccine applications as a booster against COVID-19 [4][5][6]. When the pandemic started, pharmaceutical enterprises suggested several drugs as treatments. However, they presented signifi cantly less effectiveness than previous data suggested in clinical trials [7]. In addition, coronavirus can rapidly mutate, which may demand new effective vaccines shortly [8].
In this context, the identifi ed variants have elevated the race for new drugs and vaccines against important VOC, such as alpha (B.1.1.7), beta (B. 1.351), gamma (P.1), and delta (B. 1.617.2). Moreover, the low vaccine adherence in several countries promoted the development of new variants. This scenario is the case of the omicron Variant (B.1.1.529), whose dissemination caused outbreaks in several countries in early 2022. Moreover, again, ringing the warnings, the fi ne line among new variants, and the risk of resistance to already developed vaccines [9,10]. In particular, the omicron variant has many mutations in the Spike protein. These mutations may contribute to the low effi cacy of current vaccines and provide virus evasion and already immunized people [11].
According to recent studies, COVID-19 vaccines approved to date produce serum IgG1 but leave nasal epithelium and upper respiratory tract unprotected. As a result, they allow the SARS-CoV-2 infection (i.e., not providing mucosal immunity) [12]. What makes necessary new pathways for COVID-19 infection therapies? In this instance, natural or synthetic compounds are an awesome font to discover new antiviral drugs. Plant compounds include an extensive list of biologically active molecules. Several of them have antiviral activity [13]. Thus, they are a good starting point for the search against those interactions with virus proteins.
The SARS-CoV-2 main protease (Mpro) is a promising drug target. This protein plays a crucial role in viral replication and transcription [14]. Mpro is conserved among coronaviruses, sharing ~76% sequence similarity with SARS-CoV-1 Mpro. In addition, there is no human host cell protease with similar substrate specifi city [15].
At last, molecular docking is an excellent methodology for predicting their protein-ligand interaction. Several endpoints, such as interactive activity, interaction energy, and ligandspecifi c interaction region, are essential in that approach. Consequently, molecular docking studies could help model the ligand-receptor interaction to identify potential SARS-CoV-2 Mpro pharmacophore sites. Herein, it aims to test 35 natural compounds as potential inhibitors of the SARS-CoV-2 major protease. In addition, it analyzed the computational pharmacokinetic parameters and predicted their toxicological effects on humans. Lastly, this study resembles information on a drug structure through standard and drug similarity.

ADME analysis
Using the canonical SMILES chemical nomenclature of each ligand under consideration, it performed Absorption, Distribution, Metabolism, and Excretion (ADME) prediction.
Then, the molecules are uploaded in SMILES format to the SwissADME server (http://www.swissadme.ch). We subsequently surveyed HIV protease inhibitors, already authorized by the FDA as positive controls. Thus, with this approach, some parameters were established among the tested molecules and some inhibitors already on the market.

Molecular docking studies
Molecular docking was performed for 35 phytochemical components against Mpro using UCSF Chimera 1.15 to identify molecular interactions between the protein active site and ligands. The binding energy (ΔG) in these complexes' best poses is shown and compared in Figure 1. The best ligands were those with the highest binding energy, including Myricitrin, Calceolarioside B, Hypericin, Epicatechin-gallate, (2S)-Eriodictyol 7-O-(6''-O-galloyl)-beta-D-glucopyranoside and Calceolarioside B.

ADMET prediction
The analysis of the physical and chemical properties of all molecules, their lipophilicity, and drug likeliness are shown in Table 1. The rule of fi ve (RO5) predicts that good absorption is more likely when the molecular weight is lower than 500, the In another inhibitor analysis, the peptide inhibitor N 3 and -ketamine interacted via hydrogen bonds with residues in the protease binding site. The interactions happen among the compounds and Thr190, His163, Cys145, Glu166, and Gly143 protein residues. These residues are also the interaction targets of the ligands tested in our study Hypericin, Epicatechin, Myricitrin, and Calceolarioside B [23].
Another study shares these interaction residues.
Interactions with Remdesivir (Cys145, Glu166, Gln192, and of Mpro. Our data indicate a ΔG close to or higher than those found in other works that tested other natural producers, such as the generous Aloe [26], Xylopic acid, Resveratrol, Ellagic acid, Kaempferol, and Quercetin [27] and diterpenoids and biofl avonoids of TorreyaNucifera leaves [28].
This rule describes molecular properties that are important for the pharmacokinetics of a drug in the human body. This  rule can be used when creating drugs. The active drug is gradually optimized to increase the activity and selectivity of the compound and to ensure that the physicochemical properties inherent in compounds obeying the Lipinski rule are maintained.
Bioavailability Score is based on a probability value of a molecule to have an optimum profile of permeability and bioavailability, where 0.55 indicates the obedience of the Lipinski rule of five. A molecule can be considered a good drug if all the computational fi lters of SwissADME -Lipinski, Ghose, Veber, Egan, and Mueggehave no violations.
Myricitrin, Calceolarioside B, and Myricetin 3-O-beta-Dglucopyranoside have several H-bond acceptors equal to 12, 11 and 13, respectively, which is more than ten acceptable. The molecular weight of (2S)-Eriodictyol 7-O-(6''-O-galloyl)beta-D-glucopyranoside is not lower than 500 g/mol and equal to 602.50 g/mol as well as Hypericin. Epicatechin-gallate showed the value of topological polar surface area equal to 177.14 Å 2 , which is not in the excellent range of 20-140 Å 2 .
The lipophilicity study revealed that values of Log Po/w (Consensus Log Po/w) of Gossypol and Oxyacanthine are out of the acceptable fi ve and equal to 5.04 and 5.12, respectively. In addition, Myricitrin, Myricetin 3-O-beta-D-glucopyranoside, Amaranthine, and Crocin have negative lipophilicity values, which is unfavorable.
Hypericin is an anthraquinone derivative naturally found in the yellow fl ower of Hypericumperforatum with an antidepressant, potential antiviral, antineoplastic and immunostimulating activities [29]. It has the highest docking score (-9.6 Kcal/mol) with the main coronavirus protease among all the phytochemicals. Flavonoid Myricitrin is found in vegetables, fruits, nuts, berries, herbs, plants, tea, wine, and medicinal plants. Myricitrin is nitric oxide (NO) and protein kinase C (PKC) inhibitor that has central nervous system activity, including anxiolytic-like action [30] docked with a target with a docking score of -9.3 Kcal/mol. Myricetin 3-O-beta-D-glucopyranoside is a compound with chemotherapeutic, chemopreventive, and antiangiogenic properties [31], a naturally occurring plant metabolite found in several food items such as blackcurrant, typical grape, highbush blueberry, and tea docked with the target with a docking score of -9.3 Kcal/mol. Epicatechin-gallate, a fl avonoid present in brewed black, green, and oolong teas, cherries, and strawberries inhibit the growth of cancer cells, and gas anti-infl ammatory effects show a docking score of -9.3 Kcal/mol [32].