Phytochemicals of Rhus spp. as Potential Inhibitors of the SARS-CoV-2 Main Protease: Molecular Docking and Drug-Likeness Study

Background The outbreak of coronavirus disease 2019 (COVID-19) induced by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originated in China and spread to cover the entire world with an ongoing pandemic. The magnitude of the situation and the fast spread of the new and deadly virus, as well as the lack of specific treatment, led to a focus on research to discover new therapeutic agents. Aim In this study, we explore the potential inhibitory effects of some active polyphenolic constituents of Rhus spp. (sumac) against the SARS-CoV-2 main protease enzyme (Mpro; 6LU7). Methods 26 active polyphenolic compounds of Rhus spp. were studied for their antiviral activity by molecular docking, drug likeness, and synthetic accessibility score (SAS) as inhibitors against the SARS-CoV-2 Mpro. Results The results show that all tested compounds of sumac provided good interaction with the main active site of SARS-CoV-2 Mpro, with better, lower molecular docking energy (kcal/mol) compared to the well-known drugs chloroquine and favipiravir (Avigan). Only six active polyphenolic compounds of Rhus spp. (sumac), methyl 3,4,5-trihydroxybenzoate, (Z)-1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)-2-hydroxyprop-2-en-1-one, (Z)-2-(3,4-dihydroxybenzylidene)-6-hydroxybenzofuran-3(2H)-one, 3,5,7-trihydroxy-2-(4-hydroxyphenyl)chroman-4-one, 2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxy-4H-chroman-4-one, and 3,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one, were proposed by drug likeness, solubility in water, and SAS analysis as potential inhibitors of Mpro that may be used for the treatment of COVID-19. Conclusion Six phenolic compounds of Rhus spp. are proposed for synthesis as potential inhibitors against Mpro and have potential for the treatment of COVID-19. These results encourage further in vitro and in vivo investigations of the proposed ligands and research on the preventive use of Rhus spp. against SARS-CoV-2.


Introduction
e coronavirus disease 2019 (COVID-19) crisis started in China in December 2019. By 30 January 2020, about 213 individuals had died and at least 9066 had been infected [1]. It also spread globally, first to a number of Asian countries, as well as to Canada, France, Germany, and the United States. As a result, due to the spread of this new and deadly virus, governments around the world put several major cities on lockdown and put aside all normal plans to deal with the crisis. In addition, on 30 January 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a global health emergency because it could spread to countries that were not prepared [1][2][3][4]. us, on 11 March 2020, the WHO characterized COVID-19 as a pandemic, which has affected more than 200 countries; by March 2020, there were 30,105 deaths and 638,146 confirmed cases around the world [3], which have increased considerably over time. Genomic and molecular-based analyses show that SARS-CoV-2 is a new type of human-infected β-coronavirus (CoV), which suggested that zoonotic hosts like bats might be the original host of this virus [5]. Situation Reports released by the WHO on 13th April revealed that SARS-CoV-2 infection can induce severe collateral disorders such as severe pneumonia, pulmonary edema, acute respiratory disease syndrome (ARDS), or multiple organ failure (such as shock, acute heart injury, or acute kidney injury), which are ultimately responsible for an increased number of deaths worldwide [6].
is may be also due to the lack of an effective specific antiviral therapy, since SARS-CoV-2 is a novel pathogen.
us, several drugs such as lopinavir/ritonavir, neuraminidase inhibitors, and other present antiviral drugs have been proposed for the treatment of COVID-19 infection [7,8].
ese drugs produce their antiviral potency via an inhibition process against SARS-CoV-2 proteases [7,8]. e magnitude of the situation and the lack of a specific therapy for the virus have led to a focus on research to find new therapeutic agents [8]. For the management of COVID-19, preventive and supportive therapies based on herbal plants may complement research on existing antiviral agents.
Medicinal herbs known in ethnopharmacology have been suggested as antiviral agents for the treatment and control of contagious diseases like COVID-19 [9][10][11][12][13][14]. Most of these studies use molecular docking analysis to identify the potential activities of phytoconstituents present in herbal plants for the treatment of diseases like COVID-19. ey focus on the activities of these plant constituents on the main proteases present in CoVs [12][13][14].
During infection, coronavirus attaches to target cells with the help of angiotensin-converting enzyme 2 (ACE2) present in the spike protein of the virus, which produces a spike protein-host cell protein interaction, whereby the virus genome with its nucleocapsid can easily release into the cytoplasm of the host cells [36,37]. Sequence analysis of the replicase polyprotein in Avian infectious bronchitis virus, another coronavirus, originally predicted the presence of the coronavirus M pro protease enzyme [38]. is enzyme was related to chymotrypsin-like cysteine proteases which significantly play a potential role in the replication and transcription of the coronavirus (SARS-CoV).
e SARS-CoV genome encodes a number of proteases. e main protease (M pro ) chymotrypsin-like protease (3CL pro ) from SARS-CoV-2 (6LU7) has an important role along with other cysteine proteases in the replication of the CoV genome.
us, synthetic or herbal-based drugs targeting the proteases of SARS-CoV-2 (6LU7) may have a considerable role in the treatment of COVID-19 [38]. Several inhibitors including boceprevir, GC-376, and calpain inhibitors II and XII were identified to have potent activity to inhibit SARS-CoV-2 viral replication in cell culture [39]. e protease enzyme (6LU7) has been successfully crystallized and deposited in the Protein Data Bank (PDB) [40,41]; thus, it is considered as a potential target for therapeutic strategies, particularly for those who use phytochemicals [7,42,43]. It was reported that an evaluation of up-to-date knowledge relating to the characteristics of COVID-19 infection and complications encourages the investigation of the effectiveness of sumac extracts for COVID-19 treatment [44][45][46][47][48]. Recently, active metabolites from 14 cooking seasonings were examined as inhibitors for SARS-CoV-2 main protease (M pro ). A high potency of salvianolic acid A and curcumin as M pro inhibitors with binding energies of −9.7 and −9.2 kcal/ mol, respectively, was identified by in silico molecular docking analysis [47]; the potential activity of salvianolic acid A and curcumin as M pro inhibitors against the SARS-CoV-2 main protease significantly depends upon forming from nine and six hydrogen bonds, respectively, with amino acids proximal to M pro 's active site [47]. Supporting review study showed that Rhus spp. (sumac) constituents could have a higher potency against the consequences produced by SARS-CoV-2 during human infection. e review study demonstrated that sumac could be useful in COVID-19 infection due to its versatile activities as anti-inflammatory, antimicrobial, antioxidant, and antimalarial effects [48]. In addition, the review article along with others mentioned that use of sumac as syrup or in capsules with different concentrations has no toxicity on human life and could be recommended in treatment protocol for COVID-19 patients [18,[48][49][50][51].

Rhus spp. Phytoconstituents.
A total of 26 compounds previously extracted from Rhus spp. [52] were selected to study their potential activity against the SARS-CoV-2 main protease enzyme. ese 26 components in Rhus spp. extracts 2 Evidence-Based Complementary and Alternative Medicine ( Figure 1) were selected based on their versatile biological activity, such as antimicrobial, antifungal, antidiabetic, antioxidant, wound-healing, and antiviral activity [26,36,52].

Molecular Docking Analysis.
e crystal structure of the protease enzyme (the molecular target) of SARS-CoV-2 (3cl pro /M pro , PDB ID: 6LU7) was retrieved from RCSB Protein Data Bank (https://www.rcsb.org/) [53]. e proposed natural components in Rhus spp. extracts (Figure 1), in addition, well-known drugs, chloroquine and favipiravir (Avigan), widely tested in clinical trials for the treatment of COVID-19 have been subjected to molecular docking [54]. e structures of the selected sumac compounds along with the proposed drugs were drawn by ChemOffice (2015) freeware and were optimized by HyperChem v.8.1 to have the conformer with minimum energy using AMBER force field [54]. According to previously reported methodology and wording [14,[53][54][55], for analysis, an open Babel 2.3.2 software was used to convert a molecular format files into pdb format. en, the ligands under investigations were loaded and their torsion along with rotatable bonds was assigned and saved as ligand PDBQT.
In our experiment by using Molegro Virtual Docker software 6.0, the binding modes of the proposed natural components in Rhus spp. extracts with the protease enzyme (the molecular target) of the SARS-CoV-2 (3cl pro /M pro ; PDB ID: 6LU7) were identified [56]. In addition, a blind docking was performed to enumerate the strength of binding interactions between the proposed ligands and the protease enzyme target [14,56]. In this step, a lowest binding energy (−kcal/mol) represents the best stable conformations of the ligand with the protease enzyme target [56]. Finally, according to the methodology previously reported [14], MGLTools-1.5.6 rc3 were used to analyze all released auto Dock output files (.dlg), whereas the docking parameters were represented as coordinates of the center of binding site with x � 126, y � 126, z � 126, and binding radius � 0.375 Ã. In addition, constituents that may hinder the simulation, such as water molecules, other heteroatoms, and native ligands attached to the target, were removed in the process of accommodating the ligands for the molecular docking study.

Drug-Likeness Analysis.
is analysis qualitatively assesses the chance for a molecule to become an oral drug with respect to bioavailability, as previously described [57]. In this analysis, structural or physicochemical inspections of the proposed herbal compounds and selected drugs chloroquine and favipiravir (Avigan) were used to assess the drug likeness to be considered as an oral drug candidate. e analysis depends on five rule-based filters, with a diverse range of properties, inside of which the molecule is defined as drug-like. e Lipinski (Pfizer) filter is the pioneer ruleof-five implemented from [57].
e original rule-of-five (RO5) deals with orally active compounds and defines four simple physicochemical parameter ranges (MWT ≤ 500, log P ≤ 5, H-bond donors ≤ 5, and H-bond acceptors ≤ 10) associated with 90% of orally active drugs that have achieved phase II clinical status. ese physicochemical parameters are associated with acceptable aqueous solubility and intestinal permeability and comprise the first steps in oral bioavailability [57][58][59][60][61][62]. is analysis is routinely performed to filtrate chemical libraries and exclude compounds with an incompatible or unacceptable pharmacokinetic profile.
In addition, the synthetic accessibility score (SAS) for each molecule extracted from sumac was calculated according to the methodology reported previously [57,58]. SAS was shown to be essential in both the early drug discovery stage and the drug manufacturing process [59,60]. To characterize the accessibility of drugs or molecules for synthesis, the synthetic accessibility score was grouped according to previous studies into three groups: easy to synthesize (SAS ≤ 3), moderately easy to synthesize (SAS � 3-4), and difficult to synthesize (>4) [57][58][59][60][61][62][63].

Molecular Docking Analysis.
e molecular docking parameters, including docking score, ligand-protein interactions, and hydrogen bonds for selected compounds and reference drugs, are provided in Table 1. e possible interactions with the active sites of protease enzyme in this experiment are discussed herein with some details. e reference drugs, Avigan and chloroquine, interact with active sites of protease enzyme 6LU7 with an energy docking score of −2.99 kcal/mol and −6.32 kcal/mol, respectively, as shown in Table 1.
All tested compounds of Rhus spp. have lower energy docking scores compared with the reference drugs Avigan and chloroquine (Table 1), as these compounds are polyphenols and can interact with 6LU7 more strongly through hydrogen bonds, pi-cation interactions, or pi-pi stacking interactions. Compound (14), although it demonstrated a good binding energy, could not form any hydrogen bond with the viral enzyme. Compound (14) showed no perceptible interactions but only electrostatics (Van der Waals) (see Supplementary file).
Due to the varied biological activity of the extracted polyphenolic compounds of Rhus spp. as potential active materials in the treatment of several diseases, several compounds are proposed: (1), (12), (13), (22), (23), and (26). ese compounds showed the lowest docking energy with efficient interaction with the active sites of protease 6LU7 of SARS-CoV-2 through many hydrogen bonds compared with Avigan and chloroquine (Tables 1 and 2 and Figures 2-8).

Drug-Likeness Analysis.
To study the availability of the proposed sumac compounds for synthesis, drug likeness, physicochemical properties, and synthetic accessibility score (SAS) were assessed according to the Lipinski rule-of-five. e drug likeness, water solubility, bioavailability scores, and SAS scores for all proposed active polyphenolic compounds of sumac and proposed Avigan and chloroquine are reported in Table 3.

Discussion
In this study, we are trying to decipher the proposed mechanism of the most potent active polyphenolic compounds extracted from Rhus spp. (sumac) in terms of binding affinity, necessary hydrogen bond formation [18,54,56], drug likeness, physicochemical properties, bioavailability, and synthetic accessibility score (SAS) [56][57][58][59][60][61][62], responsible for inhibition of target enzyme of the SARS-CoV-2. is study might lead to clinical trials for the treatment of infections caused by coronaviruses. us, a total of 26 known active polyphenolic compounds extracted from Rhus spp. (sumac) were measured for their activity as potential inhibitors against the SARS-CoV-2 main protease enzyme.
Molecular docking, drug-likeness analysis, and synthetic accessibility score (SAS) were performed for each active polyphenolic compound and compared with the results of Avigan and chloroquine as reference control drugs. All studied active polyphenolic compounds showed interactions with more hydrogen atoms to the main active site of the SARS-CoV-2 protease enzyme. is fits the complexation or interaction performed at lower energy docking score (kcal/mol) compared to that when Avigan and chloroquine interacted with the same active site. e polyphenolic nature of the extracted sumac compounds plays a pivotal role in their versatile biological activity, including antioxidant, antimicrobial, wound-healing, and antiviral activity [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]64]. e presence of polyphenolic active groups with more hydrogen atoms in sumac compounds [57][58][59][60] easily facilitates the orientation and tight interaction with different amino acids present in the core of the main active site of the SARS-CoV-2 protease enzyme. In addition, it was reported previously that the antiviral activity of most herbal plants, including sumac, is related to the presence of active compounds such as phenolics, organic acids, proteins, fibers, volatile oils, fatty acids, vitamins, and minerals [33,34].
is may be due to the fact that, in most proteins, almost all distances between two atoms are longer than the covalent bond length, so they tend to cause steric hindrance which may affect hydrogen bond energy during interaction with the main active site of the SARS-CoV-2 protease. In addition, all H atoms are donor rotatable, which could be sterically hindered by the surrounding atoms or molecules, thus resulting in the minimization of bond energies of H-bonds with longer lengths.

Conclusion
e antiviral activity of 26 active polyphenolic compounds of Rhus spp. (sumac) was analyzed by molecular docking, drug likeness, and synthetic accessibility score (SAS). e results showed that all tested compounds provided good interaction in the main active site of the SARS-CoV-2 protease enzyme 6LU7 with lower molecular docking energy (kcal/mol) compared to two drugs already under clinical tests. Together, the molecular docking, drug likeness, and synthetic accessibility score (SAS) data proposed six active polyphenolic compounds of Rhus spp. for the synthesis of potential inhibitors against the main active site of the SARS-CoV-2 protease enzyme 6LU7, which may be candidates for the treatment of COVID-19. e data herein provide new directions for in vitro and in vivo investigations of the proposed ligands to develop new inhibitors as SARS-CoV-2 antivirals.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.