Potential activity of a selected natural compounds on SARS-CoV-2 RNA-dependent-RNA polymerase, and binding anity of the receptor-binding domain (RBD)

Coronavirus disease (COVID-19) is caused by SARS-CoV-2 and represents the causative agent of a potentially lethal disease. COVID-19 has been described as a signicant global public health pandemic by the World Health Organization due to its high mortality rate, rapid spread, and the lack of drugs and vaccines for it. Active antiviral drugs are desperately needed to combat the potential return of severe acute respiratory syndrome (SARS). In this study, we selected 39 natural compounds present in plants, algae, and sponges with antiviral activity. Molecular docking was used to screen the compounds’ activity on SARS- CoV-2 RNA-dependent-RNA polymerase, receptor-binding domain (RBD), and the human ACE2 receptor. Compounds with binding energy ≤ -6.5 kcal/mol enter pre-clinical testing using in silco ADME/Tox (absorption, distribution, metabolism, excretion, and toxicity). We found eight potential SARS-CoV-2 inhibitors: (glycyrrhizin, rutin, baicalin, 1, 6-di-O- galloyl-beta-D-glucose, pyropheophorbide A, pheophorbide A, beta-Sitosterol, and vitexin). These outcomes indicate that these compounds could be potential candidates to be utilized in lead optimization for the design and production of the anti-SARS-CoV-2 drug.


Introduction
In COVID-19 is the name given to this novel coronavirus (CoV), spread throughout the world. Coronaviruses are enveloped RNA that belongs to the subfamily Coronavirinae of the family Coronaviridae; they broadly commonly spread by humans, birds, and other mammals, causing respiratory and intestinal infections in both animals and humans 3 . Six species of coronavirus are known to cause human disease 4  COVID-19 is a β-coronavirus (CoV) that causes self-limiting upper infections in immunocompetent hosts. Severe symptoms such as breathing di culty and pneumonia occur in immunocompromised and elderly persons and those with chronic underlying diseases. Moreover, the touching of contaminated objects seems to characterize the propagation of COVID-19 8 .
The angiotensin-converting enzyme 2 (ACE2) is an enzyme that transforms angiotensin II into angiotensin (1-7) 9, 10 . Angiotensins are peptides involved in maintaining blood pressure control and arterial hypertension by increasing the secretion of aldosterone 11 and promoting sodium retention by the kidneys 12 . ACE2 receptors are attached to the surfaces of the lungs, heart, and kidney cell membranes 13,14 .
The spike (S) glycoprotein of SARS-CoV-2 contains the receptor-binding domain (RBD) that recognize speci c cell receptors for its attachment. Angiotensin is peptides involved in membrane fusion and entry process through endocytosis 15 . It has been reported that ACE2 is the receptor of the SARS-CoV-2 S glycoprotein 16,17 due to the presence of a nity domains 18 ..
The interaction of this spike protein with ACE2 could be responsible for lung damage 19 . Therefore, the RBD of spike glycoprotein could be a candidate for drug targets for inhibiting the initiation process of virus infection 20 .
In the absence of antiviral treatment, infected patients receive oxygen therapy and immunoglobulin G for critical cases 21 . Antiviral drugs were previously tested in clinical practice, including nucleoside analogs acting as polymerase inhibitors, such as ribavirin (hepatitis C virus), acyclovir (herpes virus), ganciclovir (cytomegalovirus), and favipiravir (Ebola and in uenza A virus); protease inhibitors, such as lopinavir and ritonavir (human immunode ciency virus, SARS, and MERS), and nafamostat (in uenza virus and Ebola); and neuraminidase inhibitors, such as oseltamivir (in uenza virus A). However, all of the tests using these molecules were invalid for COVID-19. By contrast, the best results were obtained with the non-antiviral drug chloroquine, which has been used since the 1950s to treat malaria 22 . Nevertheless, its antiviral mechanism is not well understood; however, we suppose that this compound inhibits pH-dependent steps of the virus replication 23 or interferes with the glycosylation of cellular receptors 24 . In vitro studies also demonstrated that chloroquine acts at both entry and post-entry stages of the COVID-19 infection on Vero cells 25 .
Remdesivir, which is an adenosine nucleotide analog prodrug, has been reported to exhibit activity against several RNA viruses 26 by inhibiting RNA polymerase activity 27 .
This study aimed to in silico evaluate the effect of 39 natural compounds, which have been reported to exhibiting in vitro antiviral activity, on the SARS-CoV-2 RNA-dependent-RNA polymerase activity as well as on their interaction with RBD and ACE2. The compounds, which displayed a binding energy more than 6.5 Å, were tested for absorption, plasma clearance, tissue distribution, metabolic effects, toxicity, and druglikeness using Pre ADMET pro ling.

Natural products and active molecules with antiviral activity
Thirty-nine natural compounds isolated from plants, algae, and sponges (Table 1) and three antiviral drugs (Velpatasvir, IDX-184, and Oseltamivir) have been tested by in silico for their inhibitory activity on SARS-CoV-2 RNA-dependent-RNA polymerase activity, as well as RBD and ACE2 interaction.

Preparation of proteins and inhibitors
The structures of RdRp, ACE2, and the SARS-CoV-2 spike protein were prepared for docking by protonation, water molecules removal, atom xation, RMSD gradients re nement, and energy minimization by the drug discovery platform Molecular Operating Environment (MOE) suite (demo version 2019; Chemical Computing Group Inc: Montreal, QC, Canada). Thirty-nine natural products compounds (Table 1) and three antiviral references (Velpatasvir, IDX-184, and Oseltamivir) structures were obtained from the PubChem database 44 .

Molecular docking
The default docking parameters in MOE were used to study the best stable conformational binding (poses with the lowest energy level) between ligands and protein receptors. Docking with root-mean-square deviation (RMSD) less than 2 Å, was considered a success 45 . Known and predicted active sites and interacting residues were used for ligand docking 17 .

Protein-Ligand interaction analysis
The binding a nities, visualization, and interactions of ligand-receptor complexes were examined by the Protein-Ligand Interaction Pro ler (PLIP) web server (Salentin et al., 2015), and Discovery Studio Visualizer 2020 (BIOVIA, 2017). Compounds with the most favorable binding energy (≤ -6.5 kcal/mol), were selected for further analysis 46 .

ADMET Pro ling
To assess the potential pharmacokinetic properties of the top eight compounds (that scored the lowest energy ≤ -6.5), we conducted a preliminary computational screening using PreADMET pro ling to estimate their absorption rate, plasma clearance, tissue distribution, metabolic effects, toxicity, and drug likeness 47-49 .

Docking and interaction analysis
From 39 natural compounds virtually screened for their activities on RdRp, ACE2, and RBD proteins, only eight hits scored binding energy ≤ -6.5 kcal/mol (Table 2). Then the promoting eight compounds were selected for further interaction analysis and PreADMET pro ling. As shown in Table 3, the docking results of glycyrrhizin show the lowest binding a nity (-7.9) among all of the screened compounds that forming many hydrogen bonds, alkyl, and carbon hydrogen bond ( Figure 1). Docking of glycyrrhizin is considered successful with RMSD 2 (dockings with RMSDs 2Å are considered a successful, docking with RMSD 2-3Å are considered a partial success) 51 . The activity of glycyrrhizin on SARS-CoV-2 polymerase, aligned with previous studies, con rmed the activity of glycyrrhizin on the replication of SARS-CoV and polymerase of in uenza virus 52,53 . Rutin with RdRp active site showed the second-lowest binding energy, at -7.8; this was a result of the formation of two conventional hydrogen bonds, two pi-alkyl, one pi-donor hydrogen bond, three pi-cation bonds, and ve carbon-hydrogen bonds with residues located at the conserved pocket of RdRp 54 . The docking of rutin is considered successful with RMSD 2 Å (Table 3) (Figure 2).
Baicalin formed a strong interaction with protein active site, which scored low binding energy (-7.6) and RMSD 1.3. Baicalin strong interaction and well con guration in protein pocket occurred due to the presence of six hydrogen bonds with distances less than 3.33 Å. In addition to the presence of H bonds, there were many other types of bonds, such as pi-alkyl, pi-sigma, Pi-cation, pi-anion, pi-pi t-shaped, pi-lone pair, carbon hydrogen bond (Table 3) (Figure 3). 1,6-di-O-galloyl-beta-D-glucose, a natural product found in an abundant amount in Acacia nilotica, has a previously reported activity on HIV-1 reverse transcriptase (RT) 29 . Our study demonstrated that this molecule shows a promising anti-SARS-CoV-2 polymerase activity, forming nine-strong H bonding, picationic, and pi-anionic interaction with four amino acids, and pi-alkyl bond (Table 3 and Figure 4).
Pyropheophorbide A and pheophorbide A are compounds found in many plants, and they have possess a signi cant inhibitory activity against H1N1, H3N2, H5, and B in uenza viruses 55 . Both compounds score low docking energy (-7.3) and form many types of interactions with the SARS-CoV-2 RdRp enzyme (Table   3) (Figures 5 and 6).
0000With low binding energy (-7), three alkyl bonds, and 2.87Å distanced hydrogen bond, beta-sitosterol, tightly blocked the active site of the RdRp enzyme (Table 3 and Figure 7). This good interaction aligned with a previously reported capacity of beta-sitosterol to inhibit the enzymatic activity of SARS 3CLpro 56 .
Vitexin interacts with RdRp through seven hydrogen bonds, two pi-cationic bonds, one pi-anionic bond, and two carbon-hydrogen bonds ( Table 3 and Figure 8).
Three antiviral agents (velpatasvir, IDX-184, oseltamivir, and ribavirine), showed a high activity against SARS-CoV-2 RdRp. Velpatasvir is a hepatitis C RNA polymerase inhibitor 57 (Table 3). Natural ligands showed numerous interactions with the following amino acids; THR345, ARG403, ASP405,   GLN409, GLY416, LYS417, ILE418, TYR421, GLY496, TYR453,   TYR495, GLY504, and TYR505 (Table 4), these residues located at the binding site of RBD 1 . Glycyrrhizin was the most active compound on RBD that scored the lowest energy -7.3 kcal/mol and an RMSD within the acceptable range (2.3Å). Glycyrrhizin forms a strong H bonding with exposed TYR505, which has known contact with the ACE2 receptor 59 . Glycyrrhizin which creates alkyl interaction with LYS417, is one of the most important mutant residues in SARS-CoV-2 RBD 1 , which may contribute to its increased binding a nity to ACE2 receptors. In addition, glycyrrhizin interacts with another important residue (TYR453) that interacts with HIS34 of ACE2. Blocking of these important residues may interfere with the virus binding to human cells (Figure 9). Rutin created two conventional hydrogen bonds with TYR453 (ACE2 binding residue) and ILE418, located at the hydrophobic pocket of RBD 59 .

Interactions of ligands and RBD
The presence of pi-alkyl and carbon-hydrogen bonds in the RBD active site make rutin the second active compound with binding energy -6.9 ( Figure 10). Pheophorbide A makes a hydrogen bond with GLN409, in addition to alkyl and pi-alkyl bonds with different aliphatic amino acids in the active site of RBD (Table 4) ( Figure 11).
Drug distribution depends on a drug's ability to penetrate the blood-brain barrier (BBB), as well as its degree of plasma protein binding (PPB) 65 . Most of our compounds, as predicted by PreADMET, have a high binding a nity to plasma protein (up to 100%), and good penetration of the BB, compared with reference antiviral agents (Table 5).  The cytochrome P450 (CYP) enzymes play a major role in the rst phase of drug metabolism and detoxi cation of harmful substances in cells, such as drugs and toxins 66 . Inhibition of CYP enzymes could lead to toxic side effects or a decrease in drug effectiveness 67 . Pheophorbide A, and pyropheophorbide A showed low toxicity to CYP enzymes, followed by glycyrrhizin, beta-Sitosterol, baicalin, and vitexin, which were inhibitors for two of six CYP enzymes predicted by PreADMET. 1, 6-Bis-O-galloyl-beta-D-glucose and rutin show the highest inhibitory effect for CYP enzymes. The nhibitory effect of rutin to CYP3A4 is in agreement with a previous study that found that rutin is one of the most potent CYP34A4 inhibitors 66 (Table 5).
These outcomes indicate that these compounds are potential candidates to be utilized in lead optimization for the development of anti-SARS-CoV-2 drugs.         Three and two-dimensional interaction of pheophorbide A ligand with Spike protein RBD. A. Display of the receptor surfaces with H-bond contacts. B. 2D structure shows the ligand surrounded by active site residues, bond types, and distances.

Figure 12
Three and two-dimensional interaction of glycyrrhizin ligand with ACE2. A. Display of the receptor surface with H bonds surrounding ligand-binding site. B. 2D structure shows the ligand surrounded by active site residues, bond types, and distances.

Figure 13
Three and two-dimensional interaction of rutin ligand with ACE2. A. Display of the receptor surface with Hbond contacts and ligand. B. 2D structure shows the ligand surrounded by active site residues, bond types, and distances.