Table 2 showed the amino acid binding sites, the number of hydrogen bonds produced and binding energy value for each ligand after docking analysis. Results of ligands docking, ranked by number of hydrogen bonds produced were; remdesivir > quercetin > naringenin, ellagic acid > gallic acid, ribavirin > oleuropein > caffeine, benzoic acid and resveratrol. It well known that the high binding affinity of the drug compounds depends on the type and amount of bonds that occurs with the target protein33. Docking results, ranked by binding energy value (ΔG) of several tested ligands were; remdesivir > gallic acid > quercetin > caffeine > ribavirin > resveratrol > naringenin > benzoic acid > oleuropein > ellagic acid. The binding energies were –8.51, - 7.55, - 7.17, –6.10, - 6.01, - 5.79, - 5.69, - 5.54, - 4.94 and –4.59 kcal/mol, respectively (Table 2). Figure 2 showing the comparative binding energy value ΔG in minus kcal/mol of several ligands.
Table 2 showed that remdesivir formed H-bonds with the COVID–19 polymerase ASN 691, CYS 622, LYS 621, TYR 619, THR 680 and THR 687 (Figure 3A), ribavirin formed H-bonds with ARG 553, ARG 555, ARG 624 and SER 681 (Figure 3B), gallic acid formed H-bonds with ARG 553, PHE 442 and ALA 547 (Figure 3C), naringenin formed H-bonds with ARG 553, ARG 555, SER 682 and THR 556 (Figure 3D), quercetin formed H-bonds with ARG 553, ARG 624, LYS 545, ASP 452, ALA 554 and SER 682 (Figure 3E), benzoic acid formed H-bonds with ARG 555 and GLN 444 (Figure 3F), ellagic acid formed H-bonds with ARG 553, ARG 555, SER 682 and THR 556 (Figure 3G), resveratrol formed H-bonds with ARG 555 and SER 682 (Figure 3H). oleuropein formed H-bonds with ASP 445, ASP 452, ASN 552 and TYR 455 (Figure 3I) and caffeine formed H-bonds with PHE 442 and GLN 444 (Figure 3J). From previous results, all tested polyphenols performed hydrogen bonds with one or two of the amino acids of nucleotide triphosphate entry channel (NTP) in COVID–19 polymerase except remdesivir, caffeine and oleuropein. Binding of polyphenols to ARG 553, ARG 555 and LYS 545 of COVID–19 polymerase may prevent the entry of the substrate and divalent cations into the central active site cavity, inhibiting the enzyme activity preventing RNA replication34. Figure 4 showed the proposed mechanism of COVID–19 inhibition by polyphenols.
The NTP entry channel of RNA dependent RNA polymerase, is formed by a set of hydrophilic residues, including LYS 545, ARG 553 and ARG555 in motif F. The RNA template is expected to enter the active site composed of motifs A and C through a groove clamped by motif F and G. Motif E and the thumb subdomain support the primer strand. The product-template hybrid exits the active site through the RNA exit tunnel at the front side of the polymerase35. Because of the structural conservation of the polymerase catalytic chamber between COVID–19 polymerase (PDB ID: 6M71) and HCV ns5b polymerase (PDB ID: 4WTG)36, a model of COVID–19 polymerase with remdesivir diphosphate is proposed with similar mechanisms of action. In this proposed model; remdesivir is likely to form hydrogen bond with THR 680 and ASN691 in motif B as well as, ASP 623 in motif A and the hydrophobic side chain of VAL 557 in motif F35. These proposed binding results are confirmed by our docking analysis that remdesivir can form hydrogen bonds with THR 680 and ASN691 like the proposed binding model but not to ASP 623 and VAL 557. This difference between results may be due to a little structural difference between COVID–19 polymerase and HCV ns5b polymerases.
Concerning to polyphenol docking to COVID–19 polymerase, it is a promising that, gallic acid and quercetin expressed high binding affinity toward COVID–19 polymerase than ribavirin standard drug with –7.55, –7.17 and –6.01 kcal/mol, respectively (Table 2).
Table 3 showed the drug likeness and pharmacokinetic properties of tested COVID–19 polymerase ligands. Concerning drug likeness properties, all tested compounds bear the Mol. Wt. range from 122.12 to 302.197 (< 500) except oleuropein. The present investigation predicted that all tested compound have number of rotatable bonds less than 15. They have less than five hydrogen bond donors (NH and OH) except quercetin and oleuropein. Besides, the numbers of hydrogen bond acceptors (O and N atoms) predicted in all compounds are less than 10 except for oleuropein (Table 3). In the same time, the number of rotatable bonds in all tested compounds ranged from 0 to 2, except oleuropein. Permeability possessions (logP) of ligands were also studied and found that, all polyphenols showed value of logP less than 5. Also, Topological Polar Surface Area (TPSA) values of all ligands are more than 140 Å for oleuropein and ellagic acid. Lipinski’s rule of five is a rule used to calculate the drug likeness of certain compound has a certain pharmacological activity that would make it alike orally active drug in humans37. The rule describes the molecular characteristics influence the drugs pharmacokinetics in the human body, including their absorption, distribution, metabolism and excretion (ADME)38.
It has previously reported that, drug molecules have low, molecular weight (<500) are transported, diffused and absorbed easily in comparison to large molecules that affects the efficiency of the drug 39. The number of rotatable bonds is a measure of molecular flexibility and is important in determining oral bioavailability of the drugs40. Regarding rotatable bonds, only 4% of the molecules in the human metabolite dataset have no rotatable bonds, whereas 32% have 1–10 rotatable bonds and 47% of the molecules have rotatable bonds in the range 36–5037. Lipinski hydrogen bond donors (LHBDs) are determined by counting the numbers of OH and NH bonds in each molecule.
Approximately 21% of the metabolites, 12% of the drugs and 34% of the toxin molecules don’t possess any LHBDs37. Hydrogen bond acceptors (LHBAs), computed by summing the numbers of nitrogen and oxygen atoms in each. Only a fraction of molecules in all the datasets (0.35% of metabolites, 0.40% of drugs and 3.6% of toxins) do not possess LHBAs, molecule37. TPSA and the value of logP are the two essential properties in analyzing bioavailability of drug molecule and permeability through bio-membranes41. Topological Polar Surface Area (TPSA) was calculated from surface areas occupied by oxygen, nitrogen and the hydrogen atoms that are attached to them. Thus, the TPSA is closely related to the H- bonding ability of a compound42. It can illustrate the drug absorption, including intestinal absorption, Caco–2 permeability, BBB penetration, and bioavailability. For the compounds with ten rotatable bonds and TPSA of ≤ 140 Å can be said to have good bioavailability40.
Pharmacokinetic properties prediction of tested polyphenols showed in (Table 3) results revealed that all polyphenols showed a high absorption rat through GI tract except oleuropein. Also, all tested ligands have no ability to pass through the blood brain barrier except benzoic acid and resveratrol. Also, naringenin only can be act as substrates for P- gp that reduced its efficacy in further clinical use43. The present results expressed that only caffeine, oleuropein and benzoic acid have no inhibitory effect for any of cytochrome P450 enzymes (CYP1A2, CYP2C19, CYP2D6 and CYP3A4). Caffeine, oleuropein, benzoic acid and gallic acid have no inhibitory activity toward CYP1A2. Also, CYP2C19 and CYP2D6 activity does not affected by any tested compounds. Furthermore, CYP2C9 activity does not affected by any of tested compounds except resveratrol. Besides, caffeine, oleuropein, ellagic acid and benzoic have no predicted inhibitory effect toward CYP3A4.
Pharmacokinetic analysis provides a mathematical basis to assess the time course of drugs and their effects in the body. It enables the following processes to be quantified: Absorption, Distribution, Metabolism, and Excretion (ADME)44. Understanding of these parameters is required to design an appropriate drug regimen for a patient45. It is known that, the gastrointestinal tract absorption affected by several factors such as physicochemical parameters of the drug, gastrointestinal motility, drug concentration at the site of absorption46. The distribution of a drug is influenced by many factors such as lipid-solubility, concentration in plasma and binding ability to plasma proteins, transport proteins 47. Also, metabolism of any drug is the process of irreversible transformation of parent compounds into daughter metabolites. The major site of metabolism in the body is the liver48. Metabolism in the liver occurs in two stages: Phase I pathways in liver microsomes, are catalyzed by a group of enzymes known as the cytochrome P450 system including aromatic hydroxylation, aliphatic hydroxylation, oxidative N dealkylation, oxidative O-dealkylation, S-oxidation, reduction and hydrolysis so the drug become more soluble, facilitating its elimination through the kidneys. Phase II pathways in liver cells where the parent or the metabolite from Phase I gets conjugated occurs by glucuronidation, sulfation, amino acid conjugation, acetylation, methylation or glutathione conjugation to facilitate elimination49. There are several factors that influence drug metabolism including route of administration, dose, genetics, disease state, and metabolic activity50. From docking analysis and prediction of drug likeness and pharmacokinetic properties of 8 tested polyphenols, gallic acid and quercetin exhibited high binding affinity than ribavirin toward COVID–19 polymerase and expressed good drug likeness and pharmacokinetic properties. Therefore, gallic acid and quercetin may represent a potential treatment option for COVID–19.
Quercetin, naringenin, ellagic acid, benzoic acid, resveratrol and gallic acid are group of polyphenols, and many of them have been found in plant-based foods. Dietary polyphenols have received tremendous attention among nutritionists, food scientists and consumers due to their roles in human health 51. Table 4 showed the natural sources of some bioactive compounds. Concerning fruit and vegetable processing, industries produced huge amount of by-products in the form of peels, cores, seeds, leaves and others that are discarded. These by-products regarded as rich sources in phenolic compounds, and they could be used as a food additives and nutraceuticals52. Quercetin, naringenin, ellagic acid and resveratrol are already present in the form of dietary supplement for public use, which reported as anti-inflammatory, antioxidant, supporting cardiovascular health and promoting heath immune function and brain function 53,54,55. From our results we recommend use of dietary supplement quercetin and producing gallic acid dietary supplement then starting evaluate its activity against COVID–19.