A brief summary of four herbs (Aegle Marmelos, Coleus Amboinicus, Aerva Lanta and Biophytum Sensitivum) referred for the present study and their biological / therapeutic value are given in Table 1. Initially by using molecular docking study we scrutinized 87 phytochemicals obtained from these herbs for finding the most effective among them. Out of these 87 constituents, appreciable LibDock scores can be obtained for Aegelinoside B, Ervoside, Epoxy aurapten, Epicatechin, Feruloyltyramine, Marmin, Anhydromarmeline and Quercetin. Moreover, the corresponding binding energies are also found to be greater than 7.5 kcal/mol. The highest LibDock score of 142.50 is observed for Aegelinoside B. Ervoside and Epoxyaurapten exhibit next best dock score values of 129.69 and 129.06 respectively. List of phytochemicals with LibDock score > 120 and their corresponding binding energy values are shown in Table 2. Complete details of all these compounds are given in the supporting information (SI). Reference compounds such as Rimonabant, Metixene, Remdesivir, Indinavir, Oxiconazole, Doxapram, Pirenepine, Pimozide, Zopiclone, Saquinavir and Tipranavir were taken for comparison [36, 37]. It can be noted that Aegelinoside B exhibit higher LibDock score than some of the popular compounds reported in the literature, such as, Rimonabant, Metixene, Oxiconazole, Doxapram, Pimozide, Zopiclone, Saquinavir and Tipranavir. Among these, Remdesivir shows a closely competitive LibDock score as compared to our study (Indinavir is the second highest). Detailed descriptions are provided in the following subsections, 3.1–3.6. From the present reference compounds, Remdesivir is the only FDA approved nucleoside COVID19 drug which inhibits the replication of RdRp of SARS-CoV-2 by terminating its chain [38]. Others are repurpose drugs used for other viral infections and some of them are used to conduct computational studies for docking purposes [39–42].
Table 1
Medicinal and physiological properties of herbal plants considered in the current study.
Medicinal plants | Family | Action |
Aegle marmelos | Rutaceae | Stomachic, asthma, antimicrobial (specific for diarrhoea, colitis, dysentery and enteric infections), digestive, astringent, spasmolytic,antiallergic activity, hypoglycaemic. |
Aerva lanata | Amaranthaceae | Anticalculus (used in lithiasis), lithontriptic, diuretic, demulcent, anthelmintic, antidiarrhoeal, anticholerin, bechic; leaf used in headache and hepatitis, root in strangury. |
Biophytum sensitivum | Oxalidaceae | Insomnia, strangury, asthma, phthisis, diabetes, convulsions, cramps, chest-complaints, inflammations, tumours, stomachache, diuretic, astringent, antiseptic, chronic skin diseases. |
Coleus Amboinicus | Lamiaceae | Epilepsy and other convulsive affections, asthma, bronchitis, cold and chronic cough, urinary diseases, vaginal discharge, colic and dyspepsia. Stimulates the function of liver. |
Table 2
Summary of phytochemicals with LibDock values and Binding energies.
Plants | Phytochemicals | LibDock | BE in kcal/mol |
Aegle marmelos | Aegelinoside B | 142.50 | -8.54 |
Epoxyaurapten | 129.06 | -7.75 |
Marmin | 122.66 | -7.84 |
Biophytum sensitivum | Epicatechin | 124.33 | -7.69 |
Aerva lanata | Ervoside | 129.69 | -7.91 |
Feruloyltyramine | 123.22 | -8.01 |
3.1 Docking analysis of the phytochemical constituents from Aegle Marmelos
The obtained binding energy for the complex structures from this category (phytochemicals of Aegle Marmelos docked with Mpro) ranges from − 8.55 kcal/mol to -7.14 kcal/mol. For these the LibDock score is between 142.00 to 63.00. Tigogenin (-8.55 kcal/mol), Aegelinoside (-8.54 kcal/mol) and Dehydromarmeli (-8.53 kcal/mol), show best binding energies obtained from DOCKTHOR. O-Prenylhalfordinol, Imperatorin, Skimmianine, N-[2-Ethoxy-2-(4-methoxyphenyl)ethyl]cinnamide, Xanthotoxin, Aeglemarmaelosine, Anhydromarmeline and Aegeline exhibit binding energy values between − 8.41 kcal/mol to -8.06 kcal/mol. Umadevi et. al.[43] reported a binding energy value of -7.2 kcal/mol for Imperatorin-Mpro complex (also known as Marmelosin) obtained from autodock which is lower than that obtained from the current study (-8.39 kcal/mol). Aegelinoside B, an alpha glucosidase inhibitor, is the phytochemical that can be extracted from the leaves of Aegle marmelos. The 3D interaction diagram shown in Fig. 2A indicates the hydrogen bond donors and hydrogen bond acceptors around Aegelinoside B. We have observed that Aegelinoside B interacts with GLU166, GIN192, THR190, ARG 188 and GLN189 through conventional hydrogen bonds. The hydrogen of GLN 192 interacts with two O3 and O5 of the ligand, while oxygen of THR 190 residue interacts with H35 and H34 of ligands. Altogether seven hydrogen bonds can be seen in the 2D figure (Fig. 2B). Numerous carbon-hydrogen bonds and two π- sulphur interactions, one π-π stacked and one π alkyl interactions can be observed. The hydrogen bond interactions of native ligands (Inhibitor N3) in the crystal structure showed GLY143, GLU166, HIS164, PHE140, GIN 189 and THR 190. However, the docking studies on the native ligand with Mpro reveals that hydrogen bond interactions involving the residues are HIS41, HIS163, HIS172, GIN189, THR190 and GLU166. The common hydrogen bond interacting residues are GLU166, THR190 and GIN189. Such interactions are also observed on the Aegelinoside B with Mpro in our docking results. The other important constituents of Aegle marmelos that exhibit good LibDock scores are shown in the Table 2. The second best scored (129.06 kcal/mol) complex, Epoxyaurapten is shown in the Fig. 3. It forms four hydrogen bonds with ASN142 (here, two hydrogen bonds), THR190 and GLU166. π- anion interactions were found between GLU166 and the aromatic part of the ligand and other π- alkyl interactions can be seen with CYS145 and HIS41. The third best, Marmin, exhibits 4 hydrogen bonds (Fig. 4). The hydrogen bonded interacting residues in this case are, TYR54, GLU166, GLY143 and SER144. GLY143 is the common active residue seen in Epoxyaurapten and Aegelinoside B whereas GLU166 active interacting residue can be seen in all three complexes and the native ligand. For further understanding, molecular dynamics (MD) studies were performed for Aegelinoside B, Epoxyaurapten and Marmin, and the results are highlighted in section 3.6.
3.2 Docking of the phytochemicalconstituents from Aerva lanata
Aerva lanata contains many alkaloids and flavonoids and hence is quite popular for various biological activities. It is used for antiurolithiatic, astringent, diuretic, antimicrobial, anti-inflammatory and hepatoprotective drugs. One of the major chemical constituents of Aerva lanta is Ervoside, which is a biologically active canthin-6-one alkaloid [44]. Our study reveals that, out of 17 phytochemicals obtained from Aerva lanata, Ervoside docks better with Mpro target, with a LibDock score of 129.69 kcal/mol (Fig. 5). The TYR54, HIS172, CYS145, SER144 and MET165 residues interact with Ervoside through hydrogen bonds. A sulphur π- interaction can be seen for MET49 residue. The second best Libdock scored (123.22) phytochemical is Feruloyltyramine. This compound contains three hydrogen bonds with the protein (Fig. 6). The active residues of Mpro are ASP776 and THR556. It is interesting to note that the binding site is slightly changed and due to this reason common active residues have not involved in the interaction. Methergine, Ervolanine, Kaempferol, Quercetin and 4-Methoxykaempferol exhibit LibDock score in the range of 110 to 130 kcal/mol. Interestingly, Quercetin has been studied for protective effects from COVID19 induced acute kidney injury [45]. In our study, Quercetin shows five Hydrogen bonds with GLY143, SER144 ARG and MET165 residues while GLY143 and SER144 residues make three hydrogen bonds with Kaempferol. Even though higher number of hydrogen bonds seen in Qucercetin, it appears that the LibDock score is close to the score of Feruloyltyramin. In this category, we have selected best two phytochemicals based on the LibDock score. Hiremath et. al. reported in-silico docking analysis of Kaempferol and Quercetin obtained from the leaves of phyllanthus amarus. The binding affinity of these compounds with Mpro in their study was − 7.70 kcal/mol and − 7.50 kcal/mol, respectively [46]. DOCKTHOR provides closely similar binding energies of -8.10 kcal/mol for Kaempferol and − 7.97 kcal/mol for Quercetin. However, Kaempferol with spike protein indicates a significant improvement in binding affinity (-9.6 kcal/mol) [46]. Proceeding with the docking studies, MD studies were also performed by us for Ervoside and Feruloyltyramine, and the results are highlighted in section 3.6.
3.3 Docking of the phytochemical constituents from Biophytum sensitivum
Maximum number of phytochemicals is seen for Biophytum Sensitivum harbs, however most of them exhibit binding energy values less than − 7 kcal/mol. Attractive among them is Epicatechin which exhibits a LibDock score of 124.33. Reports indicate that Epicatechin mediates reverse transcriptase inhibiting activity for HIV [47, 48]. The green tea also contains Epicatechin and its derivatives such as, epigallocatechin gallate epicatechin gallate and gallocatechin-3-gallate. These compounds and their interactions with Mpro were studied previously by Ghosh et. al. [49]. The binding energy of Epicatechin was found to be -7.20 kcal/mol which is similar to our estimation (-7.69 kcal/mol). The hydrogen bond interactions occurred between HIS164, HIS163, SER144, PHE140 and ASN142 residues with the ligand. Numerous van der Waals and two π alkyl interactions can also be seen in Fig. 7. The other phytochemical is Stigmast-4-en-3-one, which exhibits a LibDock score of 118.74. It has only two hydrogen bonds between SER144 and HIS163 residues with oxygen ligands. Gamma Sitosterol and 4-hydroxy-3,5-dimethoxy hydrazid Benzoic acid have shown similar LibDock scores of 111.12 and 111.15, respectively. However, Dicyclohexyl phthalate, Gamma Sitosterol and Stigmast-4-en-3-one show a binding energy value greater than − 8 kcal/mol. The exact values observed for Dicyclohexyl phthalate, Gamma Sitosterol and Stigmast-4-en-3-one, respectively, -8.84 kcal/mol, -8.47 kcal/mol and − 8.31 kcal/mol. The Mpro – Epicatechin complex was further explored by MD study and the results are provided in section 3.6.
3.4 Docking of the phytochemical constituents from Coleus Amboinicus
Coleus Amboinicus is also known as Indian borage and is used for respiratory diseases like sore throat, cold, bronchitis, asthma, etc [50]. Recent studies indicate its antiproliferative effect against cancer cell lines [51]. Maste and Saxena studied the possibility of multi target response of the chemical constituents of Coleus Amboinicus [52]. They considered only four ligands and all these exhibit less than − 8.00 kcal/mol which agrees well with our observations. Here, we considered a maximum of 24 chemical constituents. None of the phytochemicals of the Coleus Amboinicus plant except N-benzoyl-L-phenylalaninol show > 100 LibDock score and > -8 kcal BE. Among all the phytochemical constituents from this set, only N-benzoyl-L-phenylalaninol shows a highest binding energy of -8.22 kcal/mol. However, as compared to chemical constituents of the other three herbs, our observation is that the docking values are not high enough. Only N-benzoyl-L-phenylalaninol exhibits over 100 LibDock score. Conventional hydrogen bonding occurs between residue GLN189 and two OH and NH bonds from the ligands and residue GLU 166 with O of N-benzoyl-L-phenylalaninol. None of the constituents are attractive candidates for the further studies since most of them show less than LibDock score of 100 (except two candidates showing LibDock score of 111 and 104). Hence, no compound in this section was subjected to MD study.
3.5 Druglikeness and ADMET screening
Assessment of Lipinski rule and pharmacokinetics are important for optimizing drugs [30]. Owing to toxicity, many drugs fail at clinical trial stage, hence it is necessary to find lead compounds with good pharmacokinetics properties [53]. Lipinski rule, also known as rule of thumb, is an algorithm based model which predicts the drug likeness of small molecules. In this study, 87 constituents did not violate more than one criterion. Stigmast-4-en-3-one and Gamma Sitosterol display highest lipophilicity (8.319 and 8.084, respectively) whereas (13R)-8,13-epoxylabd-14-ene, Aurapten and 3-Methoxyamphetamine are in the border line. Only 3-Hydroxy-4-methoxybenzoic acid violates the Veber rule (PSA 2D of 151.42) whereas the remaining compounds were accepted as oral bioavailability of potential drugs by means of Veber's rule.
Compounds were scrutinized for their pharmacokinetic properties and toxicity using in-silico method as implemented in Discovery studio. After administrations all the phytochemicals show good absorption and moderate absorption except 3-Hydroxy-4-methoxybenzoic acid, Gamma Sitosterol and Stigmast-4-en-3-one. Bioavailability of drugs depends on the solubility level and the majority of the constituents tested in this study exhibits good and optimal solubility in water at 25°C. (13R)-8,13-epoxylabd-14-ene, Tigogenin, Gamma Sitosterol exhibit very low solubility. BBB model describes the blood brain barrier penetration level of the drug to the central nervous system of the brain and spinal cord. Note that, small lipophilic potential drug molecules should enter the nervous system and stay there a long time for the desired action. In this study, majorities of drugs are in very high, high, medium and low level of BBB except those for 4′,7-Dimethoxykaempferol, Gamma Sitosterol, Stigmast-4-en-3-one, 3-Hydroxy-4-methoxybenzoic acid, dl-Phenylephrine, Ervoside and Methoxykaempferol. Aegelinoside B is at the border level and all constituents are non-inhibitors except Aeglemarmaelosine, oct-1-en-2-ol and 3-Butylindolizidine. Eight, twelve, eight and eleven constituents of Coleus Amboinicus, Aerva Lanta, Aegle Marmelos and Biophytum Sensitivum, respectively, are toxic in nature. The binding of a drug with plasma protein was described using PPB Tight binders, as shown by 49 compounds (< 1) remaining indicating the weak binders. Gamma Sitosterol, Anhydromarmeline and Stigmast-4-en-3-one show a value of greater than 7. All the well docked ligands are acceptable as a drug in terms of pharmacokinetics. All chemical constituents provided in the SI exhibits good druglikeness and pharmacokinetics properties.
3.6 Molecular dynamics simulation
In order to evaluate the stability of ligand bound protein, conformations, flexibility and compactness, we also performed MD simulations. For this, we selected the top six ligand docked protein complexes with Mpro and also evaluated the ligand induced changes on the protein structure. Figure 8A represents the root-mean-square deviation (RMSD) profile of docked complexes, which indicates the stability of the protein ligand complex. RMSD values were gradually increased till 7 ns time scale except Epicatechin, which stabilized quickly after about 4 ns for the first run. Proteins with Aegelinosides B, Ervoside, Feruloyltyramine, Epicatechin and Marmin were quite rigid with RMSD < 0.45 nm after 17 ns. On the other hand, Mpro-Epoxyaurapten complex show rigidity with RMSD < 0.55 nm. Initially all complexes with ligands were flexible in nature and then they reached a stable state. Epicatechin bound complex show some marginal fluctuation compared to others in the first run. However, it attains stability quickly in second and third run simulation (SI). Three replicas of MD simulation have been performed for the all six docked protein complexes for 100 ns. All complexes show a similar trend in RMSD for at least two run simulations whereas, Epoxyaurapten and Marmin exhibit similar RMSD plot for three replica calculations. The complexes achieved stability after 25 ns (SI). We observed that this system is highly stable as compared to the Mpro-Lopinavir complex [54]. The average RMSD values for Mpro-Aegelinoside B, Mpro-Ervoside, Mpro-Epoxyaurapten, Mpro-Epicatechin, Mpro-Feruloyltyramine, Mpro-Marmin and Mpro were found to be 0.32 nm, 0.31 nm, 0.339 nm, 0.390 nm, 0.327 nm, 0.310 nm and 0.320 nm, respectively. In general, it can be said that all complexes attain good stability and similar behavior till the end of the simulations. RMSD of ligands as a function of simulation time is plotted (Fig. 8B) and it indicates that all ligands are located well within the active site of protein. All ligands rapidly reached the dynamic equilibrium after 20 ns. Feruloyltyramine shows a small increment in RMSD (0.3 nm) after 7 ns and then reaches an earlier equilibrium state after 13 ns. In the case of Aegelinoside B, a dynamic equilibrium persists up to 15 ns and thereafter shows many fluctuations in RMSD. It can be suggested that Marmin adopted a small change in the conformation, but later on it reverts to stable state [55]. Similar case is also possible for the conformational change of Epoxyaurapten. Remaining ligands show minimal fluctuations and they maintained RMSD value within 1 nm till the end of MD simulation. Epicatechin and Feruloyltyramine show lowest fluctuations as compared to other ligands, indicating the highest stability of the simulation system [56].
The root-mean-square fluctuation (RMSF) of Cα carbon atoms for Mpro and Mpro-Ligands was estimated for analyzing the flexibility of residues of protein (Fig. 9). A significant fluctuation has been seen in the region of residue THR169 of Mpro-Epoxyaurapten and Mpro-Epicatechin. The RMSF value of these complexes is 0.42 nm and the corresponding residue is one of the pocket atoms having no interaction with ligands. Mpro-Epicatechin also shows a strong RMSF fluctuation at MET6 and LYS12 in comparison to other complexes. Another strong fluctuation (RMSF = 0.40 nm) was observed for Mpro-Aegelinoside B and Mpro-Marmin of residue ASN142. The residue of ALA193 fluctuations in Mpro was not observed for Mpro-Aegelinoside B, Mpro-Epicatechin, Mpro-Epoxyaurapten, Mpro-Marmin and Mpro-Ervoside. The average value of RMSF for Mpro and all complexes are in the range of 0.15 nm to 0.20 nm. Residues of complex of protein with Epoxyaurapten, Epicatechin, Feruloyltyramine and Marmin did not show much flexibility in comparison to Mpro for at least two run [SI]. Hence, these complexes could be regarded as stable systems.
In addition, we evaluated the intermolecular hydrogen bonds formed between protein and ligands during the simulation. In fact, H-bond is a main factor in determining selectivity, stabilization and binding affinity. The Mpro-Aegelinoside B contains a maximum of five hydrogen bonds, out of which two hydrogen bonds were consistently seen during the simulation. Moreover, within 0.35 nm we identified a maximum of 8 hydrogen bonds. Mpro-Epicatechin, Mpro-Epoxyaurapten, Mpro-Ervoside, Mpro-Feruloyltyramine and Mpro-Marmin contains an average of 1, 1, 2, 2, and 1 hydrogen bonds, respectively, for MD runs (SI). The constant range of intermolecular hydrogen bonding of Mpro-Epoxyaurapten persists throughout the simulation indicating highest stability compared to other ligands and no change in the conformation [57]. Solvent accessible surface area (SASA) and radius of gyration (Rg) are used to analyze solvent accessibility of all complexes and structural compactness of protein. We observed that the average value of Rg for Mpro and Mpro-Marmin has almost similar Rg value (2.13 nm) and others show values in the range of 2.10 to 2.16 nm for three MD run. Mpro- Feruloyltyramine (Rg = 2.15 ns) shows highest Rg and this suggests slightly less compactness in comparison to other complexes and free Mpro (Fig. 10A). Khan et. al. reported that Remdsivir, Saquinavir and Darunavir provide average Rg score values (2.2 ± 0.1 nm) which are at the upper range of obtained score of our drugs (2.10 nm – 2.16 nm) [58]. Mpro-Epoxyaurapten shows highest compactness and stable folding due to being lowest in the Rg value, which was maintained till the end of the simulation for three MD runs [59]. Moreover, Rg of all complexes decrease over the simulation time (SI), meaning that binding of ligands helps stabilization of the whole complex [60]. The effect of solvent molecules on the residues of Mpro when it is bound with ligand is confirmed from SASA versus MD simulation time. All Mpro-Ligands complexes and Mpro show SASA values in the range of 115 nm2 to 145 nm2 (Fig. 10B). For Mpro-Marmin, a decrease in SASA value in comparison to other complexes over the simulation time is observed, indicating the shrinkage of surface area upon binding with Marmin. All other Mpro-Ligand complexes and Mpro show a comparable behavior till the end of MD simulation. Mpro-feruloyltyramine, Mpro-Aegelinoside B, Mpro-Ervoside exhibit an average SASA value of 130 nm2 whereas, Mpro-Marmin exhibit a lowest average SASA value of 125 nm2. Trajectory of Mpro-Epoxyaurapten complex suggests the highest average SASA (157 nm2) which indicates the high solvation effect and high molecular size of Epoxyaurapten. Total volume of all systems and Mpro lies in between 56 nm3 to 59 nm3 and the density lies between 951 g/l to 1200 g/l (SI). Similar volume and density of all systems were seen for other two MD runs. Mpro-Marmin, Mpro-Epicatechin and Mpro-feruloyltyramine show a slight increment of 200 g/l as compared to other Mpro-Ligands. In the case of Mpro also we observed an increment of 1050 g/l towards the end of the MD.
We have evaluated the interacting residues of protein with six ligands after MD simulations. Table SI14 indicates the interacting residues of protein with ligands after the MD simulation. Aegelinoside B shows two hydrogen bonds with two pi-alkyl interactions, one anion-pi and one cation-pi interactions. The crucial residues of proteins which involve hydrogen bonds with ligands are, SER46 and GLY143. However, the docked systems show five hydrogen bonds and the pocket atoms of proteins are changed after MD. In Epoxyaurapten and Marmin no hydrogen bonds could be observed. Interestingly, Ervoside shows seven hydrogen bonds and other non-bonds are pi-sulphur, pi-pi stacked and pi-alkyl interactions. Hydrogen bonds are found in ASN119, ASN142, SER144, SER46, THR24, THR26 and CYC44 with Ervoside ligands (All show H-bond distances less than 3 Å). Epicatechin and Feruloyltyramin exhibit, three and two hydrogen bonds, respectively. Epicatechin exhibits Hydrogen bond interaction with GLU166, GLN192 and GLN189 residues of protein, whereas Feruloyltyramin show hydrogen bond interaction at GLY143 and HIS165 residues of protein. Upon comparing structures of docked systems and systems after MD simulations (Figure SI13 to SI17) it is vividly observable that a slight rearrangement in terms of confirmation of ligands and pockets of proteins occurs in order to interact each of them effectively during the movement.