Turk J Pharm Sci 2022;19(4):431-441

DOI: 10.4274/tjps.galenos.2021.83548 ORIGINAL ARTICLE

Molecular Docking Study of Several Seconder

Metabolites from Medicinal Plants as Potential
Inhibitors of COVID-19 Main Protease
Sinan BİLGİNER1*, Sefa GÖZCÜ2, Zuhal GÜVENALP3

1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Erzurum, Türkiye

2Erzincan Binali Yıldırım University, Faculty of Pharmacy, Department of Pharmacognosy, Erzincan, Türkiye
3Atatürk University, Faculty of Pharmacy, Department of Pharmacognosy, Erzurum, Türkiye

ABSTRACT

Objectives: Coronaviruses (CoVs) cause infections that affect the respiratory tract, liver, central nervous, and the digestive systems in humans and
animals. This study focused on the main protease (Mpro) in CoVs (PDB ID: 6LU7) that is used as a potential drug target to combat 2019-CoV. In this
study, a total of 35 secondary metabolites from medical plants was selected and docked into the active site of 6LU7 by molecular docking studies
to find a potential inhibitory compound that may be used to inhibit Coronavirus Disease-2019 (COVID-19) infection pathway.
Materials and Methods: The chemical structures of the ligands were obtained from the Drug Bank (https://www.drugbank.ca/). AutoDockTools
(ADT ver. 1.5.6) was used for molecular docking studies. The docking results were evaluated using BIOVIA Discovery Studio Visualizer and PyMOL
(ver. 2.3.3, Schrodinger, LLC).
Results: Pycnamine, tetrahydrocannabinol, oleuropein, quercetin, primulic acid, kaempferol, dicannabidiol, lobelin, colchicine, piperidine,
medicagenic acid, and narcotine is found to be potential inhibitors of the COVID-19 Mpro. Among these compounds, pycnamine, which was evaluated
against COVID-19 for the first time, showed a high affinity to the COVID-19 Mpro compared with other seconder metabolites and reference drugs.
Conclusion: Our results obtained from docking studies suggest that pycnamine should be examined in vitro to combat 2019-CoV. Moreover,
pycnamine might be a promising lead compound for anti-CoV drugs.
Key words: COVID-19, molecular docking, pycnamine, seconder metabolites

INTRODUCTION Currently, there are several vaccines for COVID-19, but no

Coronaviruses (CoVs) cause disorders in both the respiratory antiviral drugs are available for specific treatment of COVID-19.
tract and the digestive system in humans and animals.1 During However, some antiviral drugs such as lopinavir, ritonavir,
an epidemic in Wuhan, China at the end of 2019, the new CoV remdesivir, and nelfinavir have been using to prevent further
strain was identified and named 2019-nCoV. In a very short complications and organ damage caused by COVID-19.4 Among
time, this newly emerging virus spread to almost all countries all these drugs, nelfinavir, which has been used in clinics, was
and the disease is officially named as Coronavirus Disease-2019 found as the most potential inhibitor drug against COVID-19
(COVID-19) by World Health Organization (WHO).2 According main protease (Mpro) based on its docking score according to
to WHO’s COVID-19 Weekly Epidemiological Update Report the docking studies conducted by Xu et al.5 In docking studies,
released on May 11, 2021, the number of confirmed cases Mpro is used as a potential drug target to combat 2019-CoV.6-8
reached 157,362,408 including 3,277,834 deaths in the world Secondary metabolites obtained from medicinal plants and
as of May 9, 2021.3 their semi-synthetic derivatives have been widely used in new

*Correspondence: [email protected], Phone: +90 536 357 40 46, ORCID-ID: orcid.org/0000-0001-5676-2045

Received: 06.08.2021, Accepted: 24.09.2021
©Turk J Pharm Sci, Published by Galenos Publishing House.

431
432 BİLGİNER et al. COVID-19: Molecular Docking Study

drug development. Therefore, the use of secondary metabolites animals.27 This study focused on the Mpro in CoVs (PDB ID:
purified from medicinal plants in drug development against 6LU7) that is used as a potential drug target to combat 2019-
severe acute respiratory syndrome (SARS)-CoV becomes CoV. 6LU7 has been structured in PDB and has been publicly
important.9 There are many studies reporting the antiviral available since early February, 2020. To date, this Mpro (6LU7)
effects of many compounds with alkaloid,10-12 flavonoid,13,14 has been studied by different groups to find potential inhibitors
monoterpene,15-19 sesquiterpene lactone,20,21 saponoside,22,23 and that can stop this enzyme activity and, thus, the replication of
aryl alkene24,25 structures. CoVs.8,27,28
In this study, the potential innhibitory effects of alkaloids Nelfinavir, lopinavir, indinavir, and ritonaviprotease inhibitory
(atropine, caffeine, castanospermine, codeine, ephedroxane, drugs, of which ritonavir and lopinavir is proposed for treating
hygrine, cuscohygrine, colchicine, lobeline, tussilagine, SARS and MERS.29 In an in vitro study by Yamamoto et al.29,
punicalagin, papaverine, pycnamine, piperidine, scopolamine, nelfinavir was reported to strongly inhibit the replication of
morphine, narcotine, pelletierine, ricinine), cannabinoids SARS-CoV in Vero E6 cells.30 However, in an in silico study by
(cannabidiol and tetrahydrocannabinol), monoterpenes (citral Xu et al.5, nelfinavir was identified as the most potent inhibitor
A, thymol, oleuropein, and harpagoside), sesquiterpene lactone, against COVID-19 with a binding free energy score. In our
e.g. artemisinin, saponins (primulic acid and medicagenic acid), study, nelfinavir, lopinavir, indinavir, and ritonavir were used
aryl alkene (aromatic ketone), e.g. gingerol, and flavonoids as standard drugs for comparison.
(quercetin and kaempferol) were investigated on 2019-CoV Pro In this study, 35 secondary metabolites from medical plants
via molecular docking studies. We hope that the findings of this were selected and docked into the active site of 6LU7. Docking
study will contribute to drug research to combat COVID-19 and studies were performed by AutoDockTools (ADT ver. 1.5.6).
direct the researchers working in this field to further designs. Table 1 shows the binding free energy scores of all selected
molecules. The native ligand for 6LU7 is n-[(5-methylisoxazol-
MATERIALS AND METHODS 3-yl) carbonyl]alanyl-l-valyl-n~1~-(1r,2z)-4-(benzyloxy)-4-oxo-
1-[[(3r)-2-oxopyrrolidin-3-yl] methyl]but-2-enyl)-l-leucinamide.
Experimental in silico part According to the results presented in Table 1, the binding
The 2019-CoV Mpro (PDB ID: 6LU7) structure was obtained free energy scores of the compounds were between -11.30
from The Protein Data Bank (PDB, https://www.rcsb.org/). The kcal/mol and -4.13 kcal/mol. We investigated pycnamine,
pdb file of the 6LU7 protein was prepared using chain A and tetrahydrocannabinol, oleuropein, quercetin, primulic acid,
transferred to AutoDockTools (ADT ver. 1.5.6). Water molecules kaempferol, cannabidiol, lobeline, colchicine, piperidine,
of the structures were removed and only polar hydrogen and medicagenic acid, and narcotine as potential inhibitors of the
Kollman charges were added to the proteins. Finally, the pdbqt COVID-19 Mpro because to the binding free energy scores of
files of the proteins were saved.26 -11.30, -9.10, -9.06, -8.94, -8.94, -8.70, -8.52, -8.30, -8.28, -7.74,
Chemical structures of the ligands were obtained from the Drug -7.71, and -7.60 kcal/mol, respectively.
Bank (https://www.drugbank.ca/). The ligands unavailable in Analysis of docking results and interactions with six of these
the Drug Bank were drawn in ChemDraw (Professional, version compounds are presented in Tables 2 and 3. Table 2 shows the
19.0.1.28), passed to ChemDraw 3D (professional, version analysis of molecular docking results (binding energy/Gibbs
19.0.1.28) and minimized. Torsion of the ligands was examined Energy, ligand efficiency, inhibition constant, intermolecular
and then the files of the ligands were saved as pdbqt format by energy, and Van der Waals-H Bond desolvation energy) for
AutodockTools (ADT ver. 1.5.6). the compounds with binding energies less than -7.60 kcal/mol,
The active site of the 6LU7 was defined using BIOVIA which is similar to the binding free energy of ritonavir.
Discovery Studio Visualizer (v20.1.0.19295). AutoDockTools Table 3 shows 2D and 3D visualizations of interactions between
(ADT ver. 1.5.6) was used for molecular docking studies. 6LU7 and the compounds presented in Table 2. According to
Lamarckian genetic algorithm with local search was used as a Table 3, which shows interactions between compounds and
search engine, with 10 runs. The active site of the protein was 6LU7, nelfinavir forms H-bonds with the amino acids Gly143,
defined by a grid box of 60 x 60 x 60 points. Ten conformers His163, Thr190, Gln189 of 6LU7. Lopinavir forms H-bonds with
of the ligands were considered to evaluate the docking results. the amino acids His41, Cys145, Gln189, and Glu166. Indinavir
Finally, the conformer with the lowest binding free energy was realizes H-bonds with the amino acid, i.e. Asn142, while
evaluated by BIOVIA Discovery Studio Visualizer and PyMOL ritonavir, the latest standard drug, forms H-bonds with the amino
(ver. 2.3.3, Schrodinger, LLC).26 acids His164 and Glu166. When the interactions of the seconder
metabolites in Table 3 are evaluated, the following results are
Statistical analysis
seen: pycnamine forms H-bond with the amino acid, i.e. Glu166.
No statistical analysis was used in this study. Tetracannabinol forms H-bonds with the amino acids Glu166,
Cys145. Oleuropein realizes H-bonds with the 6LU7 amino
RESULTS acids, e.g. His41, Thr26, Gly143, Glu166, and Thr190. Quercetin
CoVs cause infections that affect the respiratory tract, liver, realizes H-bonds with 6LU7 amino acids, e.g. Glu166, Thr190,
central nervous and the digestive systems in humans and and His164.
 BİLGİNER et al. COVID-19: Molecular Docking Study 433

Table 1. Binding free energy scores of the compounds

Compounds Binding free energy (kcal/mol) Compounds Binding free energy (kcal/mol)
Pycnamine -11.30 Harpagoside -6.82
Tetrahydrocannabinol -9.10 Atropine -6.70
Oleuropein -9.06 Punicalagin -6.63
Quercetin -8.94 Couscohygrin -6.41
Primulic acid -8.94 Gingerol -6.27
Kaempferol -8.70 Ephedroxan -5.96
Cannabidiol -8.52 Tussulagin -5.91
Lobeline -8.30 Castanospermine -5.90
Colchicine -8.28 Pelletierin -5.30
Piperidine -7.74 Citral-A -4.98
Medicagenic acid -7.71 Thymol -4.95
Narcotine -7.60 Caffeine -4.64
Butylscopolamine -7.42 Hygrin -4.55
Hyoscyamine -7.39 Ricinin -4.51
Reticuline -7.29 Ivermektin -4.13
Papaverine -7.16 Native ligand -7.96
Codeine -7.07 Nelfinavir* -10.70
Artemisinin -7.03 Lopinavir* -8.95
Scopolamine -6.97 Indinavir* -8.73
Morphine -6.88 Ritonavir* -7.81
*Nelfinavir, lopinavir, indinavir, and ritonavir are HIV protease inhibitor drugs

Table 2. Molecular docking results analysis of compounds with low binding energy scores and the drugs used in clinic
Binding energy Van der Waals-H Bond
Compounds Ligand efficiency Inhibition constant Intermolecular energy
(kcal/mol) desolvation energy
Pycnamine -11.30 -0.25 5.21 nM -12.20 -11.85
Tetrahydrocannabinol -9.10 -0.40 214.2 nM -10.59 -10.58
Oleuropein -9.06 -0.25 229.87 nM -13.23 -12.96
Quercetin -8.94 -0.41 277.84 nM -9.24 -9.13
Primulic acid -8.94 -0.12 279.96 nM -14.01 -13.70
Kaempferol -8.70 -0.41 422.9 nM -8.99 -8.88
Cannabidiol -8.52 -0.37 570.21 nM -10.01 -9.97
Lobeline -8.30 -0.33 821.52 nM -10.09 -9.98
Colchicine -8.28 -0.29 -856.99 nM -9.77 -9.66
Piperidine -7.74 -0.37 2.11 µM -8.64 -8.58
Medicagenic acid -7.71 -0.21 2.21 µM -9.50 -9.66
Narcotine -7.60 -0.25 2.69 µM -8.79 -8.42
Nelfinavir -10.70 -0.27 14.45 nM -12.78 -12.72
Lopinavir -8.95 -0.19 275.32 nM -13.72 -13.55
Indinavir -8.73 -0.19 400.34 nM -12.90 -12.33
Ritonavir -7.81 -0.16 1.9 µM -13.47 -13.39
434 BİLGİNER et al. COVID-19: Molecular Docking Study

Table 3. Two-dimensional and three-dimesnsional interaction diagrams for several compounds

2D representation of 3D representation of
No Compound
compounds-6LU7 interactions compounds-6LU7 interactions

1 Pycnamine

2 Tetrahydrocannabinol

3 Oleuropein

4 Quercetin
 BİLGİNER et al. COVID-19: Molecular Docking Study 435

5 Primulic acid

6 Kaempferol

7 Cannabidiol

8 Lobeline
436 BİLGİNER et al. COVID-19: Molecular Docking Study

9 Colchicine

10 Piperidine

11 Medicagenic acid

12 Narcotine
 BİLGİNER et al. COVID-19: Molecular Docking Study 437

13 Nelfinavir

14 Lopinavir

15 Indinavir

16 Ritonavir
438 BİLGİNER et al. COVID-19: Molecular Docking Study

Primulic acid forms H-bonds with the amino acids, e.g. Ser46, 1, HSV-2), and respiratory syncytial virus, influenza virus
Ser144, Glu166, Gln189, Asn142, Cys145, and Leu141. Kaempferol strain, parainfluenza virus type-3, sindbis virus, rhinovirus,
forms H-bonds with the 6LU7 amino acids, e.g. Tyr54, Glu166, echovirus (types-7, -11, -12, and -19), coxsackievirus (A21 and
and Gln192. Dicannabidiol realizes H-bond with the 6LU7 amino B1), poliovirus (type-1 Sabin) and grouper iridovirus in cell
acid, i.e. Glu166. Lobelin realizes H-bonds with the amino acids, cultures.49-53 Early in vivo studies showed that oral treatment with
e.g. Gly143 and Glu166. Colchicine realizes H-bonds with the quercetin-protected mice from lethal Mengo virus.54 In mice
amino acids, e.g. Gly143, Thr190, Gln189, and Gln192. Piperidine infected with rhinovirus, quercetin treatment decreased viral
forms H-bonds with the amino acids, e.g. Gly143 and Asn142. replication and attenuates virus- induced airway cholinergic
Medicagenic acid forms H- bonds with the amino acids, e.g. hyperresponsiveness.55
Thr26, Cys145, Glu166, and His164. Narcotine realizes H- bonds
Kaempferol is another flavonoid derivative found in most edible
with the 6LU7 amino acids, e.g. Cys145 and Glu169. The results
plants such as tea, fruits, and vegetables consisting of Allium
presented in Table 3 suggest that the Mpro Glu166 plays a crucial
cepa L., Camellia sinensis (L.) Kuntze, Citrus paradisi Macfad.,
role in drug interactions. Besides, the other amino acids, e.g.
Fragaria vesca L., Lactuca sativa L., and in medicinal plants
Asn142, Gln189, Cys145, and Thr26 are also predicted to play
such as Tilia tomentosa Moench., Aloe vera L., Crocus sativus
roles in drug interactions, as reported in previous studies.8,27
L., Vitis vinifera L., Ginkgo biloba L., Hypericum perforatum L.,
According to the results in Tables 1 and 2, the most impressive Phyllanthus acidus L., Ribes nigrum L., Rosmarinus officinalis
compound of our study is pycnamine with a score of -11.30 kcal/ L., Hippophae rhamnoides L., and Sambucus nigra L.56 Antiviral
mol, which is higher than that of nelfinavir. When the results
activity of kaempferol on the influenza viruses (H1N1 and
in Table 2 are evaluated, it is seen that pycnamine has a
H9N2), HIV-1, flavivirus, two RNA viruses (murine norovirus
predicted inhibition constant value (5.21 nM) approximately
and feline calicivirus), and human cytomegalovirus were
3 times lower than that of nelfinavir (14.45 nM). According to
mentioned.14,48,51,57,58
pycnamine-6LU7 complex presented in Table 3, hydroxy moiety
of pycnamine forms a hydrogen bond with the side chain of Primulic acid is a saponin found in some species of Primulaceae
Glu166. Additionally, pycnamine forms π-cation, π-sulfur, [Primula officinalis L., P. elatior (L.) Hill, P. veris L.] and Poaceae
π-sigma, and several hydrophobic interactions with the active (Panicum repens L.),59-63 and was reported to have antiviral
site of 6LU7, as shown in Table 3. activity by Helal and Melzig.58
Finally, cannabidiol, the potential inhibitor of COVID-19 Mpro,
DISCUSSION purified from the C. sativa L.,64,65 and was reported to show high
Pycnamine is an alkaloid found in some species of efficacy against viral hepatitis in previous studies.64
Menispermaceae (Triclisia patens Oliv., T. dictyophylla
Diels, Pycnarrhena manillensis Vidal, P. ozantha Diels) and CONCLUSION
Ranunculaceae families (Thalictrum cultratum Wall., Isopyrum At currently, there is no antiviral drug for specific treatment of
thalictroides L.).31-36 Pycnamine was reported to be a potential COVID-19, which is still a threat to global health. Mpro was used
antimalarial, antiplasmodial, antiamoebic, and antimicrobial in as a potential drug target to combat 2019-CoV. In this study,
previous studies.36-40 It was evaluated against COVID-19 for the we evaluated several secondary metabolites obtained from
first time in this study. medicinal plants against COVID-19 Mpro by molecular docking
Tetrahydrocannabinol, which has the second lowest binding studies to identify a potential inhibitory compound that may be
free energy score (-9.10 kcal/mol) in this study, purified from used to inhibit COVID-19 infection pathway. According to the
Cannabis sativa L. was reported to inhibit macrophage extrinsic results, pycnamine, tetrahydrocannabinol, oleuropein, quercetin,
antiherpesvirus activity.41 primulic acid, kaempferol, cannabidiol, lobeline, colchicine,
Oleuropein, a secoiridoid monoterpene and the main component piperidine, medicagenic acid, and narcotine are found to be
of Olea europaea L., is a potential inhibitor of the COVID-19 Mpro potential inhibitors of COVID-19 Mpro. Among these compounds,
due to it is binding free energy score of -9.06 kcal/mol. It has pycnamine, which was evaluated against COVID-19 for the first
antiviral activity against mononucleosis herpes, hepatitis, time, showed high affinity to COVID-19 Mpro compared with other
rota, bovine, parvo, HIV, leukemia, respiratory syncytial, seconder metabolites and reference drugs. According to the
parainfluenza-3, and salmonid rhabdoviruses.42-46 In hepatitis B results in this study, pycnamine has a binding free energy score
virus infected ducks, oleuropein reduced the virus entering the of -11.30 kcal/mol, which is higher than nelfinavir used in clinics
bloodstream.47 as the most potent inhibitor drug against COVID-19 Mpro. As a
Quercetin, a flavonoid, is found abundantly in fruits and conclusion, this study has clearly shown that pycnamine may
vegetables including onions, broccoli, buckwheat, peppers, strongly inhibit COVID-19 Mpro. Our results obtained from the
Brassica species, apples, grapes, berries, tea, and wine as well docking studies suggest that pycnamine should be examined
as many nuts, seeds, barks, flowers, leaves, and spices.48 in vitro to combat 2019-CoV. Moreover, pycnamine might be a
promising lead compound for anti-CoV drugs.
Quercetin also demonstrated a dose-dependent antiviral
activity against poliovirus type 1, Herpes simplex virus (HSV-
 BİLGİNER et al. COVID-19: Molecular Docking Study 439

Ethics virus and human severe acute respiratory syndrome coronavirus.

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