Potential Inhibitor of COVID-19 Main Protease (M pro ) From Several Medicinal Plant Compounds by Molecular Docking Study

TLDR: Assessing bioactive compounds found in medicinal plants as potential COVID-19 Mpro inhibitors, using a molecular docking study, found nelfinavir and lopinavir may represent potential treatment options, and kaempferol, quercetin, luteolin-7-glucoside, demethoxycurcumin, naringenin, apigenin- 7-gl Sucoside appeared to have the best potential to act as COV inhibitors.

Abstract: COVID-19, a new strain of coronavirus (CoV), was identified in Wuhan, China, in 2019. No specific therapies are available and investigations regarding COVID-19 treatment are lacking. Liu et al. (2020) successfully crystallised the COVID-19 main protease (Mpro), which is a potential drug target. The present study aimed to assess bioactive compounds found in medicinal plants as potential COVID-19 Mpro inhibitors, using a molecular docking study. Molecular docking was performed using Autodock 4.2, with the Lamarckian Genetic Algorithm, to analyse the probability of docking. COVID-19 Mpro was docked with several compounds, and docking was analysed by Autodock 4.2, Pymol version 1.7.4.5 Edu, and Biovia Discovery Studio 4.5. Nelfinavir and lopinavir were used as standards for comparison. The binding energies obtained from the docking of 6LU7 with native ligand, nelfinavir, lopinavir, kaempferol, quercetin, luteolin-7-glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, epicatechin-gallate, zingerol, gingerol, and allicin were -8.37, -10.72, -9.41, -8.58, -8.47, -8.17, -7.99, -7.89, -7.83, -7.31, -7.05, -7.24, -6.67, -5.40, -5.38, and -4.03 kcal/mol, respectively. Therefore, nelfinavir and lopinavir may represent potential treatment options, and kaempferol, quercetin, luteolin-7-glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, and epicatechin-gallate appeared to have the best potential to act as COVID-19 Mpro inhibitors. However, further research is necessary to investigate their potential medicinal use.

Figures

Table 3. Molecular docking analysis of several compounds against 6LU7

Figure 1. Histogram showing molecular docking results between 6LU7 and several drug candidate compounds (the binding energy value ΔG is shown in minus kcal/mol)

Figure 3. Luteolin-7-glucoside (aglycone) (a) and kaempferol (b) mapped to the pharmacophore model [48]

Table 1. Protein target structures and active site amino acids (Biovia Discovery Studio 4.5, 2019) and the native ligand structure

Table 2. Properties of COVID-19 Mpro potential inhibitor candidates

Table 4. Source of several compounds belong to medicinal plants

Full text

Article
Potential Inhibitor of COVID-19 Main Protease (M
pro
)
from Several Medicinal Plant Compounds by
Molecular Docking Study
Siti Khaerunnisa
1,
*, Hendra Kurniawan
2,3
*, Rizki Awaluddin
4
, Suhartati Suhartati
5
, Soetjipto
Soetjipto
1,
*
1
Departement of Medical Biochemistry, Faculty of Medicine, Airlangga University, Surabaya, East Java,
Indonesia, 60132; st.khaerunnisa@fk.unair.ac.id; soetjipto@fk.unair.ac.id
2
Departement of Medical and Surgical Nursing, Faculty of Health Science, University of Muhammadiyah
Jember, Jember, East Java, Indonesia, 68121; hendrakurniawan@unmuhjember.ac.id
3
PhD Student, Tropical Disease Research Center, Faculty of Medicine. Khon Kaen University, Khon Kaen,
Thailand, 40002; hendrakurniawan@unmuhjember.ac.id
4
Departement of Pharmacy, Faculty of Health Science, University of Darussalam Gontor, Ponorogo, East
Java, Indonesia, 63471; awaluddinrizki@gmail.com
5
Departement of Medical Biochemistry, Faculty of Medicine, University of Wijaya Kusuma Surabaya, East
Java, Indonesia, 60225; tati_biokim@yahoo.co.id
* Correspondence: st.khaerunnisa@fk.unair.ac.id ; Tel.: +6281233118194 (S.K.); soetjipto@fk.unair.ac.id;
Tel.: +6281331340518 (S.S.); hendrakurniawan@unmuhjember.ac.id ; Tel.: +628113572277 (H.K.)
Abstract: COVID-19, a new strain of coronavirus (CoV), was identified in Wuhan, China, in 2019.
No specific therapies are available and investigations regarding COVID-19 treatment are lacking.
Liu et al. (2020) successfully crystallised the COVID-19 main protease (M
pro
), which is a potential
drug target. The present study aimed to assess bioactive compounds found in medicinal plants as
potential COVID-19 M
pro
inhibitors, using a molecular docking study. Molecular docking was
performed using Autodock 4.2, with the Lamarckian Genetic Algorithm, to analyse the probability
of docking. COVID-19 M
pro
was docked with several compounds, and docking was analysed by
Autodock 4.2, Pymol version 1.7.4.5 Edu, and Biovia Discovery Studio 4.5. Nelfinavir and lopinavir
were used as standards for comparison. The binding energies obtained from the docking of 6LU7
with native ligand, nelfinavir, lopinavir, kaempferol, quercetin, luteolin-7-glucoside,
demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin,
epicatechin-gallate, zingerol, gingerol, and allicin were -8.37, -10.72, -9.41, -8.58, -8.47, -8.17, -7.99, -
7.89, -7.83, -7.31, -7.05, -7.24, -6.67, -5.40, -5.38, and -4.03 kcal/mol, respectively. Therefore, nelfinavir
and lopinavir may represent potential treatment options, and kaempferol, quercetin, luteolin-7-
glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin,
and epicatechin-gallate appeared to have the best potential to act as COVID-19 M
pro
inhibitors.
However, further research is necessary to investigate their potential medicinal use.
Keywords: COVID-2019; M
pro
; 6LU7; Medicinal Plant Compounds; Docking
1. Introduction
Coronaviruses (CoVs) are an etiologic agent of severe infections in both humans and animals,
which can cause disorder not only in the respiratory tract but also in the digestive tract and
systemically. Previous studies of CoVs have reported that CoVs can infect certain species of animals,
including mammals, avian species, and reptiles [1].
The new strain of CoV was identified at the end of 2019, initially named 2019-nCoV, and
emerged during an outbreak in Wuhan, China [2]. The Emergency Committee of the World Health
Organization (WHO) declared an outbreak in China on January 30, 2020, which was considered to be
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© 2020 by the author(s). Distributed under a Creative Commons CC BY license.

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a Public Health Emergencies of International Concern (PHEIC) [3]. Officially, WHO named this CoV
COVID-19 (coronavirus disease 2019), on February 11, 2020, based on consultations and
collaborations with the World Organization for Animal Health and the Food and Agriculture
Organization of the United Nations [4].
According to the current situational report from WHO, released on February 11, 2020, 43,103
COVID-19 cases have been confirmed globally, including 2,560 new cases. In China, the number of
confirmed cases reached 42,708, including 2,484 new cases, 7,333 severe cases, and 1,017 deaths.
Outside of China, 395 cases were confirmed in 24 countries, with 1 death [4].
Currently, no specific therapies for COVID-19 are available and investigations regarding the
treatment of COVID-19 are lacking [3]. However, the measures that have been implemented remain
limited to preventive and supportive therapies, designed to prevent further complications and organ
damage [3]. Some preliminary studies have investigated potential combinations that include the
protease inhibitor lopinavir/ritonavir, which is commonly used to treat human immunodeficiency
virus (HIV)/acquired immunodeficiency syndrome patients, for the treatment of COVID-19-infected
patients [5]. Other reported antiviral treatments form human pathogenic CoVs include nucleoside
analogues, neuraminidase inhibitors, remdesivir, umifenovir (arbidol), tenofovir disoproxil (TDF),
and lamivudine (3TC) [5]. A separate investigation performed by Xu et al. (2020) indicated that
among 4 tested drugs (nelfinavir, pitavastatin, perampanel, and praziquantel), nelfinavir was
identified as the best potential inhibitor against COVID-19 M
pro
, based on binding free energy
calculations using the molecular mechanics with generalised Born and surface area solvation
(MM/GBSA) model and solvated interaction energy (SIE) methods [6].
The results from preliminary studies remain unapproved for therapeutic use in clinical settings
for the treatment of COVID-19-infected patients [5, 7]. Liu et al. (2020) have successfully crystallised
the main protease (M
pro
)/chymotrypsin-like protease (3CL
pro
) from COVID-19, which has been
structured and repositioned in the Protein Data Bank (PDB) and is accessible by the public. This
protease represents a potential target for the inhibition of CoV replication [6].
Environmental factors can greatly influence the secretion of secondary metabolites from tropical
plants. Therefore, great attention has been paid to the secondary metabolites secreted by plants in
tropical regions that may be developed as medicines [8, 9]. Several compounds, such as flavonoids,
from medicinal plants, have been reported to have antiviral bioactivities [10–12]. In the present study,
we investigated kaempferol, quercetin, luteolin-7-glucoside, demethoxycurcumin, naringenin,
apigenin-7-glucoside, oleuropein, curcumin, catechin, epicatechin-gallate, zingerol, gingerol, and
allicin as potential inhibitor candidates for COVID-19 M
pro
. The findings of the present study will
provide other researchers with opportunities to identify the right drug to combat COVID-19.
2. Experimental Section
Proteins/Macromolecules
COVID-19 3cl
pro
/M
pro
(PDB ID: 6LU7) [13] and 3cl
pro
/M
pro
(PDB ID: 2GTB) [6] structures were
obtained from PDB (https://www.rcsb.org/), in .pdb format. PDB is an archive for the crystal
structures of biological macromolecules, worldwide [14].
The 6LU7 protein contains two chains, A and B, which form a homodimer. Chain A was used
for macromolecule preparation. The native ligand for 6LU7 is n-[(5-methylisoxazol-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.
Ligand and Drug Scan
The 3-dimensional (3D) structures were obtained from PubChem
(https://pubchem.ncbi.nlm.nih.gov/), in .sdf format. PubChem is a chemical substance and biological
activities repository consisting of three databases, including substance, compound, and bioassay
databases [15]. Several ligands for which the active compound can be found in herbal medicine were
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downloaded from Dr. Duke’s Phytochemical and Ethnobotanical Databases
(https://phytochem.nal.usda.gov/phytochem/search/list). The compounds used in the present study
were nelfinavir (CID_64143), lopinavir (CID_92727), luteolin-7-glucoside (CID_5280637),
demethoxycurcumin (CID_5469424), apigenin-7-glucoside (CID_5280704), oleuropein
(CID_56842347), curcumin (CID_969516), epicatechin-gallate (CID_107905), zingerol (CID_3016110),
gingerol (CID_442793), catechin (CID_9064), and allicin (CID_65036), quercetin (CID_5280343),
kaempferol (CID_5280863) and naringenin (CID_439246).
Drug-like properties were calculated using Lipinski’s rule of five, which proposes that molecules
with poor permeation and oral absorption have molecular weights > 500, C logP > 5, more than 5
hydrogen-bond donors, and more than 10 acceptor groups [16, 17] Adherence with Lipinski’s rule of
five as calculated using SWISSADME prediction (http://www.swissadme.ch/).
Determination of Active Sites
The amino acids in the active site of a protein were determined using the Computed Atlas for
Surface Topography of Proteins (CASTp) (http://sts.bioe.uic.edu/castp/index.html?201l) and Biovia
Discovery Studio 4.5. The determination of the amino acids in the active site was used to analyse the
Grid box and docking evaluation results. Discovery Studio is an offline life sciences software that
provides tools for protein, ligand, and pharmacophore modelling [18].
Molecular Docking
Ligand optimisation was performed by Avogadro version 1.2, with Force Field type MMFF94,
and saved in .mol2 format. Autodock version 4.2 used for protein optimisation, by removing water
and other atoms, and then adding a polar hydrogen group. Autodock 4.2 was supported by Autodock
tools, MGL tools, and Rasmol. Autogrid then determined the native ligand position on the binding
site by arranging the grid coordinates (X, Y, and Z). Ligand tethering of the protein was performed
by regulating the genetic algorithm (GA) parameters, using 10 runs of the GA criteria. The docking
analyses were performed by both Autodock 4.2, Pymol version 1.7.4.5 Edu and Biovia Discovery
Studio 4.5.
3. Results
Table 1 shows the structures and amino acids found in the active site pockets of 6LU7 and 2GTB.
6LU7 is the main protease (M
pro
) found in COVID-19, which been structured and repositioned in PDB
and can be accessed by the public, as of early February 2020.
2GTB is the main protease found in the CoV associated with the severe acute respiratory
syndrome (SARS), which can be accessed in PDB and was suggested to be a potential drug target for
2019-nCov [6]. Xu et al. (2020) mentioned that the main protease in 2019-nCov shares 96% similarity
with that in SARS.
Table 1. Protein target structures and active site amino acids (Biovia Discovery Studio 4.5,
2019) and the native ligand structure
No
PDB
ID
Macromolecule
Native Ligand
Active site
1
6LU7
THR24, THR26, PHE140,
ASN142, GLY143, CYS145,
HIS163, HIS164, GLU166,
HIS172
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2
2GTB
LYS5, ALA7, THR25, HIS41,
MET49, TYR54, VAL125,
TYR126, GLY127, PHE140,
LEU141, ASN142, GLY143,
SER144, CYS145, HIS163,
HIS164, MET165, GLU166,
LEU167, PRO168, HIS172,
ASP187, ARG188, GLN189,
GLN192, ALA198, LYS236,
TYR237, GLN273
Ligands and several drug candidate compounds have been previously selected, based on
adherence to Lipinski’s rule of five. The selected ligands that did not incur more than 2 violations of
Lipinski’s rule could be used in molecular docking experiments with the target protein. The drug
scanning results (Table 2) show that all tested compounds in this study were accepted by Lipinski’s
rule of five.
Table 2. Properties of COVID-19 M
pro
potential inhibitor candidates
No
Compound
Molecular
formula
Molecular structure and
Interaction with 6LU7
Lipinski’s rule of five
Properties
Value
1
Lopinavir
C37H48N4O
5
Molecular weight (<500
Da)
628.8
LogP (<5)
4.37
H-Bond donor (5)
4
H-bond acceptor (<10)
5
Violations
1
2
Nelfinavir
C32H45N3O
4S
Molecular weight (<500
Da)
567.78
LogP (<5)
4.33
H-Bond donor (5)
4
H-bond acceptor (<10)
5
Violations
1
3
Luteolin-7-
glucoside
C21H20O11
Molecular weight (<500
Da)
448.38
LogP (<5)
0.16
H-Bond donor (5)
7
H-bond acceptor (<10)
11
Violations
2
4
Demethoxycur
cumin
C20H18O5
Molecular weight (<500
Da)
338.35
LogP (<5)
3
H-Bond donor (5)
2
H-bond acceptor (<10)
5
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Violations
0
5
Apigenin-7-
glucoside
C21H20O10
Molecular weight (<500
Da)
432.34
LogP (<5)
0.55
H-Bond donor (5)
6
H-bond acceptor (<10)
10
Violations
1
6
Oleuropein
C19H22O8
Molecular weight (<500
Da)
378.37
LogP (<5)
1.57
H-Bond donor (5)
3
H-bond acceptor (<10)
8
Violations
0
7
Epicatechin-
gallate
C22H18O10
Molecular weight (<500
Da)
442.37
LogP (<5)
1.23
H-Bond donor (5)
7
H-bond acceptor (<10)
10
Violations
1
8
Catechin
C15H14O6
Molecular weight (<500
Da)
290.27
LogP (<5)
0.85
H-Bond donor (5)
5
H-bond acceptor (<10)
6
Violations
0
9
Curcumin
C21H20O6
Molecular weight (<500
Da)
368.38
LogP (<5)
3.03
H-Bond donor (5)
2
H-bond acceptor (<10)
6
Violations
0
10
Zingerol
C11H16O3
Molecular weight (<500
Da)
196.24
LogP (<5)
1.86
H-Bond donor (5)
2
H-bond acceptor (<10)
3
Violations
0
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Frequently asked questions

Q1. What are the contributions mentioned in the paper "Potential inhibitor of covid-19 main protease (mpro) from several medicinal plant compounds by molecular docking study" ?

The present study aimed to assess bioactive compounds found in medicinal plants as potential COVID-19 Mpro inhibitors, using a molecular docking study. Therefore, nelfinavir and lopinavir may represent potential treatment options, and kaempferol, quercetin, luteolin-7glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, and epicatechin-gallate appeared to have the best potential to act as COVID-19 Mpro inhibitors. However, further research is necessary to investigate their potential medicinal use. 

Q2. What have the authors stated for future works in "Potential inhibitor of covid-19 main protease (mpro) from several medicinal plant compounds by molecular docking study" ?

Therefore, the authors suggested that nelfinavir and lopinavir may represent potential treatment options, and kaempferol, quercetin, luteolin-7-glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, and epicatechin-gallate were the most recommended compounds found in medicinal plants that may act as potential inhibitors of COVID-19 Mpro. However, further research is necessary to investigate the potential uses of the medicinal plants containing these compounds. 

Q3. What is the binding energy of kaempferol?

Hydroxy groups (-OH), ketone groups (=O) and ether groups (-O-) in luteolin and kaempferol compounds are predicted to play roles amino acid residue interactions at the active site of COVID-19 Mpro [50]. 

Q4. What is the role of proteases in the development of antiviral drugs?

In many viruses, proteases play essential roles in viral replication; therefore, proteases are often used as protein targets during the development of antiviral therapeutics [22]. 

Q5. What are the recommended compounds for COVID-19 inhibitors?

quercetin, luteolin-7-glucoside, apigenin-7-glucoside, naringenin, oleuropein,demethoxycurcumin, curcumin, catechin, and epigallocatechin were the most recommended compounds found in medicinal plants as potential inhibitors of COVID-19 Mpro, which should be explored in future research. 

Q6. what is the molecule that is used in the study?

The present study focused on the main proteases in CoVs (3CLpro/Mpro), especially PDB ID 6LU7, as potential target proteins for COVID-19 treatment. 

Q7. how many violations of lipinski’s rule could be used in molecular docking?

The selected ligands that did not incur more than 2 violations of Lipinski’s rule could be used in molecular docking experiments with the target protein. 

Q8. What is the main protease in the CoV associated with SARS?

2GTB is the main protease found in the CoV associated with the severe acute respiratorysyndrome (SARS), which can be accessed in PDB and was suggested to be a potential drug target for 2019-nCov [6]. 

Q9. What are the recommended compounds in medicinal plants?

the authors suggested that nelfinavir and lopinavir may represent potential treatment options, and kaempferol, quercetin, luteolin-7-glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, and epicatechin-gallate were the most recommended compounds found in medicinal plants that may act as potential inhibitors of COVID-19 Mpro. 

Q10. What are the recommended compounds for COVID-19?

lopinavir, kaempferol, quercetin, luteolin-7-glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, and epicatechin-gallate have the lowest binding energies and inhibition constants. 

Q11. What are the potential inhibitors of COVID-19?

In the present study, the authors investigated kaempferol, quercetin, luteolin-7-glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, epicatechin-gallate, zingerol, gingerol, and allicin as potential inhibitor candidates for COVID-19 Mpro. 

Q12. What are the proteinsequences of the 2019-nCoV Mpro?

Proteases represent potential targets for the inhibition of CoV replication, and the proteinsequences of the SARS-CoV Mpro and the 2019-nCoV Mpro are 96% identical, and the active sites in both proteins remain free from mutations. 

Q13. What is the name of the new strain of cov?

Previous studies of CoVs have reported that CoVs can infect certain species of animals, including mammals, avian species, and reptiles [1]. 

Q14. What are the potential inhibitors of the COVID-19 Mpro?

The authors investigated kaempferol, quercetin, luteolin-7-glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, epicatechingallate, zingerol, gingerol, and allicin as potential inhibitors of the COVID-19 Mpro. 

Q15. How many cases of COVID-19 have been confirmed in China?

According to the current situational report from WHO, released on February 11, 2020, 43,103COVID-19 cases have been confirmed globally, including 2,560 new cases. 

Q16. what is the binding energies of 6LU7 with the native ligand?

The binding energies obtained from docking 6LU7 with the native ligand, nelfinavir, lopinavir,kaempferol, quercetin, luteolin-7-glucoside, demethoxycurcumin, naringenin, apigenine-7-glucoside, oleuropein, curcumin, catechin, epicatechin-gallate, zingerol, gingerol, and allicin were -8.37, -10.72, -9.41, -8.58, -8.47,-8.17, -7.99, -7.89, -7.83, -7.31, -7.05, -7.24, -6.67, -5.40, -5.38, -5.40, and -4.03 kcal/mol, respectively (see Table 3 and Figure 1). 

Q17. What is the affinity of compounds in the present study?

The docking analysis in the present study showed the inhibition potential of several compounds,ranked by affinity (ΔG); nelfinavir > lopinavir > kaempferol > quercetin > luteolin-7-glucoside > demethoxycurcumin > naringenin > apigenine-7-glucoside > oleuropein > curcumin > catechin > epigallocatechin > zingerol > gingerol > allicin.