Heliyon 10 (2024) e25837

Contents lists available at ScienceDirect

Heliyon
journal homepage: www.cell.com/heliyon

Research article

An in silico approach to develop potential therapies against Middle

East Respiratory Syndrome Coronavirus (MERS-CoV)
Suvro Biswas a, 1, Mohasana Akter Mita b, 1, Shamima Afrose b, 1, Md. Robiul Hasan b, 1,
Mst. Sharmin Sultana Shimu b, Shahriar Zaman a, Md. Abu Saleh a, *
a
Microbiology Laboratory, Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi, 6205, Bangladesh
b
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi, 6205, Bangladesh

A R T I C L E I N F O A B S T R A C T

Keywords: A deadly respiratory disease Middle East Respiratory Syndrome (MERS) is caused by a perilous
Phytoconstituents virus known as MERS-CoV, which has a severe impact on human health. Currently, there is no
MERS-CoV approved vaccine, prophylaxis, or antiviral therapeutics for preventing MERS-CoV infection. Due
3CLpro
to its inexorable and integral role in the maturation and replication of the MERS-CoV virus, the
Molecular docking
ADMET
3C-like protease is unavoidly a viable therapeutic target. In this study, 2369 phytoconstituents
Molecular dynamics simulation were enlisted from Japanese medicinal plants, and these compounds were screened against 3C-
like protease to identify feasible inhibitors. The best three compounds were identified as Kiha­
danin B, Robustaflavone, and 3-beta-O- (trans-p-Coumaroyl) maslinic acid, with binding energies
of − 9.8, − 9.4, and − 9.2 kcal/mol, respectively. The top three potential candidates interacted
with several active site residues in the targeted protein, including Cys145, Met168, Glu169,
Ala171, and Gln192. The best three compounds were assessed by in silico technique to determine
their drug-likeness properties, and they exhibited the least harmful features and the greatest drug-
like qualities. Various descriptors, such as solvent-accessible surface area, root-mean-square
fluctuation, root-mean-square deviation, hydrogen bond, and radius of gyration, validated the
stability and firmness of the protein-ligand complexes throughout the 100ns molecular dynamics
simulation. Moreover, the top three compounds exhibited better binding energy along with better
stability and firmness than the inhibitor (Nafamostat), which was further confirmed by the
binding free energy calculation. Therefore, this computational investigation could aid in the
development of efficient therapeutics for life-threatening MERS-CoV infections.

1. Introduction

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an unprecedented virus that caused Middle East respiratory syn­
drome (MERS), a highly deadly respiratory disease reported in June 2012 in the Arabian Peninsula and subsequently expanded
globally [1]. It is the first lineage 2C Betacoronavirus with severe pathogenicity, imposing a dangerous impact on human health with a
35% fatality rate [2]. MERS-CoVs have several natural hosts, including Camelus dromedarius (dromedary camel), Vespertilio superans
and Neoromicia capensis (two species of bats), and Erinaceus europaeus (European hedgehog). It is believed that MERS-CoV has

* Corresponding author.
E-mail address: saleh@ru.ac.bd (Md.A. Saleh).
1
Equal contributing author.

https://doi.org/10.1016/j.heliyon.2024.e25837
Received 25 May 2023; Received in revised form 2 February 2024; Accepted 2 February 2024
Available online 9 February 2024
2405-8440/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
S. Biswas et al. Heliyon 10 (2024) e25837

originated from bats, and as human-to-bat transmission is limited, humans likely acquired this virus from the dromedary camels, acting
as an intermediate host [3,4]. MERS-CoV belongs to the family Coronaviridae, sub-family Orthocoronavirinae, which includes four
genera (Alpha, Beta, Gamma, and Delta) and the order Nidovirales. Six diverse coronaviruses (CoVs) have been identified as
disease-causing agents in humans, with MERS causing strongly damaging and intensely severe respiratory tract infections [2,5].
MERS-CoV genome is a single-stranded positive-sense RNA with a size of about 30 kb. Phylogenetic analysis revealed two clades- A and
B, with clade A having only few strains, while clade B contains most strains [2].
The length of the MERS-CoV genome is over 30,000 nucleotides (nt). The RNA genome of this virus has a 5′ untranslated region
(UTR) with a length of 278 nt and a 3′ untranslated region with a length of 300 nt [6]. The first two-thirds of the 5′ UTR of the
MERS-CoV genome comprise the replicase complex, which includes ORF1a and ORF1b. The remaining one-third of the 3′ UTR encodes
four structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N), as well as accessory proteins (ORF3, ORF4a,
ORF4b, ORF5, and ORF8b), which are not necessary for genome replication but are likely implicated in pathogenesis [7,8]. These
accessory proteins of MERS-CoV do not share homology with any known virus or host [8]. Therefore, the order of the MERS-CoV
genome is 5′ UTR-ORF1ab (Open reading frames)-S-ORF3-ORF4a-ORF4b-E-M-N-3′ UTR-poly(A) tail [7,9]. The spike (S) protein is
located on the surface of beta-coronaviruses, playing a crucial role in transmission between different species by mediating
receptor-virus recognition and activating the viral infection procedure [10]. The 5’ end of the MERS-CoV genome is translated to
generate a large polyprotein, which is subsequently cleaved in cis into 16 functional nonstructural proteins by two viral proteases.
These proteases collaborate in a complex process for viral RNA recombination and RNA synthesis [6].
In MERS-CoV, the S protein, containing 1353 amino acids, is a type I transmembrane glycoprotein comprising two subunits: S1 and
S2. The S1 subunit contains the receptor binding domain (RBD), responsible for attaching to the target cell receptor and determining
cellular tropism. On the other hand, the S2 subunit includes the fusion peptide (FP), a lengthy heptad repeats 1 domain (HR1), and a
short heptad repeat 2 domains (HR2), which mediate membrane fusion [5,11,12]. A pivotal host cellular receptor for MERS-CoV is
Dipeptidyl peptidase-4, also known as adenosine deaminase (ADA)-complexing protein-2 or CD26. It is an idiosyncratic 766-amino
acid prominent type II transmembrane glycoprotein exposed as a homodimer on the cell surface, responsible for dipeptide cleavage
[13,14]. The viral fleck of MERS-CoV enters the host cell by engaging with the dipeptidyl peptidase-4 (DPP4) cell receptor via the RBD
in the spike protein on the host cell surface. Subsequently, the conformation of the viral virion particle is altered by the S2 subunit and
embedded into the endosomal membrane or plasma membrane. Later, a fusion core, which is a six-helix bundle (6-HB), is formed by
attaching HR2 with HR1, bringing the cell and viral membranes into close proximity for viral fusion [12,15,16]. MERS-CoV’s genome
consists of ORF1a and ORF1b, which produce two large polyproteins. The first, pp1a, contains 4382 amino acids, and the second,
pp1ab, holds 7073 amino acids following a − 1 ribosomal frameshift mechanism [17].
The 3C-like protease (3CLpro), also known as the main protease, Mpro, or nsp5, is a dimer with a Cys-His dyad that can cleave
polyproteins at the eleventh individual region, producing several non-structural proteins crucial in the viral replication process [18,
19]. The Mpro is associated with two proteins, nsp4 and nsp6, and along with some parts of nsp3, forms the replication or transcription
complex on the double-membrane vesicle originating from the endoplasmic reticulum membrane during infection. The 3CLpro qua­
ternary structure consists of each monomer formed by three structural domains. Among these domains, domain I and domain II form a
catalytic cysteine prominent chymotrypsin-like architecture linked to the third C-terminal domain via an extended loop [20–22]. The
Mpro cleavage substrate follows a typical pattern, such as (small)-X-(L/F/M)-Q↓(G/A/S)-X, where X denotes any amino acid, and
represents the cleavage site. In the P1 position of the substrate, the glutamine (Q) residue is essential, while the P2 position strongly
prefers a leucine residue [23,24]. Unlike the tightly associated dimer of SARS-CoV 3CLpro, a ligand is necessary to form a loosely
associated dimer of MERS-CoV 3CLpro. In the presence of a ligand, MERS-CoV 3CLpro structures exhibit a similar shape to SARS-CoV
3CLpro, with a backbone root-mean-square deviation (RMSD) of 1.06 Å over 232 Cα atoms in the protomers [25,26]. The 3C-like
protease (3CLpro) has significant potential as an effective drug target against viral infections, given its auto-cleavage procedure
catalyzed by an enzyme that is essential for viral replication and maturation [27].
Several studies have been conducted to develop an efficient strategy to halt MERS-CoV. Among them, an in vitro comparative study
was undertaken for the treatment of Ribavirin, Chloroquine, Toremifene, and Chlorpromazine [28,29]. Additionally, numerous
comparative studies have been carried out on rhesus monkeys, Ad5-hCD26-transduced mice, and twelve healthy common marmosets
[30,31]. Retrospective cohort and case report studies have also been performed to explore potential antiviral therapies [31]. To date,
no vaccines, antiviral therapeutics, or prophylaxis have been approved for preventing or treating MERS-CoV infections. Despite this,
numerous therapeutics have been demonstrated in preclinical studies, and several of them have undergone clinical testing [32,33].
Several potential vaccines are currently undergoing clinical and preclinical trials. MVA-MERS-S and ChAdOx1 MERS vaccines are in
phase I clinical trials [34,35]. Additionally, MVvac2-MERS-N, RLP3-GEM, rNTD, VSVΔG-MERS, pcDNA3.1-S1, and rAd/Spike vac­
cines are in the preclinical stages [36–39]. This investigation aims to identify potential inhibitors against the 3CLpro of MERS-CoV by
screening 2369 phytoconstituents obtained from Japanese medicinal plants, using an extensive array of in silico techniques.

2. Materials and methods

2.1. Protein preparation

The 3D (three-dimensional) crystal structure (PDB ID: 5WKK; Method: X-ray diffraction; Resolution: 1.55 Å; Organism: MERS) of
the MERS 3CL protease was obtained from the Protein Data Bank (PDB) database maintained by RCSB (Research Collaboratory for
Structural Bioinformatics) [40]. Pymol [41], in conjunction with Discovery Studio software [42], was used to prepare the selected
protein structure initially by excluding heteroatoms along with water molecules. YASARA tools [43] were employed to perform the

2
S. Biswas et al. Heliyon 10 (2024) e25837

energy-minimization process on the prepared clean protein structure to obtain a validated, optimized, and energy-minimized protein.
The minimized protein structure was saved for executing additional molecular docking and molecular dynamics (MD) simulation
schemes.

2.2. Ligand preparation

About 2369 phytochemical constituents extracted from Japanese medicinal plants were listed after an extensive literature search
(Supplementary files 1–5). Google Scholar (https://scholar.google.com/), ResearchGate (https://www.researchgate.net/), PubMed
(https://pubmed.ncbi.nlm.nih.gov/), Dr. Duke’s Phytochemical and Ethnobotanical database (https://phytochem.nal.usda.gov/),
along with various web tools, were utilized for phytochemical screening. Gas chromatography-mass spectroscopy, in conjunction with
liquid chromatography-mass spectroscopy data, was considered for additional validation of the phytochemical dataset. Furthermore,
the phytochemical dataset was rechecked to ensure accuracy, and duplicate entries were removed. The 3D structure of the listed
phytochemicals was retrieved from the PubChem database [44]. The phytochemicals identified as potential ligands were optimized
through a cleaning and energy minimization process using the mmff94 force field [45], with approximately 2000 minimization steps
and the sheared gradient optimization algorithm. Additionally, a dataset of MERS-CoV inhibitors (10 inhibitors) was prepared based
on literature studies [46–49].

2.3. Active site prediction

To predict the active sites, we utilized the PDBsum database (https://www.ebi.ac.uk/pdbsum/) and the Protein-Ligand Interaction
Profiler (PLIP) database (https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index). These databases offer a pictorial overview of
each 3D structure available in the Protein Data Bank (PDB). Consequently, we conducted a search in the database using the PDB code
(5WKK) to retrieve the active sites of the 3CLpro protein. The database provides schematic diagrams that accurately predict the
functional and active sites of the protein [50].

2.4. Molecular docking study

The computer-based screening approach, molecular docking, was implemented to estimate the systematic binding affinity and
interaction mode between the listed phytochemicals and the MERS 3CL protease, utilizing the ’Autodock Vina’ program [51]. The
structural optimization of the phytochemical-based ligands was completed using the Universal Force Field (Uff). After conversion into
PDBQT format, the phytochemical-based ligands were made accessible to Autodock for executing the docking method, with unstable
bonds being functionally eliminated. A grid box with center points specified as X = − 26.99, Y = 12.6039, Z = 58.9455, and dimensions
noted as X = 50.3334, Y = 67.2744, Z = 59.2586 (each in Angstrom) was created after ligand and protein preparation using Autodock
Vina. In the case of the 3CL protease, the cocrystallized fixed protein structure obtained as (PDB: 5WKK) was prepared in a similar
protocol for convenient conversion in Autodock scheme for the docking method. Thus, the molecular docking study between the ligand
and protein is implemented as a control system in the docked research. Binding affinities of the ligands were assessed in kcal/mol based
on negative values, where higher negativity indicates more competent binding. MERS-CoV inhibitors (10 inhibitors) were also used for
docking with the 3CLpro protein.

2.5. ADMET

Several reliable online servers, including admetSAR [52], SwissADME [53], and pKCSM [54], were employed to assess the
pharmacokinetic features based on ADMET predictions. These predictions encompass distribution, excretion, absorption, metabolism,
and toxicity. The Canonical SMILES (simplified molecular-input line-entry system) for the screened plausible antiviral phytochemicals
were retrieved from the PubChem database. These Canonical SMILES, which lack stereochemical and isotopic information and are
derived from chemical structures, were used as input for the mentioned web servers to obtain ADMET predictions that can reveal the
drug-likeness features of the phytochemicals.

2.6. Molecular dynamics simulations

The molecular dynamics simulation study was conducted using the YASARA dynamics software package [43], assisted by the
AMBER14 force field [55]. The docked complexes were initially cleaned, optimized, and hydrogen bond networks were oriented. The
TIP3P water solvation model was applied with periodic boundary conditions [56]. The simulations were conducted under physio­
logical conditions set at 298K, pH 7.4, and 0.9% NaCl. Initial energy minimizations were performed using the steepest gradient al­
gorithms with the simulated annealing method (5000 cycles). Long-range electrostatic interactions were calculated using the Particle
Mesh Ewald (PME) methods [57–59]. The time step for the simulations was set to 2.0fs, and trajectory snapshots were saved every
100ps. The simulations were extended for 100ns following constant pressure and Berendsen thermostat protocols [60]. The simulation
trajectories were utilized to calculate the root mean square deviations (RMSD), root mean square fluctuations (RMSF), solvent
accessible surface area (SASA), radius of gyrations (Rg), and hydrogen bonds [61–70]. Additionally, the Molecular
Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) approach, along with the AMBER14 force field, was employed in the YASARA
dynamics software package to compute the binding free energy [71]. For the computation of binding free energy, the default macro file

3
S. Biswas et al. Heliyon 10 (2024) e25837

was modified, and the following equation was used:

ΔGbind = ΔGcomplex(minimised) – [ΔGligand(minimised) + ΔGreceptor(minimised)]

ΔGbind = ΔGMM + ΔGPB + ΔGSA − TΔS

Here, ΔGMM denotes the sum of electrostatic and van der Waals interactions; ΔGSA and ΔGPB indicate the non-polar and polar
solvation energies, respectively; and TΔS denotes the entropic contribution [72]. A flow chart showing the main steps of the study is
depicted in Fig. 1.

3. Results

3.1. Active site prediction

From the PDBsum database, the active groove of the 3CLpro from MERS-CoV was identified. The red dots in Fig. 2, retrieved from
the PDBsum database, indicate the active site of the 3CLpro protein. A total of twenty-one active sites were determined by the PDBsum
database, including Met6, His8, His41, Leu49, Phe143, Leu144, Cys145, Gly146, Ser147, Cys148, His166, Gln167, Met168, Glu169,
Ala171, His175, Asp190, Qln192, Val193, His194, and Met298. The active site residues obtained from the PDBsum database
encompass all the active site residues found in a previous study [73].
On the contrary, a total of ten active sites were determined (Fig. 3) by the PLIP database: His41, Phe143, Cys145, Gln167, Met168,
Glu169, Ala171, His175, Gln192, and His194. Therefore, ten amino acid residues are considered active sites of the 3CLpro protein for
this study, based on the amino acid residues obtained from both databases (PDBsum & PLIP). To evaluate the non-bonding interactions
from the molecular docking study, these ten amino acid residues from the 3CLpro were carefully considered.

3.2. Molecular docking analysis

A total of 2369 phytochemicals were docked with the 3CLpro protein, with 25 being selected as the top binding molecules (Sup­
plementary File 6; Supplementary Table 1). Re-docking was performed to ensure accuracy and avoid errors after selecting the top 25
molecules with higher binding affinities (Supplementary File 6; Supplementary Table 2). After redocking the top 25 compounds, the
top 10 phytochemicals were selected based on docking energy (Supplementary File 6; Supplementary Table 3), and finally, the top
three [Fig. 4(A–C)] compounds were chosen for further study.
The selected 10 inhibitors were also docked against the MERS-CoV 3CLpro protein (Table 2). Nafamostat exhibited the highest
binding affinity as − 8.1 kcal/mol and for this reason, only Nafamostat is selected and used for further studies. Nafamostat expressed
eight interactions with 3CLpro having one Electrostatic Bond at GLU294 position, four Conventional Hydrogen Bonds at PRO135,
GLY112, ASN206, VAL246 positions, one Pi-Sigma Bond at VAL205 position, one Pi-Alkyl Bond PRO111 position, and one

Fig. 1. Flow chart showing the main steps of the study.

4
S. Biswas et al. Heliyon 10 (2024) e25837

Fig. 2. The red dots indicate the active site of the MERS-CoV 3CLpro protein which was retrieved from the PDBsum database. (For interpretation of
the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Unfavorable Bond at LYS201 position (Table 3, Fig. 6).

3.3. ADMET

The drug-likeness properties of the three selected compounds were evaluated using ADMET calculations (Table 4). According to
Lipinski’s rule of 5, which indicates drug-likeness, the standard values for molecular weight, hydrogen bond donors, and hydrogen
bond acceptors should be less than 500 g/mol, less than 5, and less than 10, respectively [74]. Kihadanin B (486.51 g/mol) with 1
hydrogen bond donor and 9 hydrogen bond acceptors, Robustaflavone (538.46 g/mol) with 6 hydrogen bond donors and 10 hydrogen
bond acceptors, and 3-beta-O-(trans-pCoumaroyl) maslinic acid (618.84 g/mol) with 3 hydrogen bond donors and 6 hydrogen bond
acceptors meet the drug-likeness criteria. It is worth noting that natural compounds, especially, may accept up to 2 violations of the
Lipinski rule [74]. In a recent analysis, the highest mean TPSA value was calculated to be 108 Å2 (95th percentile: 202 Å2), and the
TPSA values for Kihadanin B (128.73 Å2), Robustaflavone (181.80 Å2), and 3-beta-O-(trans-pCoumaroyl) maslinic acid (104.06 Å2) are
below the 95th percentile value. Concerning human intestinal absorption, the mean experimental value for drugs is 75.97% [75].
Therefore, Kihadanin B (100%), Robustaflavone (90.473%), and 3-beta-O-(trans-p-Coumaroyl) maslinic acid (78.769%) exhibit
satisfactory human intestinal absorption. Toxicity, which indicates potential damage to cells and organs, is a crucial parameter for drug
development [76]. As all of the compounds are noncarcinogens, they exhibit no AMES toxicity, hepatotoxicity, and skin sensitization
[77,78].

5
S. Biswas et al. Heliyon 10 (2024) e25837

Fig. 3. The active sites of the MERS-CoV 3CLpro protein were retrieved from the PLIP database.

3.4. Molecular dynamics simulation

The molecular dynamics simulation study aimed to understand the structural stability of the docked complexes. The root mean
square deviations (RMSD) of the C-alpha atoms of the docked complexes were examined to illustrate the binding stability. Fig. 7 (a)
indicates that the CID-156766 (Kihadanin B)-3CLpro, CID-5281694 (Robustaflavone)-3CLpro, CID-14335962 (3-beta-O-(trans-p-Cou­
maroyl) maslinic acid)-3CLpro, and Nafamostat-3CLpro complexes initially exhibited an upper trend in RMSD, indicating the flexible
nature of the complexes at the beginning phase. However, the top three complexes began to stabilize after 25ns and maintained
stability for the rest of the simulation periods with minor fluctuations. In contrast, the Nafamostat-3CLpro complex showed increased
flexibility in the final 30 ns of the simulation time. Overall, the RMSD of the complexes remained below 2.5 Å, defining the stable
nature of the complexes [64].
Furthermore, the solvent accessible surface area (SASA) of the complexes was analyzed to understand changes in the surface area,
where higher SASA defines the extensions of the surface area, and a lower SASA value relates to the truncated nature of the complexes
[68]. Fig. 7 (b) indicates that the CID-14335962 (3-beta-O-(trans-p-Coumaroyl) maslinic acid)-3CLpro complex had extensions in the
surface upon the binding of the ligand molecules, whereas the other two complexes had a stable SASA profile. The higher SASA values
of the Nafamostat-3CLpro complex signify the expansion of the protein surface area. The radius of gyrations profile of the simulated
complexes was also analyzed, where higher Rg relates to the mobile nature of the complexes, and lower Rg relates to the stable nature
of the complexes [65]. Fig. 7 (c) indicates that the CID-14335962 (3-beta-O-(trans-p-Coumaroyl) maslinic acid)-3CLpro complex
exhibited a higher Rg and more flexibility, whereas the other two complexes demonstrated the rigid nature of the complexes.
Furthermore, compared to the top three complexes, the Nafamostat-3CLpro complex showed a higher average Rg value, indicating its
greater flexibility.
The hydrogen bonds in the simulation complexes play a crucial role in determining stability in macromolecular systems [79]. All
four complexes formed a substantial quantity of hydrogen bonds, which were essential for the integrity and stability of the complexes
(see Fig. 7d). Root mean square fluctuations (RMSF) define the flexibility across the amino acid residues. Fig. 7e indicates that most
residues had RMSF values lower than 2.5 Å, with minor fluctuations, signifying the stability of the complexes [68]. Moreover, the

6
S. Biswas et al. Heliyon 10 (2024) e25837

Fig. 4. Two-dimensional (2D) chemical structures of (A) Kihadanin B, (B) Robustaflavone, and (C) 3-beta-O-(trans-p-Coumaroyl)maslinic acid. The
structures were drawn using MarvinSketch software.

CID-156766 (Kihadanin B)-3CLpro, CID-5281694 (Robustaflavone)-3CLpro, CID-14335962 (3-beta-O-(trans-p-Coumaroyl)maslinic

acid)-3CLpro, and Nafamostat-3CLpro complexes exhibited average binding free energies of − 86.54 kJ/mol, − 73.82 kJ/mol, − 67.08
kJ/mol, and − 61.79 kJ/mol, respectively (see Fig. 8). In the MM-PBSA computation, the top three drug complexes exhibited higher
average binding free energy, suggesting more efficient binding than the inhibitor (Nafamostat).

4. Discussion

Since the detection of MERS-CoV in 2012, the ominous fatality rate of 35% and transmission via close contact with an R0 (the basic
reproduction number) varying from 0.8 to 1.3 in the scarcity of infection control measures have raised concerns about a potential
global pandemic, especially during the Hajj Pilgrimage [80,81]. Additionally, available data-derived evidence of adaptive evolution in
MERS-CoV-related viruses via the heptad repeat region, along with in vitro-based confirmation for boosting MERS-CoV infection
efficacy through mutations like T1015 N, has underscored the necessity for designing antiviral therapy against MERS-CoV [82].
However, currently, there is neither an approved vaccine for preventing MERS-CoV infection nor any specific treatment for Middle East
respiratory syndrome caused by MERS-CoV [83]. Therefore, computer-based virtual screening through molecular docking and mo­
lecular dynamics simulation can be a dependable approach to uncover plausible inhibitors or repurposable drugs against MERS-CoV,
as the three-dimensional crystal structure of the MERS 3CL protease is available to reveal receptor− ligand interactions with elaborated
investigations.
According to the crystal structure of 3CLpro, each monomer remains perpendicular in the homodimer consisting of a core
chymotrypsin-like fold assembled by two domains, designated domains I and II, consisting of residues 1–187, a prolonged connecting
loop consisting of residues 188–204, and domain III or C-terminal alpha-helical domain consisting of residues 205–306 [21,84]. In the
case of the proteolytic site, glutamine, leucine, basic residues, and small hydrophobic residues at P1, P2, P3, and P4 positions,
respectively, are preferred by the 3CLpro. Additionally, small residues are required at P1′ and P2′ positions [21,85]. As 3CLpro is
responsible for cleavage at 11 sites during the production of 16 nonstructural proteins, it holds significance in the autocleavage process
during the proteolytical process of the polyproteins named pp1a and pp1ab. Thus, it is required for viral replication and maturation to
conduct the coronaviral life cycle, indicating it to be a reliable target for inhibition and making it suitable as a substrate against
plausible phytochemicals or repurposable drugs [86,87].
In this investigation, approximately 2369 phytochemical components extracted from Japanese medicinal plants were utilized as
data to be operated as ligands. The 3D or three-dimensional crystal structure of the MERS 3CL protease had been retrieved from the
PDB databank to be used as the protein target for MERS CoV inhibition. Later, after the protein and ligand preparation, a molecular
docking approach was executed to assess the systematic binding affinity, along with the interaction mode between the MERS 3CL
protease and enlisted phytochemicals for prompt recognition of potential inhibitors within a short period. Based on binding affinity,
the top 25 phytochemicals were re-docked, among which the top 3 compounds were determined as Kihadanin B, Robustaflavone, and

7
S. Biswas et al. Heliyon 10 (2024) e25837

Fig. 5. Different binding modes for the selected compounds within the MERS-CoV 3CLpro protein’s active and catalytic sites; (A) Kihadanin B, (B)
Robustaflavone, (C) 3-beta-O-(trans-p-Coumaroyl)maslinic acid.

3-beta-O-(trans-p-Coumaroyl)maslinic acid with binding energies of − 9.8, − 9.4, and − 9.2 kcal/mol, respectively, for further inves­
tigation. Besides, Nafamostat exhibited the highest binding affinity at − 8.1 kcal/mol among the selected 10 inhibitors of MERS CoV.
Kihadanin B, a limonoid extracted from the immature peel of Citrus unshiu, Araliopsis soyauxii Engl. plant, Phellodendron
amurense bark, and Dictamnus dasycarpus’s root bark, has demonstrated various medicinal properties. These include suppressing
adipogenesis through the repression of the Akt-FOXO1-PPARγ axis [88], significant cytotoxicity against nine tested cancer cell lines
[89], notable antifeedant activity tested against Reticulitermes speratus [90], and cytotoxicity against human cancer cell lines [91].
Robustaflavone, a biflavonoid extracted from N. domestica fruits, Garcinia latissima Miq. leaves, and Rhus succedanea, exhibits
several activities, including inflammation inhibition [92], antibacterial activity [93], and potential inhibition of hepatitis B virus
replication [94]. 3-beta-O-(trans-p-Coumaroyl)maslinic acid, a strictinin isomer isolated from the root of Rosa roxburghii Tratt (Ci Li
Gen), demonstrates an antimicrobial mechanism [95].
Then, ADMET predictions for the identified phytochemical hits were conducted using multiple online-based servers to ensure drug-
likeness features based on pharmacokinetic estimations. Subsequently, molecular dynamics simulations were executed to confirm
binding rigidity and stability, relying on multiple descriptors of the simulation trajectories. A more stable binding was observed for the
three hit phytochemicals compared to the inhibitor (Nafamostat), which was further confirmed by the calculation of binding free
energy. nHowever, the active site for the 3CLpro from MERS-CoV was obtained from the PDBsum database, which mentioned twenty-
one active sites. Among the three hit phytochemicals, Kihadanin B binds to 3CLpro through six non-bond interactions, three of which
involve the residues Glu169, Met168, and Cys145 located in the active site of the 3CLpro of MERS-CoV. Robustaflavone is bound by
seven non-bond interactions, four of which involve the residues Gln192, Ala171, Cys145, and Met168, located in the 3CLpro active site.
Similarly, 3-beta-O-(trans-p-Coumaroyl) maslinic acid binds with 3CLpro via six non-bond interactions, two of which involve the
residues Glu169 and Met168, situated in the active site of the MERS-CoV 3CLpro. As several amino acid residues involved in the non-

8
S. Biswas et al. Heliyon 10 (2024) e25837

Table 1
Non-bond interactions between the top three compounds (selected based on docking scores) and the MERS-CoV 3CLpro protein.
Compounds PubChem Binding Affinity (kcal/ Residues in Interaction type Distance in
CID mol) contact Å

Kihadanin B 156766 − 9.8 GLU169 Conventional Hydrogen Bond 1.93232

MET168 Carbon Hydrogen Bond 2.76511
HIE194 Pi-Alkyl Bond (Hydrophobic) 4.00511
HIS166 Carbon Hydrogen Bond 2.44893
CYS145 Carbon Hydrogen Bond 2.79336
GLY146 Conventional Hydrogen Bond 1.76957
Robustaflavone 5281694 − 9.4 HIE41 Conventional Hydrogen Bond 2.1817
GLN192 Conventional Hydrogen Bond 1.68389
VAL193 Conventional Hydrogen Bond 1.74401
ALA171 Carbon Hydrogen Bond 2.66935
CYS145 Pi-Alkyl Bond (Hydrophobic) 5.11343
MET25 Pi-Alkyl Bond (Hydrophobic) 4.63918
MET168 Pi-Alkyl Bond (Hydrophobic) 5.42348
3-beta-O-(trans-p-Coumaroyl) maslinic 14335962 − 9.2 GLU169 Conventional Hydrogen Bond 1.99887
acid ASP190 Conventional Hydrogen Bond 2.78672
MET168 Carbon Hydrogen Bond 2.47132
HIE194 Carbon Hydrogen Bond 3.09155
HIE41 Pi-Pi Stacked Bond 4.14442
(Hydrophobic)
LEU49 Pi-Alkyl Bond (Hydrophobic) 4.97781

With absolute ensuring on proper running in the analyzing tool tilled as ‘Autodock vina’, the top three ‘Kihadanin B’, ‘Robustaflavone’, and ‘3-beta-O-
(trans-p-Coumaroyl)maslinic acid’ showed the binding affinity as − 9.8, − 9.4, and − 9.2 kcal/mol respectively. On the contrary, ‘BIOVIA Discovery
Studio’ explored the non-binding interaction between these three drug candidates and the 3C-like protease (3CLpro). Preliminary, the first drug
candidate, Kihadanin B expressed six interactions with 3CLpro where one Pi-Alkyl bond at HIE194, two Conventional Hydrogen bonds at GLU169, and
GLY146, and three Carbon Hydrogen bonds at MET168, HIS166, and CYS145 was observed. Robustaflavone also provided seven interacting sites
where one was at ALA171 with Carbon Hydrogen bond, three Conventional Hydrogen bonds at HIE41, GLN192, VAL193 positions, and three Pi-Alkyl
bonds at CYS145, MET25, MET168 positions. On the other hand, the last drug candidates showed complex interaction having one Pi-Pi Stacked bond
at the HIE41 residue position, one Pi-Alkyl bond at the LEU49 residue position, and two Conventional Hydrogen bonds at GLU169 and ASP190
residue position, and two Carbon Hydrogen bonds at MET168 and HIE194 residue position (Table 1), [Fig. 5(A–C)].

Table 2
Binding Affinities of the selected 10 inhibitors against MERS-CoV 3CLpro protein.
Inhibitor Names PubChem CID Binding Affinity (kcal/mol)

Nafamostat 4413 − 8.1

Nelfinavir 64143 − 7.8
Camostat 2536 − 7
Resveratrol 445154 − 6.7
Nitazoxanide 41684 − 6.4
Ribavirin 37542 − 6.3
Alisporivir 11513676 − 6.1
Cyclosporin A 5284373 − 6
Ritonavir 392622 − 5.9
Mycophenolate 446541 − 5.7

Table 3
Non-bond interactions between MERS-CoV 3CLpro and the top inhibitor (Nafamostat).
Inhibitors PubChem CID Binding Affinity (kcal/mol) Residues in contact Interaction type Distance in Å

Nafamostat 4413 − 8.1 GLU294 Electrostatic Bond 4.73878

PRO135 Conventional Hydrogen Bond 2.19994
GLY112 Conventional Hydrogen Bond 2.72113
ASN206 Conventional Hydrogen Bond 2.5218
VAL246 Conventional Hydrogen Bond 2.23728
VAL205 Pi-Sigma Bond 2.38199
PRO111 Pi-Alkyl Bond 4.90979
LYS201 Unfavorable Bond 3.82713

bond interactions are located within the active site, the three top phytochemicals can be considered for potential use as a drug for
antiviral therapy concerning MERS-CoV.
An investigation of docked compounds from the ZINC database against the 3CLpro protein of MERS-CoV uncovered three hit

9
S. Biswas et al. Heliyon 10 (2024) e25837

Fig. 6. Two-dimensional (2D) view of the interactions between MERS-CoV 3CLpro and the top inhibitor (Nafamostat).

Table 4
Pharmaceutical profiles derived from the SwissADME, admetSAR, and pKCSM webservers for the top three potential candidates obtained from
docking.
Parameters Kihadanin B Robustaflavone 3-beta-O-(trans-p-Coumaroyl)maslinic acid

Molecular Weight 486.51 g/mol 538.46 g/mol 618.84 g/mol

Num. H-bond acceptors 9 10 6
Num. H-bond donors 1 6 3
TPSA (S) 128.73 Å2 181.80 Å2 104.06 Å2
Human Intestinal Absorption 100% 90.473% 78.769%
AMES Toxicity No No No
Hepatotoxicity No No No
Skin sensitization No No No
Carcinogens Non-carcinogens Non-carcinogens Non-carcinogens

compounds, including ZINC ID: 75121653, 41131653, and 67266079, possessing binding energies of − 7.12, − 7.1, and − 7.08 kcal/
mol, respectively [96]. In another prior study, compounds available in the National Cancer Institute (NCI) database were docked
against the MERS-CoV 3CLpro protein. They identified five hit compounds, including NSC648199, NSC159375, NSC29007,
NSC335985, and NSC337571, with binding energies of − 8.7, − 8.3, − 8.1, − 8.1, and − 7.8 kcal/mol, respectively. These values were
lower than the binding energies uncovered in our current study, which found the top three compounds Kihadanin B, Robustaflavone,
and 3-beta-O-(trans-p-Coumaroyl)maslinic acid with binding affinities of − 9.8, − 9.4, and − 9.2 kcal/mol, respectively [97].
In another previous study, several flavonoids utilized as MERS-CoV 3C-like protease inhibitors showed favorable binding affinity.
However, among them, Helichrysetin, Herbacetin, and isobavachalcone did not bind with the residues of the active site. Only
Quercetin 3-β-D-glucoside was found to bind with Gln 169 residues at the active site [21]. In a different analysis, two potent and
permeable inhibitors were synthesized through structure-based design to inhibit MERS Coronavirus 3C-like protease. Among them,
10a bound with one residue (Cys148), and 10c bound with five residues at the active site (His41, Phe143, Gln192, Gln167, and
Glu169) [98]. Contrastingly, in our findings, all the hit compounds interacted with the amino acid residues at the active site. For
instance, Kihadanin B interacted with Glu169, Met168, and Cys145; Robustaflavone interacted with Gln192, Ala171, Met168, and
Cys145; and 3-beta-O-(trans-p-Coumaroyl) maslinic acid interacted with Glu169 and Met168. Thus, our hit compounds have more
potential to block the active site, inhibiting the function of the 3C-like protease, which is crucial for viral protein maturation and
replication.
In addition, plant-derived phytochemicals, including Kaempferol (− 9.3 kcal/mol), Resveratrol (− 9.31 kcal/mol), Quercetin
(− 11.88 kcal/mol), and Theaflavin (− 14.35 kcal/mol), showed the highest binding affinity for the 3CLpro protein of MERS-CoV.
However, the relevant study lacks an analysis of the binding mode stability of the docked complex through molecular docking
simulation [99]. Conversely, all the hit compounds in our study, including Kihadanin B, Robustaflavone, and 3-beta-O-(­
trans-p-Coumaroyl) maslinic acid, exhibited the structural stability and firmness of the docked complexes over 100 ns of molecular
dynamics simulation. After a thorough literature review, it is noted that Kihadanin B exhibits suppression of adipogenesis by reducing
lipid accumulation and suppressing the Akt-FOXO1-PPARγ axis in 3T3-L1 adipocytes [88]. Likewise, Robustaflavone manifests
antibacterial activity, anti-inflammatory activity, potent inhibition of hepatitis B virus replication in the 2.2.15 cell line,
anti-angiogenic and pro-apoptotic impacts, cytotoxic effect on cancer cells, and antioxidant effect [100]. Similarly,

10
S. Biswas et al. Heliyon 10 (2024) e25837

Fig. 7. The molecular dynamics simulation of the top three compound-MERS-CoV 3CLpro complexes along with Nafamostat-MERS-CoV 3CLpro
complex, here (a) root mean square deviation of the c-alpha atoms, (b) solvent accessible surface area, (c) radius of gyration, (d) hydrogen bonding
of the complexes, (e) root mean square fluctuation of the complexes.

Fig. 8. The binding-free energy of the top three drugs and inhibitor complexes calculated by the MM-PBSA method.

3-beta-O-(trans-p-Coumaroyl) maslinic acid is found to be associated with an antimicrobial mechanism [95].

The top three phytoconstituents evaluated in this study may be able to prevent the 3CLpro of MERS-CoV from performing its
function, as determined by a combination of computational techniques, including molecular docking and molecular dynamics in­
vestigations. These three plausible candidates exhibited higher binding affinity, showed interaction with the active sites of the protein,
displayed lower toxic characteristics with maximum drug-likeness properties, and also exhibited the structural stability and firmness of
the docked complexes over 100 ns of simulation time. Therefore, these three phytochemicals can be potential candidates as antiviral
therapeutics for combating MERS-CoV. Besides, the creation of these new phytoconstituents datasets will allow researchers to carry out

11
S. Biswas et al. Heliyon 10 (2024) e25837

investigations against other specific viral and bacterial proteins or other disease-causing molecules. Despite the verification of this
study’s results using a variety of bioinformatics techniques, further in vitro assessment will be required to confirm the accuracy of the
results.

5. Conclusion

In this study, 2369 phytoconstituents from Japanese medicinal plants were identified through extensive literature mining. These
compounds underwent screening against the 3CLpro of MERS-CoV to identify potential inhibitors of 3CLpro. Molecular docking analysis
facilitated the selection of the three best compounds based on their binding energy, namely Kihadanin B, Robustaflavone, and 3-beta-
O-(trans-p-Coumaroyl)maslinic acid. These three hit molecules predominantly interacted with the active site of 3CLpro, playing a
crucial role in inhibiting the function of the 3CLpro protein. The ADMET profiling results for these three lead molecules confirmed their
lower toxicity and maximum drug-likeness properties. The stability and rigidity of these protein-ligand complexes throughout the
simulation period were confirmed through a molecular dynamics simulation study. In comparison to the inhibitor Nafamostat, the top
three compounds exhibited superior binding energy, stability, and firmness, as further validated by the calculation of binding free
energy. This study relies entirely on computational screening and simulation programs; therefore, additional in vitro assessments will
be necessary to validate the efficacy of these compounds against MERS-CoV.

Funding

This research work receives no external funding.

Data availability

All data generated or analyzed during this study are included in this published article.

CRediT authorship contribution statement

Suvro Biswas: Writing – original draft, Investigation, Data curation, Conceptualization. Mohasana Akter Mita: Writing – original
draft, Investigation, Formal analysis, Data curation, Conceptualization. Shamima Afrose: Writing – original draft, Methodology,
Investigation, Formal analysis, Data curation. Md Robiul Hasan: Writing – original draft, Methodology, Investigation, Formal
analysis, Data curation. Mst Sharmin Sultana Shimu: Formal analysis, Data curation. Shahriar Zaman: Writing – review & editing,
Resources, Project administration. Md Abu Saleh: Writing – review & editing, Supervision, Resources, Project administration,
Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e25837.

References

[1] A.N. Alshukairi, J. Zheng, J. Zhao, A. Nehdi, S.A. Baharoon, L. Layqah, A. Bokhari, S.M. Al Johani, N. Samman, M. Boudjelal, P. Ten Eyck, M.A. Al-Mozaini,
J. Zhao, S. Perlman, A.N. Alagaili, High prevalence of MERS-CoV infection in camel workers in Saudi Arabia, mBio 9 (2018) 1–10, https://doi.org/10.1128/
MBIO.01985-18.
[2] Z. Zhang, L. Shen, X. Gu, Evolutionary dynamics of MERS-CoV: potential recombination, positive selection and transmission, Sci. Rep. 6 (2016) 25049, https://
doi.org/10.1038/srep25049.
[3] B.L. Haagmans, S.H.S. Al Dhahiry, C.B.E.M. Reusken, V.S. Raj, M. Galiano, R. Myers, G.J. Godeke, M. Jonges, E. Farag, A. Diab, H. Ghobashy, F. Alhajri, M. Al-
Thani, S.A. Al-Marri, H.E. Al Romaihi, A. Al Khal, A. Bermingham, A.D.M.E. Osterhaus, M.M. AlHajri, M.P.G. Koopmans, Middle East respiratory syndrome
coronavirus in dromedary camels: an outbreak investigation, Lancet Infect. Dis. 14 (2014) 140–145, https://doi.org/10.1016/S1473-3099(13)70690-X.
[4] F. Li, L. Du, MERS Coronavirus: an emerging zoonotic virus, Viruses 11 (2019) 1–6, https://doi.org/10.3390/v11070663.
[5] N. Masood, S.S. Malik, M.N. Raja, S. Mubarik, C. Yu, Unraveling the epidemiology, geographical distribution, and genomic evolution of potentially lethal
coronaviruses (SARS, MERS, and SARS CoV-2), Front. Cell. Infect. Microbiol. 10 (2020) 1–8, https://doi.org/10.3389/fcimb.2020.00499.
[6] V.S. Raj, A.D.M.E. Osterhaus, R.A.M. Fouchier, B.L. Haagmans, MERS: emergence of a novel human coronavirus, Curr. Opin. Virol. 5 (2014) 58–62, https://
doi.org/10.1016/j.coviro.2014.01.010.
[7] R. Lu, Y. Wang, W. Wang, K. Nie, Y. Zhao, J. Su, Y. Deng, W. Zhou, Y. Li, H. Wang, W. Wang, C. Ke, X. Ma, G. Wu, W. Tan, Complete genome sequence of
Middle East respiratory syndrome coronavirus (MERS-CoV) from the first imported MERS-CoV case in China, Genome Announc. 3 (2015) 2014–2015, https://
doi.org/10.1128/genomeA.00818-15.
[8] A. Chafekar, B.C. Fielding, MERS-CoV: understanding the latest human coronavirus threat, Viruses 10 (2018), https://doi.org/10.3390/v10020093.

12
S. Biswas et al. Heliyon 10 (2024) e25837

[9] M.M. Ba Abduallah, M.G. Hemida, Comparative analysis of the genome structure and organization of the Middle East respiratory syndrome coronavirus
(MERS-CoV) 2012 to 2019 revealing evidence for virus strain barcoding, zoonotic transmission, and selection pressure, Rev. Med. Virol. 31 (2021) 1–12,
https://doi.org/10.1002/rmv.2150.
[10] Z. Qian, S.R. Dominguez, K.V. Holmes, Role of the spike glycoprotein of human Middle East respiratory syndrome coronavirus (MERS-CoV) in virus entry and
syncytia formation, PLoS One 8 (2013) 1–12, https://doi.org/10.1371/journal.pone.0076469.
[11] J.K. Millet, G.R. Whittaker, Physiological and molecular triggers for SARS-CoV membrane fusion and entry into host cells, Virology 517 (2018) 3–8, https://
doi.org/10.1016/j.virol.2017.12.015.
[12] R. Liang, L. Wang, N. Zhang, X. Deng, M. Su, Y. Su, L. Hu, C. He, T. Ying, S. Jiang, F. Yu, Development of small-molecule MERS-CoV inhibitors, Viruses 10
(2018), https://doi.org/10.3390/v10120721.
[13] V.S. Raj, H. Mou, S.L. Smits, D.H.W. Dekkers, M.A. Müller, R. Dijkman, D. Muth, J.A.A. Demmers, A. Zaki, R.A.M. Fouchier, V. Thiel, C. Drosten, P.J.M. Rottier,
A.D.M.E. Osterhaus, B.J. Bosch, B.L. Haagmans, Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC, Nature 495 (2013)
251–254, https://doi.org/10.1038/nature12005.
[14] T. Pillaiyar, Middle East respiratory syndrome-coronavirus (MERS-CoV): an updated overview and pharmacotherapeutics, Med. Chem. (2015), https://doi.
org/10.4172/2161-0444.1000287.
[15] S. Xia, Q. Liu, Q. Wang, Z. Sun, S. Su, L. Du, T. Ying, L. Lu, S. Jiang, Middle East respiratory syndrome coronavirus (MERS-CoV) entry inhibitors targeting spike
protein, Virus Res. 194 (2014) 200–210, https://doi.org/10.1016/j.virusres.2014.10.007.
[16] L. Lu, Q. Liu, Y. Zhu, K.H. Chan, L. Qin, Y. Li, Q. Wang, J.F.W. Chan, L. Du, F. Yu, C. Ma, S. Ye, K.Y. Yuen, R. Zhang, S. Jiang, Structure-based discovery of
Middle East respiratory syndrome coronavirus fusion inhibitor, Nat. Commun. 5 (2014), https://doi.org/10.1038/ncomms4067.
[17] M.A. Alamri, M. Tahir ul Qamar, O. Afzal, A.B. Alabbas, Y. Riadi, S.M. Alqahtani, Discovery of anti-MERS-CoV small covalent inhibitors through
pharmacophore modeling, covalent docking and molecular dynamics simulation, J. Mol. Liq. 330 (2021) 115699, https://doi.org/10.1016/j.
molliq.2021.115699.
[18] K. Shirato, M. Kawase, S. Matsuyama, Middle East respiratory syndrome coronavirus infection mediated by the transmembrane serine protease TMPRSS2,
J. Virol. 87 (2013) 12552–12561, https://doi.org/10.1128/jvi.01890-13.
[19] V. Kumar, J.S. Shin, J.J. Shie, K.B. Ku, C. Kim, Y.Y. Go, K.F. Huang, M. Kim, P.H. Liang, Identification and evaluation of potent Middle East respiratory
syndrome coronavirus (MERS-CoV) 3CLpro inhibitors, Antivir. Res. 141 (2017) 101–106, https://doi.org/10.1016/j.antiviral.2017.02.007.
[20] D. Needle, G.T. Lountos, D.S. Waugh, Structures of the Middle East respiratory syndrome coronavirus 3C-like protease reveal insights into substrate specificity,
Acta Crystallogr. Sect. D Biol. Crystallogr. 71 (2015) 1102–1111, https://doi.org/10.1107/S1399004715003521.
[21] S. Jo, H. Kim, S. Kim, D.H. Shin, M.S. Kim, Characteristics of flavonoids as potent MERS-CoV 3C-like protease inhibitors, Chem. Biol. Drug Des. 94 (2019)
2023–2030, https://doi.org/10.1111/cbdd.13604.
[22] K. Knoops, M. Kikkert, S.H.E. Van Den Worm, J.C. Zevenhoven-Dobbe, Y. Van Der Meer, A.J. Koster, A.M. Mommaas, E.J. Snijder, SARS-coronavirus
replication is supported by a reticulovesicular network of modified endoplasmic reticulum, PLoS Biol. 6 (2008) 1957, https://doi.org/10.1371/journal.
pbio.0060226. –1974.
[23] F. Wang, C. Chen, W. Tan, K. Yang, H. Yang, Structure of main protease from human coronavirus NL63: insights for wide spectrum anti-coronavirus drug
design, Sci. Rep. 6 (2016) 1–12, https://doi.org/10.1038/srep22677.
[24] R. Hilgenfeld, From SARS to MERS: crystallographic studies on coronaviral proteases enable antiviral drug design, FEBS J. 281 (2014) 4085–4096, https://doi.
org/10.1111/febs.12936.
[25] J. He, L. Hu, X. Huang, C. Wang, Z. Zhang, Y. Wang, Since January 2020 Elsevier Has Created a COVID-19 Resource Centre with Free Information in English
and Mandarin on the Novel Coronavirus COVID- 19 . The COVID-19 Resource Centre Is Hosted on, Elsevier Connect , the company ’ s public news and
information, 2020.
[26] S. Tomar, M.L. Johnston, S.E.S. John, H.L. Osswald, P.R. Nyalapatla, L.N. Paul, A.K. Ghosh, M.R. Denison, A.D. Mesecar, Ligand-induced dimerization of
Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 protease (3CLpro): implications for nsp5 regulation and the development of antivirals, J. Biol.
Chem. 290 (2015) 19403–19422, https://doi.org/10.1074/jbc.M115.651463.
[27] A. Bahadur Gurung, M. Ajmal Ali, J. Lee, M. Abul Farah, K. Mashay Al-Anazi, Structure-based virtual screening of phytochemicals and repurposing of FDA
approved antiviral drugs unravels lead molecules as potential inhibitors of coronavirus 3C-like protease enzyme, J. King Saud Univ. Sci. 32 (2020) 2845–2853,
https://doi.org/10.1016/j.jksus.2020.07.007.
[28] D. Falzarano, E. De Wit, C. Martellaro, J. Callison, V.J. Munster, H. Feldmann, Inhibition of novel β coronavirus replication by a combination of interferon-α2b
and ribavirin, Sci. Rep. 3 (2013) 1–6, https://doi.org/10.1038/srep01686.
[29] Y. Cong, B.J. Hart, R. Gross, H. Zhou, M. Frieman, L. Bollinger, J. Wada, L.E. Hensley, P.B. Jahrling, J. Dyall, M.R. Holbrook, MERS-CoV pathogenesis and
antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells, PLoS One 13 (2018) 1–17, https://doi.org/10.1371/journal.
pone.0194868.
[30] Y. Li, Y. Wan, P. Liu, J. Zhao, G. Lu, J. Qi, Q. Wang, X. Lu, Y. Wu, W. Liu, B. Zhang, K.Y. Yuen, S. Perlman, G.F. Gao, J. Yan, A humanized neutralizing antibody
against MERS-CoV targeting the receptor-binding domain of the spike protein, Cell Res. 25 (2015) 1237–1249, https://doi.org/10.1038/cr.2015.113.
[31] H. Momattin, A.Y. Al-Ali, J.A. Al-Tawfiq, A systematic review of therapeutic agents for the treatment of the Middle East respiratory syndrome coronavirus
(MERS-CoV), Trav. Med. Infect. Dis. 30 (2019) 9–18, https://doi.org/10.1016/j.tmaid.2019.06.012.
[32] Y. Zhou, S. Jiang, L. Du, Prospects for a MERS-CoV spike vaccine, Expert Rev. Vaccines 17 (2018) 677–686, https://doi.org/10.1080/
14760584.2018.1506702.
[33] L. Du, Y. Yang, Y. Zhou, L. Lu, F. Li, S. Jiang, MERS-CoV spike protein: a key target for antivirals, Expert Opin. Ther. Targets 21 (2017) 131–143, https://doi.
org/10.1080/14728222.2017.1271415.
[34] L.M. Weskamm, A. Fathi, M.P. Raadsen, A.Z. Mykytyn, T. Koch, M. Spohn, M. Friedrich, E. Bartels, S. Gundlach, T. Hesterkamp, V. Krähling, S. Lassen, M.L. Ly,
J.H. Pötsch, S. Schmiedel, A. Volz, M.E. Zinser, B.L. Haagmans, S. Becker, G. Sutter, C. Dahlke, M.M. Addo, Persistence of MERS-CoV-spike-specific B cells and
antibodies after late third immunization with the MVA-MERS-S vaccine, Cell Reports Med 3 (2022), https://doi.org/10.1016/j.xcrm.2022.100685.
[35] M. Bosaeed, H.H. Balkhy, S. Almaziad, H.A. Aljami, H. Alhatmi, H. Alanazi, M. Alahmadi, A. Jawhary, M.W. Alenazi, A. Almasoud, R. Alanazi, M. Bittaye,
J. Aboagye, N. Albaalharith, S. Batawi, P. Folegatti, F. Ramos Lopez, K. Ewer, K. Almoaikel, M. Aljeraisy, A. Alothman, S.C. Gilbert, N. Khalaf Alharbi, Safety
and immunogenicity of ChAdOx1 MERS vaccine candidate in healthy Middle Eastern adults (MERS002): an open-label, non-randomised, dose-escalation,
phase 1b trial, The Lancet Microbe 3 (2022) e11–e20, https://doi.org/10.1016/S2666-5247(21)00193-2.
[36] N. Zhang, J. Shang, C. Li, K. Zhou, L. Du, An overview of Middle East respiratory syndrome coronavirus vaccines in preclinical studies, Expert Rev. Vaccines 19
(2020) 817–829, https://doi.org/10.1080/14760584.2020.1813574.
[37] L. Jiaming, Y. Yanfeng, D. Yao, H. Yawei, B. Linlin, H. Baoying, Y. Jinghua, G.F. Gao, Q. Chuan, T. Wenjie, The recombinant N-terminal domain of spike
proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection, Vaccine 35 (2017) 10–18, https://doi.org/
10.1016/j.vaccine.2016.11.064.
[38] E. Li, H. Chi, P. Huang, F. Yan, Y. Zhang, C. Liu, Z. Wang, G. Li, S. Zhang, R. Mo, H. Jin, H. Wang, N. Feng, J. Wang, Y. Bi, T. Wang, W. Sun, Y. Gao, Y. Zhao,
S. Yang, X. Xia, A novel bacterium-like particle vaccine displaying the MERS-CoV receptor-binding domain induces specific mucosal and systemic immune
responses in mice, Viruses 11 (2019), https://doi.org/10.3390/v11090799.
[39] B.S. Bodmer, A.H. Fiedler, J.R.H. Hanauer, S. Prüfer, M.D. Mühlebach, Live-attenuated bivalent measles virus-derived vaccines targeting Middle East
respiratory syndrome coronavirus induce robust and multifunctional T cell responses against both viruses in an appropriate mouse model, Virology 521 (2018)
99–107, https://doi.org/10.1016/j.virol.2018.05.028.
[40] P.W. Rose, A. Prlić, A. Altunkaya, C. Bi, A.R. Bradley, C.H. Christie, L. Di Costanzo, J.M. Duarte, S. Dutta, Z. Feng, R.K. Green, D.S. Goodsell, B. Hudson,
T. Kalro, R. Lowe, E. Peisach, C. Randle, A.S. Rose, C. Shao, Y.P. Tao, Y. Valasatava, M. Voigt, J.D. Westbrook, J. Woo, H. Yang, J.Y. Young, C. Zardecki, H.

13
S. Biswas et al. Heliyon 10 (2024) e25837

M. Berman, S.K. Burley, The RCSB protein data bank: integrative view of protein, gene and 3D structural information, Nucleic Acids Res. 45 (2017)
D271–D281, https://doi.org/10.1093/nar/gkw1000.
[41] D. Wl, The PyMOL molecular graphics system, CCP4 Newsl. Protein Crystallogr. 40 (2002) 82–92.
[42] D. Studio, Dassault Systemes BIOVIA, Discovery Studio Modelling Environment, Release 4.5, Accelrys Softw. Inc., 2015.
[43] H. Land, M.S. Humble, in: U.T. Bornscheuer, M. Höhne (Eds.), YASARA: A Tool to Obtain Structural Guidance in Biocatalytic Investigations BT - Protein
Engineering: Methods and Protocols, Springer New York, New York, NY, 2018, pp. 43–67, https://doi.org/10.1007/978-1-4939-7366-8_4.
[44] S. Kim, J. Chen, T. Cheng, A. Gindulyte, J. He, S. He, Q. Li, B. Shoemaker, P. Thiessen, B. U, L. Zaslavsky, J. Zhang, E. Bolton, PubChem substance and
compound databases, Nucleic Acids Res. 47 (D1) (2019) D1202–D1213. D11.
[45] T.A. Halgren, Performance of MMFF94*, scope, parameterization, J. Comput. Chem. 17 (1996) 490–519.
[46] S.C. Lin, C.T. Ho, W.H. Chuo, S. Li, T.T. Wang, C.C. Lin, Effective inhibition of MERS-CoV infection by resveratrol, BMC Infect. Dis. 17 (2017) 1–10, https://
doi.org/10.1186/s12879-017-2253-8.
[47] L. Sauerhering, A. Kupke, L. Meier, E. Dietzel, J. Hoppe, A.D. Gruber, S. Gattenloehner, B. Witte, L. Fink, N. Hofmann, T. Zimmermann, A. Goesmann, A. Nist,
T. Stiewe, S. Becker, S. Herold, C. Peteranderl, Cyclophilin inhibitors restrict Middle East respiratory syndrome coronavirus via interferon-λ in vitro and in
mice, Eur. Respir. J. 56 (2020), https://doi.org/10.1183/13993003.01826-2019.
[48] J.A. Al-Tawfiq, Z.A. Memish, Update on therapeutic options for Middle East respiratory syndrome coronavirus (MERS-CoV), Expert Rev. Anti Infect. Ther. 15
(2017) 269–275, https://doi.org/10.1080/14787210.2017.1271712.
[49] M. Yamamoto, S. Matsuyama, X. Li, M. Takeda, Y. Kawaguchi, J.I. Inoue, Z. Matsuda, Identification of nafamostat as a potent inhibitor of middle east
respiratory syndrome Coronavirus s protein-mediated membrane fusion using the split-protein-based cell-cell fusion assay, Antimicrob. Agents Chemother. 60
(2016) 6532–6539, https://doi.org/10.1128/AAC.01043-16.
[50] T.A.P. De Beer, K. Berka, J.M. Thornton, R.A. Laskowski, PDBsum additions, Nucleic Acids Res. 42 (2014), https://doi.org/10.1093/nar/gkt940.
[51] oleg Trott, Arthur J. Olson, AutoDock Vina, Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and
multithreading, J. Comput. Chem. 31 (2010) 455–461.
[52] F. Cheng, W. Li, Y. Zhou, J. Shen, Z. Wu, G. Liu, P.W. Lee, Y. Tang, AdmetSAR: a comprehensive source and free tool for assessment of chemical ADMET
properties, J. Chem. Inf. Model. 52 (2012) 3099–3105, https://doi.org/10.1021/ci300367a.
[53] A. Daina, O. Michielin, V. Zoete, SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small
molecules, Sci. Rep. 7 (2017) 1–13, https://doi.org/10.1038/srep42717.
[54] D.E.V. Pires, T.L. Blundell, D.B. Ascher, pkCSM: predicting small-molecule pharmacokinetic and toxicity properties using graph-based signatures, J. Med.
Chem. 58 (2015) 4066–4072, https://doi.org/10.1021/acs.jmedchem.5b00104.
[55] J. Wang, R.M. Wolf, J.W. Caldwell, P.A. Kollman, D.A. Case, Development and testing of a general Amber force field, J. Comput. Chem. 25 (2004) 1157–1174,
https://doi.org/10.1002/jcc.20035.
[56] M.F. Harrach, B. Drossel, Structure and dynamics of TIP3P, TIP4P, and TIP5P water near smooth and atomistic walls of different hydroaffinity, J. Chem. Phys.
140 (2014) 174501.
[57] U. Essmann, L. Perera, M.L. Berkowitz, T. Darden, H. Lee, L.G. Pedersen, A smooth particle mesh Ewald method, J. Chem. Phys. 103 (1995) 8577–8593,
https://doi.org/10.1063/1.470117.
[58] E. Krieger, J.E. Nielsen, C.A.E.M. Spronk, G. Vriend, Fast empirical pKa prediction by Ewald summation, J. Mol. Graph. Model. 25 (2006) 481–486, https://
doi.org/10.1016/j.jmgm.2006.02.009.
[59] M.J. Harvey, G. De Fabritiis, An implementation of the smooth particle mesh Ewald method on GPU hardware, J. Chem. Theor. Comput. 5 (2009) 2371–2377,
https://doi.org/10.1021/ct900275y.
[60] E. Krieger, G. Vriend, New ways to boost molecular dynamics simulations, J. Comput. Chem. 36 (2015) 996–1007.
[61] A. Mahfuz, M.A. Khan, S. Biswas, S. Afrose, S. Mahmud, N.M. Bahadur, F. Ahmed, In search of novel inhibitors of anti-cancer drug target fibroblast growth
factor receptors: insights from virtual screening, molecular docking, and molecular dynamics, Arab. J. Chem. 15 (2022) 103882.
[62] S. Mahmud, M.R. Hasan, S. Biswas, G.K. Paul, S. Afrose, M.A. Mita, M.S. Sultana Shimu, M.M. Promi, U. Hani, M. Rahamathulla, M.A. Khan, S. Zaman, M.
S. Uddin, M. Rahmatullah, R. Jahan, A.M. Alqahtani, M.A. Saleh, T. Bin Emran, Screening of potent phytochemical inhibitors against SARS-CoV-2 main
protease: an integrative computational approach, Front. Bioinforma. 1 (2021) 1–15, https://doi.org/10.3389/fbinf.2021.717141.
[63] S. Mahmud, M.A. Mita, S. Biswas, G.K. Paul, M.M. Promi, S. Afrose, R. Hasan, S.S. Shimu, S. Zaman, S. Uddin, T.E. Tallei, T. Bin Emran, A. Saleh, Molecular
docking and dynamics study to explore phytochemical ligand molecules against the main protease of SARS-CoV-2 from extensive phytochemical datasets,
Expet Rev. Clin. Pharmacol. 14 (2021) 1305–1315, https://doi.org/10.1080/17512433.2021.1959318.
[64] S. Mahmud, S. Biswas, G. Kumar Paul, M.A. Mita, S. Afrose, M. Robiul Hasan, M. Sharmin Sultana Shimu, M.A.R. Uddin, M. Salah Uddin, S. Zaman, K.
M. Kaderi Kibria, M. Arif Khan, T. Bin Emran, M. Abu Saleh, Antiviral peptides against the main protease of SARS-CoV-2: a molecular docking and dynamics
study, Arab. J. Chem. 14 (2021), https://doi.org/10.1016/j.arabjc.2021.103315.
[65] S. Mahmud, S. Biswas, G.K. Paul, M.A. Mita, M.M. Promi, S. Afrose, M.R. Hasan, S. Zaman, M.S. Uddin, K. Dhama, T. Bin Emran, M.A. Saleh, J. Simal-Gandara,
Plant-based phytochemical screening by targeting main protease of sars-cov-2 to design effective potent inhibitors, Biology 10 (2021), https://doi.org/
10.3390/biology10070589.
[66] S. Mahmud, M.O. Rafi, G.K. Paul, M.M. Promi, M.S.S. Shimu, S. Biswas, T. Bin Emran, K. Dhama, S.A. Alyami, M.A. Moni, Designing a multi-epitope vaccine
candidate to combat MERS-CoV by employing an immunoinformatics approach, Sci. Rep. 11 (2021) 15431.
[67] S. Mahmud, G.K. Paul, S. Biswas, S. Afrose, M.A. Mita, M.R. Hasan, M.S.S. Shimu, A. Hossain, M.M. Promi, F.K. Ema, K. Chidambaram, B. Chandrasekaran, A.
M. Alqahtani, T. Bin Emran, M.A. Saleh, Prospective role of peptide-based antiviral therapy against the main protease of SARS-CoV-2, Front. Mol. Biosci. 8
(2021), https://doi.org/10.3389/fmolb.2021.628585.
[68] S. Mahmud, G.K. Paul, M. Afroze, S. Islam, S.B.R. Gupt, M.H. Razu, S. Biswas, S. Zaman, M.S. Uddin, M. Khan, N.A. Cacciola, T. Bin Emran, M.A. Saleh,
R. Capasso, J. Simal-Gandara, Efficacy of phytochemicals derived from avicennia officinalis for the management of covid-19: a combined in silico and
biochemical study, Molecules 26 (2021), https://doi.org/10.3390/molecules26082210.
[69] S. Biswas, S. Mahmud, M.A. Mita, S. Afrose, Molecular Docking and Dynamics Studies to Explore Effective Inhibitory Peptides against the Spike Receptor
Binding Domain of SARS-CoV-2, vol. 8, 2022, pp. 1–10, https://doi.org/10.3389/fmolb.2021.791642.
[70] G.K. Paul, S. Mahmud, A.A. Aldahish, M. Afroze, S. Biswas, S.B.R. Gupta, M.H. Razu, S. Zaman, M.S. Uddin, M.H. Nahari, Computational screening and
biochemical analysis of Pistacia integerrima and Pandanus odorifer plants to find effective inhibitors against Receptor-Binding domain (RBD) of the spike
protein of SARS-Cov-2, Arab. J. Chem. 15 (2022) 103600.
[71] I. Massova, P.A. Kollman, Combined molecular mechanical and continuum solvent approach (MM- PBSA/GBSA) to predict ligand binding, Perspect. Drug
Discov. Des. 18 (2000) 113–135, https://doi.org/10.1023/A:1008763014207.
[72] A. Rakib, Z. Nain, S.A. Sami, S. Mahmud, A. Islam, S. Ahmed, A.B.F. Siddiqui, S.M.O.F. Babu, P. Hossain, A. Shahriar, F. Nainu, T. Bin Emran, J. Simal-Gandara,
A molecular modelling approach for identifying antiviral selenium-containing heterocyclic compounds that inhibit the main protease of SARS-CoV-2: an in
silico investigation, Briefings Bioinf. 22 (2021) 1476–1498, https://doi.org/10.1093/bib/bbab045.
[73] A.C. Galasiti Kankanamalage, Y. Kim, V.C. Damalanka, A.D. Rathnayake, A.R. Fehr, N. Mehzabeen, K.P. Battaile, S. Lovell, G.H. Lushington, S. Perlman, K.
O. Chang, W.C. Groutas, Structure-guided design of potent and permeable inhibitors of MERS coronavirus 3CL protease that utilize a piperidine moiety as a
novel design element, Eur. J. Med. Chem. 150 (2018) 334–346, https://doi.org/10.1016/j.ejmech.2018.03.004.
[74] M. Tareq Hassan Khan, Predictions of the ADMET properties of candidate drug molecules utilizing different QSAR/QSPR modelling approaches, Curr. Drug
Metabol. 11 (2010) 285–295, https://doi.org/10.2174/138920010791514306.
[75] D. Lagorce, D. Douguet, M.A. Miteva, B.O. Villoutreix, Computational analysis of calculated physicochemical and ADMET properties of protein-protein
interaction inhibitors, Sci. Rep. 7 (2017) 46277.

14
S. Biswas et al. Heliyon 10 (2024) e25837

[76] G. Klopman, L.R. Stefan, R.D. Saiakhov, ADME evaluation: 2. A computer model for the prediction of intestinal absorption in humans, Eur. J. Pharmaceut. Sci.
17 (2002) 253–263, https://doi.org/10.1016/S0928-0987(02)00219-1.
[77] L. Guan, H. Yang, Y. Cai, L. Sun, P. Di, W. Li, G. Liu, Y. Tang, ADMET-score-a comprehensive scoring function for evaluation of chemical drug-likeness,
Medchemcomm 10 (2019) 148–157, https://doi.org/10.1039/C8MD00472B.
[78] Y. Han, J. Zhang, C. Hu, A systematic toxicity evaluation of cephalosporins via transcriptomics in zebrafish and in silico ADMET studies, Food Chem. Toxicol.
116 (2018) 264–271, https://doi.org/10.1016/j.fct.2018.04.046.
[79] S. Islam, L. Mahmud, W.H. Almalki, S. Biswas, A. Islam, Cell-Free Supernatants (CFSs) from the Culture of Bacillus Subtilis Inhibit Pseudomonas Sp, Biofilm
Formation, 2022, pp. 1–14.
[80] K. Ramanathan, D. Antognini, A. Combes, M. Paden, B. Zakhary, M. Ogino, G. Maclaren, D. Brodie, Middle East respiratory syndrome coronavirus:
quantification of the extent of the epidemic, surveillance biases, and transmissibility, Lancet Infect. Dis. 14 (2014) 50–56.
[81] A. Sharif-Yakan, S.S. Kanj, Emergence of MERS-CoV in the Middle East: origins, transmission, treatment, and perspectives, PLoS Pathog. 10 (2014), https://
doi.org/10.1371/journal.ppat.1004457.
[82] D. Forni, G. Filippi, R. Cagliani, L. De Gioia, U. Pozzoli, N. Al-Daghri, M. Clerici, M. Sironi, The heptad repeat region is a major selection target in MERS-CoV
and related coronaviruses, Sci. Rep. 5 (2015) 1–10, https://doi.org/10.1038/srep14480.
[83] A. Mubarak, W. Alturaiki, M.G. Hemida, Middle east respiratory syndrome coronavirus (mers-cov): infection, immunological response, and vaccine
development, J. Immunol. Res. 2019 (2019), https://doi.org/10.1155/2019/6491738.
[84] J. Shi, J. Sivaraman, J. Song, Mechanism for controlling the dimer-monomer switch and coupling dimerization to catalysis of the severe acute respiratory
syndrome coronavirus 3C-like protease, J. Virol. 82 (2008) 4620–4629, https://doi.org/10.1128/jvi.02680-07.
[85] C.P. Chuck, H.F. Chow, D.C.C. Wan, K.B. Wong, Profiling of substrate specificities of 3C-like proteases from group 1, 2a, 2b, and 3 coronaviruses, PLoS One 6
(2011), https://doi.org/10.1371/journal.pone.0027228.
[86] S.E. St John, S. Tomar, S.R. Stauffer, A.D. Mesecar, Targeting zoonotic viruses: structure-based inhibition of the 3C-like protease from bat coronavirus HKU4–
The likely reservoir host to the human coronavirus that causes Middle East Respiratory Syndrome (MERS), Bioorg. Med. Chem. 23 (2015) 6036–6048, https://
doi.org/10.1016/j.bmc.2015.06.039.
[87] K. Anand, J. Ziebuhr, P. Wadhwani, J.R. Mesters, R. Hilgenfeld, Coronavirus main proteinase (3CLpro) Structure: basis for design of anti-SARS drugs, Science
300 (2003) 1763–1767, https://doi.org/10.1126/science.1085658.
[88] S. Baba, Y. Ueno, T. Kikuchi, R. Tanaka, K. Fujimori, A limonoid Kihadanin B from immature Citrus unshiu peels suppresses adipogenesis through repression of
the Akt-FOXO1-PPARγ Axis in adipocytes, J. Agric. Food Chem. 64 (2016) 9607–9615, https://doi.org/10.1021/acs.jafc.6b04521.
[89] A.T. Mbaveng, C.G.T. Noulala, A.R.M. Samba, S.B. Tankeo, G.W. Fotso, E.N. Happi, B.T. Ngadjui, V.P. Beng, V. Kuete, T. Efferth, Cytotoxicity of botanicals and
isolated phytochemicals from Araliopsis soyauxii Engl.(Rutaceae) towards a panel of human cancer cells, J. Ethnopharmacol. 267 (2021) 113535.
[90] H. Kawaguchi, M. Kim, M. Ishida, Y.-J. Ahn, T. Yamamoto, R. Yamaoka, M. Kozuka, K. Goto, S. Takahashi, Several antifeedants from Phellodendron amurense
against Reticulitermes speratus, Agric. Biol. Chem. 53 (1989) 2635–2640.
[91] X.-X. Guo, L.-N. Zhao, J. Wang, S. Liu, Q.-R. Bi, Z. Wang, N.-H. Tan, Chemical constituents from root barks of Dictamnus dasycarpus and their cytotoxic
activities, Zhongguo Zhong Yao Za Zhi= Zhongguo Zhongyao Zazhi= China J. Chinese Mater. Medica. 43 (2018) 4869–4877.
[92] A. Jo, H.J. Yoo, M. Lee, Robustaflavone isolated from nandina domestica using bioactivity-guided fractionation downregulates inflammatory mediators,
Molecules 24 (2019) 2–13, https://doi.org/10.3390/molecules24091789.
[93] N.S.S. Ambarwati, B. Elya, A. Malik, H. Omar, M. Hanafi, I. Ahmad, New robustaflavone from Garcinia latissima Miq. leave and its antibacterial activity,
J. Adv. Pharm. Technol. Res. 13 (2022) 50.
[94] D.E. Zembower, Y.M. Lin, M.T. Flavin, Fa-Ching Chen, B.E. Korba, Robustaflavone, a potential non-nucleoside anti-hepatitis B agent, Antivir. Res. 39 (1998)
81–88, https://doi.org/10.1016/S0166-3542(98)00033-3.
[95] Y. Ma, Y. Wang, H. Zhang, W. Sun, Z. Li, F. Zhang, H. Zhang, F. Chen, H. Zhang, J. An, C. He, Antimicrobial mechanism of strictinin isomers extracted from the
root of Rosa roxburghii Tratt (Ci Li Gen), J. Ethnopharmacol. 250 (2020) 112498, https://doi.org/10.1016/j.jep.2019.112498.
[96] M.M. Rahman, M.B. Hosen, M.Z.H. Howlader, Y. Kabir, Lead molecule prediction and characterization for designing MERS-CoV 3C-like protease inhibitors: an
in silico approach, Curr. Comput. Aided Drug Des. 15 (2019) 82–88.
[97] A.A. Radwan, F.K. Alanazi, In silico studies on novel inhibitors of MERS-CoV: structure-based pharmacophore modeling, database screening and molecular
docking, Trop. J. Pharmaceut. Res. 17 (2018) 513–517, https://doi.org/10.4314/tjpr.v17i3.18.
[98] A.C. Galasiti Kankanamalage, Y. Kim, V.C. Damalanka, A.D. Rathnayake, A.R. Fehr, N. Mehzabeen, K.P. Battaile, S. Lovell, G.H. Lushington, S. Perlman, K.
O. Chang, W.C. Groutas, Structure-guided design of potent and permeable inhibitors of MERS coronavirus 3CL protease that utilize a piperidine moiety as a
novel design element, Eur. J. Med. Chem. 150 (2018) 334–346, https://doi.org/10.1016/j.ejmech.2018.03.004.
[99] C. Ghosh, M. Saha, S. Kulavi, S. Chatterjee, In-silico screening of some important bioactive compounds as potential inhibitors of 3CLpro protein of SARS-CoV-2
and MERS-CoV virus, Int. J. Pharm. Sci. Nanotechnol. 15 (2022) 6043–6054.
[100] W.K. Sim, J.H. Park, K.Y. Kim, I.S. Chung, Robustaflavone induces G0/G1 cell cycle arrest and apoptosis in human umbilical vein endothelial cells and exhibits
anti-angiogenic effects in vivo, Sci. Rep. 10 (2020) 1–12, https://doi.org/10.1038/s41598-020-67993-5.

15