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Volume 65, Issue 5, 2021
Journal of Scientific Research
of
The Banaras Hindu University
96
DOI: 10.37398/JSR.2021.650511
Abstract: Coronavirus disease 2019 (COVID-19) pandemic has
infected billions and has killed millions of people. Despite the
advancement in drugs and vaccines, the COVID-19 pandemic rages
on. Bioactive compounds of medicinal plants with antiviral
properties such as flavonoids might be useful as phytotherapy for
COVID-19. The present study aims to evaluate 16 flavonoids and
identify a potential inhibitor for the main protease of severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2 3CL pro) which
is responsible for replication of the viral genome in a host cell.
Among the 16 flavonoids, molecular docking simulation studies
reveal that rutin has the highest binding affinity towards chain A,
chain B, chain C, and chain D of SARS-CoV-2 3CL pro. Rutin
formed conventional hydrogen bonds and non-covalent
hydrophobic bonds with important amino acid residues at the
active binding site of SARS-CoV-2 3CL pro. Also, rutin was
computed to be non-toxic. Rutin had a better drug-likeness score
and protease inhibition than the co-crystal inhibitor of SARS-CoV-
2 3CL pro. The structure-activity relationship revealed important
moieties of rutin that might be essential for the inhibition of SARS-
CoV-2 3CL pro. The in-silico evidence suggests rutin may inhibit
SARS-CoV-2 3CL pro and advocates its use as a phytotherapy for
COVID-19.
Index Terms: COVID-19, Molecular docking, Rutin, SARS-CoV-
2, Structure-activity relationship
I. INTRODUCTION
At present, the world is facing an unprecedented physical,
mental and emotional burden due to the ongoing coronavirus
disease 2019 (COVID-19) pandemic that is caused by severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Hu et
al., 2020). As of 11th May 2021, the COVID-19 pandemic has
*
Corresponding Author
infected 1,58,651,638 people and has killed more than 3.29
million people (WHO, 2021a). Many drugs are currently being
used to treat COVID-19 (Singh et al., 2021). Also, many
vaccines are currently being used to treat COVID-19 infection
(WHO, 2021b).
Despite the recent advancements in pharmacotherapy and
vaccines for COVID-19, several countries remained severely
affected by COVID-19. Problems such as adverse effects,
toxicity, and drug interactions seem to be associated with
repurposed drugs that are intended for the treatment of COVID-
19 (Shende et al., 2020). Therefore, additional therapies for
COVID-19 that are safe and effective seem to be the need of the
hour (Shende et al., 2020). Interestingly, bioactive compounds
from medicinal plants might be able to offer a safe and effective
therapy for COVID-19 (Islam et al., 2020; Antonio et al., 2020).
This is because bioactive compounds have been successfully
used to treat many viral diseases (Narkhede et al., 2020; Kim et
al., 2014; Tahir Ul Qamar et al., 2019). Thus, medicinal plants
and their bioactive compounds offer promising phytotherapy for
COVID-19 (Narkhede et al., 2020).
The main protease of SARS-CoV-2 is one of the most
favorable drug targets to treat COVID-19 (Jin et al., 2020;
Prajapat et al., 2020; Robson, 2020). This is because the main
protease of SARS-CoV-2 is essential for the replication of viral
RNA within the host cell (Jin et al., 2020). Once the viral RNA
of SARS-CoV-2 enters a host cell, the polyproteins that are
translated from the viral genome are cleaved by the main
protease of SARS-CoV-2 which is followed by transcription and
replication of the viral genome (Huang et al., 2020; Jin et al.,
2020; Gildenhuys, 2020).
Flavonoids have been extensively studied for their antiviral
properties (Zakaryan et al., 2017; Ninfali et al., 2020). In the
current study, the inhibitory potential of 16 flavonoids against
the main protease of SARS-CoV-2 was evaluated with in-silico
Molecular Docking Simulation Studies,
Toxicity Study, Bioactivity Prediction, and
Structure-Activity Relationship Reveals Rutin
as a Potential Inhibitor of SARS-CoV-2 3CL pro
James H. Zothantluanga*1
1Department of Pharmaceutical Sciences, Faculty of Science and Engineering, Dibrugarh University, Dibrugarh 786004, Assam, India,
jameshztta@gmail.com
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techniques.
II. MATERIALS AND METHODS
A. Retrieval of a target protein
The main protease of SARS-CoV-2 was used as the target
protein in the study. The crystal structure of SARS-CoV-2 3CL
pro with a protein data bank (PDB) ID of 6M2N was retrieved
from the Research Collaboratory for Structural Bioinformatics
(RCSB)-PDB website (RCSB-PDB, 2021). The main protease of
SARS-CoV-2 was found to have four chains (chain A, chain B,
chain C, and chain D). The co-crystal inhibitor (CCI) was also
identified and retrieved from the PubChem database (PubChem,
2021).
B. Pre-processing of protein
The target protein was pre-processed with BIOVIA Discovery
Studio Visualizer v20.1.0.19295 software. All the chains (A, B,
C, and D) of the target protein were individually processed.
Initially, water was removed from the target protein. Then, the
active binding site was defined with the ‘Define and edit binding
site’ feature of the Discovery Studio visualizer software. The 3-
dimensional attributes (XYZ coordinates) of the active binding
site were identified and saved for further use. Then, the CCI of
the target protein was removed. Finally, polar hydrogen was
added to the target protein with Discovery Studio visualizer
software. The processed target protein was saved in PDB format
for future use.
C. Preparation of compound library
The 3-dimensional (3D) structures of 16 flavonoids were
retrieved from the PubChem database (PubChem, 2021). The
structure of each flavonoid was saved in structure-data file
format and their PubChem compound ID (CID) was also saved.
D. Energy minimization of ligands
Energy minimization of ligands was carried out on the PyRx
0.8 tool. To minimize the energy of the ligands used in the
present study, the default parameters of the PyRx 0.8 tool was
used (Force field = Universal force field; Optimization algorithm
= Conjugate gradients; Total no. of steps = 200; No. of steps for
update = 1; Stop if energy difference is less than = 0.1).
E. Detection and coordinates of the active binding site
In all the four chains (A, B, C, and D) of SARS-CoV-2 3CL
pro, a CCI was found to be present at the active binding site. At
first, chain A of the pre-processed target protein that was
initially prepared with the Discovery Studio visualizer software
was loaded onto the PyRx virtual screening platform. This
particular protein was then converted into ‘pdbqt.’ Format with
the PyRx software.
Then, the original target protein complexed with the CCI
which did not undergo any processing was also loaded onto the
PyRx virtual screening platform. The chains that made up
protein complexed with the CCI were revealed by expanding the
protein. Initially, chain B, chain C, and, chain D were removed
from the scene. Then, chain A of the pre-processed target protein
and chain A of the target protein complexed with the CCI was
automatically made to superimpose by the PyRx tool.
Following this, the chain A of the target protein complexed
with the CCI that was not pre-processed was expanded and the
sequence of all the amino acids residues that made up the chain
including the CCI (3WL) was revealed. The location of the CCI
present at the active binding site of the target protein complexed
with the CCI was detected by labelling the atoms of the CCI.
Then, the 3D affinity gird box was carefully made to align
with the central part of the CCI to cover the entire active site
residues by considering (i) the active binding site coordinates of
the pre-processed target protein previously identified with the
Discovery Studio visualizer software and (ii) the location of the
CCI that was made visible on the protein. In this way, the entire
amino acid residues present at the active binding site of the
protein were covered by the 3D affinity grid box.
Finally, the chain A of the target protein complexed with the
CCI that was not pre-processed was removed from the scene.
However, the chain A of the pre-processed target protein was
kept for MDSS. The active binding site of chain B, chain C, and,
chain D of SARS-CoV-2 3CL pro was detected similarly. The
size of the 3D affinity grid box was kept default at 25 Å for the
entire simulation process. The coordinates of the active binding
site of each chain are described below.
1) Chain A of SARS-CoV-2 3CL pro
In the Vina search space, the coordinates at the active binding
site of the protein were x = -33.9203241113, y = -
64.4918230137, z = 40.5838306859.
2) Chain B of SARS-CoV-2 3CL pro
In the Vina search space, the coordinates at the active binding
site of the protein was x = -48.4728236411, y = -
1.05170930781, z = -6.07895092506.
3) Chain C of SARS-CoV-2 3CL pro
In the Vina search space, the coordinates at the active binding
site of the protein were x = -40.7113525071, y = -
21.7108783003, z = 55.108989351.
4) Chain D of SARS-CoV-2 3CL pro
In the Vina search space, the coordinates at the active binding
site of the protein were x = -61.8523694685, y = -
35.3575321951, z = 22.9711975727.
F. Molecular docking simulation studies
In the present study, molecular docking simulation studies
(MDSS) were carried out for 16 flavonoids and the CCI using
AutoDock Vina (Trott & Olson, 2010) on a virtual screening
tool known as PyRx 0.8 tool. Other than those specified, the
standard operating procedure of PyRx tool was followed for the
MDSS (Dallakyan & Olson, 2015).
The flavonoid which showed the highest binding affinity
towards chain A, chain B, chain C, and chain D of SARS-CoV-2
3CL pro was selected for further analysis.
G. Visualization and analysis of ligand interactions
The ligand interactions formed by the flavonoid with the
amino acid residues at the active binding site of SARS-CoV-2
3CL pro were analyzed with BIOVIA Discovery Studio
Visualizer v20.1.0.19295 software. Visualization of the ligand
interactions was done in a 2-dimensional (2D) and 3D view. The
3D binding pose of the flavonoid and the CCI at the active
binding site of the target protein was generated using PyMOL
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molecular graphics system, Version 2.4.1 Schrodinger, LLC
software. The ligand interactions between the CCI and SARS-
CoV-2 3CL pro were also visualized and analyzed using the
same technique as described above.
H. Validation of molecular docking via re-docking,
superimposing, and RMSD calculation
The molecular docking protocol used in the present study was
validated by a re-docking method (Shivanika et al., 2020). To
validate the docking protocol, the CCI was re-docked into the
active binding site of each chain of the target protein using the
same protocols that were previously used for docking the
flavonoids to the target protein. After re-docking was completed,
the ‘pdbqt.’ output of the re-docked ligand was opened with a
text document and all the information on the ligand was copied.
The ‘pdbqt.’ output of the target protein was opened with a text
document and the information on the re-docked ligand was
pasted at the end. This text document was saved for further use.
Finally, the re-docked protein-CCI complex was converted to
PDB file format from the ‘pdbqt.’ file format. This process was
carried out for all the chains of the target protein.
At the same time, the original protein-CCI complex was
processed with the Discovery Studio Visualizer software to
obtain chain A complexed with the CCI, chain B complexed
with the CCI, chain C complexed with the CCI and chain D
complexed with the CCI. It may be noted that all the chains of
the target protein are originally complexed with CCI. The
original protein-CCI complex (chain A) and the re-docked
protein-ligand complex (chain A) were opened simultaneously
on the Discovery Studio visualizer software. The sequence
alignment of the target proteins was created following which
they were made to superimpose on each other. The root mean
square deviation (RMSD) between the original protein-CCI
complex and the re-docked protein-CCI complex was calculated.
Also, the superimposed protein-CCI image was generated with
the Discovery Studio Visualizer software. This process was
repeated for all the chains of SARS-CoV-2 3CL pro.
I. Toxicity analysis and drug-likeness
The toxicity and drug-likeness of the flavonoid were
evaluated with Data Warrior v.5.2.1 software. Toxicities such as
mutagenicity, tumorigenic, reproductive effective and irritant of
the flavonoid were analyzed. The toxicity and drug-likeness of
the CCI were also analyzed.
J. Protease inhibition
The protease inhibition of the flavonoid with the highest
binding affinity towards SARS-CoV-2 3CL pro was predicted
with the Molinspiration Chemoinformatics web tool
(Molinspiration, 2021). The protease inhibition of the CCI was
also calculated.
K. Structure-activity relationship
The contribution of chemical groups, atoms, and sugar moiety
of the flavonoid towards protease inhibition was studied. The
structure of the flavonoid was modified at certain points using
Marvin Sketch 20.10 software. The SMILES ID of each
modified flavonoid structure was generated and its protease
inhibition was evaluated with the Molinspiration
Chemoinformatics web tool (Molinspiration, 2021). In this way,
a preliminary structure-activity relationship (SAR) study was
carried out for rutin.
III. RESULTS AND DISCUSSION
A. Features of the target protein
The crystal structure of the SARS-CoV-2 3CL pro is available
from the RCSB-PDB website. It is made up of four chains i.e.
chain A, chain B, chain C and chain D. The sequence length of
SARS-CoV-2 3CL pro is 306. All the chains are complexed with
a co-crystal inhibitor viz. 3WL. 3WL is a flavonoid known as
baicalein and bears a PubChem CID of 5281605. In the present
study, 3WL was used as a standard drug whenever and wherever
necessary.
B. Details of the flavonoid library
A total of 16 flavonoids were selected to be screened against
SARS-CoV-2 3CL pro. The library of flavonoids to be used in
the study included apigenin (5280443), aromadendrin (122850),
eriodictyol (440735), fisetin (5281614), hesperetin (72281),
isorhamnetin (5281654), kaempferol (5280863), luteolin
(5280445), myricetin (5281672), naringenin (932), pachypodol
(5281677), quercetin (5280343), rhamnazin (5320945), rutin
(5280805), tangeretin (68077) and taxifolin (439533). The co-
crystal inhibitor that is complexed with the target protein is also
a flavonoid.
C. Molecular docking and binding affinities of flavonoids
Molecular docking is a computational technique that predicts
possible interactions between a drug and a protein. It gives an
idea of the inhibitory potential of a drug against a protein
involved in a disease network (Meng et al., 2012). When MDSS
is carried out on a PyRx tool, it provides a binding affinity value
(kcal/mol) for each ligand so that the binding potential of ligands
toward a protein can be ranked (Dallakyan & Olson, 2015).
The binding affinities of all the flavonoids towards chain A,
chain B, chain C, and chain D of SARS-CoV-2 3CL pro are
given in Table I. The binding affinity of the 3WL is also
included in Table I. For each ligand, PyRx tool generates a total
of 9 poses at the active binding site of the target protein. A more
negative binding affinity value suggests a better binding between
a compound and a protein (Dallakyan & Olson, 2015). A low
binding affinity value also indicates the low energy requirement
for protein-ligand binding (Azam & Abbasi, 2013). In all cases,
the first pose is considered the best pose since it has the highest
binding affinity towards the target protein. The ninth pose has
the lowest binding affinity towards the target protein. Based on
this, the first pose and its binding affinity value were considered
for the study.
Table I. Binding affinities of the flavonoids and the CCI towards the
active binding site of different chains of SARS-CoV-2
Compounds
Binding affinity (kcal/mol)
Chain A
Chain B
Chain C
Chain D
3WL
-7.3
-7.5
-7.4
-7.3
Apigenin
-7.3
-7.6
-7.1
-7.2
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Aromadendrin
-7.5
-7.5
-7.0
-6.9
Eriodictyol
-7.6
-8.2
-7.4
-7.4
Fisetin
-7.7
-8.6
-7.1
-7.3
Hesperetin
-7.6
-8.0
-7.0
-7.1
Isorhamnetin
-7.5
-7.5
-7.2
-7.2
Kaempferol
-7.3
-7.4
-7.1
-6.8
Luteolin
-7.8
-8.6
-7.3
-7.4
Myricetin
-7.6
-8.4
-7.4
-7.6
Naringenin
-7.2
-7.6
-6.6
-7.1
Pachypodol
-7.0
-7.0
-7.3
-7.5
Quercetin
-7.9
-8.7
-7.4
-7.4
Rhamnazin
-7.4
-7.2
-7.1
-7.5
Rutin
-8.5
-9.3
-8.7
-9.7
Tangeretin
-7.1
-6.4
-6.4
-6.9
Taxifolin
-7.6
-8.1
-7.3
-7.1
Among all the flavonoids, rutin has the highest binding
affinity towards chain A (-8.5 kcal/mol), chain B (-9.3 kcal/mol),
chain C (-8.7 kcal/mol), and chain D (-9.3 kcal/mol) of SARS-
CoV-2 3CL pro. This suggests that in comparison to other
flavonoids, rutin will bind easily to the active binding site of
different chains of the target protein with minimum energy
involvement.
3WL is the CCI that is present at the active binding site of all
the chains of the target protein. In comparison to 3WL, it was
also observed that rutin had a better binding affinity towards all
the chains of SARS-CoV-2 3CL pro. Thus, the MDSS study
revealed that rutin has the best potential to bind and interact with
the amino acid residues at the active binding site of the target
protein. Based on this evidence, rutin was selected for further
analysis.
D. Molecular interactions between different chains of SARS-
CoV-2 3CL pro and rutin
A good understanding of the interaction between a protein and
a ligand is important in the field of drug development (Du et al.,
2016). The 2D and 3D ligand interactions along with the 3D
binding pose of rutin and 3WL at the active binding site of chain
A, chain B, chain C, and chain D of SARS-CoV-2 3CL pro are
given in Fig. 1, 2, 3, and 4 respectively. The ligand interactions
of rutin and 3WL with the amino acid residues at the active
binding site of different chains of SARS-CoV-2 3CL pro are
given in Table II. Generally, rutin was found to interact with
more amino acid residues at the active binding site than 3WL.
Table II. Interacting active site residues of SARS-CoV-2 3CL pro with
3WL and rutin
SARS-CoV-2
3CL pro
Ligand interactions
3WL
Rutin
Chain A
GLY143, GLU166,
MET165, CYS145,
ASN142, HIS163
MET49, GLU166,
ASP187, ARG188,
GLN189, THR190,
GLN192, HIS41, CYS44
Chain B
LEU141, SER144,
CYS145, GLU166,
HIS41, CYS44, MET49
HIS41, THR24, CYS44,
MET49, CYS145,
ASN142
Chain C
ASN142, GLY143,
SER144, GLU166,
CYS145, MET165
LEU141, SER144,
CYS145, MET165,
GLU166
Chain D
GLY143, SER144,
HIS163, GLU166,
CYS145, MET165
THR26, HIS41, TYR54,
LEU141, ASN142,
GLU166, ASP187,
MET49, CYS44,
HIS164, MET165,
ARG188
In chain A, rutin interacted with 9 amino acid residues
(MET49, GLU166, ASP187, ARG188, GLN189, THR190,
GLN192, HIS41, and CYS44) while 3WL interacted with 6
amino acid residues (GLY143, GLU166, MET165, CYS145,
ASN142, and HIS163) (Fig. 1, Table II). Both rutin and 3WL
interacted with GLU166 (Table II). The hydroxyl (-OH) group at
position 13 of rutin interacted with MET49 and ASP187 through
a conventional hydrogen bond. The -OH at position 19 of rutin
interacted with GLU166 through a conventional hydrogen bond.
The -OH group at positions 28 and 29 of rutin interacted with
ARG188 and GLN192 through conventional hydrogen bonds
respectively. The -OH group at position 30 of rutin interacted
with GLN189 and THR190 through a conventional hydrogen
bond. The A-ring of the C6-C3-C6 scaffold of rutin formed pi-
sigma interaction with MET49, pi-pi interaction with HIS41, and
pi-sulfur interaction with CYS44. The C-ring of the C6-C3-C6
scaffold of rutin also formed pi-alkyl interaction with MET49
and pi-pi interaction with HIS41.
In chain B, rutin (HIS41, THR24, CYS44, MET49, CYS145,
ASN142) and 3WL (LEU141, SER144, CYS145, GLU166,
HIS41, CYS44, MET49) interacted with 6 and 7 amino acid
residues respectively (Fig. 2, Table II). Both rutin and 3WL
interacted with CYS44, HIS41, MET49, and CYS145 (Table II).
The -OH group at position 13 and the oxygen atom at position
43 of rutin interacted with HIS41 through a conventional
hydrogen bond. The -OH group at position 40 of rutin interacted
with THR24 through a conventional hydrogen bond. The A-ring
and C-ring of the C6-C3-C6 scaffold of rutin formed pi-pi
interaction with HIS41. The A-ring of the C6-C3-C6 scaffold of
rutin formed pi-alkyl interaction with MET49 while the B-ring
and C-ring of rutin formed pi-alkyl interaction with CYS145.
The A-ring of rutin formed pi-sulfur interaction with CYS44.
In chain C, rutin interacted with 5 amino acid residues
(LEU141, SER144, CYS145, MET165, GLU166) while 3WL
interacted with 6 amino acid residues (ASN142, GLY143,
SER144, GLU166, CYS145, MET165) (Fig. 3, Table II). Both
rutin and 3WL interacted with SER144, CYS145, MET165, and
GLU166 (Table II). The -OH group at position 19 of rutin
interacted with SER144 through a conventional hydrogen bond.
The -OH group at position 20 of rutin interacted with LEU141
through a conventional hydrogen bond. The B-ring and C-ring of
the C6-C3-C6 scaffold of rutin formed pi-alkyl interaction with
CYS145 and MET165 respectively.
In addition to hydrogen bonds, non-covalent bonds such as
hydrophobic interactions and electrostatic interactions are also
considered important for the stabilization of protein-ligand
complexes (de Freitas & Schapira, 2017; Kumar & Nussinov,
2002). Rutin showed non-covalent interactions (pi-pi, pi-alkyl,
and pi-sulfur) with different amino acid residues at the active
binding site of different chains of SARS-CoV-2 3CL pro (Fig. 1,
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Fig. 2, Fig. 3 and Fig. 4). Thus, rutin might be able to inhibit
SARS-CoV-2 3CL pro as it was found to have a good binding
affinity towards all the different chains. In comparison to 3WL,
rutin showed better molecular interactions with different amino
acid residues at the active binding site of SARS-CoV-2 3CL pro.
Based on the above findings, rutin was subjected to further
analysis.
Fig. 1. Chain A: 2D ligand interactions (a, c), 3D ligand interactions (b,
d), and 3D binding pose (e) (blue=3WL, red=Rutin) of 3WL (a, b, e)
and rutin (c, d, e) at the active binding site of SARS-CoV-2 3CL pro.
Fig. 2. Chain B: 2D ligand interactions (a, c), 3D ligand interactions (b,
d), and 3D binding pose (e) (blue=3WL, red=Rutin) of 3WL (a, b, e)
and rutin (c, d, e) at the active binding site of SARS-CoV-2 3CL pro.
Fig. 3. Chain C: 2D ligand interactions (a, c), 3D ligand interactions (b,
d), and 3D binding pose (e) (blue=3WL, red=Rutin) of 3WL (a, b, e)
and rutin (c, d, e) at the active binding site of SARS-CoV-2 3CL pro.
Fig. 4. Chain D: 2D ligand interactions (a, c), 3D ligand interactions (b,
d), and 3D binding pose (e) (blue=3WL, red=Rutin) of 3WL (a, b, e)
and rutin (c, d, e) at the active binding site of SARS-CoV-2 3CL pro.
E. Validation of docking
The purpose of this study was to evaluate the efficiency of the
docking protocol used in the present study. The docking protocol
was validated through re-docking of the CCI to the active
binding site of the target protein using the same parameters that
were used to dock the flavonoids. The re-docked protein-ligand
complex was then superimposed to the original protein-ligand
complex. The superimposed image of the protein-ligand
complex is given in Fig. 5.
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The RMSD of the re-docked protein-ligand complex was
calculated by using the original protein-ligand complex as the
reference protein. Several researchers had also validated their
docking protocol with this method (Shivanika et al. 2020). A
low RMSD value is preferred over a higher value to provide
validation to the docking protocol.
In the present study, an RMSD value of 0.00 was obtained for
all the docking procedures used for docking the CCI towards the
active binding site of chain A, chain B, chain C, and chain D.
Re-docking showed that the CCI had a binding affinity of -7.3
kcal/mol, -7.5 kcal/mol, -7.4 kcal/mol, and -7.3 kcal/mol
towards chain A, chain B, chain C, and chain D respectively.
The binding affinity values obtained from re-docking are similar
to the value previously obtained in the first docking (Table I).
Also, the docking protocol used for the present study allowed the
binding of CCI into the active binding site of the target protein
as evidence by re-docking (Fig. 5). This proves the efficiency
and validity of the docking protocol followed in the present
study.
Fig. 5. Superimposition of re-docked protein-3WL complex (3WL=red
color) with the original co-crystallized complex (3WL=blue color) for
chain A (a), chain B (b), chain C (c) and chain D (d) of SARS-CoV-2
3CL pro.
F. Toxicity analysis and drug-likeness
Toxicity is one of the main reasons behind the withdrawal of
many drugs from the market (Guengerich, 2011). Drug-likeness
is a parameter that indicates the possibility of a molecule to
become an orally bioavailable drug (Daina et al., 2017).
Therefore, it may be considered logical to assess the potential
toxicity and drug-likeness of a compound at the initial stage of a
study. The potential mutagenicity, tumorigenic, reproductive
effective and irritant of rutin and 3WL are given in Table III.
The drug-likeness of rutin and 3WL are also included in Table
III. It was observed that rutin was free from all possible
toxicities. Also, the drug-likeness of rutin was higher than 3WL.
Based on these findings, rutin was subjected to further analysis.
Table III. Toxicity and drug-likeness of 3WL and rutin
Property
3WL
Rutin
Mutagenicity
None
None
Tumorigenic
None
None
Reproductive effective
None
None
Irritant
None
None
Drug-likeness
0.28194
1.9337
G. Protease inhibition
The protease inhibition of rutin was predicted and it was
compared to that of 3WL (Table IV). SARS-CoV-2 3CL pro is a
viral protease (Prajapat et al., 2020). Although the predicted
protease inhibition might not necessarily correlate to the
inhibition of the main protease of SARS-CoV-2, it provides a
preliminary idea of the potential bioactivity of rutin. With a
bioactivity score of -0.07, rutin has a better protease inhibition
than 3WL (-0.35). Based on these findings, the structure-activity
relationship of rutin concerning its protease inhibition was
studied.
Table IV. Protease inhibitory potential of 3WL and rutin
Compound
Protease inhibition
3WL
-0.35
Rutin
-0.07
H. Structure-activity relationship
Preliminary SAR studies can be used to identify potential
chemical groups that might be responsible for eliciting the
bioactivity of a compound (McKinney et al., 2000). The SAR of
rutin concerning its protease inhibition was studied with the
Molinspiration Chemoinformatics web tool. The contribution of
different atoms, groups, and the sugar moiety towards protease
inhibition of rutin are given as numerical value in Table V.
Table V. SAR of rutin
Structure No.
Structure
Protease inhibition
1 (Rutin)
-0.07
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2
-0.06
3
-0.25
4
0.05
5
-0.08
6
-0.03
7
-0.04
8
-0.05
From structures no. 2 and 3 (Table V), the sugar moiety of
rutin might be necessary for its protease inhibition activity. Also,
the -OH groups of the sugar moiety of rutin were significantly
involved in the formation of conventional hydrogen bonds with
the amino acid residues at the active binding site of different
chains of SARS-CoV-2 3CL pro (Fig. 1, 2, and 4). The removal
of an oxygen atom at position 43 attached to the C-ring of rutin
significantly increases the protease inhibition activity (Table V,
Structure no. 4). Also, the oxygen atom at position 43 attached
to the C-ring of rutin was not involved in any molecular
interactions (Fig. 1, 3, and 4). In structures no. 5 to 8 (Table V),
the removal of -OH groups from different positions does not
seem to have a positive effect on the protease inhibition activity
of rutin. The aglycone moiety (C6-C3-C6 scaffold) was actively
involved in the formation of different types of protein-ligand
interactions (Fig. 1, 2, 3, and 4).
Based on these findings, it may be assumed that the sugar
moiety and the aglycone moiety of rutin are essential for binding
to the active binding site of SARS-CoV-2 3CL pro. The sugar
moiety of rutin may also be essential for the protease inhibition
activity of rutin (Table V). On the other hand, the oxygen atom
at position 43 attached to the C-ring of rutin does not seem to
contribute to the formation and stabilization of the protein-ligand
complex (Fig. 1, 2, 3, and 4).
Interestingly, molecular docking and molecular dynamics
simulation studies also showed rutin as a potential inhibitor of
SARS-CoV-2 3CL pro (Cherrak et al., 2020). In the study, chain
A of SARS-CoV-2 3CL pro was used. Cherrak et al. (2020)
showed that the sugar moiety of rutin formed two hydrogen
bonds with GLU166. Cherrak et al. (2020) also showed that the
A-ring of rutin formed hydrophobic interactions with MET49.
Similarly, in our study, rutin formed a conventional hydrogen
bond with GLU166. Rutin also formed conventional hydrogen
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bonds and non-covalent interactions with MET49 of chain A of
SARS-CoV-2 3CL pro. In addition, our study also provides data
for chain B, chain C, and chain D.
Rutin is a citrus flavonoid (Kreft et al., 1997) that is
abundantly found in apples, tea, buckwheat, and passionflowers
(Harborne, 1986). Rutin exerts several pharmacological
activities such as anticarcinogenic, antioxidant, vasoprotective,
cytoprotective, cardioprotective, and neuroprotective activities
(Ganeshpurkar & Saluja, 2017). An analog of rutin known as
sodium rutin sulfate showed anti-retroviral activity against
various strains of human immunodeficiency virus 1 (Tao et al.,
2007). Rutin also showed antiviral activity against vesicular
stomatitis virus (Wacke & Eilmes, 1978), canine distemper virus
(Carvalho et al., 2013), and avian influenza (H5N1) virus
(Ibrahim et al., 2013). The literature review revealed the potent
anti-viral activity of rutin against different pathogens of viral
origin. Considering the anti-viral activity of rutin against other
RNA viruses, there is a possibility that rutin might elicit anti-
viral activity against SARS-CoV-2 by inhibiting its main
protease (3CL pro) which is a key component for viral
replication.
CONCLUSION
The present in-silico study revealed rutin as a potential
inhibitor of SARS-CoV-2 3CL pro. Rutin showed a good
binding affinity at the active binding pocket of all the four chains
of SARS-CoV-2 3CL pro. Rutin also showed better ligand
interactions with SARS-CoV-2 3CL pro than 3WL. In many
cases, rutin and 3WL showed similar molecular interactions with
the same amino acid residues at the active binding site of SARS-
CoV-2 3CL pro. Rutin formed more conventional hydrogen
bonding than 3WL. Rutin is non-toxic and had a better drug-
likeness score than 3WL. Also, rutin was predicted to be a better
protease inhibitor than 3WL. SAR revealed that the aglycone
moiety and the sugar moiety of rutin might be essential for its
protease inhibition activity. SAR also revealed that the oxygen
atom at position 43 of rutin does not contribute to protein-ligand
binding and protease inhibition activity. Therefore, the present
in-silico study advocates rutin as a potential inhibitor of SARS-
CoV-2 3CL pro and as promising phytotherapy for COVID-19.
The C6-C3-C6 scaffold of rutin may also be used to design and
develop potent anti-viral agents against SARS-CoV-2.
ACKNOWLEDGMENTS
The author acknowledges the Department of Pharmaceutical
Sciences, Dibrugarh University for providing the necessary
facilities to carry out the in-silico work.
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