Targeting SARS-CoV-2 spike protein of COVID-19 with naturally occurring
Phytochemicals: an In silco study for drug development
Jitendra Subhash Rane
1
, Aroni Chatterjee
2#
, Abhijeet Kumar
3#
and Shashikant Ray
4 *
1
Department of Biosciences & Bioengineering, Indian Institute of Technology Bombay, Mumbai
-400076, India
2
Indian Council of Medical Research (ICMR)—Virus Research Laboratory, NICED, Kolkata,
India
3
Department of Chemistry, Mahatma Gandhi Central University Motihari-845401, India
4
Department of Biotechnology, Mahatma Gandhi Central University Motihari-845401, India
#
Both authors contributed equally to this work.
*Correspondence may be addressed to this author
4*
. Shashikant Ray, Assistant Professor, Department of Biotechnology, Mahatma Gandhi Central
University Motihari-845401, India, E-mail: shashikantray@mgcub.ac.in
Keywords: COVID-19, Molecular Docking, Phytochemicals, flavonoids and non-flavonoids
Abbreviations: COVID-19, Coronavirus Disease 2019, SARS-CoV-2S, Severe Acute
Respiratory Syndrome Coronavirus 2 Spike Protein, HCQ, Hydroxychloroquine, CQ,
Chloroquine, ACE2, Angiotensin Converting Enzyme-2, MERS-CoV, Middle East Respiratory
Syndrome coronavirus, PDB, protein data bank, ADME, absorption, distribution, metabolism
and excretion
Competing financial interests: We report no conflicts of interest
Abstract
Spike glycoprotein found on the surface of SARS-CoV-2 (SARS-CoV-2S) is a class I fusion
protein which helps the virus in its initial attachment with human Angiotensin converting
enzyme 2 (ACE2) receptor and its consecutive fusion with the host cells. The attachment is
mediated by the S1 subunit of the protein via its receptor binding domain. Upon binding with the
receptor the protein changes its conformation from a pre-fusion to a post-fusion form. The
membrane fusion and internalization of the virus is brought about by the S2 domain of the spike
protein. From ancient times people have relied on naturally occurring substances like
phytochemicals to fight against diseases and infection. Among these phytochemicals, flavonoids
and non-flavonoids have been found to be the active source of different anti-microbial agents.
Recently, studies have shown that these phytochemicals have essential anti-viral activities. We
performed a molecular docking study using 10 potential naturally occurring flavonoids/non-
flavonoids against the SARS-CoV-2 spike protein and compared their affinity with the FDA
approved drug hydroxychloroquine (HCQ). Interestingly, the docking analysis suggested that C-
terminal of S1 domain and S2 domain of the spike protein are important for binding with these
compounds. Kamferol, curcumin, pterostilbene, and HCQ interact with the C-terminal of S1
domain with binding energies of -7.4, -7.1, -6.7 and -5.6 Kcal/mol, respectively. Fisetin,
quercetin, isorhamnetin, genistein, luteolin, resveratrol and apigenin on the other hand, interact
with the S2 domain of spike protein with the binding energies of -8.5, -8.5, -8.3, -8.2, -8.2, -7.9,
-7.7 Kcal/mol, respectively. Our study suggested that, these flavonoid and non-flavonoid
moieties have significantly high binding affinity for the two main important domains of the spike
protein which is responsible for the attachment and internalization of the virus in the host cell
and their binding affinities are much higher compared to that of HCQ. In addition, ADME
(absorption, distribution, metabolism and excretion) analysis also suggested that these
compounds consist of drug likeness property which may help for further explore as anti-SARS-
CoV-2 agents. Further, in vitro and in vivo study of these compounds will provide a clear path
for the development of novel compounds that would most likely prevent the receptor binding or
internalization of the SARS-CoV-2 spike protein and therefore could be used as drugs for
COVID-19 therapy.
Introduction: The world population is currently facing a severe challenge for survival due to the
rise of a global pandemic named Coronavirus Disease 2019 (COVID-19) [1]. This pandemic is
caused by a positive sense RNA virus belonging to the β-coronaviridae genera called severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [2, 3]. As per the reports provided by
the World Health Organization (WHO) currently there exists no effective treatment regime
including antivirals or vaccines against SARS-CoV-2. Infected cases along with mortality are
increasing rapidly day by day and scientists all over the world are desperately looking for
effective compounds which can be used as antivirals to use against this virus. Natural
compounds with high bioavailability and low cytotoxicity seem to be the most efficient
candidates in this regard. Since ancient times, humans have resorted to the use of natural
compounds especially phytochemicals for the treatment of different diseases and disorders [4].
Flavonoids are secondary metabolites produced by plants and play very important roles in plant
physiology, possessing a variety of potential biological benefits such as antioxidant, anti-
inflammatory, anticancer, antibacterial, antifungal and antiviral activities [5, 6]. Different
flavonoids including both flavones and flavonols have been quite thoroughly investigated for
their potential antiviral properties and many of them showed significant antiviral response
in both in vitro as well as in vivo studies [7]. Curcumin, a component of turmeric, has been
described to exhibit enhanced antiviral activity against diverse viruses such as dengue
virus (serotype 2) herpes simplex virus, human immunodeficiency virus, Zika and chikungunya
viruses among others [8]. Apigenin isolated from the sweet basil (Ocimum basilicum) plant has
shown potent antiviral activity against hepatitis B virus, adenoviruses, african swine fever virus
and some RNA viruses in vitro [9]. Luteolin, another acclaimed flavone has shown significant
antiviral effects on both HIV-1 reactivation and inhibition of Epstein-Barr virus (EBV)
reactivation in cells. Besides these antiviral activities, luteolin also showed antiviral effects
against Chikungunya virus, Japanese encephalitis virus [10], severe acute respiratory syndrome
coronavirus (SARS-CoV) and rhesus rotavirus. Among flavonols the antiviral effect of quercetin
has been most extensively investigated. Quercetin has been found to demonstrate an in-vitro
dose-dependent antiviral activity against respiratory syncytial virus, poliovirus type 1, HSV-1
and HSV-2. It was also reported that quercetin has potential ability as a prophylactic against
Ebola virus infection [11]. Kaempferol, another flavonol extracted from Ficus benjamina leaves
have shown inhibitory activity against HCMV, HSV-1, HSV-2 and influenza A virus [12].
Fisetin a modified flavonol has shown to inhibit CHIKV infection as well as the HIV-1 infection
by blocking viral entry and virus-cell fusion [12]. Resveratrol and pterostibenes have been found
to exhibit anti-viral activities against a wide range of viruses including HIV-1 [13]. Resveratrol
has been shown to inhibit the replication of pseudorabies virus (PVR) and Middle East
Respiratory Syndrome coronavirus (MERS-CoV) and also reduced the expression of MERS-
CoV nucleocapsid (N) protein [14-17]. Pterostilbene, a natural dimethylated analogue of
resveratrol found in berries and grapes has been found to inhibit the replication of several
viruses, including herpes simplex viruses (HSVs) 1 and 2, varicella-zoster virus, influenza virus
and human papillomaviruses. Considering the contagiousness of the COVID-19 and its
consequences, there is an imperative need to develop an efficient therapy to curtail the spread of
this deadly virus and safely treating the infected individuals. In that direction, the repurposing of
the FDA approved existing drugs like chloroquine (CQ) and HCQ either alone or in combination
with other known drugs are currently being attempted [18, 19]. Preliminary in vitro studies and
clinical trials carried out by scientists on COVID-19 patients disclosed the effectiveness of HCQ,
an anti-malarial drug in combination with azithromycin, a broad spectrum anti-bacterial drug in
reducing the viral load [20]. Although some of these initial studies disclosed promising results, a
lot still remains to be done to analyse their compatibility, cost, accessibility, side effects, dosages
etc. Currently scientists are indulging themselves to identify ideal natural compounds that can
target and modulate unique or novel sites like the spike glycoprotein (S) on the surface of SARS-
CoV-2 [21, 22]. In this in-silico study, we have performed molecular docking experiments to
ascertain the most potent natural compounds (flavonoids) that can bind to the functional domains
of the SARS-CoV-2S protein, a viral surface glycoprotein required for initial attachment and
internalisation within host cells [1]. We found that about 10 of these compounds effectively
binds to the C-terminal region of either the S1 domain or the S2 domain of SARS-CoV-2S and
their binding interaction are more stable than with that of HCQ. These natural compounds are
capable of binding to either the S1 or S2 domains of the SARS-CoV-2S protein and most
probably prevent it from binding to the ACE receptor or internalization during fusion [23, 24].
The ADME analysis also suggested that most of these compounds have potential to function as
effective anti-SARS-CoV-2 agents and needs further scientific explorations.
Material and methods:
Preparation of Ligands and Receptor:
The 3-dimensional structure of all ligands (Kaempferol, Curcumin, Pterostilbine,
Hydroxychloroquine, Fisetin, Quercetin, Isorhamnetin, Genistein, Luteolin, Resveratrol and
Apigenin) was downloaded from the PubChem database and then these structures were
converted in pdb format by using PyMol [25]. The structure of SARS-CoV-2S protein (Fig. 1)
was downloaded from the RCSB protein data bank (PDB-ID: 6VYB) [21]. Structure of all the
ligands have been provided in table S1
Molecular docking of Phytochemicals on SARS-CoV-2 S (spike protein):
The cryo-electron microscopic structure of SARS-CoV-2S was used for the docking analysis.
SARS-CoV-2S protein is a heterotrimer consisting of chain A, B and C [21]. For the docking
experiment, chain A of the spike protein was used. First, the SARS-CoV-2S, Curcumin and HCQ
were converted into pdbqt format using autodoc tools. Polar hydrogens and gasteiger charges
were added to SARS-CoV-2S, curcumin and HCQ structure before docking. The molecular
docking tool autodoc vina [26] was used to study the binding of curcumin and
hydroxychloroquine on the SARS-CoV-2S. Further, blind docking of curcumin was performed
to know the probable binding sites. For this, entire SARS-CoV-2S molecule was covered with
the grid box of dimension 76 Å × 92 Å × 160 Å with grid spacing 1 Å. The SARS-CoV-2S was
kept rigid while the curcumin was kept flexible. The four sets of docking were performed with
exhaustiveness 100. Each set of autodoc vina produced 9 conformations, among them, 6
conformations were docked at one domain of SARS-CoV-2S, that domain is used for the local
docking. For local docking, SARS-CoV-2S was covered with the grid box of dimension 60 Å ×
54 Å × 66 Å with grid spacing 1 Å. The exhaustiveness was kept at 100. Four sets of local
docking were performed for each ligand and the site where the maximum number of
conformations bind was determined as a binding site. The conformations with high negative
binding energy are represented in the figures. The docking results were analyzed using MGL tool
1.5.6 [27] and the hydrophilic and hydrophobic interactions were determined using PyMol [25].
