Journal of Biomolecular Structure and Dynamics

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Dietary factors and SARS-CoV-2 contagion: in silico

studies on modulation of viral and host proteins
by spice actives

Kottapalli Srividya, Snober S. Mir, Saravanamuthu Thiyagarajan & Aamir

Nazir

To cite this article: Kottapalli Srividya, Snober S. Mir, Saravanamuthu Thiyagarajan & Aamir
Nazir (2021): Dietary factors and SARS-CoV-2 contagion: in�silico studies on modulation of
viral and host proteins by spice actives, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2021.1948448

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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2021.1948448

Dietary factors and SARS-CoV-2 contagion: in silico studies on modulation

of viral and host proteins by spice actives
Kottapalli Srividyaa, Snober S. Mirb, Saravanamuthu Thiyagarajanc and Aamir Nazira
a
Division of Neuroscience and Ageing Biology, CSIR-Central Drug Research Institute, Lucknow, India; bDepartment of Bioengineering,
Integral University, Lucknow, India; cInstitute of Bioinformatics and Applied Biotechnology (IBAB), Bengaluru, India
Communicated by Ramaswamy H. Sarma

ABSTRACT ARTICLE HISTORY

The SARS-CoV-2 contagion has had a huge impact on world population. It has been observed that Received 18 July 2020
despite massive spread of the contagion in India particularly during the second wave, the overall case Accepted 22 June 2021
fatality rates remain low. This prompted us to look into dietary factors that can possibly modulate the
KEYWORDS
viral impact and/or host response. In silico studies were carried out on forty-two commonly used
SARS-CoV-2; Nsp13; TBK1;
spices and their 637 known active compounds with an aim of identifying such compounds that may virtual screening; spices;
have propensity to reduce viral impact or boost host immune response. We chose to study SARS-Cov- phytochemicals; immunity
2 helicase on account of its functional importance in maintaining viral load within the host, and the
human tank binding protein (TBK1) for its important role in host immunity. We carried out in silico vir-
tual screening, docking studies with 637 phytochemical against these two proteins, using in silico
methods. Upon assessing the strength of the ligand-target interactions and post simulation binding
energy profile, our study identifies procyanidin-B4 from bay leaf, fenugreekine from fenugreek seed
and gallotannin from pomegranate seed as active interactors that docked to viral helicase. Similarly,
we identified eruboside B from garlic, gallotannin from pomegranate seed, as strong interacting part-
ners to human TBK1. Our studies thus present dietary spice constituents as potential protagonists for
further experimentation to understand how spices in the diet might help the hosts in countering the
viral assault and mount a robust protective response against COVID and other infections.

Introduction India, the second-most populous country of the world, has

The severe acute respiratory syndrome–Coronavirus-2 (SARS-
CoV-2, SCV) is the seventh Coronavirus to infect the human
race after HCoV-NL63, HCoV-229E, HCoV-OC43, HKU1, SARS-
CoV, and MERS-CoV (Andersen et al., 2020). Over the past
two decades, SARS-CoV hit humans in 2002 infecting 8,000
people with a case fatality rate of
10%, followed by the
MERS-CoV contagion in 2012 which was contracted by 2,500
people with a case fatality rate of 36%. SARS-CoV and MERS-
CoV caused severe respiratory syndrome and exhibited
higher case fatality rates, but their spread was contained
thus minimizing the effect on human life (Gordon et al.,
2020). The newly evolved sub-type SARS-CoV-2 (SCV),
reported to have originated in December 2019, spread to
223 countries, infecting 169 million and proving fatal for
over 3.5 million people worldwide (WHO, 2021). SCV is a
large, single-stranded, positive-sense RNA virus. The cleavage
of polyproteins by viral proteinases results in the generation
of a total of 16 non-structural proteins (Nsp’s) (Adedeji et al.,
2012; Keum & Jeong, 2012; Seth et al., 2006). An intriguing
factor has been the varying percentage of death rates
between various countries and populations. Specifically,
seen lower case fatality rates. As of 30th May 2021, according
to WHO report, India has reported 27,894,800 cases and
325,972 deaths (WHO, 2021), with the case fatality rates
being 1.16% against the average of 2.26% for the rest of the
world. Amongst the reasons for how the Indian population
has been mounting such protective response, various possi-
bilities including higher basal immunity, immunization pro-
files including BCG vaccine, and its resulting immune
memory have become subjects of discussion and research.
We endeavoured to look at the aspect of ’what the Indian
population eat’ towards aiming to identify possible routine
dietary ingredients that may have the potential of either lim-
iting the viral impact or via activating the host immune sys-
tem towards mounting a robust response. Amongst various
dietary ingredients, ones that are consumed routinely are
spices and condiments that form an ingredient of every meal
an Indian consumes. Constituent flavonoids, terpenoids, and
other metabolites from spices are known to protect by anti-
oxidant, anti-inflammatory, antiviral and anti-microbial activ-
ities. We carried out in silico studies using 637 active
constituents within 42 commonly used spices and condi-
ments with a two-fold aim of (a) identifying such

CONTACT Aamir Nazir anazir@cdri.res.in Division of Neuroscience and Ageing Biology, CSIR-Central Drug Research Institute, Lucknow 226001, India;
Saravanamuthu Thiyagarajan sthiyaga@ibab.ac.in Institute of Bioinformatics and Applied Biotechnology (IBAB), Behind 3M and next to Arvind Mills,
Electronics City Phase I, Bangalore 560100, Karnataka, India
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2021.1948448
ß 2021 Informa UK Limited, trading as Taylor & Francis Group
2 K. SRIVIDYA ET AL.
constituents that can be predicted to exhibit a propensity of
checking the SCV replication or viral load (via studying SCV
protein Nsp13), and (b) identifying the constituents that can
boost the host immune system towards mounting an effect-
ive response (via studying host immune response protein
TBK1), until the viral load is cleared.
Materials and methods
1. Mining of literature towards the identification of target pro-
teins from SCV and its human host and identification of
commonly used spices in India: The selection of proteins
for the study was carried out in a hypothesis-driven man-
ner of studying a protein that plays a critical role in viral
replication and proliferation thus being important for
maintaining viral load within the host. Similarly, literature
was mined for identifying host (human) protein, related
to the immune pathways that are activated in the case
of SARS-CoV-2 infections. The spices were selected from
the Food Safety and Standards, Regulations document
based on the common usage across various parts of
India overlapping these spices with Spice Board of India’s
cultivation data.
2. Ligand preparation: 3D structures of metabolites from 42
spices were procured using ChemSpider (https://www.
chemspider.com/), and PubChem (https://pubchem.ncbi.
nlm.nih.gov/) databases. Along with these structures,
known inhibitors for each protein were also added to
the list for screening. The downloaded structures were
further processed using the LigPrep module of the
Schro€dinger suite (Schro€dinger LLC, 2020) in the Maestro
builder panel to add appropriate number of hydrogen
atoms to satisfy the valency requirements of non-hydro-
gen atoms, ensure proper ionic states and 3D geometry
of the compounds at a pH 7.0. The structures were opti-
mized by energy minimization using the all-atom force-
field OPLS3e.
3. Preparation of protein structures and grid generation: Viral
protein Nsp13 (Helicase) and host human TBK1 (Tank
binding protein) protein structures were downloaded
from PDB (Protein Data Bank, 2019). Hydrogen atoms,
missing side-chain atoms and non-standard residues
were fixed in these structures by using the protein prep-
aration wizard (Schro €dinger LLC, 2020). Potential binding
sites were identified in these proteins using the SiteMap
module (Schro €dinger LLC, 2020). Sites having scores
greater than one were used for downstream virtual
screening and docking studies. XYZ-Coordinates for the
selected sites were given as input, for making a grid
using the ‘Glide’s Receptor Grid Generation’ module. A
cubic box was then generated around the active site of
the target molecules. The grid box dimension was then
adjusted to completely encompass the active site region
of the target protein molecules.
4. Virtual screening and docking: The optimized structures of
the ligand set were then docked using both SP and XP
scoring functions using Glide (Schro €dinger LLC, 2014).
This Virtual screening using the natural compounds
against the chosen target proteins (Host: TBK1; Virus:
Nsp13) was then ranked based on the glide score. First,
the ligands were subjected to standard precision
(Friesner et al., 2004) docking. Resulting 32 hit ligands
were further subjected to extra precision docking.
OPLS3e force field with a pH ¼ 7 was used in all these
studies. GlideScore is an empirical scoring function that
approximates the ligand binding free energy. It is com-
posed of many terms, including force field (electrostatic,
van der Waals), GScore is the short form for GlideScore,
used for comparing poses of different ligands which uses
the equation below. vdW is Scaled van der Waals energy,
Coul is Scaled Coulomb energy, Site is Polar interactions
in the active site, CvdW is Non-bonded ligand/receptor
interactions (Coul þ vdW), not included in GlideScore
(though Coul and vdW are included individually, with
separate weights), Intern is Internal energy of ligand, not
included in GlideScore, Emodel is combination of GScore,
CvdW, and Internal energy. These contributions and the
terms rewarding or penalizing inter and intra molecular
interactions known to influence ligand binding constitute
the GScore. (Friesner et al., 2006).
XPGlideScore ¼ Ecoul þ EvdW þ Ebind þ Epenalty
Ebind ¼ Ehydenclosure þ Ehbn nmotif þ Ehbc cnotif þ Ehbpair þ Ephobicpair
X
Ephobicpair ¼ f ðrij Þ
ij
Epenalty ¼ Edesolv þ Eligandstrain
5. MD simulation and MM-GBSA analysis: Molecular dynamic
simulations were undertaken on the docked poses of the
hit ligands for Nsp13 and TBK1, using the Desmond
module (D.E. Shaw group) interfaced with the Maestro
tool of the Schrodinger suite (Schro €dinger LLC, 2018).
The receptor-ligand complexes were minimized after sol-
vating with SPC water molecules in a box whose dimen-
sions covered 10Å from the surface of the molecule in all
directions. The solvated systems were energy minimized
using steepest descent strategy until a gradient thresh-
old of 1.0 kcal/mol/Å was approached with a minimum
step size of 10. Periodic boundary conditions were used
along with a 9.0 Å cut-off for electrostatic interactions.
The MD simulation run consists of eight stages, including
the pre-relaxation phase of the protein-ligand complexes.
Unrestrained production runs were conducted at a tem-
perature of 300 K and 1.01325 bar pressure, for 100ns.
Constant pressure and temperature were maintained
using Berendsen coupling method (Berendsen et al.,
1981). No additional restraints were given for the hydro-
gen atoms of the protein or the ligand molecules.
Coordinates and energy parameters were saved at every
25ps. The resultant 4000 frame trajectories of the simu-
lated systems were analysed and visualized using the in-
built tool ‘simulation interaction diagrams’ of the
Maestro/Desmond module of the Schrodinger Suite. For
analysing the hydrogen bonding interactions from the
simulated trajectories, the following geometric criteria for
a protein-water or water-ligand H-bond are used: a dis-
tance of 2.8 Å between the donor and acceptor atoms
(D—HA); a donor angle of 110 between the donor-

hydrogen-acceptor atoms (D—HA); and an acceptor
angle of 90 between the hydrogen-acceptor-bonded
atom atoms (HA—X), while for hydrogen bond is dis-
tance of 2.5 Å between the donor and acceptor atoms
(D—HA); a donor angle of 120 between the donor-
hydrogen-acceptor atoms (D—HA); and an acceptor
angle of  90 between the hydrogen-acceptor-bonded
atom atoms (HA—X).Binding energy calculations were
performed using MM-GBSA using the Prime (Schro €dinger
LLC, 2014) module of Schrodinger software on the snap-
shots taken at every nanosecond, where the dynamic
shift in energy was measured between the unbound and
bound state of the complex. For the run, simulation
report was generated. The binding energy was calculated
according to the equation:
DGbind ¼ Ecomplex ðminimizedÞEligand ðminimizedÞ
Ereceptor ðminimizedÞ
Results
1. Identification of target proteins from SARS-CoV-2 and its
human host and identification of commonly used spices in
India: In context with viral machinery, the replicase/
transcriptase complex was chosen as a target in the
virus system (human-SARS CoV2 interactome – refer-
ence). Further understanding of helicase Nsp13 gave
enthralling reasons for its vital existence in the virus. As
its functionality varies from contributing to the cytoskel-
eton, signalling, vesicle trafficking to epigenetic, and
gene expression regulation (Fung & Liu, 2014; Gordon et
al., 2020). Similarly, human host proteins interacting with
Nsp13 were procured from BioGrid (https://thebiogrid.
org/), amongst the list TBK1; a Serine/Threonine kinase
that plays an essential role in regulating inflammatory
responses to foreign agents. To lead the idea of immun-
ity and immune response to host-virus interaction
TBK1was identified as target host protein. Especially,
when virus hijacks host the ER system for cargo facility
and amplification that in return creates stress response
which activates the defence (https://thebiogrid.org/).
Depending on age, gender, and other individual attrib-
utes this immune response can vary from being insensi-
tive to moderately responsive by stimulating a defence
against the cytokine storm. The Food safety and stand-
ards, regulations document was used as a primary criter-
ion to select spices. By overlapping these spices with
Spice Board of India’s cultivation data (http://www.indi-
anspices.com); forty-two most commonly used/cultivated
spices that are rooted in India’s geography were identi-
fied and selected for further studies (Table 1).
2. Ligands from edible parts of spices:
Ligands exclusively from edible parts that are used in
culinary practices were collected with reference of FSSAI
enlisted forty-two original Indian spices. The individual
plant metabolite list were collected from Dr. Duke’s
phytochemical and ethnobotanical database (https://
phytochem.nal.usda.gov). 3D coordinates of 637 unique
JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 3
metabolites were collected from open source databases
viz PubChem and ChemSpider (See supplementary file 1
for a complete list of compounds with their source
plants). In addition, adamantane derivatives that have
been used clinically for many years as antiviral treat-
ments and as muscle relaxants were also included in the
ligands to be used in the screening. In a different study,
a demonstrated class of pyridoxal-conjugated trioxaada-
mantanes, known as the bananins, was shown to inhibit
both the ATPase and helicase activities of the SARS
Coronavirus helicase (Tanner et al., 2005). A representa-
tive of that class, bananins was taken to be a potent
inhibitor for Nsp13 as a positive control. Whereas for
TBK1 a known inhibitor from Gordon et al. (2020).
ZINC95559591 was taken as screening/control.
3. Protein preparation and grid generation: Structure of
Nsp13 (PDB code 6JYT) and human TBK1 (PDB code
4IM0) were downloaded from the PDB (www.rcsb.org).
These structures were fixed for downstream computa-
tional studies using the Protein Preparation Wizard of
the Schrodinger suite (Schro €dinger LLC, 2020). The struc-
tures were energy minimized to remove short contacts
and to ensure proper stereochemistry. Potential binding
sites in the proteins were identified using the SiteMap
module (Schro €dinger LLC, 2020). Sites having scores
more than 1 were taken for grid calculations as enlisted
in Table 2. Five sites were identified in each of the pro-
teins TBK1and Nsp13 and were ranked according to
their score. Analysis of the 3D structure revealed that
these sites are populated with residues that can poten-
tially make electrostatic, hydrogen bonds as well as
hydrophobic interactions.
4. Hit ligands from Virtual screening: With ligands and pro-
teins prepared for docking, each ligand was docked
against the receptor using standard precision (SP) dock-
ing for each grid generated from SiteMap results. From
this pipeline, the 32 hit ligands obtained from SP dock-
ing were then subjected to extra precision (XP) docking
for the same receptor protein and grid. The results are
categorized into two clades one with Nsp13-metabolites
from spices and the other one being TBK1-metabolites
from spices docking. To begin with, results obtained
from Nsp13 docking with Site ID: Site-A are as follow:
our study identified procyanidin B4 from Bay leaf with
Glide score 11.8, interacting with protein residues that
participated in hydrogen Bonding with ligand as ARG-
178, ARG339, ASN-361, CYS-309, THR-410, and LYS-146
with distances 2.93, 2.88, 3.21, 2.77, 3.10 and 3.17 & 3.12
Å respectively (Figure 1), fenugreekine (Glide score:
11.7 from fenugreek seed, gallotannin (Glide score:
11.4) from pomegranate seed, and eruboside B (Glide
score: 11.3) from garlic bulb (See Supplementary file 2,
Figure S1), which has efficient binding with helicase
domain in viral target Nsp13. The next part of the study
consists of docking results of human host protein TBK1,
wherein two sets of results have been obtained.
Docking sites were mainly localized in the kinase
domain [9-310] and Ubiquitin-like domain (309–385)
4 K. SRIVIDYA ET AL.

Table 1. List of major spices of India.

Sr.No. Common name in English Scientific name Name of the family Plant parts used
1 Ajowan Trachyspermumammi L. Apiaceae Fruit
2 Aniseed Pimpinella anisum L. Apiaceae Fruit
3 Asafoetida Ferula asafoetida L Apiaceae Oleogum resin from rhizome
& thickened root
4 Basil Ocimum basilicum L. Lamiaceae Leaf
5 Bay Leaf Laurus nobilis L. Lauraceae Leaf
6 Camboge Garcinia cambogia [Gaertn]. Desr Clusiaceae Rind
7 Caraway Carum carvi L. Apiaceae Fruit
8 Cardamom [Large] Amomum subulatum Roxb Zingiberaceae Fruit, Seed
9 Cardamom [Small] Elettaria cardamomum Maton Zingiberaceae Fruit, Seed
10 Cassia Cinnamomum cassia. Blume Lauraceae Bark
11 Chilli: Bird’s Eye Capsicum frutescens L. Solanaceae Fruit
12 Chilli: Capsicum Capsicum annuum L. Solanaceae Fruit
13 Cinnamon Cinnamomum zeylanicum Breyn Lauraceae Bark
14 Clove Syzygium aromaticum [L] Merr. & Perry Myrtaceae Unopened Flower bud
15 Coriander Coriandrum sativum L. Apiaceae Leaf & Fruit
16 Cumin Cuminum cyminum L. Apiaceae Fruit
17 Curry leaf Murraya koenigii [L] Sprengel Rutaceae Leaf
18 Dill Anethum graveolens L. Apiaceae Fruit
19 Fennel Foeniculum vulgare Mill. Apiaceae Fruit
20 Fenugreek Trigonella foenum-graecum L. Fabaceae Seed
21 Garlic Allium sativum L. Alliaceae Bulb
22 Ginger Zingiber officinale Rosc. Zingiberaceae Rhizome
23 Greater Galanga Alpinia galanga Willd. Zingiberaceae Rhizome
24 Horse Radish Armoracia rusticana Gaertn. Brassicaceae Root
25 Hyssop Hyssopus officinalis L. Lamiaceae Leaf
26 Juniper berry Juniperus communis L. Cupressaceae Berry
27 Kokam Garcinia indica Choisy Clusiaceae Rind
28 Mace Myristica fragrans Houtt. Myristicaceae Aril
29 Marjoram Marjorana hortensis Moench. Lamiaceae Leaf
30 Mint Mentha piperita L. Lamiaceae Leaf
31 Mustard Brassica juncea L. Czern Brassicaceae Seed
32 Nutmeg Myristica fragrans Houtt. Myristicaceae Seed
33 Pepper Piper nigrum L. Piperaceae Fruit
34 Pepper Long Piper longum L. Piperaceae Fruit
35 Pomegranate Punica granatum L. Punicaceae Seed
36 Poppy seed Papavers somniferum L. Papaveraceae Seed
37 Saffron Crocus sativus L. Iridaceae Stigma
38 Star Anise Illicium verum Hook. Illiciaceae Fruit
39 Sweet flag Acorus calamus L. Araceae Rhizome
40 Tamarind Tamarindus indica L. Caesalpiniaceae Fruit
41 Tejpat Cinnamomum tamala [Buch Ham] Nees & Eberum Lauraceae Bark&Leaf
42 Turmeric Curcuma longa L. Zingiberaceae Rhizome

Table 2. SiteMap coordinates for sites with score >1.

Sr.No. Protein SiteMap coordinates [X,Y,Z] Volume [Å3] Site score Site ID
1 Nsp13 428.1, 29.1, 69.9 715.5 1.09 Site-A
2 TBK1 92.4, 31.8, 9.7 694.2 1.049 Site-B
3 103.4, 12.3, 32.2 483.5 1.052 Site-C

(Figure 2). There were two binding sites; each site has
its own distinct set of ligand interactors. Site ID: Site-B
in TBK1 had eruboside B with Glide score: 12.1, inter-
acting with protein residues that participated in hydro-
gen Bonding with ligand as GLN-274, PRO-264, SER-260
with distances 2.99, 2.84, 2.59 and 3.22 Å respectively
(Figure 2) along with inulin (Glide score: 11.2) from
garlic, hesperidin (Glide score: 10.0) from hyssop, capsi-
cum, and chilli (See Supplementary file 2, Figure S2) as
selective interactors. Site ID: Site-C in TBK1 had
Leuteoin-7-glucoside with a Glide score of 12.1, inter-
acting with ASN-140, GLY-139, MET-142, GLU-87, CYS-89,
PRO-90 making hydrogen bonding with ligand as with
distances 3.20, 2.80-2.90, 3.33, 3.12, 2.76, 3.01 Å respect-
ively (Figure 3) and cosmosiin (Glide score: 11.7) from
cumin, cyanin (Glide score: 11.8) from ginger, as select-
ive interactors (Figure S3). Whereas gallotannin (Glide
score: 11.1 and 11.5) (See Supplementary file 2,
Figure S2 and S3) from pomegranate seed was able to
bind at both the sites. The results of these docking stud-
ies with Glide/docking score along with interacting resi-
dues are listed in Table 3.
5. MD Simulation and MM-GBSA analysis: Unrestrained
molecular dynamic simulations studies were undertaken
using the Desmond module of the Schrodinger Suite
and analysed using the in-built modules of maestro/
Desmond. The RMSD plots (Figure 4) show that the pro-
tein-ligand complexes were stable during most of the
100 ns simulation time. At a few snapshots, the ligands
have changed their pose but stayed within the binding
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 5

Figure 1. Nsp13 docked with Procyanidin B4.(A) 3D Structural representation of SARS-Nsp13 in complex with Procyanidin B4 (in stick) from bay leaf predicted to
be docked in the Site ID – Site A with a Docking score: 11.8. The Receptor structure: SARS-Nsp13, is made of a ZBD (Zinc Binding Domain), the stalk, and inserted
domain, RecA-like domains that contribute to helicase activity.

site (protein-ligand contact map is provided in
Supplementary file 3), which can be observed from the
RMSD plot where the RMSD trajectories of the protein
and its respective ligand overlap. MM-GBSA analysis
shows that the binding is highly stable for 4 out of 5
ligands that we have studied with high binding energy
values. Table 4 lists total binding energy along with the
components of the energy terms from MM-GBSA ana-
lysis of the systems under study. MM-GBSA analysis,
(Table 4) showed that for helicase Nsp13, eruboside B
(31 kcal/mol) has weak binding while gallotannin
(73.5 kcal/mol) is the strongest. Similarly, for TBK1 eru-
boside B (120.7 kcal/mol) and gallotannin (120.6 kcal/
mol) have strong binding with the receptor respectively.
MM-GBSA analyses also show that eruboside B and gal-
lotannin have strong binding affinity towards TBK1,
while gallotannin has the highest affinity for Nsp13 fol-
lowed by fenugreekine and procyanidin B4 (Figure 4).
Eruboside B seems to be a potential candidate to inhibit
both the viral helicase and the human TBK1 protein,
though its interaction with the human TBK1 is stronger
than that with the viral nsp13.
These can be comprehended by the schematic represen-
tation (Figure 5). Here it can be seen that there were hydro-
gen bonds that were present in the maximum time of the
6 K. SRIVIDYA ET AL.

Figure 2. TBK1 docked with Eruboside B from Site ID – Site B with Dscore: 12.1.(A) The tripartite structure of Human-TBK1 in complex with Eruboside B (in stick)
from garlic. The structure of Human-TBK1 is made of a Kinase activity domain, Ubiquitin like domain, Dimerization domain, and C-terminal domain.

simulation run, but also water bridges were formed. Here,
only those residues were taken into account which stayed in
interaction for more than 50% of the run. It can be observed
that water bridges contributed by polar amino acid residues
like (HIS, ASN, THR) contributed to the interactions for longer
durations. Apart from these interactions there were Pi-
Cationic and ionic contacts were also seen.
Discussion
In the current study, we have undertaken studies on Nsp13
as well as TBK1 protein using the dietary metabolites from
spices to decipher if any of the spice constituents can act as
stimulants for immunity towards providing rescue and pro-
tection from viral pathology. Structural features of TBK1 con-
tain a predicted Ubiquitin-like domain (ULD) that is located
between the N-terminal kinase domain (KD) and the C-ter-
minal scaffolding/dimerization domain (SDD), a domain
arrangement that appears to be shared among the IKK family
of kinases (Figure 2) (Shi et al., 2018). TBK1 triggered
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 7

Figure 3. TBK1 docked with Leuteoin-7-glucoside from Site ID: Site C Dscore: 12.099.(A) Overall tripartite structure of Human-TBK1 in complex with Leuteoin-7-
glucoside (in stick) from cumin. The structure of Human-TBK1 is made of a Kinase activity domain, Ubiquitin ike domain, Dimerization domain, and C-ter-
minal domain.

immune signalling cascade directs host’s defence towards
viral invasion. Here we present two sets of potential candi-
dates, based on docking studies, that can function either in
boosting immune response or providing protection in case
of SARS-CoV-2 infection. After docking studies in Set-A
metabolites (Site ID: Site-B), eruboside B and inulin from gar-
lic, gallotannin from pomegranate seed, and hesperidin from
hyssop, capsicum, and chili, were identified. Inulin is a non-
digestible carbohydrate also used as a prebiotic which may
have an impact on the immune system, especially in the
area of the gut-associated lymphoid tissue [GALT]. Recent
studies show that it modulates the immune system by
activating immune cells in Peyer’s patches including IL-10
production and natural killer [NK] cell cytotoxicity (Watzl et
al., 2005). Hesperidin is a flavanone glycoside comprised of
the flavanone hesperetin and the disaccharide rutinose.
Various studies have reported it to defend airway hyper-
responsiveness, inhibiting inflammatory cell infiltration, and
mucus hyper-secretion. Also, it enhances mucosal and
humoral immunity by increasing intestinal intraepithelial
lymphocyte numbers and lymphoid organ indices. It is a
strong chain-breaking antioxidant that provides potent cellu-
lar antioxidant defence against the damaging effects(Xiao et
al., 2018). The second set of hit metabolites interacting with
8 K. SRIVIDYA ET AL.

Table 3. Results from XP docking.

Receptor Site ID Ligand Glide score H-bond Hydrophobic interactions Source plant
Nsp13 Site-A Procyanidin B4 11.8 LYS-139, ARG-178, Bay leaf
GLY-142, SER-310,
ASN-179, CYS-309,
MET-378, ALA-407,
ASP-534 THR-410,
GLU-143,
THR-380,
LYS-146,
TYR-382,
ASP-383,
LEU-138
Fenugreekine 11.7 LYS-139, THR-231, Fenugreek
ASN-361, HIS-230,
ARG-339, ARG-390,
ALA-336, PRO-335,
CYS-309 GLU-197,
VAL-181,
HIS-311,
SER-310,
MET-233
Gallotannin 11.4 ASN-179, ARG-178, Pomegranate
MET-378, SER-310,
ASP-534, LYS-309,
PRO-408, ALA-407,
GLY-142, THR-410,
LYS-139 GLU-143,
THR-380,
LYS-146,
TYR-382,
ASP-383,
LEU-138
Eruboside B 11.3 TYR-382, ARG-339, Garlic
ARG-390, THR-228,
LYS-139, ALA-336,
HIS-230, SER-229,
THR-231, MET-233
ASN-361
TBK1 Site-B Eruboside B 12.1 GLN-274, ARG-271, Garlic
PRO-264, VAL-265,
SER-260 CYS-267,
MET-263,
LYS-251,
ILE-397,
SER-398,
GLU-395,
ASP-26,
ILE-393
Inulin 11.2 MET-263, ASP-262, Garlic
GLN-274, ARG-271,
PRO-264, LEU-392
VAL-265,
ILE-293
Gallotannin-Site-2 11.1 LYS-251, ASP-262, Pomegranate
GLU-395, MET-263,
GLN-274 ILE-393,
VAL-265
Hesperidin 10.0 LYS-251, GLY-395, Hyssop,
ASP-258, ILE-393, Capsicum,
GLN-274 VAL-265, Chilli
PRO-264,
MET-263,
ASP-262,
SER-260
Site-C Leuteoin-7-glucoside 12.1 ASN-140, ASN-157, Cumin
GLY-139, GLY-18,
MET-142, THR-156,
GLU-87, VAL-123,
CYS-89, ALA-36,
PRO-90 PHE-88,
LEU-15,
GLY-92,
VAL-68
(continued)
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 9

Table 3. Continued.
Receptor Site ID Ligand Glide score H-bond Hydrophobic interactions Source plant
Cyanin 11.8 GLN-17, LYS-137, Ginger
GLU-87 TYR-95,
SER-93,
GLY-139,
LEU-15,
THR-96,
MET-142,
VAL-168,
ALA-36,
PHE-88,
THR-156,
ASP-157,
GLY-16,
GLY-18
Cosmosiin 11.7 GLU-87, ASP-157, Cumin
ASN-140, GLY-18
GLY-139, VAL-23,
MET-142, THR-156,
CYS-89 VAL-68,
ALA-36,
GLY-92,
PHE-88,
LEU-15
Gallotannin-Site-1 11.5 GLU-87, CYS-89, THR-96, SER-93, GLY-139, VAL-23, Pomegranate
ASP-157, ASN-140 THR-156,
MET-86,
ALA-19,
GLY-18,
GLY-92,
MET-142,
LEU-15,
VAL-68,
PHE-88,
ALA-36

TBK1 are listed within Set-B [Site ID: Site-C], in order as the
first hit being luteolin-7-glucoside from cumin; a glycosyloxy-
flavone that is luteolin substituted by a beta-D-glucopyrano-
syl moiety at position 7 via a glycosidic linkage. It plays a
role in the modulation of Reactive Oxygen Species [ROS] and
immune response involving NF-jB pathways (Jin et al., 2011;
Maatouk et al., 2017). We also identified a metabolite – cya-
nin, from ginger that boosts host immunity and creates a
defense response to allergies and inflammation (Mashhadi et
al., 2013). The third metabolite is Cosmosiin also known as
apigetrin from cumin which participates in rescuing inflam-
matory response in the host that has lost its balance(Liu et
al., 2017). With the results, it could be stated that flavonoids,
terpenoids, and saponins are the major class of plant metab-
olites participating in interactions with the host and viral
proteins. The experimentally known inhibitors used as con-
trols for virtual screening did not dock in the initial standard
precision mode for both the proteins. Thus, we are anticipat-
ing significantly higher activity than the control molecules
used. It has been established that the Nsp13 has a putative
binding site comprising of TYR-277, ARG-507 and LYS-508
wherein the inhibitor SSYA10-001 binds. Interestingly this (A
O Adedeji et al., 2014) overlaps within our chosen dock site
from SiteMap results. The hit metabolites were interacting in
the same docked region if not directly to the same residues
as before mentioned.
MD simulation and the MM-GBSA analysis show that the
results of docking studies are not just pertaining to a specific
snapshot of the interaction profile, but there is a strong
energy dynamics involved in the active site that in return
contributes to the stability of the protein-ligand complexes.
Starting with the Nsp13 (helicase) complex, it is quite evident
with the results that eruboside B is the weakest of all though
it is strong enough to be considered as a potential candidate
with a binding energy 31 kcal/mol; while gallotannin, fenu-
greekine and procyanidin B4 are strongly bound proving to
be better ligands from the list. While in case of TBK1 com-
plex eruboside B and gallotannin were seen to be positioned
with strong interactions in the binding pocket as per MM-
GBSA energy profile. From the root mean square deviation
(RMSD) plots; it can be comprehended that the ligands that
are bound to Nsp13 or TBK1 stayed in the binding pocket
when the system is subjected to 100 ns simulation. The bind-
ing energy as reported by MM-GBSA run for Nsp13 from
31.0 kcal/mol to 73.5 kcal/mol. Eruboside B interaction
with Nsp13 is primarily of Vander Waals interactions (VDW)
in nature. Whereas for fenugreekine, gallotannin and procya-
nidin B4 the dominating donor was electrostatic/columbic
interactions followed by VDW. The RMSD plots indicate that,
in these systems too, though there are some frames along
the trajectory where the binding orientations have changed,
the drugs mostly remained in the tightly bound state. TBK1
where the average NBE of each ligand was around
121
k/cal/mol. These results can be further supported with the
help of simulation interaction diagram, there it can be seen
that hydrogen bonds and water bridges comprise majority of
10 K. SRIVIDYA ET AL.

Figure 4. MD simulation and MM-GBSA analyses of Nsp13 and TBK1 in complex with ligands. The Root Mean Square Deviation (RMSD) gives the deviation of Ca
atoms of the protein superposed to those of the starting structure (teal lines) and all atoms in the respective ligand (magenta lines) The structures are as
follows(A) Nsp13-Fenugreekine complex, (B) Nsp13-Gallotannin complex, (C) Nsp13-Procyanidin B4 complex, (D) TBK1-Eruboside B complex, and (E) TBK1-
Gallotannin complex. In the MM-GBSA plot (F), the DG (kcal/mol, Y-axis) value is plotted as a function of time (nanoseconds, X-axis).

Table 4. MM-GBSA calculation profile of binding energy for the receptor (Nsp13, TBK-1) and ligand (Eruboside B, Fenugreekine, Gallotannin, Procyanidin B4)
complexes respectively.
Helicase TBK1
Energy terms
DG in kcal/mol represented as Mean(SD) Eruboside B Fenugreekine Gallotannin Procyanidin B4 Eruboside B Gallotannin
Binding energy 31.0 (20.7) 59.3 (6.8) 73.5 (5.7) 56.0 (9.4) 120.7 (9.4) 120.6 (10.6)
Coulomb energy 19.9 (15.1) 182.7 (17.3) 42.5 (8.5) 175.5 (16.8) 64.8 (45.7) 74.7 (44.6)
Covalent binding energy 2.4 (4.0) 4.6 (1.5) 0.7 (2.4) 6.0 (1.7) 4.2 (3.2) 4.9 (3.3)
Hydrogen binding correction 2.5(1.6) 4.92 (1.1) 6.4 (0.7) 5.2 (0.8) 5.2 (1.5) 6.4 (1.3)
Lipophilic energy 8.2 (5.3) 14.6 (1.2) 16.1 (1.3) 14.2 (1.4) 30.0 (2.2) 28.2 (1.7)
Vander Waals energy 35.6 (20.0) 55.7 (3.2) 62.9 (3.4) 56.6 (3.5) 99.9 (7.4) 94.7 (5.1)
Binding energy (Docking_XP) 9.7 4.7 0.3 1.3 12.1 2.71

the interactions in the run along with few ionic and pi-cat-
ionic interactions. As it is known, that when a ligand is
strongly bound to the receptor it can either induce physico-
chemical alterations or prevent the natural substrate from
interacting with the receptor, thus affecting the downstream
biological processes. Here, it can be understood in terms of
two distinct modes of action hypothetically. The first one is
with helicase protein, the bound ligand can alter the func-
tion viral protein, next is TBK1; a host protein that is respon-
sible for immune response. Thus, it can contribute to the
host defense system upon infection or as a preventive meas-
ure for the same.
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 11

Figure 5. A schematic of detailed ligand atom interactions with the protein residues. Interactions that occur more than 30.0% of the simulation time in the
selected trajectory (0.00 through 110.01 nsec), are shown. Arrows represent H-bond conserved in the simulation run, they are represented with the % simulation
time and the residues highlighted in box are those participating in the water bridge during the run. The simulation interaction diagrams that are presented are in
the order as mentioned: (A) Helicase-Gallotannin, (B) Helicase-Fenugreekine, (C) Helicase-Procyanidin B4, (D) TBK1-Gallotannin, and (E) TBK1-Eruboside B.

Although this study opens up a fresh perspective, in silico
results are subject to laboratory validation. Furthermore, this
study performed is just a primary staging of wonders Indian
spices sustain. As they are being part of the population’s
daily diet, they can be employed for the wellness of the soci-
ety as a nutraceutical supplement. Especially with the grow-
ing development in the fields of using medicinal properties
of hydrolyzing tannins, gallotannins can be further studied as
a mutual target. While eruboside B for host protein and
fenugreekine, procyanidin B4 for viral protein can be further
validated at individual experimental studies to validate the
precise mechanism involved and what are the upshots in
terms of immunity and health of an organism. We propose
potential nourishment in dietary spice metabolites such as
Cumin, Garlic, Ginger, Hyssop, Capsicum, Chili, Bay leaf, and
Pomegranate seeds, which could potentially strengthen
immunity when taken in diet. Since most of these com-
pounds are used as flavours, these can be added to the cur-
rent medical ingredients used in the steam inhalation
mixture which may yield faster relief. This has to be tested
using appropriate cohorts in the study. With the support of
free energy profile and docking results, garlic, pomegranate,
fenugreek and bay leaf stand as key modulators concerning
viral-host interactions. The current study revealed the thera-
peutic wealth of these spices. The enriched components of
Indian spices like hydrolysable tannins (gallotannin), PADs
(Pyridines and derivatives like fenugreekine); saponins
(eruboside B) and flavonoids (procyanidin B4) can be theoret-
ically used to manage the outbreak of COVID-19, or any simi-
lar RNA virus or the new strains of SARS-CoV-2. We
anticipate that the comprehensions gained in the current
study may support the valued contribution for employing
the spice components as a part of a fortified Nutraceuticals
herbal immune boosting supplement, towards the produc-
tion of natural origin supplements in fight against COVID-19.
Such a development might add up to the ease in the
restriction of infection and for support of the susceptible
host health. Henceforth, this study proves to be a precious
addition in the fight toward the viral infection front where
an individual standing globally can have access to the kit-
chen spices and sustain their response to the viral
concurrence.
Conclusion
For understanding the relationship and modulated dynamics
of Nsp13 and TBK1 upon interaction with metabolites gener-
ated by the study, we present dietary spice actives as protag-
onists for further experimentation to understand how spices
can potentially stimulate host’s immune system and at the
same time if these are also modulating the pathogenicity, or
can these compounds be potential leads to inhibit viral repli-
cation and its consequent propagation, thus modulating
effect of the virus on exposed human population. A
12 K. SRIVIDYA ET AL.
combination of these spice actives could have translational
potential as immunity-boosting health supplement.
Acknowledgments
We are grateful to CSIR mission directorate for initiating us in this area
by funding the projects under Nutraceutical and Immunity Missions. We
are also grateful to the chairman of the CSIR Mission mode monitoring
committee Dr. V. Prakash, CSIR Mission on Nutraceuticals and
Nutritionals/Immunity Mission; Dr. Sanjay Kumar, Director of IHBT,
Palampur and all the members of the committee for their valuable
advice and initiating this new idea. Our gratitude for encouraging us in
its periodical review of the project with innovative ideas about the
active constituents of spices and their potential beneficial effects and
fruitful discussions; held as part of the monitoring committee meetings
that periodically reviewed our project on spices. Those learnings and
top of the line suggestions eventually motivated us to carry out the
studies reported in this manuscript. An institutional grant to IBAB from
the department of IT, BT, Science, and Technology, the Government of
Karnataka is acknowledged.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The authors of this manuscript wish to acknowledge gratefully the top
driven project concept on Mission Mode from CSIR on this idea of spices
in the prevention of diseases (HCP0019WP7 and HCP0035). IBAB is also
supported by DST-FIST vide sanction no SR/FST/LSI-536/2012. CSIR-CDRI
manuscript number is 10260.
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