Mitochondrion 61 (2021) 138–146

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Mitochondrion
journal homepage: www.elsevier.com/locate/mito

Molecular docking and simulation studies of phytocompounds derived from

Centella asiatica and Andrographis paniculata against hexokinase II as
mitocan agents
Geeta Swargiary, Shalini Mani *
Centre for Emerging Diseases, Department of Biotechnology, Jaypee Institute of InformationTechnology, Noida, India

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

Keywords: Hexokinase II (HK2), a glycolytic enzyme is commonly overexpressed in most cancer types. The overexpression
Hexokinase II of HK2 is reported to promote the survival of cancer cells by facilitating the constant ATP generation and pro­
Andrographis paniculata tecting the cancer cell against apoptotic cell death. Hence, HK2 is considered as potential target of many
Centella asiatica
mitochondria targeting anticancerous agents (referred to as mitocans). Most of the existing mitocans are syn­
Molecular docking
Molecular dynamics simulation
thetic and hence such compounds are observed to exhibit adverse effects, witnessed through many experimental
Asiatic acid outcomes. These limitations necessitates hunting for an alternative source of mitocans with minimum/no side
Andrographolide effects. The need for an alternative therapy points towards the ethnomedicinal herbs, known for their minimal
Bayogenin side effects and effectiveness. Henceforth recent studies have put forth the effort to utilize anticancer herbs in
formulating naturally derived mitocans as an add-on to improve cancer therapeutics. So, our study aims to
explore the HK2 targeting potential of phytocompounds from the selected anticancerous herbs Andrographis
paniculata (AP) and Centella asiatica (CA). 60 phytocompounds collectively from CA and AP were docked against
HK2 and drug-likeness prediction of the selected phytocompounds was performed to screen the best possible
ligand for HK2. Furthermore, the docked complexes were subjected to molecular dynamics simulations (MDS) to
analyse the molecular mechanism of protein-ligand interactions. The results of the study suggest that the natural
compounds asiatic acid and bayogenin (from CA) and andrographolide (from AP) can bepotential natural
mitocans by targeting HK2. Further experimental studies (in-vitro and in-vivo) are required to validate the results.

1. Introduction
Mitocans are a novel class of drugs that are exclusively designed to
target the mitochondrial metabolism of cancer cells by targeting the
mitochondrial electron transport chain, tricarboxylic acid (TCA) cycle,
glycolysis, oxidative phosphorylation (OXPHOS), mitochondrial redox
signalling pathways including ROS homeostasis and other signalling
pathways that affect the mitochondrial functions (Dong et al., 2020).
Moreover, Warburg in his hypothesis explained that the cancer cells
undergo a major metabolic shift which is due to the prominently higher
metabolic rate in the cancer cells in comparison to the normal cells. The
metabolic shifting or the metabolic reprogramming in cancer cells is the
switch from OXPHOS to glycolysis for the fulfilment of their metabolic
requirements. So, agents that can target both glycolysis and OXPHOS
may play a significant role as potentially efficient anticancer therapeu­
tics. The very first enzyme of the metabolic pathway is the HK2 enzyme
that is overexpressed in the cancer cells and plays a vital role in main­
taining the glycolytic activity (Diaz-Ruiz et al., 2011; Guo et al., 2020;
Ma et al., 2021). The overexpression of HK2 is reported to promote the
survival and proliferation of cancer cells by facilitating the conversion of
highly up taken glucose to glucose 6 phosphate (G6P) that enters the
metabolic machinery to yield a higher amount of ATP (energy) in the
cancer cells (Mathupala et al., 2006; Nowak et al., 2018). HK2 also as­
sociates with voltage-dependent anion channel (VDAC) located on the
outer membrane of the mitochondria and plays a crucial role in
apoptosis. Normally, the VDAC is one of the exit channels for the cyto­
chrome c, but the highly interacting HK2-VDAC in cancer cells inhibits
the cytochrome c release from the mitochondria and thereby protecting
the cancer cell against apoptotic cell death (Arzoine et al., 2009; Ma
et al., 2021). Therefore, inhibition of HK2 may suppress or inhibit
glycolysis or suppress the anti-apoptotic effect of HK2-VDAC interac­
tion. For example, HK2 inhibitor FV-429 induced apoptosis in the cancer

* Corresponding author.
E-mail address: shalini.mani@jiit.ac.in (S. Mani).

https://doi.org/10.1016/j.mito.2021.09.013
Received 28 June 2021; Received in revised form 13 September 2021; Accepted 29 September 2021
Available online 2 October 2021
1567-7249/© 2021 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
G. Swargiary and S. Mani
cells by both inhibitions of glycolysis via suppression of HK2 and acti­
vation of mitochondria-mediated apoptosis via interfering with the
HK2-VDAC interaction (Zhou et al., 2016). Similarly, benitrobenrazideis
a selective novel inhibitor of HK2 that inhibited glycolysis, inhibiting
the proliferation of cancer cells and mediated apoptosis via mitochon­
drial intrinsic pathway by targeting HK2 (Zheng et al., 2021). Moreover,
D-Mannoheptulose treated in synergy with Newcastle disease virus
reduced the activity of HK2 that lead to a reduction in the pyruvate, ATP
and acidity of the tumor microenvironment in the human breast cancer
cells. As a result, the proliferation of the cancer cell was inhibited and
induced apoptosis (Al-Ziaydi et al., 2020). Thus, collectively different
mitochondria mitocans (compounds targeting mitochondria of cancer
cells) such as 3-bromopyruvate, 2-deoxyglucose, metformin,
mitochondria-targeted carboxy-proxyl (Mito-CP) have shown to inhibit
HK2 in cancer cells (Dong et al., 2020). These studies indicate that HK2
may be considered an important target in cancer therapeutics. Addi­
tionally, the overexpression of HK2 is not reported in association with a
specific cancer type, rather most of the cancer types, hence targeting
such an enzyme may help propose an alternative therapy for several
cancer types. But the use of these synthetic mitocans has the possibility
of exhibiting adverse effects as observed in most synthetic drugs.
Therefore, this possibility of limitation demands a need for alternative
mitocans with minimum or no side effects. The use of ethnomedicinal
herbs is highly known for their lower side effects and effectiveness.
Therefore, recent studies have put forth the effort to utilize anticancer
herbs in formulating naturally derived mitocans as an add-on to improve
cancer therapeutics. Taking a lead from the studies on the importance of
HK2 as a target in cancer therapy and natural anticancer herbs as a
potential healer, our study aims to explore naturally derived mitocans
by targeting HK2. While exploring a plethora of anticancer herbs, we
selected Centella asiatica (CA) and Andrographis paniculata (AP) as their
mitochondria targeting and/or HK2 targeting ability is not studied
thoroughly.
CA, also known as Gotu Kola is a medicinal plant from the Apiaceae
family and is a folklore medicine used for the ailment of several diseases
such as wound healing, burns, psoriasis and scleroderma. CA is majorly
found in the wetlands of Asian countries including India, Pakistan,
Bangladesh, Madagascar and some parts of Africa and America. The
active compounds of CA include the pentacyclic triterpenes majorly
comprising of asiaticoside, madecassoside, asiatic and madecassic acids.
The medicinal properties of CA are due to the presence of these active
compounds. However, the past few decades of studies havealso focused
on the anticancer potential of CA. CA has exhibited its anticancer effect
lung cancer cells (Aizad et al, 2021; Han et al, 2020), lung tumour
induced mice (Hamid et al., 2016) and breast cancer cells via induction
of apoptosis (Babykutty et al., 2009) Also CA has shown its effectiveness
in colorectal cancer cells via reduction of MMP-9 invasive protein and
migration of the cancer cells during metastasis (Manmuan et al., 2021).
Moreover, silver nanoparticle biosynthesised by CA extract has shown
anti-proliferative and caspase dependant apoptosis of breast cancer cells
(Fard et al., 2018). On the other hand, AP is a perennial herbthat is
commonly known as the Kalmegh, Greenchiretta and the ‘king of bitters’
that belongs to the Acanthaceae family. Similar to CA, AP is also native
to Asian countries including India, Sri Lanka, China and Thailand. AP is
the traditional medicinal plant used for the treatment of diseases like
diabetes, cancer, influenza, high blood pressure, skin diseases, malaria,
leprosy and ulcer (Okhuarobo et al., 2014). The medicinal property of
AP is mainly due to the presence of an abundant amount of terpenoid
comprising of andrographolide (Okhuarobo et al., 2014). Anticancer
effect of AP is reported for various human cancer cells including breast
cancer (Sholihah et al., 2019), colon cancer (Shimura et al., 2021),
cervical cancer, ovarian cancer, liver cancer (Singh et al., 2013a; Singh
et al., 2013b), human lymphocytes culture (Ahmad et al., 2014) and in
mouse melanoma cells (Paul et al., 2019) and bone marrow cells of al­
bino mice (Ahmad et al., 2014). Therefore, CA and AP were selected for
the study of their phytocompounds that may potentially target the HK2
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Mitochondrion 61 (2021) 138–146
and contribute as an alternative natural mitocan for cancer therapy.
2. Methods
2.1. Selection of receptor and ligands
There are four isoforms of hexokinase, but HK2 is overexpressed in
cancer cells and are reported as an important target for many anticancer
agents. HK2 is the rate-limiting enzyme of glycolysis and in normal
physiological conditions, it catalyses the phosphorylation of glucose to
glucose-6-phosphate (G6P) via ATP. Therefore, glucose is the most
important substrate of HK2 and G6P is the important product. HK2
undergoes an induced-fit conformational change when it binds to
glucose which ultimately prevents the hydrolysis of ATP. And HK2 is
allosterically inhibited by the physiological concentrations of its im­
mediate product G6P. HK2 is bound to the mitochondria via interaction
with VDAC that provides ATP for its phosphorylation. When the HK2 is
bound to VDAC, the HK2 is not inhibited by G6P. Therefore, dissociation
of HK2-VDAC is necessary for the inhibition of HK2 by G6P. In the case
of cancer cells, the highly expressed HK2 functions to enhance the
glycolysis rate. So, our study considered HK2 as the prime target and
aimed to explore the potential of natural compounds that may inhibit
glycolysis by inhibiting HK2. Therefore, G6P was considered as the
control and the natural compounds were docked at the G6P binding site
of the HK2. The structure of HK2 co-crystallized with G6P and glucoseis
available in the PDB with entry ID 2NZT. However, this structure con­
sists of missing residues and to incorporate those missing residues, the
structure was regenerated by using homology modelling. On the other
hand, the phytocompounds of CA and AP were retrieved from the
curated database of Indian Medicinal Plants, Phytochemistry And
Therapeutics (IMPPAT) (Mohanraj et al., 2018).
2.2. Homology modelling of HK2 receptor
The 3D structure of HK2 was obtained by homology modelling using
Modeller 10.1. Modeller is a python based tool that requires the align­
ment of a sequence to be modelled with known related structures and
automatically calculates a model containing all non-hydrogen atoms
(Fiser and Šali, 2003; Webb and Sali, 2016). The query sequence of HK2
was retrieved from UniProtKB (https://www.uniprot.org/) with acces­
sion ID P52789 and the template was selected by running a protein
BLAST (BLASTp) search (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The
BLASTp results showed that 5HEX has 100% identity, 98% query
coverage and maximum accession length (Supplementary Fig. S1). So,
5HEX was considered as the template to generate the HK2 model.
Further, the HK2 model was energy minimized by using Swiss-
PdbViewer (http://www.expasy.org/spdbv/) and its quality was vali­
dated by different programs of SAVES v6.0 (https://saves.mbi.ucla.edu/
) including the Verify 3D, ERRAT and PROCHECK. Moreover, the HK2
model was superimposed with the template by using PyMol (The PyMol
Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC).
2.3. Receptor and ligand preparation
The modelled HK2 structure was visualized in Autodock Tools
(Morris et al., 1998) and hydrogen atoms were added. Furthermore,
modelled HK2 was converted to pdbqt format and used for docking in
Autodock Vina1.1.2. In case of the ligand, the 3D structure of G6P was
retrieved from the co-crystal structure 2NZT and pre-processed by
converting it from pdb to pdbqt by using Autodock Tools. For the 60
phytocompounds collectively from CA and AP, the IMPPAT database
provides 3D structures of phytocompounds that are pre-processed to
pdbqt format and easily downloadable that makes the docking with
Autodock Vina (Trott and Olson, 2010) convenient.
G. Swargiary and S. Mani
2.4. Binding pocket identification
Since the structures of receptor and ligands are ready, the next step is
to define the binding pocket for the ligands. The binding pocket for our
study is the binding site of G6P (control). G6P binds to the residues Asp-
657, Thr-680 and Ser-897 of HK2 (Lin et al., 2016; Miao et al., 2019;
Nawaz et al., 2018; Salani et al., 2013). Therefore, these residues were
the primary focus around which the grid box of dimension 16 × 16 × 20
Å centred at x = 104.842, y = 85.718 and z = − 93.97 was generated by
using Autodock Tools.
2.5. Molecular docking in Autodock Vina
The 60 phytocompounds and control (i.e. G6P) was docked against
HK2 by using Autodock Vina that considers the receptor and ligand as
rigid. G6P was re-docked with HK2 to compare the results of the un­
known ligands. The information of the receptor, ligands, grid box
dimension and centre was defined within a configuration file to run the
command for Autodock Vina. The docking resulted in the binding en­
ergies of various poses of the ligands concerning the HK2. The phyto­
compounds with the binding energy lower than the control (i.e. G6P)
were selected for further analysis. The post docking analysis including
the hydrogen bonds and hydrophobic bond interactions between the
HK2 and the ligands were performed for those selected phytocompounds
that showed drug-like properties. The interactions were visualized in
PyMol and LigPlot+ (Laskowski and Swindells, 2011).
2.6. Prediction of drug-likeness properties
The drug-like properties of the selected 47 phytocompounds were
predicted by using Molinspiration (https://www.molinspiration.com).
The canonical SMILES notations for the selected phytocompounds were
retrieved from the IMPAAT database and used as the input for Molins­
piration for thecalculation of important molecular properties including
the miLogP, polar surface area (TPSA), number of hydrogen donors and
acceptors, molecular weight, molecular volume, TPSA, number of
rotatable bonds and number of atoms. Moreover, Molinspirationpredicts
the bioactivity score for the most important drug targets comprising the
GPCR ligands, kinase inhibitors, ion channel modulators, nuclear re­
ceptors, protease inhibitors and enzyme inhibitors). The results were
filtered based on the collective rules of Veber (Veber et al., 2002), Ghosh
(Ghose et al., 1999). Pfizer (Hughes et al., 2008) and Lipinski Rule of five
(Lipinski et al., 1997) and their bioactivity score was noted.
2.7. Molecular dynamics simulation
The calculations were performed with GROMACS 5.1 (Pronk et al.,
2013) package, using the OPLS (Jorgensen et al., 1996) force field. The
box dimensions ensured that any protein atom was at least 1 nm away
from the wall of the box with periodic boundary conditions and solvated
by simple point charge (spc) (Berendsen et al., 1987) water molecules.
NaCl counter ions were added to satisfy the electro-neutrality condition.
Energy minimization was carried out using the steepest descent method.
Berendsen temperature coupling (Berendsen et al., 1984) and Parinello-
Rahman pressure coupling (Parrinello and Rahman, 1981) were used to
keep the system in a stable environment (300 k, 1 bar), and the coupling
constants were set to 0.1 and 2.0 ps for temperature and pressure,
respectively. The partial mesh Ewald (PME) algorithm (Darden et al.,
1993) was employed for electrostatic and Van der Waals interactions;
cut-off distance for the short-range VdW (rvdw) was set to 1.4 nm, where
Coulomb cut-off (r coulomb) and neighbour list (rlist) were fixed at 0.9
nm. All the bond lengths were constrained using the LINCS algorithm
(Xie et al., 2014) and the time step was set to 0.002 ps. The complexes in
a medium were equilibrated for 100 ps in NVT and NPT ensembles,
respectively. Finally, 20 ns molecular dynamics simulations were car­
ried out for all complexes. All trajectories were stored every 2 ps for
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Mitochondrion 61 (2021) 138–146
further analysis.
2.8. Analysis of molecular dynamics simulation
Structural analysis of the HK2 with G6P, bayogenin, andrographo­
lide and asiatic acid was carried from the trajectory files with the built-in
function of GROMACS 5.1. Structural analysis, such as root-mean-square
deviation (RMSD) and radius of gyration (Rg) were analysed through
built-in functions of GROMACS. The number of hydrogen bond formed
between the protein and ligands during the simulation was calculated.
The number of hydrogen bond determined based on donor-acceptor
distance smaller than 3.6 Å and of donor-hydrogen acceptor angle
larger than 90◦ . Solvent accessible surface area (SASA) analysis formed
in hexokinase was analysed by using the GROMACS tool. To generate
the plot for the three-dimensional backbone, gyration of backbone and
SASA analysis, we used Graphing, Advanced Computation and Explo­
ration (GRACE) program (Turner, 2005).
3. Results and discussion
3.1. Homology modelling and validation of HK2
The results of Verify 3D revealed that modelled HK2 passed with
96.73% of its residues scoring less than or equal to 0.2 (Supplementary
Fig. S2). ERRAT on the other hand provided an overall quality factor
that is expressed as the percentage of the protein for which the calcu­
lated error value falls below the 95% rejection limit. Moreover, good
high-resolution structures generally produce overall quality values
around 95% or higher. For lower resolutions (2.5 to 3A) the average
overall quality factor is around 91%. Therefore, the overall quality
factor of the modelled HK2 as predicted by ERRAT is 93.333 (Supple­
mentary Fig. S3) that indicates the average resolution of the HK2 model.
In addition to that, the Ramachandran plotby PROCHECK also indicates
that the maximum of the residues are in the allowed regions (i.e. 85.8%
in the most favoured region, 13.3% in the additional allowed region,
0.6% in the generously allowed region and only 0.2 % in the disallowed
region) (Fig. 1). Further, the structural variation of the model from the
crystal structure was verified by the superimposition of modelled HK2
structure to the template. The superimposition of the modelled HK2 to
the template 5HEX has shown an RMSD of 0.667 Å as shown in Fig. 2.
Therefore, these results show the good and reliable quality of the
modelled HK2 for further studies.
3.2. Molecular docking and drug-likeness prediction
From the docking results, it was confirmed that the missing residues
observed in the crystal structure 2NZT and 5HEX do not lie within the
vicinity of 5 Å distance from the binding site of G6P (i.e control) as well
as they do not fall within the defined grid box used for docking. Thus, the
missing residues were not involved in the active site region where the
phytocompounds bound to HK2. The docking of 60 phytocompounds
resulted in their binding energies towards HK2 (Supplementary
Table S4). The binding energy of the control (G6P) was − 5.2 Kcal/mol
and therefore, this value was considered as the cut-off for comparing the
binding energy of the phytocompounds towards HK2. Therefore, selec­
tion based on the binding energycutoff was used as the first criteria to
narrow down the number of phytocompounds for further analysis. A
lower value of binding energy indicates the higher binding affinity and
stability of the receptor-ligand complex. So, 47 out of 60 phyto­
compounds had better binding energy than G6P and were considered for
the implementation of second criteria of elimination by prediction of
druglikeness properties. Further, the prediction of drug-likeness prop­
erties of the selected 47 phytocompounds enabled the eliminationof
those phytocompounds that do not have significant drug-like properties.
The collective implementation of the laws of Lipinski Rule of 5, Veber,
Ghosh and Pfizerenabled to set elimination criteria as follows: partition
G. Swargiary and S. Mani
Fig. 1. Ramachandran plot of the modelled HK2 (The red zone indicates the most favourable region, the yellow zone is the additionally allowed region, the light
yellow zone is the generously allowed region and the white zone is the disallowed region of the residues). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
coefficient (miLogP) < 5, topological surface area (TPSA) = 75–140,
number of atoms (natoms) = 20–70, molecular weight (MW) < 500,
number of hydrogen donors (nON) <= 10, number of hydrogen accep­
tors (nOHNH) <= 5, nviolations = 0 or 1 andnrotb <= 10. As a result, 11
out of 47 phytocompounds followed the rules and were selected and
considered for further studies (Table 1). Additionally, Molinspiration
calculated bioactivity score of these 11 phytocompounds showed
bioactivity score more than zero towards the class of enzyme inhibitors
(Table 2). Since our study aims to explore the HK2 enzyme inhibition
potential of the selected phytocompounds, hence the primary focus was
on the bioactivity score towards the class of enzyme inhibitors. The
bioactivity score of more than zero indicates that the compound is
active, − 5.0 to 0.0 is moderately active and less than − 5.0 is inactive
(Singh et al., 2013a; Singh et al., 2013b; Veber et al., 2002). Therefore,
our result suggests that the selected 11 phytocompounds can be
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Mitochondrion 61 (2021) 138–146
potential drugs to target the HK2enzyme. To strengthen the interacting
potential of these phytocompounds with HK2, further, the HK2-ligand
interaction was analysed.
3.3. HK2-ligand interactions
According to the UniProtKB database, G6P binds to the residues Ser-
657, Thr-680 and Ser-897 of the C-terminal domain of HK2. Studies have
also shown that these residues are involved in the hydrogen bond in­
teractions between G6P and HK2 (Lin et al., 2016; Miao et al., 2019;
Nawaz et al., 2018; Salani et al., 2013). Our result showed that these
residues (Ser-657, Thr-680 and Ser-897) are involved in either hydrogen
or hydrophobic bonds interactions with the selected 11 phyto­
compounds (Bayogenin, Asiatic acid, Andrographolide, 14-Deoxy-11-
oxoandrographolide, MLS001143515, Dehydroandrographoline,
G. Swargiary and S. Mani Mitochondrion 61 (2021) 138–146

strengthen the selection of these phytocompounds for the molecular

dynamics simulations study. The superimposition of the G6P-HK2 with
Bayogenin-HK2, Andrographolide-HK2 and Asiatic acid-HK2 is shown
in Fig. 3, indicating that bayogenin, andrographolide and asiatic acid
share common binding positions while interacting with HK2.

3.4. Molecular dynamics simulation

The molecular dynamics simulations were performed for the four

Fig. 2. Superimposition of HK2 model with the template 5HEX (RMSD of
0.667 Å) visualized in PyMol.
Kaempferol, 5-hydroxy-7,8,2′ ,3′ -tetramethoxyflavone, Apigenin, 5-Hy­
droxy-3,7,8-trimethoxy-2-(2-methoxyphenyl)-4H-chromen-4-one and
Andrographin), suggesting the potential of these phytocompounds to
bind to HK2 at its G6P binding site and may compete with G6P to bind to
HK2. The hydrogen and hydrophobic bond interactions are shown in
Table 3 and Supplementary Figs. S5 and S6. However, to narrow down
the number of ligands and to strengthen the study, the top three ligands
(from the 11 phytocompounds) with the minimum binding energy i.e.
Bayogenin (− 7.2 Kcal/mol), Asiatic acid (− 7 Kcal/mol) and Androgra­
pholide (− 6.9 Kcal/mol) were selected for the molecular dynamics
simulations. Bayogenin is found in CA, yet it is also a common bioactive
compound found in other herbs as well. However, bayogenin has min­
imum binding energy towards HK2. Moreover, asiatic acid and
andrographolide are abundantly found in CA and AP respectively that
HK2 complexes which include G6P-HK2, Bayogenin-HK2,
Andrographolide-HK2 and Asiatic acid-HK2. The average value of RMSD
for backbone atoms was 1.24 nm in the G6P-HK2 complex. Whereas, the
average values of RMSD for the backbone atoms in Bayogenin-HK2,
Andrographolide-H2K and Asiatic acid-HK2 complexes were 0.79 nm,
0.74 nm and 1.12 nm respectively (Fig. 4). The Rg value of protein for
the G6P-HK2 complex varied between 1.69 nm and 2.28 nm. For
Bayogenin-HK2, Andrographolide-HK and Asiatic acid-HK the Rg value
varied between 1.74 nm and 2.29 nm, respectively. The average values
of gyration (Rg-protein) for G6P-HK2 and the other three complexes
were 1.89 nm, 2.15 nm, 2.15 nm and 1.92 nm, respectively (Fig. 5). The
lower values of Rg in protein G6P-HK2 complexsuggested it to be more
compact. SASA was calculated for all the four proteins trajectories for
each residue and values averaged. The average SASA of G6P-HK2,
Bayogenin-HK2, Andrographolide-HK2 and Asiatic acid-HK2 were
126.25 nm2, 126.30 nm2, 130.56 nm2 and 125.73 nm2, respectively
(Fig. 6). The SASA of G6P-HK2 varied from 119.59 nm2 to 143.09 nm2
and that of other complexes Bayogenin-HK2, Andrographolide-HK2 and
Asiatic acid-HK2 differed from nm2 to nm2, nm2 to nm2 and nm2 to nm2,
respectively. The number of hydrogen bonds between protein and ligand
was calculated for all four complexes to understand the strength of the
four compounds interaction with HK2. The G6P and other compounds
(bayogenin, andrographolide and asiatic acid) formed average hydrogen
bonds of 3, 4, 3, and 2, respectively (Fig. 7). The molecular dynamics

Table 1
Drug-likeness properties of the selected 11 phytocompounds that passed the Lipinski Rule of 5, Veber’s rule, Ghosh’s rule and Pfizer rule.
S. Phytocompounds miLogP TPSA natoms MW nON nOHNH Nviolations nrotb volume
No. (<5) (75–140) (20–70) (<500) (<=10) (<=5) (0 or 1) (<=10) (<500)

1. 14-Deoxy-11-oxoandrographolide 0.62 83.83 25 348.44 5 2 0 4 332.47

2. 5-Hydroxy-3,7,8-trimethoxy-2-(2-methoxyphenyl)-4H- 3.26 87.38 26 358.35 7 1 0 5 310.2
chromen-4-one
3. 5-hydroxy-7,8,2′ ,3′ -tetramethoxyflavone 3.09 87.38 26 358.35 7 1 0 5 310.2
4. 4zAndrographin 3.27 78.14 24 328.32 6 1 0 4 284.65
5. Andrographolide 1.05 86.99 25 350.45 5 3 0 3 338.33
6. Apigenin 2.46 90.89 20 270.24 5 3 0 1 224.05
7. Asiatic acid 4.7 97.98 35 488.71 5 4 0 2 487.79
8. Bayogenin 4.63 97.98 35 488.71 5 4 0 2 487.44
9. Dehydroandrographoline 2.39 90.89 25 348.44 5 3 0 3 332.12
10. Kaempferol 2.17 111.12 21 286.24 6 4 0 1 232.07
11. MLS001143515 1.42 76 26 364.48 5 2 0 5 355.86

Table 2
Bioactivity score of the selected 11 phytocompounds.
S. Phytocompounds GPCR Ion channel Kinase Nuclear receptor Protease Enzyme
No. ligand modulator inhibitors ligand inhibitors inhibitors

1. 14-Deoxy-11-oxoandrographolide 0.18 0.08 − 0.47 0.5 − 0.01 0.71

2. 5-Hydroxy-3,7,8-trimethoxy-2-(2-methoxyphenyl)- − 0.16 − 0.08 0.05 0.03 − 0.31 0.22
4H-chromen-4-one
3. 5-hydroxy-7,8,2′ ,3′ -tetramethoxyflavone − 0.11 − 0.01 0.08 0.13 − 0.24 0.2
4. Andrographin − 0.11 − 0.03 0.12 0.16 − 0.26 0.24
5. Andrographolide 0.32 0.17 − 0.01 0.94 0.26 0.81
6. Apigenin − 0.07 − 0.09 0.18 0.34 − 0.25 0.26
7. Asiatic acid 0.2 − 0.19 − 0.46 0.91 0.28 0.66
8. Bayogenin 0.2 − 0.22 − 0.38 0.8 0.2 0.63
9. Dehydroandrographoline − 0.03 − 0.18 − 0.32 0.59 − 0.07 0.49
10. Kaempferol − 0.1 − 0.21 0.21 0.32 − 0.27 0.26
11. MLS001143515 0.4 0.17 − 0.26 − 0.88 0.22 0.74

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G. Swargiary and S. Mani Mitochondrion 61 (2021) 138–146

Table 3
Binding energy, hydrogen bond and hydrophobic interactions of the selected 11 phytocompounds against HK2.
S. Compounds Binding energy H-bonds from PyMol H-bonds from Hydrophobic bonds from LigPlot+
No. (Kcal/mol) LigPlot+

1. Bayogenin − 7.2 Arg-539, Asn-683 Arg-539, Asp-861 Gly-679, Gly-681, Ile-677, Asn-683, Lys-621, Asp-
657, Gly-534, Gly-896
2. Asiatic acid − 7 Arg-539 Arg-539, Asp-861, Ser-682, Gly-681, Lys-621, Asp-657, Gly-534, Gly-
Glu-742 896, Gly-679, Ile-677
3. Andrographolide − 6.9 Asn-683, Arg-539, Arg-539, Asn-683, Gly-896, Asp-861, Ile-677, Gly-681, Gly-679, Ser-
Asp-532 Asp-532 682, Thr-680, Asp-657, Lys-621
4. 14-Deoxy-11-oxoandrographolide − 6.8 Asn-683, Asp-861, Ser-897, Asp-861, Gly-896, Gly-681, Thr-680, Lys-621, Ser-682, Ile-
Arg-539, Ser-897 Asn-683, Asp-532 677, Arg-539, Asp-657
5. MLS001143515 − 6.8 Asp-532, Arg-539, Arg-539, Asp-532, Ser-897, Asp-657, Asp-861, Gy-862, Thr-680, Ile-
Ser-897, Gly-681 Gly-681 677, Lys-621, Ser-603, Leu-533
6. Dehydroandrographoline − 6.6 Lys-866, Gly-681, Lys-866, Gly-681, Thr-680, Asp-657, Asp-861, Asp-895, Gly-896,
Arg-539 Arg-539 Gly-862, Ile-677, Gly-679
7. Kaempferol − 6.6 Lys-621 Lys-621, Asp-861 Gly-862, Asp-657, Ser-603, Asn-683, Ser-682,
Glu-742, Ile-677
8. 5-hydroxy-7,8,2 ,3 -tetramethoxyflavone
′ ′
− 6.5 Asn-683, Gly-681, Thr-680, Gly-681, Asp-861, Ser-603, Asp-657, Lys-621, Ser-897,
Thr-680 Asn-683 Glu-708, Ser-682, Thr-658, Ile-677, Gly-679
9. Apigenin − 6.5 Asn-683, Glu-742 Asn-683, Ser-897 Ser-682, Ile-677, Glu-742, Gly-681, Gly-679, Thr-
680, Asp-861, Asp-657
10. 5-Hydroxy-3,7,8-trimethoxy-2-(2- − 6 Lys-621, Gly-681, Lys-621, Gly-681, Asp-861, Gly-679, Ile-677, Asp-657, Ser-603, Thr-
methoxyphenyl)-4H-chromen-4-one Thr-680 Thr-680 658, Asn-683, Ser-897
11. Andrographin − 5.9 Gly-681, Thr-680 Gly-861 Thr-658, Asp-657, Lys-621, Gly-679, Ile-677, Gly-
862, Asp-861, Thr-680, Asn-683

Fig. 3. Superimposition of the complexes G6P-HK2 with Bayogenin-HK2, Andrographolide-HK2 and Asiatic acid-HK2. The white colour compound represents G6P,
the blue colour is the bayogenin, the green colour is andrographolide and yellow is asiatic acid. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)

Fig. 4. The average value of RMSD for backbone atoms in simulated G6P-HK2,
Fig. 5. The average values of gyration (Rg-protein) for G6P-HK2, Asiatic acid-
Asiatic acid-HK2 and Andrographolide-HK2 complexes.
HK2 and Andrographolide-HK2 complexes.

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G. Swargiary and S. Mani Mitochondrion 61 (2021) 138–146

Fig. 7. Average hydrogen bonds of G6P-HK2, Bayogenin-HK2,

Fig. 6. The average SASA of G6P-HK2, Bayogenin-HK2, Andrographolide-HK2
Andrographolide-HK2 and Asiatic acid-HK2 complexes.
and Asiatic acid-HK2 complexes.

of HK2 may lead to either inhibition of glycolysis or dissociation of HK2-

simulations analysis showed bayogenin could be the best compound to
VDAC interaction. Most likely the HK2 inhibitors have shown to disso­
target HK2 and another compound andrographolide could also perform
ciate HK2 and VDAC that in turns suppress the anti-apoptotic effect of
a similar kind of effect like the native (G6P) compound. Asiatic acid
the HK2-VDAC complex (Zhou et al., 2016). Moreover, it is also possible
could also have good interaction with HK2 but maybe not as effective as
that the dissociation may lead to opening the gate for cytochrome c via
other native and other two novel compounds.
VDAC and resulting in apoptosis of the cancer cell. Few shreds of evi­
Cancer is known to be a multifactorial and complex disease that
dence are suggesting the anticancer effect of bayogenin, asiatic acid and
functions via various mechanisms depending on the type of cancer.
andrographolide. Bayogenin is the phytocompound of CA that has
Therefore, the treatments are mostly designed based on the cancer types.
shown to reduce the proliferation of HeLa cells and MCF-7 cells (Avato
The development in the current cancer research has highlighted the role
et al., 2017). Asiatic acid is another phytocompound of CA that is
of bioenergetics of cancer cells which is due to the higher energy
abundantly present in CA and exhibited anticancer effect in different
requirement of cancer cells that is a common nature of all cancer types.
types of cancer cells including breast cancer, lung cancer, melanoma,
Therefore, mitochondria being the hub of energy production in the cells
colon cancer, nasopharyngeal carcinomaand cholangiocarcinoma (Gou
is an important target in cancer cells. Mitocansare a novel class of drugs
et al., 2020; Wu et al., 2017; Park et al., 2005; Hao et al., 2018; Liu et al.,
that were designed to target the mitochondrial metabolism of cancer
2020; Sakonsinsiri et al., 2018). On the other hand, andrographolide is a
cells (Ralph et al., 2006). The research on mitocansare still ongoing and
bioactive compound that is found abundantly in AP. The anticancer
in addition to that, natural agents are being studied for their mito­
effect of AP was observed in the cell lines of breast cancer, prostate
chondria targeting potential in cancer therapeutics (Dong et al., 2020;
cancer, colon cancer, gastric cancer, lung cancer, ovarian cancer, renal
Mani et al., 2020). Based on their mode of action, the mitocans are
cancer, melanoma and glioblastoma (Ajaya Kumar et al., 2004; Beesetti
classified into different class including the hexokinase inhibitors. com­
et al., 2019; Dai et al., 2017; Khan et al., 2018; Mir et al., 2016).
pounds targeting Bcl-2 family proteins, thiol redox inhibitors plus
However, the HK2 targeting potential of bayogenin, asiatic acid and
VDAC/ANT targeting drugs, electron redox chain targeting drugs,
andrographolide are not yet studied and therefore, our study for the first
lipophilic cations targeting the inner membrane, drugs targeting the
time show the preliminary evidence of bayogenin, asiatic acid and
tricarboxylic acid cycle and drugs targeting mtDNA (Neuzil et al., 2013).
andrographolide as potential mitochondria targeting compounds via
However, concerning Warburg’s effect, the hexokinase inhibitors are
targeting HK2 and may benefit as an alternative in cancer therapeutics.
closely associated with glycolysis which is highly active in the cancer
cells. There are four isoforms of hexokinase, but the HK2 is highly
4. Conclusion
overexpressed in cancer cells (Mathupala et al., 2006). Therefore, the
primary aim of our study was to inhibit the HK2 by occupying the
HK2 is a crucial target in cancer therapeutics and the overexpression
inhibitory G6P binding site of HK2. HK2 is not inhibited by G6P when it
of HK2 is one of the characteristics of cancer cells. Therefore inhibition
is intact to VDAC and therefore, glycolysis is highly elevated in cancer
of HK2 is an important approach in cancer therapy. Bayogeninand
cells. Inhibition of HK2 is an important aspect of cancer therapy. So, the
andrographolide could be the best lead compounds to target HK2.
molecular docking and drug-likeness prediction in the study suggested
Moreover, asiaticacid could interact with HK2, but may not be as
that 11 phytocompounds can potentially bind to HK2 with better
effective as G6P, bayogenin and andrographolide. The HK2 targeting
binding energies than the control G6P and they have drug-like proper­
potential of these three compounds is previously never been reported.
ties that collectively highlight them to be potential lead compounds for
Therefore, for the first time, our study suggests that bayogenin,
HK2. However, molecular dynamics simulation conducted for the top 3
andrographolide and asiatic acid may be novel anticancer agents by
phytocompounds selected based on their binding energies i.e. bayogenin
targeting HK2. Further experimental studies (in-vitro and in-vivo) are
(− 7.2 Kcal/mol), asiatic acid (− 7 Kcal/mol) and andrographolide (− 6.9
required to understand the underlying mechanism of action and the
Kcal/mol) confirms that these compounds can be the better and poten­
results of this study.
tial ligand for HK2 that may function as the inhibitors of HK2 in cancer
cells. However, as per the results of molecular dynamics simulations,
Declaration of Competing Interest
bayogenin seems to the most potent compound to target HK2. Inter­
estingly, andrographolide may also exhibit a similar effect on HK2 as
The authors declare that they have no known competing financial
G6P which suggest the potentiality of andrographolide to inhibit HK2 by
interests or personal relationships that could have appeared to influence
occupying the G6P binding site. Asiatic acid may alsopotentially interact
the work reported in this paper.
with HK2 but maybe not as effective as G6P, bayogenin and androgra­
pholide. Possibly bayogenin, andrographolide and asiatic acid may be
potential HK2 inhibitors contributing to cancer therapeutics. Inhibition

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G. Swargiary and S. Mani Mitochondrion 61 (2021) 138–146

Acknowledgement Han, A.-R., Lee, S., Han, S., Lee, Y.J., Kim, J.-B., Seo, E.K., Jung, C.-H., 2020.
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ogy, Noida, for providing the suitable infrastructure to complete this Hao, Y., Huang, J., Ma, Y., Chen, W., Fan, Q., Sun, X., Shao, M., Cai, H., 2018. Asiatic
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