Journal of Theoretical Biology 461 (2019) 254–267

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Journal of Theoretical Biology

journal homepage: www.elsevier.com/locate/jtb

In silico analysis of plasmodium falciparum CDPK5 protein through

molecular modeling, docking and dynamics
Subhashree Rout, Rajani Kanta Mahapatra∗
School of Biotechnology, KIIT University, Bhubaneswar-751024, Odisha, India

a r t i c l e i n f o a b s t r a c t

Article history: Calcium-Dependent Protein Kinase 5 (CDPK5) protein is one of the family members of a calcium-
Received 21 August 2018 dependent protein kinase that is found in plants and some species of protozoa which includes Plasmod-
Revised 15 October 2018
ium falciparum (Pf), the pathogen responsible for malaria. CDPKs regulate many biological processes in
Accepted 22 October 2018
Apicomplexans such as Plasmodium, Toxoplasma or Cryptosporidium. The study addresses the similarity in
Available online 27 October 2018
sequences and evolutionary relationship of CDPK5 across Apicomplexans. Further, the three-dimensional
Keywords: structural conformation of PfCDPK5 is generated through homology modeling. Molecular dynamics sim-
CDPK5 protein ulation of the homology model for a time interval of 40 ns resulted in a stable conformation of the
Homology modeling PfCDPK5 protein. Inhibitor identification was carried out from computational screening of known anti-
Molecular dynamics malarial compounds. The reliability of the binding mode for the best inhibitor compound MMV687246
Molecular docking was validated through a complex molecular dynamics study. This findings advocates that MMV687246
from Pathogen Box as the best inhibitor against PfCDPK5 protein and can be considered for experimental
validation study in future.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction the discovery of novel inhibitors specific for P. falciparum. Plants

Plasmodium falciparum is known for its most lethal and debili-
tating form of malaria worldwide. Resistance to the most promis-
ing drug artemisinin is now been reported in Plasmodium species
(Mbengue et al., 2015; Birnbaum et al., 2017). This emergence has
led to the identification of new drug targets and development of
new therapies.
Protein kinases have been reported as a valuable drug target for
new chemotherapeutics against the malaria parasite (Zhang et al.,
2012). Calcium-dependent protein kinases (CDPKs) are predomi-
nantly functional in plants and apicomplexans including Plasmod-
ium species (Miller et al., 2013). Plasmodium CDPKs share no sim-
ilarity with host proteome (Doerig and Meijer, 2007; Lucet et al.,
2012; Aher and Roy, 2016). Apicomplexan CDPKs have a struc-
tural architecture that closely resembles to that of plant CDPKs
(Lucet et al., 2012). CDPK family protein performs many metabolic
and cellular functions (Brochet et al., 2016). Eukaryotic CDPK binds
with calcium that effectively controls many critical events includ-
ing secretion, motility and development process in the life cy-
cle of apicomplexans (Billker et al., 2009; Kadian et al., 2017).
The presence of these kinases and kinase families could facilitate
∗
Corresponding author.
E-mail addresses: rmahapatra@kiitbiotech.ac.in, rkmahapatra83@rediffmail.com
(R.K. Mahapatra).
and alveolates canonical CDPKs contain one short N-terminal do-
main region followed by kinase domain and four calmodulin-like
EF-hands. CDPKs also contains an auto inhibitory junction region
which functions as pseudo substrate (Billker et al., 2009; Chandran
et al., 2006; Harper and Harmon, 2005; Aher and Roy, 2016).
CDPK5 is one isoform of CDPK in apicomplexan which is ex-
pressed in the asexual stage of parasite development specifically
in the mature schizonts and mature merozoites, playing a crucial
role in egress of mature merozoite from infected RBCs and inva-
sion of healthy RBCs of host (Harper and Harmon, 2005; Dvorin
et al., 2010; Lucet et al., 2012; Lasonder et al., 2015). Experimental
studies suggest that the parasites lacking with CDPK5 protein ar-
rest in the mature schizonts form and fail to develop further into
the invasive merozoite form of the parasite. Furthermore, a recent
study proposed that CDPK5 is an essential genes as well as target
considered to be high value for drug development (Zhang et al.,
2018).
Another role of PfCDPK5 protein is probably associated with
blockage of an ion transporter protein of the parasite. The P-type
ATPase 4 protein of the parasite transports and maintains the ef-
flux of Na+ and H+ ions within the parasitophorous vacuole. PfAT-
Pase performs the role of the proton pumping by maintaining
the metabolic requirements and guard against the rupture of the
plasma membrane of the parasite (Rottmann et al., 2010; Vaidya
et al., 2014; Spillman and Krik, 2015). PfATPase protein is highly

https://doi.org/10.1016/j.jtbi.2018.10.045
0022-5193/© 2018 Elsevier Ltd. All rights reserved.
 S. Rout and R.K. Mahapatra / Journal of Theoretical Biology 461 (2019) 254–267
believed drug target for pharmacokinetic development but unfor-
tunately, the structural conformation of the protein still remains
unclear and the molecular mechanism behind the role of PfCDPK5
against PfATP4 needs to be illustrated (Goldgof., 2016; Rottmann
et al., 2010). The regulation of PfATPase 4 activity by PfCDPK5 pro-
tein could lead to a comparative valedictory study focusing on the
binding potential of the proposed lead compound against PfCDPK5
protein and also with PfATPase protein.
PfCDPK5 is believed to be essential while completing the asex-
ual stage of parasite development demonstrate the characteris-
tics of a valuable drug target (Dvorin et al., 2010; Lucet et al.,
2012). PfCDPK5 contains a multi-domain architecture with a ser-
ine/threonine kinase domain and C-terminal calmodulin-like do-
main comprising four Ca2+ -binding EF hands (Dvorin et al., 2010).
In order to develop novel inhibitor and searching available chem-
ical libraries against PfCDPK5, reliable molecular structures are
needed. However, no experimentally validated structural informa-
tion of PfCDPK5 is available. Therefore, our present study involves
the prediction of the three-dimensional molecular structure of
PfCDPK5 and inhibitor screening using computational methodolo-
gies.
2. Materials and methods
2.1. Sequence analyses and phylogenetic tree construction
The primary sequence of PfCDPK5 protein was retrieved
from UniProt knowledgebase database (Q8IDV5) (Bairoch et al.,
2005), interlinked with PlasmoDB database (PF3D7_1,337,800)
(Aurrecoechea et al., 2008). Additionally, Pfam database
(Sonnhammer et al., 1997), SMART database (Schultz et al.,
20 0 0) and InterPro database (Hunter et al., 2008) were used to
get the predicted domain information of our target protein. The
orthology of PfCDPK5 with host proteome was checked using Or-
thoVenn web-server (Wang et al., 2015). This web-server allows for
genome-wide comparison of 272 species and provides interactive
graphical Venn diagram that defines orthologous clusters.
Multiple sequence analysis was carried out using Clustal Omega
(Sievers et al., 2011) and evolutionary relationship among Api-
complexan CDPKs was analyzed through phylogenetic tree using
MEGA7 (Kumar et al., 2016) with different phenetic and cladistic
methods (Tamura et al., 2011).
2.2. 3D model prediction and model optimization
BLASTP search (Altschul et al., 1997) was performed to iden-
tify a suitable template for model prediction of the PfCDPK5 pro-
tein against the PDB database (Bernstein et al., 1978) with de-
fault parameters. The identity percentage of our query protein with
known PDB template structures was below 50%. So, initially, the
tertiary structure of the PfCDPK5 protein was predicted using dif-
ferent model predicting web-servers namely, Phyre2 (Kelley et al.,
2015), Swiss-model (Schwede et al., 2003) and I-TASSER (Roy et al.,
2010). These three web-servers predicted the 3D structure of pro-
tein using a protein kinase template from Plasmodium berghei (PDB
ID- 3Q5I, chain A) with a query coverage of 79% and an iden-
tity percentage of 42%. Each model was evaluated through Ra-
machandran plot analysis. However, due to some constraints and
structural errors, these structures were not optimized by molec-
ular dynamics simulation. Finally, computational homology mod-
eling approach was used to generate the possible tertiary struc-
ture of our target PfCDPK5 protein. The template structure con-
tains 4 calcium ions. Calcium triggers many activities regulated by
CDPKs. Therefore, ions from the template were also used for ho-
mology model generation of PfCDPK5. The model of the PfCDPK5
protein was generated using Modeller v9.13 software (Webb and
255
Sali, 2014) using template 3Q5I. The best-modeled structure of
PfCDPK5 was selected considering the lowest DOPE score out of
10 generated models. The stereo-chemical quality of the mod-
eled protein was evaluated using PROCHECK (Laskowski et al.,
1993). The loop conformation of the best model was improved
using loop refinement tool of MODELLER software. The model
was checked for non-bonded contacts and clashes using Chimera
software (Pettersen et al., 2004). Different model validation tools
and programs namely, Verify3D (Eisenberg et al., 1997), ERRAT
(Colovos & Yeates, 1993) and ProSA web-server (Wiederstein and
Sippl, 2007) were used to validate the quality of the generated
model. 3dLigandSite server (Wass et al., 2010) was employed for
prediction of possible active site pockets in our target protein. It
predicts the possible binding residues based on a number of clus-
ters formed by the complex crystal structures.
The structure of PfATPase 4 protein was determined using I-
TASSER web-server (Roy et al., 2010); as the structure was reported
with acceptable validation parameters. The structure was predicted
using SERCA Ca2 + -ATPase protein structure (PDB ID: 3BA6 chain
A) as a template. The active site residues information of the protein
was predicted by taking the corresponding residues information
from P-Type ScATPase 4 protein (Goldgof et al., 2016). The binding
site residues of PfATPase are predicted as Glu154, Thr416, Lys436,
and Pro437.
2.3. Molecular dynamics simulation
The modeled protein was subjected to protein molec-
ular dynamics simulation using GROMACS v5.0.7 software
(Berendsen et al., 1995). Energy minimization of the protein
was carried out in an aqueous medium with OPLS-AA/L (Opti-
mized Potentials for Liquid type Simulation) all-atom force field
(Jorgensen et al., 1996). A protein centered cubic box was defined
with a grid cell distances of 36 nm x 36 nm x 36 nm and solvated
with a total of 36,944 SOL molecules in the defined box followed
by neutralization of the charged protein with Na+ and Cl− ions.
Long-range electrostatic interactions were calculated using the
Particle-Mesh-Ewald (PME) method (Darden et al., 1993). Energy
minimization of the system was performed with the steepest
descent method and lowest potential energy of the system was
calculated. Isothermal and isobaric equilibrium of the system was
maintained using Berendsen thermostat temperature coupling and
Parrinello-Rahman pressure coupling methods till every 100 ns
run in order to maintain the equilibrated environment at 300 K
and 1 bar respectively. The leap-frog algorithm was used for inte-
grating Newton’s equation in MD simulation. All the bond lengths
were constrained using the LINCS algorithm (Hess et al., 1997).
Finally, MD run was carried out for 40 ns. Trajectory analysis
was performed to check the stability of the protein for 40 ns
using the GROMACS package followed by molecular visualiza-
tion of the simulated protein structure of PfCDPK5 using Pymol
(DeLano, 2002).
2.4. Virtual screening of compounds
The SMILE formats of each ligand molecules were used to gen-
erate the 3D structure using Open Babel software (O’ Boyle et al.,
2011). Open Babel was also used to compute and determine the
lowest energy conformation of the lead molecules with conjugate
gradient algorithm and MMFF94 force field. A total of 27, 283
ligand molecules were retrieved for virtual screening against the
PfCDPK5 protein. Virtual screening against PfCDPK5 protein was
performed using GOLD software suite (Verdonk et al., 2003; Ver-
donk et al., 2004). These molecules were grouped into 5 data
sets; 1st set of molecules consists of approximately 13,451 com-
pounds deposited by Glaxo Smith Kline’s Tres Cantos Antimalar-
256 S. Rout and R.K. Mahapatra / Journal of Theoretical Biology 461 (2019) 254–267
ial Set (TCAMS) (Crowther et al., 2016). The cell activity of these
compounds was tested against five different protein kinases and
screened in biochemical assays of P. falciparum calcium-dependent
protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated
protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and pro-
tein kinase 7 (PK7). The compound list was retrieved from ChEMBL
database (Gaulton et al., 2016). The 2nd set of molecules consists of
178 compounds deposited by MMV (Lucantoni et al., 2013); these
compounds were screened against blood-stage P. falciparum. The
hits were fully profiled in additional Plasmodium assays, as well
as in-vitro distribution metabolism pharmacokinetic (DMPK) as-
says. The 3rd set of compounds contained approximately 13,519
compounds deposited by TCAMS (Almela et al., 2015). The dataset
listed structures with confirmed inhibition of the growth of the
parasite by more than 80% at 2 μM concentration. The 4th set of
compounds comprises of 125 compounds retrieved from Pathogen
Box repository of MMV (Spangenberg et al., 2013). Lastly, the
5th set of compounds comprised of 10 best compound out of
200 molecules based on structure-based drug identification using
Dockblaster server (Irwin et al., 2009). The target protein struc-
tural confirmation is utilized by this server to perform computa-
tional screening against the library of the ZINC database (Irwin and
Shoichet 2005).
2.5. Molecular docking studies
Molecular docking of the best 10 compounds based on GOLD
score from each set was studied using AutoDock Vina software
(Trott and Olson, 2010). Broyden-Fletcher-Goldfarb-Shanno algo-
rithm and grid-based energy evaluation method are used by
AutoDock Vina. Protein and ligand molecules were separately pre-
pared using Auto Dock Tool (ADT) graphical user interface. Po-
lar hydrogen atoms were assigned and non-polar hydrogen’s were
merged before performing docking steps. Atomic potential grid
map was calculated by Auto Grid with 0.375 Å spacing in a box
of 26.25 Å × 19.5 Å × 26.25 Å dimensions. A box was centered at the
predicted active site residues with dimensions (x, y, and z): 63.568,
57.498 and 56.66 respectively. Docking parameters were enabled
with default values. The configuration file for each docking run was
prepared using receptor, ligand and grid information. AutoDock
Vina docking was performed using the command-line interface.
Best binding mode and conformations of ligand compounds were
selected with lowest binding energy for analysis and further stud-
ies.
The potential activity of the compounds can be studied by in-
hibition study against PfATPase protein. This study could help to
illustrate the binding efficiency of the compound against the pro-
tein that is highly regulated by PfCDPK5 protein.
The binding efficacy and potent activity of the best-docked
compound were again evaluated by docking it with the analogous
and orthologous proteins of P. falciparum CDPK5. For molecular
docking study, the analog and ortholog CDPK protein was selected
from the consensus result of the phylogenetic tree generated. The
phylogenetic tree indicates close resembles of PfCDPK5 protein
with C. parvum CDPK1 protein. Therefore, the crystal structure of
C. parvum CDPK1 (PDB ID: 3IGO) was taken as ortholog protein
structure (Wernimont et al., 2010). The structure was solved with
X-ray resolution of 2.25 Å and co-crystallized with a phosphate
ion, glycerol, calcium, tartaric acid and Phosphoaminophosphonic
acid-adenylate ester. For analog structure, the crystal structure of T.
gondii CDPK1 (PDB ID: 5W8R) was selected. The TgCDPK1 structure
was solved with X-ray resolution 2.2 Å, bounded with 3CIB-PPI in-
hibitor. The docking studies were carried out using AutoDock Vina
software. The protein structures were initially prepared for docking
studies and the compound was allowed to dock in the pre-defined
binding site of each protein. This study could highlight the broad
spectrum multi-target ability of the compound and could help in
understanding the specificity of the compound for PfCDPK5 pro-
tein.
2.6. Complex molecular dynamics simulation
Complex MD simulation was performed to check the binding
mode and confirmation of docked ligand molecules by GROMACS
v5.0.7 software package (Berendsen et al., 1995). Protein struc-
ture topology was generated using GROMOS96 43a1 force field
(Van Der Spoel et al., 2005) and ligand topology was prepared us-
ing PRODRG server (SchuEttelkopf and Van Aalten, 2004) and cor-
rections were made to check the charges of some functional groups
of small molecules in GROMOS compatible format (Lemkul et al.,
2010). The complex was set for simulation procedure in an aque-
ous environment and neutralized system with certain parame-
ters as mentioned in protein simulation. In total, 48,123 solvent
molecules were filled in the cubic box. The equilibrated complex
system was allowed for a simulation run till 20 ns of the time in-
terval. All graphs were generated using the Xmgrace tool of Grace
Software (Vaught, 1996).
3. Results and discussion
3.1. Primary sequence analysis of PfCDPK5 protein
The primary sequence of PfCDPK5 was retrieved from UniProt
database (Uniprot consortium, 2014) with ID- Q8IDV5 interlinked
with its PlasmoDB ID- PF3D7_1,337,800. This gene is expressed on
chromosome No. 13. The protein is of 568 amino acids in length
and the molecular weight is 66 kD. PfCDPK5 protein is orthologous
to putative CDPK5 protein of other species of Plasmodium namely,
P. berghei, P. chaubadi, P. knowlesi, P. vivax, P. yoelii, P. fragile, P. gal-
linaceum, P. reichenowi, P. goboni and other strains of the species
indicating the degrees of conservation of the protein among the
different species of parasite. However, no structural studies have
been performed on any of the CDPK5 proteins. The protein consists
of one protein kinase domain starting from residue 125–379 amino
acid in length and two EF-hands from 424–490 and 497–564
amino acids in length respectively as per the Pfam database and
sequence-based domain analysis search. InterPro database analy-
sis suggested that the protein consists of one protein kinase do-
main with 125–379 amino acids and four EF-hand domains from
425–460, 462–495, 496–531 and 534–568 amino acids in length.
Furthermore, study by SMART database reported that the protein
consists of Ser/Thr protein kinase catalytic domain (124–379 amino
acids) and 4 EF-hands (429–457 amino acids, 464–492 amino
acids, 500–528 amino acids and 538–566 amino acids) (Supple-
mentary Fig. 1). Different domain databases have provided domain
information in which few start and end sequence position of pos-
sible domains are similar particularly for protein kinase domain,
while EF-hand motif residue positions are highly divergent. But,
consensus analysis of these databases suggested that PfCDPK5 con-
tains domain architecture with one Ser/Thr protein kinase domain
(125–376 amino acids) and four EF-hands like motifs. However,
Billker et al. (2009) in a phylogenetic analysis of apicomplexan
CDPK proteins described the domain architecture of PfCDPK5 con-
sists of a relatively short N-terminal domain, Ser/Thr protein ki-
nase domain and four EF-hands and orthologous to TgCDPK5 and
CpCDPK5 proteins. Similar domain architecture was also suggested
by Dvorin et al. (2010) that PfCDPK5 exhibits N-terminal of 100
amino acids followed by a canonical multi-domain structure with
a Ser/Thr kinase domain and C-terminal calmodulin-like domain
comprising four EF-hands. PfCDPK5 consists of a multi-domain
structure of protein kinase and calcium binding sites was clearly
 S. Rout and R.K. Mahapatra / Journal of Theoretical Biology 461 (2019) 254–267
revealed by this study. The orthology study using OrthoVenn web-
server clearly suggested that the target protein (PfCDPK5) shares
no similarity with the host proteome (Supplementary Fig. 2).
3.2. Multiple sequence alignment and phylogenetic analysis
From different domain database analysis, the estimated domain
architecture of the PfCDPK5 protein was found to be a multi-
domain structure. The upstream protein kinase domain is known
to be associated with CDPKs and the downstream of the cat-
alytic region with four helix-loop-helix type EF-hand motif are
most similar to calcium-binding proteins (Wernimont et al., 2011).
A protein that contains four EF-hands-like motifs may undergo
major conformational changes upon calcium binding. In order to
predict the possible EF-hands amino acid residues and binding
site of the calcium ion, multiple sequence alignment of PfCDPK1,
PfCDPK2, PfCDPK3, PfCDPK4 and PfCDPK5 protein was performed
using Clustal Omega program of EBI database. The EF-hand cal-
cium binding region consists of 12 amino acids where six residues
are actively involved in calcium binding with positions 1, 3, 5, 7,
9 and 12. Glu (E) or Asp (D) is conserved at position 12 which
provide oxygen for binding calcium (Wernimont et al., 2011). In
our study, multiple sequence analysis clearly reported the con-
served calcium-binding region of each EF-hand motif. The EF-hand
1 residue position in PfCDPK5 is from 429–457 amino acids and
the calcium binding residue arrangement is DHNGDGVLTISE. The
EF-hand 2 in PfCDPK5 (464–492 amino acid) calcium binding re-
gion is DTDGNGLIDYTE. Similarly, DLDGDGVITKDE and DSNNDG-
FIDYDE are calcium binding region of EF-hand 3 (500–528 amino
acids) and EF-hand 4 (538–566 amino acids) respectively (Fig. 1).
The calcium binding sequence arrangement of each EF-hand mo-
tifs reported glutamate amino acid at position 12 as typical EF-
hand motifs. The structural arrangement of the PfCDPK5 protein
with conserved domain architecture is clearly demonstrated by this
study.
The phylogenetic analysis of protein sequences of PfCDPKs,
TgCDPKs, and CpCDPKs suggested the close resembles of CDPK
family protein among apicomplexans (Fig. 2). The Phenetic meth-
ods (NJ and UPGMA methods) are mainly used to compute the ge-
netic distances from given multiple sequence alignment but do not
consider evolutionary prediction. PfCDPK5 shares higher sequence
identity with TgCDPK5 and CpCDPK1 than other PfCDPK proteins
(Supplementary Fig. 3A, B); the revelation is made by these above
stated phylogenetic trees. The cladistic method (maximum likeli-
hood and maximum parsimony) of phylogenetic tree construction
was also used with bootstrap statistical analysis. Bootstrap is a sta-
tistical method for obtaining a non-parametric estimate of errors in
which taxa are held as constant and the sequence data are resam-
pled randomly with replacements (Felsenstein, 1985; Efron, 1982).
The maximum parsimony method constructed trees using an im-
plicit model of evolution and reported that the parsimonious tree
has a length of 2820. The consistency index is (0.724925), the re-
tention index is (0.474249) and the composite index is (0.350641)
for all sites and parsimony informative sites. The percentage of
the replicate trees in which the associated taxa clustered together
in the bootstrap test was shown next to the branches is also re-
ported by this tree (Supplementary Fig. 2C). This tree also showed
a resemblance of PfCDPK5 protein with 98% similarity with T.
gondii CDPK5 and C. parvum CDPK1 protein (Supplementary Fig.
3C). However, the maximum likelihood method, considered as an
advanced method for phylogenetic analysis, uses optimality cri-
teria and applies an explicit model of evolution for phylogenetic
tree construction. The tree is constructed by applying two inde-
pendent assumptions; first, different sites were evolved indepen-
dently. Second, diverged sequences were evolved independently af-
ter diverging. The tree is observed with the highest log likelihood
257
(−13,548.58) score. In this case, a close identity (94%) was ob-
served with T. gondii CDPK5 and C. parvum CDPK1 (Fig. 2).
3.3. 3D model generation and energy minimization
The knowledge of protein 3D (three-dimensional) structures is
vitally important for rational drug design. Although X-ray crystal-
lography is a powerful tool in determining protein 3D structures,
it is time-consuming and expensive, and not all proteins can be
successfully crystallized. Membrane proteins are difficult to crystal-
lize and most of them will not dissolve in normal solvents. There-
fore, so far very few membrane protein structures have been de-
termined. The recent breakthroughs indicate that NMR is indeed
a very powerful tool in determining the 3D structures of mem-
brane proteins (Oxenoid et al., 2016; Dev et al., 2016; Schnell and
Chou, 2008; Berardi et al., 2011; OuYang et al., 2013; Fu et al.,
2016), but it is also time-consuming and costly. To acquire the
structural information in a timely manner, a series of 3D protein
structures were developed by means of homology technique (Chou
et al., 1997; Chou et al., 20 0 0; Chou, 20 05; S.Q. Wang et al., 2007;
J.F. Wang et al., 2007; Wang et al., 2009; Li et al., 2011; Ma et al.,
2012; K.C. Chou, 2004), and were found very useful for drug devel-
opment. In view of this, the homology technique was also adopted
to develop the relevant protein 3D structures for the current study.
Initially, different molecular modeling web-servers were used
for 3D-model prediction of PfCDPK5 namely, Phyre2, Swiss-model
and I-TASSER servers. The possible PfCDPK5 structural conforma-
tion was predicted by each server considering PDB ID: 3Q5I as a
template structure. The target model structure was taken for model
evaluation through Ramachandran plot. I-TASSER generated struc-
ture reported 68.6% residue in the most favored region and 13
residues in the disallowed region. Phyre2 provided a structure with
89.8% residues in the most favored region and not a single residue
in the disallowed region. In parallel, the structure predicted us-
ing Swiss-model reported 90.2% residues in the core region and
3 residues in the disallowed region. This study clearly suggested
that the models predicted using Swiss-model and Phyre2 server
possess accurate structural coordinates. However, molecular opti-
mization of these structures through molecular dynamics simula-
tion got terminated prematurely as these protein structures con-
tained a number of missing atom coordinates and were associated
with poor quality parameters.
Under such circumstances, we chose a homology model based
method to predict the possible 3D structural conformation of the
protein. The first hit was crystal structure of PfCDPK2 (4MVF, chain
A) with 45% identity and 79% query coverage, suggested by BLASTP
search. The second hit was identified with the crystal structure
of CpCDPK1 (3IGO, chain A) with 42% identity and 76% query
coverage. In contrast to the above facts, all the model predicting
web-servers predicted the model considering 3Q5I as the template
structure. The PfCDPK5 protein shares 41% identity and 76% query
coverage with template protein (Supplementary Fig. 4). The model
with the lowest DOPE score was further refined using loop re-
finement tool of MODELLER. The final model was selected based
on the lowest DOPE score (−599,660.78125). The stereo chemical
quality of the protein was evaluated using a Ramachandran plot
which suggested that the protein showed 85.3% residues in most
favored region and 0.6% residues in the disallowed region. Ver-
ify 3D program reported the compatibility of the 3-dimensional
structure to its 1-dimensional conformation and revealed 62.50% of
residues are compatible. ProSA analysis reported that protein fold-
ing energy of the modeled structure (Z-score) is −7.41, which is
well within the range of native conformation of crystal structure.
The ERRAT server gives the overall quality factor (expressed as the
percentage of the protein for which the calculated error values falls
the 95% rejection limit) of the model as 55.759. This study clearly
258 S. Rout and R.K. Mahapatra / Journal of Theoretical Biology 461 (2019) 254–267

Fig. 1. Multiple sequence alignment of protein sequences of CDPK1, CDPK2, CDPK3, CDPK4 and CDPK5 of P. falciparum. The calcium binding region of the four helix-loop-helix
motifs are indicated in square boxes. The conserved residues are highlighted in red color.
 S. Rout and R.K. Mahapatra / Journal of Theoretical Biology 461 (2019) 254–267
Fig. 2. Phylogenetic analysis of CDPK family proteins. Comparison of PfCDPK5 protein with other CDPKs of P. falciparum and with TgCDPKs and CpCDPKs. Phylogenetic tree
constructed using maximum likelihood method.
suggested that the predicted model is of good quality as per model
validation results and can be considered for structure-based drug
design studies.
Since the pioneer paper entitled ’The Biological Functions of
Low-Frequency Phonons’ (Chou and Chen, 1977) was published in
1977, a series of investigations into biomacromolecules from dy-
namic point of view have been stimulated. These studies have sug-
gested that low-frequency (or terahertz frequency) collective mo-
tions do exist in proteins and DNA (Zhou, 1989; Chou and Mao,
1988; Chou et al., 1989; Martel, 1992; Chou, 1988). Furthermore,
many important biological functions in proteins and DNA and their
dynamic mechanisms (Wang and Chou, 2009), such as cooperative
effects (K.C. Chou, 1989), allosteric transition (Chou et al., 1981;
Wang and Chou, 2010), intercalation of drugs into DNA (Chou and
Mao, 1988), and assembly of microtubules (Chou et al., 1994), can
be revealed by studying the low-frequency internal motions as
summarized in a comprehensive review (Chou, 1988). Some sci-
entists even applied this kind of low-frequency internal motion for
medical treatments (Gordon, 2007; Gordon, 2008; Madkan et al.,
2009). Actually, investigation into the internal motion in biomacro-
molecules and its biological functions is deemed as a "genuinely
new frontier in biological physics", as announced in the mission
of some biotech companies (see, e.g., Vermont Photonics). In view
of this, to really understand the action mechanisms of biomacro-
molecules, we should consider not only the static structural infor-
mation but also the dynamical information acquired by studying
their internal motions. To realize this, the MD simulation is one of
the feasible tools.
Therefore, the protein structure was subjected to energy min-
imization through molecular dynamics study using GROMACS
v5.0.7 software. Molecular dynamics simulation allowed for the de-
termination of interaction of atoms of the system under Newtonian
equations of motion where forces and potential energies between
the particles are calculated using molecular force fields for a spe-
259
cific time period (Andersen, 1980). The dynamic behavior of the
protein was analyzed in a solvated system with OPLS-AA/L (Opti-
mized Potentials for Liquid type Simulation) all-atom force field for
40 ns simulation run. The RMSD plot showed stable deviation after
20 ns up to 40 ns and maintained at ∼0.45 nm (Fig. 3A). The back-
bone RMSD graph suggested that the simulated protein trajectory
has acquired a stable conformation during 40 ns simulation run
an equilibrated system. Thereafter, the simulated protein confor-
mation was retrieved and the overall quality of the structure was
confirmed from Ramachandran plot analysis. Ramachandran plot
of this simulated structure suggested that 99.1% residues are in
the favored region and 0.9% residues are in the disallowed region
(Supplementary Fig. 5). As compared to the Ramachandran plot of
target PfCDPK5 modeled structure before simulation, most of the
residues are found in the additionally allowed region (19.4%) and
generously allowed region (2.6%) and only 2 residues are found in
the disallowed region. In the absence of any experimentally deter-
mined structural co-ordinates of PfCDPK5, the predicted structure
could help us to understand the possible structural conformation
of the PfCDPK5 protein along with probable position of binding site
of the protein, and interaction with small chemical entities.
The secondary structure arrangement of the CDPK5 protein con-
sists of β -sheets: 8.1%, α -helices: 24.1%, short 3–10 helix: 4.9% and
turns: 62.9%. In total, the structure of the CDPK5 protein consists
of 7 beta-sheets and 24 alpha-helices (Fig. 3B). The Ser/Thr protein
kinase domain of the protein contains most of the beta-sheets (S1-
S7) and alpha-helices (H1-H13) of the protein. The four EF-hand
loops are located in H16-H17, H18-H19, H21-H22 and H23-H24 re-
spectively (Fig. 4).
The information of a binding pocket of a receptor for its lig-
and is very important for drug design, particularly for conducting
mutagenesis studies (K.C. Chou, 2004). In the literature, the bind-
ing pocket of a protein receptor to a ligand is usually defined by
those residues that have at least one heavy atom (i.e., an atom
260 S. Rout and R.K. Mahapatra / Journal of Theoretical Biology 461 (2019) 254–267

Fig. 3. Plasmodium falciparum calcium-dependent protein kinase (CDPK5). A- Backbone RMSD plot of the CDPK5 plot up to 40 ns. B- Three dimensional structure of PfCDPK5.
Secondary structural arrangement protein showing helix-sheet-loop colored in cyan-red-magenta.

other than hydrogen) within a distance of 5 Å from a heavy atom of
the ligand. Such a criterion was originally used to define the bind-
ing pocket of ATP in the Cdk5-Nck5a complex (Chou et al., 1999)
that has later proved very useful in identifying functional domains
and stimulating the relevant truncation experiments (Zhang et al.,
2002). The similar approach has also been used to define the bind-
ing pockets of many other receptor-ligand interactions important
for drug design (Chou et al., 1997; Chou et al., 20 0 0; Chou et al.,
20 03; Huang et al., 20 08; J.F. Wang et al., 2007; K.C. Chou, 2004;
Li et al., 2011; Wang and Chou, 2011; Wang and Chou, 2012; Pielak
et al., 2009)
In the absence of an experimentally validated structure of the
PfCDPK5 protein, the possible binding pocket residues (active site
residues) were predicted using the 3DLigandSite server. The pos-
sible binding site residues were predicted based on the clusters
made by ligand of homologous proteins deposited in the PDB
database (Bernstein et al., 1978). In case of our target protein,
10 ligand clustered near residue Lys133, Tyr136, Gly137, Val139,
Ala152, Lys154, Ile183, Glu200, Leu201, Cys202, Glu206, Glu249,
Asn250, Leu252, Ile265 and Asp266 (Fig. 5). These predicted active
site residues were arranged in a pocket-like arrangement within
the Ser/Thr protein-kinase domain of the protein that covers most
of the beta-sheet regions and few alpha helices of the protein. The
predicted active site of the protein is considered for interaction of
ligand compounds through molecular docking simulation analysis.
3.4. Virtual screening and docking studies
In the present study, GOLD docking suite was used for
virtual screening against the PfCDPK5 protein. The entire
 S. Rout and R.K. Mahapatra / Journal of Theoretical Biology 461 (2019) 254–267
Fig. 4. Cartoon and surface representation of the PfCDPK5 protein highlighting the
domain architecture of the protein (yellow color). The Ser/Thr protein kinase (124–
379 amino acids) domain of the protein is colored in blue. The four EF-hand motifs
of the protein namely, EF hand 1 (429–457 aa), EF hand 2 (464–492 aa), EF hand 3
(500–528 aa) and EF hand 4 (538–566 aa) are indicated in red, green, magenta and
orange color respectively.
Fig. 5. Cartoon representation of the predicted binding site of the PfCDPK5 protein.
Surface view of the predicted residues of the possible binding site. These residues
are present in the Ser/Thr protein kinase domain (ribbon view in light pink color).
known compound lists were retrieved from ChEMBL database
(Gaulton et al., 2016). These compounds were grouped into multi-
ple sets based on the source and activity against the pathogen. The
3-dimensional conformation of these compounds was generated
including energy minimization of each compound using the Open-
babel software. A total of 27,283 ligand molecules were allowed to
dock against the predicted binding site of the protein and ranked
based on GOLD fitness score. The first compound set comprises of
approximately 13,451 known inhibitors of CDPK family protein of
P. falciparum. The second set of compounds consists of 178 com-
pounds which have shown inhibition activity against asexual-stage
of parasite development. The third compound set comprises of
13,519 potent inhibitors of P. falciparum deposited by TCAMS. The
fourth sets of compounds were retrieved from Pathogen Box that
contains 125 active antimalarial compounds against the pathogen.
Additionally, the fifth set of compounds was generated from
Dockblaster server (Irwin et al., 2009) through structure-based
virtual screening of compounds against the ZINC database library
261
(Irwin and Shoichet, 2005). Afterwards, based on the GOLD fitness
score, 10 compounds from each set were ranked (Supplementary
Table 1). The study revealed that some of the H-bond interactions
were found with predicted binding site residues Lys154, Cys202,
Glu206, Asn250 and Asp266. Most of the polar interactions were
also observed with residues other than predicted binding residues
viz. Lys131, Lys133, Ser135, Lys141, Gln150, Leu201, and Leu252.
The binding affinity of these 50 compounds was predicted
through molecular docking studies using AutoDock Vina soft-
ware. Based on lowest binding affinity 10 compounds are listed
in (Table 1). The compound C1 (MMV687246) (Supplementary
Fig. 6) contains the lowest binding energy (−11.6 kcal/mol) and
three polar interaction was found with Gly134 (N2H2—O = C,
2.50 Å, Glu200 (C = O—HN22, 2.42 Å) and Lys247 (NH—O9S8, 2.12 Å)
(Fig. 6). These residues are part of the predicted binding residues
except Lys247. Compound C2 (TCMDC-138,644) has a binding affin-
ity −11.0 kcal/mol and molecular interaction with Glu249, one of
the predicted binding site residues. Compound C3 has a bind-
ing affinity of −10.8 kcal/mol having two polar interactions with
Arg166 and Cys202. Compound C3, C4, C5 and C6 binding affinities
range from −10.8 to −10.1 kcal/mol and H-bond interaction with
Ser135, Lys141, Lys154, Cys202, and Asp266. Compound (C3, C4, C6,
and C9) belongs to the 178 compounds with known anti-malarial
activities. Compound C7, C8, C9, and C10 have binding affinities be-
tween −9.7 kcal/mol to −9.6 kcal/mol. Polar interactions of these
compounds were found mainly with Lys154 and Asp266 predicted
binding site residues.
3.5. MMV687246 as a lead molecule against PfCDPK5 drug target
The compound MMV687246 (N-(2H-benzotriazol-5-yl)−7-
[4-(4-methylpiperazin-1-yl) sulfonylphenyl] quinolin-4-amine)
(CHEMBL ID- CHEMBL3637881 and ZINC ID- ZINC473170876)
belongs to Pathogen Box or Malaria Box, a library of compounds
deposited by Medicines for Malaria Venture (MMV). The diverse
chemical activity of the compounds was tested against blood-stage
of the parasite (Avery et al., 2014). The efficacy of Pathogen
Box (MMV687246) compounds was also tested against parasitic
nematodes C. elegans (Partridge et al., 2017).
The PfCDPK5 protein activity is monitored in the rupture of par-
asite’s membrane at the schizont stage and the protein is highly
responsible in regulating and blocking the functional activity of
the PfATPase protein in the transport of nutrients to the plasma
membrane of the parasite and guards against the rupture during
the blood stage (Drovin et al; 2010; Spillman and Krik, 2015). So,
the activity of the compound was studied against PfATPase pro-
tein. The inhibitory activity of the compound was reported previ-
ously against the PfATPase4 protein (EC50 = 0.5140839317 and log
(ec50 = −0.288966) (Golgof et al., 2016; Rottmann et al., 2010). The
binding affinity of the compound with PfATPase 4 protein was re-
ported to −7.9 kcal/mol. There was a remarkable difference in the
comparable binding score of the compound against PfCDPK5 and
PfATPase (Table 2). The docking interaction showed one hydrogen
bond interaction with Ser175, few hydrophobic bonds, and van der
Waals interactions (Supplementary Fig. 7(a-b)). Interestingly, these
interactions are formed with other surrounding residues rather
than predicted active site residues. This study revealed that the
compound has no specificity in binding affinity towards the PfAT-
Pase protein.
The ability of compound MMV687246 to analyze the effective
binding affinity for PfCDPK5 can be evaluated by docking the com-
pound with analogous and orthologous structures of PfCDPK5. The
comparative binding affinity of the compound against PfCDPK5,
analog TgCDPK1 and ortholog CpCDPK1 was listed in Table 2. The
compound MMV687246 was docked in the specified active site of
the targeted analog and ortholog protein. The binding site residues
262 S. Rout and R.K. Mahapatra / Journal of Theoretical Biology 461 (2019) 254–267

Table 1
Docking result of best 10 compounds with lowest binding energies and H-bond interactions, predicted active site residues are highlighted in bold.

Sl.No Source Compound Id Gold score Autodock vina result (kcal/mol) H-bond interactions

C1 Pathogen Box MMV687246 69.01 −11.6 Gly 134, Glu200, Lys247

C2 TCMDC TCMDC-138,644 98.68 −11.0 Glu249
C3 178 MMV 80.92 −10.8 Arg166, Cys202
C4 178 MMV 77.82 −10.2 Ser135, Lys141
C5 TCMDC TCMDC-137,320 95.82 −10.1 Gly134, Ser135, Lys141, Lys154, Cys202, Glu249, Asp266
C6 178 MMV 87.53 −10.1 Lue131, Gly134, Ser135, Lys141, Asp266
C7 178 MMV 85.49 −9.7 Ser135, Asp266
C8 Pathogen Box MMV020512 61.83 −9.7 Ser135, Lys154
C9 178 MMV 76.81 −9.6 Lys247, Asp266
C10 Pathogen Box MMV026020 64.46 −9.6 Lys141, Lys154

Fig. 6. Binding conformation of the MMV687246 compound with PfCDPK5 protein (green color in cartoon representation). 2-Dimensional representation of MMV687246
(yellow stick) interactions with residues of target protein. Hydrogen bonds are shown in green colors, electrostatic bonds in orange color, hydrophobic bonds such as pi-alkyl
in light pink color and pi-sigma in purple color.

Table 2
Comparative docking result of compound MMV687246.

Sl.No Compound Id Protein Autodock vina result (kcal/mol) H-bond interactions

1 MMV687246 PfCDPK5 −11.6 Glu200, Lys247, Gly134

2 TgCDPK1 −5.1 Arg 192
3 CpCDPK1 −9.8 Ile150
4 PfATPase −7.9 Ser175

of TgCDPK1 consist of two amino acid residues Glu129 and Tyr131.
The docking result showed the binding affinity of the compound
MMV687246 with TgCDPK1 protein is −5.1 kcal/mol (Table 2) and
a single hydrogen bond interaction was observed with residue
Arg192 (bond length, 3.4 Å) (Supplementary Fig. 8A). The interact-
ing residue Arg192 is not reported to be the active site residue
of TgCDPK1 protein. Similarly, in case of ortholog CpCDPK1 pro-
tein, the structural coordinates of the protein is solved experi-
mentally and its active site residue position was referred from
the PDB database for molecular docking study. Amino acid like
Ser86, Phe87, Val90, Glu153, Tyr155, Leu205, and Asp219 are the
key residues of the crystal structure (3IGO) of CpCDPK1 pro-
tein. The compound MMV687246 docked with a binding affinity
of −9.8 kcal/mol and a single H-bond interaction was observed
with residue Ile150 (bond length, 2.7 Å). There was no interac-
tion observed with known active site residues of the ortholog pro-
tein structure. The comparative docking study conclusively sug-
gested that the lead molecule (MMV687246) has a high affinity
for PfCDPK5 protein followed by the ortholog protein CpCDPK1 and
least affinity for analog TgCDPK1 protein.
3.6. Complex molecular dynamics simulation
The stability of the binding conformation and binding mode
of the MMV687246 compound was checked after a molecular dy-
namics simulation of the protein-ligand complex for time duration
of 20 ns in a solvated and equilibrated system. The RMSD plot of
the complex was obtained by taking a docked conformation of the
protein-ligand as reference structure (Fig. 7A). The backbone RMSD
of the complex maintained at ∼0.5 nm till 10 ns, followed by a
gradual decrease in the deviation of RMSD plot was observed from
12 ns and finally the plot maintained stability in deviation slightly
less than ∼0.4 nm from 16 ns to 20 ns . The converged RMSD plot
clearly indicates the stability of protein-ligand complex till 20 ns
time interval. The fluctuation of amino acid residues of protein
during 20 ns is represented by the RMSF plot (Fig. 7B). However,
some fluctuations are observed in the residues from 0–50 in the
 S. Rout and R.K. Mahapatra / Journal of Theoretical Biology 461 (2019) 254–267 263

Fig. 7. Trajectory analysis till 20 ns. A- RMSD plot of PfCDPK5-MMV687246 complex B- RMSF plot of protein-ligand complex.

Fig. 8. Molecular interaction anlysis of MMV687246 with PfCDPK5 residues after MD simulation till 20 ns time interval. Trajectories were analyzed at each 5 ns time interval.
Two-dimensional representation of molecular interactions at (a) 5 ns (b) 10 ns (c) 15 ns and (d) 20 ns.

RMSF plot. Nonetheless, the residues forming molecular interaction
with the ligand and other predicted binding site residues main-
tained stability during simulation time interval. There trajectory
frames were extracted for each 5 ns interval for detailed molecu-
lar interaction analysis (Fig. 8).
Molecular interaction of the compound at a different time in-
terval (5 ns each) was listed in Tables 3 and 4. Hydrogen bond
interaction, ionic interaction and hydrophobic interactions of the
compound with most of the predicted binding site residues are
suggested by the complex molecular dynamics study. Molecu-
264 S. Rout and R.K. Mahapatra / Journal of Theoretical Biology 461 (2019) 254–267

Table 3
Docking results and interactions analysis of compound MMV687246. Active site residues forming hydrogen bonds are indicated in Bold.

Ligand Docking score (kcal/mol) H-bond formation before MD simulation Distance (Å) H-bond formation after MD simulation Distance (Å)

MMV687246 −11.6 N2H2—O = C Gly134 2.50 N29—HN Ala152 1.90

N22H—O = C Glu200 2.42 N27H27—O = C Asp203 2.10
S8O9—HN Lys247 2.12 S8O9 —O = C Arg244 3.03

Table 4
Comparative docking interaction analysis of compound MMV687246 before and after simulation at different time intervals of
MD simulation.

MMV687246 Hydrogen bond Distance (Å) Ionic bond Distance (Å) Hydrophobic bond Distance (Å)

Docking interaction Gly134 2.50 Cys202 5.80 Leu131 5.40

Glu200 2.42 Asp266 4.21 Lys133 4.71
Lys247 2.12 Asp266 4.42 Val139 4.56
Ala152 4.93
Leu201 3.82
Leu201 3.90
Ile265 3.80
Ile265 5.10
5 ns Gln150 5.46 Glu249 8.03 Leu131 5.65
Cys202 3.96 Val139 6.51
Lys141 6.23
Ala152 4.17
Ala152 4.38
Ala152 6.60
Leu201 5.78
Ile265 5.51
10 ns Ser135 3.95 Glu249 6.17 Val139 7.08
Cys202 4.89 Ala152 5.31
Lys247 4.29 Cys202 6.50
Lys247 4.87 Cys202 3.84
Lys247 4.83
Leu252 5.66
Leu252 5.73
Ile265 5.41
15 ns Gly134 4.48 Val139 6.97
Ser135 3.25 Lys141 5.87
Gln150 5.18 Arg151 7.22
Arg151 5.43 Ala152 4.92
Ala152 3.86 Leu199 4.37
Cys202 3.46 Leu199 5.02
Cys202 3.46
Lys247 4.67
Ile265 5.19
Ile265 5.52
20 ns Arg151 5.00 Gly134 3.54
Ala152 1.90 Lys141 4.53
Leu201 4.93 Arg151 3.32
Cys202 2.47 Ala152 2.47
Cys202 2.93 Ala152 4.01
Asp203 2.10 Glu200 3.69
Arg244 3.03 Leu201 4.93
Lys247 5.25
Ile265 5.30

The predicted active site residues of the protein are highlighted in bold.

lar interaction with amino acid residues other than the pre-
dicted binding site of the PfCDPK5 protein was also observed. Af-
ter 20 ns simulation, MMV687246 formed three hydrogen bonds
with Ala152 (NH—N29, 1.9 Å), Asp203 (C = O—H27N27, 2.10 Å) and
Arg244 (C = O—O9S8, 3.03 Å) (Table 3). Nine hydrophobic interac-
tions were also found with residue Gly134, Lys141, Arg 151, Ala152,
Glu200, Leu201, Lys247 and Ile265 (Table 4). A comparative bind-
ing conformation analysis of the docked ligand revealed a slight
bend in its conformation and maintained rest of the docked posi-
tion within the protein (Fig. 9).
As pointed out in (Chou and Shen, 2009), user-friendly and
publicly accessible web-servers represent the future direction for
reporting various important computational analyses and findings
(Chen et al., 2014; Feng et al., 2013; W. Chen et al., 2016; W. Chen
et al., 2016; Feng et al., 2017; Chen et al., 2017; X. Cheng et al.,
2017; Qiu et al., 2017; Zhang et al., 2016; Jia et al., 2016; Cheng
et al., 2016; X. Cheng et al., 2017; Cheng et al., 2018; X. Cheng
et al., 2017). Actually, they have significantly enhanced the impacts
of computational biology on medical science (Chou, 2015), driving
medical science into an unprecedented revolution (Chou, 2017). In
 S. Rout and R.K. Mahapatra / Journal of Theoretical Biology 461 (2019) 254–267
Fig. 9. Superimposition of PfCDPK5- MMV687246 complex before (in green color)
and after (in magenta color) simulation.
our future work we shall strive to establish a web-server for the
findings presented in this paper.
4. Conclusion
In the present study, PfCDPK5 was taken as the valuable tar-
get for anti-malarial drug development. P. falciparum kinomes are
highly similar to the P. berghei kinomes (Lucet et al., 2012). The
molecular model of PfCDPK5 was predicted using PbCDPK1 as
a template. The possible structural conformation and secondary
structural arrangement of the PfCDPK5 are indicated by the homol-
ogy modeled protein which can be used in the lack of any struc-
tural conformation of the PfCDPK5 protein. The conserved domain
architecture of the protein provided information about the struc-
tural arrangements and its resemblance with the CDPK family. Five
different sets of compounds were considered for molecular dock-
ing studies out of which the Pathogen Box compound MMV687246
was found to have the highest binding affinity with PfCDPK5. The
binding reliability was further validated by complex molecular dy-
namics simulation for 20 ns. This study suggests that MMV687246
is a lead molecule to bind in the PfCDPK5 active site and arrest its
function and cease the parasite growth.
Acknowledgments
The authors would like to acknowledge the Bioinformatics Lab
Facility of School of Biotechnology, KIIT University, during the
course of the work. Miss Subhashree Rout would like to acknowl-
edge DST, Government of India, for the financial support to pur-
sue her Ph.D. work through INSPIRE fellowship. SR and RKM thank
Prof. Mrutyunjay Suar, Director of School of Biotechnology in KIIT
University, for his encouragement and support during the course
of study.
Conflict of interest
The authors declare that they have no conflict of interest.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.jtbi.2018.10.045.
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