J Mol Model (2017) 23:239
DOI 10.1007/s00894-017-3396-7
ORIGINAL PAPER
Investigation of naphthofuran moiety as potential dual inhibitor
against BACE-1 and GSK-3: molecular dynamics simulations,
binding energy, and network analysis to identify first-in-class dual
inhibitors against Alzheimers disease
Akhil Kumar 1 & Gaurava Srivastava 1 & Swati Srivastava 1 & Seema Verma 2 &
Arvind S. Negi 2 & Ashok Sharma 1
Received: 13 December 2016 / Accepted: 25 June 2017
# Springer-Verlag GmbH Germany 2017
Abstract BACE-1 and GSK-3 are potential therapeutic
drug targets for Alzheimer's disease. Recently, both the targets
received attention for designing dual inhibitors for
Alzheimers disease. Until now, only two-scaffold triazinone
and curcumin have been reported as BACE-1 and GSK-3
dual inhibitors. Docking, molecular dynamics, clustering,
binding energy, and network analysis of triazinone derivatives
with BACE-1 and GSK-3 was performed to get molecular
insight into the first reported dual inhibitor. Further, we de-
signed and evaluated a naphthofuran series for its ability to
inhibit BACE-1 and GSK-3 with the computational ap-
proaches. Docking study of naphthofuran series showed a
Electronic supplementary material The online version of this article
(doi:10.1007/s00894-017-3396-7) contains supplementary material,
which is available to authorized users.
* Ashok Sharma
ashoksharma@cimap.res.in
Akhil Kumar
akhil2687@gmail.com
Gaurava Srivastava
gaurava1708@gmail.com
Swati Srivastava
swati23.srivastava@gmail.com
Seema Verma
seemaverma12@gmail.com
Arvind S. Negi
arvindcimap@rediffmail.com
1
Biotechnology Division, CSIR-Central Institute of Medicinal and
Aromatic Plants, P.O. CIMAP, Lucknow, UP 226015, India
2
Chemical Sciences Division, CSIR-Central Institute of Medicinal
and Aromatic Plants, P.O. CIMAP, Lucknow, UP 226015, India
good binding affinity towards both the targets. Molecular dy-
namics, binding energy, and network analysis were performed
to compare their binding with the targets and amino acids
responsible for binding. Naphthofuran series derivatives
showed good interaction within the active site residues of both
of the targets. Hydrogen bond occupancy and binding energy
suggested strong binding with the targets. Dual-inhibitor bind-
ing was mostly governed by the hydrophobic interactions for
both of the targets. Per residue energy decomposition and
network analysis identified the key residues involved in the
binding and inhibiting BACE-1 and GSK-3. The results in-
dicated that naphthofuran series derivative 11 may be a prom-
ising first-in-class dual inhibitor against BACE-1 and GSK-
3. This naphthofuran series may be further explored to de-
sign better dual inhibitors.
Keywords Alzheimers disease . BACE-1 and GSK-3 .
Dual inhibitor . Molecular dynamics simulation . Binding
energy analysis . Network analysis
Introduction
Alzheimer disease (AD) is the sixth leading cause of death,
currently affecting more than 44 million people worldwide
[13]. AD gradually develops and shows symptoms like neu-
ral loss, neurotransmitter depletion, and synaptic dysfunction
in patients [2]. The most common findings in an AD brain are
deposition of senile neuritic plaques (SNPs) and neurofibril-
lary tangles (NFTs) [3]. SNPs are the result of an extracellular
accumulation of amyloid protein (A), which is generated by
the cleavage of amyloid precursor protein (APP) by the -
secretase (BACE-1) [4]. On the other hand, NFTs are gener-
ated by the hyperphosphorylation of tau protein by the kinase
239 Page 2 of 16
enzyme, glycogen synthase kinase-3 (GSK-3) [57].
These abnormalities lead to the neurotoxic cascades and cyto-
skeletal changes that cause synaptic dysfunction and neural
cell death [3]. The synaptic dysfunction and AD pathology
have been described by the various hypothesis in which am-
yloid, cholinergic, glutamatergic, oxidative stress, and metal
hypothesis are the most accepted ones [8, 9].
The currently used drugs based on the cholinergic hypoth-
esis are hardly able to modify or halt the disease progression
[10] and most of the current treatments are purely symptom-
atic based. There is no FDA-approved drug available that
completely halts the diseases progression. Several reports
are available on targeting single AD targets based on various
disease causing hypothesis to block the disease progression.
[8, 9, 1114]. However, one target-one inhibitor strategies
seem to be less effective in fighting against multifactorial
complex diseases like AD [1517]. A new concept multi-
targeted directed ligand (MTDL) has recently emerged for
neurodegenerative diseases like AD [15]. Computational
screening, docking, and molecular dynamics simulations have
been used extensively for the identification of new leads for
drug discovery [1821]. For multi- or dual-inhibitor design,
various AD target combinations have been explored [2227].
Recently, 3,4-dihydro-1,3,5-triazin-2(1H)-ones chemical moi-
ety was reported as a first dual inhibitor (DI1) for BACE-1 and
GSK-3 [28]. These dual inhibitors simultaneously inhibit
two of the most validated drug targets in AD drug discovery.
GSK-3 has been proposed as the possible link between A
and tau. Further inhibition of GSK-3 downregulates the
amyloidogenic cleavage of APP which suggests the cross talk
between these two disease pathways. [1, 6, 17]. Inhibition of
GSK-3 kinase decreases A production and A-induced neu-
rotoxicity by reducing BACE-1 cleavage of APP [29]. Thus,
these two targets are now considered as potential and the most
promising dual targets for AD [1, 28]. It was observed that multi-
target agents provide a better efficacy profile and effective dis-
ease treatment than the single-target inhibitors [30, 31]. The hy-
pothesis of dual inhibitor for BACE-1 and GSK-3 is challeng-
ing and underexplored until now. Only two scaffolds viz.,
triazinone [28] (Fig. 1a) and curcumin [1], have been identified.
Thus, there is a need to discover new scaffolds that inhibit both
therapeutic targets. Small molecules and various databases have
been successfully screened with the help of an in silico approach
[32, 33]. In silico methods have been applied successfully to find
dual inhibitors against different targets [21, 34] as well as against
AD [35, 36]. Computational methods have earlier not been used
to identify dual inhibitors for BACE-1 and GSK-3. In this
study, we applied computational methods to obtain molecular
insight into first reported dual inhibitor DI for BACE-1 and
GSK-3.
We screened various chemical moieties in which
naphthofuran showed better binding scores towards both the
targets. 1,4-naphthoquinon combined with the tryptophan
J Mol Model (2017) 23:239
moieties have shown complete inhibition of A oligomerization
and fibrillization [37]. Similarly, arylquinone is also known to
inhibit BACE-1, aggregation of -amyloid peptide, and a
destabilizer of preformed -amyloid fibrils [38]. In one of the
studies, furan moiety was reported to inhibit GSK-3 in human
leukemia cancer cells [39]. Another study reported furo-
pyrimidines as the potent GSK-3 inhibitor [40]. Furan-based
derivatives are also reported to penetrate the bloodbrain barrier
[41]. We therefore, combined the two series naphtho and furan
and selected it for further exploration. We rationally designed
naphthofuran series derivatives and identified first-in-class dual
BACE-1 and GSK-3 inhibitor NS11 (Fig. 1b). NS11 showed
good binding scores as compared to the first reported dual inhib-
itor. It was thus taken as a lead molecule for further study.
Interaction and molecular dynamics studies for BACE-1 and
GSK-3 were performed both in the absence of ligand and in
the presence of first reported dual inhibitor DI1 and NS11.
Hydrogen occupancy and binding energy of ligands were calcu-
lated to estimate the affinity towards both of the targets. Free-
energy landscape was derived to check the stability of the sys-
tems. Networking and clustering methods were also used to
study the induced conformational changes and interaction be-
tween protein and dual inhibitors. Our study integrates the con-
cept of dual targeting with the help of established computational
methods to identify the new chemical moiety that might act as a
dual inhibitor for BACE-1 and GSK-3. This model may help in
developing a potent dual inhibitor for BACE-1 and GSK-3.
Materials and methods
Protein 3D structure preparation
X-ray crystal structures of BACE-1 and GSK-3 complexes
with inhibitor were obtained from the Protein Data Bank, i.e.,
1FKN for BACE-1 and 1Q5K for GSK-3. Prior to docking,
bond orders were assigned and hydrogen atoms were added to
the crystal structures and protein structures were prepared by
AutoDock 4.2 software [42]. The solved structure of BACE-1
has been (PDB ID 1FKN) previously used in molecular dy-
namics and binding energy analysis [43]. 1Q5K is also report-
ed to be successfully used for pose prediction accuracy in
docking study for GSK-3 [44]. We set the protonation states
of Asp32 and Asp228 dyad of BACE-1 according to one of
the state of art study. Which recommended that protonation
state of these residues are important for effective virtual
screening [45].
Ligand preparation and grid generation
Reference compounds, i.e., first reported dual inhibitor, for
BACE-1 and GSK-3 and naphthofuran molecules referred
to as an (NS) were drawn in 2D with ChemSketch software
J Mol Model (2017) 23:239
Fig. 1 2D structures of the
studied inhibitors against BACE-
1 and GSK-3 a 3,4-dihydro-
1,3,5-triazin-2(1H)-ones (DI1)
first reported dual inhibitor of
BACE-1 and GSK-3 and b 1-
(3,4,5 trimethoxyphenyl)-
naphtho [2, 1-b]-furan-2-
aldoxime acetate(NS11)
and saved in a .mol file. These molecules were then converted
into 3D .pdb file format with the help of Open Babel software
[46]. Avogadro software was used for structure minimization
using MM3 force field as per the earlier studies [47, 48]. The
size of the grid box was 80 80 80 3 with spacing a of
0.375 , so that the active site of both receptors may fit well
into the grid.
Docking parameters
AutoDock had been successfully used earlier for both of the
targets BACE-1 [24] and GSK-3 [49]. BACE-1 is a protease
enzyme with Asp dyad and GSK-3 is a Ser/Thr kinase en-
zyme. Their active sites for both the targets are well charac-
terized. The grid box was placed around flap and active site
residues of BACE-1, and the grid box dimensions were
X = 80, Y = 80, Z = 80 and coordinates were X = +10.069,
Y = 2.417, Z = 0.366 to cover active site residues. For
GSK-3 grid was placed around the active site as well as
ATP binding site residues. For GSK-3, the grid box dimen-
sions were X = 80, Y = 80, Z = 80 and coordinates were
X = 22.868, Y = 27.175, Z = 8.107 to cover active site resi-
dues. Parameters used during docking experiments with
AutoDock 4.2 were - number of GA runs 10, population size
150, mutation rate 0.02, and crossover rate 0.8. Grid spacing
and other parameters were used as default. Docking simula-
tions were carried out up to 2.5 million energy evaluations
steps with a maximum of 270,000 generations. In
AutoDock, docking simulation produces ten docked confor-
mations. They were clustered on the basis of RMSD. The
lowest energy conformations generated by AutoDock were
regarded as the best binding conformations between ligands
and the protein. The 3D coordinates of the best conformation
from the docking experiment were taken for the proteinli-
gand MD simulations.
Pharmacophore search with PharmaGist
Pharmacophore, which is the spatial arrangement of features
that is essential for a molecule to interact with the receptor, is
Page 3 of 16 239
important for designing new inhibitors. PharmaGist [50] is a
freely available web server for pharmacophore detection. It
computes candidate pharmacophore by multiple flexible
alignments of the input ligands. The main innovation of this
approach is that the flexibility of the input ligands is handled
explicitly and in a deterministic manner within the alignment
process. Another important characteristic of the method is the
capability of detecting pharmacophore shared by different
subsets of input molecules. This capability is a key advantage
when the ligands belong to different binding modes or when
the input contains outliers. For multiple alignments, a
curcumin molecule was also incorporated into the input file
for PharmaGist.
Molecular dynamics simulations for protein ligand
complex
Molecular dynamics simulations were performed using
Gromacs 4.5.5 [51], which was earlier successfully applied
in various studies including BACE-1 [52] and GSK-3 [53]
using force field GROMOS 53a6 [54]. The simple point
charge (SPC) model was used for water molecules and 11
Na+ ions were added in the BACE-1 system and 8 Cl ions
were added in GSK-3). For BACE-1, 20,707 water mole-
cules whereas were added, for GSK-3 24,517 water mole-
cule were added in the system. Each system was subjected to
energy minimization using the steepest descent integrator
without constraints for 1000 steps. Periodic boundary condi-
tions were imposed on the system and canonical ensemble
(NVT) and isothermal-isobaric ensemble (NPT) were used
with normal temperature (300 K) and 1 bar of pressure for
1 ns. Berendsen temperature control [55] (a coupling constant
of 0.1 ps) and ParrinelloRahman pressure coupling [56] (a
coupling constant of 2.0 ps) were used for keeping the system
in a stable environment (300 K, 1 bar). The particle mesh
Ewald algorithm [57] was employed to deal with the long-
range Coulomb interactions with Fourier grid spacing of
0.12 nm. Other parameters cutoff such as short range
neighborlist cutoff (rlist = 1.0 nm), short-range electrostatic
cutoff (rcoulomb = 1.0) nm and short-range van der Waals
239 Page 4 of 16
cutoff (rvdw = 1.0 nm) were set. The LINCS [58] algorithm
was used to constrain all the bonds. For, each system MD
simulation has been performed for 25 nanosecond (ns) and
trajectories were collected. An identical simulation strategy
was used in all the cases. In the first set, we performed molec-
ular dynamics simulations of unbound BACE-1, BACE-1
with dual inhibitor (DI 1), and BACE-1 with NS11 and in
the second set unbound GSK-3, GSK-3 with dual inhibitor
(DI 1) and GSK-3 with NS11. In total, we had six systems
and a total of 150 ns molecular dynamics simulations were
performed to evaluate the binding of NS11 towards both of the
targets. A 2-fs time step was applied and 2-ps final coordinates
were saved. Most of the analyses were performed using
Gromacs inbuilt tools. All visualizations were performed
using Chimera (http://www.rbvi.ucsf.edu/chimera), and
PyMOL (The PyMOL Molecular Graphics System, Version
0.99 Schrdinger, LLC). Further, hydrogen bonds were
calculated by proton donor and acceptor distance 3.5 and
the angle between acceptordonor hydrogen 30. Hydrogen
bond occupancy between protein and ligands molecule were
calculated via a Python script.
Binding energy calculation MM_PBSA
Gibbs free energy was calculated using Gromacs and the
method developed by Rashmi kumari (http://rashmikumari.
github.io/g_mmpbsa/) [59]. Interaction energy for BACE-1
with DI 1, BACE-1 with NS11, GSK-3 with DI 1 and
GSK-3 with NS11 were calculated via MM_PBSA methods
for 20 to 25 ns. For each simulated system, total snapshots
were taken from the last 5 ns of the trajectory at the interval of
20 ps. This methodology includes electrostatic interactions,
van der Waals interactions, polar solvation energy, and non-
polar solvation energy. The MM_PBSA analysis also offers
an individual contribution to the binding energy and per-
residue contribution that provides the important amino acid,
which can be helpful in designing dual inhibitors.
Clustering analysis
In order to select a reduced set of representative models for all
six systems, root-mean-square deviation (RMSD) conforma-
tional clustering was performed using the gromos option [60]
implemented in GROMACS (g_cluster) with a 0.1-nm cutoff
value for RMSD. The RMSD clusters analysis uses the pro-
cess of structural-fitting, which includes superimposition (to
remove overall translation) and a least-squares-fitting proce-
dure (to remove overall rotations). The RMSD matrices were
computed on each of the trajectories by the least square fitting
of backbone atoms and the matrices were generated. Then,
based on RMSD, similar conformations were clustered into
groups.
J Mol Model (2017) 23:239
Network analysis
The study of individual amino acid residues and their molec-
ular interactions in protein structures were studied with anoth-
er computational approach, viz., residue networks analysis
with the help of RINalyzer [61] and was further visualized
with the Cytoscape platform [62]. The geometry-based resi-
due network was derived from 3D protein structures, which
were taken after post molecular dynamics simulation cluster-
ing. RINalyzer was used to generate a 2D visualization of
network and residue interaction. In this visualization, network
nodes represent amino acid residues and edges depict non-
covalent residue interactions. To visualize this non-covalent
interaction, UCSF Chimera [63] was used. Communication
between Cytoscape and UCSF Chimera was handled by struc-
ture, viz., the structure-based layout algorithm, which is im-
plemented in RINalyzer, and uses structureViz to retrieve the
current spatial coordinates.
Principal component analysis (PCA) and the Gibbs
free-energy landscape
PCA was performed on each of the BACE-1 and GSK-3
system trajectories obtained from the MD simulation. PCA
is based on the construction of a mass-weighted covariance
matrix of the atom displacement, and this covariance matrix is
diagonalized to extract a set of eigenvectors and eigenvalues
that reflect the concerted motion of the molecule. First, the
g_covar tool was used to extract eigenvectors and its corre-
sponding eigenvalues of Calpha atoms of each unbound pro-
tein and protein ligand complex trajectories obtained from
MD. To view the most dominant mode and 2D projection of
eigenvector1 with eigenvector 2, we used the g_anaeig tool.
First, two dominant modes were used to plot Gibbs free ener-
gy by applying Boltzmann relation using g_anaeig and
g_sham module of Gromacs.
Blood-brain barrier (BBB) prediction and drug-likeness
prediction
Bloodbrain barrier prediction of NS11 was performed by
applying two algorithms, viz., AdaBoost and SVM, combined
with four different fingerprints (MACCS, Open Babel (FP2),
Molprint 2D, PubChem). Both AdaBoost and SVM are ad-
vanced machine learning algorithms used earlier to detect
Alzheimers disease [64]. The online BBB predictor [65] pre-
dicts if a compound can pass the BBB(BBB+) or cannot pass
the BBB (BBB-). Drug-likeness prediction was performed
with the help of the PreADMET server [66]. PreADMET is
a web-based application for predicting ADME data and build-
ing the drug-like library using in silico method [67].
J Mol Model (2017) 23:239
Results
Interaction study through molecular docking of DI1
and NS11 with BACE-1 and GSK-3
The AutoDock docking results analysis showed that the estimat-
ed binding energy for BACE-1 with DI1 and NS11 was 6.70
kcal/mol and 8.98 kcal/mol, respectively. It was found that DI1
interacts with BACE-1 through a hydrogen bond with one of the
catalytic dyad (Asp228) and other amino acids Tyr71, Thr72,
Gly230, Thr231, and Ile118 through hydrophobic interaction
(Fig. 2a). NS11 formed three hydrogen bonds with Arg235 (with
different atoms of Arg235) and one hydrogen bond with Gln 73.
BACE-1 residues (Phe108, Ile226, and Tyr198) showed hydro-
phobic interaction with NS11. Superimposition of protein-ligand
docked complex generated with the help of ligplot+ suggested
that Tyr71, Thr 72, Asp228, Gly230, Thr231 were the common
amino acid residues for binding of both the ligand DI1 and NS11
with BACE-1(Fig. 2a). These residues might be the core residues
for the binding of BACE-1 and can be further considered for the
design of dual inhibitors. Binding of peptide inhibitor OM992
with BACE-1 has already revealed various subsites for binding
like S1, S2, S3, and S4 [68]. Our docking results also showed
NS11 binding at S1 (Asp32, Tyr71, Gln73, Phe108, Asp228,
Gly230), S2 (Tyr71, Thr72, Gln73, Gly230, Thr231, Arg235)
subsites and interaction with few residues of the S3 subsite.
These BACE-1 amino acid interactions with NS11 suggested
Fig. 2 2D protein (BACE-1 and GSK-3) ligand (DI 1 and NS11)
interaction plot were generated by LigPlot+ and superimposed with
each other. a Superimposed plot of BACE-1 with DI 1 and NS11. b
Page 5 of 16 239
the strong binding and support of our docking result as one of
the earlier reports on the study of inhibitors of BACE-1 had
revealed that residues Tyr71, Thr72, Thr231, Thr232, and
Gln73 provide van der Waals and Arg235, Arg128, and
Lys224, and Lys321 provides electrostatic contributions for the
binding of the ligand [69, 70].
For another target (GSK-3), estimated binding energy
from docking of DI 1 and NS11 was found to be 5.84 kcal/
mol and 9.73 kcal/mol, respectively. Docking of DI 1
showed a total of three hydrogen bonds (two with Asp200
and one with Ser66). Amino acid residues Gly68, Gly65,
and Phe67 were involved in hydrophobic interaction with
the DI 1 inhibitor. In the case of NS11, four hydrogen bonds
were predicted in the docking studies with Lys85, Asp200,
Cys199, and Phe 67. Gly65, Gly68, Ser66, Lys85, Asp200,
and Phe67 were found to be common amino acid residues for
binding of DI 1 and NS11 (Fig. 2b). Strong binding of GSK-
3 with pyrimidylpyrrole, oxadiazole, and halomethyl ke-
tones was reported and they formed a hydrogen bond with
Asp200 and showed interaction with Cys199 located in ATP
binding pocket [13, 71]. In GSK-3, Ile62, Gly63, Phe67, and
Val70 formed a hydrophobic pocket and docking study re-
vealed that NS 11 was located in this pocket. In the docking
results, it was found that ligand NS11 also interacted with the
polar region formed by the Lys85, Cys199, and Asp200.
Previous studies had revealed that the ATP-competitive inhib-
itors establish hydrogen bonds with the backbone atoms of
Superimposed plot of GSK-3 with DI 1 and NS11. Red circle shows
shared/common amino acid residues present in the binding of ligand DI1
and NS11 with BACE-1 and GSK-3
239 Page 6 of 16
Asp133 and Val135 [72] and interactions with these ami-
no acids are key for enhancing affinity to GSK-3, but it
does not provide the selectivity over other kinases [71,
72]. Ligand NS11 did not show any interaction with these
residues. Another region of GSK-3 was also reported to
enhance activity and selectivity [71, 72] and this region is
characterized by the amino acids Lys85, Glu97, and
Asp200 where ligand NS11 binds. Interaction of an inhib-
itor with this region increases activity and selectivity for
GSK-3. Thus, the ligand might behave as a selective and
potent kinase inhibitor.
Alignment of DI1 and NS11 to study the common spatial
arrangement of pharmacophore in both DI1 and NS11
Alignment of two reported scaffold triazinones and
curcumin with NS11 was performed to know the similar
pharmacophores among these compounds. The results
revealed common pharmacophoric features, i.e., one
aromatic, one hydrogen bond acceptor, and one hydro-
gen bond donor in both of the ligands. Distances of
hydrogen bond acceptor and donor from the aromatic
center were 5.91 and 3.66 , respectively, and
distance between donor and acceptor was 3.97 (Fig.
3b). Alignment of DI1, NS11, and curcumin (Fig. 3a)
suggested the same features as reported earlier where
one of the pharmacophore analyses of B ACE-1
suggested similar distance required to inhibit BACE-1
[73, 74]. In the marine natural-derived inhibitors of
glycogen synthase kinase-3 phenylmethylene
hydantoins, pharmacophoric study also showed nearly
similar distance. [75].
Fig. 3 Common feature after alignment from PharmaGist. a Spatial arrangement of three features. b Alignment of DI1 and NS 11 with common
pharmacophore features aromatic (light blue) acceptors (green) and donor (yellow)
J Mol Model (2017) 23:239
Molecular dynamics simulations
Structure and stability analysis of the MD-simulated BACE-1
and GSK-3 systems
Despite the initial structural rearrangements, the trajectories
eventually stabilized as indicated by the RMSD plot of back-
bone atoms for all the simulated systems. Average RMSD for
unbound BACE-1 backbone atoms was 0.21 nm. In an earlier
report of BACE-1 (1FKN) 20-ns simulation, it was observed
that the system equilibrated well after 5-ns simulation and the
RMSD was reported to be 0.2 nm [76]. Molecular dynamics
of DI1 and NS11 with BACE-1 showed the average value of
RMSD to be 0.20 and 0.23 nm, respectively. These results
were in agreement for structural stability of the system. In
GSK-3 systems, average RMSD for unbound GSK-3
backbone atoms was 0.23 nm. Molecular dynamics of GSK-
3 with DI1 and NS11 showed that the average values of
RMSD were 0.260 and 0.267 nm, respectively, which was
slightly higher than unbound protein. The RMSD for all
BACE-1 and GSK-3 systems was nearly similar, thus indi-
cating the stable trajectory of both of the complexes. The
radius of gyration (Rg) provides insight into the overall di-
mension and the shape of the protein. The average value of Rg
was higher for BACE-1 with DI12.14 nm than unbound
BACE-1 protein - 2.13 nm, and BACE-1 with NS11
2.11 nm. The average value of Rg for unbound GSK-3 pro-
tein was 2.14 nm, with DI12.14 nm and with NS11 was
2.18 nm. Average RMSD and Rg values for the BACE-1
system were nearly comparable and RMSD and Rg plot ver-
sus time indicated no structural instability or uncoiling for the
backbone of any of the systems. Thus, both RMSD (Fig. 4a)
and Rg (Fig. 4b) indicated structural stability.
J Mol Model (2017) 23:239 Page 7 of 16 239

Fig. 4 RMSD profile of protein backbone and Rg profile of whole (green). a RMSD of backbone of unbound BACE-1 and with DI and
protein calculated over the course of 25-ns molecular dynamics simula- NS11. b Rg of backbone of unbound BACE-1 and with DI and NS11.
tion of unbound BACE-1 and GSK-3 (black)/ BACE-1 and GSK-3 c RMSD of backbone of unbound GSK-3 and with DI and NS11. d Rg
bound with DI 1(red)/ and NS11 bound with BACE-1 and GSK-3 of backbone of unbound GSK-3 and with DI and NS11

Comparison of mobility of various flexible regions of unbound
BACE-1/ BACE-1 with DI1 and NS11 and unbound GSK-3 /
GSK-3 with DI1 and NS11
BACE-1 is a flexible protein and its loop region and various
inserts (i.e., A, B, C, D, E, and F) show considerable mobility
in molecular dynamics simulations and substrate binding [76].
Careful observation of the flap region and inserts showed
various conformational changes (Fig. 5). Binding of NS11
showed higher fluctuation in inserts B, F, E, D, C, whereas
the third strand and insert A showed less fluctuation as com-
pared to BACE-1 and bound with DI1 (SF1). In GSK-3 loop
C (8995), C helix region (96104), activation loop (200
226), and hinge region (133138) were considered as impor-
tant regions for substrate binding. RMSF plot for GSK-3
indicated that hinge domain and C helix showed a similar

Fig. 5 RMSF of protein amino

acid residue calculated over the
course of 25-ns molecular
dynamics simulation of unbound
BACE-1 and GSK-3 (black), /
DI1 with BACE-1 and GSK-3
(red)/, and NS11 with BACE-1
and GSK-3 (green). a BACE-1
RSMF plot with time. b GSK-3
RSMF plot with time
239 Page 8 of 16
pattern of fluctuation for DI1 and NS11 binding (SF. 2).
Activation and C loop were more fluctuating in unbound
GSK-3 as compared to bound with DI1 and NS11, suggest-
ing that both of the ligands interact with hinge domain and
provide stability to the DI1 and NS11. It is therefore suggested
that hinge domain may provide a platform for inhibitors to
bind. Studies of RMSF fluctuations suggested that binding
of both the ligands stabilize the movement of loop A, C, and
hinge domain as compared to unbound GSK-3.
Possible mechanism of BACE-1 inactivation upon ligand
binding: Dynamics of flap region for unbound BACE-1/ bond
with DI1 and NS11
The flap is an important aspect for protease inhibitor design [77].
The movement of the flap can be monitored by the changes in the
three interatomic distances C(Thr72)- C(Asp32),
C(Thr72)- C(Thr329) and OG1(Thr72)- NH1(Arg235))
[76]. The distance between C(Thr72)-C(Asp32) defines the
motion of the flap and substrate recognition [76]. Unbound
BACE-1 C(Thr72)-C (Asp32) distance indicate that the flap
was switching between open and closed conformations through-
out the simulation run. It was earlier reported that the flap of HIV
protease and BACE 1 switch between open and closed confor-
mations [77, 78]. The average C(Thr72)-C(Asp32) distance
was 1.24 nm (Table 1), representing the close conformation of
BACE-1 [29] as previously reported in dynamic studies of flaps
[52]. In the case of the BACE-1 binding with DI1, the mean value
of distance was 1.29 nm, indicating that the flap also adopts stable
close conformation. Binding of NS11 with BACE-1 increased
the distance further and the mean value for C (Thr72)-
C(Asp32) was 1.64 nm. This indicates that the flap adopts an
open conformation when binding to NS11 and a closed confor-
mation with DI1 (Fig. 6). Adaptation of open conformation of
flap upon inhibitor binding was earlier reported in BACE- SC6,
aminopyridine and 2-aminoquinazole inhibitor binding [79, 80].
Docking results showed that flap residue Tyr71 interacted with
van der Waals interaction, resulting in an open flap after NS11
binding, whereas DI1 interacted with hydrogen bound with
Tyr71, resulting in a closed conformation of the flap. Hydrogen
bond was earlier observed to stabilize the closed conformation of
flap [81]. The variation in the extent of the flap closing and open-
ing was caused by the differences in interaction pattern with the
Table 1 Computed interatomic
distance (nm) between amino acid C (Thr72-C(Asp32)
(C (Thr72-C(Asp32), C
(Thr72)- C(Thr329) and Distance (nm)
OG1(Thr72)- NH1(Arg235))
from molecular simulation data to BACE-1 1.24
monitor the movement of the flap BACE-1 with DI 1 1.29
along 25 ns of simulations BACE-1 with NS11 1.64
J Mol Model (2017) 23:239
flap. Van der Waals and hydrogen bond interaction between the
inhibitors and Tyr71 plays a key role in flap movement [79]. The
C (Thr72)- C(Thr329) and (Thr72)- NH1(Arg235) distance is
important, as it has been observed earlier that these two residues
are involved in ligand BACE-1 interaction [76]. The distance
between OG1(Thr72)- NH1(Arg235) did not show any signifi-
cant change. The distance of C (Thr72-C(Asp32), C
(Thr72)- C(Thr329) was comparable to earlier reports [76],
whereasthedistanceofOG1(Thr72)-NH1(Arg235))waslonger,
suggesting thatthe active site was more open uponNS11 binding.
Possible mechanisms of GSK-3 inactivation upon ligand
binding: Dynamics of spine residue and distance
between Lys85-Glu97 and Asp200-Gly 202 of GSK-3
We analyzed the RMSD of hydrophobic spine residues, the
distance between hydrogen bond (Asp200-Gly202) and salt
bridge (Lys85-Glu97) within the active site of unbound GSK-
3 and bound with DI1 and NS11. In earlier reports, it has
been suggested that if spine residue RMSD is very low, hy-
drogen bond and salt bridge exist between residues Asp200-
Gly202 and Lys85-Glu97, respectively. If these three condi-
tions exist then GSK-3 may be considered in an active state
or active conformation [82]. Spine residue showed a higher
RMSD value when bound with both of the inhibitors. A large
distance gap between (Asp200-Gly202) and (Lys85-Glu97)
was observed, indicating that the there was no possibility to
form a hydrogen bond as well as the salt bridge between the
above-mentioned residues (Fig. 7). The average value of
RMSD and distance between (Asp200-Gly202) and salt
bridge (Lys85-Glu97) in the active site along 25 ns of simu-
lations suggested the inactivation of GSK-3 after binding of
NS11 and DI1 [83].
Hydrogen bond network and occupancy during MD
simulation for ligand DI1 and NS11 with the target BACE-1
and GSK-3
Hydrogen bond numbers were plotted against the time for DI1
and NS11 for BACE-1 and GSK-3 complex (SF3). To de-
termine the overall residual contribution in the formation of
hydrogen bond between protein and ligand, we calculated the
hydrogen bond occupancy. The percentage existence of
C(Thr72)- (Thr329) OG1(Thr72)-NH1(Arg235)
St. Dev. Distance (nm) St. Dev. Distance (nm) St. Dev.
0.18 1.29 0.17 0.90 0.17
0.12 1.38 0.13 0.95 0.16
0.12 1.45 0.17 0.91 0.18
J Mol Model (2017) 23:239 Page 9 of 16 239

Fig. 6 Dynamics of flap region

for unbound BACE-1 (black)/
bond with DI 1 (red) and NS11
(green) over the course of 25-ns
molecular dynamics simulation.
Interatomic distance between
(C(Thr72-C(Asp32)) atom was
plotted against time to monitor the
flap movement

hydrogen bond in the case of DI1 for Gln12, Val31, Asp32,
Thr72, and Gly230 was 17%, 11%, 16%, 4%, and 2%,
respectively, whereas for NS11 hydrogen bond with Thr232
showed existence of 30%, Asn233 16%, Arg235 23%,
Thr72 10%, Trp115 6%. NS11 showed better binding in
terms of a hydrogen bond with BACE-1. Earlier comparison
of the binding modes of the BACE-1 inhibitors suggested that
hydrogen bonds with the backbones of Gly230, Arg 235,
Thr72, or Gln73 were frequently present and this interaction
appears to be vital for high BACE-1 inhibitory activity [79].
These results suggest the strong inhibitory activity of NS11
against BACE-1. Similarly, number of hydrogen bond and
occupancy calculated for GSK-3. NS11 showed better hy-
drogen bond profile with GSK-3. The hydrogen bond occu-
pancy for both of the ligands revealed that DI1 showed Lys85,
Phe67, Arg141, and Thr138 as the major residues involved in
a hydrogen bond. In the case of NS11, Arg96, Lys85, Phe67,
and Phe201 and Asp200 made a good contribution to hydro-
gen bond interactions. Percentage existence of hydrogen bond
between GSK-3 and DI1 was 11% Arg141, 5% Thr138,
4% Phe67,4% Tyr 134, and 4% Lys 85, whereas for NS11
it was 76% Phe201, 48% Lys85, 11% Asp200, and 5% Arg96.
These interaction results with GSK-3 were in accordance of
earlier findings [84].
MM_PBSA analysis for BACE-1 and GSK-3 complex
with DI 1 and NS11
To estimate the strength of interaction between target proteins
and inhibitors, energy contribution of binding for each amino
acid was calculated. MM_PBSA method was applied to DI1
and NS11 complexes. Binding energy calculations were per-
formed from 20 to 25 ns. Binding energies for DI1 and NS11
ligands were 72.15 kJ/mol and 99.34 kJ/mol, respec-
tively. Energy decomposition to individual component
showed that van der Waals and electrostatic forces were the
most dominant contributors towards energy contribution
(Table 2). The van der Waals force was more prominent in

Fig. 7 Structural analyses of functional residues in the active site of GSK-3 along 25 ns of simulations. a RMSD of hydrophobic spine residues. b
Distance between hydrogen bond (Asp200-Gly202). c Salt bridge (Lys85-Glu97) forming residues
239 Page 10 of 16 J Mol Model (2017) 23:239

Table 2 Binding energy values and individual component energy calculated with MM-PBSA method for BACE-1 with DI 1 and NS11

Ligands van der Waals energy Electrostatic energy Polar solvation energy SASA energy Binding energy
(kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol)

BACE-1 with DI1 -106.107 9.843 -48.2510.33 94.3918.84 -12.190.67 -72.159.40

BACE-1 with NS11 -164.8916.72 -34.8512.86 118.1319.25 -17.721.07 -99.3414.96
GSK3 with DI1 -95.799.35 -28.4916.92 78.8516.02 -11.370.65 -56.809.06
GSK3 with NS11 -218.4013.26 -33.638.57 134.6919.92 -19.871.01 -137.2019.97

NS11 binding, showing the hydrophobic interaction being a
key player in effective binding. Predicted binding energy
through MM-PBSA suggested the strong binding of DI 1
and NS11 with both the targets. In MM-PBSA analysis
NS11 showed better binding than DI1 towards both the
targets.
Finally, energy partition for individual residues of the
BACE-1 and GSK-3 was calculated to identify the contribu-
tions to the total energy (Fig. 8). Mostly residues belonged to
the active site and flap region of BACE-1. Asp228 (4.44kJ/
mol), Trp115 (4.22kJ/mol), Ile110 (3.68kJ/mol), Leu30 (2.67
kJ/mol), Thr231 (2.44kJ/mol) and Phe108 (1.24kJ/mol), Tyr71

Fig. 8 Binding energy graph for individual amino acid contributing to binding energy y-axis show the energy contribution per residue in kJ/mol and x-
axis residue number: a for BACE-1 with DI 1(blue) and NS11 (red) and b GSK-3 with DI 1(blue) and NS11 (red)
J Mol Model (2017) 23:239
(1.88kJ/mol), Gly230 (1.07kJ/mol), Gln12 (1.75kJ/mol)
were observed to be involved in effective energy con-
tribution in BACE-1 with DI1 complex. Similarly effec-
tive energy contribution, per residue analysis of NS11
with BACE-1 revealed, Asp228 (1.12kJ/mol), Trp115
(1.01kJ/mol), Ile110 (3.62kJ/mol), Leu30 (2.30kJ/mol)
Thr231 (2.52 kJ/mol ), Phe108 (1.45 kJ/mol ), Tyr71
(4.88kJ/mol), Gly230 (0.18kJ/mol), Gln12 (0.91kJ/mol).
Binding energy for interacting BACE-1 amino acids
with 5HA (isophthalamide) were in accordance with
earlier reports [71, 82]. Residue analysis indicated that
binding was majorly governed by the amino acid with
hydrophobic and polar uncharged side chain. It was ear-
lier suggested that Asn233, Arg235, and Ser325, which
was located in the S2 open region, a hydrophobic phe-
nyl ring of the inhibitors can interact with these sur-
rounding residues to improve the inhibitory activity
[79]. In binding of DI1 with GSK-3, Leu188
(6.12 kJ/mol ), Val70 (6.01 kJ/mol ), Ile62 (2.59kJ/mol ),
Thr138 (2.40 kJ/mol ), Tyr134 (2.35 kJ/mol ), Asp200
(2.09kJ/mol), Leu132 (2.09kJ/mol) were major contribu-
tors to the binding energy. Val70 (10.47kJ/mol), Leu188
(8.45kJ/mol), Cys199 (6.46kJ/mol), Leu132 (5.79kJ/mol),
Val110 (4.18kJ/mol), Ile62 (3.05kJ/mol) were major con-
tributors in NS 11 binding with GSK-3. The energy
contributing per residues was similar to previous molec-
ular dynamics studies of various inhibitors against GSK-
3 [84]. These interactions with amino acid and indi-
vidual amino acid energy contribution were in accor-
dance with our results, further suggesting the good
binding against GSK-3.
Fig. 9 2D visualization and interactive analysis of residue interaction,
BACE-1 with DI1 (a) and BACE-1 with NS11 (b), and GSK-3 binding
with DI1 (c), and NS11 (d). In this visualization, network nodes represent
Page 11 of 16 239
Network analysis of BACE-1, GSK-3, with DI1 and NS11
to determine the important residues essential for dual
inhibition activity
The protein ligand network was generated from the structure
obtained from post molecular dynamics run after the clustering.
The most representative structure of the cluster was taken and the
ligand was retrieved from the trajectory for BACE-1 and GSK-
3 (Fig. 9). BACE-1 with DI1 complex network showed that
residues Gly11, Gln12, Gly13, Leu 30, Gln73, Gly74, Trp115,
and Thr231 were non-covalently interacting with the target pro-
tein. BACE-1 with NS11 complex network showed more inter-
action as compared to DI1. In this network, Val69, Tyr71, Gln73,
Trp76, Lys107, Phe108, Ile110, Trp115, Gly230, Thr231,
Thr232, Asn233, and Arg235 were non-covalently interacting
with BACE-1. Network analysis also supported that docking
interaction at subsite S1, S2, and S3 residues [85, 86] were pres-
ent in the network of BACE-1 with NS11. One of the state-of-the-
art studies, encompassing BACE-1 flexibility and ensemble
docking-based hybrid QSAR approach, suggested the essential
interacting residues [87]. Our results were also in agreement with
the earlier study. These results were also supported by the binding
energy contribution to binding for each residue from molecular
dynamics and docking results. The DI1 network showed direct
interaction with the flap residues Gln73 and Gly74 and this may
be the reason for the closed flap conformation while binding to
DI1. Third strand residue Trp115 also showed interaction with
DI1 as shown in an RMSF plot and clustering.
BACE-1 is a protease enzyme having a large pocket with
hydrophobicsurfaces.Thus, hydrophobic interactionsare impor-
tant for proteinligand interactions. Dispersion forces, pipi
amino acid residues and edges depict non-covalent residue interactions.
To visualize this non-covalent interaction, UCSF Chimera was used (yel-
low line showed non-covalent residue interactions)
239 Page 12 of 16
stacking, and Hpi stacking are an important force in
the development of BACE-1 inhibitors [88]. These in-
teractions are non-covalent interactions and may occur
between aromatic rings since they contain pi bonds.
The interaction depended upon the arrangement of
rings such as sandwich, parallel displaced, T-shaped,
face to face, or edge to face. In this case, the aromatic
ring of Tyr71 and aromatic ring of NS11 were arranged
in parallel displaced orientation that stabilizes the
interaction. As per earlier reports, these interactions
are most abundant in arginine and tyrosine among
BACE-1 inhibitors. In acylguanidine-based inhibitors
pyrrole ring formed a Pi stacking interaction with the
flap Tyr 71 residue and stabilize the enzyme in an open
conformation [12, 88]. Interaction of Tyr71 with NS11
was also supported by the free energy per residue con-
tribution graph (Fig. 9). Residues Lys107, Phe108,
Ile110, and Trp115 showed interaction with the NS11
and this interaction may explain the shifting of the third
strand towards active site. Thus it can be said that bind-
ing of DI1 and NS11 involved third-strand shifting
towards the active site. Substrate binding in BACE-1
was guided by the third strand and downwards move-
ment of A and F strand [76]. GSK-3 DI 1 complex
n e t w o r k s h o w e d , I l e 6 2 , G l y 6 3 , Va l 7 0 , A l a 8 3 ,
Asp133, Tyr134, Val135, Pro136, Thr138, Leu 188,
whereas the GSK-3 NS11 complex showed interaction
with Ile62, Gly63, Gly68, Val70, Ala83, Lys85, Glu97,
Val110, Leu132, Asp133, Val135, Gln185, Asn186,
Leu188, Cys 199, and Asp200. These results were in
accordance with the docking and free-energy contribu-
tion of each residue in binding energy. The network
showed the hinge domain residues interacting through
both of the ligands. These network results were also
supported by the RMSF and clustering results as
RMSF of hinge domain decrease and clustering
Fig. 10 Superimposed backbone structure of the various mobile region of free (a) BACE-1 (black), BACE-1 with DI1 (red) BACE-1 with NS11 (green)
and (b) GSK-3 (black) and bound with DI 1 (red) and NS11 (green) obtained after clustering of 25-ns simulation trajectory
J Mol Model (2017) 23:239
showed stable hinge backbone atoms after ligand
binding.
Ligand-induced conformation changes during MD
simulation: clustering of MD trajectory of mobile region
of BACE-1 and GSK-3 before and after binding of DI1
and NS11
We studied the top clusters for all the systems to observe the
structural changes in backbone atoms of BACE-1 and GSK-3
in unbound and bound with DI and NS11. The most representa-
tive structure of cluster 1 for all the protein and protein-ligand
complex ligand system was retrieved and aligned (Fig. 10). The
alignment showed that the flap region adopted a relatively closed
conformation in unbound BACE-1 and bound with DI1, whereas
it was open in NS11. Binding NS11 shifted the third strand
(Lys107- Gly117) into the active site and that shifting was oppo-
site with respect to what was observed in unbound BACE-1 and
bound with DI1. Insert A (Gly158-Leu167) showed extended
conformation with both the DI 1 and NS11. Insert B (Lys218-
Asn221) E (Glu290-Ser295), and C (Ala251-Pro258) followed a
similar pattern as observed with unbound BACE-1. Insert D
(Trp270- Thr274) showed slight shifting in the case of NS11.
Insert F (Asp311- Asp 317) showed considerable shifting in the
DI 1 binding. NS11 binding affected the insert A, D, and the third
strand whereas in DI 1 major changes occurred in insert F. In
DI1, slight shifting of insert A and third strand was also observed
when compared to free BACE-1.GSK-3 was made up of N-
lobe (35138) and large C-lobe (139386). For an enzymatic
activity majorly three regions i.e., P loop (6469), activation loop
(200226) and C-helix (96104) were responsible which also
contain basic grove (Arg 96, Arg180, and Lys205), Glu97,
Lys85, Tyr216 present at the catalytic site [83]. The activation
loop was highly dislocated when it was bound to DI-1 and NS11
as compared to free GSK-3. C- helix adopted a similar pat-
tern, but Arg 96 seems to shift away in the NS11 in the
J Mol Model (2017) 23:239
alignment. Shifting of P-loop was observed for DI 1 and NS11.
The most prominent sign for activation of GSK-3 was where
C-helix came closer to both the P-loop and activation loop [83].
P-loop and activation loop shifted away from the active site in a
ligand-bound state in comparison to unbound state of GSK-3,
which indicate the inactivation of protein. Hinge domain (133
138) was reported to provide stability to binding ligand following
a similar pattern of backbone.
Principal component analysis (PCA) and the Gibbs
free-energy landscape
PCA of apo and docked systems of both BACE-1 and GSK-3
beta have shown that more than 60% of total fluctuations in the
systems were governed by initial ten eigenvectors (SF4).
Calculated PC1 and PC2 (SF5) used to derive free-energy land-
scape (SF6) indicated that all protein ligand systems were able to
fold smoothly and achieved stable conformation, and indicated
an arrangement of amino acid on the surface, which narrows the
free-energy landscape, leading to convergence of landscape at
the bottom. All the systems were in their stable form.
The bloodbrain barrier (BBB) prediction
Any molecule working in central nervous system (CNS) drug
discovery project has to be an optimal candidate that can cross
the BBB. The bloodbrain barrier (BBB) is the separation of
circulating blood and cerebrospinal fluid (CSF) in the CNS. It
consists of tight junctions around the capillaries formed by endo-
thelial cells that specifically restrict the diffusion of microscopic
objects and large or hydrophilic molecules into the CSF, while
allowing the diffusion of small hydrophobic molecules. Cells of
the barrier actively transport metabolic products such as glucose
across the barrier with specific proteins.NS11,the leadcandidate,
was checked for its ability to cross the bloodbrain barrier.
AdaBoost and SVM algorithms were used, combined with
MACCS, Open Babel (FP2), Molprint 2D, PubChem finger-
prints to predict the NS11 bloodbrain permeability. All methods
showed +BBB predictions of NS11 except the SVM combine
with the MACCS fingerprint (SF7, SF8). PreADMET prediction
suggested that NS11 had good drug-like properties, as it followed
the CMC, MDDR, and WDI like rule (SF9).
Discussion
AD is a multifactorial neurodegenerative disease and various
reports indicate that amyloid and tau hypothesis are not mutually
exclusive [1, 15, 28]. Thus, the development of dual inhibitors for
these targets might be an important therapeutic strategy.
Recently, the first dual inhibitors for BACE-1 and GSK-3 have
been reported. The present study was performed to screen and
identify the potential of various chemical moieties as dual
Page 13 of 16 239
inhibitors for BACE-1 and GSK-3. We combined two
fragments- first naptho - which are already reported to inhibit
BACE-1 and second- furan reported to inhibit GSK-3.
Computational studies of dual inhibitor DI1 and NS11 bearing
naphthofuran moiety against BACE-1 and GSK-3 showed po-
tential to inhibit both the targets. Various computational methods
were applied to compare the results between unbound target pro-
teins and bound structure of BACE-1 and GSK-3 with DI1 and
NS11. The use of docking with molecular dynamics, free-energy
calculation lead to identification of a new series of first-in-class
dual inhibitor (1-(3,4,5 trimethoxyphenyl)-naphtho [2,1-b]-
furan-2 aldoxime acetate) of BACE-1 and GSK-3. The docking
of both the DI and NS11 with BACE-1 was found to share com-
mon binding residues Asp32, Tyr71, Thr72, Asp228, Gly230,
and Thr231, whereas in binding with GSK-3, Gly65, Gly68,
Lys85, Asp200, Ser 66, Phe67 were found as common amino
acid residues. These common residues were previously reported
in binding of BACE-1 and GSK-3 with potent inhibitors [71,
78, 79]. Thus these residues can be target for dual inhibitor design
for BACE-1 and GSK-3.
Initial conformation obtained by docking experiments was
subjected to molecular dynamics simulation. NS11 bound
with BACE-1 showed hydrogen bond percentage existence
with Thr232 > Arg 235 > Asn233 > Thr72 > Trp115 and
hydrophobic interaction with Gly74, Lys107, Phe108,
Ile226, Asp32, Asp228, Gly34, Tyr198, Thr72, Val332,
Thr231, and Tyr71. Similarly, NS11 interacted with GSK-3
through Phe201 > Lys85 > Asp 200 > Arg96 residues with
hydrogen bond. In GSK-3 binding with NS11 residues
Gly65, Ser 66 Gly68, Val70, Gly97, Met 101, Val110,
Leu130, Thr138, lys183, Gln185, Leu188 showed hydropho-
bic interaction. These interactions suggested that Gly, Lys,
Thr, Val, and Arg were common amino acids for both the
targets and may be further used to design dual inhibitors.
The binding energies MM-PBSA suggested that binding of
NS11 was good with BACE-1 and GSK-3. Analysis of the
individual components of energy contribution to binding en-
ergy suggested that binding of DI 1 and NS11 with BACE-1
and GSK-3 was mostly governed by the van der Waals en-
ergy. Van der Waals interactions were governed by the inter-
action of hydrophobic active site residues with both the inhib-
itors DI1 and NS11. Van der Waals interaction is prominent in
BACE-1 and GSK-3 as reported in earlier docking and dy-
namics studies [89, 90]. Further, per-residue energy contribu-
tion for NS11 and DI1 in binding with BACE-1 showed sim-
ilar residues, except Tyr71, Asp32, and Asn233, as these were
found more prominent in energy contribution with NS11 bind-
ing. These residues may provide the high binding affinity of
NS11 with BACE-1. Similarly, per-residue energy contribu-
tion for NS11 and DI1 binding in GSK-3 showed that Val70,
Leu132, Leu188, Cys199 were more prominent in NS11. The
network between inhibitors and both the targets mostly
showed hydrophobic and polar amino acid residues. These
239 Page 14 of 16
interacting amino acid residues as well as shared common
amino acid residues involved in binding of both BACE-1
and GSK-3, together with the spatial arrangement of
pharmacophore, may be useful in designing new potent inhib-
itors against BACE-1 and GSK-3. The observed results in-
dicate the potential of NS11 as a lead molecule against AD.
These shared residues between both the targets and their phys-
iochemical properties may be used to design potent inhibitors
for both the targets belonging to different families, i.e., prote-
ase and kinase. These computational studies provide a plat-
form to design potent and selective inhibitors against these
two targets.
Acknowledgements The research work was carried out at the
Bioinformatics laboratory, Biotechnology Division of the CSIR-
CIMAP. Akhil Kumar is grateful to the Council of Scientific &
Industrial Research (CSIR), India, for the Senior Research Fellowship,
Gaurava Srivastava and Swati Srivastava to BTISnet, DBT, New Delhi.
Author contributions A.K., A.S. and A.S.N. conceived the study, de-
signed the methodology and wrote the initial draft of the paper, G.S.
performed PCA and FEL analysis, S.S. performed network analysis.
A.K., S.V., A.S.N., A.S. contributed to interpreting results, discussion
of the associated dynamics, and improvement of this paper.
Compliance with ethical standards
Competing financial interests The authors declare no competing fi-
nancial interests.
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