Acetyl-cholinesterase enzyme (AChE) is a known target for identifying potential inhibitors against Alzheimer diseases (AD). Therefore, it is of interest to screen AChE with the CNS-BBB database. An AChE enzyme is a member of hydrolase family is activated by acetylcholine (ACh), so, targeting the AChE enzyme with the potential inhibitor may block the binding of the ACh. In this study we carried out virtual screening of drug-like molecules from Chemical Diversity Database particularly CNS-BBB compounds, to identify potential inhibitors using Glide docking program. Top ranking ten compounds, which have lower Glide Score when compared to known drugs (Tacrine and Galantamine) for AChE. For top three molecules MD simulation was carried out and calculated binding free energy. We report the best binding compounds with AChE compared to known drugs (Taine and Galantamine) for AD. We further document the salient features of their molecular interaction with the known target. Three molecules (1-benzyl-3-(2- hydroxyethyl)-N-[2-(3-pyridyl)ethyl]-3-pyrrolidinecarboxamide, N-{3[benzyl(methyl)amino]propyl}-1,5-dimethyl-4-oxo-4,5-dihydro- 1H-pyrrolo[3,2-c]quinoline-2-carboxamide, and 6-chloro-N-[2-(diethylamino)-2-phenylethyl]-4-oxo-4H-chromene-2-carboxamide) have -196.36, -204.27, -214.40 kJ/mol, binding free energy values respectively which are much lower than values calculated for the reference ligands Tacrine and Galantamine having -119.65 and -142.18 kJ/mol respectively. Thus these molecules can be very novel potential inhibitors against AChE involved in Alzheimer's disease.
Acetyl-cholinesterase enzyme (AChE) is a known target for identifying potential inhibitors against Alzheimer diseases (AD). Therefore, it is of interest to screen AChE with the CNS-BBB database. An AChE enzyme is a member of hydrolase family is activated by acetylcholine (ACh), so, targeting the AChE enzyme with the potential inhibitor may block the binding of the ACh. In this study we carried out virtual screening of drug-like molecules from Chemical Diversity Database particularly CNS-BBB compounds, to identify potential inhibitors using Glide docking program. Top ranking ten compounds, which have lower Glide Score when compared to known drugs (Tacrine and Galantamine) for AChE. For top three molecules MD simulation was carried out and calculated binding free energy. We report the best binding compounds with AChE compared to known drugs (Taine and Galantamine) for AD. We further document the salient features of their molecular interaction with the known target. Three molecules (1-benzyl-3-(2- hydroxyethyl)-N-[2-(3-pyridyl)ethyl]-3-pyrrolidinecarboxamide, N-{3[benzyl(methyl)amino]propyl}-1,5-dimethyl-4-oxo-4,5-dihydro- 1H-pyrrolo[3,2-c]quinoline-2-carboxamide, and 6-chloro-N-[2-(diethylamino)-2-phenylethyl]-4-oxo-4H-chromene-2-carboxamide) have -196.36, -204.27, -214.40 kJ/mol, binding free energy values respectively which are much lower than values calculated for the reference ligands Tacrine and Galantamine having -119.65 and -142.18 kJ/mol respectively. Thus these molecules can be very novel potential inhibitors against AChE involved in Alzheimer's disease.
Alzheimer's disease (AD) is an old age illness affecting many
people over the age of sixty and becoming the 7th leading cause
of death in all over the world. Just in the United States alone
there is an increasing trend in the number of people being
diagnosed with AD, the chance of being diagnosed with
Alzheimer's increases exponentially and women's are at a larger
risk. As per statistics released from the "2018 Alzheimer's Disease
(AD), Facts and Figures" report, AD accounts for all types of
dementia nearly 60-70% of the cases and an estimated 5.5 million
Americans of all ages have Alzheimer's disease and almost 47.5
million people are living with dementia around the world by this
disease and it is estimated by 2050 more than 115 million people
will have dementia (https://www.alz.org/documents_custom/
2016-facts-and-figures.pdf) [1-2]. The early diagnosis and the
treatment of AD is now an emerging research field, although
presently there is no cure for the disease with the existing anti-
Alzheimer's drugs, and only moderately effected treatment is
possible. At present study on the AD the disability weight (DW),
of this disease in people older than 60 years is greater than other
lethal disease as like cardiovascular disease (CVD), Stroke,
Cancer, and Muscular skeletal disorders. Cholinesterase is the
family of the enzymes that catalysis the neurotransmitter
acetylcholine (ACh) through hydrolysis into the choline (Ch) and
acetic acid [3]. In AD, acetylcholinesterase (AChE) inhibitors,
leading to inhibition of acetylcholine (ACh), breakdown and
make a way for disease, several strategies to elaborate the disease
on the characteristics of symptoms, although the one of that has
been most successful till is "cholinergic hypothesis" strategies.
(https://jnnp.bmj.com/content/jnnp/66/2/137) [4], the current
FDA approved mostly drugs are follows the cholinergic
hypothesis, the ACh deficit is; they try to enhance the ACh level
in the diseased brain. So, inhibition of AChE level plays an
important role for enhancing the cholinergic transmission in the
diseased brain. Currently, available AChEI, such asTacrine (year,
1993) [5], first FDA approved drug used for AD treatments,
another Donepezil (year, 1996) [6], Rivistigmine (year, 2000) [7],
and Galantamine (year, 2001) [8], are found moderately effected
to treat the level of the diseased brain, but approximately, 40-70%
patients are beneficial from AChEI. AChE [E.C. 3.1.1.7] is the
found many types of transporting in tissues likewise nerve and
muscle, central and peripheral, sensory fibers and motor, and
cholinergic and non-cholinergic fibers, the level of activity of
AChE is much higher in motor neurons than in sensory neurons
[9, 10, 11].
AChE also present in Red blood cells (RBCs) membranes,
where is consisting Yt blood group antigen and has similar
catalytic properties [12]. More about ACh neurotransmitter as the
neuromuscular junction between the skeletal muscle and motor
nerve, in the central nervous system (CNS), ACh primarily found
in the intravenous and few important long-axon cholinergic
pathways identified, and degeneration of this pathway is one of
the crucial pathologies and closely related to Alzheimer's
diseases (AD) [13, 14]. But non-selectivity of the drugs and
limited efficacy, poor bioavailability, other side effects in the
periphery, hepato-toxicity are some of the limitations for their
success. So, far we need some other effected small molecules,
those shown more effected to the elaborate the moderate effect to
the best way for the cure of the diseased brain. Thus, the present
study was carried out in order to find effective molecules,
analysis the interactions mechanism [15]. In about blood brain
barrier (BBB), it is a crucial investigation in pharmacological
studies under pharmaceutical umbrella in respect to CNS; CNSactive
compounds must be crossed BB barrier to interact their
specific targets. BBB blocks majority of chemicals do not target
the CNS because of unusual side effects [16].
Methodology
The workflow used in this study is shown in Figure 5.
Figure 5
Flowchart for the study from start to end.
Target protein structure
Human acetyl cholinesterase (h-AChE) is a most significant drug
target for the therapeutic drugs. Here, we have used high
resolution crystal structure of h-AChE a (PDB ID: 4PQE). While
preparation of the receptor, all water molecules were deleted and
missing hydrogen atoms were added, and prepared via
Schrodinger, maestro [17]. It was followed by retrained energy
minimization by fixing the residues 543 and remove steric clashes
between side chains. Two approved drugs Tacrine &
Galantamine used in treatment for Alzheimer's diseases were
used as a reference compounds. All other important active site
residues were identified using online Coach Server (A Meta-
server based approach to protein-ligand binding site prediction
(http://zhanklab.ccmb.med.umich.edu/COACH/) [18, 19]. The
receptor grid was generated using Glide module [20], of the
Schrodinger suite. The receptor grid was generated using the
Acyl binding pocket (ABP), Catalytic triad (CT), Peripheral
anionic site (PAS), Oxyanionic site, and Anionic sub-site as a grid
centre, and the grid boundary was defined between any atoms of
ligand and grid boundary is at least 5Å.
Ligand library preparation
A library of 23,731 CNS_BBB compounds was obtained from the
ChemDiv database (http://www.chemdiv.com/cns-bbblibrary/)
[21]. CNS-BBB ChemDiv database compound is a
known as antioxidant and anti-inflammatory nature, and the 2D
structures in Sdf format were downloaded from the ChemDiv
database, and created a 3D-Phase database from Phase module of
the Schrodinger, phase version 3.2. [22]. In this h-AChE receptor
cavity have Catalytic triad (CT), Anionic sub-site (AS), Peripheral
Anionic site (PAS), Oxyanion sites, and Acyl binding pocket
(ABP) is the main binding sites. Here, docking study is done with
the reference compounds of FDA approved anti-Alzheimer's
drugs [5, 6,
7, 8]. And other ChEMBL approved under clinical trials
compounds also docked the same active/binding site of the
receptor, and calculate the binding energy profile of the under
clinical trials approved drugs shown in (Table 5 and Figure 6).
CNS_BBB data were filtered for Lipinski Rule of Five, using
phase module version with few default parameters. Bond orders
were assigned and various others states, likewise, tautomer's,
stereochemistry, and ring conformations were produced for each
input structure. All the structures were minimized using OPLS
force field, OPLS force field was used by Glide (grid-based ligand
docking with energetics) docking [20].
Table 5
CHEMBL clinical approved drugs on trials, showed binding Energy profile of five-top compound as referenced: (CHEMBL ID)
Parameters (kJ/mol)
CHEMBL-1200541
CHEMBL-1555
CHEMBL-1678
CHEMBL-211471
CHEMBL-1128
van der Waal energy
-260.10+/- 13.44
-155.13 +/- 6.46
-202.97 +/- 78.47
-157.09 +/- 7.59
-123.37 +/-7.01
Electrostattic energy
-34.22+/- 9.48
-2.57+/- 0.909
-1.07+/- 1.57
-12.9 +/-8.40
-0.14+/- 1.70
Polar solvation energy
118.42+/-15.36
36.29+/-10.09
37.08+/-11.00
50.61+/- 9.99
35.89+/- 5.58
SASA energy
-23.98 +/- 1.278
-15.63+/- 0.86
-19.17+/-7.12
-14.04+/- 0.64
-11.52+/- 0.63
Binding energy
-199.88 +/-14.15
-137.05+/-10.62
-186.13+/-82.56
-133.45+/-11.12
-99.16+/- 9.31
Figure 6
2D-Structure of CHEMBL approved clinical trial drugs.
Receptor grid generation
A receptor grid was created around the protein binding residues
(W86, G121, G122, Y124, E202, S203, A204, W236, F295, F297
Y337, F338, and H447). The reference ligand were sketched by
Marvin sketch software and ligand were prepared in LigPrepmodule
of Schrodinger suite, which generates all possible states
with the neutral pH and generated ionized and tautomer state for
the ligands. After protein preparation, Grid generation and
LigPrep preparation, ligands were used for molecular docking
suite Glide version 9.2 in Schrodinger maestro suite with the
extra-precision mode for docking [23].
Virtual Screening
Here, in this study, we have used CNS-BBB database and
followed a cut-off for virtual screening as HTVS-10%, SP-10%,
and XP-10%, respectively. The fast & accurate prediction of a
ligand tightly and specifically binding to a target protein is a
crucial step for computational virtual screening [20].
Glide Extra-Precision Mode (XP)
The extra-precision (XP) mode of Glide combines a powerful
sampling protocol with the use of a custom scoring function
designed to identify ligand poses that would be expected to have
unfavourable energies, based on well-known principles of
physical chemistry. Glide-Score is based on Chem-Score, but
includes a steric-clash term and adds buried polar terms devised
by Schrodinger to penalize electrostatic mismatches. Experiments
were performed using the program GLIDE (Grid-based Ligand
Docking) module version 5.6, Schrodinger, LLC, New York, NY,
2011 (Schrodinger Inc.) [23, 24]
ADME properties
The predicted ADMET properties showed that these compounds
could be potent & effective inhibitor against protein ligand
interaction b/w Acetyl-cholinesterase and the FDA approved
moderately effective drugs. In current study we have identified
potential-lead molecules which can be taken for in-vitro studies.
The ADME properties of the ligands were predicted using
QikProp the compounds prepared were subjected to druglikeness
filter. The acceptance criteria of the filter includes
Molecular weight (< 500), Q Plog BB (-3.0 to 1.2), Donar HB (0-6),
% HOA ( 80% high < 25-poor, > 500-Good), PSA (7-200), Q P log
S (-6.5 to 0.5), Metabolism (1-8), Accept HB (2-20), Log P Value
o/w (-2.0 to 6.5), CNS (-2 to +2) [25]. All the ligands confirmed to
the above mentioned acceptance criteria and they were evaluated
for docking using extra precision GLIDE dock module and the
results are shown in Table 1, and (Table 3,
Table 4, Table 5),
respectively.
Table 1
Protein-ligand interactions for Top hit compounds and referenced FDA approved drugs Tacrine and Galantamine. (NOH: Number of Hydrophobic Interactions, NIB: Number of Non-Bonded Interactions).
S.No
Compound Name & PubChem CID
IUPAC Name
G-Score Kcal/mol
Number of residues, H-Bonds, & ∏-∏ Bonds Interactions
ADMET Property profile of the top ten hits of CNS-BBB of ChemDiv database compounds
Compound Ida
Molecular Weight1
SASA2
QPLogS3
QPlog- HERG4
QPlog-BB5
% HOA6
Lipinski's rule of five7
M1
353.463
687.395
-2.623
-5.667
-0.689
79.012
0
M2
353.463
688.923
-2.655
-5.697
-0.671
79.542
0
M3
421.984
769.171
-4.25
-5.211
-0.384
83.29
0
M4
353.463
686.182
-2.467
-5.649
-0.719
77.768
0
M5
353.463
688.27
-2.503
-5.682
-0.704
78.257
0
M6
313.374
635.854
-4.673
-6.707
0.106
100
0
M7
313.374
634.414
-4.655
-6.705
0.101
100
0
M8
416.522
777.376
-5.225
-7.703
-0.458
96.477
0
M9
439.571
794.701
-4.39
-7.341
-0.575
95.746
0
M10
398.888
689.205
-4.179
-6.483
-0.319
91.284
0
aChemDiv ID of the compound, 1Molecular weight of the molecule, 2Surface area, 3Predicted aqueous solubility, 4Predicted IC50 value for blockage
Table 4
H-Bonds Occupancy profile of the referenced ligands and potential compounds from CNS-BBB data of ChemDiv database
Interacting residues
H-bond in FDA drug-h-AChE docking
H-Bond in CNS-BBB docking
% Occupancy of H-Bond in MD simulations
FDA drug h-AChE MD Simulation
CNS-BBB Compounds
M1
M8
M10
D74
No
No
-
-
30
-
N83
No
Yes
-
-
-
-
S125
Yes
No
-
-
-
-
S203
Yes
No
-
-
-
-
W86
Yes
Yes
-
-
-
-
W286
No
No
1.8
30
-
-
F295
No
Yes
-
-
-
50.8
Y124
No
No
2.5
26.5
19.9
Y133
Yes
No
-
-
-
-
Y337
Yes
Yes
12.7
-
-
-
Y341
No
Yes
-
-
-
-
Protein –Ligand docking
All the docking calculations were performed using the "Extra
Precision (XP)" mode of docking via Schrodinger maestro suite of
Glide. A scale factor of 0.8 and partial atomic charge of less than
0.15 was applied to the atoms of both proteins for van der Waals
radii. The number of poses generated for each ligand was set
criteria to 10,000 and out of them 10 best poses per ligand. The
best docked structure from each of the ligand docking calculation
was chosen based on XP-Glide Score, Glide energy, and Glide
emodel value and interaction of the relative docked complexes
were further studied for MD simulations package of GROMACS
5.1.2 and Binding energy (MM-PBSA), calculation, Hydrogen
bond Occupancy, with FEL Analyses.
Molecular dynamics simulations
GROMACS 5.1.2 molecular dynamics package [26] was used to
know the structural stability of the selected protein-ligand
complexes. All the generated protein structures were processed
further on WhatIf server for completing the structures [27], and
ligand topology was generated using the PRODRG server [28].
Further, proteins were solvated by SPC216 with Spce-ignh water
model in the triclinic-box size of 1.0 nm distance. The bond angles
and geometry of the water molecules were constrained with
LINCS [29], and SETTLE [30]. The van der Waals and electrostatic
long-range interactions were applied by using fast Particle-Mesh
Weald electrostatics (PME) [31]. Additional, Parrinello-Rahman
[32] method was used to regulate the pressure, whereas modified
weak Coupling Bearsden thermostat and V-rescale algorithm
were used to regulate the temperature of the system. NVT and
NPT were accomplished for 100ps and monitored for their
equilibration status. Finally, the system was subjected to 30ns of
production MD simulation run with a time frame of 2fs [33].
Binding energy calculation was performed by MM-PBSA [34].
FEL analysis was performed to check the quantification of the
trajectory changes. The cosine value less than 0.5 are considered
favourable to generate the good plots of FEL analysis [35, 36].
Results
Virtual screening
The virtual screening workflow consists of three important steps
for good selectivity criteria of the compounds, as first step was
high throughput virtual screening (HTVS), which was followed
by standard precision (SP) docking & finally extra-precision (XP)
docking. The top 10% of the compounds identified in the first
step (23,731-HTVS-2373) were selected and moved to the second
step of the virtual screening workflow and follow the same 10%
for second step for (2,373-SP-237), and once again the same for
10% for (237-XP-23) screening, respectively. Virtual screening
against AChE, three top scoring compounds were screened from
CNS-BBB data with the higher XP glide score and binding free
energy as compared to the known FDA approved Alzheimer's
inhibitor drugs selected for further study shown in Table 1.
Molecular Docking Studies
Among all the docked compounds, three top scoring
Molecule21878, Molecule10520 and Molecule13123 showed good
interaction and binding energy against human-AChE. In order to
understand the binding orientation and non-bonding interaction,
that performed well in docking and MD simulations, this study
were executed and compared with the binding mode of
interaction of the known FDA approved and under trial drugs
ligands. Examine of the binding of approved inhibitor ligand
shown that Tacrine, and Galantamine (both are well-known FDA
approved drugs), have Tyr 133 (2.64 Å) and Ser203 (3.01 Å)
interaction in the cavity of the human-AChE enzyme, and
docking amino acid interactions between ligand and receptor
shown in Figure 2A, 2B and 2C.
Figure 2
Docking view of the M1 (2A), M8 (2B), and M10 (2C) docked ligand in the binding site of protein. Dotted green lines shows
H-bonds in LigPlot view, and ligand in sphere view in the active site cavity of surface view of PyMol.
Post docking analysis
The highest XP Glide score of -15.19 kcal/mol, -13.70 kcal/mol,
and -13.57 kcal/mol were found for molecule21878 (M1),
molecule10520 (M8), and molecule13123 (M10), respectively
shown in Table 1. Whereas, other compounds are also showed
good XP Glide score value with AChE enzymes, but the binding
free energies predictions from MD simulations were also high for
these top three compounds. All the hits compounds were docked
into the active binding site of the protein and the top scoring pose
for ligand was saved for further analysis.
Binding mode of screened compounds
Molecule 21878 (M1)
This compound bind within the active site
of AChE specially in Acyl binding pocket (ABP) and Peripheral
anionic site (PAS) with a XP Glide score -15.19 kcal/mol and
binding free energy -196.36 kJ/mol (Table 1 & Table 2). It
formed three hydrogen bonds with the amino acids Asn87,
Phe295 and Tyr337 with the distance of 3.06Å and 2.85 Å
respectively shown in Figure 2A. Total 14 hydrophobic & 69
Non-bonded interactions were exhibited by the Asp74, Gly82,
Thr83, Trp86, Gly121, Tyr124, Glu202, Trp296, Phe297, Phe338,
Tyr341, Trp439, and His447. Mostly, hydrophobic interactions
were observed in ABP and PAS region with Oxyanionic site,
anionic sites and catalytic triads.
Table 2
Binding free energy of selected ligands against AChE enzyme protein.
Parameters (KJ/mol)
Tacrine
Galantamine
M1
M8
M10
van der Waal energy
-129.14+/14.55
-157.44 +/- 9.36
-226.71+/-10.17
-249.37+/-44.47
-252.75 +/-13.60
Electrostattic energy
-4.76 +/- 4.81
-1.66 +/- 1.74
-18.90 +/- 4.60
-17.55 +/- 6.67
-13.84 +/- 5.47
Polar solvation energy
26.49 +/- 10.94
31.65+/- 9.00
69.73 +/- 10.43
82.62 +/- 10.77
74.75 +/- 14.79
SASA energy
-12.24 +/- 1.45
-14.73+/-0.81
-20.47 +/- 1.00
-19.96 +/- 3.99
-22.55+/- 0.96
Binding energy
-119.65+/14.60
-142.18+/-11.36
-196.36+/-12.19
-204.27+/-50.94
-214.40 +/12.67
Molecule 10520 (M8)
The binding free energy from this
compound was -204.27 kJ/mol shown in Table 2, and XP Glide
score value was -13.70 kcal/mol. One hydrogen bond was
observed with Tyr337 from ABP site with the distance of 2.82 Å
shown in Figure 2B. In addition, 12 hydrophobic and 92 Nonbonded
interactions were evolved in stabilizing the complex.
Amino acids Tyr72, Asp74, Trp86, Gly121, Gly122, Trp124,
Ser203, Trp286, Phe297, Phe338, and Tyr341 were involved in
several hydrophobic interactions within ABP, catalytic triad, PAS
and Oxyanion site shown in Figure 2B.
Molecule 13123 (M10)
This compound made a complex with XP
Glide score value of -13.57 kcal/mol, and has a binding free
energy -214.40 kJ/mol calculated from g_MMPBSA shown in
Table 2. This compound was found to have one H-bond with
Tyr337 has distance with 3.18 Å shown in Figure 2C. Moreover,
complex was also stabilized with 14 hydrophobic and 76 Nonbonded
interactions with residues Tyr72, Asp74, Trp86, Gly121,
Gly122, Tyr124, Ser203, Trp286, phe297, Phe338, Tyr341, Trp439,
and His447. Compounds ID & IUPAC name list with detailed
shown in (Table 6), and all combined docking related poses of
M1, M8 and M10 are shown in (Figure 7,
Figure 8, Figure 9) respectively.
Table 6
CNS-BBB, (ChemDiv database) Molecule Name with ID Number, &IUPAC Name
Docking pose view of the M1 ligands and their amino acid interactions in the active/binding site of the h-AChE enzyme,
views are in LigPlot, Ligand with receptor, Ligand in active site of the gorge in surface view in (lavender-color) & full surface view in
(cyan-color).
Figure 8
Docking pose view of the M8 ligands and their amino acid interactions in the active/binding site of the h-AChE enzyme,
views are in LigPlot, Ligand with receptor, Ligand in active site of the gorge in surface view in (lavender-color) & full surface view in
(cyan-color).
Figure 9
Docking pose view of the M10 ligands and their amino acid interactions in the active/binding site of the h-AChE enzyme,
views are in LigPlot, Ligand with receptor, Ligand in active site of the gorge in surface view in (lavender-color) & full surface view in
(cyan-color).
Molecular dynamics and post-dynamic analysis
On the basis of best-docked ligand enzyme complexes that
resulted compounds were them to expose further to MD
simulations using the GROMACS 5.1.2 package [33]. And
followed the procedure explanations in our computational
methodology part of this manuscript. Here, we examine the postdynamic
nature of the how ligand interacted with the h-AChE
target receptor within a range as of 5Å, depicted by H-bonds and
Hydrophobic interactions using LigPlot (v.1.4.5) and PyMol
version of Schrodinger (v.1.3). And other graphs were prepared
by used Xmgrace tools. In our study, MD simulations of 30ns
were performed for the top ten, score ligand-complexes shown in
Figure 3, to ensure the stability of the ligand within the h-AChE
active site asCAS, PAS and Oxyanions sites. Here, interestingly
for all the ligand-enzyme complexes the average RMSD values
were coming to the range between 1.5Å to 3.50Å, in addition,
the gyrate or radius of gyration is showed good compactness in
the range between 2.32 nm to 2.27 nm, and also RMSF graph
shows well compare to the reference ligands, fluctuations is not
much observed in hit complexes, H-bonds shown in Figure 3D,
and H-bonds with H-bond-Occupancy shown in (Table 4). We
took our study ahead by obtaining a top three CNS-BBB chemDiv
compounds out of 23,731, through virtual screening, these are
identified leads have the propensity to be considered as a
potential Human-AChE anti-Alzheimer's compounds. All the
insilico information gained from this study could be shed light on
the new series of lead compounds as potential h-AChE inhibitors.
Figure 3
Molecular dynamics simulations Trajectory-graph for (A) RMSD, (B) RMSF, Gyrate (C), and H-bonds (D), for all three hit
compounds along with both references (Tacrine & Galantamine). The time period scale used is 30ns.
Molecular dynamic simulations analysis
A 30ns of molecular dynamic simulations was performed for
each complex to access the stability of the enzyme-ligand
complexes shown in Figure 3A, the root mean square deviation
(RMSD) value of the AChE-Ligand complex over the simulation
of time. RMSD plot shown stable backbone trajectories of M1
(average b/w 2.25 Å -2.30 Å), M8 (average b/w 1.5 Å - 2.0 Å),
and M10 (average b/w 2.20 Å - 2.30 Å ) as compared to
reference h-AChE+Tacrine (R1) (average 3.5 Å). Whereas
binding of second reference h-AChE+Galantamine (R2) (average
2.48 Å), have shown slightly stable trajectories comparatively
than R1. Here, it can be concluded that these molecules, M1, M8,
and M10 are the most preferable compounds for h-AChE
inhibitors. In the root mean square fluctuations (RMSF) profile,
five to six peaks observed high, but one high peak was found
between the residues 380-390 where it was observed that only M8
restricted little movement, while in all other ligand and R1 and
R2 ( reference ligand) complexes, high fluctuations was observed
in this region shown in Figure 3B. Here, our results suggested
that h-AChE enzyme and selected hit compounds were able to
maintain their structural integrity during the simulations. In the
radius of gyration of an object describes its dimension, calculated
as root mean square distance between centre of gravity and its
ends. Radius of gyration is indicative of level of compaction in
the structures. In context of Radius (Rg) value is measure of the
compactness of a protein complex, radius is a measurement of the
stability of the folded protein. Radius of the initial starting
structure was 2.32 nm and the value goes too decreased to 2.27
nm at the end of the 30 ns and MD simulations showed that
protein ligand complexes were stable and well folded shown in
Figure 3C. In h-bond analyses profile of h-AChE docked systems
showed consistent h-bond trajectory in M1, M8, and M10 average
2-5 h-bonds were found throughout the trajectory, where in case
of reference R1, one h-bond found throughout the time scale
shown in Figure 3D. On the basis of MD simulation studies,
among the three screened compounds, compound M10 have
shown highest binding free energy against h-AChE, with -214.40
kJ/mol. Shown in Table 2.
Free Energy Land Scape (FEL) Analysis
In free energy land scape analyses, all the possible state of
minimum energy conformations that a protein can adopt during
simulation has been studied. This analysis is based on Gibbs free
energy. In the lowest energy conformations of Tacrine, and
Galantamine (as reference ligand inhibitors) and top three
binding energy ligated complexes M1, M8 and, M10 were
retrieved and their interactions were analysed. Where, Tacrine-
AChE, and Galantamine-AChE maintained its H-bonds and ∏-∏
interactions with Ser125 H-bond side chain in Tacrine, and Trp86
have two ∏-∏ interactions, and in GalantamineTyr133 and Ser203
have H-bonds, respectively. Moreover, hydrophobic interaction
with Asp74, Asn87, Glu202 and His447. Further, in M1 residues
Asn87, Phe295, and Tyr337 maintained H-bonds with Trp86 have
one ∏-∏ interactions, and other formations like hydrophobic with
Thr83, Glu203, and His447. In M8, residues Tyr337 maintained Hbond,
and Trp286 with Tyr341 have ∏-∏ stacking interactions, also
Asp74, Gly120, Gly121, and Glu203 were involved in
hydrophobic interaction, and finally M10, residue Tyr337 shows
H-bond interaction with Tyr124 have one ∏-∏ stacking
interactions, and Asp74, Gly121, Gly122, Gly448, Ser203 and
His447 shows hydrophobic interaction observed shown in Figure 4. Thus, the FEL analysis conveyed the importance of residues
Asn87, Ser125, Phe295, and Tyr337 are forming hydrogen bonds
in the binding site of the gorge with Trp86, Tyr124, Trp286, and
Tyr314 shows ∏-∏ stacking interactions. All the figures of
receptor-ligand complexes by depicted by PyMol v.1.3 [37], and
LigPlot v.1.4.5 [38].
Figure 4
Two dimensional FEL Plots of top three scoring ChemDiv hits ligands (A) M1, (B) M8, and (C) M10 retrieved at 21, 10 and
13ns, respectively, and Ligand Interaction with residues (i, ii, and iii) along with surface view poses, respectively.
Discussion
Based on this CNS-BBB, Standard Data Format (SDF) data,
provided by the ChemDiv Data base server, finding the top three
ligand inhibitors as M1, M8, and M10 shows a good G-Score
value, H-Bonds, and ∏-∏ interactions, hydrogen bond occupancy
and binding energy with MM-PBSA, Free energy land scape, and
residue Tyr337 is playing an important role interacting to all top
hits compounds with the CAS and PAS active site, and hydrogen
bond occupancy shows a good % (percentage) of H-bonds
contributions shown in (Table 4) in interaction analysis by MD
simulation studies. In context of the Tacrineas Cognex (year,
1993), was the first FDA approved AChEI, but unfortunately, it
has toxicity towards the liver etc. [5], others like Donepezilas
Aricept (year, 1996), [6] second anti-Alzheimer's drug,
Rivistigmineas Exelon (year, 2000), [7], Galantamineas Nivalin
(year, 2001), [8], all are based on cholinergic hypothesis and
Memantine was approved (year, 2003), and it is based on
amyloid hypothesis, since then no new treatments have been
developed for Alzheimer's. Glide score of the top three ligands
M1, M8, and M10 are -15.19 kcal/mol, -13.70 kcal/mol, and -13.57
kcal/mol, respectively. While binding free energies (MM-PBSA)
for reference ligand Tacrine is -119.65 kJ/mol, Galanatamine is -
142.18 kJ/mol, and M1 have -196.36 kJ/mol, M8 have -204.27
kJ/mol, and M10 have -214.40 kJ/mol, respectively a good score
value of free energy, comparatively to the reference ligand, and
follows the Lipinski rule of five criteria, so all of them indicate
that they can be potential active inhibitors. In the light of the MD
simulations results of the Binding free energy calculations,
hydrogen bonds, ∏-∏ stacking and physiochemical properties, we
conclude that the ligand M1, M8, and M10 to be a potential h-
AChE inhibitors.
Conclusion
We used virtual screening, molecular docking and, MD
simulations analysis, and FEL analysis to identify inhibitors
against AChE. Compounds were ranked based on glide score and
their binding mode. H-bonds interactions, and Binding free
energy calculations with Free energy landscape analyses were
also reported. We found that all the top ranked molecules
interacted with the catalytic triad, peripheral anions site, Anionic
sub-site, Oxyanion site, and Acyl binding pocket, of the h-AChE
enzyme region with aromatic amino acid residues asTyr124, and
Tyr337, And Phe295, Asn87 residues are the key residues
involved in the binding interactions. We document the top three
molecules ligands as M1, M8, and M10, as potential h-AChE
inhibitors based on MD simulations, binding free energy and
physiochemical properties.
Authors: Richard A Friesner; Jay L Banks; Robert B Murphy; Thomas A Halgren; Jasna J Klicic; Daniel T Mainz; Matthew P Repasky; Eric H Knoll; Mee Shelley; Jason K Perry; David E Shaw; Perry Francis; Peter S Shenkin Journal: J Med Chem Date: 2004-03-25 Impact factor: 7.446
Authors: Richard A Friesner; Robert B Murphy; Matthew P Repasky; Leah L Frye; Jeremy R Greenwood; Thomas A Halgren; Paul C Sanschagrin; Daniel T Mainz Journal: J Med Chem Date: 2006-10-19 Impact factor: 7.446
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