Discovery of dual cation-π inhibitors of acetylcholinesterase: design, synthesis and biological evaluation

Article abstract of DOI:10.1007/s43440-020-00086-2

Background: Alzheimer's disease (AD) is a widespread dementia-related disease affecting mankind worldwide. A cholinergic hypothesis is considered the most effective target for treating mild to moderate AD. Present study aims to identify new scaffolds for

Full text of DOI:10.1007/s43440-020-00086-2

Pharmacological Reports
https://doi.org/10.1007/s43440-020-00086-2
ARTICLE
Discovery of dual cation‑π inhibitors of acetylcholinesterase: design,
synthesis and biological evaluation
Naresh Damuka · Kurumurthy Kammari · Angamba Meetei Potshangbam · Ravindranath Singh Rathore3 ·
1
1
1,2
1
1
Anand K. Kondapi · Vaibhav Vindal
Received: 18 May 2019 / Revised: 18 February 2020 / Accepted: 25 February 2020
Maj Institute of Pharmacology Polish Academy of Sciences 2020
©
Abstract
Background Alzheimer’s disease (AD) is a widespread dementia-related disease afecting mankind worldwide. A choliner-
gic hypothesis is considered the most efective target ꢀor treating mild to moderate AD. Present study aims to identiꢀy new
scafolds ꢀor inhibiting acetylcholinesterase activity.
Methods To ꢁnd Acetylcholinesterase (AChE) inhibitors, we computationally designed and chemically synthesized a series
oꢀ cation-π inhibitors based on novel scafolds that potentially block AChE. The cytotoxic efect oꢀ inhibitors were deter-
mined by MTT. AChE inhibition experiment was perꢀormed by Ellman and the Amplex red method in the SH-SY5Y cell
line. Further, the experimental data on designed compounds corroborate with various computational studies that ꢀurther
elucidate the binding mode oꢀ interactions and binding aꢂnity.
Results The inhibitors were designed to promote dual binding and were incorporated with groups that may ꢀacilitate any oꢀ
the cation- π, hydrophobic and hydrogen-bonding interactions with the conserved and hot-spot residues in the binding site.
The inhibitors possessing pyridine-N-methylated pyridinium group and thereby involved in cation- π interactions are highly
active relative to the marketed drug Donepezil as well as the designed analogs that lack the group. In vitro enzymatic Ellman
assay and Amplex red assay on SH-SY5Y cell line estimated IC oꢀ the designed compounds in nM range with one having
5
0
binding aꢂnity higher than Donepezil. Compounds exhibit no signiꢁcant toxicity up to µM range.
Conclusions Compounds possessing methylidenecyclohexanone scafolds, with characteristic dual-binding and involving
strong cation-π interactions, serves as new leads ꢀor AChE and opens a new direction ꢀor drug discovery eforts.
Keywords Drug design · 2,6-dimethylenecyclohexanone · AChE · Cation-π interactions
Introduction
Alzheimer׳s disease (AD) is characterized by a progressively
degenerative disorder in the brain, having multiꢀactorial
syndromes such as memory loss, progressive impairment in
cognitive ꢀunctions and behavioral impairment.
Electronic supplementary material The online version oꢀ this
article (https://doi.org/10.1007/s43440-020-00086-2) contains
supplementary material, which is available to authorized users.
Several studies have been undertaken to comprehend the
molecular pathogenesis oꢀ AD. However, the most signiꢁ-
cant outcome ꢀrom these studies regarding the pathogenesis
oꢀ AD, and which are implicated in the drug development. In
AD, the main pathological ꢀeatures are grouped into the ꢀol-
lowing categories such as genetic ꢀactors [1], aggregation oꢀ
beta-amyloid in senile plaques [2], neuron-immune dysꢀunc-
tion [2, 3], cerebrovascular dysꢀunction [4] and cholinergic
neurotransmission [5, 6]. Oꢀ all these pathogenic events, the
cholinergic neurotransmission also known as a cholinergic
hypothesis is considered as the utmost efective target ꢀor
*
Vaibhav Vindal
vaibhav@uohyd.ac.in
1
2
3
Department oꢀ Biotechnology and Bioinꢀormatics,
University oꢀ Hyderabad, Hyderabad 500046, India
Department oꢀ Biotechnology, Manipur University,
Canchipur, Imphal, Manipur 795003, India
Department oꢀ Bioinꢀormatics, School oꢀ Earth, Biological
and Environmental Sciences, Central University oꢀ South
Bihar, Gaya 824236, India
Vol.:(0
1
123456789)
3

N. Damuka et al.
treating AD. The cholinergic hypothesis is based on the
observation that AD patients showed a loss oꢀ cholinergic
activity in their brain. Thereꢀore, it was postulated that the
cognitive weakening experienced by AD patients is due to
the lack oꢀ acetylcholine resulting in ineꢂcient cholinergic
neurotransmission. Hence, the inhibition oꢀ Acetylcholinest-
erase (AChE) was considered the most natural therapeutic
strategy to reverse the acetylcholine deꢁciency in AD. Four
cholinergic AChE inhibitors such as donepezil, galantamine,
rivastigmine, and tacrine are commonly prescribed ꢀor the
symptomatic treatment oꢀ AD.
ꢀor designing new drugs. The X-ray crystal structures oꢀ
AChE with diverse inhibitor complexes revealed signiꢁcant
insight into structural ꢀeatures critical ꢀor designing new
drug molecules.
In this study, we have designed a series oꢀ inhibitors based
on novel scafolds that potentially block AChE in dual bind-
ing ꢀashion incorporating ꢀunctional groups that promote
cation-π, hydrogen bonding and hydrophobic interactions
with the conserved and hot-spot residues.
The active pocket oꢀ AChE consists oꢀ two regions the
Materials and methods
‘esteratic’ and ‘anionic’ subsites [7]. The esteratic sites ꢀorm
the catalytic machinery oꢀ the enzyme, while the anionic
regions are involved in guiding the acetylcholine (ACh)
substrate ꢀor binding to the conserved residues [8]. At the
esteratic subsite, the substrate undergoes hydrolysis by the
catalytic triads consisting oꢀ Ser203, His447 and Glu334
residues [9]. Additionally, AChE possesses several subsites
such as Peripheral Anionic Subsite (PAS) [10, 11] compris-
ing oꢀ Asp74, Tyr124, Ser125, Trp286, Tyr337 and Tyr341,
oxyanion hole [12] (Gly121, Gly122, Ala204), anionic sub-
site [13] (Trp86, Tyr133, Glu202, Gly448, Ile451) and acyl
binding pocket [14] (Trp236, Phe295, Phe297, Phe338).
PAS provides a transitional binding site oꢀ the ACh sub-
strate [15–17]. The residue Trp286 plays a critical role in
ligand binding using a combination oꢀ steric and electro-
static blockade via the gorge by altering the catalytic rate
and catalytic triad conꢀormation [18]. The anionic pocket is
located next to the catalytic site, where the conserved residue
Trp86 plays a very signiꢁcant role in guiding the substrate
to the active site [14, 19]. The acyl binding pocket binds to
the acetyl group oꢀ AChE is ꢀormed by the residues Phe295,
Trp236, Phe297 and Phe338 [14]. The positively charged
quaternary ammonium group oꢀ the substrate ꢀorms cation-π
interactions [14]. These interactions lead to substrate recog-
nition and catalytic mechanism oꢀ the enzyme [9].
The Ala scan was perꢀormed on AChE-Donepezil complex
(
PDB ID: 4EY7) using the Residue Scanning module in
Bioluminate (Schrödinger). The method calculates relative
binding aꢂnity values (Δ G ) between the mutant and
bind
the wild type using implicit solvation based MM-GBSA
approach and stability (ΔG
) is calculated using the
bind
stability
thermodynamic cycle approach. ΔG
or Δ Aꢂnity is the
change in binding aꢂnity between binding partners upon
mutation. A negative value reꢀers that the mutant binds bet-
ter. The calculations were carried out using the Schrodinger
Prime program with an implicit solvent term [23]. ΔG
stability
or Δ Stability (solvated) is the change in the stability oꢀ the
protein upon mutation, calculated using the Prime energy
ꢀ
unction with an implicit solvent term. The stability was
deꢁned as the diference in ꢀree energy between the ꢀolded
state and the unꢀolded state. A negative value reꢀers that the
mutant is more stable [23].
Chemical synthesis and characterization
All reagents and solvents were obtained ꢀrom Aldrich and
Merck and were spent on chemical synthesis and characteri-
13
1
zation. The C-NMR and H-NMR spectrum were recorded
at Bruker 500 MHz and 400 MHz spectrometer, respectively.
The chemical shiꢀts are reported in parts per million (ppm).
Tetramethylsilane (TMS) and DMSO-d6 used as an inter-
nal standard and solvent, respectively. Platinum-ATR-IR,
Bruker spectrometer, Germany managed by Bruker OPUS
7.0 soꢀtware, was used to record Inꢀrared spectra (IR), in the
AChE is also involved in several noncatalytic activities
[20, 21], ꢀor example, the development oꢀ AD plaque by pro-
moting Aβ accumulations [15]. Further, it has been reported
that AChE inhibitors capable oꢀ interacting with both the
catalytic sites and PAS were able to prevent Aβ accumula-
tion [15, 22]. Thereꢀore, dual binding AChE inhibitors may
provide efective treatment oꢀ AD, eventually leading to the
development oꢀ Donepezil and other drugs currently preva-
lent in the market.
−
1
range oꢀ 500–4000 cm . Approximately 2 mg oꢀ the com-
pound was used ꢀor spectra recording and absorbance was
generated by signal averaging 64 scans with a resolution oꢀ
−
1
Despite the availability oꢀ Rivastigmine (Exelon),
Memantine (Namenda), Galantamine (Razadyne), Done-
pezil (Aricept) and Tacrine, the prolonged treatment, multi-
ple side efects and various studies questioning the sustained
eꢂcacy oꢀ these drugs necessitated exploration oꢀ a new set
oꢀ potent and efective drugs. Towards this direction, the
knowledge oꢀ protein–ligand interactions could be exploited
4 cm and the spectra were obtained as wave number ver-
sus absorbance units. High-resolution mass spectra (HRMS)
were noted on micromass ESI-TOF MS. TLC was done on
percolated silica gel plates (Kieselgel 60,254, E.Merck, Ger-
many). Chromatograms were analyzed by UV at 254 nm and
365 nm, ꢀollowed by iodine vapors. Koꢃer hot stage device
was used to analyse melting points.
1
3

Discovery of dual cation‑π inhibitors of acetylcholinesterase: design, synthesis and…
General method for the chemical synthesis of compounds
–3
water. Each oꢀ the molecules was diluted to various con-
1
centrations immediately beꢀore use. Donepezil was used as
a positive control. Ellman’s modiꢁed colorimetric method
was used to evaluate AChE inhibitory activity [27]. The
assay procedure contained sodium phosphate bufer (0.1 M),
DTNB (0.3 mM), AChE enzyme ꢀrom Electrophorus elec-
tricus (0.01 U) and ATCI (0.5 mM). Various concentrations
were evaluated ꢀor each chemically synthesized compound
in triplicate to get the range oꢀ 15% and 90% inhibition ꢀor
AChE. Each tested compounds were mixed to the assay solu-
tion and incubated at 37 °C ꢀor 10 min. Aꢀter that period,
ATCI substrate was added. The rate oꢀ absorbance change
was measured at 405 nm ꢀor 5 min on a microplate reader
Tecan inꢁnite M 200 pro. The values oꢀ IC were calculated
The chemical synthesis was perꢀormed as previously
described [24]. Brieꢃy, Pyridine aldehyde (4 mmol) and
cyclohexanone (2 mmol) were mixed in 20 ml distilled
water. The solution was then stirred in 2 ml oꢀ ice-cold
Sodium hydroxide (10%) ꢀor 10 h. Further, the solution was
neutralized with dilute HCl leading to the ꢀormation oꢀ a
precipitate that was ꢁltered careꢀully. Ethanol was used to
recrystallize the precipitate and the pure compound was
recovered by column chromatography using ethyl acetate/
hexane solution [25].
50
General method for the chemical synthesis of compounds
ꢀrom the absorbance at 405 nm.
1A, 2A and 3A
MTT assay
The compounds were synthesized as per the
protocol described elsewhere [26]. Brieꢀ ly,
To investigate cellular toxicity, 10,000 SH-SY5Y cells were
cultured in 96-well plate. Aꢀter 24 h, diferent concentra-
tions oꢀ test ligands were added and maintained at 37 °C
2
,6-Bis(pyridinylmethylene)cyclohexanone (2 mmol) deriv-
atives were dissolved in acetonitrile (10 ml) and immediately
cooled to 5 °C in a 25 ml round bottom ꢃask. 5 ml oꢀ Methyl
Iodide solution was injected into the ꢃask using a syringe.
The entire mixture was stirred ꢁrst at 5 °C ꢀor 12 h, ꢀollowed
by RT ꢀor 72 h. The compound precipitate was ꢁltered and
washed with little acetonitrile.
and 5% CO ꢀor 24 h. Aꢀter the test ligands treatment, the
2
remaining media, including test compounds, were discarded.
Again 100 μl oꢀ media and 20 μl oꢀ 5 mg/ml MTT solution
were added to each well and ꢀurther maintained ꢀor 4 h at
37 °C. The media was careꢀully discarded and 200 μl oꢀ
DMSO was added. MTT ꢀormazan product was ascertained
by calculating absorbance with an ELISA reader at 570 nm
wavelength by Tecan inꢁnite M 200 pro. The untreated cells
were deꢁned as 100%.
In vitro activity and toxicity Assays
Chemicals
F12 medium, Eagle’s minimum essential medium (EMEM),
pen-strep solution and FBS, were obtained ꢀrom Gibco-
Thermo Fisher Scientiꢁc. The Amplex® Red Acetylcholine/
Acetylcholinesterase assay kit (A12217) was obtained ꢀrom
Thermo Fisher Scientiꢁc. Acetylcholinesterase (AChE),
Amplex red cellular assay
Amplex red Acetylcholinesterase inhibition assay was
evaluated by using the Amplex® Red Acetylcholine/Ace-
tylcholinesterase Assay Kit (A12217) method obtained
ꢀrom Thermo Fisher Scientiꢁc. Brieꢃy, 6000 viable SH-
SY5Ycells were seeded into black 96 well plate and main-
tained at RT with a 200 µl reaction mixture possessing
100 μM ACh, 50 μM Amplex Red; 0.25 U/mL choline oxi-
dase, 1.0 U/mL horseradish peroxidase. The rate oꢀ Fluo-
rescence oꢀ resoruꢁn (λex= 544 nm; λem = 590 nm) was
calculated at 60 min with a microplate reader Tecan inꢁnite
M 200 pro.
5
,5-Dithiobis-2-nitrobenzoic acid (DTNB) and acetylthi-
ocholine iodide (ATCI). MTT and resoruꢁn sodium salt
were procured ꢀrom Sigma-Aldrich. Dipotassium hydrogen
phosphate (K HPO ) and Potassium dihydrogen phosphate
2
4
(
KH PO ) were purchased ꢀrom Molychem.
2 4
Cell culture
SH-SY5Y neuroblastoma cell vial was taken ꢀrom NCCS
(
National Center ꢀor Cell Science, Pune, India). The SH-
In silico study
SY5Y cells were cultured in a 45% EMEM medium, F12
medium (45%) and 10% FBS, at 37 °C, 5% CO
Molecular docking
2
.
Enzymatic Ellman’s assay
Molecular docking investigation was done using Glide
program [28] oꢀ Schrödinger Suite. Protein structure com-
plex was pre-processed beꢀore docking study by Protein
Preparation Wizard. Since no conserved water molecules
Compounds 1, 2, 3 were dissolved in dimethyl sulꢀoxide
DMSO) and 1A, 2A, 3A compounds were prepared in
(
1
3

N. Damuka et al.
were observed in the crystal structure water molecules
were completely deleted. To maintain the suitable ioniza-
tion states ꢀor the basic and acidic amino acid residues,
hydrogen atoms were included at pH 7.0. Further, the
maximum possible positions ꢀor − SH and − OH hydro-
gen atoms were selected and proper assignment oꢀ the
protonation states and tautomers oꢀ His as well as Chi
Molecular dynamic simulation
The molecular dynamics simulations oꢀ the Apoenzyme
and AChE-inhibitor complexes were done using Desmond
[29]. OPLS-AA 2005 ꢀorce ꢁeld was used in molecu-
lar dynamics simulations [29, 30]. The apoenzyme and
complexes were solvated with TIP3P water model as the
protein and ligands were already prepared in the dock-
ing process. An orthorhombic periodic boundary box was
chosen ꢀor subsequent MD simulations. To preserve charge
neutrality oꢀ the system, proper numbers oꢀ counter ions
were included. The distance between the wall and box was
ꢁxed greater than 10 Å to prevent direct binding with its
own protein complex. The steepest descent process was
implemented to reduce the potential energies oꢀ the sys-
tems by setting greater than 5000 steps with a 25 kcal/
mol/Å gradient threshold. This ensued through L-BFGS
minimizer until 1 kcal/mol/Å convergence criteria were
attained.
‘
ꢀlip’ ꢀor His, Gln and Asn amino acid residues were also
perꢀormed. RMSD (Root mean square deviation) value
oꢀ 0.30 Å was taken to avoid steric collisions among the
residues. The OPLS-2005 ꢀorce ꢀield was used ꢀor energy
minimization.
The inhibitors were made using the Maestro builder
panel. 3D structures were made using the Ligprep unit
in Schrödinger. The OPLS-2005 ꢀorce ꢀield was used
ꢀ
or computing partial atomic charges. The correct bond
length and angles, as well as chiralities, tautomers, stereo
chemistries, ring conꢀormation and ionization state at pH
7
.0, were obtained using Ligprep. To generate energy-
minimized 3D structures, the OPLS-2005 ꢀorce ꢀield was
used with 0.01 Å RMSD cutoꢀꢀs.
Deꢀault parameters provided in Desmond were used ꢀor
pressure and temperature equilibrations. The MD simula-
tions were carried out ꢀor 50 ns at 300 K constant tem-
perature and 1 atm constant pressure with a 2 ꢀs time step
oꢀ on the equilibrated systems. Particle-mesh Ewald PME
method (particle-mesh Ewald) [31] was used to give 0.8 Å
grid spacing ꢀor long-range electrostatic interactions.
Short-range electrostatic and Van der Waals interactions
were eꢂciently shortened at 9.0 Å.
Molecular docking oꢀ the designed inhibitors was per-
ꢀormed on the human AChE crystal structure in complex
with Donepezil (PDB ID: 4EY7 resolution, 2.35 Å). There
was 88% sequence identity and an RMSD oꢀ 0.394 Å at
the binding site (within a distance oꢀ 5 Å ꢀrom ligand
binding site) (supplementary Fig. 1) with the other PDB
ID (1C2O, 1C2B and 1EEA ꢀrom Electrophorus electri-
cus) having a resolution oꢀ 4.2–4.5 Å.
Grid box was generated around the co-crystallized
drug molecule oꢀ AChE (PDB ID: 4EY7) active site with
a grid box size oꢀ 20 × 20 × 20 Å. The van der Waals scal-
ing was kept as deꢀault and the partial charges were given
Binding free energy
The Prime MM-GBSA technique, implemented in
Schrödinger [23, 32], was used to analyze the ꢀree energy
oꢀ the simulation trajectories oꢀ the complexes. The bind-
ing energy was estimated ꢀor an entire oꢀ 100 ꢀrames oꢀ
the MD trajectory. The calculation oꢀ the MD trajectory
started ꢀrom the last 25 ns stable simulation until the ꢁnish
oꢀ the trajectory. According to the ꢀollowing equation, the
binding ꢀree energy was estimated.
ꢀ
rom the ꢀorce ꢀield. All the designed inhibitors interacted
into the active pocket described by the Grid generation
protocol implementing Glide XP (extra precision). Glide
scoring ꢀunction is used to estimate the binding aꢀꢀinity oꢀ
protein–ligand docking poses. The ꢀunnel-type approach
was included in the glide algorithm to carry out an eꢀꢀi-
cient search oꢀ orientations, conꢀormations, and positions
oꢀ the ligand in the active site oꢀ the protein. Maestro’s
pose viewer was used to analyze the output ꢀiles.
ΔGBind = ΔEMM + ΔGSolv + ΔGSA
Re-docking, the extracted co-crystallized drug ꢀrom the
binding site serves as one way oꢀ validating the reliabil-
ity oꢀ the molecular docking procedure, implemented in
the present study. The RMSD between the co-crystallized
Donepezil and the binding orientation obtained ꢀrom its
re-docking into AChE active site was ꢀound to be less
than 1 Å. It results in a similar binding pose as shown in
supplementary Fig. 2, validating the reproducibility oꢀ
the docking procedure.
•
•
•
∆E
is the energy change between the ligand–protein
MM
complexes and sum oꢀ the energies oꢀ the protein and
ligand.
∆G
is the change in GBSA solvation energies oꢀ the
Solv
ligand–protein complex and the sum oꢀ the solvation
energies ꢀor the protein and unbound ligand.
∆G is the change in surꢀace area energy ꢀor the
SA
ligand–protein complex and sum oꢀ the surꢀace area
energies ꢀor protein and ligand.
1
3

Discovery of dual cation‑π inhibitors of acetylcholinesterase: design, synthesis and…
Statistical evaluation
inhibitors without positive ions were prepared ꢀollowing
the procedure in Scheme 1 routine. Compounds 1–3 were
prepared by aldol condensation oꢀ various pyridine alde-
hydes with cyclohexanone in the presence oꢀ 10% aqueous
sodium hydroxide. Compound 1A–3A was prepared by pyri-
dine N-methylation oꢀ Scheme 1 products in the presence oꢀ
methyl iodide, as depicted in Scheme 2.
The IC inhibitory concentrations were ascertained using
5
0
GraphPad Prism soꢀtware v. 7 by studying the log oꢀ the
dose–response curves by nonlinear regression analysis.
Results
The new type oꢀ proposed inhibitors are shown in Table 3.
The biological activities oꢀ each oꢀ the molecules were eval-
uated by Ellman’s and amplex red biological assay. Further,
molecular docking and molecular dynamics studies were
held to understand the molecular mechanism oꢀ the binding
interaction oꢀ the inhibitors at the AChE active site.
Hot‑spot residues in binding site and design
of cation‑π inhibitors
To design the new series oꢀ inhibitors, initially, hot-spot
residues were identiꢁed using the ALA scanning approach
implemented in Schrödinger-Bioluminate [23]. The quan-
titative assessment oꢀ the contribution oꢀ each residue to
the binding aꢂnity and stability is shown in Table 1. A
residue was considered a hot-spot residue iꢀ its mutation to
alanine yielded loss in binding aꢂnity greater than 2 kcal/
mol [33]. The substantial losses oꢀ aꢂnity upon ALA muta-
tions were observed ꢀor residues Trp286, Tyr341, Phe338
and Trp86 (supplementary Fig. 3). These hot-spot residues
were targeted primarily ꢀor designing the new inhibitors.
Further, the X-ray structure oꢀ AChE-ACh complexes (PDB
ID: 2HA5 and 2HA4) revealed that the quaternary ammo-
nium oꢀ acetylcholine not only has cation-π interaction with
Trp86 at the anionic site but also with Trp286 at the PAS
as shown in supplementary Fig. 4. This signiꢁes the criti-
cal role oꢀ Trp86 and Trp286 in enzyme catalytic activity
through cation-π interactions. Another important criterion
that we considered ꢀor designing the new cation-π inhibitors
was inspired by the characteristic dual binding drug such as
Donepezil, which mainly interacts with the enzyme through
hydrophobic groups. Accordingly, the two ends oꢀ the inhibi-
tors were prepared using pyridine-N-methylated pyridinium
moiety, thereby introducing positive group as well as the
hydrophobic moiety. The linker group prominently as hydro-
phobic was constructed with 2,6-dimethylenecyclohexanone
moieties. This group is intended to bind with the Acyl bind-
ing pocket oꢀ the enzyme.
Molecular activity
To determine the inhibitory efect oꢀ the designed and syn-
thesized novel type ligands, In vitro AChE inhibition assays
were perꢀormed according to Ellmann’s method. Donepezil
was utilized as a reꢀerence compound ꢀor the inhibitory eval-
uation. The initial screening oꢀ the AChE inhibitory efects
oꢀ the synthesized compounds was perꢀormed at 100 µM
concentration. Compounds 1, 2, 3 have ꢀailed to exhibit
AChE inhibitory activity, as shown in Fig. 1.
The lack oꢀ inhibitory activity oꢀ chemically synthesized
compounds 1, 2 and 3 is attributed to the lack oꢀ positive
charge group in pyridinyl moieties, which is necessary ꢀor
cation-π interactions at the enzyme active site. However,
when the positive group is introduced in compound 1A, 2A
and 3A, the inhibitory activity was observed in the nanomo-
lar range. The quaternary ammonium at the Para position oꢀ
compound 1A has IC =20.9 nM, arguably due to cation-π
5
0
Overall, inhibitors have pyridine-N-methylated pyridin-
ium moiety similar to acetylcholine quaternary ammonium
moiety. It also possesses aromatic groups at both ends and
a linker group in the middle. To understand the critical role
oꢀ positive ions in the molecule, a series oꢀ inhibitors similar
to the cation- π inhibitors were also designed, without the
positive group.
Synthetic chemistry
Fig. 1 The efect oꢀ compounds 1–3 and 1A–3A on AChE inhibitory
activity. All experiments on three diferent test materials were con-
ducted at the same time using Donepezil as a single positive control.
Error bars represent mean± SD; n=3
The pyridine-N-methylated oꢀ 2,6-bis(pyridinylmethylene)
cyclohexanone derivatives were prepared by implement-
ing the synthetic route, as shown in Schemes 1 and 2. The
1
3

N. Damuka et al.
interactions with the conserved residues Trp86 and Trp286
at the enzyme active site. Compound 2A with the Meta
positioned quaternary ammonium has the highest activity
Table 1 Relative binding aꢂnity and stability between wild type and
mutated residues
Sl. No
Residuea
Mutatedb
∆Aꢂnityc
∆Stability
(solvated)d
(
IC =6.2 nM), again due to proximity and thereby strong
50
interaction with the conserved residues (Trp86 and Trp286).
The quaternary ammonium at ortho position (in compound
1
2
3
4
5
6
7
8
9
Trp286
Tyr341
Phe338
Trp86
ALA
ALA
ALA
ALA
ALA
ALA
ALA
ALA
ALA
ALA
ALA
ALA
ALA
ALA
ALA
ALA
ALA
ALA
ALA
11.55
10.18
5.39
3.96
3.12
2.87
2.10
2.01
1.33
1.05
0.74
0.21
0.17
11.60
11.67
12.10
12.63
9.39
14.01
6.23
9.54
10.72
0.21
12.27
18.53
29.15
13.06
0.91
21.78
11.22
-3.41
-8.99
3
A), wherein the cation group ꢀaces the opposite side oꢀ π
moiety in Trp86 and Trp286 ꢀails to participate in cation- π
interaction leading to lesser activity.
Tyr72
In addition to Ellmann’s method, which was carried out
in the presence oꢀ a pure enzyme, the potency oꢀ the 1A, 2A,
and 3A were also assessed by Amplex red assay in neuronal
SH-SY5Y cell lines. In the amplex red assay, AChE activity
is monitored by highly ꢃuorescent product resoruꢁn, which
is easily measured in ꢃuorescence emission spectroscopy.
According to Amplex red AChE inhibitory assay, 1A and
Phe297
Tyr337
Tyr124
Ser203
His447
Val294
Ile451
Arg296
Leu289
Ser293
Tyr133
Phe295
Asp74
Glu202
10
11
12
13
14
15
16
17
18
19
2
A compounds showed high activity ꢀor AChE inhibition
in the nanomolar range and 3A compound demonstrated
a less inhibitory activity that corroborates with Ellman’s
data. Compound 2A was discovered to have the most potent
inhibitory activity with IC values oꢀ 4.4 nM and notice-
0.02
−0.06
−0.43
−1.89
−2.44
−5.25
5
0
able observation is that its activity is even higher than that
oꢀ Donepezil. (IC =9.0 nM). The IC values oꢀ 1A, 2A,
5
0
50
and 3A are represented in Table 2.
a
Original residues
Mutated residues
Calculated binding aꢂnity
Calculaed stability
b
c
d
Molecular toxicity
The cytotoxic efect oꢀ the newly designed and synthe-
sized compounds was determined in SH-SY5Y, a human
neuroblastoma cell line. In the MTT assays, the treatment
oꢀ SH-SY5Y with 1A, 2A and 3A at increasing concentra-
tions (0.6 µM to 8 µM) ꢀor 24 h did not show signiꢁcant
cytotoxicity. Compounds 1, 2, and 3 have demonstrated con-
siderable cytotoxicity compared to their pyridine-N-methyl-
ated pyridinium analogs 1A-3A (Fig. 2a, b).
Fig. 2 a, b Efect on Cell viability oꢀ SH-SY5Y Cells at diferent
concentrations (0.06 µM–8 µM) oꢀ compounds 1–3 (a) and 1A–3A
experiment was perꢀormed in triplicates. The data represent the per-
centage oꢀ viability cells (means± SD)
(
b) ꢀor 24 h. Cell viability was determined with MTT assay. The
1
3

Discovery of dual cation‑π inhibitors of acetylcholinesterase: design, synthesis and…
O
O
1
.10%NaOH solution
0
0
5
C-25 C,10h
2
R-CHO
R
R
2
.Dilute HCl
(
Cyclohexanone) (Aldehyde)
O
1
.
R =
R =
N
N
N
2
,6-bis(pyridin-4-ylmethylene)cyclohexanone
O
N
N
2.
N
N
2
,6-bis(pyridin-3-ylmethylene)cyclohexanone
O
N
N
R =
3.
2
,6-bis(pyridin-2-ylmethylene)cyclohexanone
Scheme 1 Preparation oꢀ 2, 6-bis(pyridinylmethylene)cyclohexanone derivatives
Binding mode and interactions
moiety oꢀ compound 1A and the backbone atom oꢀ Phe295
(
O…HN distance: 1.93 Å).
Docking analysis
Similarly, ꢀor designed molecule 2A (with the highest XP
docking score oꢀ −13.727 kcal/mol), at the anionic sub site,
acyl binding pocket and PAS the meta-positioned quater-
nary ammonium is stacked with the aromatic residues Trp86
(Pyrrole: 5.55 Å, Benzene: 6.38 Å), Phe338 (5.46 Å) and
Tyr337 (4.19 Å) making cation-π interactions with the posi-
tively charged nitrogen and the aromatic groups, respectively
(Fig. 4). Further, at the acyl binding site, similar hydrogen
bond interaction was ꢀound between the carbonyl oxygen
oꢀ the linker moiety and the backbone oꢀ Phe295 (O…HN,
distance: 2.00 Å).
Molecular docking studies oꢀ the designed molecules were
carried out to delineate a key set oꢀ interactions that govern
the activity oꢀ AChE. The XP docking score and binding
residues oꢀ the inhibitors are given in Table 3.
Molecule 1A has a high XP docking score oꢀ
-
13.398 kcal/mol and the binding orientation to the criti-
cal residues oꢀ AChE active site is presented in Fig. 3. The
Para positioned pyridine-N-methylated pyridinium group is
ꢁ
rmly bound to the anionic sub site, acyl binding pocket
and PAS by stacking against Trp86 (Pyrrole: 4.83 Å, Ben-
zene: 5.91 Å), Phe338 (6.65 Å) and Tyr337 (4.81 Å) ꢀorm-
ing cation-π interactions with the aromatic residues, respec-
tively. Hydrogen bond interaction was also observed at the
acyl binding site between the carbonyl group oꢀ the linker
The pyridine-N-methylated pyridinium group oꢀ 2A
ꢀurther interacts at the PAS by stacking against the residue
Trp286. It allows the positive charge meta-substituted nitro-
gen oꢀ quaternary ammonium to ꢀorm cation- π interaction
as well as π—π interaction, which ꢀurther stabilizes the
complex.
1
3

N. Damuka et al.
O
O
H
C
I
R
R
H
H
R1
R1
CH CN
5 C-12h,RT-72h
3
0
2
,6-bis(pyridinymethylene)cyclohexanone
derivatives
(
Methyl iodide)
Pyridine-N-methylated
2
,6-bis(pyridinylmethylene)cyclohexanone
derivatives
O
I-
1
A .
I-
N
N
4
,4'-(1E,1'E)-(2-oxocyclohexane-1,3-diylidene)bis(methan-1-yl-1-ylidene)bis(1-
methylpyridinium) iodide
O
N
N
I-
2
A .
I-
3,3'-(1E,1'E)-(2-oxocyclohexane-1,3-diylidene)bis(methan-1-yl-1-ylidene)bis(1-methylpyridinium) iodide
O
I-
I-
N
N
3
A .
2,2'-(1E,1'E)-(2-oxocyclohexane-1,3-diylidene)bis(methan-1-yl-1-ylidene)bis(1-methylpyridinium) iodide
Scheme 2 Pyridine-N-methylation oꢀ 2,6-Bis(pyridinylmethylene)cyclohexanone
The higher inhibitory activity oꢀ 2A as compared to 1A,
observed in both types oꢀ in vitro assays, can be explained
according to the predicted interactions at the active site. The
meta-positioned quaternary methylated pyridinium allows
the binding oꢀ the molecule with the key aromatic residues
directed away ꢀrom the crucial residue Trp86 (Fig. 5),
which may provide the reason ꢀor lower inhibitory activ-
ity as observed in the inhibitory assay. However, cation–π
as well as π–π interactions with residues Tyr337 (cation-
π distance: 4.49 Å and π–π distance: 5.12 Å) and Phe338
(cation- π, distance: 4.28 Å) were observed at PAS and
acyl binding pocket, respectively. Further, at the acyl
binding pocket site, the same hydrogen bond interaction
is ꢀormed between the carbonyl oxygen and Phe295 (O…
HN, distance: 1.95 Å). At the PAS, another π–π interac-
tion was observed with pyridine-N-methylated pyridinium
group by stacking against Trp286 with a distance oꢀ 4.3 Å
(
Trp86, Phe295 and Trp286) at the anionic sub site, acyl
binding pocket and PAS. However, Para positioned directs
the quaternary methylated pyridinium away ꢀrom the aro-
matic residue Trp286 at the PAS, resulting in less interaction
and thus less experimental inhibitory activity.
In the case oꢀ molecule 3A, at the anionic subsite, the
ortho-positioned quaternary methylated pyridinium is
1
3

Discovery of dual cation‑π inhibitors of acetylcholinesterase: design, synthesis and…
Table 2 Acetylcholinesterase inhibitory activities oꢀ 1A, 2A and 3A
Dynamics of the enzyme‑inhibitor complex: RMSD curves
compound
The molecular dynamics study was carried out to study the
behavior oꢀ the predicted complexes, considering the inher-
ent ꢃexibility involved in the enzyme. Further, the stabil-
ity oꢀ the molecular interactions predicted by the docking
method was examined by monitoring the percentage occur-
rence oꢀ the interactions during the simulation period.
The stability oꢀ the simulation system was explored
by analyzing the RMSDs oꢀ the proteins ꢀrom the start-
ing structure. As shown in Fig. 7, aꢀter initial ꢃuctuation,
Compounds
AChE inhibition IC50
AChE inhibition IC50
a
b
(
nM) (Ellman’s method) (nM) (Amplex red
assay)
1A
2A
3A
20.9± 4.2
6.2± 2.06
N/A
67.9± 2.2
4.4± 0.8
N/A
Donepezil
8.5± 0.8
9.0± 2.3
a
The inhibition activities oꢀ AChE were screened by Ellman’s
method. IC50 values are means± standard deviation oꢀ three inde-
pendent experiments
α
the C -RMSD value oꢀ Apo-enzyme (in Donepezil, and
b
designed Para, Meta and Ortho positioned quaternary pyri-
dinium inhibitor complexes) reached a relatively stable
conꢀormation aꢀter 25 ns, with an average RMSD value
oꢀ 2–2.4 Å. Similarly, ꢀor the ligands bound to the protein,
namely Donepezil, 1A-3A, the RMSDs are shown in Fig. 8.
Para and Meta positioned quaternary pyridinium compounds
(1A and 2A respectively) showed small ꢃuctuations initially
but stabilized at around 2.5 Å throughout the simulation. The
study indicates the preserved binding mode oꢀ interactions
arising out oꢀ stable and strong interaction throughout. In the
case oꢀ Donepezil, the RMSD value was stabilized at around
3 Å at the initial 30 ns, but it ꢀurther increased to 4.5 Å. The
most ꢃuctuation was seen in Ortho positioned quaternary
pyridinium compound (3A), where three major spikes can be
Amplex red Assay perꢀormed using SH-SY5Y cells. IC50 values are
means± standard deviation oꢀ duplicate independent experiments
ꢀ
rom the rings. Molecule 3A also has a lower docking
score (− 10.29 kcal/mol) as compared to 1A and 2A.
The docked conꢀormations obtained ꢀrom molecular
docking ꢀor molecules 1, 2, and 3, are shown in Fig. 6.
Molecule 1, 2, and 3 ꢀailed to demonstrate the dual bind-
ing mode. However, they are able to participate in hydro-
gen bonding interactions at the acyl binding pocket with
Phe295. The absence oꢀ cation–π interactions ꢀor these
compounds might have resulted in the loss oꢀ activity in
the experimental assays.
Table 3 Docking result oꢀ the
a
b
No
Compound
XP GScore Binding residues
−1 a
synthesized molecules with
AChE
(
kcal mol )
O
-
9.272
PHE295
PHE295
1
2
3
N
N
N
N
O
-
9.442
O
N
N
-9.536
PHE295
PHE295,
O
-
13.398
1
2
3
A
TRP86,TYR337,PHE338
N
N
O
PHE295,TRP286,TRP86,
PHE338
-
-
13.727
10.219
N
N
A
A
O
N
N
PHE295,TRP286
o
-
11.234
PHE295,TRP286,TRP86,
PHE338
o
N
Donepezil
o
a
Glide XP Docking score oꢀ the compounds
AChE residues which were predicted to be interacting with the compounds
b
1
3

N. Damuka et al.
Fig. 4 Binding interaction oꢀ compound 2A at AChE active site
shown in yellow-green color. Red, green and yellow dotted lines rep-
resent cation- π interactions, π–π interactions and hydrogen bonds,
respectively. Distance in Å is shown in green
Fig. 3 Binding interaction oꢀ compound 1A at AChE active site
shown in turquoise color. Red and yellow dotted lines represent cat-
ion- π interactions and hydrogen bonds, respectively. Distance in Å is
shown in green
The least active molecule 3A having Ortho substituted
quaternary ammonium had π–π interaction with Trp86 pre-
served over 51% oꢀ the simulation time, comparatively less
percentage oꢀ the time with respect to 1A and 2A (supple-
mentary Fig. 7).
seen reaching around RMSD oꢀ 6–7 Å; however, it remained
approximately about 5 Å throughout the simulation. The
increased ꢃexibility oꢀ Donepezil and 3A could be attributed
to ꢀewer interactions with AChE and thereby be highlighting
weak binding as compared to 1A and 2A, as also observed
in both enzymatic and cell line assays.
Free energy analysis using MM‑GBSA
Binding interactions analysis
The predicted binding ꢀree energy obtained ꢀrom docking
and 100 molecular dynamics snapshots complexes calcu-
lated using an implicit salvation based MM/GBSA approach
are given in Table 4. The plot oꢀ approximate binding ꢀree
In the docking study, the Para substituted methylpyridinium
group oꢀ molecule 1A was stacked against Trp86, Tyr337,
and Phe338, ꢀorming cation-π interactions with the aromatic
residues. The Trp86 was preserved in 80% oꢀ the simulation
time (supplementary Fig. 5). A new hydrophobic π–π inter-
action was also observed ꢀor 47% oꢀ the simulation period
with Tyr341. In the case oꢀ 2A, the Meta positioned quater-
nary ammonium showed cation-π interactions with Trp86,
which was also preserved ꢀor 79% along the simulation
energies (ΔG ) versus the 100 complexes is shown in sup-
bind
plementary Fig. 8. As expected, the most ꢀavored binding
was exerted by the molecules 1A and 2A, having averaged
−
1
binding ꢀree energies (ΔG ) oꢀ -96.84008 kcal mol
bind
−
1
and − 94.9527 kcal mol respectively. The Donepezil-
AChE complex has slightly lower average binding ener-
−
1
gies (−89.25343 kcal mol ) than 1A and 2A. The Ortho
positioned pyridine-N-methylated pyridinium molecule
(
supplementary Fig. 6). Further, due to the Meta substituted
position, the distance between Trp86 and methylpyridinium
group oꢀ 2A was closer at 4 Å (Fig. 9), which seems to sta-
bilize the complex considerably as compared to 1A, thus
resulting in more biological activity.
3A has the least average binding ꢀree energy (ΔG
)
bind
−
1
−63.75939 kcal mol , perhaps due to the loss oꢀ cation-π
interactions. Predicted binding ꢀree energy (ΔG ) is shown
bind
in Table 4.
1
3

Discovery of dual cation‑π inhibitors of acetylcholinesterase: design, synthesis and…
Fig. 6 Binding interaction oꢀ compound 1–3 at AChE active site rep-
resented as a thick stick in grey color. Yellow dotted lines represent
hydrogen bonds
Fig. 5 Binding interaction oꢀ compound 3A at AChE active site
shown in plum color. Red, green and yellow dotted lines represent
cation- π interactions, π–π interactions and hydrogen bonds, respec-
tively. The distance in Å is shown in green
(
2H), 9.00 (2H), 8.734 (2H), 8.230 (2H), 7.68 (2H); 4.41
1
3
Active AChE inhibitors 1A, 2A and 3A spectral
characterization
(6H); 3.01 (4H); 1.78 (2H); C- NMR (100 MHz, DMSO-
d δ ppm): 188.44, 146.63, 145.50, 144.98, 141.48, 135.06,
6
,
1
29.64, 127.92, 48.80, 27.86,22.00; HRMS(ESI+): m/z: cal-
2
+
2+
Compound 1A
culated ꢀor C H N O (M ): 153.0850; ꢀound: 153.0877.
20 22 2
The spectral ꢁgures are given in supplementary section
(Figs. S13–S16).
4
1
6
3
,4′-(1E,1′E)-(2-oxocyclohexane-1,3-diylidene)bis(methan-
-yl-1-ylidene)bis(1 methylpyridinium)iodide (1A): Yield:
−
1
10 mg (99%); m.p: 196–198 °C; ATR-FTIR (ν cm ):
Compound 3A
1
455.97, 3334.04, 3014.65, 1672.74,1637.77; H–NMR
(
(
400 MHz, DMSO-d , δ ppm): 9.03 (4H), 8.26 (4H), 7.71
2,2′-(1E,1′E)-(2-oxocyclohexane-1,3-diylidene)bis(methan-
1-yl-1-ylidene)bis(1-methylpyridinium)iodide (3A):
Yield: 479 mg (78%), m.p: 187–189 °C; ATR-FTIR
6
13
2H), 4.37 (6H), 3.01 (4H), 1.80 (2H); C–NMR (100 MHz,
DMSO-d , δ ppm): 188.72, 151.01, 145.65, 144.26, 131.09,
6
−
1
1
28.23, 48.14, 28.02, 21.63; HRMS (ESI+): m/z: calculated
(ν cm ):3600.41,3434.18, 3019.39, 2997.49,1682.61,
2
+
2+
1
ꢀ
or C H N O (M ): 153.0850; ꢀound: 153.0861.
1621.76; H-NMR (400 MHz,DMSO-d , δ ppm): 9.19 (2H),
2
0
22
2
6
The spectral ꢁgures are given in supplementary section
8.64 (2H), 8.15 (4H), 7.66 (2H), 4.29 (6H);2.85 (4H); 1.77
1
3
(
Figs.S9–S12).
(2H); C-NMR (100 MHz, DMSO-d , δ ppm): 187.78,
6
1
50.65, 147.55, 145.57, 144.90, 129.55, 127.51, 126.43,
Compound 2A
46.75, 28.06, 21.82; HRMS (ESI +): m/z: calculated ꢀor
2
+
2+
C H N O (M ): 153.0850; ꢀound: 153.0892.
2
0
22
2
3
1
,3′-(1E,1′E)-(2-oxocyclohexane-1,3-diylidene)bis(methan-
-yl-1-ylidene)bis(1-methylpyridinium)iodide (2A):
Note: Signal multiplicity could not be shown in spectral
data. We assumed that due to the di- positivity oꢀ mole-
1
Yield: 602 mg (98%); m.p: 230–232 °C; ATR-FTIR
cules, splitting oꢀ H-NMR signals was not observed in the
−
1
(
ν cm ): 3451.9,3410.8,3026.5, 2956.4, 2919.7,2866.7,
spectrum.
1
1
675.0,1628.8; H-NMR (400 MHz,DMSO-d δ ppm): 9.54
6,
1
3

N. Damuka et al.
Fig. 7 Root mean square deviation (RMSD) oꢀ AChE in the absence (Blue) and presence oꢀ bound compounds; Donepezil (Orange), 1A (Grey),
2
A (Yellow), and 3A (Dark blue) during the simulation period
Fig. 8 Ligand Root means square deviation (RMSD) oꢀ Donepezil (Orange), 1A (Grey), 2A (Yellow), and 3A (Dark blue) with respect to AChE
during the simulation period
Fig. 9 Plot oꢀ cation- π interaction distance (in Å) oꢀ compound 1A (Orange) and 2A (Blue) with residue Trp86 during the simulation time
1
3

Discovery of dual cation‑π inhibitors of acetylcholinesterase: design, synthesis and…
Table 4 Predicted binding ꢀree energy (ΔGbind) using MM/GBSA
existing drugs available in the market such as Rivastigmine
Exelon®), Memantine (Namenda®), Galantamine (Raza-
method
(
ΔGbind (Docking)a
−101.568 kcal mol
ΔGbind (MD)b
dyne®), Donepezil (Aricept®) and Tacrine. These deriva-
tives promote characteristic dual-binding as well as strong
cation-π interactions, hydrophobic and hydrogen bonding
interactions with hot-spot residues. Thus, these compounds
can serve as novel leads ꢀor AChE.
Compounds
−
1
1
−1
1
2
3
A
A
A
−96.84008 kcal mol
−
−1
−89.074 kcal mol
−94.9527 kcal mol
−63.75939 kcal mol
−89.25343 kcal mol
−57.701 kcal mol−
−89.299 kcal mol−
1
−1
−1
1
Donepezil
a
Acknowledgements This work was supported in part by UPE Phase-II.
Financial support ꢀrom the DBT ꢀellowship is grateꢀully acknowledged
by DN. Financial support ꢀrom the DBT-RA Program in Biotechnology
and Liꢀe Sciences is grateꢀully acknowledged by PAM. Authors also
thank ꢀunding support ꢀrom DST-PURSE, UGC-SAP to Department oꢀ
Biotechnology and Bioinꢀormatics, University oꢀ Hyderabad.
ΔGbind obtained ꢀrom docking single inhibitor-enzyme complex
average ΔGbind obtained ꢀrom 100 molecular dynamics snapshots
b
The spectral ꢁgures are given in the supplementary sec-
tion (Fig. S17–S20).
Compliance with ethical standards
Conflict of interest There are no conꢃicts oꢀ interest declared by the
authors.
Discussion
To discover novel and efective drugs ꢀor treating Alzhei-
mer’s disorder, a series oꢀ methylidenecyclohexanone com-
pounds were designed, synthesized. By exploring the active
site environment, hot-spot residues oꢀ AChE, and exploiting
the characteristic dual binding observed in Donepezil, the
inhibitors were designed to promote strong cation-π inter-
actions with the conserved residues (Trp86 and Trp286)
as well as hydrophobic and hydrogen bonding interactions
with important residues. Accordingly, positively charged
pyridine-N-methylated pyridinium group was introduced at
both the ends oꢀ the three inhibitors 1A, 2A and 3A. AChE
inhibitory activity oꢀ these designed compounds was per-
References
1
.
Richens JL, Bramble JP, Spencer HL, Cantlay F, Butler M, O’Shea
P. Towards deꢁning the mechanisms oꢀ alzheimer’s disease based
on a contextual analysis oꢀ molecular pathways. AIMS Genet.
2
016;3:25–48.
2
3
.
.
Yoon S-S, Jo SA. Mechanisms oꢀ amyloid-β peptide clearance:
potential therapeutic targets ꢀor Alzheimer’s disease. Biomol Ther.
2012;20:245–55.
Lombardi V, Garcıa M, Rey L, Cacabelos R. Characterization
oꢀ cytokine production, screening oꢀ lymphocyte subset patterns
and in vitro apoptosis in healthy and Alzheimer’s disease (AD)
individuals. J Neuroimmunol. 1999;97:163–71.
ꢀormed with the pure enzyme (Ellman’s assay) and with
4. Marlatt MW, Lucassen PJ, Perry G, Smith MA, Zhu X. Alzhei-
mer’s disease: cerebrovascular dysꢀunction, oxidative stress, and
advanced clinical therapies. J Alzheimers Dis. 2008;15:199–210.
SH-SY5Y neuroblastoma cell line (Amplex red assay).The
designed inhibitors involving pyridine-N-methylated pyri-
dinium group to ꢀacilitate cation-π interactions demonstrated
AChE activity in nM ranges, while compound 1, 2 and 3
lacking such group were ꢀound to be inactive at 100 µM or
lower concentration.
5
6
7
8
.
.
.
.
Whitehouse PJ, Price DL, Struble RG, Clark AW, Coyle JT, Delon
MR. Alzheimer’s disease and senile dementia: loss oꢀ neurons in
the basal ꢀorebrain. Science. 1982;215:1237–9.
Francis PT, Palmer AM, Snape M, Wilcock GK. The cholinergic
hypothesis oꢀ Alzheimer’s disease: a review oꢀ progress. J Neurol
Neurosurg Psychiatry. 1999;66:137–47.
Compound 2A with the Meta substituted quaternary
ammonium has the highest activity (IC =6.2 nM), while
Nachmansohn D, Wilson IB. The enzymic hydrolysis and syn-
thesis oꢀ acetylcholine. Adv Enzymol Relat Subj Biochem.
5
0
1
951;12:259–339.
the Para substituted compound having compound 1A has
Quinn DM. Acetylcholinesterase: enzyme structure, reac-
tion dynamics, and virtual transition states. Chem Rev.
IC =20.9 nM and the least active compound is 3A. Further,
50
docking, molecular dynamics studies helped elucidate bind-
ing mode and the key set oꢀ molecular interactions responsi-
ble ꢀor AChE activity. Compounds 1A–3A, possessing posi-
tive charged, may not cross the Blood–brain barrier (BBB).
However, there are reports ꢀor the successꢀul implementation
oꢀ biocompatible nanoparticles that can overcome the BBB
issues oꢀ the ligands. One oꢀ the latest reports demonstrated
that Fe3O4@C nanoparticle linked with small molecules
were efectively delivered into a speciꢁc subcellular com-
ponent oꢀ the brain and restore the memory in mice [34, 35].
The Compounds 1A-3A, possessing novel 2,6-dimethyl-
ene-cyclohexanone scafolds are entirely diferent ꢀrom the
1
987;87:955–79.
9. Sussman JL, Harel M, Frolow F, Oeꢀner C, Goldman A, Toker
L, et al. Atomic structure oꢀ acetylcholinesterase ꢀrom Torpedo
californica: a prototypic acetylcholine-binding protein. Science.
1
991;253:872–9.
1
0. Taylor P, Lappi S. Interaction oꢀ ꢃuorescence probes with ace-
tylcholinesterase. The site and speciꢁcity oꢀ propidium binding.
Biochemistry. 1975;14:1989–97.
1
1. Amitai G, Taylor P. Characterization oꢀ peripheral anionic site
peptides oꢀ AChE by photoaꢂnity labeling with monoazidopro-
pidium (MAP). In: Massouli J, Bacou F, Barnard EA, Chatonnet
A, Doctor BP, Quinn DM, editors. Cholinesterases: structure,
ꢀunction, mechanism, genetics and cell biology. American Chemi-
cal Society; Washington, DC; 1991: p. 285.
1
3

N. Damuka et al.
1
1
1
2. Harel M, Quinn DM, Nair HK, Silman I, Sussman JL. The X-ray
structure oꢀ a transition state analog complex reveals the molecu-
lar origins oꢀ the catalytic power and substrate speciꢁcity oꢀ ace-
tylcholinesterase. J Am Chem Soc. 1996;118:2340–6.
25. Vatsadze S, Manaenkova M, Sviridenkova N, Zyk N, Krut’ko
D, Churakov A, et al. Synthesis and spectroscopic and structural
studies oꢀ cross-conjugated dienones derived ꢀrom cyclic ketones
and aromatic aldehydes. Russ Chem Bull. 2006;55:1184–94.
26. Katritzky AR, Fan WQ, Jiao XS, Li QL. Bridged cyanine dyes.
Part 3. Zwitterionic bis-4-pyridyl and cationic bis-dimethylanilino
derivatives. J Heterocycl Chem. 1988;25:1321–5.
3. Weise C, Kreienkamp HJ, Raba R, Pedak A, Aaviksaar A, Hucho
F. Anionic subsites oꢀ the acetylcholinesterase ꢀrom Torpedo cali-
ꢀ
ornica: aꢂnity labelling with the cationic reagent N, N-dimethyl-
2
-phenyl-aziridinium. EMBO J. 1990;9:3885–8.
27. Ellman GL, Courtney KD, Andres V, Featherstone RM. A new
and rapid colorimetric determination oꢀ acetylcholinesterase activ-
ity. Biochem Pharmacol. 1961;7:88–95.
4. Ordentlich A, Barak D, Kronman C, Flashner Y, Leitner M, Segall
Y, et al. Dissection oꢀ the human acetylcholinesterase active center
determinants oꢀ substrate speciꢁcity. Identiꢁcation oꢀ residues
constituting the anionic site, the hydrophobic site, and the acyl
pocket. J Biol Chem. 1993;268:17083–95.
28. Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz
DT, et al. Glide: a new approach ꢀor rapid, accurate docking and
scoring. 1. Method and assessment oꢀ docking accuracy. J Med
Chem. 2004;47:1739–49.
1
5. Alvarez A, Opazo C, Alarcón R, Garrido J, Inestrosa NC. Ace-
tylcholinesterase promotes the aggregation oꢀ amyloid-β-peptide
29. Bowers KJ, Chow E, Xu H, Dror RO, Eastwood MP, Gregersen
BA, et al. (2006) Scalable algorithms ꢀor molecular dynamics
simulations on commodity clusters. In: ACM/IEEE SC 2006
Conꢀerence.
ꢀ
ragments by ꢀorming a complex with the growing ꢁbrils. J Mol
Biol. 1997;272:348–61.
1
1
6. Radić Z, Quinn DM, Vellom DC, Camp S, Taylor P. Allosteric
control oꢀ acetylcholinesterase catalysis by ꢀasciculin. J Biol
Chem. 1995;270:20391–9.
30. Jorgensen WL, Maxwell DS, Tirado-Rives J. Development
and testing oꢀ the OPLS all-atom ꢀorce ꢁeld on conꢀormational
energetics and properties oꢀ organic liquids. J Am Chem Soc.
1996;118:11225–36.
7. Barak D, Kronman C, Ordentlich A, Ariel N, Bromberg A, Marcus
D, et al. Acetylcholinesterase peripheral anionic site degeneracy
conꢀerred by amino acid arrays sharing a common core. J Biol
Chem. 1994;269:6296–305.
31. Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Ped-
ersen LG. A smooth particle mesh Ewald method. J Chem Phys.
1995;103:8577–93.
1
1
2
2
8. Bourne Y, Taylor P, Radić Z, Marchot P. Structural insights into
ligand interactions at the acetylcholinesterase peripheral anionic
site. EMBO J. 2003;22:1–12.
32. Lyne PD, Lamb ML, Saeh JC. Accurate prediction oꢀ the rela-
tive potencies oꢀ members oꢀ a series oꢀ kinase inhibitors using
molecular docking and MM-GBSA scoring. J Med Chem.
2006;49:4805–8.
9. Dorronsoro I, Castro A, Martinez A. Peripheral and dual binding
site inhibitors oꢀ acetylcholinesterase as neurodegenerative disease
modiꢀying agents. Expert Opin Ther Pat. 2003;13:1725–32.
0. Cousin X, Strähle U, Chatonnet A. Are there non-catalytic ꢀunc-
tions oꢀ acetylcholinesterases? Lessons ꢀrom mutant animal mod-
els. Bioessays. 2005;27:189–200.
33. Bogan AA, Thorn KS. Anatomy oꢀ hot spots in protein interꢀaces.
J Mol Biol. 1998;3(280):1–9.
34. Chaturbedy P, Kumar M, Salikolimi K, Das S, Sinha SH, Chat-
terjee S, Suma BS, Kundu TK, Eswaramoorthy M. Shape-directed
compartmentalized delivery oꢀ a nanoparticle-conjugated small-
molecule activator oꢀ an epigenetic enzyme in the brain. J Control
Release. 2015;217:151–9.
1. Inestrosa NC, Alvarez A, Perez CA, Moreno RD, Vicente M,
Linker C, et al. Acetylcholinesterase accelerates assembly oꢀ
amyloid-β-peptides into Alzheimer’s ꢁbrils: possible role oꢀ the
peripheral site oꢀ the enzyme. Neuron. 1996;16:881–91.
2. Galdeano C, Viayna E, Arroyo P, Bidon-Chanal A, Ramon Blas J,
Munoz-Torrero D, et al. Structural determinants oꢀ the multiꢀunc-
tional proꢁle oꢀ dual binding site acetylcholinesterase inhibitors
as anti-Alzheimer agents. Curr Pharm Des. 2010;16:2818–36.
3. Impact Schrödinger, LLC, New York, NY, 2016; Prime,
Schrödinger, LLC, New York, NY, 2016; BioLuminate,
Schrödinger, LLC, New York, NY, 2016. https://www.schrodinge
r.com/. Accessed: 2019.
35. Chatterjee S, Cassel R, Schneider-Anthony A, Merienne K, Cos-
quer B, Tzeplaef L, Sinha SH, Kumar M, Chaturbedy P, Eswar-
amoorthy M, Le Gras S. Reinstating plasticity and memory in
a tauopathy mouse model with an acetyltransꢀerase activator.
EMBO Mol Med. 2018;11:1–10.
2
2
2
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional aꢂliations.
4. Banerjee K, Roy S, Biradha K. Design, synthesis, and photolu-
minescence properties oꢀ one-, two-, and three-dimensional coor-
dination polymers: anion-assisted argentophillic interactions as
building blocks. Cryst Growth Des. 2014;22:5164–70.
1
3