Novel hits for acetylcholinesterase inhibition derived by docking-based screening on ZINC database

Article abstract of DOI:10.1080/14756366.2018.1458031

The inhibition of the enzyme acetylcholinesterase (AChE) increases the levels of the neurotransmitter acetylcholine and symptomatically improves the affected cognitive function. In the present study, we searched for novel AChE inhibitors by docking-based virtual screening of the standard lead-like set of ZINC database containing more than 6 million small molecules using GOLD software. The top 10 best-scored hits were tested in vitro for AChE affinity, neurotoxicity, GIT and BBB permeability. The main pharmacokinetic parameters like volume of distribution, free fraction in plasma, total clearance, and half-life were predicted by previously derived models. Nine of the compounds bind to the enzyme with affinities from 0.517 to 0.735 μM, eight of them are non-toxic. All hits permeate GIT and BBB and bind extensively to plasma proteins. Most of them are low-clearance compounds. In total, seven of the 10 hits are promising for further lead optimisation. These are structures with ZINC IDs: 00220177, 44455618, 66142300, 71804814, 72065926, 96007907, and 97159977.

Full text of DOI:10.1080/14756366.2018.1458031

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Novel hits for acetylcholinesterase inhibition
derived by docking-based screening on ZINC
database
Irini Doytchinova, Mariyana Atanasova, Iva Valkova, Georgi Stavrakov,
Irena Philipova, Zvetanka Zhivkova, Dimitrina Zheleva-Dimitrova, Spiro
Konstantinov & Ivan Dimitrov
To cite this article: Irini Doytchinova, Mariyana Atanasova, Iva Valkova, Georgi Stavrakov,
Irena Philipova, Zvetanka Zhivkova, Dimitrina Zheleva-Dimitrova, Spiro Konstantinov & Ivan
Dimitrov (2018) Novel hits for acetylcholinesterase inhibition derived by docking-based screening
on ZINC database, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 768-776, DOI:
10.1080/14756366.2018.1458031
To link to this article: https://doi.org/10.1080/14756366.2018.1458031
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2018
VOL. 33, NO. 1, 768–776
https://doi.org/10.1080/14756366.2018.1458031
RESEARCH PAPER
Novel hits for acetylcholinesterase inhibition derived by docking-based screening
on ZINC database
Irini Doytchinovaa,b , Mariyana Atanasovaa, Iva Valkovaa,b, Georgi Stavrakova,c, Irena Philipovac,
Zvetanka Zhivkovaa, Dimitrina Zheleva-Dimitrovaa, Spiro Konstantinova and Ivan Dimitrova
b
aFaculty of Pharmacy, Medical University of Sofia, Sofia, Bulgaria; Drug Design and Development Lab, Sofia Tech Park, Sofia, Bulgaria;
cInstitute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria
ABSTRACT
ARTICLE HISTORY
Received 17 February 2018
Revised 23 March 2018
Accepted 23 March 2018
The inhibition of the enzyme acetylcholinesterase (AChE) increases the levels of the neurotransmitter
acetylcholine and symptomatically improves the affected cognitive function. In the present study, we
searched for novel AChE inhibitors by docking-based virtual screening of the standard lead-like set of ZINC
database containing more than 6 million small molecules using GOLD software. The top 10 best-scored
hits were tested in vitro for AChE affinity, neurotoxicity, GIT and BBB permeability. The main pharmacoki-
netic parameters like volume of distribution, free fraction in plasma, total clearance, and half-life were pre-
dicted by previously derived models. Nine of the compounds bind to the enzyme with affinities from
0.517 to 0.735 mM, eight of them are non-toxic. All hits permeate GIT and BBB and bind extensively to
plasma proteins. Most of them are low-clearance compounds. In total, seven of the 10 hits are promising
for further lead optimisation. These are structures with ZINC IDs: 00220177, 44455618, 66142300,
71804814, 72065926, 96007907, and 97159977.
KEYWORDS
Virtual screening; molecular
docking; ITC; PAMPA;
Neuro 2A
Introduction
3D-QSAR, high-throughput screening, and/or machine learning
methods30. Recently Chen et al. performed virtual screening on
263,146 entries from Specs database using a structure-based
pharmacophore (SBP) derived on two tacrine hybrids with nano-
molar affinity to AChE31. The top 50 compounds were docked in
the AChE and the best 15 of them were tested in vitro by Ellman’s
method32. Ten hits showed IC50 values below 10 mM. Dhanja et al.
derived a ligand-based pharmacophore using the 3D structures of
16 known AChE inhibitors33. The pharmacophore was used to
screen 50,000 small molecule natural compounds from ZINC data-
base, followed by docking studies. The best two binders were ana-
lysed for molecular interactions with AChE, but have not been
tested experimentally. Chen et al. conducted a virtual screening
on Chemdiv compound collection, which contains 1,293,896 mole-
cules34. Initially, the collection was screened by rapid overlay of
chemical structures using the AChEI donepezil as a template.
Then, the top 1% of the most similar structures was screened by
SBP generated on the basis of donepezil-AChE X-ray complex.
Finally, 24 compounds of the best hits were tested for AChE and
BuChE activity in vitro by Ellman’s method. Among them, five new
inhibitors were discovered.
The enzyme acetylcholinesterase (AChE) is a serine protease (EC
3.1.1.7) catalysing the hydrolysis of the neurotransmitter acetylcho-
line (ACh) to choline and acetic acid. Low levels of ACh lead to
cognitive impairment and dementia1. The inhibition of AChE
increases the ACh levels and symptomatically improves the
affected cognitive function2. AChE inhibitors (AChEIs) are the main
drugs currently in use for treatment of Alzheimer’s disease (AD),
the most common form of dementia3. Donepezil, galantamine,
and rivastigmine are AChEIs. They have moderate affinity to AChE
and provide delay in AD progression.
The binding site on AChE is deep and narrow gorge and con-
sists of several domains: catalytic, anioinic, acylic, oxyanionic, and
peripheral anionic4–7. The most important of them are the cata-
lytic active site (CAS) where ACh hydrolysis happens and the per-
ipheral anionic site (PAS) placed near the entrance of the gorge
and associated with the formation of amyloid plaques8. Thus,
AChE is a target with dual functionality: ACh hydrolysis and amyl-
oid beta (Ab) peptide aggregation. Because of its importance,
AChE is a focus of many intensive and extensive drug discovery
studies during the last two decades9–11. These studies could be
grouped into three directions: lead optimisation of known
AChEIs12–16, hybrids between them17–24 and search for new scaf-
folds25–28. A wide range of computational tools are involved in
these studies29.
In the present study, we conducted a docking-based virtual
screening on ZINC database containing over 6 million biologically
active small molecules in order to identify novel hits binding to
AChE. The top 10 best-scored binders to AChE were tested in vitro
for affinity to AChE, neurotoxicity, blood-brain barrier (BBB), and
gastrointestinal (GI) permeability. Predictions of their physicochem-
One of the most useful structure-based computational methods
in the discovery of novel hits binding to a specific target is the
molecular docking used solely or in combination with 2D- and ical and ADME properties also were performed.
CONTACT Irini Doytchinova
idoytchinova@pharmfac.mu-sofia.bg
Faculty of Pharmacy, Medical University of Sofia, Sofia 1000, Bulgaria
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
769
wavelength analysed 250–500 nm, pH 7.4, temperature 25 ꢀC, per-
meation time 4 h, lipid formulation GIT-0 and stirring 60 rpm and
BBB-1 and no stirring, respectively. The intestinal permeability was
tested at three pH values: 5.0, 6.2, and 7.4, while the BBB perme-
Materials and methods
Database and docking protocol
ZINC (www.zinc.docking.org) contains several databases of biologic-
ally active structures. We selected the Standard Lead-like database ability was tested at pH 7.4. The permeability was presented as
which consists of 6,053,287 small molecules with molecular weights ꢁlogPe, where Pe is the permeability coefficient (10ꢁ6 cm/s).
between 250 and 350 g/mol, log P up to 3,5 and up to 7 rotatable
Compounds with ꢁlogPe below 5 are considered as highly perme-
bonds. The set was downloaded in March 2015. The molecules
able, with ꢁlogPe between 5 and 6 – as medium permeable and
with above 6 – as low permeable41. Carbamazepine, ketoprofen,
were docked into the X-ray structure of human recombinant acetyl-
cholinesterase (rhAChE, pdb id: 4EY6, R ¼ 2.40Å)35. The docking sim-
and ranitidine were used as controls for intestinal permeability.
ulations were performed by GOLD v. 5.1. [CCDC Ltd., Cambridge,
UK] using a protocol previously optimised in terms of scoring func-
tion, rigid/flexible ligand and binding site, radius of the binding site,
the presence/absence of a water molecule (HOH846) within the
binding site, number of genetic algorithm (GA) runs36–39. The dock-
ing simulations in the present study were performed at the follow-
ing settings: scoring function ChemPLP, flexible ligand, rigid protein,
radius of the binding site 6 Å, no water molecule, 10 GA runs. The
top 20 best-scored compounds were selected and docked five
times with 100 GA runs each. The top 10 best-scored compounds
from all runs were selected for tests.
Theophylline, progesterone, and propranolol were used as controls
for BBB permeability41.
Neurotoxicity test
Murine neuroblastoma NEURO-2 A cells (German collection DSMZ,
Braunschweig, Germany) were cultivated under standard conditions:
complete medium (90% DMEM, 10% heat-inactivated FBS, and
1 ꢂ non-essential amino acids); 37 ꢀC and5% CO2 in fully humidified
atmosphere. The cell line was kept in the logarithmic growth phase
by splitting 1:4 once a week using trypsin/EDTA. About 30% of the
cells grow like neuronal cells. For the experimental evaluation of
the cytotoxicity NEURO-2A, cells were plated in 96-well flat-bot-
tomed cell culture plates at the recommended density of 1 ꢂ 106
cells/25 cm2. After 24 h, the cells were treated with various concen-
trations of the investigational compounds and after 72-h incubation,
a MTT-dye reduction assay was performed42. Briefly, at the end of
incubation, a MTT stock solution (10 mg/ml in PBS) was added
(10 mL/well). Plates were further incubated at 37 ꢀC for 4 h. Next, the
formazan crystals were dissolved by the addition of 110 mL/well 5%
formic acid in 2-propanol (v/v). Absorption was measured at 580 nm
wavelength on an automated ELISA reader Labexim LMR1. At least
six wells per concentration were used, and data were processed
using the GraphPad Prism 5.0 software 2.
Compounds
N-Methyl-3-(2-oxo-1-pyridyl)-N-[(2-phenylphenyl)methyl]propana-
mide (1, ZINC72065926) and N-[3-(benzimidazol-1-yl)propyl]-3-
indol-1-yl-propanamide (2, ZINC71804814) were purchased from
AKos GmbH, Germany. 3-(6-Oxo-3-phenylpyridazin-1(6H)-yl)-N-
phenethylpropanamide (3, ZINC00220177), 1-[3-(2-fluorophenyl)-
1-methyl-pyrazol-4-yl]-N-(6-quinolylmethyl)
ZINC23159164),
methanamine
(4,
N1-((1-methyl-3-phenyl-1H-pyrazol-4-yl)methyl)
piperidine-1,4-dicarboxamide (6, ZINC96007907), N-((1-methyl-3-
(pyridin-3-yl)-1H-pyrazol-4-yl)methyl)-2-(quinolin-8-yl)ethanamine (7,
ZINC97159977), 2–(2,4-dioxoquinazolin-1-yl)-N-[2-(3-fluorophenyl)
ethyl]acetamide (9, ZINC08993868) and N-[2-(4-hydroxyphenyl)
ethyl]-2-(1-isopropylindol-3-yl)acetamide (10, ZINC96116182) were
obtained from MolPort SIA, Latvia. The purchased compounds
arrived with analytical data for identity and purity. Compounds 2-
indol-1-yl-N-[2-(8-quinolyloxy)ethyl]acetamide (5, ZINC44455618)
and N-((1S,2S)-1-(4-aminophenyl)-1,3-dihydroxypropan-2-yl)-2-(4-
methoxyphenyl)acetamide (8, ZINC66142300) were synthesised.
Calculation of physicochemical properties and prediction of
pharmacokinetic (PK) parameters
The main physicochemical properties pKa, logP, logD7.4, polar sur-
face area (PSA), number of hydrogen-bond donors (HBD), and
hydrogen-bond acceptors (HBA) in the molecules of the tested
compounds were calculated using ACD/logD v. 9.08 (ACD Inc.,
Canada). The fraction ionised as a base at pH ¼ 7.4 (fB) was calcu-
lated according to the equation:
Isothermal titration calorimetry (ITC) protocol
The ITC measurements40 were performed on NanoITC tool (TA
Instruments, Lindon, UT) with 190 mL sample cell and 50 mL syringe.
The lyophilised AChE from Electrophorus electricus (electric eel)
(Sigma Aldrich, St. Luis, MO) was reconstructed in 50mM TRIS-HCl
pH 7.4 buffer to obtain ca. 1140 U/mL with the addition of 0.1%
BSA as an enzyme stabilising factor, according to the manufacturer’s
instructions. The tested compounds were prepared in 5 mM stock
solutions in ethanol and diluted to 0.5mM in 50 mM Tris-HCl pH 7.4
buffer. All samples were degassed prior the experiments. The AChE
solution was placed into the sample cell and titrated by the tested
compounds in 25 steps of 2 mL at 5 min intervals at 25 ꢀC. The blank
samples (buffer lacking AChE) were titrated at the same conditions.
The corresponding Kd values were calculated using NanoAnalyze
software (TA Instruments, Lindon, UT).
1
fB ¼
:
1 þ 10ð7:4ꢁpK Þ
a
The key PK parameters were predicted by quantitative structur-
e–activity relationships (QSPkRs) models, derived previously43–46
.
Briefly, the PK parameters of 145 neutral and/or 262 basic drugs47
were used to derive QSPkR models by multiple linear regression
(MLR) with MDL QSAR v. 2.2 (MDL Information Systems Inc., San
Leandro, CA). Three PK parameters were modelled: the steady-
state volume of distribution (VDss), free fraction of drug in plasma
(fu), and unbound clearance (CLu). The total clearance (CL) and
half-life (t1/2) were calculated following the equations.
CL ¼ CLu ꢂ fu
ln2 ꢂ VDss
t1=2
¼
CL
Parallel artificial membrane permeability assay (PAMPA)
The intestinal and BBB permeabilities were measured by PAMPA good performing QSPkRs48,49. The AChEI galantamine (GAL) is
Permeability Analyzer (pION Inc.) at the following settings: given as a reference compound.
The models are statistically significant and meet the criteria for

770
I. DOYTCHINOVA ET AL.
Compounds 1–4, 6, 7, 9, and 10 were purchased; compounds
5 and 8 were synthesised.
Results
Docking-based screening of ZINC database on rhAChE
The top 10 best-scored hits by ChemPLP from the docking of
6,053,287 molecules from ZINC database on rhAChE are given in
Figure 1. The dockings were performed with flexible ligands and
rigid binding site lacking the water molecule necessary for the
binding of GAL. The ChemPLP scores are given in Table 1.
All compounds consisted of two aromatic moieties connected
by a linker of 3–7 carbon chain containing NH or NHCO group. An
exception is made by compound 6 which contain one non-aro-
matic piperidine ring in the linker. The docking poses showed that
the first aromatic moiety binds in CAS, the aliphatic chain is
stretching along the binding gorge and the second aromatic ring
binds in PAS (Figure 1).
Synthesis of compounds 5 and 8
The synthetic strategy towards the synthesis of compound 5 was
based on the formation of amide bond between 2-indoleacetic
acid 11 and quinoline derived amine 12 (Scheme 1).
The desired acid 11 was synthesised via nucleophilic substitu-
tion of methyl 2-bromoacetate with deprotonated indole, followed
by hydrolysis of the resulting methyl ester50. Reaction of 8-hydrox-
yquinoline with N-Boc protected 2-bromoethanamine in the pres-
ence of Cs2CO3 as a base, and subsequent deprotection of the
amine afforded 2-(quinolin-8-yloxy)ethanamine 12 in excellent
yield. The coupling between the two building blocks was achieved
Figure 1. Structures of the top 10 best-scored hits. The fragments binding in CAS are given by ellipse.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
771
Table 1. Docking score, eeAChE affinity, neurotoxicity, BBB, and GIT permeability of the tested compounds.
ID
ZINC ID
ChemPLP score
Kd lM ITC
IC50 lM Neuro2A
pPe pH 7.4 BBB PAMPA
pPe pH 6.2 GIT PAMPA
1
2
3
4
5
6
7
8
72065926
71804814
00220177
23159164
44455618
96007907
97159977
66142300
08993868
96116182
104.58
101.17
101.11
100.79
99.89
99.64
99.62
99.02
98.69
0.525
0.618
0.555
0.618
0.517
0.613
0.577
0.682
>100
>100
>100
35
4.540
4.452
4.294
4.258
4.146
5.008
4.267
4.731
4.628
4.311
5.06039
4.268
4.339
4.207
4.251
4.230
4.428
4.246
4.469
4.361
4.202
4.268
>100
>100
>100
>100
>100
53
9
10
GAL
Non-binder
0.735
388.2
98.64
74.56039
>5039
Compounds with pPe < 5 are considered as highly permeable, with pPe between 5 and 6 – as medium permeable and with pPe > 6 – as
low permeable.
The Kd values of the tested compounds are given in Table 1.
Compound 9 does not interact with eeAChE. For the rest, the Kds
are in the range of 0.517–0.735 mM.
Estimation of GIT and BBB permeability by PAMPA
The permeability of the compounds tested by PAMPA shows that
all of them are highly permeable through the GIT and BBB
(Table 1). Only compound 6 has intermediate BBB permeability
with ꢁlogPe ¼ 5.008. The ꢁlogPe for the rest compounds ranges
from 4.146 to 4.731 for the BBB permeability and from 4.202 to
4.469 for the GIT permeability at pH ¼ 6.2. For comparison, GAL
has ꢁlogPe 5.060 and 4.268 for BBB and GIT permeability, respect-
ively39, which corresponds well to its low ability to penetrate BBB
by passive diffusion51 and to 90% oral bioavailability52. Moderate
correlations exist between logD7.4 and BBB permeability (r ¼ 0.693)
and between logD7.4 and GIT permeability (r ¼ 0.637) of the
tested compounds.
Estimation of neurotoxicity on neuro-2A cells
The neurotoxicity of the compounds was tested on NEURO-2A
cells as described in “Materials and methods” section. All of them
are non-toxic at concentrations up to 100 mM, apart from com-
pounds 4 and 10. Compound 4 has IC50 ¼ 35 mM and compound
10 has IC50 ¼ 53 mM.
Scheme 1. Synthesis of compound 5.
applying 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
and 1-Hydroxybenzotriazole hydrate (HOBT) in dichloromethane
(Scheme 1).
Compound 8 was synthesised by coupling of the commercially
available (1S,2S)-2-amino-1-(4-nitrophenyl)propane-1,3-diol with 4-
methoxyphenylacetic acid and subsequent catalytic hydrogenation
of the nitro group to the corresponding amine (Scheme 2).
The detailed synthesis, analytical data (incl. LC-MS and elemen-
tal analyses) and copies of 1H and 13C NMR spectra for com-
pounds 5 and 8 are given in the Supplementary File.
Physicochemical properties and PK parameters
The molecular weights of the tested compounds vary in a short
range from 330.38 to 347.41 g/mol. The pKa values are from ꢁ0.69
to 8.14, logPs are between 0.13 and 3.65, logD7.4 s – between 0.13
and 3.42 (Table 2). According to fB, compounds 4 and 7 are mod-
erate bases, partially ionised at physiological pH, and the rest are
neutral molecules. Most of the compounds are lipophilic, only
compounds 6 and 8 are rather hydrophilic with PSAs above 80 Å2.
The number of HBDs in the molecules is up to 5 and the number
of HBAs – up to 10.
The predicted VDss values of the bases 4 and 7 are 3.17 L/kg
and 3.67 L/kg, respectively. They are considerably higher than the
VDsss of the neutral molecules, which range from 0.73 to 1.61 L/kg.
This is in a good agreement with the distribution of VDss of the
Estimation of binding affinity by ITC
The binding affinity of the best-scored compounds was tested in
vitro by ITC as described in “Materials and methods” section. AChE
from electric eel (eeAChE) was used in the measurements. The
UniProt alignment of rhAChE (UniProt: P22303) and eeAChð
(UniProt: O42275) have showed that all 17 residues forming the
binding gorges are identical39. Our previous experience has shown
drugs from the training set of bases used to derive the model41
.
The VDsss in the training set range between 0.073 and 140 L/kg,
with mean 5.90 L/kg and median 2.45 L/kg. The lower VDsss of the
neutral molecules also is in accordance with the VDsss of the train-
that the docking scores predicted for rhAChE correlate well with ing set of neutral molecules, which vary between 0.16 and 25 L/kg,
the experimental binding affinity measured on eeAChE36–39 with mean 1.94 L/kg and median 1 L/kg44. As logP is one of the
.

772
I. DOYTCHINOVA ET AL.
Scheme 2. Synthesis of compound 8.
Table 2. Calculated physicochemical properties and predicted PK parameters of the tested compounds. VDss is the volume of distribution in steady state, fu – the
fraction of free (unbound) compound in plasma, CL – total plasma clearance, t1/2 – half-life.
ID
MW (g/mol)
pKa
logP
logD7.4
PSA Å2
HBD
HBA
fB
VDss (L/kg)
fu
CL (mL/min/kg)
t1/2 (h)
1
2
3
4
5
6
7
8
346.42
346.43
347.41
346.40
345.39
341.41
343.43
330.38
341.34
336.43
287.35
0.14
5.71
2.69
3.18
1.96
3.65
2.82
0.15
1.98
0.13
1.79
3.18
1.75
2.69
3.17
1.96
3.42
2.82
0.15
1.21
0.13
1.79
3.18
1.12
40.62
51.85
61.77
42.74
56.15
93.25
55.63
104.81
78.51
54.26
41.93
0
1
1
1
1
3
1
5
2
2
1
4
5
5
4
5
7
5
6
6
4
4
0.00
0.02
0.00
0.44
0.00
0.00
0.85
0.00
0.00
0.00
0.77
1.09
1.34
1.29
3.17
0.96
0.84
3.67
0.73
1.35
1.61
2.3047
0.04
0.02
0.16
0.03
0.01
0.27
0.06
0.14
0.09
0.02
0.8347
0.59
142.66
2.48
5.47
9.82
1.28
5.45
0.59
1.68
21.23
0.11
6.02
6.69
1.13
7.57
7.78
14.27
9.27
2.24
5.3047
ꢁ0.69
7.29
3.25
2.09
8.14
4.46
ꢁ0.83
ꢁ0.67
7.92
9
10
GAL
8.30
5.6047
descriptors with high positive effect on VDss43, the hydrophilic cells, permeates easily the gastric mucosa and the BBB, 96% of the
compounds 6 and 8 have reasonably the lowest VDsss.
molecules are bound to plasma proteins. Although the VDss is
1.09 L/kg, compound 1 is cleared very slowly (CL ¼ 0.59 mL/min/
kg) and has long half-life (t1/2 ¼ 21.23 h). The docked pose of 1
into rhAChE shows that the biphenyl fragment binds in CAS, while
the oxo-pyridyl moiety is placed in PAS (Figure 2). Hydrogen
bonds are formed between Tyr337 and carbonyl oxygen atom
from the linker and between His447 and the carbonyl oxygen
atom from the oxo-pyridyl moiety. Hydrogen–p interactions exist
between Trp86, Gly121, and biphenyl, and between Phe295 and
oxo-pyridyl. Trp286, Val294, Phe297, Phe338, and His447 take part
in a network of hydrophobic interactions with the linker and the
oxo-pyridyl fragment.
Compound 2 is a weak base with logD7.4 of 3.17, high affinity
to AChE (Kd ¼ 618 nM), good permeability and absence of toxicity.
The predicted ADME properties show that 98% of compound 2
are bound to plasma proteins and the VDss is 1.34 L/kg.
Compound 2 has very intensive probably overpredicted clearance
(CL ¼143 mL/min/kg) and ultra-short half-life (t1/2 ¼ 0.11 h). The
indole moiety stacks with Trp86 in CAS, while the benzimidazol
fragment is positioned in PAS making a hydrogen bond with
Phe295 (Figure 2). Another hydrogen bond is formed between
Tyr337 and the carbonyl oxygen atom from the linker. Phe297,
Tyr341, and Phe338 are involved in hydrophobic interactions with
the linker.
The tested compounds bind extensively to plasma proteins
(PP). The predicted fus are between 0.01 and 0.27, i.e. 73–99% of
the compounds are bound to plasma proteins. Again, lipophilicity
is the major governing factor for high PP binding44. The hydro-
philic compounds 6 and 8 have the highest fus.
The predicted CL values vary between 0.59 and 9.82 mL/min/
kg. The CL of compound 2 is overpredicted, as it exceeds the max-
imum total body blood flow (80 mL/min/kg). Most of the tested
compounds are low-clearance compounds with CLs below 30% of
hepatic blood flow (QH ¼ 21 mL/min/kg)53,54. Only compounds 5
and 10 are moderate-clearance drugs with CLs around 50% of QH.
The half-lives t1/2 were predicted from the corresponding VDss
and CL values. Expectedly, the t1/2 of compound 2 is underpre-
dicted. Compounds 5 and 10 have very short t1/2 due to the low
VDss and relatively high CL. The rest of compounds have moderate
t1/2 in the range of 6–21 h, allowing convenient multiple-
dose regimens.
Discussion
The standard lead-like set of ZINC database was virtually screened
by molecular docking on rhAChE and the 10 best-scored structures
were tested in vitro for binding affinity to the enzyme, neurotox-
icity, permeability across GIT and BBB. ADME properties were pre-
dicted. Nine of the compounds bind well the enzyme with Kd in
nanomolar range, eight of them are non-toxic at concentrations
up to 100 mM. All of the tested compounds are highly permeable
across the GIT and BBB, have MWs up to 350 g/mol, logD7.4 up to
3.5 and bind extensively to plasma proteins. Most of them are
low-clearance compounds.
Compound 3 is a high binder to AChE with Kd ¼ 555 nM,
logD7.4 of 1.96, VDss of 1.29 L/kg, moderate clearance (CL
¼15.48 mL/min/kg) and half-life of 6 h. It is permeable and non-
toxic. The single phenyl ring of 3 binds in CAS stacking with
Trp86, while the phenylpiperazine moiety is placed in PAS interact-
ing with Trp286 and Tyr341 and forming a hydrogen bond with
Phe295 (Figure 2). Compound 4 binds well to the enzyme with
Compound 1 is a neutral molecule with logD7.4 of 2.69 and Kd ¼ 618 nM but because of its toxicity (IC50 ¼ 35 mM), it is not con-
high affinity to AChE (Kd ¼ 525 nM). It is non-toxic on Neuro-2 A sidered as a perspective hit for further optimisation.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
773
Figure 2. Interactions between rhAChE residues and (1) compound 1, (2) compound 2, (3) compound 3, (4) compound 5, (5) compound 6, (6) compound 7, (7) com-
pound 8, and (8) GAL in the docked complex.

774
I. DOYTCHINOVA ET AL.
Figure 2. Continued.
Compound 5 is a neutral molecule with logD7.4 of 2.82 and Kd plasma, distributed extensively with VDss of 2.30 L/kg, has moder-
of 517 nM. It is non-toxic, easily permeable with VDss ¼ 0.96 L/kg, ate clearance (CL ¼ 5.60 mL/min/kg) and half-life of 5.30 h47. GAL
binds extensively (99%) with plasma proteins, is cleared with mod-
easily crosses the intestinal mucosa (pPe GIT ¼5.060) which corre-
sponds well to its oral bioavailability of 90%52. The value of 5.060
erate rate (CL ¼9.82 mL/min/kg) and has short half-life
for pPe BBB, found in our previous study39, indicates for moderate
(t1/2 ¼ 1.13 h). The indolyl fragment inhibits CAS making p–p inter-
action with Trp86 and hydrogen–p bond with Gly121, while the
quinolyl moiety binds in PAS forming hydrogen–p bond with
Phe297 (Figure 2). The carbonyl oxygen from the linker makes
hydrogen bonds with Tyr124 and Ser125.
ability to cross the BBB by passive diffusion but mediation by cho-
line transport system has been suggested55. GAL binds mainly in
CAS; only the methoxy group interacts with Phe295, Phe297 and
Phe338 from PAS. The pose presented in Figure 2 has RMSD of
1.0396 Å from the X-ray data of the complex GAL-AChE35 which is
a good validation of the docking protocol used in the pre-
sent study.
Compound 6 binds tightly to eeAChE with Kd of 613 nM. It is a
neutral molecule with logD7.4 of 0.15, VDss ¼ 0.84 L/kg, 73% is
bound to plasma proteins, has low clearance (CL ¼1.28 mL/min/
kg) and half-life of 7.57 h. The phenyl-pyrazolyl moiety is bound in
CAS making hydrogen bond with Ser203, while the piperidine
fragment is oriented towards the PAS and binds hydrophobically
to Trp286 and Val294 (Figure 2).
Compound 7 is a weak base partially protonated at pH 7.4
with logD7.4 of 1.21. Binds to AChE with Kd of 577 nM. It absorbs
easily through the intestinal mucosa, crosses the BBB, distributes
extensively with VDss of 3.67 L/kg, is cleared moderately
(CL ¼ 5.45 mL/min/kg) and has half-life of 7.78 h. The quinolyl
fragment enters CAS interacting with Tyr341, while the pyridyl-
pyrazolyl moiety makes hydrogen bond with Ser203 near PAS
(Figure 2).
Conclusions
The virtual screening on ZINC database and the following in vitro
tests and PK predictions featured seven new hits for acetylcholin-
esterase inhibition (compounds 1, 2, 3, 5, 6, 7, and 8). The hits are
non-toxic, GIT and BBB permeable and bind the enzyme AChE
with nanomolar affinity. They could be considered for further lead
optimisation.
Disclosure statement
Compound 8 binds to eeAChE with Kd ¼ 682 nM. It is a neutral
molecule with small VDss, slow clearance (CL ¼ 0.59 mL/min/kg)
and half-life of 14.27 h. The aminophenyl binds in CAS, while the
methoxy-benzene is placed in PAS. Hydrogen bonds are formed
between Ser125 and oxygen from the CH2OH group and between
Tyr124 and the carbonyl oxygen from the linker (Figure 2).
Compound 9 is a non-binder and compound 10 is toxic on
Neuro-2A (IC50 ¼ 53 nM). Both of them are not considered as pro-
spective hits for AChE inhibition.
The authors report no declarations of interest.
Funding
This work was supported by the Medical Research Council,
Medical University of Sofia, Bulgaria [Grant 10-S/2016] and the
Bulgarian National Science Fund [Grant DN03/9/2016].
GAL is a well-known AChE inhibitor. The Kd found by ITC is
388.2 mM. It is a weak base with pKa ¼ 7.92, partially protonated at
ORCID
pH 7.4. Most of it (83%) exists as a free, non-bound fraction in Irini Doytchinova
http://orcid.org/0000-0002-1469-1768

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
775
quinone-tryptophan hybrid aggregation inhibitor. PLoS One
2010;5:e11101.
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