Molecules 23 02192

molecules
Article
The Targeted Pesticides as Acetylcholinesterase
Inhibitors: Comprehensive Cross-Organism
Molecular Modelling Studies Performed to Anticipate
the Pharmacology of Harmfulness to Humans In Vitro
Milan Mladenović 1, *, Biljana B. Arsić 2,3 ID , Nevena Stanković 1 , Nezrina Mihović 1 ,
Rino Ragno 4,5 ID , Andrew Regan 6 , Jelena S. Milićević 7 ID , Tatjana M. Trtić-Petrović 7 and
Ružica Micić 8
1 Kragujevac Center for Computational Biochemistry, Faculty of Science, University of Kragujevac, Radoja
Domanovića 12, P.O. Box 60, 34000 Kragujevac, Serbia; nevena.stankovic@pmf.kg.ac.rs (N.S.);
nezrina.mihovic@pmf.kg.ac.rs (N.M.)
2 Department of Mathematics, Faculty of Sciences and Mathematics, University of Niš, Višegradska 33,
18000 Niš, Serbia; biljana.arsic@pmf.edu.rs
3 Division of Pharmacy and Optometry, University of Manchester, Oxford Road, Manchester M13 9PT, UK
4 Rome Center for Molecular Design, Department of Drug Chemistry and Technologies, Faculty of Pharmacy
and Medicine, Sapienza Rome University, P.le A. Moro 5, 00185 Rome, Italy; rino.ragno@uniroma1.it
5 Alchemical Dynamics srl, 00125 Rome, Italy
6 School of Chemistry, University of Manchester, Oxford Road, Manchester M13 9PL, UK;
Andrew.Regan@manchester.ac.uk
7 Vinča Institute of Nuclear Sciences, University of Belgrade, P.O. Box 522, 11001 Belgrade, Serbia;
jdjordjevic@vin.bg.ac.rs (J.S.M.); ttrtic@vinca.rs (T.M.T.-P.)
8 Faculty of Sciences and Mathematics, University of Priština, Lole Ribara 29, 38220 Kosovska Mitrovica,
Serbia; ruzica.micic@pr.ac.rs
* Correspondence: mmladenovic@kg.ac.rs; Tel.: +381-34-336-223
Academic Editor: Diego Muñoz-Torrero

Received: 6 August 2018; Accepted: 24 August 2018; Published: 30 August 2018
Abstract: Commercially available pesticides were examined as Mus musculus and Homo
sapiens acetylcholinesterase (mAChE and hAChE) inhibitors by means of ligand-based (LB) and
structure-based (SB) in silico approaches. Initially, the crystal structures of simazine, monocrotophos,
dimethoate, and acetamiprid were reproduced using various force fields. Subsequently, LB alignment
rules were assessed and applied to determine the inter synaptic conformations of atrazine, propazine,
carbofuran, carbaryl, tebufenozide, imidacloprid, diuron, monuron, and linuron. Afterwards,
molecular docking and dynamics SB studies were performed on either mAChE or hAChE, to predict
the listed pesticides’ binding modes. Calculated energies of global minima (Eglob_min ) and free
energies of binding (∆Gbinding ) were correlated with the pesticides’ acute toxicities (i.e., the LD50
values) against mice, as well to generate the model that could predict the LD50 s against humans.
Although for most of the pesticides the low Eglob_min correlates with the high acute toxicity, it is the
∆Gbinding that conditions the LD50 values for all the evaluated pesticides. Derived pLD50 = f (∆Gbinding )
mAChE model may predict the pLD50 against hAChE, too. The hAChE inhibition by atrazine,
propazine, and simazine (the most toxic pesticides) was elucidated by SB quantum mechanics (QM)
DFT mechanistic and concentration-dependent kinetic studies, enriching the knowledge for design of
less toxic pesticides.
Keywords: pesticides; AChE; conformational analysis; QSAR; molecular docking; molecular

dynamics; quantum-chemical studies; concentration-dependent kinetic studies
Molecules 2018, 23, 2192; doi:10.3390/molecules23092192 www.mdpi.com/journal/molecules

Molecules 2018, 23, 2192 2 of 37
1. Introduction
Pesticides [1] are either chemical or biological agents, widely used in agriculture [2], that
deter, incapacitate, kill, or otherwise discourage pests. Their inappropriate administration leads to
contaminated food while intentional release pollutes the environment, especially the soil, groundwater
and natural waterways [3]. The uptake of contaminated food and water is potentially toxic to humans
and other species. Pesticides mostly, exert toxicity by inhibiting the enzyme acetylcholinesterase
(AChE) [4] which degrades acetylcholine (ACh), an essential neurotransmitter in the central nervous
system (CNS) of insects, rodents, and humans [5]. AChE competitive inhibitors interrupt the
physiology of autonomic ganglia, as well as, neuromuscular, parasympathetic and sympathetic
effector junctions [6], controlled by ACh. However, little is known about the pharmacology of
pesticides acting as AChE inhibitors. This report considers the pharmacology of common agricultural
pesticides used in particular in the Western Balkans region [7,8]: atrazine, simazine, propazine,
carbofuran, monocrotophos, dimethoate, carbaryl, tebufenozide, imidacloprid, acetamiprid, diuron,
monuron, and linuron (Table 1). Despite their widespread usage, experimental crystal structures
deposited at Cambridge Crystallographic Data Centre (CCDC) are available only for simazine [9],
monocrotophos [10], dimethoate [11], and acetamiprid [12], highlighting the poor availability of either
ligand-based (CCDC deposition) or structure-based (Protein Data Bank (PDB) deposition) information
about these compounds.
Triazines, such as atrazine, simazine, and propazine [13], are used as herbicides in crabgrass
supression. Carbamates (carbofuran and carbaryl) are used against potato beetle; carbofuran was
found extremely toxic for humans [14]. Organophosphate pesticides (monocrotophos and dimethoate)
are used as insecticides [15]. Dimethoate is widely used to kill insects and as an insecticide for
crops, vegetables, orchards, as well as for residential purposes [16]. Tebufenozide (a dialkylhydrazine
pesticide) is used for plant protection [17]. Imidacloprid and acetamiprid, as neonicotinoids, are
prescribed for the control of sucking insect pests (aphids, whiteflies, plant-hoppers, and thrips)
and a number of coleopteran pests [17,18]. Diuron (a methylurea pesticide) is used to protect fruit,
cotton, sugar cane and wheat [13]. Linuron and monuron (phenylurea pesticides) are plant protection
agents [19]. Lethal doses for house and field mouse Mus musculus, for all of the above-mentioned
pesticides, are known (Table 1) [20–31]. Lethal doses for Homo sapiens were calculated (Table 1)
according to the recommendations of Reagan-Shaw et al. [32], using Equation (1):
animal Km ,
HED = animal dose (1)
human Km
where HED is human equivalent dose in mg/kg of body weight, animal dose is used dosage for the
specific model organism in mg/kg of body weight, animal Km factor is equal to 3, human Km factor is
equal to 37.
The overexposure to pesticides results in the AChE inhibition at brain and nervous system nerve
endings, as well as with other types of AChE found in the blood [33]; as a consequence, the ACh
concentration increases its effects. Although, the AChE inhibition by pesticides listed in Table 1 is
firmly established and the lethal doses are known, their behaviour in the inter-synaptic space and their
interaction within the AChE active site, are still poorly investigated. Bearing in mind that different ACh
conformations can trigger different receptors (nicotinics and muscarinics, respectively), conformational
analysis (CA, ligand-based (LB) approach) could be considered as a computational tool to predict
pesticides behaviour before their interaction with AChE [34]. Due to the lack of crystal structures of
any pesticides co-crystallized with AChE, CA can be used to gather pesticide conformations that are,
upon the interaction with AChE, going to be transformed into the bioactive ones [35].
Molecules 2018, 23, 2192 3 of 37
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Table
Table
Table1. Acetylcholine
Table
1. andand
1. Acetylcholine
Acetylcholine
1. Acetylcholinetraining
andand
Acetylcholine and set pesticides
training
training
training structures
set pesticides
set pesticides
set as well
structures
structures
pesticides as their
as well
structures oraloral
as their
as well
well acute toxicities
acute
as their
as well
well their
as (LD(LD
toxicities
oraloral
their acute
oral toxicities
50 50
acute (LD(LD
toxicities (LD
50
Table
Table
Table
Table Acetylcholine
1. 1. Acetylcholine
1.
1. and
Acetylcholine training
training
and training
training setpesticides
set pesticides
set
set pesticides
pesticides structures
structures
structures
structuresas as
as
as well
as
well astheir
as
as their
oral
their oral
acute acute toxicities
toxicities
acute (LD
toxicities 50 (LD
50
50
oraloral acute toxicities (LD
Table 1. Acetylcholine and training set pesticides structures as well as their oral acute toxicities (LD 50
50 50
Table
Table
values)
Table
Table1.against
values)
values) 1.
values)
Table Acetylcholine
1.
against Mus
against
Mus
Acetylcholine
1.
1. against Musand
Acetylcholine
and
Acetylcholine training
and
musculus
Mus
musculus (upper
musculus
training
and training
musculus
Acetylcholine and setvalues);
training
(upper
(upper set
(upper
training pesticides
set
values);
pesticides
set
setvalues); structures
pesticides
training
values); structures
training
training set
set
structures
training
pesticides as pesticides
as
set
structures well
as
pesticides
set
pesticides
well
as as calculated
well
as
well
pesticides
as well their
as
their
as
as their
oral
their acute
oral
calculated
calculated
calculated
their oral
oral
acute
oral
oral toxicities
acute
acute
oral
acute
oral
acute (LD
toxicities (LD
toxicities
acute toxicities
toxicities
toxicities
acute (LD
toxicities
acute
toxicities 50
(LD 50
toxicities
(LD
50 50
values)
values)
values)
values) against
against
values) Mus
Mus
against
against
against Mus musculus
musculus
Mus
Mus (upper
musculus
musculus
musculus (upper
(upper
(upper
(upper values);
values);training
values);
values);
values); trainingset
training
training
training set pesticides
set
set
set pesticides
pesticides
pesticides
pesticides calculated
calculated
calculated
calculated oral
calculated
oral acute
oral
oraloral toxicities
acute
acute
acute
acute toxicities
50
toxicities
toxicities
50
toxicities
values)
(LD
values)
(LD 50against
values)
50 values)
(LD(LD
50 values)
values)against
values)
50 values) Mus
against
against
values) Mus
against musculus
Mus
Homo
against
against musculus
against
against Mus
Mus Homo (upper
musculus
sapiens
Homo
Homo (upper
(upper
musculus
musculus (lower
sapiens
sapiens
sapiens
(upper values); training
values);
values,
(lower
(lower values,
values);
(lower
(upper training
bold
values,
bold
training
values,
values);
values); setand
and
bold
and
set
bold
training
training pesticides
set pesticides
italic)
italic) [32].
setitalic)
pesticides
and
set italic) calculated
[32].[32]. calculated
calculated
[32].
pesticides
calculatedoraloral
oral acute
oral
acute
oral toxicities
acute toxicities
toxicities
acute
acute toxicities
toxicities
(LD
(LD (LD values)
values)
50
50(LD
against
values)
against
50 values) Homo
against
Homo
against sapiens
Homo
Homosapiens (lower
sapiens
sapiens (lower values,
(lower
(lower bold
values,
values,
values, and
bold
bold italic)
and
and [32].
italic) [32].
italic) [32].
(LD
(LD 50 values)
50
50 values)
(LD
(LD(LD
against
against
50 values)
Homo
Homo
against sapiens
sapiens
Homo (lower
(lower
sapiens values,
values,
(lower boldbold
bold
values, andand
and
bold italic)
italic)
italic)
and [32].[32].
[32].
italic) [32].
50 values)
(LD
50 50 against
50values)
values) Homo
against
against sapiens
Homo
Homo (lower
sapiens
sapiens
LD values,
(lower
(lower
LD boldbold
values,
values, andand
bold italic)
and [32].[32].
italic)
italic) [32]. LD LD
Pesticide
Pesticide Structure
Structure LD50
LD
50
LD50
50 50 Ref. Pesticide Structure LD50
LD
50
LD50
50 50 Ref.
Pesticide
Pesticide
Pesticide
Pesticide Structure
Structure (mg/kg)
Structure
Structure LD
LD
LD
LD
50
LD
50
50
(mg/kg) Ref.Ref.
Ref.Ref.
Pesticide
Pesticide
Ref. Pesticide
Pesticide
Pesticide
Structure
Structure
Structure
Structure
Structure LD LD
LD
50
(mg/kg)
50
50
(mg/kg)
LD LD Ref.Ref.
Ref.Ref.
Ref.
Pesticide
Pesticide
Pesticide
Pesticide Structure
Structure
Structure
Structure (mg/kg)
LD
LD
(mg/kg)
50
LD
50
50
LD5050 Ref.
(mg/kg)
(mg/kg)
50
50 Ref.Ref.
Ref. Pesticide
Pesticide
Pesticide
Pesticide Structure
Structure
Structure
Structure (mg/kg)
LD 50
LD
(mg/kg)
50
50
(mg/kg)
50 50
50 Ref.
50 Ref.
(mg/kg)
LD Ref.Ref.
Pesticide
Pesticide
Pesticide
Pesticide Structure
Structure
Structure (mg/kg)
Structure (mg/kg)
(mg/kg)
(mg/kg) Ref.
Ref.Ref.
Ref. Pesticide
Pesticide
Pesticide
Pesticide Structure
Structure
Structure (mg/kg)
(mg/kg)
(mg/kg)
(mg/kg) Ref. Ref.
Ref.
Ref.
(mg/kg)
(mg/kg)
(mg/kg)
(mg/kg) (mg/kg)
(mg/kg)
(mg/kg)
(mg/kg)
0.850
0.850
0.8500.850 5000
50005000
5000
atrazine
atrazine
atrazine
atrazine 0.850
0.850
0.850
0.850 [20]
[20][20] tebufenozide
tebufenozide
tebufenozide
tebufenozide
[20]tebufenozide 5000
50005000
5000 [29]
[29][29]
[29]
atrazine
atrazine
atrazine
atrazine 0.0689
0.0689
0.850
0.06890.850
0.0689
0.850
0.850 [20]
[20][20]
[20] tebufenozide
tebufenozide
tebufenozide 405.40
405.40
50005000
405.40
405.40
50005000 [29]
[29][29]
[29]
atrazine
atrazine 0.850
0.850
0.0689
0.0689
0.0689 [20][20] tebufenozide
tebufenozide 5000
405.40
405.40
405.40 [29][29]
atrazine
atrazine
atrazine
atrazine 0.0689
0.0689
0.0689 [20] [20]tebufenozide
[20]
[20] tebufenozide
tebufenozide
tebufenozide 405.40
405.40
405.40 [29][29]
[29]
[29]
0.0689
0.0689
0.0689
0.0689 405.40
405.40
405.40
3.18
3.183.18
3.18 131
131131
131
propazine
propazine
propazine
propazine 3.18
3.183.18
3.18 [21]
[21][21] imidacloprid
imidacloprid
imidacloprid
imidacloprid
[21]imidacloprid 131
131 131
131 [30]
[30][30]
[30]
propazine
propazine
propazine
propazine 0.258
3.180.258
3.18
0.2583.18
0.258
3.18 [21]
[21][21]
[21] imidacloprid
imidacloprid
imidacloprid 10.62
13110.62
10.62
131 131
10.62
131 [30]
[30][30]
[30]
propazine
propazine 3.18
0.258 3.18
0.258
0.258 [21][21] imidacloprid
imidacloprid 10.62131
10.62
10.62 [30][30]
propazine
propazine
propazine
propazine 0.258
0.2580.258 [21] [21]imidacloprid
[21]
[21] imidacloprid
imidacloprid
imidacloprid 10.62
10.62
10.62 [30][30]
[30]
[30]
0.258
0.258
0.258
0.258 10.62
10.62
10.62
5 5 18
18 18
simazine
simazine
simazine
simazine 555 555 [24]
[24][24] acetamiprid
acetamiprid
acetamiprid
acetamiprid
[24]acetamiprid 18
18
18
18
18 [31]
[31][31]
[31]
simazine
simazine
simazine 0.405
5550.405
0.405 555 [24][24]
[24] acetamiprid
acetamiprid 14.92
1814.92
18
14.92 18 [31][31]
simazine
simazine
simazine
simazine
simazine
simazine
0.405
0.405
0.405
0.405
0.405
[24]
[24][24]
[24]
[24]
acetamiprid
acetamiprid
acetamiprid
acetamiprid
[24]acetamiprid
[24] acetamiprid
acetamiprid
14.92
14.9218
18
14.92
14.92
14.92 [31][31]
[31]
[31][31]
[31]
[31]
[31]
0.405
0.405
0.405 0.405
0.405
0.405 14.92
14.92
14.92
14.92
14.92
O
O O
O O O
O O
N O NO O O 2 2 500
carbofuran
carbofuran
N
H OO
O HN OOO
2222 222 [25] diuron
diuron 500500
500 500 [22]
carbofuran
carbofuran
carbofuran
N
H N
N
H
N
H
O
H
O O O O 0.162 [25][25]
[25][25] diuron
diuron
diuron 500
40.54
500
500 [22][22]
[22][22]
carbofuran
carbofuran
carbofuran
carbofuran
carbofuran
N H
H
N
H N
O
N
O
N O
H
O
O
O
O
O
O
O 22 0.162
0.162
0.162 222
0.162
0.162 [25][25]
[25]
[25]
[25][25]
diuron
diuron
diuron
diuron
diuron
diuron
500
50040.54
40.54500
40.54
40.54500
500
40.54 [22]
[22]
[22]
[22]
[22]
[22]
carbofuran
carbofuran
carbofuran H HH O O 0.162
0.162
0.162 0.162
0.162 [25][25]
[25] diuron
diuron
diuron 40.54
40.54
40.54
40.54 [22][22]
[22]
O O
O 0.162 0.162 40.54
40.54
0.162 40.54
14
14 1414 1700
17001700
1700
monocrotophos
monocrotophos
monocrotophos
monocrotophos 14 14
14 14
14 [26]
[26][26]
[26] monuron
monuron
monuron
monuron 1700
17001700
1700 [23]
[23][23]
[23]
monocrotophos
monocrotophos
monocrotophos
monocrotophos
monocrotophos 1.135
141.135
1.135
14 14
1.135
14 [26]
[26][26]
[26]
[26] monuron
monuron
monuron
monuron
monuron 437.84
437.84
17001700
437.84
437.84
17001700 [23]
[23][23]
[23]
[23]
monocrotophos
monocrotophos 1.135
1.135 14
1.135
1.135 [26][26] monuron
monuron 1700
437.84
437.84
437.84 [23][23]
monocrotophos
monocrotophos
monocrotophos 1.135
1.1351.135 [26][26]
[26] monuron
monuron
monuron 437.84
437.84
437.84 [23][23]
[23]
1.135
1.135
1.135 437.84
437.84
437.84
60
60 60
60 60 2400
24002400
2400
dimethoate
dimethoate
dimethoate
dimethoate 60
60 60
60 [27]
[27][27]
[27]
[27] linuron
linuron
linuron
linuron
linuron 2400
24002400
2400 [19]
[19][19]
[19]
[19]
dimethoate
dimethoate
dimethoate 4.86
60
4.864.86
4.8660 [27][27] linuron
linuron
linuron 194.59
2400
194.59
2400
194.59
194.59 [19][19]
dimethoate
dimethoate
dimethoate 60
4.864.86
60
60
4.86 [27][27]
[27][27] linuron
linuron
linuron 24002400
2400
194.59
194.59 [19][19]
[19][19]
dimethoate
dimethoate
dimethoate 4.864.86
4.86
4.864.86
4.86
4.86
[27][27]
[27] linuron
linuron
linuron 194.59
194.59
194.59
194.59
194.59
194.59
194.59
[19][19]
[19]
100
100100
100 100
carbaryl
carbaryl
carbaryl
carbaryl 100
100 100
100 [28]
[28][28]
[28] acetylcholine
acetylcholine
acetylcholine
acetylcholine
acetylcholine
[28]acetylcholine
carbaryl
carbaryl
carbaryl 8.11
8.11
100
8.118.11
100 [28][28] acetylcholine
acetylcholine
carbaryl
carbaryl
carbaryl 100
8.118.11
100
100
8.11 [28][28]
[28][28] acetylcholine
acetylcholine
acetylcholine
carbaryl
carbaryl
carbaryl 8.118.11
8.11
8.118.11
8.11
8.11
[28][28]acetylcholine
[28] acetylcholine
acetylcholine
For either
For either mouse mouse or humans
or
or humans as aas model organism, AChE is
is aaaishomodimer (Figure 1a), formed of
For For
For
For
either
For
either
either
For
For
either
either
either
either
mouse
mouse
mouse
mouse
mouse
mouse
mouse
or
or
humans
orhumans
or or
or
humans
humans
humans
humans
humans
asas
as
as aaaas
as
as aaaaamodel
model
model
model
model
model
model
model
organism,
organism,
organism,
organism,
organism,
organism,
organism,
organism,
AChE
AChE
AChE
AChE
AChE
AChE
AChE
AChE is
is a
is
is
is
aa homodimer
homodimer
homodimer
homodimer
homodimer
aaaishomodimer
a homodimer
homodimer
(Figure
(Figure
(Figure
(Figure
(Figure
(Figure
(Figure
1a),
1a),
1a),
1a),
(Figure formed
1a),
formed
1a),
1a),
formed
formed
formed
1a),
formed
formed of
of
of
offormed
of
of
of
two
two two
twoFor
For either
For
independent
either
For
For either
independent
independent either
either
independent mouse
mouse mouse
units,
units,
mouse
mouse or
or
units, humans
or
humans
or
or humans
comprising
units, comprising
comprising
humans
humans
comprising as
as aa
614
614 as
asmodel
model
as614aa
amino
614
amino
a model
amino
model
model organism,
organism,
acids
acids
organism,
amino [36].
acids
[36].
organism,
organism,
acids AChE
The
[36].
The
AChE
[36]. AChE is
AChE
The
AChE
AChE
AChE
Theis aa
AChEis
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pesticides’
pesticides’
from pesticides’ acute
acute
pre-bound acute
acute toxicity
toxicity
toxicity
conformations,
toxicity be
be be be quantified
quantified
quantified
be before
quantified from
from from
the
from the
thethe
actual thebound
bound
the bound
interaction
bound
interaction
interaction
interaction with
withAChE? AChE?
AChE? (2) can
(2) can
canthe thepesticides’
pesticides’
pesticides’ acute acute
acute toxicity
toxicity
toxicity be quantified
be quantified
quantified from from
from the bound
the bound
bound
with AChE? (2) can the pesticides’ acute toxicity be quantified from the bound conformations, upon
the actual interaction with AChE? To the best of our knowledge, there are no previous reports on
Molecules2018,
Molecules 2018,23,
23,2192
x 44 of
of 37
37
conformations, upon the actual interaction with AChE? To the best of our knowledge, there are no
either LB or
previous SB efforts
reports to predict
on either LB orthe
SBbioactive
efforts toconformations of investigated
predict the bioactive pesticides
conformations of (Table 1) for
investigated
both mouse and human AChEs, as well as to enumerate their acute toxicities by means
pesticides (Table 1) for both mouse and human AChEs, as well as to enumerate their acute toxicities of free energy
of
byformation
means of free (Eglob_min
energy) in
ofthe aqueous
formation (Esolution
glob_min) inand/or free energy
the aqueous solutionof and/or
bindingfree
(∆G binding )of
energy tobinding
AChE.
All
(∆Gthe cheminformatics
binding ) to AChE. All the findings were externally
cheminformatics validated
findings on the series
were externally of other
validated commonly
on the series of used
other
pesticides (Table 2), also known as AChE inhibitors (i.e., the test set).
commonly used pesticides (Table 2), also known as AChE inhibitors (i.e., the test set).
Figure 1. The
Figure 1. The crystal structure of
crystal structure hAChE (PDB
of hAChE (PDBID:
ID:4EY5)
4EY5)in incomplex
complexwith withhuperzine
huperzineAA(a).(a).Subunit
SubunitA
A is depicted in pink ribbons whereas the B subunit is coloured in orange. Huperzine A
is depicted in pink ribbons whereas the B subunit is coloured in orange. Huperzine A is presented in is presented
in black
black and
and itsitsstructure
structureisishighlighted
highlightedwith
withdashed
dashedlines.
lines. The
The anionic
anionic sub-site
sub-site is
is described
described byby blue
blue
spheres, while the esterase sub-area is encircled by red spheres. For the sake of clarity,
spheres, while the esterase sub-area is encircled by red spheres. For the sake of clarity, hydrogen hydrogen
atoms
atoms were
were omitted.
omitted. TheThe graphic
graphic was
was generated
generated using
using the
the UCSF
UCSF Chimera
Chimera software
software (Resource
(Resource for
for
Biocomputing, Visualization, and Informatics (RBVI), University of California, San
Biocomputing, Visualization, and Informatics (RBVI), University of California, San Francisco, CA, Francisco, CA,
USA).
USA). Biochemical
Biochemical mechanism
mechanismof ofACh
AChhydrolysis
hydrolysisby
byAChE
AChE(b).(b).
The test
The test set
set was
was compiled
compiled of of organophosphates,
organophosphates, carbamatescarbamates and
and structurally
structurally non-classified
non-classified
pesticides.
pesticides. Regarding organophosphates, chlorpyrifos [37], DDVP [14], fenitrothion [38], azinphos-
methyl [39],
methyl [39], naled
naled (dibrom)
(dibrom) [40],
[40], TCVP
TCVP [41],[41], parathion [39], methyl parathion [42], diazinon [43],
phosmet [14],
phosmet [14], azamethiphos
azamethiphos [44],
[44], and
and terbufos
terbufos [45],
[45], respectively,
respectively, are
are used
used as
as insecticides.
insecticides. Moreover,
glyphosate is
glyphosate is aa broad-spectrum
broad-spectrum systemic
systemic herbicide
herbicide and and crop
crop desiccant
desiccant [46],
[46], malathion
malathion isis mostly
mostly used
used
for mosquito
for mosquito eradication
eradication [47],
[47], parathion
parathion isis used
used asas ananacaricide.
acaricide. Phosmet
Phosmet is is mainly
mainly used
used onon apple
apple
trees to
trees to control
control codling
codling moth
moth and
and onon aa numerous
numerous fruitfruit crops,
crops, ornamentals,
ornamentals, andand wines
wines for
for the
the control
control
of aphids,
of aphids, suckers,
suckers, mites,
mites, and
and fruit
fruit flies.
flies. Terbufos
Terbufosisisaa nematicide
nematicideusedused on
on corn,
corn, sugar
sugar beets
beets and
and grain
grain
sorghum,and
sorghum, andit it is extremely
is extremely toxictoxic to birds.
to birds. On theOn thehand,
other othercarbamate
hand, carbamate
pesticides,pesticides, such as
such as methomyl
methomyl
and and methiocarb
methiocarb are used
are used against against
insects; insects;
oxamyl oxamyl
is an is an extremely
extremely toxicto
toxic pesticide pesticide
humans, tofish,
humans,
and
fish, and birds [48–50], whereas methiocarb is additionally used as a bird repellent, acaricide and
Molecules 2018, 23, 2192 5 of 37
birds [48–50], whereas methiocarb is additionally used as a bird repellent, acaricide and molluscicide.
Molecules
Molecules 2018,
2018, 23,
Molecules 23, x 23,
2018, 23, x 5 of 37 555 of
of 37 37
Molecules 2018,
Molecules
Molecules xxx
2018,
2018, 23, xxxx 5555 of
of 37
of 37
37
Among
Molecules
Molecules
Moleculesthe structurally
2018,
2018,
Molecules
2018, 23,
23,
2018,
23, x 23,
23, non-classified pesticides, 2,4-D is widely used as a herbicide, which 37 55 of
ofmimics
of 37
37 of 37 the
action of the plant
molluscicide. growth
Among hormone
the auxin,
structurally resulting
non-classified in the uncontrolled
pesticides, 2,4-D is growth
widely and
used eventual
as death in
molluscicide.
molluscicide.
molluscicide. Among
Among
molluscicide.
molluscicide.
molluscicide. Among
molluscicide.
molluscicide. Among
Among
Among
Among
Among
the
the
the the
the structurally
structurally
the structurally
the structurally
structurally
the structurally
structurally
non-classified
non-classified
non-classified
structurally non-classified
non-classified
non-classified
non-classified
pesticides,
pesticides,
pesticides,
non-classified 2,4-D
2,4-D
pesticides,
pesticides,
pesticides, 2,4-D
is
is
2,4-D2,4-D
2,4-D
pesticides,
pesticides, is
2,4-D
is
widely
is widely
2,4-D widely
used
used
is widely
is
widely
is
widely widely
used
widely
used
as
as
used
used used
as
used
as as aaaaa herbicide,
as
aaa herbicide,
a herbicide,
as
herbicide,
as
herbicide,
herbicide,
herbicide,
herbicide,
herbicide,
susceptible
which
which which
which plants
mimics
mimics mimics
mimics
the
the [14];
the
the
action DDT
action
action
of is
the originally
of
of the
the
plant plant
plantdeveloped
growthgrowth
growth
hormone as
hormone
hormonean
auxin,insecticide,
auxin,
auxin, used
resulting
resulting
resulting in the to
in
in control
the
the malaria
uncontrolled
uncontrolled
uncontrolled growth and
growth
growthtyphus,
which which
whichwhich
mimics
which
mimics the action
mimics
mimics
the
mimics the
action
the
action of
of the
the action
action
of
actionthe ofplant
theof
of the
plant
the
plant growth
the plant
plant
growth
plant
growth hormone
growth
growth
hormone
growth
hormonehormoneauxin,
hormone
hormoneauxin, resulting
auxin,
auxin,auxin,
resulting
auxin, in
in the
resulting
resulting
in
resulting
resulting thein
the uncontrolled
in the uncontrolled
the uncontrolled
uncontrolled
in the uncontrolled
uncontrolled growth
growth
growth growth
growth
growth
and
and
andas and
a
and
eventual
eventualeventual
contact
eventual
death
death
and eventual
eventual death
poison
death
in
in
death in
in susceptible
against
susceptible
susceptible several
susceptible
in susceptibleplants
plants
susceptible plants
plants
[14];
[14];
plants [14];
arthropods
[14];
DDT
DDT DDT
DDT
is
is
[14]; is
DDT is
[51];
is originally
originally
originally DEET
originally is developed
developed
developed
is originally
originally the most
developed
as
as
developed an
an as an
common
as an insecticide,
insecticide,
insecticide,
as active
insecticide,
an insecticide,used
used
insecticide, used
ingredient
used
to
to
used to to
to
to
and and
and eventual
and
eventual deathdeath
death
eventual in
death in
in susceptible
in plantsplants
plants
susceptible
susceptible [14];
plants [14];
[14]; DDT
[14];
DDT DDT
DDT
is is
originally
is originally
originally developed
developed as
developed
developed an as
as an as an
insecticide,
an used
used to
insecticide,
insecticide, used
used
to to
control
control
incontrol
insect
control malaria
control malaria
repellents
malaria
control and
malaria
and
malaria for and
typhus,
and
typhus,
and typhus,
and
typhus,
protection
and
typhus, and
as
as a
and
a
and as a
againstcontact
contact
as a
contact
as a poison
contact poison
against
poison
mosquitoes,
poison
contact against
poison against
several
against
ticks,
several
against several
fleas, arthropods
arthropods
several chiggers,
arthropods
several [51];
arthropods
[51];
arthropods [51];
DEET
[51];
leeches
DEET
[51]; DEET
is
DEET
is the
and
DEET the is the
most
is the
many
most
is the most
most
mostbiting
control
control malaria
control
malariamalaria
and
malaria and
and typhus, typhus,
and
and as
and typhus,
typhus, and
asand as a contact
aa contact poison
as a contact
contact poison poison
against
poison
against against
several
against
severalseveral
several arthropods
arthropods [51]; [51];
[51]; DEET
arthropods
arthropods DEET
[51]; DEET
DEET is
is the
the mostisis the most
the most
most
common
common
insects common
common
active
[52].
common active
activeactive
active
ingredient
active ingredient
ingredient in
ingredient in
in insect
ingredient insect
in insect repellents
repellents
insect
repellents
in insect
insect for
repellents for
for protection
for protection
repellentsprotection against
protection
for protectionagainst
protection against
against
against mosquitoes,
mosquitoes,
mosquitoes,
mosquitoes, ticks,
mosquitoes, ticks,
ticks, ticks,
fleas,
ticks,
fleas,
ticks, fleas,
chiggers,
fleas,
chiggers,
fleas, chiggers,
chiggers,
chiggers,
common
commoncommonactive
common ingredient
activeactive ingredient
ingredient in
ingredient in
in insect
in
insect insect repellents
repellents for
repellents
repellents for
for protection
for against
protection
protection againstagainst
against mosquitoes,
mosquitoes, ticks,
mosquitoes,
mosquitoes, fleas, fleas,
ticks, fleas,
ticks, chiggers,
fleas,
chiggers, chiggers,
chiggers,
leeches
leechesleeches
and
leeches and
and many
leeches many
and many
many
biting
and many
many biting
bitingbiting
insects
biting
insects
biting insects
insects
[52].
insects [52].
[52]. [52].
[52].
[52].
leeches
leechesleeches
and
leeches and
and many
and
many biting
many insects
bitingbiting
insects insects
[52].
insects
[52]. [52].
Table 2. Test set pesticides structures and their oral acute toxicities (LD50 values) against Mus musculus
Table
Table Table
2.
2. Test
Table
Test
Table 2.
set
2.
set
2. Test set pesticides
pesticides
Test set
pesticides
Test setset structures
structures
pesticides
structures
pesticides and
structures
and
structures and
their
and
their their
oral
oral
andoral oral
acute
their oral
acute
their oral acute
acute
acute toxicities
toxicities (LD
toxicities
toxicities (LD 50
toxicities (LD
(LD 50 values)
50 values)
50
values)
(LD50
50 values) against
against Mus
against
against
values) Mus Mus
Mus musculus
musculus
musculus
musculus
against Mus musculus
TableTableTest2. Test set pesticides structures and their oral acute toxicities (LD 50 values) against Mus musculus
50
(upper
Table
Table values);
2.
2. Test
Test
Table
2. set
set
2.
set test
pesticides
pesticides
Test set pesticides
structures
structures
pesticides
and
and
structures
structures their
and their
and
their oral
oral
oral acute
acute
acute
their oral
acute toxicities
toxicities
toxicities
acute
toxicities (LD
(LD
(LD5050(LD 50
values)
50 values)
toxicities
50 (LD50
values)
50 values)
against
against
values) against
Mus
Mus Homo
musculus
musculus
against
against Mus Mus
musculus sapiens
musculus
(upper
(upper(upper
values);
(upper
values);
(upper values);
test
values);
test
values); set
settest set pesticides
pesticides
test set
pesticides
test set calculated
calculated
pesticides
calculated
pesticides oral
calculated
oral
calculated oral
acute
oral
acute
oral acute toxicities
toxicities
acute
toxicities
acute (LD
toxicities
(LD 50
50
toxicities (LD
values)
(LD
(LD 50
50
values)
50 values)
values) against
against Homo
against
against
values) Homo
against Homo
Homo sapiens
sapiens
sapiens
Homo sapiens
sapiens
(upper
(upper
(lower (upper
values);
values);
(uppervalues, values);
values);bold
(upper test
test
values); test
set
set set pesticides
pesticides
pesticides
andpesticides
test settest calculated
calculated
calculated
italic) [32].
set pesticides oral
oral
calculated oral
acute
acute
oral acute toxicities
toxicities
toxicities
acute (LD
(LD 50
toxicities
50 (LD 50
values)
values)
(LD 50
50 values) against
against
against
values) Homo
Homo
against
calculated oral acute toxicities (LD50 values) against Homo sapiens
50
50 50 Homo sapiens
sapiens
sapiens
Homo sapiens
(lower(lower
(lower(lower values,
values,values,
(lower
values,
(lower bold
values, bold
bold and
values,and
bold and
italic)
and
italic)
bold italic)
[32].
italic)
[32].
and italic) [32].
[32].
italic) [32].
[32].
(lower
(lower values,
values,
(lower(lower bold
bold bold
and
bold and
values,values,and and
italic)
italic)
bold and
italic) [32].
[32].
italic)
[32]. [32].
LD 50 LD
LD50 LD50
50 LD 50 LD
LD50
50
Pesticide
Pesticide Structure
Structure LD
LD 50 LD
5050 LD
50
50
50
50Ref. Ref. Pesticide
Pesticide Structure
Structure LD
LD 50 LD
50 LD
50 50
50
50
50
50Ref. Ref.
Pesticide
Pesticide
Pesticide Structure
Structure
Pesticide Structure LD50
Structure (mg/kg) 50 LD50Ref. Ref.Ref.
Ref. Pesticide
Pesticide
Pesticide
Ref. Structure
Structure
Pesticide Structure LD5050 LD50Ref.
Structure (mg/kg) Ref.
Ref.
Ref.
Pesticide
Pesticide
Pesticide
Pesticide
Pesticide Structure
Structure
Structure
Structure (mg/kg) (mg/kg)
(mg/kg) Ref.
Ref.
Ref. Ref. Pesticide
Pesticide
Pesticide
Pesticide
Pesticide Structure
Structure
Structure
Structure (mg/kg)
(mg/kg)
(mg/kg) Ref. Ref.
Ref.
Ref. Ref.
(mg/kg)
(mg/kg)
(mg/kg)
(mg/kg) (mg/kg)
(mg/kg)
(mg/kg) (mg/kg)
(mg/kg)
(mg/kg)
(mg/kg) (mg/kg)
(mg/kg)
(mg/kg)
1040
1040 1040
1040
1040 1040
1040 [44] 113 113
113 113
113
113 [53] [53]
azamethiphos
azamethiphos
azamethiphos
azamethiphos
azamethiphos 1040
1040 1040
1040 84.324 [44]
[44] [44]
[44] [44] phosmet
phosmet
[44] phosmet
phosmet
phosmetphosmet 113
113 113
113
113 9.162 [53] [53]
[53]
[53]
azamethiphos
azamethiphos
azamethiphos
azamethiphos
azamethiphos
azamethiphos 84.324
84.324 84.324 [44]
[44]
[44] phosmet
[44] phosmet
phosmet
phosmetphosmet 9.162 9.162
9.162 [53]
[53] [53]
[53] [53]
84.324 84.324 9.162 9.162
84.32484.324
84.324
84.324 84.324 9.162 9.162
9.162
9.162 9.162
azinphos-
azinphos- 7 465
azinphos-
azinphos-
azinphos-
azinphos-
azinphos-
azinphos-
azinphos-methyl
azinphos-
7777 7777 [54]
7 0.567 [54] [54]
[54] [54]TCVPTCVP
TCVP
TCVPTCVP
465
465
465
465
465
465
465
465
465 37.702 [55]
[55] [55]
[55] [55]
[55]
methyl
methyl
methyl 0.567
0.567 0.567 [54]
[54] [54]TCVP
[54] [54]
[54]TCVP TCVP
TCVPTCVP
TCVP 37.702 37.702
37.702 [55]
[55] [55]
[55] [55]
methyl
methylmethyl
methyl
methyl
methyl
methyl 0.567
0.567
0.567
0.567 0.567
0.567
0.567 37.702
37.702
37.702
37.702 37.702
37.702
37.702
2000
2000 2000
2000 2000
2000
2000 [56] 1.6
1.6 1.6
1.6
1.6
1.6
chlorpyrifos
chlorpyrifos
chlorpyrifos
chlorpyrifos
chlorpyrifos 2000
2000 2000
2000162.162 [56] [56]
[56] terbufos
terbufos
terbufos
[56] terbufos
terbufos 1.6
1.6
1.6 1.6
1.6 [57]
[57]
[57] [57]
[57] [57]
chlorpyrifos
chlorpyrifos
chlorpyrifos 162.162 [56]
[56] [56]
[56] terbufos
terbufos
terbufos 0.129[57]
0.129 [57]
chlorpyrifos
chlorpyrifos
chlorpyrifos 162.162
162.162
162.162
162.162
162.162
162.162
162.162
162.162
162.162
[56] terbufos
[56] terbufos
terbufos 0.129
0.129
0.129
0.129
0.129 0.129[57]
0.129
0.129
0.129
[57] [57]
17 17
1717 17
17
17 [58] 350 350
350 350
350
350
350
DDVPDDVP
DDVPDDVP
DDVPDDVP
DDVP 17
17 17
17 1.378 [58] [58]
[58] [58] methiocarb
methiocarb
methiocarb
methiocarb
methiocarb
[58] methiocarb 350
350 350
350 28.378 [59] [59]
[59] [59]
[59]
[59]
[59]
DDVP
DDVP
DDVPDDVP 1.378
1.378
1.378 1.378
1.378 [58] [58]
[58]
[58] [58] methiocarb
methiocarb
methiocarb
methiocarb
methiocarb 28.378
28.378 28.378
28.378[59]
[59]
[59] [59]
1.378
1.378 1.378
1.378 1.378 28.378
28.37828.378
28.37828.378
66
66
66 6666
66
66 [60] [60] 12
12 12
12
12 [61] [61]
diazinon
diazinon
diazinon 66
66 66 methomyl
methomyl
methomyl 12
12 12
12
diazinon
diazinon
diazinon
diazinon 66 [60] [60]
[60]
[60] methomyl
[60] methomyl
methomyl
methomyl
methomyl 12 0.972 [61]
[61] [61]
[61]
[61]
diazinon
diazinon
diazinon
diazinon 5.351
5.351
5.351
5.351
5.351
5.351
5.351
5.351
5.351 [60] [60]
[60] methomyl
[60] methomyl
methomyl 0.972 0.972
0.972
0.972
0.972
0.972
0.972
0.972 [61] [61]
[61] [61]
5.351 5.351 0.972 0.972
50
50 50
50
50 [62] [62] 5.4
5.4 5.4
5.4
5.4 [63] [63]
fenitrothion
fenitrothion
fenitrothion 50 50
50
50
50 50 [62] [62] oxamyl
oxamyl
oxamyl
oxamyl 5.4
5.4
5.4 5.4
5.4 [63] [63]
fenitrothion
fenitrothion
fenitrothion
fenitrothion
fenitrothion
fenitrothion 40.540 [62]
[62] [62]
[62] [62] oxamyl
oxamyl
oxamyl
oxamyl
oxamyl 0.437 [63] [63]
[63]
[63] [63]
fenitrothion
fenitrothion 40.540
40.54040.540
40.540
40.540
40.540
40.540 40.540
40.540
40.540
[62] [62] oxamyl
oxamyl 0.437
0.437
0.437
0.437
0.437 0.437[63]
0.437
0.437
0.437
[63]
5000
5000 5000
5000
5000[64] [64] 113
113 113
113
113 [14] [14]
glyphosate
glyphosate
glyphosate 5000 5000
5000
5000
5000 5000 [64] DDT DDT
DDT 113
113
113 113
113 [14]
glyphosate
glyphosate
glyphosate
glyphosate
glyphosate
glyphosate
glyphosate
glyphosate 405.405
405.405
405.405[64]
[64] [64]
[64]
[64]
[64] [64] DDT
[64]DDT
DDT
DDT DDT
DDT
DDT
DDT 9.162 9.162
9.162 [14]
[14]
[14] [14]
[14]
[14] [14]
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[36].
[36].
involves [36].
[36]. Moreover,
Moreover,
Moreover,
[36]. Moreover, methylcarbamate
methylcarbamate
methylcarbamate
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[74]. [74].
analogues
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analogues
imidacloprid [74].
analogues
analogues [74].
[74]. [74].
Molecules 2018, 23, 2192 6 of 37
molecular docking (SB) studies on Nephotettix cincticeps AChE. Docking studies were also performed
for imidacloprid analogues [74].
Therefore, to enrich the information related to the pharmacology of commonly used commercial
Molecules
Molecules 2018,
2018, 23,23,
23, xxx of 666of
ofof373737
Molecules
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Molecules 2018, 23, x 6 of 37
Molecules
by means of 2018, 23, x and reversible-like (for organophosphorus pesticides only, docking performed
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Therefore,
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2. Results and Discussion
pesticide-AChEinteractions
pesticide-AChE interactionsfor forthe thepurpose
purposeof ofdesigning
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2.
2.
2. 2.Results
Results
Results
Results and
and
andand Discussion
Discussion
Discussion
Discussion
2.1. The Conformational Analysis of Pesticides in Aqueous Solution
2.2.Results
Resultsand andDiscussion
Discussion
2.1.
2.1.2.1.The
Conformational
2.1. The
TheThe Conformational
Conformational analysis
Conformational
Conformational Analysis
(CA)of
Analysis
Analysis
Analysis ofof Pesticides
studies
Pesticides
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arein Aqueous
Aqueous
in Aqueous
Aqueous Solution
reported
Solution
Solution
Solution for simazine [9], monocrotophos [10],
2.1.
dimethoate
2.1. TheThe Conformational
[11], and
Conformational acetamiprid Analysis
Analysis of
[12]
of Pesticides
(Table
Pesticides inin
3), Aqueous
as
Aqueous Solution
pesticides
Solution with known [9], crystal structures,[10], as well
Conformational
Conformational
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Conformational analysis
analysis
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analysis (CA)
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(CA)(CA) studies
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studies
studies are
are
are are herein
herein
herein
herein reported
reported
reported
reported for
for
forfor simazine
simazine
simazine
simazine [9],
[9], [9],monocrotophos
monocrotophos
monocrotophos
monocrotophos [10],
[10],
[10],
as for atrazine
dimethoate
dimethoate
dimethoate and
Conformational
Conformational
dimethoate
carbofuran,
[11],
[11],
[11],
[11], and
and
and and analysis as
acetamiprid
acetamiprid
acetamiprid
analysis
acetamiprid
pesticides
(CA)
(CA)[12] studies
[12]
[12] (Table
(Table
[12]
studies
that
(Table
(Table are 3),
3),
are3),
exert
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as
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known
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[9],
crystal
crystal
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crystal
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monocrotophos
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4). as
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[10],
well
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[10],
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dimethoate
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atrazine
atrazine [11],
atrazine
for atrazine [11],and and
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and
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carbofuran,
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other (Table
pesticides
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as [12] pesticides
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pesticides
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pesticides 3),
that
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3), asexertexert reported
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crystal(Table (Table Material.
4).
4).
structures, To
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as for
crystalredundancy,
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redundancy,atrazine
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applied
simazine
applied
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and carbofuran,
studies
[9],
studies
studies
carbofuran,
studies to
to
toas as
to the
the
the pesticides
monocrotophos
the other
other
other
pesticides
other that
pesticides
pesticides exert
[10],
pesticides
that
pesticides are
exertare the
are
are
the highest
reported
dimethoate
reported
reported
highest
reported acute
as
[11],
as toxicity
Supplementary
and
Supplementary
as as
acute Supplementary (Table
acetamiprid 4).
Material.
toxicity (TableMaterial.
Supplementary To
Material.
[12]
Material.
4). To avoid avoid
The
(Table
The
The The 3,
redundancy,
crystal
crystal
Tablescrystal
S1–S3)
crystal
redundancy, structures
structures
were applied
used
structures
structures appliedofof
of of asstudies
simazine
simazine
starting
simazine
studies
simazine to
[9],
[9],
to [9],
[9], the other
monocrotophos
monocrotophos
conformational
the monocrotophos pesticides
other pesticides
monocrotophos [10],
[10], are
geometries
[10],
[10],are reported
dimethoate
dimethoate
dimethoate
reported as
dimethoate for as
[11],
[11],
theSupplementary
[11], and
and
CA.
and
Supplementary
[11], and acetamiprid
acetamiprid
For
acetamiprid Material.
pesticides
acetamiprid [12]
[12]
Material.
[12][12] (Table
(Table The
without
(Table
(TableThe
crystal
3,3,
3,
3, Tables
Tables
crystalTables
Tables structures
S1–S3)
S1–S3)
S1–S3)
S1–S3)
structures were
were
ofof
were
were simazine
used
used
usedused
simazine asas
as
as [9],
starting
[9], monocrotophos
starting
starting
starting conformational
conformational
conformational
conformational
monocrotophos [10],
[10], dimethoate
geometries
geometries
geometries
geometries
dimethoate for [11],
for
for for the
the
the
[11], and
the CA.
CA.
CA.
and CA. acetamiprid
For
For
For For pesticides
pesticides [12]
pesticides
pesticides
acetamiprid [12] (Table
without
without
without
without
(Table
experimental data (Table 4, Tables S1–S3), starting conformations were built from scratch and energy
3,3,
minimized.
TablesS1–S3)
experimental
experimental
experimental
Tables
experimental S1–S3)
For thedata
data
data were
data
were
sake
(Table
(Table
(Table
of
used
(Table
used 4, as
4,4,as
clarity,
starting
Tables
Tables
4, Tables
starting
Tables S1–S3),
S1–S3),
pesticides
conformational
S1–S3),
S1–S3), starting
starting
starting
conformational
starting
were
geometrieswere
conformations
conformations
conformations
conformations
divided geometries
into four
for
were
were
forwerethe
the
groups
CA.
built
built
built
CA.
built from Forscratch
from
fromfrom
For
based
pesticides
scratch
scratch
scratch
pesticides and
and
and
on their andwithout
energy
energy
withoutenergy
energy
common
experimental
minimized.
minimized.
minimized.
experimental
minimized. For
For
For data
For
data the
the
the (Table
the sake
sake
(Table
sakesake 4,of
of
of
4,
of Tables
clarity,
clarity,
clarity,
Tables
clarity, S1–S3),
pesticides
pesticides
pesticides
S1–S3),
pesticides starting
startingwere
were
werewere conformations
divided
divided
divided
conformations
divided into
into
into
into were
four
fourfour
were
four builtfrom
groups
groups
groups
built
groups from
based
based
based
based scratch
on
on on
scratch
on and
their
their
their
theirand energy
common
common
common
energy
common
structural patterns:
minimized. For 2-chloro-1,3,5-triazine
the sake of clarity, derivatives:
pesticides were simazine,
divided into fouratrazine,
groups and
based propazine
on their (Tables 3
common
structural
structural
structural patterns:
patterns:
patterns: 2-chloro-1,3,5-triazine
2-chloro-1,3,5-triazine
2-chloro-1,3,5-triazine derivatives:
derivatives:
derivatives: simazine,
simazine,
simazine, atrazine,
atrazine,
atrazine, and
and and propazine
propazine
propazine (Table
(Table
(Table 333and
3and
and and
minimized.
and 4, structural
Table
Table
Table
S1,
structural
4,4,
For
patterns:
patterns:
Table
Table
the 2-chloro-1,3,5-triazine
respectively);
S1,
S1,
sake of clarity, pesticides
amide derivatives:
respectively);
respectively); amide
amide
were divided
derivatives:
derivatives:
derivatives:
derivatives:
simazine,
monocrotophos, into atrazine,
simazine,
monocrotophos,
monocrotophos,
four groups
dimethoate,
atrazine,
and
and
based
propazine
and
dimethoate,
dimethoate,
on their
propazine and
and
(Table
carbofuran common
(Table
carbofuran
carbofuran
(Tables
3 and 3
Table
structural
Table 4, 4, Table
patterns:
Table S1,S1, respectively);
respectively); amide
amide derivatives:
derivatives:
derivatives: monocrotophos,
simazine,
monocrotophos, atrazine, dimethoate,
and
dimethoate, propazine andand
(Tablecarbofuran
carbofuran 3 and
and 4, Table
Table S2, respectively), carbaryl and tebufenozide (Table S2); 6-chloropyridine-3-yl)methanamine
(Table
(Table
(Table
Table
(Table 334,
4,3and
andTable
and
3Table
and Table
Table S1,4,
Table
TableS1, 4,respectively);
Table
4,respectively);
Table
4, Table
Table S2,
S2,
S2, S2, amide
respectively),
respectively),
respectively),
amide
respectively), derivatives:
carbaryl
carbaryl
carbaryl
derivatives:
carbaryl monocrotophos,
and
and and tebufenozide
tebufenozide
tebufenozide
monocrotophos,
and tebufenozide dimethoate,
(Table
(Table
(Table
dimethoate,
(Table S2);
S2);
S2); and carbofuran
carbofuran
6-chloropyridine-
6-chloropyridine-
S2); 6-chloropyridine-
and
6-chloropyridine-
derivates:
(Tableacetamiprid
3 and
3-yl)methanamine
3-yl)methanamine
3-yl)methanamine
(Table Table
3 and Table 4,
3-yl)methanamine (Table
4, Table3,
derivates:
derivates:
derivates:
Table
derivates: Table
S2, S3)
acetamiprid
acetamiprid and
respectively),
acetamiprid
S2, acetamiprid
respectively), imidacloprid
carbaryl
(Table
(Table
(Table
carbaryl
(Table 3,
3, and
Table
3, 3,
and (Table
Table
Table
Table S3)
S3)S3)S3);
tebufenozide
S3) and
and
tebufenozide
and and1-(4-chlorophenyl)-3-methylurea
(Table
imidacloprid
imidacloprid
imidacloprid
(Table
imidacloprid S2); 6-chloropyridine-
(Table
(Table
(Table S3);
S3);
S2); 6-chloropyridine-
(Table S3);S3); 1-(4-
1-(4-
1-(4-1-(4-
3-yl)methanamine
condensates: diuron,
chlorophenyl)-3-methylurea
3-yl)methanamine derivates:
monuron, and acetamiprid
linuron
condensates:
condensates:
condensates:
derivates:condensates:
acetamiprid (Table
(Table
diuron,
diuron,
diuron,
diuron,
(Table 3,
S3). Table
monuron,
monuron,
3,monuron,
monuron,
Table S3) S3)
and
and
andand and
linuron
linuron imidacloprid
linuron
linuron
and (Table
(Table
(Table
(Table
imidacloprid S3).
S3).
S3).
S3). (Table S3);
(Table S3); 1-(4- 1-(4-
chlorophenyl)-3-methylureacondensates:
chlorophenyl)-3-methylurea condensates:diuron, diuron,monuron,
monuron,and andlinuron
linuron(Table(TableS3). S3).
Table 3. Table
Table
Training 3.
3. Training
Training
set
Table3. 3.Training
Table pesticides set
set pesticides
pesticides
with
Trainingsetsetpesticides the
pesticideswith with
with
known the
the
crystal
withthetheknown known
known crystal
crystal
structures:
knowncrystal structures:
structures:
chemical
crystalstructures: chemical
chemical
structures,
structures:chemical chemical structures,
structures,
conformational
structures,
structures,
analysis, Table 3.
conformational
conformational
superposition
conformational
conformational Traininganalysis,
of set
analysis,
the
analysis,
analysis, pesticides
superposition
superposition
experimental
superposition with of
of the
the
conformation
of the known
experimental
experimental
and
experimentalcrystal
generatedstructures:
conformation
conformation global
conformation and
and chemical
and generated
generated
minima structures,
using
generated global
global
the
global best
Table 3. Training set superposition
pesticides with of the experimental
known crystal conformation
structures: and generated
chemical global
structures,
performing conformational
minima
minima
minima
minima using
using
using
force fields.
using the
the
the analysis,
the best
best
best superposition
performing
performing
best performing
performing force
force
force
force of
fields.
fields. the
fields.
fields. experimental conformation and generated global
conformational analysis, superposition of the experimental conformation and generated global
minima using the best performing force fields.
minima using the best
Pesticide
Pesticide FFperforming
FF a
aa a
force
EEglob_min
glob_minbfields.
bb NGMS
NGMS c
cc c NF
NF dd d Pesticide
Pesticide CAA
CAA f
ff f
Pesticide
Pesticide FF FF E Eglob_min b NGMS
glob_min NGMS NF NF
d Pesticide
Pesticide CAACAA
a b NGMSc c
Pesticide
Pesticide FFFFa a E(kJ/mol)
E(kJ/mol)
(kJ/mol)
glob_minb
glob_min
b
(kJ/mol) NGMS NGMS c NFd d
NF Pesticide
Alignment
Alignment
Alignment
Alignment
e
ee e CAA CAA
RMSD
RMSD
RMSD
RMSD
f f
(Å)
(Å) gg g
f (Å)
Pesticide FF E glob_min NF d Pesticide CAA(Å) g
simazine
simazine
simazine
simazine MM3
MM3
MM3MM3 (kJ/mol)
NA
NA
(kJ/mol)
NA
h
NA
h
h h NA
NANANA NA
NA
NA NA Alignment
Alignmente e
e RMSD
EC/MMFF
EC/MMFF (Å)
EC/MMFF
EC/MMFF
RMSD (Å)(Å)g gg
(kJ/mol) Alignment RMSD
simazine MM3
AMBER94
AMBER94
AMBER94
AMBER94 NA
NA
NA NA
h
NA
NANANA NA
NA
NA NA EC/MMFF
0.667
0.667
0.667
0.667
simazine
simazine MM3
MM3 NA
NA
hh
NA NA EC/MMFF
EC/MMFF
MMFF
AMBER94
MMFF
MMFF
MMFF −1022.18
NA
−1022.18
−1022.18
−1022.18 136
NA
136
136 136 2782
NA
2782
2782
2782 0.667
AMBER94
AMBER94 NA
NA NA NA NANA 0.667
0.667
MMFF −1022.18 136 2782
MMFF
MMFF
MMFFs
MMFFs −1022.18
− 1022.18
NA
NA 136
NA 136
NA 2782
2782
NA
NA
MMFFs
MMFFs NA NA NA NA NA NA
MMFFs
MMFFs NA
NA NA
NA NA
NA
MMFFs
OPLSAA
OPLSAA
OPLSAA NA
−−793.61
−793.61
793.61 NA158
158
158 NA
2306
2306
2306
OPLSAA
OPLSAA −793.61
−793.61 158 158 2306
2306
monocrotophos
monocrotophos
monocrotophos
monocrotophos OPLSAA
MM3
MM3
MM3MM3 −793.61
NA
NANA
NA NA
NA158
NANA 2306
NA
NA
NA NA EC/MMFF
EC/MMFF
EC/MMFF
EC/MMFF
monocrotophos OPLSAA
MM3 −793.61
NA 158
NA 2306
NA EC/MMFF
monocrotophos MM3
AMBER94
AMBER94
AMBER94
AMBER94 NA
NA
NA NA NA
NANANA NA
NA
NA NA EC/MMFF
0.620
0.620
0.620
0.620
monocrotophos AMBER94
MM3 NA NA NA 0.620
EC/MMFF
MMFF
AMBER94
MMFF
MMFFMMFF
MMFF −−307.36
NA
−307.36
−307.36
−307.36
307.36 241
NA
241
241 241
241 62
NA
62
62 62
62 EC/MMFFs
0.620
EC/MMFFs
EC/MMFFs
EC/MMFFs
EC/MMFFs
AMBER94 NA NA NA 0.620
MMFF
MMFFs
MMFFs
MMFFs
MMFFs − −307.36
−310.26
−310.26
310.26
−310.26 241
218
218
218 62
61
6161 EC/MMFFs
3.521
3.521
3.521
MMFFs
MMFF −310.26
−307.36 241218
218 62 61
61 3.521
3.521
EC/MMFFs
OPLSAA
MMFFs
OPLSAA
OPLSAA
OPLSAA NA
−310.26
NA
NA NA NA NA
218
NANA NA
61
NA
NA NA 3.521
OPLSAA
MMFFs NA
−310.26 NA
218 NA
61 3.521
dimethoate
dimethoate
dimethoate
dimethoate OPLSAA
MM3
MM3
MM3
MM3MM3 NA
NA
NA
NA NA NA
NANA
NA
NA NA
NA
NANA
NA EC/MMFF
EC/MMFF
EC/MMFF
EC/MMFF
EC/MMFF
OPLSAA
dimethoate AMBER94
MM3
AMBER94
AMBER94
AMBER94
AMBER94 NA
NA
NA
NA NA NA
NANA
NA
NA NA
NA
NANA
NA 0.1480.148
EC/MMFF
0.148
0.148
0.148
dimethoate MM3 EC/MMFF
MMFF
AMBER94
MMFF
MMFF
MMFF
MMFF −−383.68
383.68
NA
−383.68
−383.68
−383.68 340
NA
340
340
340 340 NA
44
4444
4444 EC/MMFFs0.148
EC/MMFFs
EC/MMFFs
EC/MMFFs
EC/MMFFs
MMFFs
MMFFs
MMFF
MMFFs −−383.68
386.71
−386.71
−386.71 215
215
340
215 40
40
44
40 3.2273.227
EC/MMFFs
3.227
MMFFs
MMFFs
MMFF −386.71
−386.71
−383.68 340215
215 44 40
40 3.227
3.227
EC/MMFFs
OPLSAA
OPLSAA
MMFFs
OPLSAA −−386.71
228.66
−228.66
−228.66 998
998
215
998 15
15
40
15 EC/OPLSAA
EC/OPLSAA
3.227
EC/OPLSAA
OPLSAA
OPLSAA
MMFFs −228.66
−228.66
−386.71 215998
998 40 15
15 EC/OPLSAA
EC/OPLSAA
3.227
OPLSAA −228.66 998 15 2.746
EC/OPLSAA
2.746
OPLSAA −228.66 998 15 2.746
2.746
2.746
EC/OPLSAA
acetamiprid
acetamiprid
acetamiprid
acetamiprid MM3
MM3
MM3MM3 NA
NA
NA NA NA
NANANA NA
NA
NA NA 2.746
EC/MMFF
EC/MMFF
EC/MMFF
EC/MMFF
2.746
acetamiprid MM3
AMBER94
AMBER94
AMBER94
AMBER94 NA
NA
NA NA NA
NANANA NA
NA
NA NA EC/MMFF
0.783
0.783
0.783
0.783
acetamiprid MM3 EC/MMFF
MMFF
AMBER94
MMFF
MMFF
MMFF −31.45
NA
−31.45
−31.45
−31.45 1916
NA
1916
1916
1916 1414
NA
14
14 EC/MMFFs
0.783
EC/MMFFs
EC/MMFFs
EC/MMFFs
MMFF −31.45 1916 14 EC/MMFFs
MMFF −31.45 1916 14 EC/MMFFs
Molecules 2018, 23, 2192 7 of 37
Table 3. Cont.
Pesticide FF a Eglob_min b NGMS c NF d Pesticide CAA f

Molecules
Molecules 2018,
2018, 23, x 7 of
37 37
Molecules 2018, 23,23,
x x 7 of7 37
of
Molecules
Molecules
Molecules 2018,
23,23,
2018,
2018, x23,x x (kJ/mol) Alignment e g ofof3737
7 of7 737
RMSD (Å)
acetamiprid MM3 NA NA NA EC/MMFF
MMFFs −31.29
MMFFs −31.29
−31.29 18161816 6 0.823
MMFFs
MMFF −31.45 1816 1916 6 6 14 0.823
0.823
EC/MMFFs
MMFFs
MMFFsMMFFs −31.29
−31.29
−31.29 1816 1816
1816 6 6 6 0.823
0.8230.823
MMFFs −31.29 1816 6 0.823
OPLSAA NA NA NA
OPLSAA
OPLSAA NA cNA NA NA NA NA
a Force field; b Energy OPLSAA NA NA NA
a Force field; of the global
OPLSAA
OPLSAA
b Energy of minimum;
the NA
global Number
NAminimum; NAof
NA ctimes
Numberthat
NANA aofsingle
times global
that minimum
a single structure
global was found
minimum
a Force
a Force field;
field; b EnergyOPLSAA
b Energy
of of
dthethe
globalNA
global minimum;
minimum; NA c Number
c Number NA
of of
times times thatthat a single
a single global
global minimum
minimum
in 10,000 processed
a aForce
Force field;
field;structures;
b bEnergy ofNumber
Energy ofthe
the of families
global
global minimum;i.e.,c different
minimum; c cNumber
Number conformations
ofof times
times found
that
that a a in 10,000
single
single processed
global
global structures;
minimum
minimum
a Force structure
field;
structure
e Red—experimental
structure was
b
waswas
Energyfound
found
found in in in
10,000 10,000
of 10,000 processed
the processed
global
processedminimum; structures;
structures;
structures; Number d Number
d Number
d Number ofBlue—MMFFs
oftimes
of of families
thati.e.,
families
families i.e.,
ai.e.,
single different
global
different
different conformations
minimum
conformations
conformations
conformation, Violet—MMFF conformation, conformation, Green—OPLSAA
structure
structure
found
structure f was
was
in found
found
10,000 inin10,000
10,000
processed processed
processed structures;
structures;
structures; d Number
d Number
e dRed—experimental ofoffamilies
families i.e., different
i.e., different
conformation, conformations
conformations
Violet—MMFF
found
found
conformation; inwas
in found
10,000
10,000
Conformation in processed
10,000
processed processed
structures;
dependent structures;
structures;
alignment Number
e eRed—experimental
accuracy; of families
g RMSD
Red—experimental i.e., different
conformation,
conformation,
measured between conformations
Violet—MMFF
Violet—MMFF
the heavy atoms of
found
found found inin
conformation,
in 10,000
10,00010,000 processed
processed
Blue—MMFFs
structures;
conformation,
structures;
e e Red—experimental
Red—experimental
Green—OPLSAA
e Red—experimental h conformation,
conformation,
conformation;
conformation, Violet—MMFF
fViolet—MMFF
Conformation
Violet—MMFF
pesticides of experimental
conformation,
conformation, and best performing
Blue—MMFFs
Blue—MMFFs conformation,
conformation, force Green—OPLSAA
field conformations;conformation;
Green—OPLSAA Not available.f Conformation
conformation; f Conformation
conformation,
conformation,
dependent Blue—MMFFs
Blue—MMFFs
alignment accuracy; conformation,
conformation,
g RMSD measured Green—OPLSAA
Green—OPLSAA
between conformation;
theconformation;
heavy atoms f f Conformation
Conformation
ofpesticides
pesticides
conformation, Blue—MMFFs conformation, Green—OPLSAA conformation; of Conformation of of of
f
dependent
dependent alignment
alignment accuracy;
accuracy; g RMSD
g RMSD measured
measured between
between thethe heavy
heavy atoms
atoms of pesticides
dependent
dependent
experimental
dependent alignment
alignment
alignment and best accuracy;
accuracy;
performing
accuracy;
g
g RMSDgRMSD
RMSD
force measured
measured
field
measured between
between
conformations;
between h
the the
Notthe heavy
heavy
available.
heavy atoms
atoms atomsof ofof pesticidesof ofof
pesticides
pesticides
inexperimental and best performing force field conformations;Not Not available.
h
CAexperimental
explicit and best performing force field conformations; available.
h
experimental
experimental solvent
andand may
best
best be used
performing
performing to
force
force predict
field
field the single
conformations;
conformations; hpesticide
Not
h Notavailable. stability prior to AChE binding.
available.
experimental and best performing force field conformations; h Not available.
Herein, CAs CAwerein tentatively
explicit solvent carried
may be out
used byto using
predict five
the different
single force
pesticide fieldsprior
stability (FFs): MM3, AMBER94,
CA CAin in explicit
explicit solvent
solvent maymay bebe used
used to to predict
predict thethe single
single pesticide
pesticide stability
stability priorprior
to to to
AChEAChEAChE binding.
binding.
binding.
MMFF, CACA
MMFFs,
Herein,
CA in in inexplicit
CAsexplicit
and
explicit were solvent
solvent
OPLSAA may
may
tentatively
solvent may be be
[35].beused
usedused
Either
carriedto to
outtopredict
predict
for
by
predict the
the
crystallized
using
the single
single
five
single pesticide
pesticide
pesticides
different
pesticide forcestability
stability
stability or
fields prior
prior
pesticides
prior(FFs):
to toto AChE
AChE
MM3,
AChE with binding.
binding.
unknown
AMBER94,
binding.
Herein,
Herein, CAsCAs were
were tentatively
tentatively carried
carried outoutbyby using
using five five different
different force
force fields
fields (FFs):
(FFs): MM3,
MM3, AMBER94,
AMBER94,
Herein,
Herein,
MMFF,
Herein,
experimental CAsCAsCAs were
werewere
MMFFs, tentatively
tentatively
and
tentatively
structures, theOPLSAA carried
carried
carried
procedure [35].
outout
out by
Either
by
was byusing
using using
for
divided five
five different
crystallized
five different
different
into the force
force
pesticides
force fields
fields
following fields
or (FFs):
(FFs):
pesticides
(FFs):
steps: MM3,
MM3, MM3,
(1) AMBER94,
with AMBER94,
AMBER94,unknown
conformational
MMFF,
MMFF, MMFFs,
MMFFs, andand OPLSAA
OPLSAA [35].
[35]. Either
Either forfor crystallized
crystallized pesticides
pesticides oror pesticides
pesticides withwith unknown
unknown
MMFF,
MMFF,
MMFF, MMFFs,
MMFFs,
experimental and
and OPLSAA
structures,OPLSAA [35].
[35].
theprocedure Either
procedure Either for
was forcrystallized
crystallized
divided into pesticides
pesticides
thefollowing or or
following pesticides
orand
pesticides
steps: with
with unknown
unknown
(1)conformational
conformational
analysis; (2)MMFFs,
experimental
experimental and
determination OPLSAA
structures,
structures, ofthe
the [35].
energy
procedure Either
of the
was for
global
was crystallized
minimum
divided
divided intointo pesticides
the (E
thefollowing pesticides
glob_min ),steps: steps:(3) with
the
(1)(1) LB unknown
superposition
conformational
experimental
experimental
analysis;
experimental (2) structures,
structures,
determination
structures, thethe
the procedure
ofprocedure
energy
procedure of
was was
was
the divided
divided
global
divided into
into
minimum
into the the
the following
(E following
glob_min),steps:
following steps:
and steps:
(3)
(1) (1)
(1)
the conformational
LB conformational
superposition
conformational
analysis;
of structures
analysis; (2)
(2)to determination
define
determination of of
the alignment energy
energy of of
the the
accuracy. global
global All minimum
steps
minimum were (E performed
(Eglob_min ), and
), and
glob_min (3)(3)
in
thethe
order
LB LB superposition
to obtain the best
superposition
analysis;
analysis;
of
analysis; (2) (2)
(2)determination
structures determination
to define
determination the
of of
of energy
energy
alignment
energy of ofof the
the global
accuracy.
the global
global Allminimum
minimum minimum
steps were
(E (E(Eglob_min
performed
),
glob_min ),),and
and and
(3) (3)
inthe the
(3)order
the
LB LBLB
to superposition
superposition
obtain
superposition the best
of of
performing structures
structures FF, to to
eitherdefine
define bythethe
means alignment
alignment of accuracy.
accuracy.
capability All
toAll
stepssteps
predict werewere
the performed
glob_min
performed
free energy in in order
orderof to to obtain
obtain
global thethe
minimum best
best or to
of of
of structures
structures
performing
structures to totodefine
FF, define
defineeither
thethe
the
by alignment
alignment
means
alignment of accuracy.
accuracy.
capability
accuracy. All All
All
to
stepssteps
steps
predict
werewere
were
the performed
performed
free
performed energy
in in order
inof
orderorder to
global
to toobtain
obtain
minimum
obtain thethe
the
bestbest
best
or to
performing
performing
superimpose FF,FF, either
either
molecules. by by
The means
means of of capability
capability to to predict
predict the thefreefree energy
energy of of global
global minimum
minimum or or
to to
performing
performing
superimpose
performing FF,FF,
FF, either
either
molecules.
either by bybyhigher
means
means
The
means
goal
of
higher
of
of CA was
ofcapability
capability
goal
capability of CA
to to to
predict
towas
predict
answer
predict
to the
the
answer
the free
the
free
free
the
question:
energy
energyenergy
question:
of ofofHow
global
global
How
global
tominimum
to
consider or
minimum
minimum consider or
the
the
to
FF of
ortoto
FF
superimpose
superimpose molecules.
molecules. The The higher
higher goal
goal of of
CA CA waswas to to answer
answer theorthe question:
question: How How to consider
tocapability?
consider thethe
FFFF
choice while
superimpose
superimpose
of choice
superimpose
treating
while pesticides:
molecules.
molecules.
treating
molecules. The The
The by
higher
pesticides:
higher
predicted
higher goal
goal goal
by of
ofCAECA
ofpredicted
CA waswas
was
glob_min totovalues
Eglob_min
to answer
answer answer thethe
values orby
the alignment
question:
question:
by
question: alignment
How How
How totoconsider
consider
tocapability?
consider thethe
FFFF
the FF
of of choice
choice while
while treating
treating pesticides:
pesticides: byby predicted
predicted Eglob_min
Eglob_min values
values oror
byby alignment
alignment capability?
capability?
of of
ofchoice
choice
choice while
while
while treating
treating
treating pesticides:byby
pesticides:
pesticides: bypredicted
predicted
predicted EEglob_min
Eglob_min values
values
values
glob_min or or
orby
by byalignment
alignment
alignment capability?
capability?
capability?
Table 4. Training
Table
Table 4. 4. set
Training
Training pesticides
setset of
pesticides
pesticides the
of ofhighest
thethe
highesttoxicity
highest toxicity
toxicity with
with
with the
the unknown
the unknown
unknown crystal
crystal
crystal
Table 4. Training set pesticides of the highest toxicity with the unknown crystal structures: chemical
structures:
structures:
structures: chemical
chemical
chemical
Table
Table
structures,
Table 4. 4.4.Training
Training
structures,
conformational
Training setset
setpesticides
pesticides
conformational
analysis,
pesticides of of
ofthe
the thehighest
analysis, highest
superposition
highest toxicity
toxicity
superposition
toxicity of with
of with
the
with the
thethe
theunknown
unknown
experimental
experimental
unknown crystal
crystalstructures:
conformation
conformation
crystal structures: andchemical
structures:
and chemical
generated
generated
chemical
structures, conformational analysis, superposition of the experimental conformation
structures, conformational analysis, superposition of the experimental conformation and generated and generated
global structures,
structures,
global
minima
structures, conformational
conformational
minima
using using
the
conformational the
best analysis,
analysis,
best superposition
superposition
performing
performing
analysis, force
force
superposition ofofthe
fields.
fields.
of the the experimental
experimental
experimental conformation
conformation
conformation andand
andgenerated
generated
generated
global
global minima
minima using
using thethe best
best performing
performing force
force fields.
fields.
global
global
global minima
minima
minima using
thethe
using
using thebest
best bestperforming
performing force
forcefields.
fields.
Pesticide a performing force b fields.
a FF Eglob_min NGMS d NF Pesticide FFDAA
c d f
Pesticide
Pesticide FFaFF a
E Eglob_min
glob_min b b NGMS
b NGMS c c NF
cc c NF d
dd d Pesticide
Pesticide FFDAA
FFDAA f f
ff f g
Pesticide
Pesticide
Pesticide
Pesticide FFFF FF
aFF aa EE E E(kJ/mol)
glob_min
glob_min
glob_min
b
bb NGMS
NGMS
NGMSNGMS c NFNF
NF
NF
d Pesticide
Pesticide
Pesticide
Alignment
Pesticide e FFDAA
FFDAA
FFDAAFFDAA
RMSD f g (Å)
(kJ/mol)
(kJ/mol)
glob_min Alignment
Alignment e ee RMSD
RMSD (Å)(Å) g
atrazine MM3 (kJ/mol)

(kJ/mol)
−1161.48
(kJ/mol)
(kJ/mol) 445 23 Alignment
Alignment
Alignment
Alignment e e e RMSD
RMSD RMSD
MM3/MMFF
RMSD (Å) (Å)
(Å)(Å)
g g g
g
atrazine
atrazine MM3MM3 −1161.48
−1161.48 445445 23 23 MM3/MMFF
MM3/MMFF
atrazine
atrazine
atrazine MM3
MM3
AMBER94
MM3 −1161.48
−1161.48
−1161.48 hNA
h
445445
445
NA NA 2323
23NA NA MM3/MMFF
MM3/MMFF
MM3/MMFF 0.892
atrazine AMBER94
AMBER94
MM3 −NA NA
1161.48 h
NA NA
445 23 0.892
0.892
MM3/MMFF
AMBER94
AMBER94
AMBER94 MMFF −1007.32 NA NA
NA hh
−1007.32
hh NA NANA
147 NA NANA62 0.892
0.892
MM3/MMFFs
0.892
MMFF
MMFF
AMBER94 −1007.32
NA 147147
NA 62
62NA MM3/MMFFs
MM3/MMFFs0.892
MMFF
MMFF
MMFFs −1007.32
−1007.32
−992.63 147
147
509 6262 MM3/MMFFs
MM3/MMFFs
MMFF
MMFF
MMFFs
MMFFs −1007.32
− 1007.32
−992.63
−992.63 147
147
509509 20 2020
6262 MM3/MMFFs
MM3/MMFFs 0.931
0.931
0.931
MMFFs
MMFFs −992.63
MMFFs
MMFFs −992.63
−992.63
−992.63 509509
509509 2020
2020 0.931
0.9310.931
0.931
OPLSAA
OPLSAA
OPLSAA −783.57
−783.57
−783.57 364364364 19 1919 MMFF/MMFF
MMFF/MMFF
MMFF/MMFF S S
S
OPLSAA
OPLSAA − 783.57
−783.57 364 19 MMFF/MMFF
OPLSAA −783.57
OPLSAA −783.57 364364
364 19 1919 MMFF/MMFF
MMFF/MMFF
MMFF/MMFF 0.343S
SS S
0.343
0.343
0.343
carbofuran MM3 29.60 3617 2 0.343
0.343
MMFF/MMFF
0.343 S
carbofuran
carbofuran
carbofuran MM3
MM3 MM3 29.60
29.60
29.60 36173617
3617 2 22 MMFF/MMFF
MMFF/MMFF
MMFF/MMFF S S
carbofuran
carbofuran MM3 29.60 3617 2NA MMFF/MMFF SS
carbofuran MM3 MM3
AMBER94
AMBER94 29.6029.60
NANA 3617 3617
NA NA 2 NA 2 MMFF/MMFF
MMFF/MMFF 0.075
0.075 S
AMBER94
AMBER94 NA
NA NA NA NA NA 0.075
0.075 S
AMBER94
AMBER94
MMFF NA
NA
20.30 NANA
3307 NA NA
NA 0.075
0.075
AMBER94
MMFF
MMFF MMFF NA20.30
20.30
20.30 NA
33073307
3307 2 22 2 MMFF/OPLSAA
0.075
MMFF/OPLSAA
MMFF/OPLSAA
MMFF/OPLSAA
MMFFMMFF
MMFF
MMFFs 20.30
20.3020.30
15.92 3307 3307
3307
3384 2 2 2 MMFF/OPLSAA
MMFF/OPLSAA
0.216
MMFFs MMFFs
MMFFs 15.92
15.92
15.92 3384
33843384 22 22 MMFF/OPLSAA0.216
0.216
0.216
OPLSAA MMFFs
MMFFs MMFFs
OPLSAA 15.92
73.27
15.9215.92
73.27 3384
3384
5621
3384 5621 2 2 275
75 0.216
0.216
MMFFs/OPLSAA
MMFFs/OPLSAA
0.216
OPLSAA
OPLSAA 73.27
73.27 56215621 75 75 MMFFs/OPLSAA
MMFFs/OPLSAA
OPLSAA
OPLSAAOPLSAA 73.27 73.27
73.27 5621 5621
5621 75 7575 MMFFs/OPLSAA
MMFFs/OPLSAA
MMFFs/OPLSAA0.239
0.239
0.239
0.239
a Force field; b Energy
a Force field; b Energy of the globalc minimum; c Number of times that a single global minimum
of the global minimum; Number of times that a single global minimum structure 0.239
0.2390.239
a Force
a Force field;
field; b Energy
b Energy of of thethe global
global minimum;
minimum; c Number
c Number of oftimestimes that
that a single
a single global
global minimumfound
minimum was
a aForce field; b bEnergy d of the global minimum; c cNumber of
in 10,000
a Force Force
structurefield;
processed
field; bwas
Energy Energy
structures;
found of in of the
Number
10,000
the global
of
processed
global minimum;
families
minimum; i.e.,
structures;
NumberNumber
c differentd Number
of oftimes
conformations
times times
of that
that
found
families
that a ai.e.,
asingle
singlesingle
in global
10,000global
different
global minimum
minimum
processed
minimum structures;
conformations
structure
structure
e Red—experimentalwaswas found
found in in 10,000
10,000 processed
processed structures;Number
structures; d d Number of of families
families i.e.,i.e., different
different conformations
conformations
structure
structure
found was
was
in conformation,
found
found
10,000 inin 10,000
10,000
processed Violet—MMFF
processed
processed conformation,
structures;
structures;
structures; d Number
d Number Blue—MMFFs
e dRed—experimental ofoffamilies
families conformation,
i.e.,
i.e.,different
different
conformation, Green—OPLSAA
conformations
conformations
Violet—MMFF
structure was
foundin fin10,000
found found in 10,000
10,000processed processed
processedstructures; structures; Number
structures; Red—experimental
e e Red—experimental of
g RMSD measured
families i.e., different
conformation,Violet—MMFF
conformation, conformations
Violet—MMFF
conformation;
found Force field dependent alignment accuracy; between thef Force
heavy atoms of pesticides
in inin 10,000 processed structures; e e Red—experimental conformation, Violet—MMFF
foundfound
conformation,
10,00010,000 processed
Blue—MMFFs
conformation,
structures; Red—experimental
Green—OPLSAA
e Red—experimental conformation,
conformation;
conformation, Violet—MMFF
field
Violet—MMFF dependent
conformation,
conformation,
experimental Blue—MMFFs
Blue—MMFFs
and bestBlue—MMFFs
performing conformation,
conformation,
force Green—OPLSAA
Green—OPLSAA
field conformations; h conformation;
conformation;
Not available.
f Force
f Force field
field dependent
dependent
conformation,
conformation,
alignment
conformation, Blue—MMFFs
accuracy;
Blue—MMFFs g RMSD conformation,
conformation,
measured
conformation, Green—OPLSAA
between Green—OPLSAA
Green—OPLSAA the heavy conformation;
atoms conformation;
of
conformation; pesticides f Force
f Forcef Forcefield
fieldfielddependent
experimental dependent
dependentand best
alignment
alignment accuracy;
accuracy; g RMSD
g RMSD measured
measured between
between thethe heavy
heavy atoms
atoms of of pesticides
pesticides experimental
experimental andand best
best
alignment
alignment
performing
alignment accuracy;
accuracy;
accuracy; force g RMSD
g RMSDmeasured
field
g RMSD measured
conformations;
measured between
hbetween
Not
between the
theheavy
available.
the heavy heavy atoms
atoms atomsof ofofpesticides
pesticides
pesticides experimental
experimental
experimental andand
and best
bestbest
performing
performing force
force field
field conformations;Not
conformations; h h Not available.
available.
performing
performing
CAperforming
results for
forceforce
force field
simazine
field field conformations;
conformations; h Not
[9], monocrotophos
conformations; h Noth Notavailable.
available.
available. [10], dimethoate [11], and acetamiprid [12] are
reportedCA in
CA CA
Tableresults
results
results 3.
forforforsimazine
MM3 simazine
simazineand[9], [9],monocrotophos
AMBER94
[9], monocrotophos
monocrotophoscould not [10],
be
[10],
[10], dimethoate
applied
dimethoate
dimethoate to all[11],
[11],
[11], ofand
and and
the acetamiprid
crystallized
acetamiprid
acetamiprid [12]
areareare
pesticides
[12]
[12]
CA
CA
reported results
CA results results
in for
Table
for for simazine
3. simazine
MM3
simazine and[9],
[9], [9], monocrotophos
monocrotophos
AMBER94
monocrotophos could [10],
not[10],
[10], be dimethoate
dimethoate
applied
dimethoate to [11],
all
[11], [11],
of and
and
the
and acetamiprid
acetamiprid
crystallized
acetamiprid [12]
[12]
pesticides
[12] are are
areas
as the reported
needed
reported in in Table3. 3.
parameters
Table MM3MM3 and
for
and AMBER94
the
AMBER94 could
optimization
could notnot
bebe
were appliedto to
allall
not available.
applied of of
thethe crystallized
Possible
crystallized pesticides
workaround
pesticides as as
could
reported
reported
the
reported in
neededinTable
in Table Table 3. MM3
3.
parameters MM3 and
and
for AMBER94
theAMBER94 could
optimizationcould not
not
were be
be
notapplied
applied toto
available. all
all of
of
Possiblethe
thecrystallized
crystallized
workaround pesticides
pesticides
could beasas
the
the
be the the needed
needed
the of3. Antechamber
parameters
utilization
needed
MM3
parameters
parameters
and
forfor AMBER94
thethe suite could
optimization
optimization were
[75], notnot
by be
were notapplied
thetogeneric
available.
available.
which allPossible
of theworkaround
Possible crystallized
workaround
AMBER pesticides
could
could
parameters bebe asthe
the
could be
the needed
utilization
theutilization
needed
utilization of
parameters
of
parameters
of Antechamber
Antechamber forfor
Antechamber for the
the
thesuite
suite
optimization
optimization
suite [75],
optimization
[75],
[75], byby by
which
were
were
which
were
which
the not
the
not
thenot available.
available.
generic
available.
generic
generic AMBERPossible
Possible
Possible
AMBER
AMBER
workaround
workaround
parameters
workaround
parameters
parameters could
could
could
could
could
be be bebe
could
be bethe
the
calculated,
calculated,
the
calculated,
calculated, but
utilization then a different protocol would have been developed instead of the one based on the
but
utilization
but
utilization
but then
then
then of aof
of Antechamber
Antechamber
different
Antechamber
a different
a accredited
different protocol
protocol
protocol
suite
suitesuite
would
[75],
would
[75],
would have
by
[75],
have
bywhich
byhave which
which
been been
been thethe
thegeneric
generic
developed
generic
developed
developed
AMBER
AMBER
instead
AMBER
instead
instead of of ofparameters
parameters
the
parameters
thetheone oneone could
basedcould
could
based
based on onon
be
the
the
be
becalculated,
the calculated,
well-known
calculated,
well-known
well-known
well-known
but
but then
then
accredited
but then aadifferent
different Schrödinger
protocol
protocol
Schrödinger
a different protocol would would
would
software software
have
have been
been[76]. MMFF
developed
developed
[76]. developed
have been MMFF showed showed
instead
instead
instead of of
totheoftheto
the
beone be
one
one
the the
based
based
only
based
only
on
on
on FF
FF
thecontaining thethe
the containing
well-known
well-known
the well-known
accreditedSchrödinger
accredited Schrödingersoftware
software[76].
[76].MMFF
MMFFshowed
showedto tobebethetheonly
onlyFFFFcontaining
containingthethe
accredited
accredited
accredited Schrödinger
Schrödinger
Schrödinger software
software
software [76].
[76].
[76]. MMFF
MMFF
MMFF showedto toto
showed
showed bebethethe
be the onlyFFFF
only
only FF containingthethe
containing
containing the
Molecules 2018, 23, 2192 8 of 37
parameterizations for all of the compounds; MMFFs failed in the optimization of simazine whereas
OPLSAA was not applicable to monocrotophos and acetamiprid.
The absolute values of Eglob_min are not necessarily good measures of FF quality [77]. FFs and are
usually parameterized to give good values of relative energies among different conformations of the
same molecule [77], so a lower value of energy for a global minimum does not necessarily mean that a
given FF would perform better [77]. The energy differences between the conformations are simple
reliable indicators of conformational analysis algorithm ability than the absolute energy values [77].
Therefore, the evaluation of conformation reproducibility was achieved by means of calculated
minima root mean square deviation (RMSD) values [78] upon conformations’ superimposition
(Tables 3 and 4, Tables S1–S3). Comparison of the experimentally available crystal structures
conformations with those obtained by CA revealed, a high level of performance by MMFF (displaying
RMSD values lower than 1 Å for all of the four considered pesticides) [78]. That was somehow expected,
given that MMFF-based optimization was applicable to all of the listed pesticides. Regarding the
MMFFs and OPLSAA, these FFs only succeeded in reproducing the crystal structure of acetamiprid.
2.2. The Prediction of Pesticides Structures in Aqueous Solution

The above assessed CA procedure was further applied to the training set pesticides with an
unknown crystal structures. The results of optimization for the pesticides with the highest acute toxicity
(i.e., the lowest LD50 values), atrazine and carbofuran, using the best performing FFs, are reported in
Table 4, while the comparison of all utilized FFs for atrazine, carbofuran, and the remaining compounds
are presented in Tables S1–S3. As expected, AMBER94 FF, as implemented in Schrödinger suite [76] was
not applicable at all, MM3, MMFF and MMFFs FFs gave similar global minima for the majority of the
listed pesticides, while OPLSAA was not applicable for all compounds. A significant conformational
alignment accuracy (RMSD < 1 Å) was achieved for atrazine (MM3/MMFF, MM3/MMFFs, and
MMFF/MMFFs combinations, Table 2, Table S1) and carbofuran, (MMFF/MMFFs, MMFF/OPLSAA,
and MMFFs/OPLSAA combinations, Table 3, Table S2). Summarizing the CA results, conclusion can
be made, despite the limited number of training set compounds, MMFF is universally applicable FF
for either Eglob_min calculation or RMSD-based alignment accuracy assessment and can be used in any
further study, describing the pesticides conformational analysis.
2.3. The Prediction of Externally Evaluated Pesticides Structures in Aqueous Solution

MMFF was thereafter used to predict the global minimum conformation of test set pesticides
(Table 2) as well. The results of the optimisation are presented as Supplementary Materials (Table S4).
Global minimum conformations were generated for by the means of the identical CA setup. Generated
conformations were then used for the purposes of training set results external validation.
2.4. The Pesticides Acute Toxicity Anticipation through the Pre-Bound Conformations
The lowest energy conformers of the 2-chloro-1,3,5-triazine group were of high stability in
the aquous solution, according to the values obtained by MMFF (Tables 3 and 4, Table S1). Being
chemically stable, pesticides may in perspective saturate the AChE active site and low values of
global minimum (Eglob_min ) represent the indicators of high acute toxicity (i.e., the low LD50 values)
against Mus musculus. For either atrazine, propazine, or simazine, low Eglob_min values are indeed
the precondition of high acute toxicity (Table 1: LD50 values equal to 0.85, 3.18, and 5 mg/kg of
body weight, respectively, Tables 3 and 4, Table S1: Eglob_min values equal to −1007.32, −1022.18, and
−992.47 kJ/mol, respectively). For much of the lasting pesticides (monocrotophos and dimethoate:
Table 1 vs. Table 3 and Table S2; carbaryl and tebufenozide: Table 1 vs. Table S2; imidacloprid: Table 1
vs. Table 3 and Table S3, and acetamiprid: Table 1 vs. Table S3), it appeared that high to medium acute
toxicities mostly correlate with low to medium Eglob_min values.
The above paradigm was confirmed for carbaryl and tebufenozide, where the high Eglob_min
values are a precondition for low acute toxicity (Table 1: LD50 values equal to 100 and 5000 mg/kg of
Molecules 2018, 23, 2192 9 of 37
body weight, respectively, Table S2: Eglob_min values equal to 20.43 and 467.38 kJ/mol, respectively), as
well as for monocrotophos and dimethoate, whose medium LD50 values (Table 1: LD50 equal to 14 and
60 mg/kg of body weight, respectively) are associated with medium Eglob_min values (Table 3: Eglob_min
values equal to −307.36 and −383.68 kJ/mol, respectively). On the other hand, the high acute toxicity of
carbofuran against Mus musculus (Table 1: LD50 = 2 mg/kg of body weight) disagreed with calculated
high Eglob_min values (Table 4: Eglob_min value equal to 20.30 kJ/mol). Interestingly, a thermodynamic
parameter calculated for carbofuran suggests some instability in aqueous solution; according to the
previous observations, a high dosage of pesticide would be required to induce mortality in Mus
musculus. Therefore, other parameters need to be considered, to explain why carbofuran acts as
an outlier.
Among the last group of pesticides, acetamiprid (Table 1 vs. Table 3 and Table S3) and imidacloprid
(Table 1 vs. Table S3) also follow the archetype that relatively low Eglob_min values are a precondition
for medium LD50 dosages. Linuron, however, behaves oppositely to 2-chloro-1,3,5-triazines group
inasmuch as with its low Eglob_min values there is a high LD50 value (Table 1: LD50 = 2400 mg/kg
of body weight, Table S3: Eglob_min value equal to −992.47 kJ/mol). Linuron’s structural analogues
diuron and monuron, despite the fact they are slightly less stable (higher Eglob_min values, Table S3:
Eglob_min values equal to −142.96 and −197.63 kJ/mol, respectively) need to be administered at high
dosages to induce mice mortality (Table 1, LD50 = 500 and 1700 mg/kg of body weight, respectively).
In order to enumerate the Mus musculus LD50 /Eglob_min interconnection, simple linear regression
(as implemented in LibreOffice Calc) was analysed considering pLD50 (calculated by converting LD50
from mg/kg of body weight to −log(mol/kg of body weight)), as dependent variable, and Eglob_min
values as calculated by MMFF (Table S5), as descriptive independent variables. The use of all the data
(model 0) did not lead to a significant statistical regression. However, the exclusion of carbofuran and
linuron as highlighted outliers (those with the worse pLD50 fitted values), led to a monoparametric
QSAR model 1 (Equation (2)):
pLD50 = −0.0021Eglob_min − 2.82 (r2 = 0.78), (2)
Additional exclusion of diuron and monuron further increased the statistical robustness leading
to a QSAR model 2 (Tables S5 and S6, Figure 2) with more than 90% of explained variance (90.5%)
(Equation (3)):
pLD50 = −0.0019Eglob_min − 3.05 (r2 = 0.905), (3)
The obtained model 2 estimated the pLD50 values of carbofuran, linuron, diuron, and monuron, as
in situ generated test set, with good accuracy (Figure 2a). On the other hand, model 2 was also used to
predict the Mus musculus pLD50 values of the test set pesticides (Tables 2 and S6, Figure 2b). However,
despite the good statistical potential of model 2 (i.e., the explained variance value), the Eglob_min values
cannot be considered as valuable indicators to anticipate the Mus musculus pLD50 values and for the
acute toxicity prediction.
Molecules 2018, 23, 2192 10 of 37
Figure 2. The plot of experimental vs. fitted pLD50 values calculated with QSAR model 2 for the
Figure 2. The plot of experimental vs. fitted pLD50 values calculated with QSAR model 2 for the
training set pesticides (training set values are depicted by blue circles; internal test set values are
training set pesticides (training set values are depicted by blue circles; internal test set values are
indicated by orange squares) (a); test set pesticides (green circles) (b) against Mus musculus. The plot
indicated by orange squares) (a); test set pesticides (green circles) (b) against Mus musculus. The plot
of
of calculated
calculated vs.
vs. fitted
fitted pLD
pLD50 values calculated with the QSAR model 4 for the training set pesticides
50 values calculated with the QSAR model 4 for the training set pesticides
(training
(training set
set values
values are
are depicted
depicted by
by blue
blue circles;
circles; internal
internal test
test set
set values
values are
are indicated
indicated by
by orange
orange squares
squares
(c); test set pesticides (green circles) (d) against Homo sapiens.
(c); test set pesticides (green circles) (d) against Homo sapiens.
Moreover, models 1 and 2 cannot be universally applied for the prediction of the Homo sapiens
Moreover, models 1 and 2 cannot be universally applied for the prediction of the Homo sapiens
pLD50 as the Eglob_min values are common to both model organisms. Therefore, different models are
pLD50 as the Eglob_min values are common to both model organisms. Therefore, different models are
required for Homo sapiens as model organism. In that sense, additional models were developed from
required for Homo sapiens as model organism. In that sense, additional models were developed from
calculated Homo sapiens pLD50 values (Equation (1)). As expected, the incorporation of all the training
calculated Homo sapiens pLD50 values (Equation (1)). As expected, the incorporation of all the training
set, pesticides did not lead to any statistically significant model. As above, exclusion of carbofuran
set, pesticides did not lead to any statistically significant model. As above, exclusion of carbofuran
and linuron led to the model 3 (Equation (4)):
and linuron led to the model 3 (Equation (4)):
= −0.0021 _ + 3.01 = 0.783 , (4)
pLD50 = −0.0021Eglob_min + 3.01 (r2 = 0.783), (4)
and further exclusion of diuron and monuron led to model 4 (Equation (5), Tables S7 and S8, Figure
2c,d):
and further exclusion of diuron and monuron led to model 4 (Equation (5), Tables S7 and S8,
Figure 2c,d): = −0.0019 _ + 4.15 = 0.91 , (5)
pLD50 = −0.0019Eglob_min + 4.15 (r2 = 0.91), (5)
Like for Mus musculus and Homo sapiens, the pLD50 values were not fully satisfactory correlated
to the global
Like for Musminima
musculusenergies,
and Homoleading to the
sapiens, thepLD
conclusion
50 values that
wereEnot fullyvalues
glob_min are not
satisfactory adequate
correlated to
indicators to anticipate
the global minima the pLD
energies, 50 against
leading to theany model organism,
conclusion the level
that Eglob_min valueswas arechanged fromindicators
not adequate LB to SB,
directing thatthethe
to anticipate pLDfree energies
50 against anyofmodel
binding could the
organism, be level
considered as appropriate
was changed from LB toacute toxicity
SB, directing
indicators.
that the freeFree energies
energies of binding
of binding couldcan be calculated
be considered separately for
as appropriate each
acute pesticide
toxicity and model
indicators. Free
organism,
energies ofby performing
binding can be molecular
calculated docking
separatelystudies,
for eacheither for Mus
pesticide andmusculus AChE orby
model organism, Homo sapiens
performing
AChE.
molecular docking studies, either for Mus musculus AChE or Homo sapiens AChE.
2.5. The Structure-Based Studies

To get more insights into the mechanistic profile of understudied pesticides, their interactions
with either mAChE and hAChE, as molecular targets, were also considered. Sequence similarity value
between mAChE and hAChE was found to be 88.44% (Figure S1), rising to the 100% identity between
Molecules 2018, 23, 2192 11 of 37
2.5. The Structure-Based Studies

To get more insights into the mechanistic profile of understudied pesticides, their interactions
with either mAChE and hAChE, as molecular targets, were also considered. Sequence similarity value
between mAChE and hAChE was found to be 88.44% (Figure S1), rising to the 100% identity between
[the active sites, indicating that similar pharmacodynamics profile should be expected with either
mAChE or hAChE by the same molecule. Therefore, the application of SB tools could represent the
computational approach to predict the biochemical mechanism and hence the level of acute toxicity.
Two SB methodologies were chosen: molecular docking and molecular dynamics. Docking and
dynamics studies were initially performed to anticipate the bioactive conformations of pesticides with
known acute toxicity and unknown binding mode into the AChE, after which the obtained free energies
of binding were correlated to the acute toxicities. Despite the fact there are no co-crystalized AChEs
in complex with pesticides as either non-covalent or covalent inhibitors, it was arbitrarily decided
to simulate reversible docking, for pure reversible AChE inhibitors, and reversible-like docking, for
covalent AChE inhibitors, followed by the appropriate, one step molecular dynamics simulations.
Reversible-like docking, was supposed to obtain the pre-covalent conformation that will lead to the
AChE covalent modification by literature-claimed [73] covalent inhibitors. The ultimate task of SB
studies was to reveal the free energies of binding and to correlate them to the pesticides acute toxicities.
2.5.1. The Alignment Rules Assessment

In order to learn how to perform the SB alignment of targeted pesticides within the AChE active
sites of either mouse or human molecular target, the 3-D coordinates of reversible AChE inhibitors
were taken from their corresponding minimized complexes and used to gain the knowledge how
to reproduce their co-crystallized conformations by means of the SB alignment assessment, as well
as to determine the best SB molecular docking methodology. Within the SB alignment assessment,
AutoDock [79], AutoDock Vina [80] (in the future text just Vina) and DOCK [81] were evaluated to
select the best docking algorithm/scoring function and to perform the SB positioning of pesticides.
The SB alignment assessment procedure was performed with the standard four step protocol [78]:
Experimental Conformation Re-Docking (ECRD), Randomized Conformation Re-Docking (RCRD),
Experimental Conformation Cross-Docking (ECCD), and Randomized Conformation Cross-Docking
(RCCD).
Thus, within the ECRD stage, Vina and DOCK possessed the highest ability to reproduce the
experimental conformations of Mus musculus and Homo sapiens AChE inhibitors. Even 75% of the
available inhibitors were correctly reproduced by Vina, whereas DOCK was slightly less potent,
reproducing the 50% of structures in either rigid or flexible manner, respectively (Tables S9 and
S10). Similarly, Vina was of the highest potency in the initial reproduction of Mus musculus AChE
inhibitors with the docking accuracy (DA) of 55.55% (Table S9). DOCK, however, completely failed
in the reproduction the experimental poses of Mus musculus AChE inhibitors (Table S9). In the
initial process, as well as in all other difficulty levels, AutoDock algorithm failed in the experimental
conformations reposition.
Similar potency for Vina and DOCK was observed within the RCRD stage in the process of human
inhibitors reproduction (Table S10, Vina DA equal to 68.75%, DOCK DA equal to 56.25%, respectively).
While processing mouse inhibitors (Table S9), Vina re-positioned the modelled inhibitors structures
with the high efficiency (DAs equal to 57.78%), while DOCK’s potency dropped below 30%.
The level of difficulty was increased during the ECCD stage where either Vina or DOCK retained
the high level of accuracy while cross-aligning of human inhibitors (Table S10). As for the other model
organism, either Vina or DOCK were inaccurate while matching the co-crystallized inhibitors with
diverse mouse enzymes (Table S9).
The decisive difference in favour of Vina, by means of the general docking accuracy, was noticed
within the RCCD stage, as the most difficult one. The cross-alignment of randomized conformations
is considered as the most important validation stage for any docking program as it represents the
Molecules 2018, 23, 2192 12 of 37
ability of a program to position a random ligand conformation in a non-native environment, i.e., into
its molecular target whose active site amino acids suffered induced fit conformational change due
Molecules
to 2018, 23, xof structurally different inhibitors. Within the RCCD stage, Vina was far superior
the presence 12 of 37
in comparison with other programs, by means of The SB alignment accuracy, and, therefore, can be
considered as
considered as aa valuable
valuable tool
tool for
for the
the prediction
prediction of
of bioactive
bioactive conformations
conformations of
of acetylcholine
acetylcholine esterase
esterase
inhibitors with the unknown binding
inhibitors with the unknown binding mode. mode.
2.5.2. The Cross-Docking and Molecular

Molecular Dynamics
Dynamics Studies
Studies of
of ACh
ACh
Both the training set or the test set pesticides were submitted to the herein Vina-based proposed
SB alignment
alignment protocols,
protocols,that
thathave
havebeen
beenvalidated by reproducing
validated by reproducing the bioactive conformations
the bioactive conformationsof PDB-
of
available inhibitors
PDB-available in complex
inhibitors with AChE
in complex (Supplementary
with AChE Materials
(Supplementary Tables Tables
Materials S9 andS9 S10,
and and Figure
S10, and
S2) andS2)
Figure applied in situ in situ
and applied predicting the bioactive
in predicting conformations
the bioactive of AChof(Figure
conformations 3).
ACh (Figure 3).
no experimental
As no experimental structures of ACh ACh inin complex
complex with
with either
either wild-type
wild-type mAChE or hAChE are
available in the Protein
available Protein Data Bank, its bioactive conformation within both enzymes was predicted by
means of Vina,
Vina, as
as the
the best performing SB alignment assessment tool (Figure 3a,b, see Supplementary
Materials Alignment
Materials AlignmentRulesRulesAssessment
Assessment section,
section, Tables
Tables S9 S10).
S9 and and S10). The stability
The stability of eitherofACh-mAChE
either ACh-
mAChE or ACh-hAChE complex was confirmed using one step molecular
or ACh-hAChE complex was confirmed using one step molecular dynamics (MD) runs, dynamics (MD) runs, upon
upon
inspection of Cα
the inspection Cα RMSD
RMSD values
values (1.2
(1.2 ns
ns simulation
simulation length,
length, Figures
Figures S7a
S7a and
and S8a),
S8a), RMSF
RMSF values
values
(Figures S7b
(Figures S7b and
and S8b),
S8b), and
and trajectories
trajectories energy
energy terms
terms (data
(data not
not shown
shown but
but available
available from
from the
the authors
authors
upon request).
request). The short-termed MD protocol was sufficient to to produce
produce stable
stable complexes.
complexes. The The two
two
ACh poses
ACh poses (in
(in mAChE
mAChE or or hAChE)
hAChE) were
were conformationally
conformationally veryvery close,
close, displaying
displaying a RMSDRMSD value
value of
of
Å.
0.043 Å.
Figure 3. The SB alignment

Figure 3. alignment of of acetylcholine into mAChE
acetylcholine into mAChE active
active site (a); hAChE
site (a); hAChE active
active site
site (b).
(b). The
The
enzyme ribbons are presented in blue and gold, respectively,
respectively, active
active site
site amino
amino acids
acids are
are depicted
depicted inin
white. For the
white. For thepurpose
purposeofofclarity,
clarity,hydrogen
hydrogenatoms
atoms areare omitted
omitted from
from thethe presentation.
presentation. The The graphic
graphic was
was generated
generated using
using the the UCSF
UCSF Chimera
Chimera software(Resource
software (Resourcefor
for Biocomputing,
Biocomputing, Visualization,
Visualization, andand
Informatics (RBVI), University of California, San Francisco, CA,
Informatics (RBVI), University of California, San Francisco, CA, USA).USA).
expected, the positive quaternary ACh amine group was located in the anionic sub-site,
As expected,
where the orientation of
where the orientation of particular
particular group
group is
is supported
supported by
by strong
strong hydrophobic
hydrophobic interactions
interactions with a
m(h)Trp86 methyl
m(h)Trp86 methyl group,
group, asas well as with m(h)Tyr124, m(h)Tyr337,
m(h)Tyr337, and
and m(h)Tyr341
m(h)Tyr341 aromatic
aromatic moieties
moieties
(Figures S7c
(Figures S7c and
and S8c).
S8c). The
The positively
positively charged
charged amine
amine was
was electrostatically
electrostatically attracted
attracted by the
the electronic
electronic
cloud of the m(h)Trp86
cloud m(h)Trp86 aromatic
aromatic group.
group. The
The acetyl
acetyl group
group occupied
occupied thethe esterase
esterase sub-site:
sub-site: the carbonyl
oxygen was
was involved
involvedininthe
theelectrostatic interactions
electrostatic with
interactions m(h)Tyr337
with m(h)Tyr337 sideside
chain hydroxyl
chain group
hydroxyl and
group
m(h)His447
and imidazole
m(h)His447 ring,ring,
imidazole while thethe
while carbonyl oxygen
carbonyl oxygenwas
wasoriented
orientedtowards
towardsthe the side
side chain of
of
m(h)Tyr133. Regarding
Regarding thethe mouse
mouseenzyme,
enzyme,during
duringthe
theMD
MDrun,
run,the
thecarbonyl
carbonyl moiety
moiety was
was observed
observedto
be involved in the hydrogen bonding with mTyr133 for 29% of simulation time (Figure S8c, dHB =
1.943–2.337 Å) whilst for human enzyme the carbonyl group was mainly involved in the electrostatic
interactions with hSer203 and hHis447 (Figure S8c). The analysis of docking and MD results agreed
with the hydrolysis reaction pathway of ACh (Figure 1a) that was also externally confirmed by
QM/MM studies elsewhere [82]. In fact, the ACh molecule had been approaching m(h)Ser203 during
Molecules 2018, 23, 2192 13 of 37
to be involved in the hydrogen bonding with mTyr133 for 29% of simulation time (Figure S8c,
dHB = 1.943–2.337 Å) whilst for human enzyme the carbonyl group was mainly involved in the
electrostatic interactions with hSer203 and hHis447 (Figure S8c). The analysis of docking and MD
results agreed with the hydrolysis reaction pathway of ACh (Figure 1a) that was also externally
confirmed by QM/MM studies elsewhere [82]. In fact, the ACh molecule had been approaching
m(h)Ser203 during the simulation for the 57% of the time, where the range of distances between the
acetyl group and the catalytic residue was 1.473–1.894 Å (Figures S7c and S8c).
2.5.3. The Cross-Docking and Molecular Dynamics Studies of Pesticides 2-chloro-1,3,5-triazine Group
Atrazine’s, simazine’s and propazine’s acute toxicity (Table 1) can be explained by means of their
docked conformations, within either mAChE or hAChE (Figures 4 and 5, respectively). The graphical
interpretation of the results of the SB and MD studies are herein shown for atrazine (Figures 4a and 5a,
Figure S9 and Figure S10, respectively) and simazine (Figures 4b and 5b, Figure S13 and Figure S14,
respectively), whereas the corresponding illustrations considering the molecular modelling studies of
propazine are reported as Supplementary Materials (Figure S3a, Figure S5a, Figure S11, Figure S12,
respectively).
The best-docked poses of the triazine-based pesticides were found to occupy the ACh binding
cavities of both the mAChE and hAChE. The main differences were not related to the predicted
pesticides bioactive conformations, but were mainly ascribed to enzymes active site residue Thr83:
the side chain methyl group of mAChE Thr83 pointed toward the anionic sub-site, whereas in the
human enzyme the corresponding group is flipped out vertically from the active site. The latter caused
the differences in the stabilization period during the MD runs: atrazine-mAChE, propazine-mAChE,
and simazine-mAChE complexes were respectively of high stability after 0.100 ns, 0.120 and 0.610 ns
(Figures S9a, S11a and S13a), whereas the complexes of human analogues equilibrated approximately
20 ps afterwards (Figures S10a, S12a and S14a).
Molecules 2018, 23, 2192 14 of 37
Figure4.4.The
Figure TheSBSBalignment of atrazine
alignment (a); simazine
of atrazine (b); carbofuran
(a); simazine (c); monocrotophos
(b); carbofuran (d); dimethoate
(c); monocrotophos (d);
(e); imidacloprid
dimethoate (f) into the mAChE
(e); imidacloprid (f) into active site. The
the mAChE enzyme
active ribbons
site. The enzyme are presented
ribbons areinpresented
blue, the in
active
site amino
blue, acidssite
the active are amino
depicted in are
acids white. For the
depicted in purpose
white. Fortothe
clarify, the hydrogen
purpose atoms
to clarify, the are omitted.
hydrogen atoms The
are omitted.
graphic The graphic
was generated usingwas generated
the UCSF Chimerausing the UCSF
software Chimera
(Resource software (Resource
for Biocomputing, for
Visualization,
Biocomputing,
and InformaticsVisualization, and Informatics
(RBVI), University (RBVI),
of California, University CA,
San Francisco, of California,
USA). San Francisco, CA,
USA).
Molecules 2018, 23, 2192 15 of 37
Figure 5. The
Figure SB alignment
5. The of atrazine
SB alignment (a); simazine
of atrazine (b); carbofuran
(a); simazine (c); monocrotophos
(b); carbofuran (d); dimethoate
(c); monocrotophos (d);
(e);dimethoate
imidacloprid(e);(f) into the hAChE
imidacloprid active
(f) into site. The
the hAChE enzyme
active site. ribbons are presented
The enzyme inpresented
ribbons are gold; the active
in gold;site
amino acids are
the active site depicted
amino acidsin white. For theinpurpose
are depicted to clarify,
white. For the hydrogen
the purpose to clarify,atoms are omitted
the hydrogen from
atoms arethe
presentation.
omitted fromThe the
graphic was generated
presentation. using thewas
The graphic UCSF Chimerausing
generated software
the (Resource for Biocomputing,
UCSF Chimera software
Visualization,
(Resource forandBiocomputing,
Informatics (RBVI), University
Visualization, andofInformatics
California,(RBVI),
San Francisco, CA,of
University USA).
California, San
Francisco, CA, USA).
Nevertheless, within either mice or human bioactive conformations, the 2-chloro-1,3,5-triazines
Nevertheless,
were parallel within
to m(h)Trp86 either
and mice or human
m(h)Tyr337, bioactive
trapped conformations,
in the hydrophobic cagethe 2-chloro-1,3,5-triazines
between two residues. The
were parallel to m(h)Trp86 and m(h)Tyr337, trapped in the hydrophobic cage between two residues.
individual contribution of these interactions was respectively −37.34 and −19.9 kcal/mol, according
The individual contribution of these interactions was respectively −37.34 and −19.9 kcal/mol,
to the Free-Energy Decomposition Analysis, FEDA (Table S11). Atrazine bioactive conformations
according to the Free-Energy Decomposition Analysis, FEDA (Table S11). Atrazine bioactive
were nearly identical in both enzymes. On the other hand, comparing simazine docked structures
(Figures 4b and 5b), the one found in hAChE (Figure 5b) was in-plane rotated for about 30◦ . The
Molecules 2018, 23, 2192 16 of 37
triazine nitrogen atom, p-positioned in relation to chlorine, was electrostatically stabilized by both
the hydroxyl group of m(h)Tyr337 and the indole nitrogen of m(h)Trp86. The individual contribution
of these interactions was respectively −34.26 and −15.56 kcal/mol, according to FEDA (Table S11).
Within the mAChE, the m-N’ atoms, holding the isopropyl function of atrazine (Figure 4a), and one of
the ethyl groups within simazine (Figure 4b), were oriented toward the mThr83 methyl group and
mTyr341 phenolic moiety. All the described interactions were confirmed during the MD simulation
(Figure S9c and Figure S13c, respectively). The atrazine m-N’ atom was involved in a strong hydrogen
bonding (HB) (dHB = 2.253–2.976 Å) with the hThr83 hydroxyl group (Figure S10c) in 56% of MD run
time. A similar interaction was not detected during the atrazine-mAChE complex MD run (Figure S9c),
suggesting that the hThr83 conformational flip and the existence of hThr86-m-N 0 HB may even increase
the acute toxicity of atrazine to humans. Regarding the simazine, as a consequence of the in-plane
rotation, its m-N’ atom created HB with hTyr341 (dHB = 2.341–3.127 Å, 76% of the time of simulation,
Figure S14c). Furthermore, the functional groups that substitute the remaining m-N” atoms were
directed towards the hTyr133, hSer203, and hHis447, and HB-attracted by hHis447 (simazine established
the strongest HB, dHB = 2.379–3.242 Å, 94% of MD time, Figure S14c). Since the precise hydrogen
bonds were not observed during the MD simulation within the mAChE, they may represent some
indicators that the atrazine and simazine could exert even higher acute toxicity against humans than
against mice. Moreover, the described docking/MD interactions were all confirmed by the FEDA
application (Table S11).
Regarding propazine, the m-N 0 atoms, holding the isopropyl units or propazin (Figure S5a) were
oriented within the mAChE towards the side chain methyl group of mThr83 and a phenolic moiety
of mTyr341. Similarly to atrazine and simazine, propazine may be even more toxic to humans than
to mice, as it exerts the toxicity against humans by establishing the hydrogen bonds with hTyr341
(dHB = 2.286–3.114 Å) and hHis447 (dHB = 2.121–3.331 Å) in the 45% and the 95% of time, respectively
(Figure S12c). Attracted to mThr83, propazine was limited with the ability to make hydrogen bonds
with mTyr341 or mHis447, as confirmed by MD studies (Figure S11c).
2.5.4. The Cross-Docking and Molecular Dynamics Studies of Pesticides Amide Group
Among the amide-based pesticides, carbofuran and monocrotophos were of the highest acute
toxicity (Table 1). Therefore, the results of carbofuran (Figures 4c and 5c, Figure S15 and Figure S16,
respectively) and monocrotophos (Figures 4d and 5d, Figures S17 and Figure S18, respectively)
docking and MD studies are herein reported and discussed, whereas the corresponding graphical
interpretations for lower toxic dimethoate (Figure 4e, Figure 5e, Figure S19, Figure S20, respectively),
carbaryl and tebufenozide are reported as a Supplementary Materials (Figure S4, Figure S5, Figures
S21–S24, respectively).
Carbofuran was the most toxic against Mus musculus (Table 1). According to the literature, this
particular pesticide is, as a carbamate derivative, proclaimed to be the reversible AChE inhibitor [73].
The results of herein conducted SB studies are in full agreement with the proposed/confirmed
reversible inhibition of AChE by carbofuran. Hence, within either mAChE or hAChE active site,
the compound’s methyl methylcarbamate scaffold contributed as follows: the N-methyl group
was positioned between the methyl group of m(h)Thr83 and m(h)Trp86 (Figures 4c and 5c), a
carbonyl group interfered with m(h)Tyr341 while the ether link was electrostatically stabilized by
hydroxyl groups of m(h)Tyr124, m(h)Tyr337, and m(h)Tyr341, respectively. However, during the
MD simulations, methylcarbamate displayed rotation towards the m(h)Tyr124 (Figures S15c and
S16c) upon which the amide carbonyl group was involved in strong HB with m(h)Tyr124 for the
40% of time (dHB = 2.478−2.783 Å, Figure S15c and Figure S16c, respectively), indicating a possible
reason for carbofuran acute toxicity. The other HB was formed between the secondary amine
and m(h)Trp86 (dHB = 2.784−3.226 Å, Figure S15c and Figure S16c, respectively). Furthermore,
the 2,2-dimethyl-2,3-dihydrobenzofuran part of carbofuran was located beneath the m(h)Trp86
and established hydrophobic interactions with m(h)Tyr337 due to the π-π stacking, as well as the
Molecules 2018, 23, 2192 17 of 37
electrostatic interactions with m(h)Tyr124 via 2,2-dimethyltetrahydrofuran oxygen atom. Moreover,

all the described docking/MD interactions were confirmed upon conducting the FEDA procedure
(Table S11).
The covalent mode of inhibition [73] and the high acute toxicity of organophosphorus-based
pesticides monocrotophos and dimethoate against Mus musculus and Homo sapiens were primarily
conditioned by the interactions of their amide groups with AChE. Hence, in the best docking pose
of monocrotophos within mAChE, the amide carbonyl was oriented toward the hydroxyl group
of mTyr341 (Figure 4d). Such interaction was a result of hydrophobic stabilization between the
pesticide’s allyl methyl group and the mTyr341 phenyl ring, as well of the N-methyl group and
mThr83. Still, within the human enzyme (Figure 5d), corresponding amide carbonyl group approached
the hThr83 hydroxyl group, confirming that the orientation of hThr83 makes a difference in SB
alignment in hAChE. The consequent important monocrotophos interactions were those generated
by the dimethylated phosphoric acid. Thus, the phosphoric acid P=O group established electrostatic
interferences with mAChE Tyr124 (Figure 4d); the precise oxygen atom served as an HB-acceptor to
mTyr124 during the MD simulation (dHB = 2.227–2.946 Å, Figure S17c). For the purpose of hAChE
inhibition, the corresponding monocrotophos carbonyl group was, due to the strong hydrophobic
attraction between the phosphoric acid methoxy groups and hTrp86, only partially directed towards
the hTyr124 (Figure 5d). However, monocrotophos adapted its conformation/binding upon 0.718 ns of
MD simulations with hAChE (Figure S18a), and, contrarily to mAChE, hTyr124 was involved in 96%
of the time in HB with the amide carbonyl (dHB = 1.984–2.117 Å, Figure S18c). Moreover, one of the
phosphoric acid oxygen atoms made HB with hTyr337 (35% of simulation time, dHB = 2.449–2.688 Å,
Figure S18c). Still, inhibiting mAChE or hAChE, the monocrotophos phosphoric acid moiety remained
situated in the proximity of m(h)Ser203 and m(h)His447 (the average distances: 1.25–1.783 Å for mAChE,
1.375–1.651 Å for hAChE, Figure S17c and Figure S18c, respectively), suggesting that the covalent
modification of mSer203 or hSer203 will occur in vivo [74]. Moreover, all the described docking/MD
interactions were confirmed upon conducting the FEDA (Table S11).
The monocrotophos structural analogue, dimethoate, is, as Mus musculus and Homo sapiens
AChE inhibitor, limited with the capacity to interfere with m(h)Tyr341 by means of steric interactions,
inasmuch as it does not contain the allyl methyl group (Figures 4e and 5e, respectively). Hence,
although dimethoate is in both active sites superimposed to monocrotophos, by means of backbone
orientation (Figures 4d and 5d, respectively), its amide carbonyl is oriented towards m(h)Tyr124 and
m(h)Tyr337. The bioisosteric replacement of the phosphoric acid carbonyl group with the thione
one influenced that the sulfur atom is within the mouse enzyme directed to the hydroxyl group of
m(h)Tyr337, while within the human one the thione moiety is in reach of the m(h)Ser203 and m(h)His447.
However, surprisingly, there was a little consistency between the best-docked structure of
dimethoate within the mouse active site (Figure 4e) and the molecular dynamics solutions (Figure S19).
Thus, pesticide’s amide carbonyl established no interactions, while the amide nitrogen was periodically
involved in hydrogen bonding with mTyr341 time (dHB = 2.971–3.557 Å, Figure S19c). Thione
functional group was HB-acceptor to mHis447 (25% of the time, dHB = 2.894–3.431 Å), while one
of the methoxy functions was in the HB-vicinity to mGly121 (27% of the time, dHB = 2.723–3.338 Å).
Those interactions allegedly provide the opportunity for mSer203 to perform the electrophilic attack on
pesticide (Figure 6f). On the other side, there was a high level of agreement between the dimethoate
rigid docking (Figure 5e) and molecular dynamics within the human enzyme, by means of the amide
carbonyl group interactions, given that this pharmacophore was for the 95% of time included in
hydrogen bonding with hTyr124 (dHB = 1.643–2.128 Å, Figure S20c). Moreover, one of the phosphoric
acid oxygens was, as for monocrotophos, used to make a hydrogen bond with hTyr337 (35% of
simulation time, dHB = 2.449–2.688 Å, Figure S20c). In general, considering the overall orientation of
the molecule, dimethoate would most likely behave as a covalent inhibitor of human AChE (Figure 6f),
as well.
Molecules 2018, 23, 2192 18 of 37
Figure 6. Schematic view of pesticides binding interactions within the hAChE active site: atrazine,
Figure 6.or
simazine Schematic
propazineview of pesticides
(a); carbofuran or binding
carbarylinteractions within the
(b); monocrotophos hAChE active
or dimethoate (c) site: atrazine,
imidacloprid
simazine or propazine (a); carbofuran or carbaryl (b); monocrotophos or dimethoate
or acetamiprid (d); diuron, monuron, or linuron (e). The hydrogen bonds are presented with pink (c) imidacloprid
or acetamiprid
dashed (d); diuron,
half arrows, monuron,interactions
the hydrophobic or linuron (e).
withThe hydrogen
a blue dot-linebonds arethe
marker, presented with pink
ionic interactions
dashed half arrows, the hydrophobic interactions with a blue dot-line marker, the
with the red dashed lines. The proposed mechanism of monocrotophos or dimethoate acute toxicity ionic interactions
with thehAChE
against red dashed
(f). lines. The proposed mechanism of monocrotophos or dimethoate acute toxicity
against hAChE (f).
Carbaryl reached the comparable bioactive conformation to carbofuran (Figures S3b and S5b),
The results of molecular docking are presented as a Supplementary Materials (Figures S35–S42).
within both species AChE active sites, with the difference that the 2,2-dimethyltetrahydrofuran scaffold
The generated conformations were used further on for the external validation of the training set
is in the structure of particular pesticide replaced with another benzene ring. Such replacement has a
results.
Molecules 2018, 23, 2192 19 of 37
direct consequence in a diminished toxicity of carbaryl in comparison with carbofuran, against either
mice or humans, since carbaryl cannot establish additional electrostatic interactions with m(h)Tyr124.
Regarding carbaryl, it is proclaimed (Table 1) that this pesticide is more than fifty times less toxic
than carbofuran when administered orally to Mus musculus, and MD revealed the reason. Hence,
within the mAChE, carbaryl’s amide carbonyl group forms a hydrogen bond, not with the mTyr124, but
with the HB-network made of ordered water molecules and mAsn87 (Figure S21c). The precise function
also establishes the pure HB with mSer125 (75% of simulation time, dHB = 2.335–2.412 Å, Figure S21c).
However, according to the MD study performed on the carbaryl-hAChE complex, there is a 36% of
probability that particular carbonyl group is oriented towards HB with hTyr124 (dHB = 2.374–2.894 Å,
Figure S22c), thus anticipating the high toxicity of this pesticide to humans.
The lowest active amide-based pesticide is tebufenozide (Table 1). Almost symmetrical
as a compound, this pesticide can be divided in two scaffolds by which it establishes the
interactions within the active site of either mAChE or hAChE: the 4-ethylbenzamide and the
N-tert-butyl-3,5-dimethylbenzamide (Figure S3c and Figure S5c, respectively). The RMDS value
between the mouse and human docked conformations amounts 0.472 Å, implying the high similarity
between the obtained conformations. Thus, the ethylbenzene scaffold of 4-ethylbenzamide was
oriented parallel to benzene core of m(h)Tyr337, establishing hydrophobic interactions. The carbonyl
group that completes the 4-ethylbenzamide was electrostatically attracted by three active site residues:
m(h)Tyr124, m(h)Tyr337 and m(h)Tyr341. The hydrophobic part of m(h)Tyr124 additionally stabilized
the tert-butyl group, incorporated in the second part of pesticide. The carbonyl group belonging to the
tert-butyl-3,5-dimethylbenzamide moiety was geared towards the amide group of m(h)Gln71 and the
hydroxyl group of m(h)Tyr124, for the purpose of forming the electrostatic interactions. The remaining
part of the distinct subsection, in the form of 3,5-dimethylbenzene, is stabilized with the hydrophobic
parts of m(h)Trp86 and m(h)Phe295. Similarly to other amide-based pesticides, this pesticide exerts
the toxicity after creating the strong hydrogen bond in-between the tert-butyl-3,5-dimethylbenzamide
moiety and either mTyr124 or hTyr124 (95% of simulation time, dHB = 1.273–1.588 Å, Figure S23c;
94% of simulation time, dHB = 1.268–1.575 Å, Figure S24c). Beside those beneficial interferences, the
pesticide owns its stability and consequent toxicity to numerous hydrophobic interactions (Figure S23c
and Figure S24c, respectively).
2.5.5. The Cross-Docking and Molecular Dynamics Studies of Pesticides (6-Chloropyridin-3-

Yl)Methanamine and 1-(4-Chlorophenyl)-3-methylurea Groups
The pesticides comprising the two remaining groups exerted the lowest acute toxicities to Mus
musculus (Table 1). The results of SB and MD studies are herein shown for imidacloprid (Figures 4f and
5f, Figure S25 and Figure S26, respectively), while the corresponding exemplifications considering the
molecular modelling studies for lower toxic acetamiprid, diuron, monuron, and linuron are available
as Supplementary Material (Figures S3–S6 and Figures S27–S34, respectively).
Regarding the best docking poses of imidacloprid within either mAChE or hAChE, these
indicated that the 2-chloro-5-methylpyridine moiety was sterically stabilized by m(h)Trp86 and
m(h)Tyr337 (Figures 4f and 5f, respectively). The difference in the alignment occurred with the
N-(imidazolidine-2-ylidene)nitramide scaffold, whose nitro group was oriented towards mSer203,
while the same function in human enzyme was in the proximity of hTyr124. Even though the
imidacloprid-mAChE complex was more stable in comparison to the human one (Figure S25a),
interactions generated within the complex were inconsistent to those that imidacloprid established
with hAChE. Thus, while interacting with mAChE (Figure S25c), the nitro group was involved in
the hydrogen bonding with mGly122 (64% of MD time, dHB = 2.483–2.788 Å) and mAla204 (59% of
MD time, dHB = 2.771–3.132 Å). The corresponding functional group in hAChE (Figure S26c) was
a HB acceptor for hTyr124 (82% of MD time, dHB = 1.664–1.738 Å) and hTyr337 (68% of MD time,
dHB = 1.825–1.931 Å), and further confirmed those residues as essential for pesticides acute toxicity in
humans. Moreover, all the described docking/MD interactions were confirmed by FEDA (Table S11).
Molecules 2018, 23, 2192 20 of 37
The important interactions anticipated by the molecular docking and molecular dynamics
(Figure 6), provided the basis for the understanding of the general mechanism of acute toxicity on the
reversible and reversible-like inhibitors level. Even though the covalent AChE inhibitors are highly
efficient as pesticides, the future pesticides production must be based on the reversible-like interactions,
avoiding the pesticide-AChE complex regeneration or ageing [73]. The obtained reversible-like docking
and MD poses for covalent AChE inhibitors clearly confirmed the potency of selected computational
tools (Vina/Desmond programmes pair) to predict the covalent binding mode of any compound
in future considered as covalent AChE inhibitor (i.e., the covalent pesticide). Therefore, there was
no point to conduct the covalent docking that would be reduced just to a conformational search
of the bonded molecules. Hereby applied docking/MD application was also aimed to assess the
docking/MD software real utility to investigate the covalent AChE inhibitors. It must be emphasized
that any inhibitor, even covalent, must undergo equilibrium between the free and the bonded stage.
Therefore, the reversible-like (pre-covalent) pose is of extreme utility to predict the ligand conformation
that will proceed to make a covalent bond. This information could be used in future works to design
new reversible inhibitors (pesticide).
Considering the docking poses of acetamiprid (Figure S3d and Figure S4d, respectively),
pesticide’s 2-chloro-5-methylpyridine moieties were aligned to each other in both enzymes and located
in-between the m(h)Trp86 and m(h)Tyr337. The toxicity of particular pesticide may be additionally seen
through the interactions of (E)-N-ethyl-N 0 -ethynyl-N-methylacetimidamide function, linked directly to
the 2-chloropyridine ring and located in the spatial pocket made of m(h)Trp86, m(h)Gly121, m(h)Tyr133,
m(h)Ser203, and m(h)His 447. However, according to the molecular dynamics results (Figure S27c
and Figure S28c, respectively), this part of the pesticide has not achieved significant interactions
with the specified site of amino acids, and the low toxicity of acetamiprid may be attributed only to
the hydrophobic interactions established between the 2-chloro-5-methylpyridine and m(h)Trp86 and
m(h)Tyr337.
Concerning the remaining compounds, diuron, linuron, and monuron, the basic scaffold they
share is the 1-(4-chlorophenyl)-3-methylurea. In the structure of diuron, the terminal amine is dimethyl
substituted while the aromatic moiety bears additional m-Cl substituent. Matching the conformations
of diuron generated in Mus musculus and Homo sapiens AChE (Figure S4a and Figure S6a, respectively),
conclusion can be made that the structures were almost perpendicular to each other, by means of
aromatic rings positioning: the aromatic scaffold in mouse enzyme active site was perpendicular
to mTrp86 and mTyr337, while in the human environment this scaffold adopted parallel orientation
related to hTrp86 and hTyr337 (analogously to previous compounds). Considering the mAChE, the
only reasonable explanation for such alignment of diuron is the interaction of mThr83 methyl group
with the pesticide aromatic scaffold; the mThr83 side chain was positioned inside the enzyme active
site and not out of it, like in humans (Figure S4a). Because of the different alignment, the carbonyl
group of diuron0 s 1,1-dimethylurea scaffold was oriented towards the mTyr124, mSer203, mTyr337, and
mHis447 (Figure S4a). In the human environment (Figure S6a), the exact carbonyl group was directed
towards the hTrp86 and hTyr133. The MD simulation of mouse complex confirmed the weak hydrogen
bonding between the urea carbonyl group and mTyr124 (31% of simulation time, dHB = 2.872–3.132 Å,
Figure S29c), while the corresponding interaction could not be established in human surroundings
(Figure S30c). The low intensity of precise hydrogen bond may be the reason of low toxicity of diuron,
latter on concluded for linuron, and monuron, too.
Speaking of the remaining derivatives, the aromatic scaffolds of monuron (Figure S4b and
Figure S6b, respectively), and linuron (Figure S4c and Figure S6c, respectively) were in a sandwich
between the m(h)Trp86 and m(h)Tyr337, in either mouse or human enzyme active site. The monuron
carbonyl group was oriented towards the m(h)Tyr133 and m(h)Ser203, whereas the particular group
in linuron is headed to m(h)Tyr 124, m(h)Tyr133 and m(h)Ser203. The nature of monuron and linuron
carbonyl groups interactions with specified amino acid had been revealed after the MD studies. Thus,
for either mouse or human AChE-based bioactive conformations, the carbonyl group of monuron
Molecules 2018, 23, 2192 21 of 37
interfered with m(h)Tyr124 in a manner of moderate strength hydrogen bonding (within the mouse
enzyme active site during the 62% of simulation time, dHB = 2.548–3.012 Å, Figure S31c; within
the human enzyme active site during the 56% of simulation time via the ordered water molecule,
dHB = 2.936–3.326 Å, Figure S32c). Similar interactions were observed for linuron as well (within
the mouse enzyme active site during the 56% of simulation time via the ordered water molecule,
dHB = 2.985–3.455 Å, Figure S33c; within the human enzyme active site during the 90% of simulation
time, dHB = 2.847–3.278 Å, Figure S34c).
2.5.6. The Cross-Docking of Externally Evaluated Pesticides

The structure-based studies by means of reversible and reversible-like bioactive conformations
generation, recommended Vina as a tool for pesticides cheminformatics treating. Therefore, Vina was
used to predict the global minimum conformation of test set pesticides (Table 2) as well.
The results of molecular docking are presented as a Supplementary Materials (Figures S35–S42).
The generated conformations were used further on for the external validation of the training set results.
2.6. The Pesticides Acute Toxicity Anticipation through the Bioactive Conformations
Upon conducting the reversible and reversible-like molecular docking and molecular dynamics
studies, for either training or test set pesticides, the acute toxicity was considered with regard to
the established bioactive conformations. Therefore, similarly as above reported for global minima
energies, monoparametric QSAR model was derived to initially describe the acute toxicity against
Mus musculus by enumerating the acute toxicities through the free energies of formation of reversible
and reversible-like binding for all the training set pesticides (model 5, Equation (6), Table S12):
pLD50 = −0.7885∆Gbinding − 2.44 (r2 = 0.95), (6)
A satisfactory model was obtained by means of the whole training set and was used to
recalculate the pLD50 values for acute toxicity against Mus musculus, for both the training and test
sets (Figure 7a,b, Tables S12 and S13). Afterwards, the model was used to predict the pLD50 values
for acute toxicity against humans on the basis of ∆Gbinding values for Homo sapiens AChE (Figure 7c,d,
Tables S14 and S15).
Thus, the QSAR model 5 described the acute toxicities against Mus musculus, by means of the free
energies of binding of reversible and reversible-like bioactive conformations for either non-covalent
or covalent pesticides, respectively. The model is also proven to be a tool for the anticipation of
acute toxicities against humans. In the end, the conduced structure-based studies unequivocally
supported pLD50 /∆Gbinding interrelation obtained by QSAR model 5. In that sense, the SB approach,
conducted through Vina as the cheminformatics engine, was proven to be the correct one for the
anticipation of acute toxicities of targeted pesticides. Consequently, the Vina-based ∆Gbinding values
can be considered in future design as valuable indicators to anticipate the pesticides LD50 values
against any model organism.
= −0.7885 binding − 2.44 = 0.95 , (6)
A satisfactory model was obtained by means of the whole training set and was used to
recalculate the pLD50 values for acute toxicity against Mus musculus, for both the training and test sets
(Figure 7a,b, Tables S12 and S13). Afterwards, the model was used to predict the pLD50 values for
acute toxicity
Molecules 2018, 23,against
2192 humans on the basis of ∆Gbinding values for Homo sapiens AChE (Figure227c,d,
of 37
Tables S14 and S15).
Figure 7. The plot of experimental vs. fitted pLD50 values calculated with QSAR model 5 for training
Figure 7. The plot of experimental vs. fitted pLD50 values calculated with QSAR model 5 for training
set pesticides (a); test set pesticides (b) against Mus musculus. The plot of calculated vs. fitted pLD50
set pesticides (a); test set pesticides (b) against Mus musculus. The plot of calculated vs. fitted pLD50
values calculated with QSAR model 5 for training set pesticides (c); test set pesticides (d) against Homo
values calculated with QSAR model 5 for training set pesticides (c); test set pesticides (d) against Homo
sapiens.
sapiens.
Thus, the QSAR model 5 described the acute toxicities against Mus musculus, by means of the
2.7. The Quantum Mechanical Studies of Acetylcholinesterase Inhibition by 2-Chloro-1,3,5-triazine-based
free energies of binding of reversible and reversible-like bioactive conformations for either non-
Pesticides
covalent or covalent pesticides, respectively. The model is also proven to be a tool for the anticipation
Thetoxicities
of acute performed molecular
against humans.docking andthemolecular
In the end, dynamics studies
conduced structure-based agreed
studies with the
unequivocally
previously reported mode of action of amide-based, 6-chloropyridine-3-yl)methanamine-based,
supported pLD50/∆Gbinding interrelation obtained by QSAR model 5. In that sense, the SB approach,
and 1-(4-chlorophenyl)-3-methylurea-based
conducted through Vina as the cheminformatics pesticides
engine, [73]. The quantum
was proven mechanical
to be the correct one for(QM)
the
calculations were only performed on hAChE, with the aim to reveal the pharmacology
anticipation of acute toxicities of targeted pesticides. Consequently, the Vina-based ∆Gbinding values of
2-chloro-1,3,5-triazine-based
can pesticides
be considered in future design and theindicators
as valuable origin for to the acute the
anticipate toxicity, givenLD
pesticides that their
50 values
pharmacology
against is the
any model least understood.
organism.
As molecular docking and molecular dynamics studies indicated the formation of hydrogen bonds
network between the atrazine, propazine, and simazine and hAChE, the HB-bearing pesticides-hAChE
MD poses were extracted from pesticides-hAChE complexes and subjected to quantum mechanical
(QM) DFT mechanistic studies. Thus, the DFT-based calculations were performed on two different
levels. The first level included the extraction of MD geometries where favourable hydrogen
bonds between the 2-chloro-1,3,5-triazine-based pesticides and hThr83, hTyr341, hTyr124, and
hHis447, respectively, were found. Subsequently, the extracted pesticide-hThr83, pesticide-hTyr124,
pesticide-hTyr341, and pesticide-hHis447 HB forming geometries were QM optimized in order to locate
the transition states (TSs) and/or the intermediary states (ISs) by which the protons transfers occur in
the particular segment of biochemical reaction. The second level of calculation attempted to find the
identical TSs and/or ISs upon the manual adjustment of starting pesticide-hThr83, pesticide-hTyr124,
pesticide-hTyr341, and pesticide-hHis447 geometries, by means of the TS-generating hydrogen atom
position between the pesticide and the regarded AChE residue, in a kind of validation process. Each of
the reaction pathways assumed the contemplation by means of Intrinsic Reaction Coordinate (IRC)
Molecules 2018, 23, 2192 23 of 37
analysis. Remarkably, the independent calculations gave similar/identical results, confirming that
each of the obtained TSs and/or ISs is not formed by chance.
Thus, the optimization of atrazine-hThr83, atrazine-hTyr124, atrazine-hTyr341, and
atrazine-hHis447 HB geometries confirmed that the proton transfer, within each of the geometries,
occurs via the adequate transition states. Hence, the inhibition of hAChE starts with the nucleophilic
attack of atrazine’s m-N0 atom hydrogen towards hThr83 (Figure 8a). The distance between the hThr83
hydroxyl group oxygen atom as a HB acceptor and m-N0 atom as a HB donor amounts 3.758 Å,
characterizing particular HB as weak, almost electrostatic by character. This is, certainly, the expected
increase in atrazine-hThr83 HB distance, arisen following the QM optimization, in comparison to the
one recorded during the MD simulations (dHB = 2.253−2.976 Å). Nevertheless, even the weak HB
is enough to increase the acute toxicity of atrazine to humans, in comparison to the propazine and
simazine, inasmuch as the latter pesticides are, according to the QM studies, unable to donate proton
for the formation of described TS1. Instead, the propazine-hThr83 and simazine-hThr83 interactions
(Figure S43a and Figure S44a, respectively) were characterized by the IS1; the optimized QM distance
between the hThr83 hydroxyl group oxygen atom and propazine and simazine m-N0 atom was 4.259 Å
and 4.281 Å, respectively, suggesting the electrostatic nature of the established bond instead of the HB
one. Back to the atrazine, upon the formation of atrazine-hThr83 TS1 (Figure 8a and Scheme 1), the
deprotonation of hThr83 side chain hydroxyl group occurs, yielding the negatively charged oxygen
atom, whereas at the same time m-N0 atom becomes positively charged. Identical TS1 conformation
and reaction pathway was obtained upon the manual setup and the optimization of the transition
state geometry. The calculated activation energy barrier for the generated TS1 is 11.2 kcal/mol and
this initial reaction is the rate-limiting step for the hAChE inhibition by atrazine (Figures 8a and 9).
The Free energy profile of all of the described reaction pathways was presented similarly to the study
describing the catalytic reaction mechanism of AChE on the ACh level [82]. The manual setup of TS1
coordinates and its optimization resulted in the fact that the 47% of IRC described the proton transfer
from TS1 to hThr83, whereas the 53% or IRC quantified the proton transfer from TS1 to atrazine m-N0
atom. This result further supported the proposed nucleophilic attack; the proton transfer is concerted,
and the characterized transition state is meaningful. The precise atrazine-hThr83 TS1/hydrogen bond
is formed and is stable at all transition and intermediate states during the inhibition process.
Molecules 2018, 23, 2192 24 of 37
Figure 8. The quantum chemical mechanism of Homo sapiens AChE inhibition by atrazine. The
Figure 8. The quantum chemical mechanism of Homo sapiens AChE inhibition by atrazine. The extracted
extracted geometry of TS1 (a); TS2 (b); TS3 (c); TS4 (d).
geometry of TS1 (a); TS2 (b); TS3 (c); TS4 (d).
The atrazine-based
The atrazine-based hAChEhAChE inhibition
inhibition further
further flows
flows towards
towards the the deprotonation
deprotonation of m-N0 atom
of m-N′ atom
(Figure 8b and Scheme 1) where hTyr341 phenoxy anion serves as a HB-acceptor.
(Figure 8b and Scheme 1) where hTyr341 phenoxy anion serves as a HB-acceptor. The deprotonation The deprotonation
concerted via
concerted viathe
theTS2,
TS2,ininwhich
which thethe distance
distance between
between thethe electronegative
electronegative atomsatoms amounts
amounts 3.2113.211
Å. TheÅ.
The similar transition states (this time the TS1s) were also observed for
similar transition states (this time the TS1s) were also observed for propazine and simazine (Figurepropazine and simazine
(Figure
S43b S43b
and and Figure
Figure S44b, S44b, respectively),
respectively), and and for peculiar
for peculiar pesticides
pesticides thisthis
waswasthe
therate-limited
rate-limited reaction
reaction
(Figure S45a,b).
(Figure The calculated
S45a,b). The calculated activation
activation energy
energy barriers
barriers for
for the
the generation
generation of of propazine-hThr83
propazine-hThr83 and and
simazine-hThr83 TS1 complexes were 8.5 and 8.9 kcal/mol, respectively (Figure
simazine-hThr83 TS1 complexes were 8.5 and 8.9 kcal/mol, respectively (Figure S45a and Figure S45b, S45a and Figure S45b,
respectively). To
respectively). Toadopt
adoptthe atrazine-hTyr341
the atrazine-hTyr341 TS2TS2geometry
geometry(Figure 8b and
(Figure 8bScheme 1), atrazine
and Scheme is slightly
1), atrazine is
in-plane rotated towards hTyr341, whereas propazine and simazine suffered
slightly in-plane rotated towards hTyr341, whereas propazine and simazine suffered more severe more severe longitudinal
movement (Figure
longitudinal S43b and
movement Figure
(Figure S44b,
S43b andrespectively).
Figure S44b, Considering that the
respectively). energy difference
Considering that thebetween
energy
the atrazine-hTyr341
difference between the reactant state and TS2
atrazine-hTyr341 is onlystate
reactant 1.6 kcal/mol
and TS2 is (Figure
only 1.6 9),kcal/mol
an extra stabilization
(Figure 9), ancomes
extra
stabilization comes with the formation of atrazine-hTyr341 TS2. The length of atrazine-hTyr124 as
with the formation of atrazine-hTyr341 TS2. The length of atrazine-hTyr124 HB characterizes it HBa
moderate one. The second level of QM optimization also confirmed
characterizes it as a moderate one. The second level of QM optimization also confirmed the the meaningfulness of TS2. As
previous, the meaningfulness
meaningfulness of TS2 isthe
of TS2. As previous, confirmed since theof56%
meaningfulness TS2ofisIRC described
confirmed thethe
since proton
56% gliding
of IRC
from TS3 to hTyr341, whereas the 44% or IRC illustrated the proton transfer
described the proton gliding from TS3 to hTyr341, whereas the 44% or IRC illustrated the proton form TS2 for atrazine m-N0
atom. Similar
transfer form trends
TS2 forwere noticed
atrazine m-N′foratom.
propazine-hTyr341
Similar trends and simazine-hTyr341
were TS1 (Figure S43b and
noticed for propazine-hTyr341 and
Figure S44b, respectively).
simazine-hTyr341 TS1 (Figure Nevertheless, duringS44b,
S43b and Figure the rotation of atrazine
respectively). (the movement
Nevertheless, duringofthepropazine
rotation
or simazine),
of atrazine (the themovement
TS1 (IS1) isof sustained
propazine(Figure S43b and the
or simazine), Figure
TS1S44b,
(IS1) respectively).
is sustained (Figure S43b and
Figure S44b, respectively).
Molecules 2018, 23, 2192 25 of 37
Scheme 1. The mechanism of Homo sapiens AChE inhibition by atrazine, propazine, and simazine,
Scheme 1. The
verified by QM studies. of Homo sapiens AChE inhibition by atrazine, propazine, and simazine,
mechanism
verified by QM studies.
In theInthird
the third
stepstep of the
of the reactionpathway,
reaction pathway,thethe electrophilic
electrophilic nature
natureofof
thethe
re-established secondary
re-established secondary
0 m-N′ amine is expressed by the release of proton towards the hTyr124 hydroxyl group anion. The
m-N amine is expressed by the release of proton towards the hTyr124 hydroxyl group anion. The proton
proton transfer takes place via the TS3, in which the distance between the electronegative atoms
transfer takes place via the TS3, in which the distance between the electronegative atoms amounts
amounts 3.522 Å (Figure 8c and Scheme 1). For the purpose of TS3 generation, atrazine is forced to
3.522 move
Å (Figure 8c and Scheme
longitudinally in order1).to For
formthe
thepurpose of TS3
tetrahedral generation,
structure with theatrazine is forced
HB-involved to move
residues.
longitudinally
Therefore, this step is slightly energetically more expensive, with the free energy difference of 4.8 this
in order to form the tetrahedral structure with the HB-involved residues. Therefore,
step iskcal/mol
slightly(Figure
energetically more expensive,
9). The characteristic of thewith the freeTS3
tetrahedral energy difference
structure is that of 4.8 kcal/mol
it holds (Figure 9).
the negatively
The characteristic of them-N′
charged secondary tetrahedral
nitrogenTS3 structure
atom. is that
The distinct it holds
state the negatively
is supported chargednature
by the aromatic of them-N0
secondary
nitrogen atom. The distinct state is supported by the aromatic nature of the atrazine’s 1,3,5-triazine
ring, which can accept the m-N0 amine negative charge by the intramolecular resonance stabilization.
As mentioned above, the meaningfulness of the TS3 is confirmed since 57% of IRC described the proton
atrazine’s 1,3,5-triazine
Molecules 2018, 23, 2192 ring, which can accept the m-N′ amine negative charge by the intramolecular 26 of 37
resonance stabilization. As mentioned above, the meaningfulness of the TS3 is confirmed since 57%
of IRC described the proton gliding from TS3 to hTyr124, whereas the 43% or the IRC illustrated the
glidingtransfer
proton from TS3 from to hTyr124, whereasm-N′
TS3 to atrazine’s the 43%
atom. or On
the the
IRCother
illustrated
hand, thethe propazine-hTyr124
proton transfer from TS3
and to
the
atrazine’s m-N 0 atom. On the other hand, the propazine-hTyr124 and the simazine-hTyr124 interactions
simazine-hTyr124 interactions are characterized by the IS2 (Figure S43c and Figure S44c,
are characterized
respectively), in whichby thethe IS2QM(Figure S43c and
optimized Figure S44c,
distances between respectively),
the hTyr124 in which
and the thepropazine
QM optimized and
distances between the hTyr124
simazine were 4.882 and 3.862 Å, respectively.and the propazine and simazine were 4.882 and 3.862 Å, respectively.
Moreover,the
Moreover, theformation
formationofof thethe atrazine-hAChE
atrazine-hAChE tetrahedral
tetrahedral TS3TS3 structure
structure appears
appears to beto thebepre-
the
pre-condition
condition for hHis447
for hHis447 to beto be jointly
jointly arranged
arranged with with the atrazine
the atrazine (Figure
(Figure 8d and 8d Scheme
and Scheme 1). In1). In last
this this
last step of the reaction pathway, the atrazine’s m-N” amine suffers
step of the reaction pathway, the atrazine’s m-N″ amine suffers the nucleophilic attack from hHis447 the nucleophilic attack from
hHis447 imidazole
imidazole ring, in which ring,the in which
protonthe proton
transfer transferout
is carried is carried
from the out from the
pesticide pesticide
towards the towards
amino acid the
amino acid via the TS4 (the HB distance was 2.699 Å). Consequently,
via the TS4 (the HB distance was 2.699 Å). Consequently, there is a localization of the negative charge there is a localization of the
negative
around thecharge
m-N″ aminearound asthe m-N”
well, which amine
can beasonce
well,again
which can be delocalized
efficiently once again by efficiently delocalized
the atrazine′s 1,3,5-
by the atrazine 0 s 1,3,5-triazine ring. The similar pathway is observed for the propazine-hHis447
triazine ring. The similar pathway is observed for the propazine-hHis447 and simazine-hTyr447
and simazine-hTyr447
interaction, with the transitioninteraction,
statewith the transition
labelled as TS2 (the stateHB labelled
distancesas TS2 were(the2.583,
HB distances
and 2.540were Å,
2.583, and 2.540 Å, respectively). In comparison with the
respectively). In comparison with the TS4 (TS2) optimized MD snapshot, the orientation TS4 (TS2) optimized MD snapshot,
of hHis447 the
orientation
needs hHis447 needs
to beof adjusted very toslightly,
be adjusted very slightly,
following which following
there is which a proton there transfer
is a proton transfer
from the
from the atrazine/propazine/simazine m-N” amine to the residue
atrazine/propazine/simazine m-N″ amine to the residue accompanied with the spontaneous break of accompanied with the spontaneous
break
the of the
scissile scissile
m-N″-H m-N”-H
bond bond
(Figure 8d, (Figure
Figure S43d 8d, Figure
and Figure S43dS44d, and Figure S44d, respectively).
respectively). As a result, a short As a
result, a short and low-barrier hydrogen bond (LBHB)
and low-barrier hydrogen bond (LBHB) is formed between the hHis447 and the is formed between the hHis447 and the
atrazine’s/propazine’s/simazine’s m-N” amine (Figure 8d,
atrazine’s/propazine’s/simazine’s m-N″ amine (Figure 8d, Figure S43d and Figure S44d, Figure S43d and Figure S44d, respectively),
and the willing
respectively), and proton transferproton
the willing is thus observed.
transfer is Energetically,
thus observed. thisEnergetically,
is the most favourable
this is the reaction
most
since the free energy differences between the reactant and transition
favourable reaction since the free energy differences between the reactant and transition states were states were 0.70, 0.68, and
0.62 0.68,
0.70, kcal/mol,
and 0.62 respectively. Alongside with
kcal/mol, respectively. the TS1,
Alongside withthethe TS4 (TS2)
TS1, theisTS4by (TS2)
far the most
is by farimportant
the most
transition state as it interrupts the hHis447 to serve as a base for
important transition state as it interrupts the hHis447 to serve as a base for hAChE catalytic triad. hAChE catalytic triad. By By all
means, the meaningfulness of TS4 is confirmed since the 51% of IRC
all means, the meaningfulness of TS4 is confirmed since the 51% of IRC described the proton gliding described the proton gliding
fromTS3
from TS3for hHis447,whereas
forhHis447, whereasthe the49%49%or orIRC
IRC illustrated
illustratedthe theproton
protontransfer
transferform formTS4 TS4for foratrazine
atrazine
m-N 0 atom (Figure 8d and Scheme 1). Similar trends were also noticed for propazine-hTyr341 and
m-N′ atom (Figure 8d and Scheme 1). Similar trends were also noticed for propazine-hTyr341 and
simazine-hTyr341 TS2
simazine-hTyr341 TS2(Figure
(FigureS43d andand
S43d Figure S44d, S44d,
Figure respectively). Nevertheless,
respectively). during the
Nevertheless, formation
during the
of the atrazine-hHis447 TS4 (propazine-hHis447 and simazine-hTyr447
formation of the atrazine-hHis447 TS4 (propazine-hHis447 and simazine-hTyr447 TS2), the TS1 (IS1), TS2), the TS1 (IS1), TS2 (TS1),
and(TS1),
TS2 TS3 (IS2)andwere
TS3 (IS2)sustained.
were sustained.
Figure
Figure9.9.Free
Freeenergy
energyprofile forfor
profile Homo sapiens
Homo AChE
sapiens inhibition
AChE by atrazine
inhibition determined
by atrazine by B3LYP
determined (6-
by B3LYP
31G*) QMQM
(6-31G*) simulations.
simulations.
The quantum mechanical studies do not provide the basis for the hSer203 covalent modification
The quantum mechanical studies do not provide the basis for the hSer203 covalent modification
by atrazine’s, propazine’s or simazine’s m-N″ atom substituent, despite the fact that in metabolic
by atrazine’s, propazine’s or simazine’s m-N” atom substituent, despite the fact that in metabolic
pathways pesticides may release the particular functional group. The literature survey supports
pathways pesticides may release the particular functional group. The literature survey supports
particular findings since the metabolites from atrazine, simazine and propazines are not able to
particular findings since the metabolites from atrazine, simazine and propazines are not able to
Molecules 2018, 23, 2192 27 of 37
establish
establish the
the interactions
interactions with
with acetylcholinesterase
acetylcholinesterase [83].
[83]. This
This statement
statement was
was further
further confirmed
confirmed in
in the
the
concentration dependent kinetic studies.
concentration dependent kinetic studies.
2.8. The Concentration-Dependent Kinetics of Human Acetylcholinesterase Inhibition by
2.8. The Concentration-Dependent Kinetics of Human Acetylcholinesterase Inhibition by
2-Chloro-1,3,5-triazine-based Pesticides
2-Chloro-1,3,5-triazine-based Pesticides
There are numerous evidences that organophosphates are irreversible AChE inhibitors whereas
There are numerous evidences that organophosphates are irreversible AChE inhibitors whereas
the carbamates acts as reversible AChE inhibitors [73]. However, studies show that atrazine decreases
the carbamates acts as reversible AChE inhibitors [73]. However, studies show that atrazine decreases
the AChE catalytic activity in Chironomus tentans [84] and Carassius auratus, applied synergistically
the AChE catalytic activity in Chironomus tentans [84] and Carassius auratus, applied synergistically
with organophosphate insecticides, [80]. Nevertheless, the data describing the atrazine’s and the
with organophosphate insecticides, [80]. Nevertheless, the data describing the atrazine’s and the
related compounds individually influence the hAChE catalytic activity is limited. Therefore, the
related compounds individually influence the hAChE catalytic activity is limited. Therefore, the 2-
2-chloro-1,3,5-triazine-based pesticides are herein evaluated as hAChE inhibitors, in the concentrations
chloro-1,3,5-triazine-based pesticides are herein evaluated as hAChE inhibitors, in the concentrations
comparable to the calculated LD50 values for Homo sapiens (Figure 10).
comparable to the calculated LD50 values for Homo sapiens (Figure 10).
The results show that all of the examined 2-chloro-1,3,5-triazine-based pesticides inhibit Homo
The results show that all of the examined 2-chloro-1,3,5-triazine-based pesticides inhibit Homo
sapiens AChE activity in a concentration-dependent manner (Figure 10). The inhibition curves analysis
sapiens AChE activity in a concentration-dependent manner (Figure 10). The inhibition curves
implies that the aging reaction, in which the hAChE releases itself from covalently bound pesticide,
analysis implies that the aging reaction, in which the hAChE releases itself from covalently bound
occurs in the presence of atrazine, propazine, and simazine at a high rate, and that the regarded
pesticide, occurs in the presence of atrazine, propazine, and simazine at a high rate, and that the
pesticides most likely do no inhibit the hAChE covalently [85]. Therefore, the protocol by which the
regarded pesticides most likely do no inhibit the hAChE covalently [85]. Therefore, the protocol by
Homo sapiens LD50 values were calculated, as well as all the performed molecular modelling studies of
which the Homo sapiens LD50 values were calculated, as well as all the performed molecular modelling
2-chloro-1,3,5-triazine-based pesticides, were experimentally validated.
studies of 2-chloro-1,3,5-triazine-based pesticides, were experimentally validated.
Figure 10.
Figure 10. Human
Human AChE
AChE inhibition
inhibition by
by 2-chloro-1,3,5-triazine-based
2-chloro-1,3,5-triazine-based pesticides.
pesticides.
3. Materials and Methods

3. Materials and Methods
3.1. Materials
3.1. Materials
Acetylcholine chloride
Acetylcholine chloride (CAS:
(CAS: 60-31-1),
60-31-1), acetylcholinesterase
acetylcholinesterase from
from human
human erythrocytes
erythrocytes (CAS:
(CAS:
9000-81-1), and
9000-81-1), and DTNB
DTNB (5,5
(5,5′-dithiobis(2-nitrobenzoic acid)) (CAS:
0 -dithiobis(2-nitrobenzoic acid)) (CAS: 69-78-3),
69-78-3), atrazine
atrazine (CAS:
(CAS: 1912-24-9),
1912-24-9),
propazine (CAS: 139-40-2), simazine (CAS: 122-34-9), and PBS (MDL: MFCD00131855)
propazine (CAS: 139-40-2), simazine (CAS: 122-34-9), and PBS (MDL: MFCD00131855) were purchased were
purchased from Sigma Aldrich (St. Louis,
from Sigma Aldrich (St. Louis, Mo., USA). Mo., USA).
3.2. The
3.2. The Generation
Generation of
of Pesticides
Pesticides Structures
Structures
The crystal
The crystalstructures
structuresofofsimazine
simazine [9],[9], monocrotophos
monocrotophos [10],
[10], dimethoate
dimethoate [11],[11],
and and acetamiprid
acetamiprid [12]
[12] were
were retrieved
retrieved fromfrom
TheThe Cambridge
Cambridge Crystallographic
Crystallographic Data
Data Centre
Centre (CCDC)(CSD
(CCDC) (CSDIDs:
IDs: KUYXIM,
ULEJIF,IPCPYB,
ULEJIF, IPCPYB, HANBAA).
HANBAA). The The remaining
remaining pesticides
structures were
were generated
generated by by drawing
drawing using
using
the MacroModel
the MacroModel version
version 88 software
software(Schrodinger,
(Schrodinger,Cambridge,
Cambridge,MA, MA,USA)
USA)[75].
[75].
3.3.
3.3. The
The Conformational
Conformational Analysis
Analysis
The
The conformational
conformational analyses
analyses were
were performed
performed by
by using
using the
the MacroModel
MacroModel version
version 88 software
software [75]
[75]
via
via the
theMonte
MonteCarlo/multiple
Carlo/multiple minimum
minimum (MC/MM) approach [76].
(MC/MM) approach [76]. The solute was described
described using
using
five different potentials (MM3, AMBER94, MMFF, MMFFs, OPLSAA) [77,85–88]. The effect of solvent
was incorporated into the MC/MM calculations using the generalized Born/surface area (GB/SA)
Molecules 2018, 23, 2192 28 of 37
five different potentials (MM3, AMBER94, MMFF, MMFFs, OPLSAA) [77,85–88]. The effect of solvent
was incorporated into the MC/MM calculations using the generalized Born/surface area (GB/SA)
continuum solvent model [89]. Cut-offs of 12.0 Å and 7.0 Å were employed for electrostatic and
van der Waals non-bonded interactions, respectively [90]. The MC simulation involved 104 steps at
310.15 K, applied to all the rotatable bonds, with random torsional rotations of up to ±180◦ . This was
combined with 103 steps of energy minimization. All the conformational calculations were performed
using the MacroModel 8.0 and BatchMin suite of programs [91].
3.4. The Mus musculus and Homo sapiens AChE Sequences Alignment
The alignment of mAChE and hAChE sequences retrieved from the UniProt database (entries
P221836 and P22303, respectively, Figure S1) was performed by means of ClustalW module [92] using
the standard setup.
3.5. The Mus musculus and Homo sapiens AChE Complexes Preparation
To the best of our knowledge, there are no crystal structures containing co-crystallized structures
of pesticides listed in Table 1, in complex with either Mus musculus or Homo sapiens AChE. Therefore,
for the purpose of targeted pesticides binding mode definition into the active sites of Mus musculus and
Homo sapiens AChEs, respectively, 59 acetylcholine esterase-inhibitor complexes (51 containing mice
inhibitors and 8 saturated with human inhibitors) were collected from Protein Data Bank and submitted
to structure-based alignment assessment protocols (see Tables S9 and S10). Initially, complexes were
prepared for molecular modelling after being loaded into the UCSF Chimera v1.10.1 software [93]
for Linux 64 bit architecture and visually inspected. The downloaded complexes were homodimers.
For the experimental purposes preparation, complexes were initially arbitrarily superimposed using
human crystal under the code name 4EY5 [94] as a template (the best-resolved complex with the
resolution of 2.3 Å) using the Matchmaker module and then separated in chains using the command
line implementation of the Chimera split command. Upon the thorough examination, the B chain of
each complex was retained for further manipulation. Compared to chains A containing complexes,
those with chains B were more complete by means of inhibitors presence. Inhibitors were extracted
from each chain B complexes after which either proteins or ligands were improved by assigning the
hydrogen atoms at pH of 7.4 and Amber parameters [95] using Antechamber semi-empirical QM
method. Complexes were the regenerated and energy minimized as follows: through the leap module
they were solvated with water molecules (TIP3P model, SOLVATEOCT Leap command) in a box
extending 10 Å in all directions, neutralized with either Na+ or Cl− ions, and refined by a single
point minimization using the Sander module with maximum 1000 steps of the steepest-descent energy
minimization followed by maximum 4000 steps of conjugate-gradient energy minimization, with a
non-bonded cutoff of 5 Å. Minimized complexes were re-aligned (4EY5 as a template), after which
all of the ligands were extracted to compose the SB aligned training set ready to be utilized for the
subsequent structure-based alignment assessment.
3.6. The Structure-Based Alignment Assessment

The SB alignment assessment for either Mus musculus or Homo sapiens AChE inhibitors was
performed by means of the four step standard protocol [78,96], by means of either AutoDock [79],
AutoDock Vina [80], and DOCK [81] docking algorithms/scoring functions.
Experimental conformation re-docking (ECRD): a procedure in which the experimental
conformations (EC) are flexibly docked back into the corresponding protein, evaluating the program
for its ability to reproduce the observed bound conformations.
Randomized conformation re-docking (RCRD): similar assessment to ECRD with a difference
that the active site of the protein is virtually occupied by conformations initially obtained from
computational random optimization of corresponding co-crystallized molecules coordinates and
Molecules 2018, 23, 2192 29 of 37
positions. Here the programs are evaluated for their ability to find the experimental pose starting by a
randomized minimized conformation.
Experimental conformation cross-docking (ECCD): comparable to ECRD, but the molecular
docking was performed on all the training set proteins except for the corresponding natives. Here the
programs are evaluated to find the binding mode of ligand in a similar but different from the native
complex active site at the same time mimicking discrete protein flexibility.
Randomized conformation cross-docking (RCCD): same as the ECCD but using RCs as starting
docking conformations. This is the highest level of difficulty since the program is demanded to dock
any given molecule into an ensemble of protein conformations not containing the native one. The
outcome is considered as the most important ability of docking program as the most accurate scoring
function is applied to any test set molecules of which the experimental binding mode is unknown. The
related docking accuracy (DA) [97] is a direct function of the program’s probability to find a correct
binding mode for an active molecule.
3.6.1. The AutoDock Settings

The prepared protein structures were imported into AutoDockTools graphical user interface.
Nonpolar hydrogen atoms were removed while Gasteiger charges and solvent parameters were
added. All of the tested compounds were used as ligands for docking. The rigid root and rotatable
bonds were defined using AutoDockTools. The docking was performed with AutoDock 4.2. The xyz
coordinates (in Ångströms) of the cuboid grid box used for the computation were: for mouse inhibitors
Xmin/Xmax = 1.420/31.800, Ymin/Ymax = 108.000/152.500, Zmin/Zmax = 8.200/43.200; for human
inhibitors Xmin/Xmax = 3.200/29.500, Ymin/Ymax = 109.200/153.800, Zmin/Zmax = 8.800/42.100,
with a purpose to embrace all the minimized inhibitors spanning 10 Å in all three dimensions. The
Lamarckian Genetic Algorithm was used to generate orientations or conformations of ligands within
the binding site. The procedure of the global optimization started with a sample of 200 randomly
positioned individuals, a maximum of 1.0 × 106 energy evaluations and a maximum of 27,000
generations. A total of 100 runs were performed with RMS Cluster Tolerance of 0.5 Å.
3.6.2. The Autodock Vina Settings

The docking simulations were carried out in the same grid space with an energy range of
10 kcal/mol and exhaustiveness of 100 with RMS Cluster Tolerance of 0.5 Å. The output comprised 20
different conformations for every receptor considered.
3.6.3. The DOCK6 Settings

During the docking simulations with DOCK program, the proteins were rigid while the inhibitors
flexible and subjected to an energy minimization process. The solvent accessible surface of each
enzyme without hydrogen atoms was calculated using the DMS program [98], using a probe radius of
1.4 Å. The orientation of ligands was described using the SPHGEN module and by sphere selector. A
box around the binding site is constructed with the accessory program SHOWBOX. The steric and
electrostatic environment of the pocket was evaluated with the program Grid using a 0.3 Å of grid
spacing. Selected spheres were within 8 Å from ligand heavy atoms of the crystal structure and for
computing the energy grids an 8 Å box margin and 6–9 VDW exponents were used.
3.7. The Ligand-Based and Structure-Based Alignment Accuracy

The alignment fitness for LB or SB approach was quantified by evaluating the RMSD values.
The alignment accuracy was initially quantified after reproducing the known crystal structures of
simazine [9], monocrotophos [10], dimethoate [11], and acetamiprid [12], with the available force fields,
to obtain the conformational alignment accuracy (CAA). Following, the superposition of pesticides
structures obtained after the conformational analysis was performed with the purpose to compare
the force fields performances, i.e., the force filed-dependent alignment accuracy, FFDAA. Finally,
Molecules 2018, 23, 2192 30 of 37
superimposition of docking poses provided the docking accuracy, DA. CAA, FFDAA or DA can be
used to test how the conformational alignment can predict the ligand pose as close as possible to the
experimentally observed one [97]. The alignment accuracy values can be calculated by the following
Equation (7):
xA = f rmsd, ≤ a + 0.5( f rmsd ≤ b − f rmsd ≤ a) (7)
In particular: xA = CAA or FFDAA, in the case of LB alignment accuracy, whereas the xA = DA,
in the case of SB accuracy. The frmsd ≤ a and frmsd ≤ b are fractions of aligned ligands showing an
RMSD value less than or equal to 2 Å (a coefficient) and 3 Å (b coefficient), respectively. The widely
accepted standard is that the correctly aligned conformations are those with RMSD less than 2 Å
on all the heavy atoms between the generated and crystallographic structure, when considering the
CAA and FFDAA, while the correctly docked structures are those with RMSD less than 2 Å on all the
heavy atoms between docked and co-crystallized ligand, when considering the DA. Structures with
the RMSD values larger than 4 Å were considered incorrectly aligned/docked. Structures with RMSD
between 2 and 3 were considered partially aligned/docked, whereas those with RMSD higher than 3
and were misaligned/mis-docked and thus not considered in the FFDAA/DA calculation.
3.8. The Structure-Based Alignment of Pesticides

The molecular docking of pesticides within either mAChE and hAChE was conducted by
the AutoDock Vina [80]. The molecular docking studies were carried out in the cuboid grid
box with the following xyz coordinates (in Ångströms): for mAChE Xmin/Xmax = 1.420/31.800,
Ymin/Ymax = 108.000/152.500, Zmin/Zmax = 8.200/43.200; hAChE Xmin/Xmax = 3.200/29.500,
Ymin/Ymax = 109.200/153.800, Zmin/Zmax = 8.800/42.100, with the energy range of 10 kcal/mol
and exhaustiveness of 100 with RMS Cluster Tolerance of 0.5 Å. The output comprised 20 different
conformations for every receptor considered.
3.9. The Pesticide-AChE Complexes Molecular Dynamics Simulations

The pesticide-AChE complexes formed after molecular docking were used to perform the explicit
solvent molecular dynamics (MD) simulations. The parallelized Desmond Molecular Dynamics
System [99] and the associated analysis tools, available within the Schrödinger suite 2009 [75] were
used for this purpose. The models were prepared using the ‘system builder’ utility. The MMFF force
field parameters were assigned to all the simulation systems. Each inhibitors-enzyme complex was
placed in the centre of an orthorhombic box ensuring 10 Å solvent buffers between the solute and the
boundary of the simulation box in each direction. The TIP3P model was used to describe the water
molecules. Moreover, Na+ and Cl− ions were added at a physiological concentration of 0.15 M. The
model systems were relaxed to 0.5 Å using the single step minimization protocol and were subjected
to the production phase, with the NPT ensemble and the periodic boundary conditions for 1.2 ns. The
pressure control was used to maintain the pressure of the cell (1.013 bars) with the isotropic coupling
method. The Nose-Hoover thermostat method was used to control the temperature at 310.15 K. The
Heavy atom-hydrogen covalent bonds were constrained using the SHAKE algorithm which allowed 2
fs integration step to be used. The integration of the motion equation using the RESPA multiple time
step scheme was used in the calculation. The Long-range electrostatic forces were obtained using the
smooth Particle-Mesh Ewald (PME) method. In order to calculate the short-range electrostatics and the
Lennard-Jones interaction, the cut-off distance was set to 9.0 Å, and the trajectories and the energies
were recorded at every 4.8 ps. The simulation quality analysis tool was used to analyse the trajectories;
RMSD and RMSF values, the hydrogen bond distances, the angles, and the van der Waals interactions
were obtained over the simulation trajectories.
Molecules 2018, 23, 2192 31 of 37
3.10. The MM-GBSA Calculations and Free Energy Decomposition

The binding free energy [100] of each system was calculated using the MM-GBSA calculations
according to the following Equation (8):
∆Gbind = ∆Eele + ∆EvdW + ∆Gsolv + T∆S, (8)
where ∆Eele corresponds to the electrostatic energy difference between the receptor-ligand bound and
the calculated unbound states using the OPLS_2005 force field, ∆EvdW corresponds to the van der
Waals contribution, while ∆Gsolv is the corresponding solvation free energy contribution of the binding
which was calculated using the GB/SA continuum model. The Embrace package implemented
in MacroModel was used for the ∆Eele , ∆EvdW , and ∆Gsolv calculations. The entropy change ∆S
was calculated using the Rigid Rotor Harmonic Oscillator (RRHO) calculations. Having used this
algorithm, the change in vibrational, rotational, and translational (VRT) entropy of the ligands on
binding was estimated. For the RRHO calculations, the representative complexes were pre-minimized
using the Desmond with the explicit solvent retained; a 2000 steps LBFGS minimization (first 10 steps
steepest descent) with the residues beyond 15 Å of ligands restrained and a convergence criterion of
0.05 kcal mol−1 Å−1 was used.
3.11. The Quantum Mechanical Mechanistic Studies

The DFT-based calculations were performed on the MD-generated pesticide-hAChE complexes
on three different levels. The first level included the extraction of hydrogen bond-forming geometries
of MD equilibrated pesticide-hThr83, pesticide-hTyr124, pesticide-hTyr341, and pesticide-hHis447
sub-complexes; subsequently, the extracted geometries were QM optimized to locate the transition
states (TSs) and/or intermediary states (ISs), by which the protons transfers occur on a particular
segment of a biochemical reaction. The second level of the calculation attempted to find the identical
TSs and/or ISs following the manual adjustment of the TS-generating hydrogen atom position,
between the pesticide and the regarded AChE residue, within the pesticide-hThr83, pesticide-hTyr124,
pesticide-hTyr341, and pesticide-hHis447 geometries. Each of the reaction pathways assumed the
contemplation by means of Intrinsic Reaction Coordinate (IRC) analysis. As water molecules are not
involved in HBs generation, they were all discarded from the system. The initiation or the manual
setup of geometries was performed by means of GaussView6 package [101] following which the B3LYP
method, as implemented in Gaussian 09 (Gaussian Inc., Wallingford, CT, USA) [102] was used to
explore the geometry of the reactants, transition states, intermediary states and products. The single
point calculations were carried out on all the geometries at the B3LYP/6-31G* level of theory [82]. The
saddle points and transition states were quantified by means of frequency calculations.
3.12. The Assay of the AChE Activity

The AChE catalytic activity was measured at 22 ◦ C by the Ellman method [103]. The assay mixture
contained the 0.25 mM ACh, 0.25 mM DTNB, and 50 mM sodium phosphate buffer. The remaining
assay conditions have been reported previously. For the selection of a suitable concentration of AChE,
at which the relationship between the initial velocity and the total enzyme concentration should be
linear, 0.20–3.20 mg/mL of AChE was assayed in vitro from 2 to 20 min under the same conditions of
the temperature and the pH. The rate of change of the substrate cleavage, determined by measuring
the absorbance of the reaction product at a wavelength of 412 nm, was performed at different time
intervals. The product was calculated for each [AChE] at 4-min incubation time intervals. To study the
effect of atrazine, simazine, or propazine on AChE substrate cleavage, the enzyme was preincubated
with each pesticide at different concentrations ranging 0–6 µg/mL (concentrations corresponding to
the LD50 values) for 10 min prior to the addition of the substrate.
Molecules 2018, 23, 2192 32 of 37
4. Conclusions
The present report describes the application of the molecular modelling techniques to shed light
on the pharmacology of the commonly used pesticides atrazine, simazine, propazine, carbofuran,
monocrotophos, dimethoate, carbaryl, tebufenozide, imidacloprid, acetamiprid, diuron, monuron, and
linuron as AChE inhibitors The pesticides’ putative pre-binding conformations in the inter-synaptic
environment were predicted by means of the conformational analysis where the determined global
minima values and the ability to reproduce the pesticides experimental conformations indicated MMFF
as the best performing FF. Having known the pesticides pre-bound conformations, their acute toxicities
were interrelated to the Eglob_min values through the QSAR studies: for the majority of pesticides, a
clear inverse correlation could be observed between the Eglob_min values and the high acute toxicities.
However, the chemometric analysis indicated that the Eglob_min values could not be used as proper
indicators for high or low LD50 values against Mus musculus or Homo sapiens.
Therefore, the pesticides acute toxicity was further on regarded trough the pesticides bound
conformations, upon the molecular docking and molecular dynamics-driven interactions with either
Mus musculus or Homo sapiens AChE. The conducted studies confirmed the binding modes and the
pharmacology of evaluated pesticides as reversible and reversible-like (i.e., future covalent) AChE
inhibitors and the determined pesticides pharmacodynamics origin of acute toxicity. The superposed
chemometric investigation revealed that for all the pesticides, a clear interrelationship could be
observed between the ∆Gbinding values and high acute toxicity, indicating that the ∆Gbinding values
can be used as indexes for high or low LD50 values against Mus musculus. Moreover, the obtained
chemometric guideline, in the form of the QSAR model pLD50 = −0.7885∆Gbinding − 2.44, is, by all
means, suitable for the prognosis of LD50 values against Homo sapiens and can be further used as a
universal tool for the acute toxicities administration of novel pesticides. Further studies which consider
the inclusion of additional parameters and machine learning algorithm application are in due course
in order to generate quantitative structure-activity relationships models able to include all considered
pesticides in a comprehensive mathematical model.
Nevertheless, it is interesting that the global minima or free energy of formation values may
indicate the level of acute toxicity; the calculations may discard the potential new pesticides even before
the actual application in agriculture. Therefore, the correlations between the pLD50 and Eglob_min or
∆Gbinding are hereby presented for the very first time as an unprecedented way to study pesticides and
to predict their acute toxicity.
Moreover, the subsequent structure-based quantum mechanical mechanistic studies for
2-chloro-1,3,5-triazine-based pesticides, as the least understood group by means of pharmacology,
revealed pathways by which considered pesticides poison Homo sapiens AChE in a reversible fashion
manner. To summarize, all the noticed interactions could further be used in the discovery of novel
pesticides with the desirable lower acute toxicity against humans. It is important to emphasize that the
conducted QM studies were performed to provide the structural basis for the pesticides’ human AChE
toxicity, not the general toxicity. The pesticide toxicity is mainly carried out by means of mechanism;
general toxicity is normally due to off target interactions or to active metabolites.
Supplementary Materials: The Supplementary Materials are available online.

Author Contributions: B.B.A. and M.M. designed the study; B.B.A., A.R., J.S.M., T.M.T.-P. and R.M. performed
conformational analyses. M.M., N.S., N.M., and R.R. performed statistical studies, molecular docking, molecular
dynamics studies and QM DFT studies.
Funding: This research was funded by the Ministry of Education and Science of Republic of Serbia (Grant Nos.
III45006, 174007, and III43004) and Progetti di Ricerca di Università 2015, Sapienza Università di Roma (Grant
Nos. C26A15RT82 and C26A15J3BB).
Acknowledgments: Gratitude is expressed to Goran Bogdanovic, Institute of Nuclear Sciences Vinča, Belgrade,
Republic of Serbia, for providing pesticides crystal structures. Special thanks are due to Jill Barber, Division of
Pharmacy and Optometry, University of Manchester, Manchester, UK for providing computational facilities and
all comments and suggestions which improved the quality of the manuscript.
Molecules 2018, 23, 2192 33 of 37
Conflicts of Interest: The authors declare no conflict of interest.
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molecules

Academic Editor: Diego Muñoz-Torrero

Keywords: pesticides; AChE; conformational analysis; QSAR; molecular docking; molecular

Molecules 2018, 23, 2192; doi:10.3390/molecules23092192 www.mdpi.com/journal/molecules

Pesticide FF a Eglob_min b NGMS c NF d Pesticide CAA f

atrazine MM3 (kJ/mol)

2.2. The Prediction of Pesticides Structures in Aqueous Solution

2.3. The Prediction of Externally Evaluated Pesticides Structures in Aqueous Solution

pLD50 = −0.0021Eglob_min − 2.82 (r2 = 0.78), (2)

2.5. The Structure-Based Studies

2.5. The Structure-Based Studies

2.5.1. The Alignment Rules Assessment

2.5.2. The Cross-Docking and Molecular

Figure 3. The SB alignment

electrostatic interactions with m(h)Tyr124 via 2,2-dimethyltetrahydrofuran oxygen atom. Moreover,

2.5.5. The Cross-Docking and Molecular Dynamics Studies of Pesticides (6-Chloropyridin-3-

2.5.6. The Cross-Docking of Externally Evaluated Pesticides

pLD50 = −0.7885∆Gbinding − 2.44 (r2 = 0.95), (6)

3. Materials and Methods

3.6. The Structure-Based Alignment Assessment

3.6.1. The AutoDock Settings

3.6.2. The Autodock Vina Settings

3.6.3. The DOCK6 Settings

3.7. The Ligand-Based and Structure-Based Alignment Accuracy

3.8. The Structure-Based Alignment of Pesticides

3.9. The Pesticide-AChE Complexes Molecular Dynamics Simulations

3.10. The MM-GBSA Calculations and Free Energy Decomposition

∆Gbind = ∆Eele + ∆EvdW + ∆Gsolv + T∆S, (8)

3.11. The Quantum Mechanical Mechanistic Studies

3.12. The Assay of the AChE Activity

Supplementary Materials: The Supplementary Materials are available online.

Conflicts of Interest: The authors declare no conflict of interest.

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