Resorcinol-, catechol- and saligenin-based bronchodilating β2-agonists as inhibitors of human cholinesterase activity

Article abstract of DOI:10.1080/14756366.2017.1326109

We investigated the influence of bronchodilating β2-agonists on the activity of human acetylcholinesterase (AChE) and usual, atypical and fluoride-resistant butyrylcholinesterase (BChE). We determined the inhibition potency of racemate and enantiomers of fenoterol as a resorcinol derivative, isoetharine and epinephrine as catechol derivatives and salbutamol and salmeterol as saligenin derivatives. All of the tested compounds reversibly inhibited cholinesterases with K_i constants ranging from 9.4 μM to 6.4 mM and had the highest inhibition potency towards usual BChE, but generally none of the cholinesterases displayed any stereoselectivity. Kinetic and docking results revealed that the inhibition potency of the studied compounds could be related to the size of the hydroxyaminoethyl chain on the benzene ring. The additional π–π interaction of salmeterol’s benzene ring and Trp286 and hydrogen bond with His447 probably enhanced inhibition by salmeterol which was singled out as the most potent inhibitor of all the cholinesterases.

Full text of DOI:10.1080/14756366.2017.1326109

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Resorcinol-, catechol- and saligenin-based
bronchodilating β2-agonists as inhibitors of
human cholinesterase activity
Anita Bosak, Anamarija Knežević, Ivana Gazić Smilović, Goran Šinko & Zrinka
Kovarik
To cite this article: Anita Bosak, Anamarija Knežević, Ivana Gazić Smilović, Goran Šinko & Zrinka
Kovarik (2017) Resorcinol-, catechol- and saligenin-based bronchodilating β2-agonists as inhibitors
of human cholinesterase activity, Journal of Enzyme Inhibition and Medicinal Chemistry, 32:1,
789-797, DOI: 10.1080/14756366.2017.1326109
To link to this article: http://dx.doi.org/10.1080/14756366.2017.1326109
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Date: 21 June 2017, At: 12:43

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 789–797
https://doi.org/10.1080/14756366.2017.1326109
RESEARCH PAPER
Resorcinol-, catechol- and saligenin-based bronchodilating b2-agonists as
inhibitors of human cholinesterase activity
a
b
b
a
a
ꢀ
Anita Bosak , Anamarija Kne ꢀz evi ꢁc , Ivana Gazi ꢁc Smilovi ꢁc , Goran Sinko and Zrinka Kovarik
a
b
-
Institute for Medical Research and Occupational Health, Zagreb, Croatia; Ruder Bo ꢀs kovi ꢁc Institute, Zagreb, Croatia
ABSTRACT
ARTICLE HISTORY
We investigated the influence of bronchodilating b2-agonists on the activity of human acetylcholinesterase
Received 31 January 2017
Revised 13 April 2017
Accepted 21 April 2017
(
AChE) and usual, atypical and fluoride-resistant butyrylcholinesterase (BChE). We determined the inhibition
potency of racemate and enantiomers of fenoterol as a resorcinol derivative, isoetharine and epinephrine
as catechol derivatives and salbutamol and salmeterol as saligenin derivatives. All of the tested compounds
KEYWORDS
i
reversibly inhibited cholinesterases with K constants ranging from 9.4lM to 6.4mM and had the highest
Acetylcholinesterase; butyr-
ylcholinesterase; adrenaline;
salmeterol; terbutaline
inhibition potency towards usual BChE, but generally none of the cholinesterases displayed any stereo-
selectivity. Kinetic and docking results revealed that the inhibition potency of the studied compounds
could be related to the size of the hydroxyaminoethyl chain on the benzene ring. The additional p–p
interaction of salmeterol’s benzene ring and Trp286 and hydrogen bond with His447 probably enhanced
inhibition by salmeterol which was singled out as the most potent inhibitor of all the cholinesterases.
Introduction
crucial in the treatment of neurodegenerative disorders such as
Myasthenia gravis, Alzheimer and Parkinson’s disease, since gener-
The transfer of nervous impulses via acetylcholine (ACh) depends
on a series of well-tuned receptors, enzymes that take part in the
synthesis and hydrolysis of ACh and a series of transporters . In
6
,8
ally all drugs in use act by inhibiting AChE and BChE activity
.
1
The activity of both enzymes depends on factors such as ethnicity,
exposure to anticholinergic reagents such as organophosphorus
and carbamate pesticides, nerve agents and anticholinergic
chronic inflammatory respiratory diseases (asthma and chronic
obstructive pulmonary disease), ACh levels increase, which leads
to the contraction of the smooth bronchial muscle tissue, intensi-
fied mucous secretion, chronic coughing and immune stimula-
4
,9,10
drugs
. BChE activity is additionally affected by flavonoids or
natural anticholinesterase toxins in food and drugs whose biocon-
version involves the action of BChE, like echothiophate, eye drops
for glaucoma treatment, anti-Alzheimer drugs or the bronchodila-
2
,3
tion . The degradation of ACh in the organism takes place
through the activity of acetylcholinesterase (AChE), which thus ful-
fils its physiological role. This role is shared by the closely related
4
,11–15
tor bambuterol
. However, the efficacy of any drug that tar-
enzyme butyrylcholinesterase (BChE), an enzyme that does not gets BChE activity can be affected by the human BCHE gene
possess a known physiological substrate, but does take part in the polymorphism. At present, more than 56 mutations of the human
1
6
bioconversion of several xenobiotics, in the metabolism of lipids BCHE gene have been identified , and different catalytic proper-
and lipoproteins and serves as a backup enzyme in acetylcholine ties or lower enzyme levels than of usual BChE have been con-
4
–7
hydrolysis when AChE is inhibited . Both AChE and BChE are firmed for several BChE variants; for example, individuals
CONTACT Anita Bosak
abosak@imi.hr; Zrinka Kovarik
zkovarik@imi.hr
Institute for Medical Research and Occupational Health, Ksaverska cesta 2, HR-10000
Zagreb, Croatia
Supplemental data for this article can be accessed here.
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

7
90
A. BOSAK ET AL.
homozygous for atypical butyrylcholinesterase can experience pro- after exposure to soman but did not influence the final lethal
2
3
longed apnoea if the muscle relaxant succinylcholine or mivacu- outcome .
1
6,17
rium are administered
.
Among the various chiral drugs, one enantiomer is often more
b-Adrenergic agonists are potent bronchodilators widely used active than another in treating a medical condition. An example is
in the management of bronchial asthma. These agents interact the (R)-enantiomer of salbutamol, which is approximately 80 times
with b2-adrenergic receptors on the smooth muscle of bronchial more potent as a b-adrenoceptor agonist than (S)-enantiomer and
tissue relieving bronchospasms and reducing airway resistance. the administration of pure (R)-salbutamol improved therapeutic
2
5
activity and reduced side effects . Similar in vivo results were
Beside the bronchial tissue b-adrenergic agonists also act on other
tissues where signals to other cells are regulated by acetylcholine,
like smooth muscles of the vascular system, intestines and uterus.
Recent studies have shown that oral intake of salbutamol and
ephedrine appeared to be effective supplement treatments in
2
6
obtained with enantiomers of terbutaline . AChE and BChE are
stereoselective enzymes in the interaction with various
. In interaction with bambuterol, which in its racemic
form is in use as a prodrug of terbutaline, both enzymes prefer
7
,9,27,28
esters
7
,27,28
1
8
(R)- over (S)-bambuterol
.
severe cases of acetylcholine receptor (AChR) , while chronic
1
9
In our previous studies, we demonstrated that some derivatives
of resorcinol were reversible inhibitors of human cholinester-
treatment with certain b2-agonists relieved neuropathic pain .
Also, recent studies demonstrated that some bronchodilators
could be used as additional antidotes in the treatment of poison-
ing by organophosphates, either in accidental poisonings by
organophosphate pesticides or following acts of terrorism using
9
,28,29
ases
. In this study, we extended our research on other b-ago-
resorcinol-, catechol- and saligenin-containing
structure. Here, we determined the inhibition potency of fenoterol
as a resorcinol derivative, isoetharine and epinephrine as catechol
derivatives, and salbutamol and salmeterol as saligenin derivatives
Figure 1) towards human AChE and three variants of human BChE
usual, atypical and fluoride resistant). The systematic structural dif-
ferences between compounds and their inhibition potency towards
certain cholinesterases were analysed by molecular modelling.
nists with
a
2
0–23
nerve agents
. Organophosphates act through the inhibition of
AChE consequently leading to a collapse of the respiratory system.
The studies of the effects of AChE and BChE inhibition in KO mice
have shown that the respiratory function is more sensitive to anti-
cholinesterases in AChE heterozygotes than in wild-type mice, sug-
gesting that BChE is an important enzyme for the respiratory
function and should be left unaffected by medications that have
(
(
2
4
the ability to reduce cholinesterase activity . Ventilatory, inflam-
matory and lethal effects of sarin may be limited by the use of
Material and methods
2
0
Chemicals
epinephrine and oxygen , while the protective activity of salbuta-
mol was demonstrated in diisopropylfluorophosphate (DFP) poi- Racemic epinephrine, isoetharine, fenoterol and terbutaline, as
2
1,22
soning
. Also, treatment with Combivent (a drug consisting of well as (R)-epinephrine, were purchased (Sigma-Aldrich, St. Louis,
ipratropium and salbutamol) improved the respiratory outcome MO) in the form of salts and converted to hydrochloride salts
Figure 1. The chemical structure of the studied resorcinol, catechol and saligenin derivatives. Asterisk denotes a chiral carbon atom.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
791
(detailed in Supplementary material). All of the chemicals, reagents with the addition of 0.01% BSA. This study was reviewed and
and solvents for the preparation of resorcinol, catechol and salige- approved by the Ethics Committee of IMROH.
nin derivatives were purchased from commercial sources and used
without further purification. Acetylthiocholine iodide (ATCh), pro- Synthesis
0
pionylthiocholine iodide (PTCh) and 5,5 -dithiobis(2-nitrobenzoic
Racemic salbutamol and salmeterol were synthesised starting from
methyl 5-acetylsalicylate (Sigma-Aldrich) according to literature
acid) (DTNB) were purchased from Sigma-Aldrich. All of the inhibi-
tors and substrates were dissolved in water, while DTNB was dis-
solved in 0.1 M sodium phosphate buffer (pH 7.4). Only salmeterol
was dissolved in ethanol due to its low solubility, while all further
dilutions were made in water.
3
2,33
procedures described previously
.
The enantiomers of salbutamol and (R)-terbutaline were
prepared from their racemic form using diastereomeric salt crystal-
32,34
lization method
. The resolving agent for (R)-terbutaline and
0
(
R)-salbutamol precursor was (þ)-O,O -di-p-toluoyl-D-tartaric acid
0
Enzymes
and (ꢀ)-O,O -di-p-toluoyl-L-tartaric acid for a precursor for (S)-sal-
butamol. Enantiomeric excess (ee) for the obtained enantiomers
was determined using a brush-type chiral stationary phase previ-
Human native serum was used as a source of BChE. Recombinant
human AChE was a gift from Dr Florian Nachon (D ꢁe partement de
Toxicologie, IRBA, France). BChE variants were confirmed earlier as
3
5
ously synthesised in our laboratory (CSP 1) . The obtained
ee were 99%, 96% and 92% for (S)-salbutamol, (R)-terbutaline and
3
0,31
described previously
. The usual BChE was collected from a
(
R)-salbutamol, respectively.
female donor at the Institute for Medical Research and Occupational
Health, Zagreb, Croatia (IMROH); the atypical BChE was a gift from
Dr Oksana Lockridge (University of Nebraska Medical Centre, Eppley
Institute, Omaha, NE), and specimens of the fluoride resistant BChE
were a gift by Dr Robert T. Evans (Cholinesterase Investigation Unit,
St’ James University Hospital, Leeds, Great Britain). Enzyme dilutions
were done in phosphate buffer and those of recombinant AChE
Enantiomers of fenoterol and salmeterol were resolved using
semipreparative chiral chromatography. The columns used were
CHIRALLICA PST-4 (AD-type column; Chirallica d.o.o., Croatia) and
CHIRALLICA PST-2 (OJ-type column; Chirallica d.o.o., Croatia) for
fenoterol and salmeterol, respectively. The obtained ee were 98%,
96%, 97% and 86% for (S,S)-fenoterol, (R,R)-fenoterol, (R)-salmeterol
and (S)-salmeterol, respectively. The absolute configuration of
enantiomers was determined by comparison of the elution order
of these compounds on AD and OJ columns with literature
3
6,37
data
(detailed in Supplementary).
Reversible inhibition
Enzyme activities were measured in 0.1 M phosphate buffer, pH
ꢁ
7
.4 at 25 C, on CARY 300 spectrophotometer equipped with tem-
perature controller (Varian Inc., Australia) by Ellman method at
3
8
4
12 nm . ATCh was used as a substrate for AChE and PTCh as a
substrate for BChE. The reaction mixture contained DTNB (final
concentration 0.3 mM), buffer, enzyme suspension, inhibitor
(0.5–10 mM) and ATCh or PTCh (0.025–1.0 mM). Inhibition of
enzymes was determined by measuring the decrease in enzyme
activity in the presence of inhibitors. The baseline activity was
determined in the presence of water since the proportion of etha-
Figure 2. Reversible inhibition of human AChE and three BChE variants (usual,
atypical and fluoride resistant) by racemic salmeterol. Points indicate the average nol in AChE and BChE activity measurement was up to 0.08% and
apparent enzyme-inhibitor constant Ki,app (±SE) determined at a given substrate
concentration (S) according to the Hunter–Downs equation. The lines are the
result of linear regression analysis where the y-intercept represents the enzyme-
0
.16%, respectively, which does not affect the enzyme activity.
The dissociation constant for an enzyme–inhibitor complex
(I) was evaluated from the impact of substrate concentrations
K
inhibitor dissociation constant, K(I)
.
[
S] on the degree of inhibition applying the Hunter–Downs
Table 1. Reversible inhibition of human AChE and three BChE variants (usual, atypical and fluoride resistant) by bron-
chodilating b2-agonists.
K(I)/mM
AChE
usual BChE
0.55 ± 0.11
atypical BChE
fluoride-resistant BChE
0.87 ± 0.18
Resorcinol derivatives
b
Metaproterenol
3.1 ± 1.2
4.8 ± 0.2
6.1 ± 0.7
3.3 ± 0.7
a
a
a
Terbutaline
Fenoterol
0.18 ± 0.15
0.30 ± 0.32
0.86 ± 0.05
0.024 ± 0.001
0.63 ± 0.03
0.061 ± 0.017
Catechol derivatives
Epinephrine
Isoproterenol
Isoetharine
Saligenin derivatives
Salbutamol
6.4 ± 1.1
2.5 ± 1.0
0.21 ± 0.04
3.7 ± 1.2
0.47 ± 0.21
0.031 ± 0.003
1.6 ± 1.0
1.6 ± 0.3
0.26 ± 0.05
3.8 ± 1.5
0.99 ± 0.12
0.086 ± 0.004
b
2.0 ± 0.2
0.030 ± 0.002
0.071 ± 0.006
0.013 ± 0.001
1.4 ± 0.2
0.086 ± 0.018
0.19 ± 0.02
0.0098 ± 0.0023
Salmeterol
The enzyme-inhibitor complex dissociation constant (K(I)± SE) was determined by linear regression from Ki,app constants
obtained from at least two experiments.
a
29
Reference .
b
9
Reference .

7
92
A. BOSAK ET AL.
7,39
1
equation
:
Molecular modelling
vi
KðIÞ
The docking of the studied compounds was performed using the
Accelrys DiscoveryStudio 2.1 CDOCKER docking protocol and
CHARMm force field. For the structural model of AChE, we used a
crystal structure deposited in the Protein Data Bank, PDB ID
Ki;app ¼
ꢂ ½Iꢃ ¼ KðIÞ
þ
ꢂ ½Sꢃ
v0 ꢀ vi
K
ðSÞ
where Ki,app is an apparent enzyme-reversible inhibitor complex
dissociation constant obtained at a given substrate concentration
S] in the presence of inhibitor concentration [I] and calculated
from the enzyme activities v and v measured without inhibitor
40
41
1
B41 and PDB ID 2PM8 as a model for usual BChE . Models for
atypical and fluoride-resistant BChE were constructed using the
PM8 structure as a model in which Asp70 was replaced with Gly
[
0
i
2
1
5
and in the presence of inhibitor concentration [I], respectively .
K(I) is the enzyme-reversible inhibitor complex dissociation con-
stant and can stand for a complex formed at the catalytic site or
for a complex formed at the peripheral site of the enzyme. K(S) is
the enzyme-substrate dissociation constant that could be approxi-
mated to the Michaelis constant or to the enzyme-substrate dis-
sociation constant when two molecules of the substrate are
bound to the enzyme.
as a model for atypical BChE, and Thr243 with Met or Gly390 with
Val as models of fluoride-resistant BChE. All of the constructed
models were minimised using the CHARMm force field according
9,42
to the docking protocol described previously . As a result of
molecular docking, a set of 20 possible poses per one compound
and enzyme pair was analysed and the pose with the highest
CDOCKER interaction energy was selected for further analysis.
Results and discussion
Table 2. The highest CDOCKER interaction energy (CIE) between 20 poses per
one bronchodilating b2-agonists ꢀ cholinesterase pair.
The tested bronchodilating b2-agonists were divided into three
groups according to the disposition of their hydroxyl groups on
the benzene ring, resembling the structural feature of resorcinol,
atypical BChE catechol and saligenin (Figure 1). The hydroxyl groups at the ben-
zene were in the meta position in the resorcinol group, and in the
—
CIE (kJ/mol)
AChE
usual BChE
Resorcinol derivatives
Metaproterenol
Terbutaline
Fenoterol
Catechol derivatives
Epinephrine
Isoproterenol
Isoetharine
Saligenin derivatives
Salbutamol
Salmeterol
178
170
245
149
168
182
178
142
231
ortho position in the catechol group. In the saligenin group, the
hydroxyl group and the hydroxymethyl group were in the ortho
position. Derivatives of each group differed in the size of the
hydroxyaminoethyl chain attached to the benzene ring.
169
186
203
146
181
212
190
138
204
Inhibition of human AChE
178
323
169
285
156
267
Kinetic studies revealed that all of the studied bronchodilating
b2-agonists reversibly inhibited AChE (Figure 2, an example of
Table 3. List of bronchodilating b2-agonists ꢀ cholinesterase interactions.
AChE
Usual BChE
Atypical BChE
Resorcinol derivatives
Metaproterenol
H-bond: Asp74, Tyr124, Ser125, Glu202,
Ser203
H-bond: Asp70, Trp82, Glu197
H-bond: Gly115, Tyr128, Glu197, Ser198,
His438
p–p: Trp86
H-bond: Asp74, Gly82, Tyr124, Tyr337,
Tyr341
p–p: Trp82
H-bond: Glu197, His438
p–cation: Trp82
H-bond: Glu197, Leu286
Terbutaline
p–p: Trp86
p–cation: Trp82
p–cation: Tyr341
H-bond: Asp74, Tyr124, Ser125, Tyr337,
Tyr341,
p–alkyl: Ala328
H-bond: Asp70, Tyr128, Glu197, His438
Fenoterol
H-bond: Thr120, Tyr128, Glu197, Tyr332,
His438
p–p: Trp86, Trp286, Tyr341
p–cation: Trp82
p–alkyl: Ala328
p–cation: Trp82, His438
p–alkyl: Ala328
Catechol derivatives
Epinephrine
H-bond: Asp74, Gly122, Tyr124, Ser125,
Glu202, Ser203, Tyr337, Tyr341
p–p: Trp86
H-bond: Glu197, His438, Gly439
H-bond: Trp82, Gly121, Tyr128, Glu197,
Ser198, His438
p–p: His438
p–p: Trp82
p–r: Gly116
H-bond: Tyr440
Isoproterenol
Isoetharine
H-bond: Asp74, Tyr124, Phe295, Tyr337,
Tyr341,
p–p: Trp286, Tyr341
H-bond: Asp70, Ser79, Glu197, Tyr332,
His438
p–cation: Trp82, His438
p–p: Trp82
p–alkyl: Ala328
H-bond: Thr120, Glu197, Ser198, His438
p–p: His438, Gly116, Gly117
p–alkyl: Trp82
H-bond: Tyr124, Tyr133, Glu202, Tyr337
p–p: Trp86
p–alkyl: Tyr124, Phe297
H-bond: Glu197, Ser198, Leu286, His438
p–p: His438
p–alkyl: Trp82
Saligenin derivatives
Salbutamol
H-bond: Asp74, Gly121, Tyr124, Tyr337
p–p: Trp86, Trp337
H-bond: Asp70, Asn83, His438
p–cation: Trp82
H-bond: Trp82, Gly121
p–p: Trp82
p–cation: Tyr341
H-bond: Asp74, Tyr124, Phe295, Arg296,
Tyr337, Tyr341, His447
p–r: Gly116
H-bond: Trp82, Thr120, Gly121, Glu197
Salmeterol
H-bond: Trp82, Thr122, Tyr128, Glu197,
His438
p–p: Trp86, Trp286
p–p: Trp82
p–cation: His438
p–p: Trp82, His438

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
793
inhibition by racemic salmeterol), with enzyme-inhibitor dissoci- epinephrine. Additional substitution on the hydroxyaminoethyl
ation constants (K(I)) ranging between 30 lM and 6.4 mM (Table 1), chain with the ethyl group, as in isoetharine, caused a further 12-
2
9,43
classifying these compounds as low-potency inhibitors
.
fold higher inhibition potency compared to isoproterenol. The
Ranges of K(I) constants of resorcinol, catechol and saligenin largest, 67-fold, increase in inhibition potency was observed in the
derivatives were very similar, indicating that the inhibition potency saligenin group between salbutamol and salmeterol, where the
of the tested bronchodilating b2 agonists towards human AChE largest difference in the size of hydroxyaminoethyl chain was.
does not depend on the disposition of hydroxyl groups on the Additionally, when compounds with identic hydroxyaminoethyl
benzene ring. However, it seems that the inhibition potency of the chain but different disposition of hydroxyl group on benzene ring
tested b2-agonists depends on the size of the hydroxyaminoethyl (metaproterenol vs. isoproterenol) were compared, no difference
chain attached to the benzene ring. For example, in the resorcinol in inhibition potency was observed. Similarly two times higher
group, metaproterenol and terbutaline differ only in the end of inhibition potency of salbutamol, compared to terbutaline, can be
hydroxyaminoethyl chain (isopropyl in metaproterenol versus tert- attributed to the substitution of one hydroxyl group on benzene
butyl in terbutaline) exhibiting very similar inhibition, while the ring with the hydroxymethyl group. From all of the tested bron-
enlargement of the hydroxyaminoethyl chain with the hydroxy- chodilating b2-agonists, salmeterol was singled out as the most
benzyl group, as it is a fenoterol, caused a fivefold increase in potent inhibitor with an inhibition potency similar to that of flavo-
4
4
inhibition potency. In the catechol group, an elongation of the noids quercetin and myricetin .
hydroxyaminoethyl chain from methyl to isopropyl lead to a 2-fold
The inhibition potency correlated well with the docking results
increase in inhibition potency of isoproterenol compared to expressed here as the interaction energy of the bronchodilating
Figure 3. Computational molecular modelling of b2-agonists and human AChE (PDB ID 1B41). Stereo view of salmeterol (A) and terbutalin (B) in the active site of
AChE. Ligands create H-bonds with residues (green-dashed lines). Hydrophobic p–p interactions with aromatic residues are represented as cyan-dashed lines and
p–cation interactions are represented as orange-dashed lines. The hydrogen atoms of the residues have been omitted for better visibility.

7
94
A. BOSAK ET AL.
Figure 4. Computational molecular modelling of b2-agonists and human BChE (PDB ID 2PM8). Stereo view of salmeterol (A), isoetharine (B) and fenoterol (C) in the
active site of usual BChE. Ligands create H bonds with residues (green dashed lines). Hydrophobic p–p interactions with aromatic residues are represented as cyan-
dashed lines and p–cation interactions are represented as orange-dashed lines. The hydrogen atoms of the residues have been omitted for better visibility.
b2-agonists ꢀ AChE complex (Table 2). The highest interaction interactions showed that, regardless of the size of the hydroxyami-
energy was that of salmeterol, the most potent AChE inhibitor. noethyl chain on the benzene ring, the chain was always directed
All of the predicted non-covalent interactions of bronchodilating to the peripheral site of the active centre gorge of the enzyme.
b2-agonists with amino acid residues defining the AChE active site This orientation was due to the formation of hydrogen bonds with
gorge are summarised in Table 3. The analysis of these Tyr124 and Asp74 (Figure 3(A,B)). These interactions, together with

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
795
Table 4. Reversible inhibition of human AChE and three BChE variants (usual, atypical and fluoride-resistant) by enantiomers of bron-
chodilating b2-agonists.
K(I)/mM
AChE
Usual BChE
0.11 ± 0.03
0.0094 ± 0.0008
0.013 ± 0.001
Atypical BChE
Fluoride-resistant BChE
Resorcinol derivatives
(
(
(
R)-terbutaline
R,R)-fenoterol
S,S)-fenoterol
2.0 ± 0.3
0.54 ± 0.03
0.35 ± 0.03
1.0 ± 0.1
0.13 ± 0.02
0.10 ± 0.00
0.22 ± 0.05
0.019 ± 0.004
0.037 ± 0.005
Catechol derivatives
(
(
R)-epinephrine
R)-isoproterenol
3.2 ± 0.4
2.0 ± 0.5
4.7 ± 1.8
0.81 ± 0.13
12 ± 2
1.4 ± 0.1
—
1.4 ± 0.2
Saligenin derivatives
(
(
(
(
R)-salbutamol
S)-salbutamol
R)-salmeterol
S)-salmeterol
4.0 ± 0.3
1.8 ± 0.1
0.033 ± 0.003
0.19 ± 0.04
0.31 ± 0.05
0.015 ± 0.001
2.3 ± 0.2
1.5 ± 0.2
0.071 ± 0.019
0.30 ± 0.06
0.30 ± 0.04
0.031 ± 0.004
0.028 ± 0.006
0.015 ± 0.001
0.067 ± 0.013
0.020 ± 0.003
—
not measured.
Enzyme-inhibitor complex dissociation constants (K(I)±SE) were determined by linear regression from Ki,app constants obtained from at
least two experiments.
interactions with tyrosines 337 and 341 and the position of the of compounds with resorcinol-, catechol- or saligenin-based com-
benzene ring relative to Trp86, were those that governed the posi- pounds towards usual BChE was primarily a consequence of cati-
tioning of the tested compounds into the active site of AChE. It on–p interaction involving Trp82 (Table 3) that enabled their
seems that epinephrine was the weakest inhibitor of AChE merely position and orientation in the active site of BChE allowing add-
because of the lack of stabilisation by hydrogen bonds with tyro- itional interactions such as hydrogen bonds with Glu197 and
sine residues 337 and 341. The difference between the inhibition His438 residues (Figure 4(A–C)). The highest inhibition potency of
potency of particular b2-agonists of the resorcinol, catechol and salmeterol could be attributed to the additional p–p interaction
saligenin groups, was attributable to additional hydrogen bonds
and hydrophobic interactions between the hydroxyaminoethyl
chain and residues (Table 3). Salmeterol’s high inhibition potency
could have been due to the additional p–p interaction of sal-
meterol’s benzene ring and Trp286 and hydrogen bond with
His447 (Figure 3(A)).
with Trp82, which anchored the saligenin part deeper in the active
site and directed the hydrocarbon chain towards the exit of the
active site of the usual BChE. This position seemed optimal for the
inhibition of BChE.
Inhibition selectivity
Due to a difference in residues lining the active site of AChE
Inhibition of human BChE variants
and BChE, these two enzymes have different reactivity toward sub-
The studied bronchodilating b2-agonists reversibly inhibited all
BChE variants (Figure 2, an example of inhibition by racemic sal-
meterol), with K(I) constants ranging between 9.8 lM and 3.8 mM
9,10,44,46
strates, inhibitors and ligands
. Therefore, it is important to
evaluate the selectivity of compounds that either targets the activ-
ity of AChE, as it is with some drugs for treating neurodegenera-
(
Table 1). Generally, atypical and fluoride-resistant variants were
46
tive disorders like Alzheimer’s disease
or prodrugs whose
up to 26 and 2.8 times, respectively, more weakly inhibited than
usual BChE. The fluoride-resistant BChE has been characterised by
an amino acid substitution changing T243M and G390V that are
far from the enzyme-active centre but could cause conformational
changes resulting in a lower inhibition potency of positively
bioconversion includes the action of BChE, as in the case of bam-
9,11
buterol . The selectivity of the studied bronchodilating b2-ago-
nists was determined from the ratio of the corresponding K(I)
constants for AChE and BChE. Generally, all of the compounds
were more potent inhibitors of BChE than of AChE. The most
selective inhibitor was fenoterol, followed by salbutamol and ter-
butaline that inhibit usual BChE 36, 28 and 27 times more potently
than AChE, respectively. It is worthy to mention that these b2-ago-
nists are currently the most frequently used bronchodilators.
Catechol derivatives were the least selective inhibitors, out of
which epinephrine showed the lowest selectivity. Salmeterol, the
most potent inhibitor of all of the tested cholinesterases, displayed
only a two times higher potency towards BChE than toward AChE.
In view of the BChE polymorphism, inhibition of fluoride-resistant
BChE is similarly selective as that of usual BChE, while no selectiv-
ity in the inhibition of the atypical variant was determined.
4
5
charged ligands . The decrease of inhibition potency towards
atypical BChE compared to usual BChE could be a consequence of
the natural mutation of aspartate 70 to glycine (D70G), a residue
located in the peripheral site of the active centre of BChE and
close to the entrance of the active centre gorge. The inhibition
potency of fenoterol, isoetharine and salmeterol towards usual
BChE was up to 23, 120 and 5.5 times higher, respectively, than
the potency of other resorcinol-, catechol- and saligenin-based
compounds. The same trend of inhibition potency was observed
for atypical and fluoride-resistant variants (Table 1). This indicates
a positive correlation between the size of the hydroxyaminoethyl
chain and the inhibition potency toward the BChE variants, simi-
larly to our observation for AChE inhibition. Out of the eight bron-
chodilating b2-agonists tested, salmeterol was the most potent
inhibitor of all BChE variants with a constant similar to that for
Stereoselectivity of human AChE and usual, atypical and
fluoride-resistant BChE
4
5
donepezil, a drug used to treat Alzheimer’s disease . The kinetic
results correlated well with the docking results (Table 2); the The tested compounds are chiral molecules (Figure 1) and the
higher the inhibitory potency of the compound, the higher the stereoselectivity of cholinesterases could have been expected as a
CDOCKER interaction energy. It seems that the inhibition potency result of
a difference in the binding of enantiomers. The

7
96
A. BOSAK ET AL.
stereoselectivity was determined from the ratio of K(I) constants
determined for the (R) and (S) enantiomers of fenoterol, salbuta-
mol and salmeterol (Table 4). For terbutaline, epinephrine and iso-
proterenol, stereoselectivity was estimated by comparing the K(I)
constants obtained for racemate with those for the (R) enantiomer
3. Pieper MP. The non-neuronal cholinergic system as novel
drug target in the airways. Life Sci 2012;91:1113–18.
4. Giacobini E. Butyrylcholinesterase: its role in brain function.
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inhibitors. 1st ed. London: Martin Dunitz; 2003:1–20.
5. Katalinic M, Hrvat NM, Karasova JZ, et al. Translation of
ꢁ
(
Table 4). Irrespectively of whether stereoselectivity was deter-
mined or just estimated, for most of the compounds, neither AChE
nor BChEs displayed any stereoselectivity; the exception was AChE
and atypical BChE preferring 2- and 3-fold (R)-terbutaline over its
racemate, respectively. The lack of cholinesterase stereoselectivity
was probably the consequence of interactions with residues that
did not involve the chiral centre of the tested compounds. Indeed,
the molecular modelling of complexes demonstrated that the sta-
bilisation of the compounds into the active site of the enzymes
was based on other interactions (Figures 3 and 4).
in vitro to in vivo pyridinium oxime potential in tabun poi-
soning. Arh Hig Rada Toksikol 2015;66:291–8.
6
.
.
Lockridge O, Norgren RB Jr, Johnson RC, Blake TA. Naturally
occurring genetic variants of human acetylcholinesterase
and butyrylcholinesterase and their potential impact on the
risk of toxicity from cholinesterase inhibitors. Chem Res
Toxicol 2016;29:1381–92.
ꢀ
7
Gazi ꢁc I, Bosak A, Sinko G, et al. Preparative HPLC separation
of bambuterol enantiomers and stereoselective inhibition of
human cholinesterases. Anal Bioanal Chem 2006;385:1513–19.
Soreq H, Seidman S. Acetylcholinesterase-new roles for an
old actor. Nat Rev Neurosci 2001;2:294–302.
8
9
.
.
Conclusions
ꢀ
Bosak A, Gazi ꢁc -Smilovi ꢁc I, Sinko G, et al. Metaproterenol, iso-
Due to the important and versatile functions of cholinesterases in
organisms, it is important to define the inhibition potency of com-
pounds that reduce the level of their hydrolytic activity, especially
for those in use as drugs. All of the tested b2-agonists are in use
as bronchodilators, except isoetharine whose use was discontin-
ued. Our results have shown that the compounds are reversible
cholinesterase inhibitors that reduce human cholinesterase
activity and therefore can affect their roles in an organism.
Cholinesterases had the lowest affinity towards epinephrine, the
hormone of adrenal glands and neurons that plays an important
role in the organism during acute stress response. Interestingly,
AChE also displayed low affinity towards terbutaline and salbuta-
mol, compounds that are active components of the most com-
monly used bronchodilators. It should also be mentioned that the
tested b2-agonists showed the highest inhibition potency towards
usual BChE among the three variants of BChE. Moreover, our
results show that the racemates of these compounds in terms of
cholinesterase inhibition are safe for use since there was no
stereoselectivity exhibited in the interaction with cholinesterases.
proterenol, and their bisdimethylcarbamate derivatives as
human cholinesterase inhibitors. J Med Chem 2012;55:6716–23.
1
0. Reiner E, Bosak A, Simeon-Rudolf V. Activity of cholinesterases
in human whole blood measured with acetylthiocholine as
substrate and ethopropazine as selective inhibitor of plasma
butyrylcholinesterase. Arh Hig Rada Toksikol 2004;55:1–4.
1. Tunek A, Svensson LA. Bambuterol, a carbamate ester pro-
drug of terbutaline, as inhibitor of cholinesterases in human
blood. Drug Metab Dispos 1988;16:759–64.
2. Ballard CL, Perry EK. The role of butyrylcholinesterase in
Alzheimer’s disease. In: Giacobini E, ed. Butyrylcholinesterase;
its function and inhibitors. London: Martin Dunitz;
1
1
2003:123–148.
1
1
1
3. Katalini ꢁc M, Bosak A, Kovarik Z. Flavonoids as inhibitors of
human butyrylcholinesterase variants. Food Technol Biotech
2014;52:64–7.
4. McGehee DS, Krasowski MD, Fung DL, et al. Cholinesterase
inhibition by potato glycoalkaloids slows mivacurium metab-
olism. Anesthesiology 2000;93:510–19.
5. Kovarik Z, Radi ꢁc Z, Grgas B, et al. Amino acid residues
involved in the interaction of acetylcholinesterase and butyr-
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Acknowledgements
The authors thank F. Nachon, O. Lockridge and R.T. Evans for their
generous gift of enzymes and V. Vinkovi ꢁc for suggestions in the
design of the new compounds.
16. Lockridge O. Review of human butyrylcholinesterase struc-
ture, function, genetic variants, history of use in the clinic,
and potential therapeutic uses. Pharmacol Therapeut
Disclosure statement
2
015;148:34–46.
ꢀ
ꢀ
1
7. Simeon-Rudolf V, Sinko G, Stuglin A, Reiner E. Inhibition of
human blood acetylcholinesterase and butyrylcholinesterase
by ethopropazine. Croat Chem Acta 2001;74:173–82.
No potential conflict of interest was reported by the authors.
Funding
18. Cruz PMR, Palace J, Ramjattan H, et al. Salbutamol and ephe-
drine in the treatment of severe AChR deficiency syndromes.
Neurology 2015;85:1043–7.
This study was supported by the Croatian Science Foundation
(Grant No. 4307).
1
9. Barrot M, Yalcin I, Choucair-Jaafar N, et al. From antidepres-
sant drugs to beta-mimetics: preclinical insights on potential
new treatments for neuropathic pain. Recent Pat CNS Drug
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