Noncovalent Inhibitors of Mosquito Acetylcholinesterase 1 with Resistance-Breaking Potency

Article abstract of DOI:10.1021/acs.jmedchem.8b01060

Resistance development in insects significantly threatens the important benefits obtained by insecticide usage in vector control of disease-transmitting insects. Discovery of new chemical entities with insecticidal activity is highly desired in order to develop new insecticide candidates. Here, we present the design, synthesis, and biological evaluation of phenoxyacetamide-based inhibitors of the essential enzyme acetylcholinesterase 1 (AChE1). AChE1 is a validated insecticide target to control mosquito vectors of, e.g., malaria, dengue, and Zika virus infections. The inhibitors combine a mosquito versus human AChE selectivity with a high potency also for the resistance-conferring mutation G122S; two properties that have proven challenging to combine in a single compound. Structure-activity relationship analyses and molecular dynamics simulations of inhibitor-protein complexes have provided insights that elucidate the molecular basis for these properties. We also show that the inhibitors demonstrate in vivo insecticidal activity on disease-transmitting mosquitoes. Our findings support the concept of noncovalent, selective, and resistance-breaking inhibitors of AChE1 as a promising approach for future insecticide development.

Full text of DOI:10.1021/acs.jmedchem.8b01060

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Article
Non-covalent Inhibitors of Mosquito Acetylcholinesterase
with Resistance-Breaking Potency
1
Sofie Knutsson, Cecilia Engdahl, Rashmi Kumari, Nina Forsgren, Cecilia Lindgren, Tomas
Kindahl, Stanley Kitur, Lucy Wachira, Luna Kamau, Fredrik J. Ekström, and Anna Linusson
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01060 • Publication Date (Web): 19 Oct 2018
Downloaded from http://pubs.acs.org on October 21, 2018
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Non-covalent Inhibitors of Mosquito
Acetylcholinesterase 1 with Resistance-Breaking
Potency
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1$
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Sofie Knutsson, Cecilia Engdahl, Rashmi Kumari, Nina Forsgren, Cecilia
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Lindgren, Tomas Kindahl, Stanley Kitur, Lucy Wachira, Luna Kamau, Fredrik
2*
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Ekström, and Anna Linusson
1Department of Chemistry, Umeå University, SE-901 82 Umeå, Sweden
2Swedish Defence Research Agency, CBRN Defence and Security, SE-906 21 Umeå,
Sweden
3Centre for Biotechnology Research and Development, Kenya Medical Research
Institute, Nairobi, Kenya
$
These authors contributed equally
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*
Corresponding
authors.
E-mail
addresses:
anna.linusson@umu.se;
fredrik.ekstrom@foi.se
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ABSTRACT
Resistance development in insects significantly threatens the important benefits obtained
by insecticide usage in vector control of disease-transmitting insects. Discovery of new
chemical entities with insecticidal activity is highly desired in order to develop new
insecticide candidates. Here, we present the design, synthesis, and biological evaluation
of phenoxyacetamide-based inhibitors of the essential enzyme acetylcholinesterase 1
(AChE1). AChE1 is a validated insecticide target to control mosquito vectors of e.g.
malaria, dengue, and Zika virus infections. The inhibitors combine a mosquito versus
human AChE selectivity with a high potency also for the resistance-conferring mutation
G122S; two properties that have proven challenging to combine in a single compound.
Structure-activity relationship analyses and molecular dynamics simulations of inhibitor-
protein complexes have provided insights that elucidate the molecular basis for these
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properties. We also show that the inhibitors demonstrate in vivo insecticidal activity on
disease-transmitting mosquitoes. Our findings support the concept of non-covalent,
selective, and resistance-breaking inhibitors of AChE1 as a promising approach for future
insecticide development.
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INTRODUCTION
The widespread usage of insecticides for control of disease-transmitting mosquitoes
(vectors) has had a profound effect on the prevention of malaria, dengue, and Zika virus
infections. The positive impact of these interventional public health measures is
manifested in the hundreds of millions of averted malaria cases in sub-Saharan Africa
1
over the last 15 years. However, the intense use of insecticides has led to the
development and spread of resistant mosquitoes to such an extent that resistant strains
have now been identified against all major classes of insecticides recommended for
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vector control by the World Health Organization (WHO). Furthermore, many of the
currently used insecticides lack specificity for the vector over other non-target species,
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which can have devastating effects on both humans and beneficial insects such as the
pollinators.3-5 Encouragingly, two agricultural insecticides re-purposed for vector control
use have recently been prequalified by the WHO.6
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The insecticides most commonly used for vector control disrupt the insect’s nervous
system by inhibiting voltage-gated ion channels (pyrethroids and organochlorines) or by
inhibiting the essential enzyme acetylcholinesterase (AChE) (organophosphates and
carbamates). The physiological role of AChE is to terminate nerve signaling by rapidly
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hydrolyzing the neurotransmitter acetylcholine. Generally, insects have two genes
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encoding AChE enzymes: ace-1 and ace-2, whilst there is only one gene in vertebrates.
In mosquitoes, AChE1 is thought to be the main catalytic enzyme.9-10 Recently, the crystal
structure of AChE1 from the malaria-transmitting mosquito Anopheles gambiae
(AgAChE1, amino acid numbering has been adapted to correspond to mAChE without
taking deletions or insertions into consideration throughout the text, see Experimental
section for details) was reported.11 Similar to AChEs from other species, the structure
shows a deep and narrow gorge with the catalytic serine at the bottom of the active site
(i.e. S202, corresponding to S203 in Homo sapiens AChE (hAChE)). The catalytic serine
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is the target for covalent insecticides, both organophosphates and carbamates. Natural
populations of mosquito species have acquired organophosphate and carbamate
resistance-conferring mutations in the ace-1 gene.10, 12-13 These mutations often occur
close to, or within the active site gorge; the most widespread mutation in disease-
transmitting mosquitoes is G122S in AChE1 of the Anopheles and Culex mosquitoes
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(corresponding to G122S and G119S in hAChE and Torpedo californica AChE,
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respectively). The crystal structure of G122S-AgAChE1 has recently become available,
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which show high similarity to the AgAChE1 structure. So far, it has proven challenging
to develop inhibitors that combine a potency for G122S-AgAChE1 with a mosquito versus
human selectivity.15-16
Current design strategies for novel insecticides include covalent inhibitors that target
either the catalytic serine residue (S202)15-23 or the cysteine residue (C289) of AChE1.24-28
As an alternative to inhibitors that react covalently with AChE1, we aim to develop non-
covalent (non-bonding) inhibitors that offer the possibility to form important interactions
with residues distant from the evolutionary conserved catalytic triad and the surrounding
residues. We have recently discovered a number of non-covalent inhibitors that
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selectively target AChE1 of the mosquitoes An. gambiae and Aedes aegypti (Ae. aegypti,
transmitting dengue, chikungunya, and Zika virus infections).29-30 Our research revealed
that the inhibitor preferences for mosquito and human AChEs were distinctly different;
only 10% of the found AChE inhibitors had a similar potency for human and mosquito
AChEs. This finding is also consistent with our studies on AChE substrate preferences
and kinetics comparing mosquito, mouse (Mus musculus, mAChE), and human AChEs.31
One of the discovered compounds, the phenoxyacetamide-based compound 1 (Figure
1), was not only a potent inhibitor of the mosquito AChE1 and selective versus hAChE,
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but also a potent inhibitor of G122S-AgAChE1 (Table 1). Herein, we report the design,
synthesis, in vitro- and in vivo evaluation of phenoxyacetamide-based analogues of 1.
The aim was to explore the molecular basis for the inhibitors’ selectivity for mosquito
AChE1 versus hAChE and their potency on G122S mutated AgAChE1. Using a
combination of X-ray crystallography (mAChE) and molecular dynamics (MD; mosquito
AChE1s and mAChE) simulations, we provide a structural understanding of these potent
and selective inhibitors of mosquito AChE1s.
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RESULTS
Design and synthesis of mosquito AChE1 inhibitors
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A set of 20 phenoxyacetamide-based compounds was designed based on 1 to explore
the structure-activity relationships (SARs) for inhibition of mosquito AChE1 and hAChE
(
Figure 1 and Table 1). Three structural elements of 1 were investigated (Figure 1a); the
importance of the ethylpiperazine group (A), the influence of the piperidine linker (B), and
the effect of the biphenyl moiety (C). These three structural elements showed specific
interactions with AChE in the 1mAChE crystal structure (Figure 1b) and a similar
interaction pattern in the 1AChE1 homology models.29 Fragment A interacts with
residues in the catalytic site (CAS, mainly W86), fragment B is positioned between the
phenols of Y124 and Y341 and C is positioned at the entrance of the active site gorge
close to W286 (Figure 1). The new compounds were designed to investigate how
changes of the interaction pattern of 1 affect the inhibition of AChEs (Table 1). Fragment
A was removed or changed to a smaller piperazine or to tertiary amines with various
electronic properties. The piperidine linker (fragment B) was changed to a more flexible
propyl chain, and the distal phenyl group of fragment C was changed for other aromatic
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groups or by exchanging the phenyl to an iodide. In the design of fragment C, the intention
was also to improve the compounds’ solubility in acetone, necessary for in vivo mosquito
experiments.
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Figure 1. The chemical structure of 1 (a) and the binding pose of 1 in mAChE (b). Three
elements (A, B and C) of the parent molecule were varied in the design of analogues.
Amino acid residues of mAChE that form the active site gorge are highlighted.
Synthesis of AChE inhibitors
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The truncated piperidine analogue 2 and the morpholino- and N-ethylpiperazine
analogues (3 and 4), which are lacking the piperidine linker, were obtained from 22 after
amide formation (Scheme 1). Amines and diamines with rigid (5 and 6) or flexible (8, 9,
and 10) linkers were prepared in an analogous fashion (Scheme 1). The synthesis of the
rigid morpholino analogue 7 was accomplished by reacting 4-morpholinopiperidine 23
with chloroacetyl chloride to give amide 24, which was substituted with 4-phenylphenol
yielding the target compound (Scheme 2). The Suzuki coupling reaction was utilized to
synthesize different para-substituted aryl ethers (15–19 and 21). For this purpose, 4-
iodophenoxyacetic acid was converted to the acid chloride and coupled with various
amines to produce the iodoaryl compounds 11–14 (Scheme S1). Suzuki coupling with 4-
methoxyphenylboronic acid proceeded smoothly for the target compounds 17 and 18.
Due to synthetic problems related to solubility issues and low yields, 15 and 16 were
produced using a variation of the route, with a reversed order of the Suzuki coupling and
amide bond formation (Scheme S2). The analogues of 1 with structural variation on the
distal aromatic ring were obtained in a similar manner, where two different aromatics were
coupled to the iodoarene to give 19 and 21 (Scheme S3). Finally, compound 20, where
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the distal phenyl group of 1 is substituted for a cyclic ketone, was accessed from 5-
hydroxy-1-indanone (Scheme S4). Several of the analogues had improved solubility in
both water and acetone compared to 1.
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Scheme 1. Synthesis of compounds 2–6 and 8–10.a
O
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OZ
1) Amide coupling
2) HCl (4-6, 8-10)
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: Y =
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aFor details, see Supporting Information.
Scheme 2. Synthesis of compound 7.a
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OH
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a
For details, see Supporting Information.
Potency of AChE inhibitors
The potency of the synthesized phenoxyacetamide-based compounds (2–21) was
evaluated by determination of their half-maximum inhibitory concentration (IC ) values
50
for AaAChE1, AgAChE1, the insecticide resistant mutant G122S-AgAChE1, and hAChE
(
Table 1 and Figure S1).
The wild type mosquito enzymes showed similar inhibition profiles when comparing
their IC50 values, and both enzymes are henceforth collectively referred to as AChE1.
Importantly, also G122S-AgAChE1 followed a similar trend and showed a similar
sensitivity as the wild type enzyme. These findings differentiate the phenoxyacetamide-
based compounds from our previous reported thiourea-based inhibitors that showed low
30
potency on the resistance-conferring mutant. Five compounds (5, 15, and 19–21) had
sub-micromolar IC50 values for inhibition of all three mosquito enzymes. The
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ethylpiperazine fragment (A) was proven crucial for an inhibitory effect; deletion of this
fragment resulted in loss of activity (2). Modification of the resulting piperidine to a
morpholine (3) or an ethylpiperazine (4) did not restore the potency. A small modification
of fragment A to methylpiperazine (5) was tolerated, resulting in sub-micromolar IC50
values, while an exchange to a piperidine (6) or a morpholine (7) led to a reduction in
potency (10- and 1000-times higher IC values, respectively). Also, the introduction of a
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
flexible propyl linker instead of the piperidine as fragment B (8–10 and 16–18) led to loss
of potency for all enzymes compared to the effects of their corresponding analogues.
Modifying fragment C from a biphenyl to a 4-iodophenyl (compounds 11–14) resulted in
a loss of potency, while other modifications of the biphenyl ring (15 and 19–21) resulted
in highly potent inhibitors, showing the importance of the aromatic ring at this position.
The 4’-methoxybiphenyl (15) led to sub-micromolar potency on all three mosquito
enzymes, and had a similar inhibition profile as the parent compound 1. The other phenyl
substituents, in combination with the piperidine-piperazine moiety (compounds 19–21),
also resulted in sub-micromolar inhibition of both AChE1 and G122S-AgAChE1.
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Table 1. Chemical structures and biochemical evaluation of the phenoxyacetamide-
based inhibitors 1–21.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
SRb
ID Structure
IC (µM)
5
0
AaAChE1
AgAChE1
G122S-
hAChE
AgAChE1
0.22
0.21
1
1.3
31
O
O
N
N
141
(
0.12–
(0.12–
0.37)
N
(0.81–2.0) (29–34)
0.38)
O
O
N
2
inactivec
inactivec
inactivec
inactivec
inactivec
inactivec
inactivec
n.a.d
n.a.d
O
O
N
3
O
inactivec
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Journal of Medicinal Chemistry
Page 14 of 72
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3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
N
n.a.d
4
5
N
>200
>200
0.24
>200
>200
10
0.25
0.54
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
N
40
N
(0.24–
(0.19–
0.32)
(0.34–
0.86)
N
(8.4–13)
0
.26)
.0
4
3.8
4.2
4.6
O
O
N
6
7
8
9
1.2
N
(2.5–6.4)
(3.5–4.2)
(3.2–5.4)
(3.4–6.3)
93
O
O
N
n.a.d
>200
>200
>200
N
O
(80–109)
3.3
3.5
16
113
O
O
N
N
H
N
32
(3.1–3.5)
(3.2–3.9)
13
(13–21)
39
(93–136)
115
12
O
O
N
N
H
N
8.8
(11–12)
(11–14)
(33–46)
(96–136)
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Page 15 of 72
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9
.9
10
23
57
O
O
N
N
H
10
5.7
5.6
1.2
(6.5–15)
(7.6–13)
3.6
(19–29)
6.2
(49–66)
20
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1.5
O
O
N
1
1
2
I
N
N
(1.3–1.7)
(2.6–5.0)
43
(5.1–7.4)
95
(18–23)
87
72
O
O
N
N
1
H
N
I
(27–190)
(19–97)
(74–123)
(74–101)
138
112
114
128
O
O
N
N
13
H
1.2
N
(106–
I
(100–126) (101–128) (104–157)
180)
36
44
31
13
O
O
N
N
14
H
0.3
45
I
(28–46)
0.23
(37–53)
(23–42)
(13–14)
O
O
N
15
N
0.40
0.96
18
N
O
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Page 16 of 72
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
0.18–
(0.37–
0.42)
(0.64–1.4) (15–21)
0
.29)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
.8
5.8
20
110
O
O
N
N
H
N
16
17
18
19
19
O
(3.7–4.0)
(4.8–7.0)
(17–24)
(98–124)
190
16
25
33
O
O
N
H
N
N
7.6
3.1
79
(116–
O
(15–17)
(24–27)
(13–80)
316)
14
3
.9
4.5
5.2
O
O
N
N
H
O
(3.7–4.1)
(3.7–5.5)
0.24
(3.7–7.3)
0.71
(13–15)
0.22
19
O
O
F
N
N
(0.18–
(0.10–
0.59)
(0.56–
0.90)
N
(15–23)
0
.26)
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Page 17 of 72
Journal of Medicinal Chemistry
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5
6
7
8
9
0
.20
0.29
0.38
4
.9
O
O
N
2
0
16
O
N
(0.18–
(0.24–
0.35)
(0.34–
0.42)
N
(4.3–5.5)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
.22)
.11
0
0.08
0.13
2.1
O
O
N
21
N
19
N
(0.096–
(0.069–
0.093)
(0.11–
0.15)
O2N
(1.4–3.2)
0.13)
a
b
95% confidence interval given in parentheses; SR = selectivity ratio, computed by
taking the compound’s IC value for hAChE and dividing by the higher of its IC values
50
50
c
d
for AgAChE1 or AaAChE1; Inactive at 1 mM; n.a. = not applicable
In general, the compounds had a lower potency on hAChE than on the mosquito
enzymes. For example, some of the most potent inhibitors of AChE1 and G122S-
AgAChE1 (5, 15 and 19) showed at least 40 times greater IC50 values for the human
enzyme. Interestingly, hAChE showed a different SAR compared to the mosquito
enzymes. The exchange of fragment A from ethylpiperazine to a piperidine (6 versus 1)
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
that led to a decrease of potency for the mosquito enzymes, resulted in an increased
potency on hAChE and consequently a complete loss of selectivity. Furthermore,
modifying fragment C from a biphenyl to a 4-iodophenyl (compounds 11–14) resulted in
loss of selectivity, mainly due to lower inhibition of the mosquito AChE1s. In fact, 14 with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4-iodophenyl in combination with a flexible linker and a piperidine resulted in the strongest
inhibition of hAChE and a reversed selectivity ratio (SR) of 0.3.
Structure-based analysis of the potency and selectivity of phenoxyacetamide-based
compounds
A combination of X-ray crystallography, structural modelling, and MD simulations was
used to elucidate the interactions and dynamics that govern the potency and selectivity
of the phenoxyacetamide-based compounds. The inhibitors 15 and 10 were analyzed in
complex with AgAChE1 (15 and 10), G122S-AgAChE1 (15), AaAChE1 (15) and mAChE
(15 and 10). Note that hAChE and mAChE are very similar in sequence and 3D structure
and the compounds showed similar potency on mAChE compared to hAChE; the IC50
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Page 19 of 72
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values were 29 µM and 18 µM for 15, and 61 µM and 57 µM for 10, on m- and hAChE
respectively (Figure S2).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Structures of phenoxyacetamide-based compounds in complex with mAChE and
AChE1s. Using X-ray crystallography, the crystal structures of the 15mAChE (PDB code
6FSE) and 10mAChE (PDB code 6FSD) complexes were both determined to a
resolution of 2.7 Å (Table S1). The electron density maps showed that 15 and 10 span
the entire active site gorge of mAChE in a similar extended conformation as previously
29
reported for 1 (PDB code 5FUM) , with the compounds forming interactions with W286
and W86 at the rim and the base of the gorge, respectively (Figure S3). The position and
geometry of the inhibitors suggest that the carbonyl oxygen of 15 forms a water-bridged
hydrogen bond with the backbone amide nitrogen of F295 while the carbonyl oxygen of
10 participates in a direct hydrogen bond with the same amide. Both complexes lack
electron density for the distal, solvent exposed phenyl ring (fragment C) and this structural
element has therefore not been modelled in the final structures. Detailed structural
descriptions of 15mAChE and 10mAChE are available in the Supporting Information.
Using the experimentally determined structures of 15mAChE, 10mAChE, and
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
AgAChE1 (PDB code 5X61) as templates, the 15AaAChE1, 15AgAChE1, 15G122S-
AgAChE1 and 10AgAChE1 complexes were modelled and used as starting structures
in the MD simulations.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Molecular dynamics of 15AChE1 and 15mAChE complexes. The conformational
landscapes of the 15mAChE, 15AaAChE1, 15AgAChE1, and 15G122S-AgAChE1
complexes were sampled by three 200 ns equilibrium MD simulations (S1–S3) for each
complex. Based on the root mean square deviation (RMSD; Figure S4) the simulations
were considered to be equilibrated after 50 ns, and thus subsequent analyses were made
using the last 150 ns of each simulation. All enzymes were stable throughout the
simulations with only small backbone structural deviations from the starting conformation
(
<2Å, Figure S4). In the simulations of the AChE1 complexes, the inhibitor 15 retained its
binding pose with deviations < 4Å from the starting structures in all three simulated
trajectories for each of the three mosquito enzymes (Figures 2a–c and 3a-c). Interestingly,
15 behaved differently in complex with mAChE, as large differences in the dynamics were
observed compared to the mosquito complexes (Figure 2d), and three different local
energy minima binding conformations were identified (Figure 3d-f). The second most
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populated conformation of 15 in 15mAChE was similar to the crystal structure (Figure
3e), while the other two showed large structural deviations compared to the crystal
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
structure, mainly in the biphenyl part of the inhibitor that interacts with residues at the
entrance of the gorge.
Figure 2. The dynamics over time of compound 15 in complex with a) AaAChE1, b)
AgAChE1, c) G122S-AgAChE1, and d) mAChE according to its RMSD after
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Journal of Medicinal Chemistry
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
superimposing the enzyme backbone; blue, red, and green curves correspond to
simulations S1, S2, and S3, respectively.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Interaction patterns between 15 and the enzymes. Contact frequencies between 15 and
residues in the binding sites were calculated to investigate the positioning of 15 in the
AChEs throughout the simulations. For all AChE complexes, the inhibitor had substantial
contacts with amino acid residues Y335/Y341 (mosquito residue numbering/mouse
residue numbering) and W87/W86 throughout the entire simulations (Figure S5). Notably,
differences in the contacts for the 15AChE1 complexes compared to 15mAChE were
observed. In the AChE1 complexes, 15 had frequent contacts also with residues Y331
and F332 at the CAS while the corresponding contacts in 15mAChE (Y337 and F338)
were infrequent. At the entrance of the gorge, 15 had frequent contacts with residue
W283/W286, although it was less frequent in AgAChE1 and mAChE compared to
AaAChE1 and G122S-AgAChE1. This was due to flipping of the W283 indole towards the
exterior solvent in one of the parallel simulations in AgAChE1, while in the case of
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Page 23 of 72
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mAChE, the biphenyl group was more dynamic resulting in fewer contacts with W286
(Figures 3e-f and S5b and d).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Binding poses of 15 in AChEs based on MD simulations. The poses were
extracted from the most populated conformations of 15 (yellow) during the MD simulations
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Journal of Medicinal Chemistry
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of the mosquito enzymes AaAChE1 (a, cyan), AgAChE1 (b, pink), and G122S-AgAChE1
(c, blue). The three most populated conformations of 15 in the mouse enzyme (grey) are
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
shown in d–f, where e is most similar to the crystal structure of 15mAChE.
The binding strengths between 15 and the residues of the identified contacts were
assessed by decomposing calculated binding energies between 15 and AChEs for
different complexes per amino acid residue using the molecular mechanics/Poisson-
Boltzmann surface area (MM/PBSA) method (Table 2; see Methods for details). The energy
values show that the binding strengths between 15 and residues Y335/Y341 and
W87/W86, respectively, were strong in all enzymes suggesting that these interactions
anchor 15 in all the AChEs in a similar fashion (Table 2).
The contact analyses revealed differences between the binding poses of 15 in the
AChE1s and mAChE, where 15 had more frequent contacts in the CAS with Y331 and
F332 in AChE1 as compared to in mAChE. The binding energy decomposition showed
that the piperidine fragment of 15 formed strong binding interactions with the aromatic
ring of Y331 throughout the simulations of the mosquito enzymes. The corresponding
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interaction in 15mAChE (15Y337) showed only weak binding strengths. Moreover, the
interactions between 15 and F332/F338 appear to be of medium strengths and
approximately similar for all complexes, showing that differences in contact frequencies
do not directly translate into differences in binding strengths. At the entrance of the gorge,
stronger binding contributions were seen between 15 and residues W283 and I73 of
AChE1 compared to corresponding interactions in 15mAChE (W286 and Y72). Taken
together, the weaker interactions in mAChE resulted in large mobility of 15 in the entire
binding site in general and at the entrance in particular (Table 2 and Figures 2 and 3).
Analyses of contact frequencies of 15G122S-AgAChE1 and minimum distance
calculations between 15 and S122 (Figures S5c and S6) showed that there were no
substantial contacts between 15 and S122 during the simulations. The strong binding
strengths between 15 and the amino acid residue Y331 contributed to binding poses of
1
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15 distant to the mutation site at position 122 of the mosquito enzyme (Table 2). This
resulted in sufficient space in 15AgAChE1 to accommodate the G122S mutation without
interrupting the binding of 15. These observations explain the high potency of 15 for
G122S-AgAChE1 (Table 1).
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Molecular dynamics of the 10AgAChE1 complex. To further elucidate the forces
governing the inhibitor potency, complex 10AgAChE1 was simulated and analyzed in
parallel with the studies of 15AgAChE1. Compared to 15, inhibitor 10 has a substantially
different chemical composition of the fragments interacting with amino acid residues in
the binding site of the AChEs, i.e. the ethylpiperazine (fragment A) and piperidine
0
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0
(fragment B) moieties of 15 have been replaced by a piperidine moiety and a flexible
propyl chain in 10 (Table 1). These chemical modifications resulted in an approximately
40 times higher IC50 value of 10 for AgAChE1 compared to 15 (Table 1). The
10AgAChE1 simulations were considered to be equilibrated after 50 ns according to the
RMSD, and the enzyme was stable throughout the simulations (Figure S7). Inhibitor 10
was more dynamic compared to 15 in the binding site of AgAChE1 (Figure S8). The
contact frequency analysis of 10AgAChE1 showed that 10 had frequent contacts with
the same residues found to substantially contribute to binding of 15 (listed in Table 2 and
Figure S9). However, the decomposition of the binding energy between 10 and AgAChE1
revealed that the interactions between 10 and amino acid residues in the binding site
were considerably weakened compared to 15AgAChE1 (Table 2). In particular, the
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binding strengths between 10 and the residues W87, Y331, and F332 were significantly
reduced, highlighting the importance of the interactions between 15 and these residues
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0
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9
0
(Table 2). It appears that the chemical differences between 10 and 15 resulted in the
weaker interactions of 10 with amino acid residues F332, Y331, and W87 thus making 10
more dynamic (Figures S8 and S10), which altogether contributed to the loss of inhibitory
potency compared to 15.
Table 2. Energy contribution of interactions between individual residues and inhibitors to
total binding energy.
Residues
Energy contribution to binding (kJ/mol)a
mosquito/mouse
15AaAChE 15AgAChE 15G122 15mACh 10AgAChE
1
1
S-
E
1
AgAChE1
W283/W286
-12.0±0.5
-7.8±0.9
-11.0±0.5 -6.1±0.8
-9.5±0.7
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2
2
2
2
2
2
2
2
2
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3
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I73/Y72
-2.8±0.3
-4.3±0.2
-13.2±0.3
-3.1±0.3
-11.5±0.4
-8.0±0.3
-10.4±0.3
-5.1±0.5
-3.6±0.3
-13.7±0.4
-2.8±0.3
-9.4±0.5
-7.0±0.3
-10.5±0.5
-4.4±0.6
-3.6±0.5
-3.0±0.3
-3.1±0.3
-3.3±0.3
F291/F297
Y335/Y341
Y124/Y124
Y331/Y337
F332/F338
W87/W86
-3.6±0.3
0
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0
-14.0±0.3 -11.7±0.5 -11.1±0.4
-2.5±0.2
-2.8±0.4
-4.5±0.3
-7.3±0.5
-5.4±0.4
-11.2±0.4 -3.8±0.3
-6.8±0.5
-6.1±0.6
-10.7±0.5 -12.6±0.5 -7.7±0.4
aThe energy of contribution was calculated by decomposing binding energy using
MM/PBSA method with solute dielectric constant of eight. Residues with less than -4
kJ/mol in at least one of the complexes were considered for the comparison.
Insecticidal effects of phenoxyacetamide-based AChE1 inhibitors on mosquitoes
The improved solubility in acetone and mosquito saline of the designed compounds
compared to parent compound 1 enabled us to evaluate in vivo potency of selected
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phenoxyacetamide-based analogues. The intrinsic insecticidal efficacy of inhibitors 19–
21 was investigated by topical application on adult female Ae. aegypti and An. gambiae
1
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mosquitoes (Figure 4).
Figure 4. The intrinsic insecticidal efficacy of compound 19-21 recorded 24 hours after
topical application on adult female Ae. aegypti (a) and An. gambiae (b) mosquitoes.
All three inhibitors showed insecticidal activity where the 2’-fluorobiphenyl compound
19 had the highest in vivo potency; a dose of 10 nmol/mosquito (4.26 µg/mosquito) killed
75% and 88% of adult Ae. aegypti and An. gambiae mosquitoes, respectively. The
mortality rates for 20 and 21 were lower when applied to Ae. aegypti mosquitoes (50%
and 31%, respectively), while only a slight decrease in mortality compared to 19 was
observed when applied to An. gambiae mosquitoes (Figure 4 and Table S3–S4). In all
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