Cysteine-Targeted Insecticides against A. gambiae Acetylcholinesterase Are Neither Selective nor Reversible Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00477

Acetylcholinesterase cysteine-targeted insecticides against malaria vector Anopheles gambia and other mosquitos have already been introduced. We have applied the olefin metathesis for the preparation of cysteine-targeted insecticides in high yields. The prepared compounds with either a succinimide or maleimide moiety were evaluated on Anopheles gambiae and human acetylcholinesterase with relatively high irreversible inhibition of both enzymes but poor selectivity. The concept of cysteine binding was not proved by several methods, and poor stability was observed of the chosen most potent/selective compounds in a water/buffer environment. Thus, our findings do not support the proposed concept of cysteine-targeted selective insecticides for the prepared series of succinimide or maleimide compounds.

Full text of DOI:10.1021/acsmedchemlett.9b00477

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Letter
Cysteine-Targeted Insecticides against A. gambiae
Acetylcholinesterase are neither Selective nor Reversible Inhibitors
Lukas Gorecki, Rudolf Andrys, Monika Schmidt, Tomas Kucera, Miroslav
Psotka, Barbora Svobodova, Veronika Hrabcova, Vendula Hepnarova,
Petr Bzonek, Daniel Jun, Kamil Kuca, Jan Korabecny, and Kamil Musilek
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00477 • Publication Date (Web): 26 Nov 2019
Downloaded from pubs.acs.org on November 27, 2019
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Cysteine-Targeted
Acetylcholinesterase are neither Selective nor Reversible Inhibitors
Insecticides
against
A.
gambiae
Lukas Gorecki,a‡ Rudolf Andrys,b‡ Monika Schmidt,a,b‡ Tomas Kucera,b Miroslav Psotka,a,b Barbora
Svobodova,a,c Veronika Hrabcova,b,c Vendula Hepnarova,a,c Petr Bzonek, b Daniel Jun,a,c Kamil Kuca,b
Jan Korabecny,a,c* and Kamil Musilek,a,b,d*
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a University Hospital Hradec Kralove, Biomedical Research Centre, Sokolska 581, 500 05 Hradec Kralove, Czech Republic;
b University of Hradec Kralove, Faculty of Science, Department of Chemistry, Rokitanskeho 62, 500 03 Hradec Kralove, Czech Republic;
c University of Defence, Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences Trebesska 1575, 500 01
Hradec Kralove, Czech Republic;
d Florida International University, Herbert Wertheim College of Medicine, Department of Cellular Biology & Pharmacology, 11200 SW
8th Street GL 495-G Miami, Florida 33199, United States.
KEYWORDS: acetylcholinesterase; inhibitor; insecticide; Anopheles gambiae; human; selectivity.
ABSTRACT: Acetylcholinesterase cysteine-targeted insecticides against malaria mosquito Anopheles gambiae and other mosquitos
have already been introduced. We have applied the olefin metathesis for the preparation of cysteine-targeted insecticides in high
yields. The prepared compounds with either a succinimide or maleimide moiety were evaluated on Anopheles gambiae and human
acetylcholinesterase with relatively high irreversible inhibition of both enzymes, but poor selectivity. The concept of cysteine binding
was not proved by several methods, and poor stability was observed of the chosen most potent/selective compounds in a water/buffer
environment. Thus, our findings do not support the proposed concept of cysteine-targeted selective insecticides for the prepared series
of succinimide or maleimide compounds.
According to the World Malaria Report 2017, there were 216
million cases of malaria with 445,000 deaths in 2016. Almost 2.7
billion people were reported at risk of malaria. Based on these
In this paper we report the optimization and total synthesis of some
of the previously reported Cys-targeted insecticides. Insecticides
targeted to free cysteine were first proposed by computational
studies in 2012 and some of them have already been
alarming data, malaria remains
a
huge threat.1 Chemical
insecticides are one of the major tools used for insect elimination
and/or prevention of the spread of vector-borne diseases.2,3
However, traditional chemical insecticides are often associated
with significant toxicity to mammals and beneficial insects such as
honey bees or bumble bees.4,5
investigated.12,15 Herein, we describe
a novel and more
straightforward synthetic strategy for Cys-targeted insecticides
starting from commercially available reagents. In other recent
work, a similar synthetic approach was applied for development of
several novel insecticides. The maleimide-containing compounds
were prepared as Cys-targeted molecules that should directly bind
to Cys447, whereas succinimide-containing compounds were
developed for comparison purposes. All of them were then
evaluated in vitro for their potency to block the action of
recombinant Anopheles gambiae acetylcholinesterase (AgAChE1),
The principal mechanism of insecticide action is the formation of a
covalent bond to acetylcholinesterase (AChE, EC 3.1.1.7), a vital
enzyme that is responsible for rapid hydrolysis of the
neurotransmitter acetylcholine (ACh).6–8 The standard
organophosphate and carbamate insecticides covalently block
Ser360 (Anopheles gambiae acetylcholinesterase, AgAChE) and
thus disable the process of ACh hydrolysis in invertebrates as well
as in vertebrates. In recent years, several compunds were
introduced having very good and selective inhibition potency
against AgAChE over human AChE.9–11 Cysteine-targeted
insecticides aimed at the Cys447 residue (full-length numbering; in
Torpedo californica the numbering is Cys286) in the peripheral
anionic site (PAS) of some insect species have been proposed to
overcome insecticide resistance.12 The uniqueness of this approach
depends on the fact that the Cys residue is missing in the
mammalian enzyme. Moreover, the Cys residue is sterically
hindered in some insects such as Drosophila melanogaster and it is
accessible to standard insecticides only with difficulty.12,13 Thus, it
is believed that Cys-targeted insecticides might possess significant
selectivity favouring Anopheles AChE over mammalian
AChE.3,11,14
human
acetylcholinesterase
(HssAChE)
and
human
butyrylcholinesterase (HssBChE).
For the chemical synthesis, the long polymethylene chains were
required as the basic scaffolds for the outlined compounds. The
methyl esters with terminal double bond (1a and 1b) were chosen
as the starting material. Olefin metathesis was applied (instead of
using Grignard reactions) with Grubbs reaction yielding dimeric
intermediates 2a and 2b in nearly quantitative yields (Scheme 1).
There was no need to use a stereoselective reaction, because
hydrogenation of the double bond took place in the next step. For
this reason, a cheaper 1st generation Grubbs catalyst was preferred
and used to obtain esters 3a and 3b instead of using one of the 2nd
generation.16 Subsequent reduction of the esters provided alcohols
4a and 4b in quantitative yields. The ester reduction can be
managed by two synthetic routes. Besides lithium aluminium
hydride, diisobutylaluminium hydride (DIBAL-H) was efficiently
applied to obtain α,ω-dihydroxy compounds 4a and 4b (Scheme 1).
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Scheme 1. The application of olefin metathesis to obtain diversely substituted long chain alkanes. Reagents and conditions: a) Grubbs 1st
gen. (0.02 eq), reduced pressure up to 2 mbar, RT up to 50 °C; b) Pd(OH)2 on C (20 %; 0.2 eq), H2, MeOH/EA (2:1), RT; c) LiAlH4 2M
solution in THF (2.5 eq), THF, RT; d) NBS (3.0 eq), PPh3 (3.0 eq), THF, RT; e) HBr 48 % solution in H2O (3.0 eq), toluene, reflux; f) furan
(3.0 eq), dioxane, 90°C; g) DIAD (1.3 eq), PPh3 (1.3 eq), THF, 0 °C – RT; h) succinimide (1.5 eq), K2CO3 (1.5 eq), DMF, 60°C; i) 7 (1.5
eq), K2CO3, (1.5 eq), DMF, 60°C.
A challenging step was the attempt at selective protection of one
hydroxyl group to successively and selectively proceed towards the
formation of alkylating agent. Initial attempts for selective
protection were not successful and are described in the Supporting
Information (SI chapter 2.4). A convenient approach consisted in
the use of HBr as a selective reagent with 4a and 4b resulting in
mono-bromide intermediates 6a and 6b.17 HBr was also employed
for the synthesis in previous work; however, the authors did not
fully describe how they obtained mono-bromide intermediates.15
6b. The intermediates 8a/b and 9a/b were obtained in two steps via
coupling reaction and subsequent bromination with N-
bromosuccinimide (NBS) in high yields (> 70 %) (Scheme 2). The
original procedure was simplified by skipping isolation of the
alcohol intermediates (17a, 17b, 18a and 18b; described in SI) by
direct bromination to get 9a and 9b. Succinimide was used for
analogous N-alkylation to obtain 8a and 8b. In this way better
yields were achieved and further reduction of maleimide into a
succinimide scaffold, as was described in the original work, was
avoided.15
Further reactions were analogous to those published.15 Diels-Alder
reaction of maleimide with furan provided imide 7.18 Imide 7, and
succinimide, were then coupled with brominated alcohols 6a and
Scheme 2. Synthetic approach for Cys-targeted insecticides. Reagents and conditions: a) succinimide (1.5 eq), K2CO3 (1.5 eq), DMF, 60°C;
b) 7 (1.5 eq), K2CO3, (1.5 eq), DMF, 60°C; c) NBS (1.5 eq); PPh3 (1.5 eq), THF, RT; d) microwave irradiation (MW), dynamic curve with
100 W and 300 PSI max cap, pyridine (2.0 eq), MeCN, 90°C; e) MW, dynamic curve with 100 W and 300 PSI max cap, piperidine (2.0 eq),
K2CO3 (3.0 eq), MeCN, 90°C; f) vacuum app. 1 mBar, 130°C.
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The final compounds containing the succinimide moiety (10a/b
14b and 15b are similar in potency to the related succinimides 10a,
10b and 11b. However, maleimide 15a was found to be several-
fold more potent than its corresponding succinimide 11a for
HssAChE. As HssAChE does not contain a free cysteine residue
which could react with maleimide 15a, the inhibitory ability of this
compound was found unexpected and cannot be rationalized by
interaction with Cys447.
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and 11a/b) were obtained using microwave irradiation. The N-
alkylation of 8a/b achieved almost quantitative yields in the case
of the piperidine analogues (11a and 11b) and around 90 % for the
the pyridine analogues (10a and 10b; Scheme 2). The final
preparation of compounds 14a/b and 15a/b containing a maleimide
residue required a slightly different approach. In the case of the
pyridinium compounds 14a and 14b, retro Diels-Alder reaction
was used prior to the N-alkylation. For the piperidine analogues 15a
and 15b, the N-alkylation of 9a/b had to precede the retro
Diels-Alder reaction to achieve the desired products. Both the
N-alkylation and the retro Diels-Alder reactions were carried out
with good yields over 58 % (Scheme 2). These final steps were
slightly distinct from those reported in the original work, by using
different conditions and by implementation of microwave (MW)
irradiation.15 Note that the compounds 14a/b correspond to
previously published compounds under codenames PM18 and
PM20, and compounds 10b, 15a and 11a are analogous to PMS20,
PY18 and PYS18, respectively (only the salts are different).15 The
rest of the compounds 10a, 11b and 15b are reported herein for the
first time.
The selectivity for AgAChE1 was rather poor and most compounds
were found relatively potent inhibitors of HssAChE. Only in the
case of 15b was the selectivity towards the insect enzyme found to
be over 10-fold. However, the prepared compounds possess only
poor inhibitory activity towards AgAChE1 compared to paraoxon
or bendiocarb.
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Further, time-dependent inhibition was used to determine
differences between both enzymes. A rapid decrease in enzyme
activity was observed within 3 min, and the activity remained
almost steady for 15 minutes for both HssAChE and AgAChE1
(Figure 1 and 2). Surprisingly, increased potency of maleimides vs
succinimides for AgAChE1 is not necessarily evidence of covalent
binding, since the same phenomenon is seen for HssAChE.
Additionally, the rate of enzyme-inhibitor complex formation
could be explained by noncovalent interaction between
pyridine/piperidine parts of the tested compounds with both
enzymes. Such interactions are in general very rapid and there is
probably no covalent bond formation involved in this short time
period.22,23 The previously-published time-dependency15 did not
prove covalent binding of the compounds, but depicted only a
slower or faster rate of binding to the enzyme.
Table 1. Inhibitory activity towards AgAChE1 and HssAChE.
IC50 ± SEM (µM)a
Selectivit
Compound
y index b
AgAChE1
HssAChE
10a ---
3.60 ± 0.095
2.06 ± 0.040
9.97 ± 0.213
25.87 ± 1.704
0.465 ± 0.020
0.810 ± 0.060
0.545 ± 0.028
2.65 ± 0.344
3.38 ± 0.143
0.94
10b PMS20
11a PYS18
3.39 ± 0.338
21.48 ± 0.843
29.30 ± 2.536
3.26 ± 0.194
2.03 ± 0.138
3.52 ± 0.523
39.50 ± 4.598
1.65
2.15
1.13
7.00
2.51
6.45
14.89
1.16
12.70
3.59
AgAChE1
14a (0.4 μM)
14b (0.8 μM)
15a (0.6 μM)
15b (2.6 μM)
100%
80%
60%
40%
20%
0%
11b
---
14a PM18
14b PM20
15a PY18
15b
---
paraoxon
0.0072 ± 0.0004 0.0084 ± 0.0004
0
3
6
9
12
15
18
bendiocarb 0.0024 ± 0.0003 0.0309 ± 0.0008
carbofuran 0.0062 ± 0.0008 0.0221 ± 0.0012
a IC50 values measured by modified Ellman’s assay 15 min after
Incubation time [min]
Figure 1. Time-dependent inhibition of maleimide derivatives on
AgAChE1.
introduction of inhibitor; b Selectivity for AgAChE1 is determined as
ratio IC50 (HssAChE) / IC50 (AgAChE1)
HssAChE
14a (3.5 μM)
14b (2 μM)
15a (3.5 μM)
15b (40 μM)
100%
80%
60%
40%
20%
0%
The crucial mechanism of action should be based on the formation
of a covalent bond between the maleimide moiety and Cys447 in
AgAChE1, resulting in irreversible inhibition. A detailed in vitro
evaluation was made by Dou et al. 2013.15 For in vitro purposes,
recombinant AgAChE1 was prepared and purified in our labs (see
SI Figure S1). The kinetic parameters of the enzyme were evaluated
(see SI Figure S2). The KM constant of AgAChE1 was found to be
54.63 μM for acetylthiocholine iodide (ATCh) as a substrate,
which is in very good correlation with the previously published
data.19,20 To verify the enzyme and assay results, the standard
inhibitors including paraoxon and carbamates (bendiocarb,
carbofuran) were evaluated.
0
3
6
9
12
15
18
Incubation time [min]
Figure 2. Time-dependent inhibition of maleimide derivatives on
HssAChE1.
The inhibitory ability of the prepared insecticides towards
HssAChE and AgAChE1 was determined using the
spectrophotometric method described by Ellman et al. (Table 1).21
HssBChE was used as a common off-target for AChE inhibitors.
As shown in the Supporting Information, the HssBChE inhibition
levels observed for the prepared compounds were over 60 μM (see
SI Table S1). Maleimides 14a, 14b and 15a were found to be the
most potent inhibitors of AgAChE1, and several-fold more potent
than corresponding succinimides. For HssAChE, maleimides 14a,
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80%
60%
40%
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0%
AgAChE1 before dialysis
AgAChE1 after dialysis
HssAChE before dialysis
HssAChE after dialysis
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Figure 3. Reversibility of inhibition on AgAChE1 and HssAChE using the dialysis method.
Thus, the mechanism of interaction is truly crucial for this series of
compounds. We have tested the reversibility of the reaction using
the removal of the selected insecticides by dialysis (Figure 3).
Apparently, none of the tested compounds showed reversible action
of inhibition for either AgAChE1 or HssAChE. Based on the
previous work,15 maleimide-harbouring compounds were
presumed to act as irreversible inhibitors and succinimide
compounds were prepared only as the controls to prove insect-
specific cysteine involvement in covalent bond formation. The
presented results are showing different findings, with all
succinimide- or maleimide-containing compounds found to be
irreversible-acting inhibitors when incubated 30 minutes with
enzymes and further dialysed overnight (Figure 3). The irreversible
inhibition was shown towards HssAChE as well, although in this
enzyme the Cys is substituted by Phe (Figure 4). It should also be
noted that maleimide is able to form a covalent bond with other
amino acid residues (except free cysteine) as was formerly
reported.24 However, the most surpring fact is that all succinimides
(10a/b, 11a/b) were found to be irreversible inhibitors of both
enzymes with recovery less than 20%. Such finding could be
plausibly explained by e.g. precipitation or denaturation of the
enzymes by the detergent-like succinimides.
conjugate with maleimide at pH values higher than 7.5; however,
for 15a this is not the case.26
Additionally,
a molecular docking study was conducted.
Compound 15a was selected from all the tested compounds for its
valuable and higher inhibition activity on AgAChE1 and moderate
effect on HssAChE/BChE. Two protein structures were used (PDB
IDs: 6ARX in Figure 4 and SI Figure S3; 5YDH only in SI Figure
S3) to reveal and better understand the binding interactions
between ligand 15a and the AgAChE1 active site.25 The top
docking-scored pose of 15a was found different in each of the used
proteins. For 6ARX with G119S mutation (Figure 4), ligand 15a
resulted in binding of the maleimide moiety close to the catalytic
triad and interaction with tyrosine residues (Tyr489 and Tyr493),
with the piperidine ring attached at the rim of the gorge (Phe449,
Phe 490 and Tyr494). Differently in the 5YDH protein (SI Figure
S3), the maleimide moiety was found interacting with the rim of
the AgAChE1 gorge (Cys447, Phe449, Tyr494), with the
piperidine scaffold attached close to the active site (Trp245,
Tyr291, Glu359). The literature was checked to elucidate these
contradictory results for the involvement of tyrosine residues in
maleimide binding, but there were no literature data supporting the
hypothesis of covalent bond formation between a hydroxyl from
either tyrosine or serine residues, and thus different binding of
ligand 15a in 6ARX (Figure 4). Apart from sulfhydryl cysteine
residues, only primary amines can be expected to possibly
Figure 4. Superimposition of compound 15a in the AgAChE1
active site (PDB ID: 6ARX, G119S mutation). The close-up view
for ligand is presented as three-dimensional (4A; 15a is presented
in green, important amino acid residues in blue, and catalytic triad
residues in yellow) and two-dimensional (4B) diagrams.
Figure 5. Reversibility of inhibition on AgAChE1 and HssAChE
using dialysis method after iodoacetamide (IAA) pretreatment.
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15a distilled water
15a 10 mM ammonium acetate, pH 7.2
15b distilled water
15b 10 mM ammonium acetate, pH 7.2
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Incubation time [hour]
Figure 6. Stability determination of 15a and 15b over time in distilled water or buffer.
The contradictory finding within in vitro evaluation and molecular
docking raised the question of binding of the presented
maleimide/succinimide molecules to AgAChE1 or HssAChE. The
enzymes were treated with 100 molar excess of iodoacetamide
(IAA) to determine the affinity of the presented inhibitors towards
Cys447. IAA is a commonly used blocking agent of free thiol
groups.27 Although IAA is not strictly thiol selective (as well as
maleimide), the reaction should result in AgAChE1 enzyme that is
resistant to irreversible inhibition by maleimide-based inhibitors.
However, both enzymes treated with IAA were found irreversibly
inhibited by selected compounds 11a (succinimide scaffold) and
15b (maleimide scaffold; Figure 5). Apparently, these results
indicate that irreversible inhibition is most probably not mediated
by the free cysteine residue Cys447 in AgAChE1 as was formerly
reported.15 The maleimide or succinimide moiety can also interact
with other amino acid residues, e.g. with histidine from the catalytic
triad of AgAChE1 or HssAChE and thus may irreversibly inhibit
both enzymes this way.
that this class of molecules will not be effective as insecticides due
to the afore-mentioned findings.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: (to be added by editorial office).
General synthetic methods; General procedure and spectral data;
HPLC analysis of final compounds; Recombinant AgAChE1
production and characterization; Enzymatic assays; Molecular
modelling studies; Time-dependent stability measurement (PDF).
AUTHOR INFORMATION
Corresponding Author
* Corresponding authors emails: kamil.musilek@gmail.com and
korabecny.jan@gmail.com
From the point of practical use for insecticidal purposes, stability
of the most potent and most selective compounds for AgAChE1
(15a and 15b) was inspected by incubation at 40°C (Figure 6). Fast
degradation of both compounds was observed in a water or buffer
environment, with the water degradation found to be apparently
more rapid. In particular, compound 15b (the most selective for
AgAChE1) was almost completely degraded within one hour in
water. Both compounds were more stable in ammonium acetate
buffer, but a degradation ratio of over 50% was observed in 3 hours.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript. ‡These authors contributed equally.
ACKNOWLEDGEMENT
The work was supported by Ministry of Health of the Czech
Republic (no. NV16-34390A), University of Hradec Kralove (no.
SV2115−2018, no. VT2019-2021 and postdoctoral job positions at
UHK) and University of Defence (Faculty of Military Health
Sciences, Long-term development plan). The authors are grateful
to Ian McColl MD PhD for assistance with the manuscript. There
are no conflicts of interest to be declared.
In summary, eight compounds with a succinimide or maleimide
moiety were prepared and evaluated as cysteine-targeted
insecticides. Some of the compounds were shown as potent
AgAChE1 inhibitors, but they were found to be potent inhibitors of
HssAChE with a limited selectivity ratio as well. The time-
dependent inhibition of the compounds was very fast, resulting in
inhibition of both enzymes within minutes. The presented
compounds acted in an irreversible manner to both AgAchE1 and
HssAChE. Molecular docking on two distinct AgAChE1 proteins
indicated contradictory results showing the possibility of different
interactions than the proposed Cys-targeted binding. This
phenomenon was further confirmed by cysteine blocking treatment
of both enzymes, after which they were still found irreversibly
inhibited by the tested compounds, suggesting a different mode of
irreverible binding. The potential use of the tested compounds
might be limited by the relatively low stability depicted within the
degradation assay. Thus, our findings do not support the proposed
concept of cysteine-targeted selective insecticides. We presume
ABBREVIATIONS
ACh acetylcholine; AChE acetylcholinesterase; ATCh
acetylthiocholine
iodide;AgAChE1
Anopheles
gambiae
acetylcholinesterase; DIBAL-H diisobutylaluminium hydride;
HssAChE human acetylcholinesterase; HssBChE human
butyrylcholinesterase; IAA iodoacetamide; IC50 median inhibitory
concentration; MW microwave irradiation; NBS N-
bromosuccinimide; PAS peripheral active site; TBAF
tetrabutylammonium
fluoride;
TBDPSCl
tert-butyl(chloro)diphenylsilane.
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