Study of para-Quinone Methide Precursors toward the Realkylation of Aged Acetylcholinesterase

Article abstract of DOI:10.1021/acsmedchemlett.7b00037

Acetylcholinesterase (AChE) is an essential enzyme that can be targeted by organophosphorus (OP) compounds, including nerve agents. Following exposure to OPs, AChE becomes phosphylated (inhibited) and undergoes a subsequent aging process where the OP-AChE adduct is dealkylated. The aged AChE is unable to hydrolyze acetylcholine, resulting in accumulation of the neurotransmitter in the central nervous system (CNS) and elsewhere. Current therapeutics are only capable of reactivating inhibited AChE. There are no known therapeutic agents to reverse the aging process or treat aged AChE. Quinone methides (QMs) have been shown to alkylate phosphates under physiological conditions. In this study, a small library of novel quinone methide precursors (QMPs) has been synthesized and examined as potential alkylating agents against model nucleophiles, including a model phosphonate. Computational studies have been performed to evaluate the affinity of QMPs for the aged AChE active site, and preliminary testing with electric eel AChE has been performed.

Full text of DOI:10.1021/acsmedchemlett.7b00037

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Letter
Study of para-Quinone Methide Precursors Towards
the Realkylation of Aged Acetylcholinesterase
Ryan J Yoder, Qinggeng Zhuang, Jeremy M Beck, Andrew Franjesevic, Travis G. Blanton,
Sydney B. Sillart, Tyler Robert Secor, Leah Guerra, Jason D. Brown, Carolyn S. Reid,
Craig A McElroy, Ozlem Dogan Ekici, Christopher S. Callam, and Christopher M. Hadad
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 08 May 2017
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Study of para-Quinone Methide Precursors towards the
Realkylation of Aged Acetylcholinesterase
Ryan J. Yoder#, Qinggeng Zhuang, Jeremy M. Beck, Andrew Franjesevic, Travis G. Blanton#, Sydney Sillart, Tyler Secor, Leah
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Guerra, Jason D. Brown, Carolyn Reid, Craig A. McElroy†, Özlem Doğan Ekici‡, Christopher S. Callam, Christopher M. Hadad*
Department of Chemistry and Biochemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210, USA
#Department of Chemistry and Biochemistry, The Ohio State University, Marion Campus, 1465 Mt. Vernon Avenue, Marion,
OH 43302, USA
†College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210, USA
‡Department of Chemistry and Biochemistry, The Ohio State University, Newark Campus, 1179 University Drive, Newark, OH
43055, USA
KEYWORDS Acetylcholinesterase, Organophosphorus chemical nerve agents, Quinone methide.
ABSTRACT: Acetylcholinesterase (AChE) is an essential enzyme that can be targeted by organophosphorus (OP) compounds, including
nerve agents. Following exposure to OPs, AChE becomes phosphylated (inhibited) and undergoes a subsequent aging process where the
OPꢀAChE adduct is dealkylated. The aged AChE is unable to hydrolyze acetylcholine, resulting in accumulation of the neurotransmitter in
the central nervous system (CNS) and elsewhere. Current therapeutics are only capable of reactivating inhibited AChE. There are no
known therapeutic agents to reverse the aging process or treat aged AChE. Quinone methides (QMs) have been shown to alkylate phosꢀ
phates under physiological conditions. In this study, a small library of novel quinone methide precursors (QMPs) has been synthesized and
examined as potential alkylating agents against model nucleophiles, including a model phosphonate. Computational studies have been
performed to evaluate the affinity of QMPs for the aged AChE active site, and preliminary testing with electric eel AChE has been perꢀ
formed.
Organophosphorus (OP) compounds containing a phosꢀ
phoryl group have been used as pesticides and chemical warꢀ
fare agents.1ꢀ4 These compounds are toxic due to their inhibiꢀ
tion of the enzyme acetylcholinesterase (AChE), a serine hyꢀ
drolase typically found in the central and peripheral nervous
system that regulates concentrations of the neurotransmitter
acetylcholine.5,6 As illustrated in Scheme 1, when the central
phosphorus atom is attacked by the catalytic serine, a leaving
group is substituted, leaving a phosphyl group covalently atꢀ
tached to the serine, which blocks the active site. Inhibition of
AChE can lead to death by respiratory failure due to overstimꢀ
ulation of muscarinic acetylcholine receptors at the neuromusꢀ
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cular junctions.7,8 There are known therapeutics, specifically
Recent studies have shown the generation of QMs from
benzyl and naphthyl derivatives via silyl cleavage 27
QMs
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7
8
oximeꢀcontaining compounds, to reverse OP inhibition of
.
AChE.9ꢀ11
have predominantly been generated by oxidative28 or photoꢀ
chemical29 means due to higher yield of the reactive intermeꢀ
diate, but for in vivo applications, the QM intermediate must
be generated thermally. Therefore, we decided to focus on a
subset of paraꢀQMPs as candidates for the realkylation of
aged AChE (Figure 1). Initial computational modeling (see
below) suggested that these QMPs would have some affinity
for the active site of aged AChE. As a proofꢀofꢀconcept, we
will focus on finding the optimum QMP through quantitative
alkylation experiments with a variety of nucleophiles, includꢀ
ing a model phosphonate, in addition to molecular modeling
and kinetic experiments with native and aged AChE.
If left untreated after OP inhibition, the Oꢀalkyl group of the
AChEꢀOP adduct can undergo a dealkylation reaction, leaving
a phosphonate (or phosphate) anion in the AChE active site in
a process known as “aging” (Scheme 1).11ꢀ14 The rate of aging
varies from minutes to hours between different OPs 15ꢀ17
Curꢀ
.
rently there is no known therapeutic to reactivate aged
AChE.17 Previous investigations utilized alkyl sulfonates19,20
and phenacyl bromides21 to alkylate a model phosphonate, but
attempts to reactivate the aged enzyme were unsuccessful.
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Scheme 1. Inhibition, aging and realkylation of AChE. The
leaving group X is substituted during inhibition. The alkyl
group R is lost during aging.
Figure 1. Target Quinone Methide Precursors
Thus, the need for an effective therapeutic to reverse the efꢀ
fects of aging in OPꢀinhibited AChE has not been met. Imꢀ
portantly, a single alkylating agent will treat the identical aged
structures of each of the common OPs, except for tabun, which
result in the same methylphosphonate structure after aging.
Consequently, the same oxime should be effective for many
realkylated OPs.
A reliable method for producing amines via reductive amiꢀ
nation has been developed (Scheme 3). In the process, secondꢀ
ary amines were reacted with the aldehyde substrate to proꢀ
duce a conjugated iminium ion which was reduced in situ by
sodium cyanoborohydride (77–86%). The resulting amines (1–
4) were isolated via column chromatography and then disꢀ
solved in methanol and toluene followed by slow addition of
methanolic hydrochloric acid to yield the corresponding hyꢀ
drochloride ammonium salts in 93–98% yield (1–4 HCl).
The preferred alkylating agent must be reactive enough to
alkylate the phosphonate anion in the aged AChE enzyme, but
selective enough to minimize alkylation of other biomolecules
in vivo. Quinone methides (QMs) have been used as alkylating
agents for other biological applications in vitro, including the
alkylation of phosphodiesters at physiological conditions.22
QMs have also been shown to reversibly alkylate DNA and
block transcription.23 Additionally, QMs have been shown to
possess highly tunable reactivity through modification of subꢀ
stituents on the aromatic ring,24 or the amine leaving group 25
.
The structure of potential quinone methide precursors (QMPs)
also strongly mimics edrophonium, an oxyaniliniumꢀbased
inhibitor of AChE that is known to bind in the (native) AChE
Scheme 3. Reductive Amination for the Preparation of
QMPs
active site 26
. Another potentially useful aspect of QMs is their
Studies to evaluate the general effectiveness of the amines
and ammonium salts towards nucleophilic substitution were
undertaken. Each of the compounds shown in Figure 1 were
separately subjected to reaction with 4ꢀmethylbenzenethiol,
piperidine/pyrrolidine and benzyl alcohol at 100 °C for 3h.
These nucleophiles were chosen as representative thiol,
amines and alcohol. We chose this high temperature because
in the absence of assistance of desolvation and proximity, the
energy barrier in bulk solvent could be higher than that at the
active site of the enzyme. Both the neutral and the protonated
forms of QMPs were tested because the active sites of enꢀ
zymes are normally highly desolvated, and can exhibit a difꢀ
ability to be delivered in an unreactive prodrug form where a
reactive QM could be generated selectively within the AChE
active site (Scheme 2).
Scheme 2. Potential Quinone Methide Precursor (QMP)
Prodrug and Quinone Methide Intermediate
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ferent pH environment due to the surrounding dissociable resꢀ observed. The major peaks observed in each reaction were a,
1
2
3
4
idues compared to that in the bulk solvent. All compounds
alkylated more than 65% of the thiol and the amine, as shown
in Table 1. None alkylated more than 20% of the alcohol. This
test implies that the candidate QMPs can react as alkylators of
nucleophiles with moderate reactivity.
c, e and f. Species c indirectly implied the formation of b via
alkylation.
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Scheme 4. Alkylation of model nucleophiles by QMPs
Table 1. Percentage yields of alkylation of model nucleo-
philes by QMPs
Nucleophile
QMP
benzyl
alcohol
4-methylbenzenethiol
piperidine*
1
2
84%
87%
83%
83%
83%
83%
85%
84%
80%
77%
81%
79%
78%
78%
81%
69%
0%
8%
11%
13%
8%
Scheme 5. Reactions between 1 or 1-HCl and the model
phosphonate (a). Protonation states are not specified.
3
4
1-HCl
2-HCl
3-HCl
4-HCl
To further examine the alkylation of the model phosphonate
by our QMPs, 31P NMR spectroscopy was used to follow the
different phosphorus species generated from the same reacꢀ
tions (Scheme 5) in D2O. The results indicated the presence of
all four different phosphorus species (aꢀd) after heating. The
major species identified in the product were a (25.1 ppm) and
d (20.6 ppm). b and c were also observed, indicating the ocꢀ
currence of alkylation.
12%
10%
16%
*3 and 3-HCl were reacted with pyrrolidine because of their piperidinyl groups in
the existing structure.
In order to test the efficacy of our precursors against a modꢀ
el phosphonate as a simple model for the aged AChE active
site, we sought to replicate a study from Steinberg21 to indiꢀ
rectly monitor alkylation by UVꢀVis spectroscopy. By appendꢀ
ing a model phosphonate with a pꢀnitrophenol leaving group,
alkylation could be indirectly monitored after addition of base
to the synthesized alkylated product.
In parallel, we used computational approaches to evaluate
this QMP approach. Past efforts towards understanding the
effect of OP nerve agents by molecular modeling studies have
been performed by our group.30 In order to better understand
the potential interaction between our QMPs and the target
aged AChE active site, molecular docking simulations were
performed using AutoDock 4.0. The ligands were initially
optimized using Gaussian 0931 at the B3LYP32,33/6ꢀ311+G**
level of theory, and then submitted to a MerzꢀKollman charge
calculation to prepare the substrates for docking. In docking
simulations with aged AChE, the receptor was kept mostly
rigid and the ligand flexible, and each of the poses was anaꢀ
lyzed to determine if it would present the potential for a favorꢀ
able interaction between the aged serine residue and the benꢀ
zylic carbon of our QMPs. Specifically, we evaluated docked
poses in which the reactive benzylic carbon was within 5.0 Å
of the anionic Oꢀ(P=O) of the aged serine residue. The aged
AChE was prepared by in silico methods from the human isoꢀ
However, after following Steinberg’s protocol, our results
proved unreliable over the course of replicate trials with large
levels of background signal (Supporting Information). Thus,
our efforts moved away from the indirect UVꢀvis screen and
were refocused on confirming the alkylation of a model phosꢀ
phonate by our QMPs.
In order to confirm alkylation of the model phosphonate by
QMPs, the reaction was monitored by tandem HPLC/ESIꢀMSꢀ
TOF. These techniques would allow for a better opportunity
to monitor the potential products generated, and perhaps other
previously unconsidered pathways that may be responsible for
the degradation of the alkylated product. A representative
QMP of the neutral (1) and protonated (1-HCl) forms were
each reacted with the model phosphonate in water at 100 °C
for 3 h and analyzed using the HPLC/ESIꢀMSꢀTOF to identify
each of the species in solution after heating. The reaction
pathways are illustrated in Scheme 5. For both 1 and 1-HCl,
only trace amounts of the intact alkylated product (b) were
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form (PDB: 1B41a) and details are provided in the supporting
information.
by establishing zoning criteria. Interestingly, once again the
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pyrrolidine (2-HCl) amine leaving group was the most promꢀ
ising ligand. Also, there was a strong preference for the proꢀ
tonated leaving groups to spend more time in the active site
while the neutral leaving groups rarely stayed in the enzyme’s
active site (see Supporting Information).
Examination of molecular docking snapshots revealed some
variability in the specific orientation of the amine leaving
group (Supporting Information). It was found that the pyrroliꢀ
dine (2/2-HCl) and piperidine (3/3-HCl) amine leaving groups
were the most promising of this library of ligands with the
highest percentage of docking poses in which the benzylic
carbon is within 5.0 Å of the aged serine residue. While the
majority of poses are outside of this distance cutoff, a signifiꢀ
cant portion do exist within this distance criterion, suggesting
that the ligands can access the aged active site. Thus, these
QMPs may allow for a therapeutic reaction to occur whether
alkylation were to take place through in situ generation of a
quinone methide or by a direct displacement at the benzylic
position. Also, among the poses not within the distance criteꢀ
rion, the vast majority were still within the enzyme itself, but
further away from the aged serine towards the gorge bottleꢀ
neck. Interestingly, the smaller dimethylamine group (1/1-
HCl) and the similarly sized morpholine group (4/4-HCl) did
not have as strong a recognition for the enzyme’s active site.
We sought to evaluate whether our QMP ligands could bind
into the active site of aged AChE; however, we were unable to
develop a protocol to evaluate that binding as the aged AChE
is not “active” to any known substrate. Instead, the binding
affinity and selectivity of QMPs to the active site of AChE can
be revealed by kinetics of reversible inhibition of native
AChE, and as measured by competition kinetics for AChE’s
substrate. Indeed, the activity of electric eel AChE was moniꢀ
tored at room temperature using Ellman’s assay in the presꢀ
ence of QMPs at varied concentrations ranging from 0 to 1.5
mM.34 Electric eel AChE is a commercially available AChE
homolog. Despite the difference between the gorge shapes of
human and nonꢀhuman AChE homologs, in this proofꢀofꢀ
concept study it is acceptable to qualitatively estimate the
binding affinity using electric eel AChE. The concentrations
of acetylthiocholine (ATC, used as the AChE substrate) were
varied from 0 to 1.1 mM independently. Acquired data were
processed with GraphPad Prism 6 for nonlinear regression.
Details of the experimental procedure and calculations are
included in the Supporting Information.
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2.98 Å
Table 2. Molecular Dynamics Results and Inhibition kinet-
ics of electric eel AChE by QMPs
QMP
1-HCl
2-HCl
3-HCl
4-HCl
44.8 (11.8)
50.5 (24.1)
44.6 (6.0)
37.4 (8.9)
MD
AS%*
Vmax
61.2 ± 2.2
104.9 ± 4.2
80.3 ± 3.9
141.0 ± 8.9
(nM/s)
Km
0.032 ± 0.014 0.079 ± 0.014 0.062 ± 0.015 0.189 ± 0.037
Figure 2. Docked pose of 3-HCl in the aged AChE active site,
displaying the putative reactive orientation of the benzylic carbon
being less than 5 Å from aged serine residue.
(mM)
Ki
(mM)
0.095 ± 0.024 0.106 ± 0.042
1.57 ± 0.83
0.080 ± 0.040
With molecular docking results in hand, molecular dynamꢀ
ics (MD) simulations were performed subsequently to examꢀ
ine how the ligands noncovalently interact with the enzyme
over the course of time. For each ligand, the three lowest enꢀ
ergy snapshots from each of the thirteen aged AChE frames
were chosen as the starting points for our MD simulations.
Once again, of interest was the interaction between the reacꢀ
tive benzylic carbon of our QMPs and the target, phosphylated
serine residue of aged AChE. Over the course of a brief 1 ns
simulation for each starting orientation from docking, the
QMP ligands could be further examined for their preference
towards the aged active site versus other areas of the enzyme
34.9 ± 22.9
0.919
8.7 ± 4.0
0.403
3.1 ± 1.7
0.227
1.24 ± 0.94
1.83
α
IC50
(mM)**
*Over a 1 ns simulation, the percentage of time ligand spent in the aged AChE
active site (defined as the benzylic carbon of the QMP being 0ꢀ5 Å from the phosꢀ
phylated serine residue (Supporting Information). Values of the corresponding neuꢀ
tral compounds are displayed in parentheses. **[acetylthiocholine] = 0.5 mM
As illustrated in Table 2, the Ki values ranged from 0.08 to
1.57 mM indicate overall weak inhibition of native AChE. The
constant α is the ratio between the dissociation equilibrium
constants of the enzymeꢀinhibitorꢀsubstrate complex and the
enzymeꢀinhibitor complex. The greater α is than 1, the closer
the inhibition mechanism is to competitive inhibition.35 All
QMPs showed α values greater than 1, especially 1-HCl and
2-HCl; thus, they are competitive inhibitors or nearꢀ
competitive mixꢀtype inhibitors35 that selectively bind to the
active site of native AChE.
a
A newer structure (PDB ID: 4EY4), which is of higher
resolution, was published after we initiated this study. Howevꢀ
er, alignment shows only trivial difference in coordinates
(backbone RMSD = 0.708 Å).
The four hydrochloride compounds were subject to an in
vitro test against methylphosphonateꢀaged AChE, which is the
common aged product of many authentic Vꢀagents and Gꢀ
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agents. Electric eel AChE (Sigma Aldrich) was used in these of poses oriented in a potentially reactive conformation. The
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studies. Each tested QMP (4 mM) was added to AChE that
was aged with a soman analogue, originally reported by Amitꢀ
ai et al.36 The negative and the positive controls prepared in
parallel were aged and native AChE without QMPs. To deterꢀ
mine the amount of residual inhibited AChE after the aging
process, a 2ꢀpralidoxime chloride (2ꢀPAM) control was also
prepared by mixing aged AChE with 4 mM 2ꢀPAM.
varied results of our experiments also suggest the tunability
within our initial scaffold to modulate reactivity. With conꢀ
firmation of the alkylated model phosphonate and preliminary
computational and kinetic data completed, the hydrochloride
QMPs described herein were further investigated to determine
their efficacy towards reactivating aged AChE. Though not
effective at present, this proofꢀofꢀconcept model demonstrates
the feasibility to develop potentially effective countermeasures
against OP exposure using QMPꢀbased AChE realkylators.
Further studies are in progress in order to optimize this potenꢀ
tial strategy and to reverse the effects of aging of AChE by OP
agents.
In a similar manner to 2ꢀPAM, fluoride ion at high concenꢀ
tration can function as a reactivator of AChE.37,38 As an anion
with simple structure, it is speculated to reactivate all inhibitꢀ
ed/realkylated AChE without bias and not to compete with
QMPs for AChE’s active site. Therefore, NH4F (4 mM) was
also added to the reaction mixture to reactivate the realkylated
AChE, in order to avoid reꢀaging after alkylation. After reacꢀ
tion for 24 h at 37 °C and pH 8.0, all samples were treated
with 4 mM of 2ꢀPAM to ensure the reactivation of any aged
AChE that was (hopefully) realkylated. The resulting AChE
activity was determined with Ellman’s assay with ATC as a
substrate. The relative activities of QMPꢀtreated samples, the
negative control and the 2ꢀPAM control compared to the posiꢀ
tive control are displayed in Figure 3 (see the Supporting Inꢀ
formation for specific procedures and additional details). Unꢀ
fortunately, none of our QMP substrates appear to realkylate
the aged form of AChE to any significant extent; indeed, the
QMPꢀtreated samples have similar activities in the Ellman’s
assay and are less than 2ꢀPAM by itself (Figure 3). (2ꢀPAM is
known not to reactivate the aged form of AChE.)11ꢀ14
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Supporting Information. Please find further experimental deꢀ
tails and computational protocols in the supporting information.
This material is available free of charge via the Internet at
http://pubs.acs.org. The content includes:
Synthesis and Characterization of QMPs
Alkylation of Model Nucleophiles
UVꢀVis Studies
HPLC Data
Computational Methods
Determination of Inhibition Characteristics of QMPs against
Native AChE
Procedures for Aging of AChE
Procedures for Realkylation and Reactivation of Aged AChE
NMR Spectra
Atom Coordinates and Parameters
AUTHOR INFORMATION
1.5
1.0
0.5
0.0
Corresponding Author
hadad.1@osu.edu
Funding Sources
National Institutes of Health (U01–NS087983) and the US Arꢀ
my (W81XWHꢀ10ꢀ2ꢀ0044).
Notes
Any additional relevant notes should be placed here.
ACKNOWLEDGMENT
Neg Ctrl 1-HCl
2-HCl
3-HCl
4-HCl
2-PAM
The authors would like to acknowledge financial support from
the National Institutes of Health (U01–NS087983) and computing
resources from the Ohio Supercomputer Center. Initial financial
support of this project was obtained with resources from the US
Army (W81XWHꢀ10ꢀ2ꢀ0044).
Figure 3. Result of AChE realkylation test. The relative activity of
2ꢀPAM control was 1.2%, implying the near completeness of
aging. Aged AChE samples treated with tested QMPs were not
obviously more active than the negative control (Neg Ctrl).
ABBREVIATIONS
In conclusion, we have shown the ability of a novel class of
Quinone Methide Precursors (QMPs) to act as alkylating
agents with a variety of model nucleophiles, including a model
phosphonate to mimic the aged AChE active site. Molecular
modeling studies confirm that this moiety has some affinity
for the aged AChE active site, with a significant contribution
AChE: acetylcholinesterase. ATC: acetylthiocholine. MD: moꢀ
lecular dynamics. OP: organophosphorus. QM: quinone methide.
QMP: quinone methide precursor. 2ꢀPAM: 2ꢀpyridine aldoxime
methyl chloride.
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