Probing the activity of a non-oxime reactivator for acetylcholinesterase inhibited by organophosphorus nerve agents

Article abstract of DOI:10.1016/j.cbi.2016.04.002

Currently fielded treatments for nerve agent intoxication include atropine, an acetylcholine receptor antagonist, and pralidoxime (2PAM), a small molecule reactivator of acetylcholinesterase (AChE). 2PAM reactivates nerve agent-inhibited AChE via direct n

Full text of DOI:10.1016/j.cbi.2016.04.002

Accepted Manuscript
Probing the Activity of a Non-Oxime Reactivator for Acetylcholinesterase Inhibited by
Organophosphorus Nerve Agents
C. Linn Cadieux, Haoyu Wang, Yuchen Zhang, Jeffrey A. Koenig, Tsung-Ming Shih,
John McDonough, John Koh, Douglas Cerasoli
PII:
S0009-2797(16)30115-6
10.1016/j.cbi.2016.04.002
CBI 7641
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 9 January 2016
Revised Date: 23 March 2016
Accepted Date: 4 April 2016
Please cite this article as: C.L. Cadieux, H. Wang, Y. Zhang, J.A. Koenig, T.-M. Shih, J. McDonough,
J. Koh, D. Cerasoli, Probing the Activity of a Non-Oxime Reactivator for Acetylcholinesterase
Inhibited by Organophosphorus Nerve Agents, Chemico-Biological Interactions (2016), doi: 10.1016/
j.cbi.2016.04.002.
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ACCEPTED MANUSCRIPT
Probing the Activity of a Non-Oxime Reactivator for
Acetylcholinesterase Inhibited by
Organophosphorus Nerve Agents
C. Linn Cadieux1*, Haoyu Wang2, Yuchen Zhang2, Jeffrey A. Koenig1, Tsung-Ming Shih1, John
McDonough1, John Koh2, Douglas Cerasoli1
1Research Division, United States Army Medical Research Institute of Chemical Defense,
Aberdeen Proving Ground, MD 21010
2Chemistry and Biochemistry Department, University of Delaware, Newark DE 19716
*Corresponding Author: Phone:410-436-2308; Email address: christena.l.cadieux.ctr@mail.mil
Keywords
Reactivator; Oxime; Organophosphorus Nerve Agent; Pralidoxime; Guinea Pig;
Acetylcholinesterase
Abstract
Currently fielded treatments for nerve agent intoxication include atropine, an acetylcholine
receptor antagonist, and pralidoxime (2PAM), a small molecule reactivator of
acetylcholinesterase (AChE). 2PAM reactivates nerve agent-inhibited AChE via direct
nucleophilic attack by the oxime moiety on the phosphorus center of the bound nerve agent. Due
to a permanently charged pyridinium motif, 2PAM is not thought to cross the blood brain barrier
and therefore cannot act directly in the neuronal junctions of the brain. In this study, ADOC, a
non-permanently charged, non-oxime molecule initially identified using pesticide-inhibited
AChE, was characterized in vitro against nerve agent-inhibited recombinant human AChE. The
inhibitory and reactivation potentials of ADOC were determined with native AChE and AChE
inhibited with tabun, sarin, soman, cyclosarin, VX, or VR and then compared to those of 2PAM.
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Several structural analogs of ADOC were used to probe the reactivation mechanism of the
molecule. Finally, guinea pigs were used to examine the protective efficacy of the compound
after exposure to sarin. The results of both in vitro and in vivo testing will be useful in the design
of future small molecule reactivators.
1. Introduction
Organophosphorus (OP) nerve agents, especially those known as chemical warfare agents
(Figure 1), are highly toxic [1]. These compounds induce toxicity by irreversibly inhibiting
acetylcholinesterase (AChE) via covalent bond formation (phosphonylation) with the active site
serine, which in turn allows the accumulation of the neurotransmitter acetylcholine [2]. This
inhibition occurs in nerve junctions in both the peripheral body systems and, once nerve agent
has crossed the blood brain barrier, in the central nervous system (CNS) [3]. If left untreated, the
excess acetylcholine swiftly causes a cholinergic crisis leading to disruption of respiratory and
cardiac function, and subsequent death. Recent use of OP nerve agents against civilian
populations [4, 5] underscores the need for novel treatments against OP intoxication as well as
increased clinical knowledge of appropriate procedures for the management of nerve agent
casualties. The current standard treatment for nerve agent intoxication includes administration of
atropine (an antimuscarinic), diazepam (an anticonvulsant), and a pyridinium oxime (in the
United States, pralidoxime [2PAM, Figure 1] which acts to reactivate phosphonylated AChE [6].
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Figure 1. The chemical structures of six organophosphorus nerve agents and two reactivator
compounds referenced in this work.
Pyridinium oximes are thought to reactivate phosphonylated AChE via a direct nucleophilic
attack of the oximate anion on the phosphorus group [7, 8]. Cleavage of the phosphorus-serine
bond releases the resulting phosphonylated oxime molecule which upon dissociation regenerates
AChE in its active form. Unfortunately, the permanent positive charge of the pyridinium moiety
found in 2PAM (and oximes such as 1-2-hydroxyiminomethyl-1-pyridino-3-4-carbamoyl-1-
pyridino-2-oxapropane [HI6] and 1,1'-oxybis(methylene) bis4-(E)-
hydroxyiminomethylpyridinium [obidoxime] which are fielded in other countries) prevents these
molecules from efficiently crossing the blood brain barrier and reactivating inhibited AChE in
the brain and CNS [6]. A tertiary oxime, monoisonitrosoacetone (MINA), which does not
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contain a quaternary amine motif, has been shown to enter the CNS and reactivate
phosphonylated acetylcholinesterase in the brain [3]. However, the absence of the pyridinium
motif in MINA means that the active oximate anion is formed much less frequently at
physiological pH, likely accounting for the overall decreased reactivation potential at pH 7.4 of
MINA as compared to 2PAM [9, 10]. A need exists for novel drugs that not only reactivate OP-
inhibited AChE but also cross the blood brain barrier to act on inhibited enzyme found in the
CNS. The permanent positive charge generally needed to lower the pKa of an oxime moiety to a
level that will be more reactive at physiological pH does not lend itself to these needs. Initially,
4-amino-2-((diethylamino)methyl)phenol (1; ADOC; Figure 1) was discovered to have
reactivation potential for AChE inhibited by the OP pesticide diisopropyl fluorophosphate (DFP)
via a high through-put screening method [11]. In the present study, the novel reactivator ADOC,
which does not utilize a standard oxime motif, was characterized for activity against OP nerve
agent-inhibited AChE. A series of structural analogs of ADOC were also tested, and
experiments designed to probe the mechanism of reactivation were conducted. Finally, the in
vivo activity of ADOC was explored using a standardized reactivation experiment [12] in guinea
pigs.
2. Materials and methods
2.1 General Materials
All inhibition and reactivation assays were conducted using recombinant human
acetylcholinesterase (RHuAChE, AA780, Chesapeake PERL, Inc., Savage, MD). Hydrolysis of
acetylthiocholine iodide (AtCh, A5751, Sigma-Aldrich Co. LLC., St. Louis, MO) was measured
using 5,5′-dithiobis-2-nitrobenzoic acid (DTNB, D8130, Sigma-Aldrich Co. LLC., St. Louis,
MO). Measurements were made using a SpectraMax Plus 384 microplate reader (Molecular
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Devices, Sunnyvale, CA) utilizing 96-well clear bottom plates (655101, Greiner Bio-One GmbH,
Frickenhausen, Germany). Unless otherwise indicated, all assays were performed in 0.1 M
potassium phosphate buffer at pH 7.0 with 1 mg/mL bovine serum albumin (BSA, A7906,
Sigma-Aldrich Co. LLC., St. Louis, MO) included to help stabilize the RHuAChE. All
organophosphorus nerve agents were obtained from the US Army Edgewood Chemical
Biological Center (Aberdeen Proving Ground, MD) at a purity >98.5% as determined by 31P
NMR.
2.2 Compounds
1: 4-amino-α-diethylamino-O-cresol (ADOC; Sigma-Aldrich Co. LLC Catalog # S561274)
2: 2-[(diethylamino)methyl]phenol (Princeton BioMolecular Research Catalog# OSSK_515806)
3: 4-aminophenol (Sigma-Aldrich Co. LLC Catalog # 60034)
4: (3-aminobenzyl)diethylamine (Sigma-Aldrich Co. LLC Catalog # CBR00121)
5: 3-[(diethylamino)methyl]-4-ethoxyaniline (Enamine Catalog# EN300-80977)
6: 4-amino-3-((diethylamino)methyl)phenol (Synthesized for this research; 1H NMR [600 MHz,
Methanol-d4] δ 6.67 – 6.53 [m, 3H], 3.53 [s, 2H], 2.53 [q, J = 7.2 Hz, 4H], 1.06 [t, J = 7.2 Hz,
6H]; 13C NMR [151 MHz, MeOD] δ 149.62, 138.39, 125.70, 117.45, 116.98, 114.30, 56.49,
46.25, 10.54)
7: 4-amino-2-((dimethylamino)methyl)phenol (Synthesized for this research; 1H NMR [400
MHz, Methanol-d4] δ 6.65 – 6.54 [m, 3H], 3.57 [s, 2H], 2.32 [s, 6H]; 13C NMR [101 MHz,
MeOD] δ 149.91, 138.71, 122.48, 117.10, 116.42, 115.54, 61.00, 43.26)
8: 4-amino-3-((dimethylamino)methyl)phenol (Synthesized for this research; 1H NMR [400
MHz, Methanol-d4] δ 6.69 – 6.50 [m, 3H], 3.37[s, 2H], 2.23 [s, 6H];13C NMR [101 MHz,
MeOD] δ 149.56, 138.31, 125.15, 117.56, 116.95, 114.65, 61.98, 43.89)
9: 4-amino-3-((diisopropylamino)methyl)phenol (Synthesized for this research; 1H NMR [400
MHz, Chloroform-d] δ 6.62 [d, J = 8.4 Hz, 1H], 6.54 [dd, J = 8.4, 2.8 Hz, 1H], 6.41 [d, J = 2.6
Hz, 1H], 3.78 [s, 2H], 3.17 [p, J = 6.7 Hz, 2H], 1.12 [d, J = 6.7 Hz, 12H]; 13C NMR [101 MHz,
CDCl3] δ 151.29, 138.04, 123.27, 116.36, 115.88, 115.48, 48.55, 47.70, 19.79)
10: 4-amino-2-((dicyclohexylamino)methyl)phenol (Synthesized for this research; 1H NMR [400
MHz, Chloroform-d] δ 7.92 [d, J = 2.8 Hz, 1H], 7.28 [s, 1H], 6.75 [d, J = 9.0 Hz, 1H], 4.05 [s,
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2H], 2.83 – 2.72 [m, 2H], 1.94 – 1.03 [multiple peaks, 20H]; 13C NMR [101 MHz, CDCl3] δ
167.19, 138.94, 125.30, 124.32, 121.87, 116.65, 58.36, 49.50, 30.06, 25.89, 25.68)
11: 2-((diethylamino)methyl)-4-(dimethylamino)phenol (Synthesized for this research; 1H NMR
[600 MHz, Chloroform-d] δ 6.73 [d, J = 8.7 Hz, 1H], 6.66 [dd, J = 8.7, 3.0 Hz, 1H], 6.47 [d, J =
2.9 Hz, 1H], 3.73 [s, 2H], 2.82 [s, 6H], 2.61 [q, J = 7.2 Hz, 4H], 1.10 [t, J = 7.2 Hz, 6H]; 13C
NMR [151 MHz, CDCl3] δ 150.43, 144.44, 122.46, 116.30, 114.82, 114.48, 57.44, 46.32, 42.17)
12: 4-acetamido-2-[(diethylamino)methyl]phenol (Sigma-Aldrich Co. LLC Catalog # S414026)
13: 2-amino-6-((diethylamino)methyl)phenol (Synthesized for this research; 1H NMR [400
MHz, Chloroform-d] δ 6.72 – 6.57 [m, 2H], 6.43 [dd, J = 7.0, 1.8, 1H], 5.32 [s, 1H], 3.75 [s, 2H],
2.64 [q, J = 7.2 Hz, 3H], 1.13 [t, J = 7.2 Hz, 6H]; 13C NMR [101 MHz, Chloroform-d] δ 145.80
, 134.80 , 121.44 , 118.88 , 118.16, 114.39 , 56.83 , 46.26)
14: 2-amino-6-((dimethylamino)methyl)phenol (Synthesized for this research; 1H NMR [400
MHz, Chloroform-d] δ 6.70 – 6.61 [m, 2H], 6.44 – 6.41 [m, 1H], 3.63 [s, 2H], 2.34 [s, 6H]; 13C
NMR [101 MHz, Chloroform-d] δ 145.54 , 134.78 , 121.34 , 119.00 , 118.11 , 114.59 , 62.77 ,
44.51)
Synthesis of compounds 6, 7, 8, 9, and 10 followed the general procedure of adding the amine
(diethylamine for 6, dimethylamine for 7 and 8, diisopropylamine for 9, dicyclohexylamine for
10) to a solution of 2-hydroxy-5-nitrobenzaldehyde (7,9,10) or 5-hydroxy-2-
nitrobenzaldehyde(6,8) in DCM. The solution was cooled to 0ºC, and 6 molar equivalents of
sodium triactoxyborohydride were added followed by stirring at room temperature for 5 hours.
Reactions were then quenched by the addition of aqueous sodium bicarbonate and extracted
using ethyl acetate after which the combined extracts were washed with brine and dried with
magnesium sulfate. Solvent was then removed via rotary evaporation, and the crude products
were isolated using flash chromatography (10% MeOH/DCM). Lithium aluminum hydride (10
molar equivalents) was added to the purified product in dichloromethane at 0ºC. The reaction
was allowed to return to room temperature and was then stirred for 5 hours. Quenching with 15%
aqueous sodium hydroxide at 0ºC was followed by a filtration through celite. Solvent was then
removed as described earlier, and the products were again purified using flash chromatography
(20% MeOH/DCM).
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Synthesis of compound 11: To a solution of ADOC in THF was added formaldehyde (10 molar
equivalents), and then triactoxyborohydride (6 molar equivalents) was added at 0ºC. The solution
was allowed to return to room temperature and stirred for 5 hours. The reaction was then
quenched by the addition of sodium bicarbonate and extracted with ethylacetate after which
combined organic layers were washed with brine and dried with magnesium sulfate. Solvent was
removed via rotary evaporation, and the products were purified by flash chromatography (20%
MeOH/DCM).
Synthesis of compounds 13 and 14: Into a solution of 3-nitrosalicylic acid was added 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC, 1.5 molar equivalent). The
solution was stirred for 15 minutes before 1.2 molar equivalents of diethylamine (13) or
dimethylamine (14) was added followed by addition of DMAP (2 molar equivalents). The
reaction was stirred for 16 hours before 1N HCl was added and the aqueous layer extracted by
DCM. The organic extracts were combined and dried over Na2SO4 and reduced in vacuo. The
product was purified by flash chromatography (10% MeOH/DCM) to give 2-hydroxy-N,N-
diethyl-3-nitrobenzamide as an oil. Into LiAlH4 (10 molar equivalents) in dry THF was added 2-
hydroxy-N,N-diethyl-3-nitrobenzamide. The resulting mixture was refluxed for 5 hours, then
quenched using the Fieser method [13], dried over Na2SO4, and filtered. The filtered solution
was reduced in vacuo to give the product as a brown oil.
2.3 Measurement of IC50
In 96-well plates, RHuAChE activity against AtCh was measured in the presence of the indicated
compound at concentrations ranging from 60 nM to125 mM. Maximum activity (100%) was
determined using a negative control containing enzyme alone, and all activities were compared
relative to this control. The resulting activities were plotted against the log value of the
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compound concentration and fit using a variable slope dose-response curve (Prism, GraphPad
Software, Inc., La Jolla, CA).
2.4 Inhibition
To determine the type of inhibition displayed by ADOC, the activity of RHuAChE was
measured in the presence of several concentrations of that compound at varying concentrations
of AtCh. Inverse velocities were plotted according to the inverse AtCh concentration and fit
using a linear regression analysis (Prism, GraphPad Software, Inc., La Jolla, CA) to determine
the nature of the inhibition [11].
2.5 Reactivation
RHuAChE was inhibited with a molar excess of nerve agent for a minimum of 10 min at room
temperature. Excess free nerve agent was removed using Centri-Sep spin columns (Life
Technologies, Grand Island, NY), and the inhibited sample was diluted to an appropriate
concentration for use in further assays. An uninhibited positive control was similarly treated
using buffer in place of nerve agent. Enzyme samples were mixed with reactivator compound to
initiate reactivation, and aliquots were removed at various time points, diluted twenty-fold and
measured for activity against AtCh. Percent reactivation was calculated by comparing each
sample to a row matched positive control of uninhibited RHuAChE in the presence of
reactivator. Residual activity at time zero was subtracted from each following time point to
account for incomplete inhibition, and then triplicates were averaged. Nonlinear regression
assuming maximal reactivation of 100% was used to determine kobs [14, 15] at each reactivator
concentration. These kobs values were plotted against reactivator concentration and fit using a
modified Michaelis-Menten equation [16]. Note that the combination of a lower pH and a lower
temperature than used in the previous studies referenced here will resulted in slower reactivation
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rates than previously reported. In some experiments, the half-time of reactivation (t1/2) was
calculated for multiple compounds at a single concentration (1 mM) using a one-phase
association fit of the percent reactivation over time (Prism, GraphPad Software, Inc., La Jolla,
CA), and these times were then compared to the spontaneous reactivation half-time of sample-
matched inhibited enzyme using a ratio t-test.
2.6 In Vivo Experimental Procedure
All testing was done as previously described in Shih et al. [12]. In short, blood was collected at
least 24 hours prior to experimentation from male Hartley guinea pigs (250 – 330 g) from
Charles River Labs (Kingston, NY). These samples were used to establish baseline
acetylcholinesterase activity in red blood cells and whole blood for each animal using a
modification of the assay described by Ellman et al. [17]. On the study day, each animal received
a pretreatment of atropine methyl nitrate (2 mg/kg) intramuscularly followed fifteen minutes
later with exposure to a 1.0 LD50 dose of sarin (GB), cyclosarin (GF), VX, or saline via
subcutaneous injection. Fifteen minutes later, when the inhibition of acetylcholinesterase activity
by these nerve agents reached maximum levels in the blood, one of 5 doses of ADOC (1.8, 10.0,
18.0, 32.0, and 56.0 mg/kg) or saline was administered intramuscularly. One hour after nerve
agent exposure and 45 minutes after reactivator administration, animals were humanely
euthanized, and all samples were collected and stored for later analysis.
3. Results
Initially, ADOC was tested in vitro for reactivation potential with recombinant human
acetylcholinesterase (RHuAChE) inhibited by sarin (GB) or VX (data not shown). While
reactivation was noted in both cases, full reactivation to uninhibited RHuAChE activity levels
was not achieved under the conditions tested. To determine if the lack of complete reactivation
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was due to direct inhibition of the enzyme by ADOC, IC50 values for ADOC (17.8 µM) and
2PAM (456 µM) were determined at a fixed concentration of RHuAChE (Figure 2A). Because
the high level of inhibition displayed by ADOC could interfere with further in vitro testing of
reactivation using established discontinuous, indirect methods, subsequent assays were
conducted to determine the type of inhibition caused by ADOC (Figure 2B).
B)
A)
1500
1000
500
0
100
90
80
70
60
50
40
30
20
10
0
2PAM = 456µM
ADOC = 17.8µM
-2
2
4
-500
-4 -3 -2 -1
0
1
2
1/[AtCh] mM
Log[Reactivator] (mM)
Figure 2. A) IC50 of ADOC(●) and 2PAM (
▲
) was determined with human recombinant
acetylcholinesterase (RHuAChE). B) Inhibition assay using ADOC in increasing concentrations
as the inhibitor and AtCh as the competitive substrate.
ADOC displayed a mixture of competitive (Ki = 6.3±0.9 µM) and noncompetitive (Ki’ =
37.4±2.1 µM) reversible inhibition, indicating the possibility of two binding sites in agreement
with the findings of Katz et al. [11]. Given these results, the inhibition of RHuAChE by
reactivator molecules was accounted for in all subsequent experiments, where the activity of the
enzyme in the presence of each concentration of each reactivator (absent any OP) was used as
the baseline activity level for the corresponding OP-inhibited sample. This was crucial to
account for the experimental parameters used to test the reactivation potential of ADOC against
nerve agent-inhibited RHuAChE.
Under these controlled conditions, the kinetic parameters of reactivation by ADOC and 2PAM
of RHuAChE inhibited by various OP nerve agents were determined using established
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reactivation methods (Figure 3) [16, 18-21]. In the cases of GB, GF, VX, and VR the rate of
reactivation (kr, Scheme 1) by ADOC (0.34±0.14 min-1, 0.13±0.03 min-1, 0.50±0.20 min-1,
4.01±2.37 min-1, respectively) was greater than that mediated by 2PAM (0.06±0.02 min-1,
0.04±0.02 min-1, 0.06±0.01 min-1, 0.06±0.04 min-1, respectively). However, the dissociation
constant (KD) of ADOC (7.61±4.07 mM, 4.45±3.37 mM, 2.08±0.94 mM, 5.46±2.02 mM
respectively) relative to 2PAM (0.71±0.13 mM, 2.43±0.65 mM, 0.54±0.15 mM, 1.34±0.79 mM
respectively) was similarly elevated. The overall rate of reactivation (kr/KD = kr2) by ADOC was
thus only significantly increased relative to 2PAM for reactivation of RHuAChE inhibited by VR
(Table 1). Of note, no evidence for cooperative behavior (as previously described during
reactivation [11]) was observed under these experimental conditions, suggesting that the
cooperativity reported before may be a condition-specific phenomenon related to the methods
used in the prior study.
Scheme 1. Reactivation from Worek et al. [19].
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8
6
4
*
2PAM
ADOC
2PAM
ADOC
15000
10000
5000
0
*
1.0
0.8
0.6
0.4
0.2
0.0
*
*
*
*
*
Figure 3. Rates (kr, min-1) and dissociation constants (KD, µM) of the reactivation of inhibited
RHuAChE by 2PAM (black) and ADOC (grey). Significant differences between 2PAM and
ADOC values are indicated by *(t-test, p<0.05).
Table 1. Overall reactivation rate for indicated agents and
reactivators.
Reactivator
kr2 (M-1 min-1)
GD GF
7.0 81.0 17.6 18.4 121
GA GB
VX
VR
46
±1.2
2-PAM
ADOC
±4.4
±16
±16
±4.2
±29
4.5 45.4 5.6
40.5 266 719*
±3.3
±7.2
±4.1
±29
±109
±386
Standard deviation of three independent experiments is indicated below
value. Statistically significant increase of reactivation rate by ADOC as
compared to 2PAM is indicated by *(t-test, p<0.05).
In an effort to expand upon the proposed mechanism of reactivation of ADOC [11], an initial
structure-activity relationship study was conducted using several ADOC structural analogs. The
importance of each substituent of the benzyl ring for reactivation of OP nerve agent-inhibited
enzyme was explored (Table 2). The removal of the aniline amine group (2) or the benzyl amine
group (3) caused a decrease in the magnitude of reactivation as compared to ADOC but also
substantially decreased the inhibitory effects on native RHuAChE. These combined results could
indicate that these substituents play a role in binding rather than reactivation chemistry,
especially in light of the fact that removal of the phenol substituent (4) obliterates all reactivation
functionality. Similarly, blocking the phenol as an ether (5) causes the loss of reactivation
functionality against all but one agent (GB), for which the function is greatly decreased as
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compared to ADOC. Taken together, these results indicate that the phenol substituent is essential
for reactivation.
Table 2. Initial Exploration of Active Moieties. Ratio
(Spontaneous/compound) of half-times (t1/2) of reactivation for
compounds (1mM) with RHuAChE after inhibition by the indicated agent.
Inhibition
of
RHuAChE
GB
GF
VX
VR
@ 1mM
Spontaneous
Reactivation
(t1/2 in days)
2.01
1.25
1.18
0.35
(±0.07)
(±0.14)
(±0.21)
(±0.07)
OH
14,723*
361*
164*
1,069*
N
60%
1
2
3
4
(6.46-
(158-824)
(97.3-277)
(393.6-2911)
(±1)
33,573,761)
NH2
OH
1.51*
1.54*
1.87*
3.65*
0%
(1.12-2.03)
(1.34-1.78)
(1.24-2.84)
(3.14-4.24)
(±6)
2.24*
1.48*
2.24*
14.3*
0%
(1.98-2.53)
(1.15-1.91)
(1.16-4.32)
(8.86-23.0)
(±6)
NH2
NH2
N
0%
0.98
0.93
0.97
0.96
(0.81-1.20)
(0.89-0.97)
(0.66-1.43)
(0.61-1.51)
(±6)
1.54*
0.89
1.11
1.25
5%
5
(1.28-1.84)
(0.77-1.03)
(0.53-2.34)
(0.90-1.73)
(±4)
* - indicates that t1/2 in presence of compound is significantly less than spontaneous using a ratio
t-test with p<0.05.
Standard deviation of at least three independent experiments is indicated for spontaneous
reactivation half-times in days. Ninety-five percent confidence intervals of ratios are indicated
below each value.
All half-times were determined using RHuAChE in the presence of 1 mM compound as the
positive control. The far right column indicates the level of inhibition of RHuAChE caused
directly by the compound at 1 mM when compared to a buffer only control of RHuAChE at
the same concentration. Standard deviation of this value determined using controls from all
independent experiments is indicated.
Given that the removal of the benzyl amine (3) had a slightly less deleterious effect on the
reactivation potential of ADOC than removal of the aniline amine (2), additional analogs of
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ADOC were used to probe the importance of this moiety (Table 3). Changing the relative
position of the benzylic diethylamine on the ring (6) reduced the reactivation potential of the
molecule but also nearly abolished inhibitory potential against naive RHuAChE. Interestingly,
reducing the bulk of the benzyl amine (7) greatly increased the inhibitory potential of the
molecule against naive RHuAChE; however, this affect could be negated by altering the relative
position of this smaller benzyl amine (8) to a configuration similar to that of 6. In the hopes of
counteracting the inhibitory effects of 7, an analog in which the benzyl amine bulk had been
increased (9) was tested and, surprisingly, was found to also have increased inhibitory potential
against naïve RHuAChE. Further increase in the bulk of the benzyl amine (10) resulted in loss of
inhibitory potential and reactivation potential, most likely from sterically driven decreased
binding of the small molecule. The inhibition and reactivation results of 9 and 10 offer some
insight into the mechanism of reactivation by helping to rule out the likelihood that quinone
methide formation is involved since these compounds would be expected to form a common
quinone methide intermediate with ADOC if this mechanism were operative [22]. Given all of
these results and the high pKa (~8 to 9) of the benzyl amine, it seems likely that this portion of
the molecule contributes mainly to the binding strength and selectivity of ADOC rather than to
the reactivation functionality of the molecule.
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Table 3. Exploring the Role of the Benzylamine Group. Ratio
(spontaneous/compound) of half-times (t1/2) of reactivation for compounds
(1mM) with RHuAChE after inhibition by the indicated agent.
Inhibition
of
RHuAChE
GB
GF
VX
VR
@ 1mM
14,723*
361*
164*
1,069*
60%
1
6
7
8
9
(6.46-
(158-824)
(97.3-277)
(393.6-2911)
(±1)
33,573,761)
1.97*
1.80*
2.76*
4.06*
1%
(1.54-2.52)
(1.46-2.22)
(1.95-3.93)
(3.01-5.49)
(±4)
95%
---
---
---
---
(±1)
2.13*
1.96*
2.72*
5.65*
18%
(1.63-2.78)
(1.92-2.01)
(2.12-3.48)
(1.20-26.5)
(±6)
94%
---
---
---
---
(±1)
9%
0.88
0.74
1.09
0.93
10
(0.65-1.20)
(0.60-0.90)
(0.60-1.98)
(0.76-1.14)
(±4)
---Reactivation could not be determined because of high level of inhibition by compound.
* - indicates that t1/2 in presence of compound is significantly less than spontaneous using a ratio t-test
with p<0.05.
Additional studies were conducted to analyze the contribution of the aniline amine substituent
to the reactivity of ADOC (Table 4). The role of the electron donating potential of the aniline
amine group was explored by both increasing (11) and decreasing (12) the electron donating
potential as guided by the reported Hammett values for these substituents [23]. While both of
these modifications displayed significantly increased reactivation compared to spontaneous
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levels for all agents, reactivation was markedly decreased compared to that of ADOC.
Additionally, the alteration of the aniline amine in both cases abolished all inhibition of
RHuAChE, raising the question of whether the decreased reactivation activity of these molecules
was a result of decreased binding or decreased reactivation rate. Determination of the
dissociation constant (KD; 11: 0.39±0.23mM; 12: 0.92±0.12mM) and reactivation rate (kr; 11:
0.07±0.02min-1; 12: 0.03±0.01min-1) for each compound supports the conclusion that the loss of
reactivation potential is due to a slowed reactivation rate rather than a decrease in binding.
Given these data, compounds that could still take advantage of the inductive electron donating
affect but were more sterically hindered about the phenol (13 and 14) were tested for reactivation
potential. While both of these compounds retained reactivation potential above that of
spontaneous rates for some agents, reactivation rates in general were greatly decreased compared
to ADOC. Taken together, the results suggest that while the para relationship of the aniline
amine relative to the phenol is important, this relationship may have more to do with positioning
the phenol upon binding to the inhibited AChE enzyme than to any inductive activation effects.
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Table 4. Exploring the Role of the Aniline Amine Group. Ratio (spontaneous/compound)
of half-times (t1/2) of reactivation for compounds (1mM) with RHuAChE after inhibition
by the indicated agent.
Inhibition
of
Hammett
RHuAChE Values[23]
GB
GF
VX
VR
@ 1mM
(σp)
OH
361*
164*
1,069*
14,723*
N
60%
(± 1)
-0.66
1
(158-824)
(97.3-277)
(393.6-2911)
(6.46-33,573,761)
NH2
6.15*
3.07*
16.5*
32.7*
0%
-0.83
0.00
11
(4.98-7.60)
(2.92-3.23)
(13.0-20.8)
(24.1-44.4)
(± 6)
OH
N
3.85*
1.72*
6.95*
19.0*
0%
12
(3.22-4.61)
(1.67-1.77)
(5.03-9.59)
(15.2-23.7)
(± 5)
HN
O
1.43*
1.09
1.24
1.65*
35%
N/A
N/A
13
14
(1.37-1.50)
(0.80-1.49)
(0.99-1.56)
(1.54-1.78)
(± 4)
1.70*
1.30
1.61*
1.59*
43%
(1.40-2.06)
(0.90-1.89)
(1.17-2.21)
(1.35-1.87)
(± 11)
* - indicates that t1/2 in presence of compound is significantly less than spontaneous using a ratio t-test with p<0.05.
Finally, ADOC was used in in vivo experiments to evaluate the compound as a potential
therapeutic for nerve agent intoxication. Utilizing methodology previously developed to quantify
the level of cholinesterase reactivation in guinea pigs after exposure to nerve agents [12], the
dose-dependent reactivation potential of ADOC in red blood cells, whole blood, brainstem,
cerebellum, cortex, mid-brain, hippocampus, striatum , spinal cord, diaphragm, heart and skeletal
muscle from animals exposed to nerve agents was tested. While a small dose-dependent
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reactivation effect was noted in the red blood cells and whole blood after exposure to VX and
GB, similar responses were not noted in any other tissue samples (Figure 4). In fact, no
significant reactivation against any agent after administration of any dose of ADOC was noted in
the tissue samples tested, suggesting that ADOC may not readily leave the bloodstream or cross
the blood brain barrier.
30
30
A)
B)
20
10
0
20
10
0
0
20
40
60
0
20
40
60
ADOC (mg/kg)
ADOC (mg/kg)
Figure 4. Dose-Dependence of In Vivo Reactivation by ADOC. A) Reactivation of red blood cell
acetylcholinesterase by ADOC administered at doses of 1.8, 10.0, 18.0, 32.0, or 56.0 mg/kg to
animals exposed to 1xLD50 GB (▲), GF (□) or VX (●). Whole blood reactivation displayed
similar trends, though absolute reactivation values differed (data not shown). B) Reactivation of
mid-brain tissue acetylcholinesterase by ADOC administered at the indicated doses to animals
exposed to GB (▲), GF (□) or VX (●). All other tissues tested (brainstem, cerebellum, cortex,
hippocampus, striatum, spinal cord, diaphragm, heart and skeletal muscle) displayed similar
reactivation trends, though absolute reactivation values differed (data not shown).
4. Discussion
To evaluate the IC50 values in a clinically relevant manner, 2PAM is administered at a dose of
145 µmol/kg [12], which translates to a maximum concentration of ~2 mM in human blood
plasma, assuming that a 70 kg adult has 5 L of blood and the full dose of administered drug
reaches the blood stream. A recent study showed that in fact only a maximum 2.5% to 3% of
2PAM can be detected in the plasma after intramuscular injection [24], which would translate to
a plasma concentration of approximately 60 µM after administration of three autoinjector doses
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[12], the current recommended emergency treatment (Mark I Nerve Agent Antidote Kit) for a
patient displaying symptoms of nerve agent exposure. While 2PAM at the above concentration
does not cause toxic effects via acetylcholinesterase inhibition, supported by the IC50 determined
in this study (456 µM), ADOC (IC50 = 17.8 µM) administered at a similar dose could result in
toxic effects from the direct inhibition of acetylcholinesterase.
While the lack of apparent reactivation of AChE by ADOC in the tissues of OP-exposed
guinea pigs was somewhat disappointing, several factors affecting in vivo reactivation can be
explored. First, the pharmacokinetic properties of ADOC were not specifically tested and, as the
compound is structurally different from the pyridinium oximes upon which the experimental
paradigm was developed, this lack of knowledge could have led to the compound being
administered at a time or via a route that was not conducive to maximal effects. Second, ADOC
is possibly not as potent a reactivator of guinea pig acetylcholinesterase as it is of the human
form of the enzyme. Both of these points could be explored by further testing both in vivo and in
vitro, including refining the animal testing parameters.
Despite the lack of evidence supporting the in vivo protective efficacy of ADOC as a
treatment for nerve agent intoxication, full characterization of the mechanism of reactivation of
this small molecule, which has in vitro reactivation effects on par with 2PAM, could lead to the
development of a new class of nerve agent intoxication treatments. The use of ADOC analogs to
explore the mechanism yielded insight into the roles of the ring substituents with both the
benzylic diethylamine and the aniline amine contributing to the binding and specificity of
ADOC, while only the phenol group appears to be critical for reactivation. Further work to
characterize the product of reactivation could lead to a definitive answer as to whether the phenol
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is essential for a direct nucleophilic attack of the phosphonylated active site or whether ADOC
mediates an indirect action via a water molecule as was postulated by Katz et al. [11].
In conclusion, while ADOC may not prove to be an ideal drug candidate to address the issue
of nerve agent intoxication, the characterization of ADOC and several analogs here can be used
to inform the design of a new class of reactivators that do not rely upon an oxime as the primary
reaction group for the regeneration of functional AChE after inhibition by organophosphorus
nerve agents.
Conflict of Interest
None.
Acknowledgements
The views expressed in this manuscript are those of the author(s) and do not reflect the official
policy of the Department of Army, Department of Defense, or the U.S. Government.
The experimental protocol was approved by the Animal Care and Use Committee at the United
States Army Medical Research Institute of Chemical Defense and all procedures were conducted
in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals
(National Research Council, 2011), and the Animal Welfare Act of 1966 (P.L. 89-544), as
amended.
This research was supported by the Defense Threat Reduction Agency – Joint Science and
Technology Office, Medical S&T Division.
C.L.C. and J.A.K. were supported in part by an appointment to the Internship/Research
Participation Program for the US Army Medical Research Institute of Chemical Defense,
administered by the Oak Ridge Institute for Science and Education through an agreement
between the US Department of Energy and the USAMRICD.
References
1. Dunn, M. A. & Sidell, F. R. (1989) Progress in medical defense against nerve agents, Jama.
262, 649-52.
2. Millard, C. B., Kryger, G., Ordentlich, A., Greenblatt, H. M., Harel, M., Raves, M. L., Segall,
Y., Barak, D., Shafferman, A., Silman, I. & Sussman, J. L. (1999) Crystal structures of aged
phosphonylated acetylcholinesterase: nerve agent reaction products at the atomic level,
Biochemistry. 38, 7032-9.
20

ACCEPTED MANUSCRIPT
3. Skovira, J. W., O'Donnell, J. C., Koplovitz, I., Kan, R. K., McDonough, J. H. & Shih, T. M.
(2010) Reactivation of brain acetylcholinesterase by monoisonitrosoacetone increases the
therapeutic efficacy against nerve agents in guinea pigs, Chem Biol Interact. 187, 318-24.
4. Rosman, Y., Eisenkraft, A., Milk, N., Shiyovich, A., Ophir, N., Shrot, S., Kreiss, Y. &
Kassirer, M. (2014) Lessons learned from the Syrian sarin attack: evaluation of a clinical
syndrome through social media, Annals of internal medicine. 160, 644-8.
5. Smithson, A. & Levy, L. (2000) Ataxia: The Chemical and Biological Terrorism Threat and
the US Response, Henry L Stimson Centre, Washington, DC.
6. Masson, P. (2011) Evolution of and perspectives on therapeutic approaches to nerve agent
poisoning, Toxicol Lett. 206, 5-13.
7. Ashani, Y. & Cohen, S. (1971) Nucleophilicity of some reactivators of phosphorylated
acetylcholinesterase, Journal of medicinal chemistry. 14, 621-6.
8. Hagedorn, I., Gundel, W. H. & Schoene, K. (1969) [Reactivation of phosphorylated
acetylcholine esterase with oximes: contribution to the study of the reaction course],
Arzneimittel-Forschung. 19, 603-6.
9. Davies, D. R. & Green, A. L. (1956) The kinetics of reactivation, by oximes, of cholinesterase
inhibited by organophosphorus compounds, The Biochemical journal. 63, 529-35.
10. Worek, F. & Thiermann, H. (2011) Reactivation of organophosphate-inhibited human
acetylcholinesterase by isonitrosoacetone (MINA): a kinetic analysis, Chem Biol Interact. 194,
91-6.
11. Katz, F. S., Pecic, S., Tran, T. H., Trakht, I., Schneider, L., Zhu, Z., Ton-That, L., Luzac, M.,
Zlatanic, V., Damera, S., Macdonald, J., Landry, D. W., Tong, L. & Stojanovic, M. N. (2015)
Discovery of New Classes of Compounds that Reactivate Acetylcholinesterase Inhibited by
Organophosphates, Chembiochem.
12. Shih, T. M., Skovira, J. W., O'Donnell, J. C. & McDonough, J. H. (2009) Evaluation of nine
oximes on in vivo reactivation of blood, brain, and tissue cholinesterase activity inhibited by
organophosphorus nerve agents at lethal dose, Toxicol Mech Methods. 19, 386-400.
13. Ho, T.-L., Fieser, M., Fieser, L., Danheiser, R., Roush, W., Fieser, L. F., Fieser, M. & Ho,
T.-L. (2006) Lithium aluminum hydride in Fieser and Fieser's Reagents for Organic Synthesis,
John Wiley & Sons, Inc.
14. Worek, F., Wille, T., Aurbek, N., Eyer, P. & Thiermann, H. (2010) Reactivation of
organophosphate-inhibited human, Cynomolgus monkey, swine and guinea pig
acetylcholinesterase by MMB-4: a modified kinetic approach, Toxicol Appl Pharmacol. 249,
231-7.
15. Sit, R. K., Radic, Z., Gerardi, V., Zhang, L., Garcia, E., Katalinic, M., Amitai, G., Kovarik,
Z., Fokin, V. V., Sharpless, K. B. & Taylor, P. (2011) New structural scaffolds for centrally
acting oxime reactivators of phosphylated cholinesterases, J Biol Chem. 286, 19422-30.
16. Aurbek, N., Thiermann, H., Szinicz, L., Eyer, P. & Worek, F. (2006) Analysis of inhibition,
reactivation and aging kinetics of highly toxic organophosphorus compounds with human and
pig acetylcholinesterase, Toxicology. 224, 91-9.
17. Ellman, G., Courtney, K., V Andres, J. & Featherstone, R. (1961) A New and Rapid
Colorimetric Determination of Acetylcholinesterase Activity, Biochemical Pharmacology. 7, 88-
95.
18. Worek, F., Reiter, G., Eyer, P. & Szinicz, L. (2002) Reactivation kinetics of
acetylcholinesterase from different species inhibited by highly toxic organophosphates, Arch
Toxicol. 76, 523-9.
21

ACCEPTED MANUSCRIPT
19. Worek, F., Thiermann, H., Szinicz, L. & Eyer, P. (2004) Kinetic analysis of interactions
between human acetylcholinesterase, structurally different organophosphorus compounds and
oximes, Biochem Pharmacol. 68, 2237-48.
20. Worek, F., Szinicz, L. & Thiermann, H. (2005) Estimation of oxime efficacy in nerve agent
poisoning: a kinetic approach, Chem Biol Interact. 157-158, 349-52.
21. Worek, F., Eyer, P., Aurbek, N., Szinicz, L. & Thiermann, H. (2007) Recent advances in
evaluation of oxime efficacy in nerve agent poisoning by in vitro analysis, Toxicol Appl
Pharmacol. 219, 226-34.
22. Ercegovic, S. M. (2011) Synthesis and Characterization of Quinone Methide Precursors as
Acetylcholinesterase Reactivators, Ohio State University.
23. Hansch, C., Leo, A. & Taft, R. W. (1991) A survey of Hammett substituent constants and
resonance and field parameters, Chem Rev. 91, 165-195.
24. Abbara, C., Rousseau, J., Lelièvre, B., Turcant, A., Lallement, G., Ferec, S., Bardot, I. &
Diquet, B. (2010) Pharmacokinetic analysis of pralidoxime after its intramuscular injection alone
or in combination with atropine-avizafone in healthy volunteers, British Journal of
Pharmacology. 161, 1857-1867.
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•
•
•
•
Non-oxime reactivator for nerve agent-inhibited acetylcholinesterase is evaluated.
ADOC reactivation efficiency similar to or better than 2PAM for all agents tested.
ADOC mechanism of reactivation probed and phenol found to be essential.
ADOC did not provide in vivo efficacy against any nerve agents in guinea pigs.