Demonstration of in Vitro Resurrection of Aged Acetylcholinesterase after Exposure to Organophosphorus Chemical Nerve Agents

Article abstract of DOI:10.1021/acs.jmedchem.7b01620

After the inhibition of acetylcholinesterase (AChE) by organophosphorus (OP) nerve agents, a dealkylation reaction of the phosphylated serine, referred to as aging, can occur. When aged, known reactivators of OP-inhibited AChE are no longer effective. Realkylation of aged AChE may provide a route to reversing aging. We designed and synthesized a library of quinone methide precursors (QMPs) as proposed realkylators of aged AChE. Our lead compound (C8) from an in vitro screen successfully resurrected 32.7 and 20.4% of the activity of methylphosphonate-aged and isopropyl phosphate-aged electric-eel AChE, respectively, after 4 days. C8 displays properties of both resurrection (recovery from the aged to the native state) and reactivation (recovery from the inhibited to the native state). Resurrection of methylphosphonate-aged AChE by C8 was significantly pH-dependent, recovering 21% of activity at 4 mM and pH 9 after only 1 day. C8 is also effective against isopropyl phosphate-aged human AChE.

Full text of DOI:10.1021/acs.jmedchem.7b01620

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Article
Demonstration of in vitro Resurrection of Aged Acetylcholinesterase
after Exposure to Organophosphorus Chemical Nerve Agents
Qinggeng Zhuang, Andrew J Franjesevic, Thomas S Corrigan, William H Coldren,
Rachel Dicken, Sydney Sillart, Ashley DeYong, Nathan Yoshino, Justin Smith, Stephanie
Fabry, Keegan Fitzpatrick, Travis G. Blanton, Jojo Joseph, Ryan J Yoder, Craig
A McElroy, Ozlem Dogan Ekici, Christopher S Callam, and Christopher M. Hadad
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01620 • Publication Date (Web): 05 Jun 2018
Downloaded from http://pubs.acs.org on June 6, 2018
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Demonstration of in vitro Resurrection of Aged Acetylcholinesterase
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after Exposure to Organophosphorus Chemical Nerve Agents
Qinggeng Zhuang1, Andrew J. Franjesevic1, Thomas S. Corrigan1, William H. Coldren1, Rachel
Dicken1, Sydney Sillart1, Ashley DeYong1, Nathan Yoshino1, Justin Smith1, Stephanie Fabry1, Kee-
gan Fitzpatrick1, Travis G. Blanton2, Jojo Joseph1, Ryan J. Yoder2, Craig A. McElroy3, Özlem
Dogan Ekici4, Christopher S. Callam1, Christopher M. Hadad1*
1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210.
2Department of Chemistry and Biochemistry, The Ohio State University–Marion, Marion, Ohio 43302.
3College of Pharmacy, The Ohio State University, Columbus, Ohio 43210.
4Department of Chemistry and Biochemistry, The Ohio State University–Newark, Newark, Ohio 43055.
ABSTRACT: After inhibition of acetylcholinesterase (AChE) by organophosphorus (OP) nerve agents, a dealkylation reac-
tion, referred to as aging, of the phosphylated serine can occur. When aged, known reactivators of OP-inhibited AChE are
no longer effective. Realkylation of aged AChE may provide a route to reverse aging. We designed and synthesized a li-
brary of quinone methide precursors (QMPs) as proposed realkylators of aged AChE. Our lead compound (C8) from an in
vitro screening, successfully resurrected 32.7% and 20.4% of the activity of methylphosphonate-aged and isopropyl phos-
phate-aged electric eel AChE, respectively, after 4 days. C8 displays properties of both resurrection (recovery from the
aged to the native state) and reactivation (recovery from the inhibited to the native state). Resurrection of
methylphosphonate-aged AChE by C8 was significantly pH-dependent, recovering 21% of activity at 4 mM and pH 9 after
only 1 day. C8 is also effective against isopropyl phosphate-aged human AChE.
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Introduction
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Acetylcholinesterase (AChE) is a serine hydrolase found in brain synapses, neuromuscular junctions and erythrocytes.
AChE selectively hydrolyzes the neurotransmitter acetylcholine with its Glu-His-Ser catalytic triad. Organophosphorus
(OP) compounds phosphylate the catalytic serine of AChE, and inhibition of AChE results in the accumulation of acetyl-
choline. OP exposure may lead to death due to seizures or respiratory failure caused by persistent nerve impulses.1-3 Thus,
OPs are toxic and have been used as pesticides and chemical warfare agents. Every year OP-based pesticides kill approxi-
mately 200,000 people worldwide, especially in rural areas of developing countries.1 OP-based chemical warfare agents
have been used in armed conflicts such as the Iran-Iraq war4 and the recent Syrian civil war,5 as well as terrorist attacks
such as the Tokyo subway sarin attack.6 OP-inhibited AChE can be reactivated by oximes (more likely, their deprotonated
form, the oximates), which nucleophilically substitute the phosphylated serine in the active site.7
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Exposure of AChE to OP compounds is complicated by an aging process in which loss of the alkyl side chain of the phos-
phylated serine produces an oxyanion of OP-poisoned AChE.8,9 Oximes, such as 2-pralidoxime (2-PAM), are ineffective
against aged AChE. Some OP compounds, such as soman, with an aging half-time (t1/2) of only several minutes, provide
only a minimal chance for medical treatment.10 After decades of research, no clinical treatment has been developed to
resurrect aged AChE.
To reverse aging, realkylation of the phosphylated oxyanion has been proposed as a strategy against this dealkylation pro-
cess,11 including unsuccessful efforts from 1970 by Steinberg et al.12 as well as more recent efforts led by Quinn.13 With the
negative charge on the phosphylated serine being neutralized by some sort of electrophilic realkylation process, oximes
should reactivate AChE again. Several types of electrophilic alkylating agents including sulfonates,11 haloketones,12 sul-
foniums,14 and methoxypyridiniums13 were evaluated as potential AChE realkylators. Recently, Khavrutskii and Wallqvist
suggested the in silico possibility to resurrect aged AChE by β-aminoalcohols, by a direct process without proceeding
through a realkylation event.15 However, prior to this report, no experimental evidence has been reported for the efficacy
of any drug to realkylate aged AChE to the best of our knowledge.16 Herein, we report the first family of compounds that
demonstrate in vitro efficacy.
Quinone methides (QMs, SCHEME 1a) can be regarded as carbocations stabilized by resonance delocalization. Quinone
methide precursors (QMPs, SCHEME 1a) are derivatives of QMs with a leaving group attached to the partially positively
charged carbon. QMPs can be attacked by nucleophiles either directly via SN2 substitution, or via the corresponding QMs
as reactive intermediates (SCHEME 1b). Protein and nucleic acid alkylation by QMPs has been reported for years.17-22
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Phosphodiesters and dibutyl phosphate, which structurally resemble the phosphyl group of aged AChE, have been suc-
cessfully alkylated by QMs, implying the possibility to realkylate and resurrect aged AChE with QMPs and QMs.22-24
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Herein we report a series of QMPs intended to be realkylators of aged AChE. Guided by in silico studies, a library of can-
didate compounds was synthesized. Their activities were characterized by Ellman’s assay, providing up to 32.7% in vitro
resurrection of electric eel AChE (eeAChE) for a methylphosphonate and up to 20.4% for a phosphate after 4 days. Resur-
rection of AChE was confirmed by bottom-up proteomics.
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SCHEME 1 Structures and substitution reaction of QMs and QMPs. (a) Typical structures of QMs (top) and QMPs (bot-
tom). (b) Nucleophiles can substitute the leaving group of QMP by either an SN2 reaction or formation of the correspond-
ing QM.
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Results
Computation-guided selection of realkylator candidates
We previously conducted molecular docking and molecular dynamics (MD) simulations to evaluate the potential orienta-
tion of QMPs at the active site of methylphosphonate-aged human AChE (huAChE).25 In this work a larger library of
QMPs was studied through this modeling approach using an in silico model of aged huAChE.
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FIGURE 1. A snapshot obtained from a 1 ns MD simulation, demonstrating a protonated QMP (shown in cyan, later called
C8) near the active site of aged AChE (wall-eyed stereo). The electrophilic carbon is ~4.2 Å from the phosphylated oxyan-
ion. Hydrogen bonds with short contact distances are shown (green dashed lines).
SCHEME 2 Structures of realkylator candidates
We determined that pyridyl compounds had a higher propensity to be bound in the active site and close to the phosphyl
oxyanion as compared to their phenyl analogues.25 Moreover, 3-hydroxypyridine-derived QMPs with the reactive benzylic
carbon attached at the 2-position displayed promising interactions. Of the 72 compounds modeled, six of the top-10 com-
pounds (FIGURE S2) were members of that specific 3-hydroxypyridine framework. The top compound had a pyrrolidine
leaving group attached to the reactive benzylic carbon (FIGURE 1).
Thirteen 3-hydroxypyridine-derived QMPs (C series, SCHEME 2) were thus synthesized via Mannich reactions, and then
evaluated by screening against aged AChE. The electrophilic benzylic methylene, hypothesized to be the site of attack by
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nucleophiles (SCHEME 1b), is attached to the 2-position of the pyridine ring, and the leaving groups were various second-
ary amines.
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Screening of Realkylator Library
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Two representative OPs (SCHEME 3) were used. PiMP (a pinacolyl methylphosphonate ester), a soman analogue, was
synthesized as reported by Amitai et al.26 The resulting methylphosphonate-aged AChE is the aging product of any
methylphosphonate nerve agent (e.g. sarin, soman, VX as well as many other V-agents and G-agents except for tabun
(GA)). We also used the commercially available pesticide DFP (diisopropyl fluorophosphate) to evaluate a phosphate at
the serine residue (SCHEME 3). We chose eeAChE as the target enzyme of these studies, considering its commercial
availability and affordable cost.
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SCHEME 3 Structures of OPs used for aging of AChE and the corresponding aged adducts
The aged enzyme was reacted with various concentrations (0.2 – 4 mM) of each QMP at 37°C, pH 8 for 1 d. Without
knowing the re-aging t1/2 of the possible realkylated adducts, NH4F (4 mM) was added as a mild and nonselective reactiva-
tor.27-30 We posited that once the aged AChE was realkylated, the newly formed “inhibited” AChE could be reactivated by
fluoride to the native AChE. The samples were eventually treated with 4 mM of 2-PAM for 1 h to ensure that any realkyl-
ated AChE was completely reactivated. Ellman’s assay,31 with acetylthiocholine as a substrate, was carried out to deter-
mine AChE activity. Three controls were prepared and analyzed in parallel: a negative control without realkylator; a 2-
PAM control with the realkylator replaced by 2-PAM; and a positive control with native AChE, rather than aged, and
without realkylator.
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FIGURE 2. Screening of three concentrations of various C series QMPs against (a) methylphosphonate-aged eeAChE and
(b) isopropyl phosphate-aged eeAChE. The horizontal solid and the dashed lines mark the negative controls and 2-PAM
controls, respectively. The error bars reflect standard deviations from four replicate efforts.
The screening results are shown in FIGURE 2a. The percentage relative activity is based on the positive control. The nega-
tive and 2-PAM controls showed negligible background signals, confirming the completion of aging (solid and dashed
horizontal lines in FIGURES 2). For this small library, the only difference was the amino leaving group. Compound C2
with an N-(methyl)ethylamino leaving group showed the highest efficacy among C1 to C7, with each of these compounds
having a noncyclic amine. Efficacy was compromised when the N-alkyl groups were shortened (C1 vs C2), lengthened (C3,
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C5 and C7 vs C2), or branched (C4 vs C2, and C6 vs C3). Overall, the pyrrolidinyl compound C8 was the most potent can-
didate. The other candidates with cyclic leaving groups showed lower or even no activity. The screening against isopropyl
phosphate-aged AChE (DFP-treated) showed a similar result (FIGURE 2b), but the trend of varying activity with the N-
alkyl groups was less obvious.
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These comparisons demonstrate that pyrrolidine is the optimum among all tested leaving groups. Hence we compared a
variety of realkylator candidates with the pyrrolidinyl leaving group (D series in SCHEME 2). The screenings were carried
out following the same procedures as for the 3-hydroxypyridine derivatives (C series). C8 and unsubstituted 3-
hydroxypyridine were used for comparison. As shown in FIGURE S10, D5 is the only active compound of these candidate
compounds. D5 is active only against isopropyl phosphate-aged AChE, and is less active than C8. This observation indi-
cates the superiority of the 3-hydroxypyridine framework over other tested scaffolds. C8 was therefore chosen as a lead
compound for subsequent investigations.
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Kinetics of Resurrection
In order to further confirm the reactivity of C8 and determine the rate of reaction, we monitored the resurrection of aged
AChE by this compound for days. Out of curiosity, neither 2-PAM nor fluoride was added to the QMP-treated samples for
reactivation of realkylated AChE. As the reaction progressed, linearly increasing activity was seen in the isopropyl phos-
phate-aged AChE sample treated with 4 mM C8 (FIGURE 3a). The negative control and 2-PAM control remained inactive.
After four days, the resurrected relative activity was as high as 20.4%. The apparent reaction rate was 0.20% per hour (r2 =
0.9958).
Resurrection kinetics of methylphosphonate-aged AChE was monitored similarly. The C8-treated sample displayed resur-
rected activity, which reached 32.7% after four days (FIGURE 3b). The apparent reaction rate was 0.32% per hour (r2 =
0.9185) and faster than against isopropyl phosphate-aged AChE.
The absence of extra reactivators (NH4F or 2-PAM, etc.) in this reaction suggests the inherent reactivation activity of C8,
that is, reactivating the realkylated AChE. This is in agreement with reports of reactivation activity of Mannich phenols by
Katz et al.,32 Cadieux et al.,33 and Bierwisch et al.34
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FIGURE 3. Kinetics of realkylation of aged eeAChE by 4 mM C8 at pH 8 over 4 days for (a) isopropyl phosphate-aged
(DFP-treated) eeAChE and (b) methylphosphonate-aged (PiMP-treated) eeAChE. The blue dashed lines illustrate the re-
sult of linear regression.
Bottom-up Proteomics
Besides determining the resurrected AChE activity by Ellman’s assay, confirmation of the reaction between the QMP and
aged AChE can also be revealed by mass spectrometry. Bottom-up proteomics35,36 was used to sequence the peptides and
differentiate enzyme species with variable modifications at the catalytic serine. We treated isopropyl phosphate-aged
eeAChE with C8 for 11 d, and digested it with trypsin. 2-PAM was not applied to the C8-treated sample. The digest was
analyzed with LC-MS/MS. The positive, negative and 2-PAM controls were also prepared in parallel, and percentages of
AChE species were obtained from the LC peak areas (TABLE 1).
TABLE 1. Percentages of eeAChE species after resurrection by C8, as determined by LC-MS/MS
Percentage (%)
Sample
Isopropyl phosphate-aged
Methylphosphonate-aged
Native Aged Realkylated Native Aged Realkylated
Positive Control
Negative Control
2-PAM Control
C8-treated
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2.1
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Peptide QVTIFGESAGAASVGMHLLSPDSRPK (residues 195–220), was observed in all samples. The catalytic serine (un-
derscored) in the positive control was completely unmodified because the enzyme was native. By contrast, the modifica-
tion observed in the negative and 2-PAM controls indicated that they were completely aged. The wild-type serine or oth-
erwise modified serine was not observed at this position. Compared to the unmodified peptide, there was a mass shift of
122.0133 Da (C3H7O3P added), which matches the added isopropyl phosphyl moiety. In the C8-treated sample, the unmod-
ified catalytic serine was observed, indicating resurrection induced by the QMP. Realkylated AChE, however, was not di-
rectly observed. This again suggests that our QMPs can resurrect aged AChE independent of extra reactivators such as
oximes due to their inherent reactivation activity. Another possibility is that the realkylated phosphyl group was lost un-
der used MS conditions. Similar results were obtained with methylphosphonate-aged AChE (TABLE 1).
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Reactivation of Inhibited AChE by Five QMPs
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Positive Control
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(0.04 mM)
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D5
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C2
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Negative Control
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FIGURE 4. Reactivation activity test of four QMPs (4 mM) against eeAChE inhibited by EMP. (a) Structure of EMP. (b)
Relative activity of eeAChE as reactivation proceeded.
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Four representative QMPs, namely C1, C2, C8, D5 and unsubstituted 3-hydroxypyridine, were reacted with eeAChE inhib-
ited with a VX analogue (EMP, an ethyl methylphosphonate compound, FIGURE 4a) to determine whether they are reac-
tivators. We chose this OP because the aging by VX is slow.37 After inhibition by EMP, the inhibited AChE was cleaned by
Sephadex spin columns, then incubated with the chosen QMPs (4 mM) at pH 8.0 and 37°C. Aliquots were taken and
cleaned also with Sephadex spin columns, and monitored by Ellman’s assay (FIGURE 4b).
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All tested QMPs exhibited obvious and similar reactivation activity, regardless of their different performances in the
aforementioned realkylation screening. After 24 h of reaction, they reactivated 64%~80% of inhibited AChE. This may
explain why C8 can efficiently resurrect aged AChE in the absence of 2-PAM and fluoride. All tested compounds reacted
slower than 3-hydroxypyridine and 2-PAM, which reached their respective plateaus within 1 h of reaction, although they
were used at only 0.4 and 0.04 mM concentrations, respectively.
pH Effect and EC50 for C8 Activity
We performed the resurrection of methylphosphonate-aged eeAChE by 4 mM C8 at four different pH values (6~9) at 37°C
for 1 d, with neither 2-PAM nor fluoride being used. The relative activity of C8-resurrected eeAChE increased dramatically
with pH (FIGURE 5a) – over 20% for 1 d at pH 9. Similar effects, but less pronounced, were also observed with isopropyl
phosphate-aged eeAChE (FIGURE 5b).
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QMP
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pH
FIGURE 5. Resurrection efficiency of 4 mM C8 against aged eeAChE increased with pH within the range of pH 6~9. (a)
Methylphosphonate-aged (PiMP-treated) eeAChE. (b) Isopropyl phosphate-aged (DFP-treated) eeAChE.
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Seven concentrations of C8 (0 – 20 mM), were therefore compared at pH 9 against methylphosphonate-aged and isopro-
pyl phosphate-aged eeAChE, in order to determine the EC50. The results (1.21 and 1.02 mM, respectively, Figure S24) illus-
trate that further optimization in binding affinity is needed for a more effective therapeutic.
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There are three heteroatoms in C8, forming multiple possible protonation states. Each of them may interact with the aged
AChE active site with different orientations, affinities and rates. 1H NMR spectra of C8 displayed shifting signals as the pH
changed (FIGURE 6a). The UV-vis spectra also dramatically changed when pH was increased from 6 to 9 (FIGURE 6b),
indicating a pKa between 7 and 8.
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pH7
pH8
pH9
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Wavelength (nm)
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FIGURE 6. Influence of C8 protonation states on spectra. (a) H NMR spectra of the aromatic protons and (b) UVꢀvis specꢀ
tra of C8 as pH is varied from 6~9.
a
b
FIGURE 7. (a) The four most probable protonation states of C8 at pH 8-9. (b) A representative snapshot in the MD simu-
lation of C8c (cyan, wall-eyed stereo). Hydrogen bonds with short contact distances are shown (green dashed lines).
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The strong pH dependence motivated us to study which isomers might be the active component(s). We compared
the four most probable states at pH 8 and 9, namely the neutral, anionic and two zwitterionic forms (C8n, C8a, C8b and
C8c, respectively, FIGURE 7a).Each protonation state was individually docked into a number of geometries of aged AChE
and subsequent MD simulations were run on the docked conformations; the location of the exchangeable proton has a
dramatic effect on the affinity of the ligand toward different zones in the AChE active site (FIGURE S3-S6). The simula-
tions suggest that both zwitterionic forms (C8b and C8c) have stable interactions and well-defined proximity to the ani-
onic oxygen of the phosphylated serine. The neutral form (C8n) also stably binds to the active site, but the benzylic car-
bon is not oriented for realkylation. The anionic form (C8a) gradually moves away from the active site during the simula-
tion. Calculations at the B3LYP/6-311+G** (SMD, water) level of theory suggest that C8c (pyrrolidinium) has a free energy
that is lower than C8b (pyridinium) by 3.1 kcal/mol, hence is a more probable species. Ser203, Tyr337, Tyr341 residues,
along with some hydrogen-bonded water molecules, contribute significantly to the binding of C8c by hydrogen bonding
and electrostatic attractions (FIGURE 7b).
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Resurrection of Aged Human AChE after exposure to DFP
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Negative Control
QMP
Positive Control
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0
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Reaction time (d)
FIGURE 8. Kinetics of resurrection of isopropyl phosphate-aged huAChE by 4 mM C8 (pH 9). The green dashed line illus-
trates the result of linear regression.
The efficacy of C8 was examined against aged huAChE. The activity of isopropyl phosphate-aged (DFP-treated) re-
combinant huAChE was monitored in the presence of 4 mM of C8 at pH 9 for a week by Ellman’s assay. After 7 d of reac-
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tion (FIGURE 8), 18% of the aged enzyme was resurrected, and the relative activity of huAChE was still increasing. The
apparent rate of resurrection was 2.5% per day, as determined by linear regression. Neither 2-PAM nor fluoride were used
in this test, again confirming that C8 alone can resurrect aged AChE to the native form.
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Discussion and Conclusions
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With in silico guidance, we synthesized 20 QMPs with various scaffolds and leaving groups, and then evaluated them
for their in vitro resurrection activity against methylphosphonate-aged and isopropyl phosphate-aged eeAChE. Multiple
QMPs with a 3-hydroxypyridine scaffold, with the reactive “benzylic” carbon attached at the 2-position, proved effica-
cious. C8 was found to be the lead compound in the screening. Used at 4 mM without the assistance of 2-PAM, C8 suc-
cessfully resurrected the relative activity of isopropyl phosphate-aged eeAChE to 20.4% and methylphosphonate-aged
eeAChE to 32.7% after 4 d of observation (FIGURE 3). This activity for C8 is also reproduced for aged human AChE
(FIGURE 8).
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The activities of C8 against isopropyl phosphate-aged and methylphosphonate-aged eeAChE were also confirmed
with bottom-up proteomics. The peptide containing the catalytic serine was sequenced to reveal the modification. In the
samples treated with C8, partial resurrection was observed as indicated by the presence of unmodified catalytic serine
from native AChE. Realkylated AChE was not directly observed, though no extra reactivator was added. These observa-
tions suggest the activity of C8 against both aged AChE and inhibited/realkylated AChE. This hypothesis was confirmed
as C8 and three other QMPs were able to reactivate inhibited AChE (FIGURE 4).
At this stage, C8 is not a practical drug for clinical post-aging treatment of OP poisoning or in vivo tests. The rate of
reaction is slower than ideal, although the concentration was on the millimolar scale. Whether this concentration will
cause toxicity also remains unknown. And, 2-4 d of reaction was needed to resurrect ~20% of activity. However, the resur-
rection rate is increased dramatically when pH is increased to 9, suggesting that structure-activity relationships and a
deeper mechanistic understanding may be effective in affording better activity. Calculations suggest that the zwitterionic
form of C8, with the pyrrolidinyl nitrogen being protonated, plays a critical role in activity.
Finally, this report provides the first in vitro success to substantially resurrect aged AChE via the use of an appropri-
ate realkylator. After almost 70 years of effort, this goal has been achieved, but further work remains to create a therapeu-
tic drug with all of the desired activity and selectivity.
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Experimental Section
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Computation-guided selection of realkylator candidates
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Optimizations, molecular docking, and MD simulations were all performed with the initial library of compounds. The
three lowest-energy docking poses of each flexible ligand across 13 rigid aged AChE structures were used as starting points
for subsequent 1 ns MD simulations. The QMPs were evaluated based on the time throughout the MD simulations in
which the reactive benzylic carbon was within close proximity to the anionic O-(P=O) of the aged serine. More experi-
mental details are described in the Supporting Information.
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LC-MS characterization of realkylator candidates
Mass spectra were obtained using Thermo LTQ Orbitrap (ESI) mass spectrometer. HPLC was performed with Agilent 1100
HPLC system equipped with Phenomenex Luna 5 µm C18 column (150 × 4.6 mm), eluted with mixtures of water and
methanol (5%~95% methanol, containing 0.1% formic acid, 0.6 mL/min), and detected by UV-vis detector at 254 nm as
well as ESI-orbitrap. The purity of each realkylator candidate was confirmed to be ≥ 95%.
Preparation of Aged AChE
Some OPs (mainly the phosphonates, which are commonly seen in G- and V-type warfare agents) are chiral, and the ste-
reoisomers can inhibit and/or age at different rates.38 Inhibited AChE may remain un-aged even after reacting for longer
than the apparent t1/2, if the enzyme is inhibited by the slower aging OP stereoisomer. Reactivation of the residual un-
aged, but inhibited, AChE can interfere with the observation of the resurrection of aged AChE and may lead to artifacts.
Thus, AChE must be thoroughly aged and free of inhibited AChE. To ensure complete aging, the methylphosphonate-
aged AChE (treated with PiMP) was prepared via two rounds of aging, considering the chirality of PiMP. We treated
AChE with 2-PAM after the first round of aging, in order to reactivate any residual inhibited AChE. Then PiMP was added
to inhibit and age the enzyme again. The fast-aging isomers thus had a second chance to compete with other isomers and
react with the enzyme. The amount of residual inhibited or native AChE in the sample was significantly minimized, often
<1% residual activity of a native AChE control. By contrast, to age AChE with achiral DFP, only one round of aging was
needed. More experimental details are described in the Supporting Information.
Ellman’s Assay
Ellman’s assay31 was carried out on clear flat-bottom 96-well microplates. The assay solution was 180 µL × 0.56 mM of
acetylthiocholine in pH 8.0 40 mM buffer, containing 0.1 g/L of BSA and 1.1 mM of 5,5’-dithio-bis-(2-nitrobenzoic acid)
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(DTNB). 20 µL of tested AChE sample was added to initiate the reaction. Samples were diluted if the activity was very
high. The absorption at 412 nm was monitored at 25°C with Molecular Devices SpectraMax i3 microplate reader. Each
sample was tested in four replicate wells. The average initial slope of the four absorbance-time plots of each sample and
the standard deviation were divided by that of the control to obtain the relative activity and error.
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Screening of Realkylator Library
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Each realkylator (4 µL × 5, 25 or 100 mM) was mixed with methylphosphonate-aged AChE (2 µL), phosphate buffer (90 µL
× 200 mM, pH 8.0 containing 1 g/L BSA and 0.02% NaN3), and NH4F (4 µL × 100 mM). After reacting at 37°C for 1 d, each
sample was treated with 2-PAM for 1 h to reactivate the realkylated AChE. Three controls were prepared in parallel. In the
positive and negative controls, the realkylator solution was replaced with blank buffer. In the 2-PAM control, it was re-
placed with 4 mM 2-PAM solution. In the positive control, the aged AChE was replaced with the same amount of native
AChE. After the reactions, all reagents were removed with a Zeba desalting spin plate (purchased from Thermo Fisher,
USA). The AChE activity of each sample was determined with Ellman’s assay.
Kinetics of Alkylation Induced by C8
Aged AChE (5 µL) was mixed with 955 µL × 200 mM of pH 8.0 phosphate buffer (200 mM, containing 1 g/L BSA and
0.02% NaN3) and C8 solution (40 µL × 100 mM) and incubated at 37°C. The negative control, positive control and 2-PAM
control were also prepared in parallel as aforementioned. Aliquots of 95 µL were taken at various time intervals (9~24 h),
all reagents were then removed by Sephadex size exclusion spin columns (filled with 0.1 g dry weight of superfine Se-
phadex G-25). AChE activity was then determined with Ellman’s assay as aforementioned. 100% was set at the t = 0 h
point of the positive control.
Bottom-up proteomics
Isopropyl phosphate-aged AChE (60 µL, aged with DFP as described above without use of BSA) was mixed with pH 8.0
phosphate buffer (36 µL × 40 mM, no BSA or NaN3) and C8 solution (4 µL × 100 mM). BSA was not used to minimize
complication in LC-MS/MS. The positive control, negative control and 2-PAM control were prepared in parallel as afore-
mentioned. After reacted for 11 d at 37°C, 100 µL acetonitrile was added to denature the enzyme. 10 min later, the solution
was washed through an Amicon Ultra centrifugal filter (3 kDa cut-off molecular weight, purchased from EMD Millipore)
and with 3 × 400 µL ammonium acetate buffer (40 mM, pH7.5) to remove organic solvent and reagents. 40 µL × 0.1 g/L
modified porcine trypsin (Arg and Lys methylated, sequencing grade, purchased from Promega) was added. After reacted
at 37°C for digestion for 7 h, the reaction was terminated by the addition of 10 µL × 1 M acetic acid.
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Methylphosphonate-aged AChE in the absence of BSA as well as the controls were similarly tested with bottom-up prote-
omics. Methylphosphonate-aged AChE (50 µL, aged with PiMP as described above without use of BSA) was mixed with
pH 8.0 phosphate buffer (45 µL × 40 mM, no BSA or NaN3) and C8 solution (12 µL × 100 µL). The samples were reacted for
5 d.
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Additional experimental details are provided in the Supporting Information.
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Reactivation of Inhibited AChE by QMPs
Electric eel AChE (~4 units in 1 µL × 50% glycerin) was mixed with BSA (96.5 µL × 1g/L in 200 mM phosphate buffer, pH
8.0, containing 0.02% NaN3) and EMP (2.5 µL × 0.2 mM in 2% DMSO). After reacted at 37°C for 1 h, the solution was
cleaned with a Sephadex spin column. Controls with EMP replaced by blank solvents were also prepared.
Each tested compound (4 µL × 100 mM, final concentrations 4 mM) was mixed with 1 µL of the freshly prepared inhibited
AChE solution and BSA (95 µL × 1 g/L in 200 mM phosphate buffer, pH 8.0, containing 0.02% NaN3). Six replicates were
made for each compound. One replicate was taken at various time intervals and cleaned again with Sephadex columns,
then analyzed with Ellman’s assay as aforementioned. For comparison, 1 mM 2-PAM and 10 mM 3-hydroxypyridine were
tested in parallel (final concentrations 0.04 and 0.4 mM). The negative and positive controls had the reagent solution re-
placed with blank water. The AChE in the positive control was native and not treated with EMP.
pH Effect in Resurrection of Aged AChE
To 94 µL × 1 g/L BSA solution (in 200 mM phosphate buffer, containing 0.02% NaN3), 2 µL of methylphosphonate-aged
AChE solution (treated with DFP or PiMP as described above) and 4 µL × 100 mM C8 solution were added. A negative
control was prepared with C8 solution replaced with blank water. A positive control was similarly prepared in parallel
with the aged AChE replaced with native AChE. Buffers at four pH values were compared: 6, 7, 8 and 9. Samples were in-
cubated at 37°C for 1 d. AChE activity was then determined with Ellman’s assay.
Measurement of EC50 of C8
Seven concentrations of C8, ranging from 0 to 20 mM, were compared against methylphosphonate-aged, combined with
2-PAM as the reactivator. NH4F was not added in this test to see whether rapid reactivation is necessary to suppress re-
aging. The specific procedures are as follows:
To 94 µL of pH 9.0 phosphate buffer (200 mM, containing 1 g/L BSA and 0.02% NaN3), 2 µL as obtained aged AChE (treat-
ed with PiMP) and 4 µL of C8 solution (neutral, 0, 25, 50, 100, 200, 300 and 500 mM) were mixed, and allowed to react at
37°C for 1 d. The final concentrations of C8 were hence 0, 1, 2, 4, 8, 12 and 20 mM, respectively. The positive control and 2-
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PAM control were also prepared in parallel following a procedure similar to that aforementioned. The reagents were re-
moved in the end by size exclusion spin columns. Ellman’s assay was carried out following the aforementioned procedure.
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Resurrection Kinetics of Aged Recombinant Human AChE
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Recombinant human AChE was provided by Dr. Douglas Cerasoli (USAMRICD, purchased from Chesapeake PERL Inc) or
by Dr. Zoran Radić (UCSD, expressed with HEK-293 cells).39
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To 97 µL of buffer at pH 7.0 (200 mM phosphate, containing 1 g/L bovine serum albumin and 0.02% NaN3), recombinant
human AChE (0.5 µL, ~50U/µL in 50% glycerol) and DFP (2.5 µL, 0.2 mM in 2% DMSO) were added. After reacting for 3 d,
the solution was washed with 3 × 400 µL pH 8.0 buffer in a centrifugal ultrafilter (Amicon Ultra 0.5 mL, cut-off molecular
weight 30 kDa). 1 µL × 2% NaN3 was added and the solution was stored at 4°C for further use. A control of native AChE
was prepared in parallel by replacing DFP solution with blank 2% DMSO.
Aged AChE (20 µL) was mixed with 172 µL × 200 mM phosphate buffer (pH 9.0, containing 1 g/L BSA and 0.02% NaN3)
and C8 solution (neutral, 8 µL × 100 mM) and incubated at 37°C. The negative control and positive control were also pre-
pared in parallel as aforementioned. Aliquots of 20 µL (10 µL from the positive control due to high activity) were taken at
various time intervals (1 or 2 d), and diluted to 100 µL. AChE activity was then determined with Ellman’s assay as afore-
mentioned. 50% was set at the t = 0 d point of the positive control (not 100% due to difference in dilution).
ASSOCIATED CONTENT
Supporting Information.
Molecular Docking and Molecular Dynamics Simulations
Synthesis of OPs and Candidate Realkylators
Enzymatic Tests
FIGURE S1 to S24
TABLE S1 to S29
NMR spectra
HPLC chromatograms
Molecular Formula Strings.csv (standalone file)
This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
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Corresponding Author
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* Email: hadad.1@osu.edu
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Author Contributions
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Q.Z. conducted all reactions involving AChE. A.J.F., T.S.C., R.D., S.S., A.D., N.Y., J.S., S.F. and C.S.C. synthesized and character-
8
9
ized the QMPs. T.S.C. synthesized and characterized PiMP. Q.Z., O.D.E. and C.A.M. contributed to the optimization of enzyme-
involving reactions and in the mass spectrometric analyses. A.J.F., W.H.C., K.F., T.G.B. and R.J.Y. conducted the in silico studies.
J.J. performed the UV-vis analyses. C.M.H. directed the project.
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ACKNOWLEDGMENT
We acknowledge financial support from the National Institutes of Health (1U01–NS087983) and generous computational re-
sources from the Ohio Supercomputer Center. We thank the OSU Campus Chemical Instrument Center, which is supported by
NIH Award Number Grant P30 CA016058 and S10 OD018056, as well as the Office of Research in The Ohio State University, for
assistance in mass spectrometric and proteomic analyses. And we thank Dr. Douglas Cerasoli at the United States Army Medical
Research Institute of Chemical Defense and Dr. Zoran Radić at University of California, San Diego, for their gifts of recombinant
huAChE. We thank Dr. Amneh Young for synthesizing EMP.
ABBREVIATIONS USED
DFP: diisopropyl fluorophosphate
DTNB: 5,5’-dithio-bis-(2-nitrobenzoic acid)
eeAChE : electric eel acetylcholinesterase
EMP: ethyl methylphosphonate
hAChE : human acetylcholinesterase
OP: organophosphorus
PiMP : pinacolyl methylphosphonate
QM: quinone methide
QMP: quinone methide precursor
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2-PAM: 2-pralidoxime
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SMD: solvation model based on density
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Resurrection of aged acetylcholinesterase
160x55mm (300 x 300 DPI)
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