New structural scaffolds for centrally acting oxime reactivators of phosphylated cholinesterases

Article abstract of DOI:10.1074/jbc.M111.230656

We describe here the synthesis and activity of a new series of oxime reactivators of cholinesterases (ChEs) that contain tertiary amine or imidazole protonatable functional groups. Equilibration between the neutral and protonated species at physiological pH enables the reactivators to cross the blood-brain barrier and distribute in the CNS aqueous space as dictated by interstitial and cellular pH values. Our structure-activity analysis of 134 novel compounds considers primarily imidazole aldoximes and N-substituted 2- hydroxyiminoacetamides. Reactivation capacities of novel oximes are rank ordered by their relative reactivation rate constants at 0.67 mM compared with 2-pyridinealdoxime methiodide for reactivation of four organophosphate (sarin, cyclosarin, VX, and paraoxon) conjugates of3 human acetylcholinesterase (hAChE). Rank order of the rates differs for reactivation of human butyrylcholinesterase (hBChE) conjugates. The 10 best reactivating oximes, predominantly hydroxyimino acetamide derivatives (for hAChE) and imidazole-containing aldoximes (for hBChE) also exhibited reasonable activity in the reactivation of tabun conjugates. Reactivation kinetics of the lead hydroxyimino acetamide reactivator of hAChE, when analyzed in terms of apparent affinity (1/K_ox) and maximum reactivation rate (k₂), is superior to the reference uncharged reactivators monoisonitrosoacetone and 2,3-butanedione monoxime and shows potential for further refinement. The disparate pH dependences for reactivation of ChE and the general base-catalyzed oximolysis of acetylthiocholine reveal that distinct reactivator ionization states are involved in the reactivation of ChE conjugates and in conferring nucleophilic reactivity of the oxime group.

Full text of DOI:10.1074/jbc.M111.230656

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2011/04/04/M111.230656.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 22, pp. 19422–19430, June 3, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
New Structural Scaffolds for Centrally Acting Oxime
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S
Reactivators of Phosphylated Cholinesterases*
Received for publication, February 13, 2011 Published, JBC Papers in Press, April 4, 2011, DOI 10.1074/jbc.M111.230656
Rakesh K. Sit‡, Zoran Radic´§, Valeria Gerardi§, Limin Zhang§, Edzna Garcia§, Maja Katalinic´¶, Gabriel Amitaiʈ,
Zrinka Kovarik¶, Valery V. Fokin‡, K. Barry Sharpless‡, and Palmer Taylor§1
From the ‡Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla,
California 92037, the §Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla,
California 92093, the ¶Institute for Medical Research and Occupational Health, HR-10001 Zagreb, Croatia, and the ʈDepartment of
Pharmacology, Israel Institute for Biological Research, Ness Ziona 74100, Israel
We describe here the synthesis and activity of a new series of
oxime reactivators of cholinesterases (ChEs) that contain terti-
ary amine or imidazole protonatable functional groups. Equili-
bration between the neutral and protonated species at physio-
logical pH enables the reactivators to cross the blood-brain
barrier and distribute in the CNS aqueous space as dictated by
interstitial and cellular pH values. Our structure-activity analy-
sis of 134 novel compounds considers primarily imidazole aldo-
ximes and N-substituted 2-hydroxyiminoacetamides. Reactiva-
tion capacities of novel oximes are rank ordered by their relative
reactivation rate constants at 0.67 mM compared with 2-pyri-
dinealdoxime methiodide for reactivation of four organophos-
phate (sarin, cyclosarin, VX, and paraoxon) conjugates of
human acetylcholinesterase (hAChE). Rank order of the rates
differs for reactivation of human butyrylcholinesterase (hBChE)
conjugates. The 10 best reactivating oximes, predominantly
hydroxyimino acetamide derivatives (for hAChE) and imida-
zole-containing aldoximes (for hBChE) also exhibited reasonable
activity in the reactivation of tabun conjugates. Reactivation
kinetics of the lead hydroxyimino acetamide reactivator of
hAChE, when analyzed in terms of apparent affinity (1/Kox) and
maximum reactivation rate (k2), is superior to the reference
uncharged reactivators monoisonitrosoacetone and 2,3-bu-
tanedione monoxime and shows potential for further refine-
ment. The disparate pH dependences for reactivation of ChE
and the general base-catalyzed oximolysis of acetylthiocholine
reveal that distinct reactivator ionization states are involved in
the reactivation of ChE conjugates and in conferring nucleo-
philic reactivity of the oxime group.
inhibited in organophosphate (OP)-intoxicated individuals
is required for sustained symptom recovery. In particular,
nerve agent OPs already used by terrorists, but also active
metabolites of OP-based pesticides, readily cross the blood-
brain barrier (BBB). The exposure to OP doses close to
lethality results in initial severe motor convulsions and epi-
leptic seizures. Accumulating evidence points to these sei-
zure events being linked to irreversible long term compro-
mise of cognitive functions and alteration of CNS electrical
excitability. Once accumulated into hydrophobic sites, OPs
that do enter the CNS are retained and partition slowly back
into the circulation. For example, victims of Tokyo subway
nerve gas attack in 1995 were found to suffer from both short
and long term symptoms of OP exposure (1–4). Accord-
ingly, comprehensive protection from and treatment of OP
intoxication to minimize the longer term consequences
require administration of antidotes capable of reactivating
OP-inhibited AChE in the CNS.
Current therapy directed to reactivating inhibited AChE is
limited to the peripheral circulation because commonly used
quaternary pyridinium aldoxime reactivators do not cross the
BBB at therapeutically relevant levels (5). Only a handful of
studies involving centrally acting reactivators have been
reported in the literature, including nonspecific acetyloxime
derivatives 2,3-butanedione monoxime (DAM) and monoiso-
nitrosoacetone (MINA) (6–8), glycosylated pyridinium oximes
(9, 10), and dihydropyridine prodrug, pro-2PAM (2-pyridineal-
doxime methiodide) derivatives (11, 12). Studies involving pro-
2PAM have yielded most promising OP protection results, yet
even with efficient or immediate conversion in the CNS, pro-
2PAM is inherently limited by reactivation efficacy of 2PAM
itself.
It has become increasingly apparent that efficient rein-
statement of CNS acetylcholinesterase (AChE)2 activity
To improve reactivation therapy in the CNS, we initiated a
program to develop, by significantly expanding existing reacti-
vator chemical space, centrally active AChE reactivators devoid
of permanent positive charge, but amenable to protonation
(Scheme A). The uncharged species of reactivators are expected
to diffuse across the BBB into the CNS, and upon establishing
the pH-dependent equilibrium between the neutral and ionized
forms in extracellular CNS space, the protonated form would
efficiently interact with the electron-rich active center environ-
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
CounterAct Program United States Public Health Service Grant U01 NS
058046 through the NINDS.
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The on-line version of this article (available at http://www.jbc.org) contains
supplemental Methods and additional references, Fig. S1, and Table S1.
1 To whom correspondence should be addressed: SSPPS, UCSD, 9500 Gilman
Dr., La Jolla, CA 92093-0657. Tel.: 858-534-4028; Fax: 858-534-8248; E-mail:
pwtaylor@ucsd.edu.
2 The abbreviations used are: AChE, acetylcholinesterase; ATCh, acetylthio-
choline;BBB, bloodbrainbarrier;ChE, cholinesterase;CuAAC, Cu-catalyzed
azidealkynecycloaddition;DAM, 2,3-butanedionemonoxime;Flu-MP, flu-
orescent methylphosphonate; hAChE, human AChE; hBChE, human
butyrylcholinesterase; MINA, monoisonitrosoacetone; OP, organophos-
phate; 2PAM, 2-pyridinealdoxime methiodide.
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Centrally Acting ChE Reactivators
SCHEME A. Generalized ionization equilibria of amino oximes with two ionizing groups proceeds through formation of a neutral or zwitterionic species.
SCHEME 1
SCHEME 4
SCHEME 2
SCHEME 3
ment of the OP-inhibited AChE. Accordingly, reactivating
SCHEME 5
amines or imines with pKa values between 7 and 10 would
exhibit optimal ionization equilibrium characteristics.
Here, we report design, synthesis, and initial functional char-
acterization of 134 novel oxime reactivators that remain largely
neutral at physiological pH values. Oxime-elicited reactivation
rates were determined at a single concentration for paraoxon-,
sarin-, VX-, and cyclosarin-inhibited recombinant human
AChE (hAChE). For the 10 best reactivators, we examined reac-
tivation of tabun-inhibited conjugates. In addition, the majority
of oximes were screened for reactivation of paraoxon-, sarin-,
VX-, and cyclosarin-inhibited human butyrylcholinesterase
SCHEME 6
(hBChE) to assess the breadth of conjugates that show signifi-
cant reactivation.
Our considerations of structure and pH dependence of reac-
tivation provide evidence that oxime access to the phosphonyl
or phosphoryl phosphorous atom within the narrow confines of
the gorge appears to be the critical criterion for efficient reac-
tivation, rather than the ionization state governing oximate
SCHEME 7
formation.
MATERIALS AND METHODS
with pCMV-N-FLAG (Sigma-Aldrich) construct of hAChE
Enzymes—Monomeric hAChE was expressed in stably trans- encoding cDNA with a stop codon truncating the sequence at
fected HEK-293 cells (American Type Culture Collection, amino acid 547. Selection of neomycin-resistant cell colonies
Manassas, VA) obtained upon calcium phosphate transfection using G418 (Invitrogen) enhanced expression. The expressed
JUNE 3, 2011•VOLUME 286•NUMBER 22
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Centrally Acting ChE Reactivators
FIGURE 1. Reactivation of VX-, cyclosarin-, sarin-, and paraoxon-inhibited hAChE by library of 134 uncharged oxime reactivators. Reactivation rates
(kobs) of 0.67 mM oximes (measured at 37 °C in 0.10 M phosphate buffer, pH 7.4) are given relative to 2PAM. Average reactivation potencies of oximes for all four
organophosphates are shown as green bars. Oxime IDs are ordered by their average reactivity (A), segregated by their general structure as acetamide,
imidazole, and other oximes (B), and segregated by length of a linker connecting an oxime nucleophile and part of the molecule “R” responsible for binding
interaction with hAChE (C). Measured rates of oximolysis (3 ϫ DA/min) and calculated fractions of oximate anion for pH 7.4 are also given as black and gray bars,
respectively. Structures and reactivation potencies of all 134 oximes are given in supplemental Table S1.
secreted protein containing the N-terminal FLAG tag was puri-
N-Substituted 2-hydroxyiminoacetamides RS41A, RS191D,
fied in several milligram quantities by an anti-FLAG affinity RS191C, RS69A, and RS69F were prepared from ethyl glyoxy-
column (Sigma-Aldrich). Purified oligomeric hBChE isolated late 2 in two steps (Scheme 2). Condensation with hydroxyla-
from human plasma was kindly donated by Drs. Douglas Cera- mine provided ethyl glyoxylate oxime 3 followed by subsequent
soli and David Lenz, United States Army Medical Research amidation with the corresponding primary amine.
Institute of Chemical Defense, Edgewood, MD.
For the preparation of RS166C, glycine ester hydrochloride
Organophosphates—Nonvolatile, low toxicity fluorescent 4 was converted into 2-chloro-2-(hydroxyimino)acetate 5
methylphosphonates (Flu-MPs) (13) were used as analogues of (Scheme 3) (14). Treatment of 5 with N-methylaniline and tri-
nerve agents sarin, cyclosarin, and VX. The Flu-MPs differ from ethylamine at room temperature afforded 2-amino-2-(hydroxy-
actual nerve agent OPs only by structure of their respective imino)acetate 6 (15). Amidation of 6 with 3-(pyrrolidinyl)pro-
leaving groups. Inhibition of ChEs by Flu-MPs results in OP- panamine delivered acetamide oxime RS166C.
ChE covalent conjugates identical to the ones formed upon
Azido intermediates 7 and 8 were synthesized from form-
inhibition with nerve agents. Paraoxon was purchased from ylimidazole 1 by alkylation with azidoalkylmethanesulfonate
Sigma-Aldrich and tabun from NC Laboratory (Spiez, followed by condensation with hydroxylamine (Scheme 4).
Switzerland).
Imidazole-1,4-triazole derivatives RS185B, RS150D, and
Oximes—2PAM, MINA (monoisonitrosoacetone), and RS210B were then subsequently obtained via a Cu-catalyzed
DAM (2,3-butanedione monoxime) were purchased from azide alkyne cyclo addition (CuAAC) (16) between azido deriv-
Sigma-Aldrich. The synthesis of imidazole aldoxime derivatives ative 7 and corresponding alkyne derivatives 9 (17), 10 (18), and
RS113A, RS113B, RS115A, RS115B, and RS115C is outlined 11, respectively.
in Scheme 1. Alkylation of 2-formylimidazole 1 with the requi-
The 1,4-triazole oxime derivatives RS146A and RS182A
site bromide followed by treatment with hydroxylamine deliv- were prepared as illustrated in Scheme 5. Amidation of ethyl
ered the desired oxime. See also supplemental Methods.
glyoxylate oxime 3 with commercially available propargyl
VOLUME 286•NUMBER 22•JUNE 3, 2011
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Centrally Acting ChE Reactivators
TABLE 1
Structures and relative rate constants for OP-hAChE reactivation (kobs) of 10 topmost uncharged reactivators (0.67 mM) compared with cationic
pyridinium aldoxime 2PAM
Nonenzymic oximolysis rates of ATCh (1 mM) by 1.0 mM oximes and calculated percentages of oximate anion and neutral species at pH 7.4 (calculated by MarvinView 5.2.6)
are also given. Experiments were performed in duplicate at 37 °C in 0.10 M phosphate buffer, pH 7.4.
* kobs values determined for 2PAM were: 0.087 minϪ1 (for POX), 0.16 minϪ1 (for sarin), 0.025 minϪ1 (for CS), 0.15 minϪ1 (for VX) and 0.0026 minϪ1 (as 1.0 mM for tabun).
** Not measured.
amine provided intermediate 13. The CuAAC reaction of 13 choline (ATCh) at 1.0 mM (hAChE) or 5.0 mM (hBChE) concen-
with 8 and 12 delivered triazole oxime derivatives RS182A and trations. OP-hAChE and OP-hBChE conjugates were prepared
RS46A, respectively.
by incubating micromolar enzyme stocks with 4-fold molar
Azido derivative 2-(azidomethyl)-4-phenyl-2H-1,2,3-tria- excess of OP for 2–3 min until inhibition exceeded 95%. Inhib-
zole 14 was prepared by treating phenyl acetylene with formal- ited enzymes and appropriate controls were passed through
dehyde and sodium azide in a one-pot synthesis (19). The imida- two consecutive size exclusion Sephadex G-50 spin columns
zole alkyne intermediates 15 and 16 were prepared in two steps (Roche Diagnostics) to remove excess inhibitor. The reactiva-
(Scheme 6) by first alkylating of formylimidazole 1 with propar- tion reaction was initiated by adding an oxime reactivator at
gyl methanesulfonate, followed by condensation with hydrox- 0.67 mM (or 1.0 mM for tabun conjugates) final concentrations
ylamine. Then CuAAC reaction between azide 14 and alkyne into a nanomolar solution of OP-conjugated enzymes. Control
16 afforded RS161B.
ChE activity was measured in the presence of oxime at concen-
To obtain the oxime derivative RS48B (Scheme 7), the trations used for reactivation. The time course of hAChE reac-
CuAAC reaction between alkyne derivative 15 and azido deriv- tivation was monitored in 96-well format by parallel consecu-
ative 17 was performed. Amidation of ethyl glyoxylate oxime 3 tive assays of hAChE activity in 10 ␮l of reactivation mixture
with 3-azidopropyl-1-amine (20) afforded N-(3-azidopropyl)- aliquots diluted 625 times into assay mixture containing ATCh
2-(hydroxyimino) acetamide 17.
and thiol detection reagent 5,5Ј-dithiobis-(2-nitrobenzoic
Oxime Reactivation Assays—hAChE and hBChE activities acid). For reactivation of tabun-inhibited hAChE conventional
were measured using a spectrophotometric assay (21) at 37 °C single cuvette spectrophotometric assay was used. The first
(or 25 °C for tabun conjugates) in 0.1 M sodium phosphate order reactivation rate constant (kobs) for each oxime ϩ OP
buffer, pH 7.4, containing 0.01% BSA and substrate acetylthio- conjugate combination was calculated by nonlinear regression
JUNE 3, 2011•VOLUME 286•NUMBER 22
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Centrally Acting ChE Reactivators
FIGURE 2. Reactivation of VX-, cyclosarin-, sarin-, and paraoxon-inhibited hBChE by a library of 100 uncharged oxime reactivators at 0.67 mM deter-
mined by QuickScreen protocol (at 37 °C in 0.10 M phosphate buffer pH 7.4). Reactivation potency is given relative to 2PAM. Average reactivation potency
of hBChE by oximes for all four organophosphates is given as yellow bars and for hAChE by red bars. Oxime IDs are ordered by their average reactivity (A) and
segregated by their general structure as acetamide, imidazole and other oximes (B). Measured nonenzymic rates of ATCh hydrolysis by oximes (3 ϫ ⌬A/min)
and calculated fractions of oximate anion for pH 7.4 are given as black and gray bars, respectively.
as described earlier (22). Reactivation of OP-hBChE conjugates between rates of nonenzymic oximolysis and reactivation rates,
was monitored using “QuickScreen” protocol by assaying
hBChE activity in the reactivation mixture in 96-well format at
a single reactivation time (5 min after initiation of reactivation).
The dependence of reactivation rates on oxime concentra-
tions and determination of maximal reactivation rate constant
k2, Michaelis-Menten type constant Kox, and the overall second
order reactivation rate constant kr were conducted as previ-
ously described (22). The nonenzymic oximolysis (general base
catalysis) reaction was measured for combinations of 1 mM
oxime and 1 mM ATCh in buffer in the absence of enzyme. The
rate was expressed from the slope of linear absorbance increase
in time.
oximolysis rates of acetamide oximes were significantly larger
than the ones measured for imidazole oximes or other oximes
(rates as ⌬A/min were 0.123 Ϯ 0.011, 0.025 Ϯ 0.002, and
0.014 Ϯ 0.007, respectively; Fig. 1B). Hence, the majority of the
most efficient reactivators were of the N-substituted 2-hy-
droxyimino acetamide structure (Table 1). Generally, com-
pound structures with their oxime moiety positioned farther
from the part of the molecule largely responsible for molecular
recognition and binding to AChE seemed to be better reactiva-
tors. The optimal separation equivalent to a five- or six-atom
linker (Fig. 1C) is consistent with structures of the lead RS41A
and other top ranking reactivators (Table 1).
Structures of nearly half of reactivators included triazole ring
(supplemental Fig. S1), but this heterocycle positioned some
distance from the oxime did not significantly influence their
reactivation potency. The average relative reactivation potency
for 64 triazoles (versus 2-PAM) was 0.15 Ϯ 0.02 (range 0.003–
0.82) and for the remaining 71 reactivators 0.11 Ϯ 0.03 (range
0–0.88), suggesting that the triazole, with its relatively strong
dipole (4–5 Debye) but absent a charge at this pH, had no
appreciable effect on reactivity. The triazole moiety was earlier
observed to contribute synergistically to the total energy of
binding of high affinity AChE inhibitors (23).
RESULTS
Reactivation of OP-hAChE Conjugates—Nearly half of the
structurally diverse library of 134 uncharged oximes (supple-
mental Table S1) demonstrated the capacity to reactivate para-
oxon-, sarin-, cyclosarin-, and VX-inhibited hAChE (Fig. 1A).
The average reactivation efficacy for the 10 best uncharged
reactivators was better than one third (34%) of 2PAM efficacy
(Table 1), and three of the oximes (RS41A, RS48B, and
RS191D) were found to be comparable with 2PAM. Reactiva-
tion trends and enhancement over 2PAM reactivation were
similar for different OP conjugates (Fig. 1) with cyclosarin and
VX conjugates being most amenable to reactivation, and para-
oxon and tabun conjugates being noticeably more resistant to
reactivation. However, the observed rank orderings were found
to parallel neither computationally predicted ionization state of
reactivator oxime nucleophiles nor their nucleophilic reactivity
reflected in nonenzymic oximolysis of ATCh. The 56 acetamide
oxime derivatives representing 41% of all tested oximes were on
average equally efficient reactivators as 41 imidazole derivatives
(30% of all oximes), whereas none of the varying structural scaf-
folds of remaining 38 oximes (29%) showed appreciable reacti-
vation activity with average respective reactivation potencies of
0.17, 0.17, and 0.016 relative to 2PAM (Fig. 1B). Although
Reactivation of OP-hBChE Conjugates—Reactivation of OP-
hBChE conjugates by a subset of 100 uncharged reactivators
revealed a larger general enhancement of reactivation rates
over those of 2PAM than observed for OP-hAChE reactivation
(0.58 Ϯ 0.04 with range 0.1–2.6 versus 0.14 Ϯ 0.02 with range
0–1.3, respectively; Fig. 2). No correlation between reactivation
of OP-hBChE and OP-hAChE conjugates was observed. This
reflects a larger structural promiscuity of hBChE for interaction
with oxime reactivators, but also a slower reactivation rate of
hBChE for the reference compound, 2PAM. Accordingly, cova-
lent OP-hBChE conjugates were reactivated, on average, simi-
larly well by acetamide, imidazole, and other oximes (Fig. 2B).
within each of structural scaffolds no correlation was observed Seven out of ten best hBChE reactivators, however, turned to be
19426 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 22•JUNE 3, 2011

Centrally Acting ChE Reactivators
TABLE 2
StructuresandrelativepotenciesforOP-hBChEreactivationof10topmostunchargedreactivators(0.67mM)comparedwithcationicpyridinium
aldoxime 2PAM determined by end- point QuickScreen procedure
Nonenzymic oximolysis rates of ATCh (1 mM) by 1.0 mM oximes and calculated percentages of oximate anion and neutral species at pH 7.4 (calculated by MarvinView 5.2.6)
are also given. Experiments were performed in duplicate at 37 °C in 0.10 M phosphate buffer, pH 7.4.
* kobs values determined in continuous reactivation assay for 2PAM were: 0.052 minϪ1 (for POX), 0.13 minϪ1 (for sarin), 0.17 minϪ1 (for CS), 0.097 minϪ1 (for VX), and
0.000461 minϪ1 (as 1.0 mM for tabun).
imidazole oximes led by simple alkyl chain derivatives (Table 2).
The rate of RS41A reactivation of cyclosarin-hAChE conju-
No correlation was observed between measured oximolysis gate in the pH range 6.4–8.4 did not vary significantly; how-
ever, oximolysis of ATCh by RS41A was 34 times faster at pH
8.4 compared with pH 6.4 (Table 4). The latter was roughly
consistent with a large increase of oximate anion calculated for
RS41A at pH 8.4 versus pH 6.4.
rates or calculated equilibrium oximate anion concentrations
with levels of hBChE reactivation.
Characterization of the Lead hAChE Reactivator RS41A—
Reactivation of OP-hAChE conjugates by the overall best reac-
tivator RS41A (Table 1) was additionally measured over a range
of oxime concentrations, from 0.1 mM to 40 mM, and compared
with uncharged (DAM and MINA) and charged (2PAM) refer-
ence compounds (Fig. 3). Although reactivation by MINA and
in particular DAM was very slow, RS41A displayed more rapid
reactivation over the entire measured concentration range, but
the rate enhancement compared with the quaternary cationic
2PAM was observed largely at high oxime concentrations. This
suggests that the maximal reactivation rate constant for RS41A
is generally superior to other oximes, whereas its binding to
covalent OP-hAChE conjugates (reflected in the constant Kox)
is not as strong as that of 2PAM (Table 3). All three uncharged
oximes were characterized with large Kox constants (i.e. low
DISCUSSION
Nerve agent OPs, as well as active forms of OP-based pesti-
cides, being small uncharged organic molecules readily diffuse
through biological membranes, quickly reach the CNS and sub-
sequently partition into brain lipids. Thus, brain AChE,
although being amenable to covalent inhibition in OP poison-
ing, is inaccessible to the ameliorating action of commonly used
pyridinium-based oxime reactivator antidotes because they are
incapable of crossing the BBB due to the positive charge.
This study demonstrates that a substantial number of novel
OP-hAChE reactivators emerged from a library of 134 novel,
structurally diverse uncharged oximes. Promising leads with
affinity) consistent with AChE having preference for binding reactivation potencies comparable with the one of commonly
cationic ligands, even in an OP-conjugated state.
used pyridinium reactivators, 2PAM, were singled out. Based
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Centrally Acting ChE Reactivators
FIGURE 3. Concentration dependence of oxime reactivation of sarin- (A), VX- (B), cyclosarin- (C), and paraoxon- (D) inhibited hAChE. Dependence for
initial lead oxime RS41A (‚) compared with reference uncharged (DAM (ꢀ) and MINA (ꢁ)) and cationic (2PAM (E)) oximes (measured at 37 °C in 0.10 M
phosphate buffer pH 7.4) is shown.
TABLE 3
Constants for reactivation of OP-hAChE conjugates by lead reactivator RS41A and reference reactivators MINA, DAM, and 2-PAM
The maximal reactivation rate constant (k2), the Michaelis-type dissociation constant of OP-hAChE-oxime complex (Kox), and overall second order reactivation rate
constant (kr) were calculated by nonlinear regression from plots presented in Fig. 3. Experiments were done in duplicate or triplicate at 37 °C in 0.10 M phosphate buffer, pH
7.4, and are summarized as shown in Fig. 3.
TABLE 4
pH dependence of reactivation of VX-inhibited hAChE by lead oxime RS41A (1.0 mM), oximolysis of 1 mM ATCh by RS41A, and calculated
concentration of oximate anion for RS41A (calculated by MarvinView 5.2.6)
Triplicate experiments (given is mean Ϯ S.D.) were done in 0.1 M phosphate buffers.
Reactivation
Oximolysis
͓Oximate͔ @pH
pH
kobs
min
Ratio
Rate
⌬A/min
Ratio
Ratio
Ϫ1
%
6.4
7.4
8.4
0.19 Ϯ 0.01
0.16 Ϯ 0.03
0.17 Ϯ 0.05
1
0.023 Ϯ 0.003
0.120 Ϯ 0.007
0.760 Ϯ 0.096
1
0.02
0.20
1.5
1
0.84
0.89
6
10
34
75
on the in vitro data, the lead reactivator, oxime RS41A, is k2; Table 3) but relatively weak binding to OP-hAChE conju-
expected to reactivate OP-hAChE conjugates in vivo in blood gates (large Kox, Table 3) indicates that the lead molecule is
and simultaneously enter the CNS and reactivate inhibited amenable to further structural refinement to gain higher reac-
brain hAChE. The concentration dependence of RS41A reac- tivation potency. Modifying the size and character of both the
tivation with its respectable maximal reactivation rates (large aliphatic linker and the heterocycle of RS41A clearly affects
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Centrally Acting ChE Reactivators
reactivation (Table 1). Several discrete structural variations reduction of seizures and status epilepticus in OP-exposed
leading to compounds RS191D, RS191C, and RS186B exhib- guinea pigs (11) upon administration of pro-2PAM, an
ited minimal or slight reductions in overall reactivation potency uncharged centrally active analog of commonly used reactiva-
compared with RS41A. However, this should enable us system- tor 2PAM. Enzymatic conversion of pro-2PAM into 2PAM,
atically to vary the structure of the heterocyclic ring and its upon its diffusion into CNS is key to functionality of the pro-
distance from the oxime and then examine the influence of the drug concept whose maximal reactivation efficacy is limited to
␣-keto and ␣-carbamoxy moieties.
the efficacy of 2PAM. Initial reports of an alternative approach
Structural diversity of OP-hAChE conjugates on the other of conjugating pyridinium aldoximes to glucose where reacti-
hand was reflected in reactivation trends, as well. Although vator molecules rely on active transport of glucose into CNS,
varying oxime structures seemed to affect reactivation of meth- instead of simple diffusion, have also proved promising for in
ylphosphonyl OP-hAChE conjugates (derived from sarin, vitro reactivation experiments (9, 10) where 2PAM reactivation
cyclosarin, and VX inhibition) in a similar way, tabun- and levels were approached but not exceeded. These oxime deriva-
paraoxon-derived conjugates with larger phosphorus substitu- tives displayed very low in vivo toxicities. Finally, cationic oxi-
ents occupying the OP-hAChE acyl pocket had different struc- mes encapsulated into nanoparticle drug delivery system capa-
tural requirements and proved more refractory to reactivation. ble of penetrating the BBB (24, 25) need to be optimized for fast
This indicates that the most effective of our reactivators pref- release of therapeutically efficient doses into CNS to prove their
erably approach the conjugated phosphorus directly from the efficiency. The maximal efficacy for all of these approaches
active center gorge opening and the acyl pocket and not from depends on the intrinsic efficacy of involved respective pyridin-
the direction of the hAChE choline binding site. In support of ium aldoximes.
this conclusion, reactivation of OP-hBChE conjugates sharing a
larger acyl pocket space and active center gorge opening did not nucleophilic molecules unrelated to pyridinium aldoximes and
indicate faster reactivation of methylphosphonates. devoid of permanent positive charge, but amenable to protona-
In contrast, our approach is based on designing small organic
The lack of noticeable influence of intrinsic nucleophilic tion. The uncharged form of reactivators should spontaneously
oxime reactivity (reflected in nonenzymic ATCh oximolysis diffuse into CNS. While in ionization equilibrium, the proto-
experiments) as well as ionization state of the oxime moiety nated amine form is efficiently attracted to the electron-rich
(reflected in pH dependence experiments) on the overall oxime- active center environment of the inhibited AChE. We are thus
assisted reactivation emphasizes a decisive influence of proper able to gauge independently two distinct molecular properties
binding complementarity of reactivator molecules with the essential for the overall efficacy, CNS penetration and intrinsic
environment of the active center gorge opening of OP-hAChE reactivation potency.
conjugates.
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