Evaluation of monoquaternary pyridinium oximes potency to reactivate tabun-inhibited human acetylcholinesterase

Article abstract of DOI:10.1016/j.tox.2006.08.003

Monoquaternary N-benzyl-4-hydroxyiminomethylpyridinium bromide (Py-4-H) and its analogous with diverse substituents introduced into the phenyl ring (Py-4-CH₃, Py-4-Br, Py-4-Cl and Py-4-NO₂) were synthesized in order to examine their

Full text of DOI:10.1016/j.tox.2006.08.003

Toxicology 233 (2007) 85–96
Evaluation of monoquaternary pyridinium oximes potency to
reactivate tabun-inhibited human acetylcholinesterase
a
b
a
ˇ
Renata Od zˇ ak , Maja Cali c´ , Tomica Hrenar ,
a
b,∗
Ines Primo zˇ i cˇ , Zrinka Kovarik
a
Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia
Institute for Medical Research and Occupational Health, Ksaverska c. 2, P.O. Box 291, HR-10001 Zagreb, Croatia
b
Received 14 June 2006; received in revised form 20 July 2006; accepted 2 August 2006
Available online 7 August 2006
Abstract
Monoquaternary N-benzyl-4-hydroxyiminomethylpyridinium bromide (Py-4-H) and its analogous with diverse substituents intro-
duced into the phenyl ring (Py-4-CH , Py-4-Br, Py-4-Cl and Py-4-NO ) were synthesized in order to examine their potency as
reactivators of tabun-inhibited human erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7). Within 24 h, the reactivation of tabun-
inhibited AChE reached 80% with Py-4-CH , Py-4-Br and Py-4-Cl, 40% with Py-4-NO , and 30% with Py-4-H. The overall
. All oximes inhibited human AChE reversibly, and the inhibition potency
increased in the following order Py-4-Br < Py-4-Cl < Py-4-CH < Py-4-H < Py-4-NO . Although oximes Py-4-H and Py-4-NO did
3
2
3
2
−1
−1
M
reactivation rate constants were up to 5.0 min
3
2
2
not show significant reactivation ability, these oximes might be of interest as pre-treatment drugs due to their high affinity for the
native AChE. Docking studies were carried out to elucidate the differences in oximes potency. The orientations of all studied oximes
in the active site of human AChE have been proposed by flexible ligand docking with AutoDock 3.0. Analyses of the obtained
complexes revealed the presence of numerous hydrogen bonds and close contacts between the oximes and the residues in the active
site. Final docked energies predicted correctly the relative order of the inhibition potency of compounds (except in the case of
Py-4-CH
atom of the tabun-phosphorylated human AChE.
2006 Elsevier Ireland Ltd. All rights reserved.
3
) as well as the most probable orientation of the best reactivator, Py-4-Br, which can result in an attack on the phosphorus
©
Keywords: Acetylcholinesterase; Tabun; Oxime; Reactivation; Protection; Docking
1
. Introduction
dinium ring is an important structural factor for reacti-
vation efficacy. There is a relationship between position
of oxime group and nerve agent, because reactivators
containing oxime group in position two or four are
more effective compared to the oximes in the position
Oximes are reactivators of phosphylated acetyl-
cholinesterase (AChE; EC 3.1.1.7) and therefore, are
used in the therapy of poisoning by organophospho-
rus compounds (Dawson, 1994; Primo zˇ i cˇ et al., 2004).
The position of the oxime group at the quaternary pyri-
ˇ
three (Pang et al., 2003; Cali c´ et al., 2006). However,
to assume the one common rule of oxime position for
all nerve agents is unrealistic. Since reactivators with
oxime group at the pyridinium ring in position four are
currently considered to be the most potent for reacti-
vation of tabun inhibited acetylcholinesterase (De Jong
∗
Corresponding author. Tel.: +385 1 46 73 188;
fax: +385 1 46 73 303.
E-mail addresses: zrinka.kovarik@imi.hr, zkovarik@imi.hr
ˇ
et al., 1989; Cabal et al., 2004; Cali c´ et al., 2006), we
(
Z. Kovarik).
0
300-483X/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2006.08.003

8
6
R. Od zˇ ak et al. / Toxicology 233 (2007) 85–96
2.2. Determination of oxime pK
Dissociation constant (K ) was determined from pH-
synthesized five monoquaternized 4-pyridinium oxime
derivatives, and investigated their ability to reactivate
human erythrocyte AChE inhibited by tabun. We also
determined their pKa values and calculated their pro-
tective potency against AChE inhibition by tabun due
to oxime reversible binding to the active site of AChE
a
a
absorbance profiles maximums (for protonated and deproto-
◦
nated ionic form) at 25 C using Eq. (1) as described earlier
ˇ
(
Cakar et al., 1999; Foreti c´ and Burger, 2004):
+
(Harris et al., 1978; Reiner, 1986; Simeon-Rudolf et
A [H ] + A K
1
2
a
A =
(1)
ˇ
ˇ
+
al., 1998; Skrinjari c´ -Spoljar et al., 1999; Lallement
et al., 2002; Hammond et al., 2003, Eckert et al.,
[H ] + K
a
The observed absorbance at a specific absorption wavelength,
A, is the sum of the absorption fractions of all oxime ionization
species present (Foreti c´ and Burger, 2004). A
and A are the
1 2
2
006).
The crystal structures of AChE (Sussman et al., 1991;
Bourne et al., 1995; Kryger et al., 2000, Ekstr o¨ m et al.,
006) provide templates for detailed structural studies
absorbance of the pure protonated and deprotonated species,
respectively. The concentration of hydrogen ions in medium is
2
+
on ligand access to the free and impacted enzyme active
center gorge and steric constraints within the active cen-
ter gorge that govern selectivity in oxime reactivation
represented by [H ].
®
A SevenEasy pH-meter with an InLab 413 electrode (both
Mettler-Toledo GmbH, Switzerland) was used for pH measure-
ments with the uncertainty of ± 0.02 pH units. The pH-meter
(Ashani et al., 1995; Grosfeld et al., 1996; Wong et al.,
◦
was calibrated at 25 C using the two-point calibration method
2
000; Kovarik et al., 2004). The site of conjugation by
with commercially available Mettler-Toledo standard buffer
solutions pH 7.00 and 9.21. Oximes spectra were recorded at
different pH on the Cary 300 spectrophotometer with tempera-
ture controller (Varian Inc., Australia) in 1 cm matched quartz
cuvette.
organophosphorus compounds lies at the base of a nar-
row and 18–20 A deep gorge. The gorge wall of AChE
is lined largely by aromatic side chains contributing to a
well-defined acyl pocket and choline binding site at the
base of the gorge (Radi c´ et al., 1993; Ordentlich et al.,
˚
1
993). The orientation of the associated oximes within
2
.3. AChE activity measurement
narrow confines of the gorge when active serine is phos-
phorylated is important determinant of the reactivation
mechanism. Therefore, docking studies were carried out
to evaluate the energy-minimized oximes conformations
in the active site of human AChE and compare it with
oxime conformations in the active site when active site
serine is phosphorylated by tabun.
The source of AChE was native intact human erythrocytes.
Blood was taken into tubes containing heparin, centrifuged
for 20 min at 2500 rpm. Erythrocytes were washed twice and
diluted with 0.9% sodium chloride to the original blood vol-
ume. The final dilution of erythrocytes during enzyme assay
was 400-fold. All experiments were done in 0.1 M phosphate
◦
buffer, pH 7.4, at 25 C and the substrate was acetylthiocholine
iodide (ATCh; Sigma Chemical Co., St. Louis, MO, USA). The
enzyme activity was measured spectrophotometrically accord-
ing to the Ellman procedure (Ellman et al., 1961) with the
thiol reagent 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB; final
concentration 0.3 mM; Sigma Chemical Co., St. Louis, MO,
USA). The increase in absorbance was read at 436 nm up to
2
. Materials and methods
ꢀ
2
.1. Synthesis of oximes
Five monoquaternary oximes were prepared by the addi-
2
min. All spectrophotometric measurements were performed
on a CARY 300 spectrophotometer with temperature controller
Varian Inc., Australia).
tion of appropriate reagents for quaternization: benzyl bro-
mide, 4-methylbenzyl bromide, 4-bromobenzyl bromide, 4-
chlorobenzyl bromide and 4-nitrobenzyl bromide; in equimo-
lar quantities to the solution of 4-hydroxyiminomethylpyridine
(
(
100 mg, 0.819 mmol) in dry acetone (2 mL). All oximes were
2.4. Oxime-assisted reactivation of tabun-inhibited AChE
newly synthesized, except oxime Py4-H that was firstly syn-
thesized by De Jong et al. (1989) using different protocol. All
reagents and chemicals were purchased from Aldrich, USA.
The reaction mixtures were kept at room temperature without
stirring in the dark for 2–3 days. The crystallization was spon-
taneous. Acetone was removed and white crystals were washed
with dry diethyl ether and obtained in very good yields. All syn-
The undiluted erythrocyte AChE was incubated with 5 ␮M
tabun(ethylN,N-dimethylphosphoroamidocyanidate;NCLab-
oratory, Spiez, Switzerland) up to 60 min achieving 100%
inhibition. The excess of tabun was extracted with a 5-fold
volume of hexane without effect on the enzyme activity. Incu-
bation mixture was diluted 10-times with 0.1 M phosphate
buffer, pH 7.4, containing the oxime to start the reactivation.
Final oxime concentrations used for the reactivation of tabun-
inhibited AChE are given in Table 2. After a specific time of
reactivation aliquots were diluted 40-times in 0.1 M sodium
1
thesized monoquaternary oximes were identified by IR, 1D ( H
1
3
and C (APT)) and 2D (HETCOR) NMR spectroscopies. Ana-
lytical and spectral data of synthesized compounds are given
in Table 1.

R. Od zˇ ak et al. / Toxicology 233 (2007) 85–96
87
Table 1
Physical data and general structure of prepared oximes
a
◦
c
−1
1
d
13
d
Compound
I
R
H
mp ( C)
Yield Elemental
IR (cm
)
H NMR (ppm)
C NMR (ppm)
b
(%)
analysis
192.5–193.5e 92
C 53.26%, H
3493, 3037,
2924, 1644,
1453, 1293,
5.82 (s, 2H, CH2 Bnl),
7.42–7.45 (m, 3H, Bnl),
7.51–7.52 (m, 2H, Bnl), 8.20 C-6 Bnl), 130.71 (C-3 and C-5
(d, J = 6.60, 2H, C-3 and C-5 Bnl), 130.99 (C-4 Bnl), 134.68
65.16 (CH2 Bnl), 125.68 (C-3
and C-5 Py), 130.21 (C-2 and
4.47%, N 9.56%;
found C 53.37%,
H 4.54%, N 9.66% 1155, 999,
50
7
Py), 8.29 (s, 1H, CH N),
.00 (d, J = 6.63, 2H, H-2
(C-1 Bnl), 145.60
9
(CH NOH), 145.97 (C-2 and
C-6 Py), 151.54 (C-4 Py)
20.83 (CH3), 64.68 (CH2 Bnl),
and H-6 Py)
2.33 (s, 3H, CH3), 5.76 (s,
II
CH3 172.5–173.3 96
C 54.74%, H
3311, 3112,
44.92%, N 9.12%; 2995, 1644,
2H, CH2 Bnl), 7.26–7.27 (d, 125.24 (C-3 and C-5 Py),
found C 55.08%,
H 5.01%, N 9.35% 1004, 765
1458, 1311,
J = 7.87, 2H, H-2 and H-6
Bnl), 7.41–7.42 (d, J = 7.74
129.82 (C-2 and C-6 Bnl),
130.88 (C-3 and C-5 Bnl),
131.16 (C-1 Bnl), 141.01 (C-4
2H, H-3 and H-5 Bnl), 8.20
(
d, J = 6.45, 2H, H-3 and H-5 Bnl), 145.20 (CH NOH),
Py), 8.29 (s, 1H, CH N),
145.43 (C-2 and C-6 Py),
8.97(d, J = 6.48, 2H, H-2 and 151.12 (C-4 Py)
H-6 Py)
III
Br
214–214.4
decomp.)
90
C 41.97%, H
3.25%, N 7.53%;
found C 41.79%,
3313, 3071,
2924, 1636,
1461, 1295,
5.75 (s, 2H, CH2 Bnl), 7.42
(d, J = 8.40, 2H, H-3 and H-5 Bnl), 125.13 (C-3 and C-5 Py),
Bnl), 7.57 (d, J = 8.41, 2H,
H-2 and H-6 Bnl), 8.18 (d,
J = 6.67, 2H, H-3 and H-5
Py), 8.26 (s, 1H, CH N),
63.70 (CH2 Bnl), 124.52 (C-1
(
131.53 (C-2 and C-6 Bnl),
133.22 (C-3 and C-5 Bnl),
133.28 (C-4 Bnl), 144.99
(CH NOH), 145.45 (C-2 and
C-6 Py), 151.13 (C-4 Py)
H 3.40%, N 7.71% 1005, 787
8.95 (d, J = 6.72, 2H, H-2
and H-6 Py)
IV
Cl
207.4–208.3 92
decomp.)
C 47.66%, H
3.69%, N 8.55%;
found C 47.61%,
3423, 3118,
2995, 1645,
1451, 1278,
5.78 (s, 2H, CH2 Bnl),
7.41–7.51 (m, 4H, Bnl), 8.19 and C-5 Py), 130.39 (C-2 and
(d, J = 6.83, 2H, H-3 and H-5 C-6 Bnl), 131.53 (C-3 and C-5
63.85 (CH2 Bnl), 125.33 (C-3
(
H 3.73%, N 8.81% 1001, 782
Py), 8.27 (s, 1H, CH N),
Bnl), 133.01 (C-1 Bnl), 136.64
8
2
.96 (d, J = 6.85 H-2 and H-6 (C-4 Bnl), 145.19
H, Py)
(CH NOH), 145.63 (C-2 and
C-6 Py), 151.33 (C-4 Py)
V
NO2 187.4–187.8 95
decomp.)
C 46.17%, H
3.58%, N 12.43%; 2959, 1643,
3411, 3017,
5.96 (s, 2H, CH2 Bnl), 7.72
(d, J = 8.67, 2H, H-3 and H-5 and C-6 Bnl), 125.98 (C-3 and
63.96 (CH2 Bnl), 125.58 (C-2
(
found C 45.96%,
H 3.69%, N
1515, 1347,
1008, 731
Bnl), 8.24 (d, J = 6.71, 2H,
H-3 and H-5 Py), 8.26 (d,
J = 8.71, 2H, H-2 and H-6
Bnl), 8.30 (s, 1H, CH N),
C-5 Py), 131.31 (C-3 and C-5
Bnl), 141.57 (C-1 Bnl), 145.70
(CH NOH), 146.51 (C-2 and
C-6 Py), 150.13 (C-4 Bnl),
152.10 (C-4 Py)
12.56%
9.02 (d, J = 6.73, 2H, H-2
and H-6 Py)
a
Melting points were determined in open capillary using a B u¨ chi B-540 melting point apparatus and are uncorrected.
Elemental analyses were performed with a Perkin-elmer PE 2400 Series II CHNS/O Analyser.
Infrared spectra (KBr pellets) were obtained on a Bruker VECTOR 22 FT-IR spectrometer.
b
c
d
e
1
13
H and C NMR spectra were recorded on a Bruker AV300 spectrometer (in MeOH-d4, internal standard Me4Si).
◦
mp 193–195 C by De Jong et al. (1981).

8
8
R. Od zˇ ak et al. / Toxicology 233 (2007) 85–96
Table 2
Reactivation of tabun-inhibited human erythrocyte acetylcholinesterase by tested oximes (constants ± S.E.)
k+2 (min 1)
−
KOX (mM)
kr (min
−1 −1
M
)
%Reactmax
Time (h)
Oxime
mM
Py-4-H
0.5, 1.0, 2.0
1.0, 2.0, 4.0
1.0, 2.0, 4.0
1.0, 2.0, 4.0
0.5, 1.0, 2.0
0.0008 ± 0.0005
0.0029 ± 0.0016
0.0030 ± 0.0004
0.0032 ± 0.0004
0.0012 ± 0.0004
0.65 ± 1.06
0.77 ± 1.48
0.60 ± 0.34
0.90 ± 0.38
0.88 ± 0.66
1.2 ± 2.1
3.8 ± 7.5
5.0 ± 2.9
3.6 ± 1.6
1.4 ± 1.1
30
80
80
80
40
23
20
16
23
25
Py-4-CH3
Py-4-Br
Py-4-Cl
Py-4-NO2
phosphate buffer containing DTNB (0.3 mM final concentra-
tion). ATCh (1.0 mM final concentration) was added, and the
enzyme activity was measured. An equivalent sample of unin-
hibited enzyme was diluted to the same extent as the inhibited
AChE, and control activity was measured in the presence of
oxime in concentrations used for reactivation. Both activi-
ties of control and reactivation mixtures were corrected for
oxime-induced hydrolysis of ATCh. Under these conditions,
the enzyme activity in the absence of the oxime was stable
and no spontaneous reactivation of the phosphorylated enzyme
took place.
where v(EP+OX)t is the activity of reactivated enzyme at time
t and v(E+OX) stands for the activity of enzyme incubated
with oxime. Since 100 − %Reactivation = 100 × [EP]
t 0
/[EP] ,
one can relate the experimental data to Eq. (2). At each oxime
concentration, kobs was calculated from the slope of the initial
portion of log(100 − %Reactivation) versus time of reactiva-
tion. Reactivation with each oxime followed Eq. (2). Therefore,
k
+2 and KOX were obtained by the non-linear fit of the relation-
r
ship between kobs versus [OX], while k was calculated using
Eq. (3).
Oxime reactivation of phosphorylated AChE proceeds
according to Scheme 1:
2.5. Reversible inhibition of AChE by oximes
where EP is the phosphorylated enzyme, [EP][OX] is the
reversible Michaelis type complex between EP and the oxime
Reversible inhibition of AChE was measured in a medium
which contained erythrocytes suspended in buffer, DTNB, the
oxime and ATCh. The concentrations of the oxime and sub-
strate are given in Table 4.
(
OX), E is the active enzyme and P-OX the phosphorylated
oxime, k+2 is the maximum first order rate constant and k is
r
the overall second order rate constant of reactivation. Scheme 1
is defined by the following equation:
The inhibition constants were evaluated from the effect of
substrate concentration (s) on the degree of inhibition accord-
ing to the equation:
[
EP]0
k
+2[OX]
ln
= K
× t = kobs
t
(2)
[
EP]t
OX + [OX]
t
and [EP] are concentrations of phosphorylated
v
i
× i
K
(I)
K
app = v
= K(I) + K × s
(5)
0
− v
i
(S)
where [EP]
0
enzyme at time zero and at time t, respectively. Kox is equal to
the ratio (k−1 + k+2)/k+1, and it usually approximates the disso-
ciation constant of the [EP][OX] complex. kobs is the observed
first order rate constant of reactivation at any given oxime
concentration. The overall second order rate constant of reac-
K
app is the apparent enzyme–oxime dissociation constant at a
given substrate concentration (s), calculated from the enzyme
activities v and v measured in the absence and in the pres-
0
ence of the oxime concentration (i), respectively. K(I) is the
enzyme–oxime dissociation constant of a complex formed
in the catalytic site (K
ꢀ
tivation (k
r
) was the ratio:
i
) or peripheral site (K ). K(S) is the
i
enzyme–substrate dissociation constant corresponding either
to the Michaelis constant (K ) or to the substrate inhibition
constant (Kss). The values of K(I) and K(S) were intercepts of
k
+2
OX
k
r
=
(3)
M
K
the line on the ordinate and abscissa, respectively.
Experimental data were presented as percentage of reactiva-
tion:
2
.6. Protection of AChE against phosphorylation
v
(EP+OX)t
%
Reactivation =
× 100
(E+OX)
(4)
v
Protective indexes for all tested oximes were evaluated the-
oretically. Such calculated protective index (PIcalc) could be
applied to any phosphylating agent, because it only depends on
the enzyme–oxime dissociation constant (Reiner, 1986). When
oxime binds only to the catalytic site, the protective index is:
i
PIcalc = 1 +
(6)
K
i
Scheme 1.
where i stands for oxime concentration.

R. Od zˇ ak et al. / Toxicology 233 (2007) 85–96
.7. Oxime catalysed hydrolysis of acetylthiocholine
The reaction of ATCh with the oxime was measured in
89
2
a medium which contained ATCh, oxime, DTNB and 0.1 M
◦
phosphate buffer, pH 7.4, at 25 C. The increase in absorbance
was read for 1 min against a blank that contained buffer and
DTNB. Concentrations of the oximes and of the ATCh are
given in Table 4. Two to four measurements were done with
each ATCh/oxime concentration pair. The second-order rate
constant of the reaction (k) was calculated according to:
c
=
k × s × i
(7)
t
Fig. 1. Two forms (protonated oxime group NOH and deprotonated
−
where c is the released thiocholine concentration, t the time of
reaction, and s and i are the initial ATCh and oxime concen-
trations. Initial concentrations were used because the time of
reaction was short and the decrease in concentration did not
need to be taken into account.
oxime group NO ) of the each ligand used for docking simulations,
with rotatable bonds highlighted with arrows.
˚
grid was at the distance of 9.3 A from the ␥-oxygen atom of
Ser203 to the rim of the gorge. The dimension of the active site
˚
˚
˚
box was set at 30 A × 30 A × 30 A, which ensured an appropri-
ate size of the oxime-accessible space. The consistencies of the
maps were ascertained by checking the minimum and maxi-
mum values of the van der Waals energies and electrostatic
potentials for each calculated grid map.
2
2
.8. Molecular modelling studies
.8.1. Structure of AChE and oximes
The coordinates for the enzyme were those deposited in the
Protein Data Bank (PDB) for the human acetylcholinesterase
ID code 1B41; Kryger et al., 2000) after eliminating the
inhibitor (Fasciculin-2) and water molecules. In the same
enzyme, tabun reaction product covalently attached to Ser203
was modelled according to the crystal structure of the non-
aged form of mouse acetylcholinesterase inhibited by tabun
Flexible ligand docking was performed for all synthe-
sized compounds. Two forms of each oxime with protonated
( NOH) or deprotonated ( NO ) oxime group were used for
(
−
docking simulations (Fig. 1). Docking calculations were car-
ried out using the Lamarckian genetic algorithm (LGA) and all
parameters were the same for each docking. We used initially
a population of random individuals (population size: 500), a
maximum number of 3,000,000 energy evaluations, a maxi-
mum number of generations of 300,000, an elitism value of 1
and a mutation rate of 0.08. For the local search, the pseudo
Solis and Wets method was used: maximum of 300 iterations
per local search, the probability of performing a local search on
an individual in the same population was 0.06, the maximum
number of consecutive successes or failures before doubling
or halving the local search step size was 4 in both cases and
the termination criterion for the local search was 0.01. At the
end of a docking procedure (100 docking runs), the resulting
positions were clustered according to a root mean square crite-
(
PDB, ID code 2C0Q; Ekstr o¨ m et al., 2006). We combined
in our docking study two crystal structures of AChE (human
and mouse) because (a) we wanted to compare our kinetic data
obtained on human AChE experiments with docking and (b)
only mouse AChE structure with tabun is available.
Using the AutoDock 3.0 suite of programs (Morris et al.,
1
998), we added polar hydrogen atoms to amino acid residues.
Gasteiger–Marsili atomic partial charges were assigned to all
atoms of the enzyme (Gasteiger and Marsili, 1980). Then,
atomic solvation parameters and fragmental volumes were
assigned to the protein atoms using AutoDock’s AddSol utility.
Quantum chemical calculations for all tested oximes were
carried out using the Gaussian 03 program (Frisch et al., 2004).
Geometries were optimized by the density functional theory
˚
rion of 0.5 A. To ensure the reliability of the results, the docking
procedures were repeated 10 independent times for each oxime
and the obtained orientations were analyzed.
(
6
DFT) method using the B3LYP functional (Becke, 1993) and
-31G(d) basis set. Further preparation of the oximes included
addition of Mulliken’s atomic charges, removal of non-polar
hydrogen atoms and addition of their atomic charges to heavy
atoms, and finally, assignment of proper atomic types. Auto-
Tors program was then used to define the rotatable bonds in
the oximes (Fig. 1).
3
. Results and discussion
The kinetic parameters for reactivation of tabun-
inhibited human AChE by the oximes are presented in
Table 2 and one representative experiment is displayed
in Fig. 2. The highest reactivation was achieved with
oximes Py-4-Br, Py-4-CH3 and Py-4-Cl. Oxime without
substituent at the benzyl ring (Py-4-H) or with nitro sub-
stituent (Py-4-NO2) had poor reactivation efficacy and
the maximum extent of the reactivation was below 40%
within 24 h. This is in accordance to previous Py-4-H
2
.8.2. Docking simulations
All docking studies were performed by using the AutoDock
.0 suite of programs(Morrisetal., 1998). AutoDock requires a
3
pre-calculated electrostatic grid map for each atom type present
˚
in the oxime molecule. All maps were calculated with 0.3 A
spacing between grid points and in all cases the center of the

9
0
R. Od zˇ ak et al. / Toxicology 233 (2007) 85–96
Fig. 3. Spectrophotometric determination of the pKa value of the
oxime Py-4-Br: (A) absorption spectra of Py-4-Br (0.1 mM) at dif-
ferent pH (6.02–10.52) and (B) determination of Py-4-Br pKa from
the peak absorbance at various pH at 281 nm (ꢁ) and 342 nm (᭹).
Solid and dotted lines represent non-linear regression by Eq. (1).
between two rings should be longer than one –CH2 group
to overcome the rigidness of the oxime molecule, and to
provide the right distance for the second ring stabiliza-
tion in the active site of AChE during reactivation. An
additionalchargeonthesecondringwouldprobablyhelp
stabilization even more.
Fig. 2. Reactivation of tabun-inhibited human erythrocyte AChE by
oxime Py-4-Br: (A) a single data point indicates calculated percent
of reactivation using Eq. (4) after designated time of oxime Py-4-Br
assisted-reactivation, (B) slopes of the reactivation curve yield kobs
constants and (C) kobs is plotted as a function of Py-4-Br and the curve
is fitted using Eq. (2).
Since the deprotonated oxime group is involved in the
nucleophilic attack during the reactivation, and its con-
centration is pH-dependant (Aldridge and Reiner, 1972;
ˇ
Sinko et al., 2006), we determined pKa values of the
assisted reactivation studies on bovine and rat AChE
inhibited by tabun (De Jong et al., 1989; Cabal et al.,
oximes. The spectrophotometric measurements of the
oximes spectra (200–400 nm, pH range between 6 and
10.5) showed two characteristic maximums in the UV
region (Fig. 3), first at 281 nm (282 for Py-4-NO2) and
second at 342 nm (346 for Py-4-NO2). These maximums
corresponded to the absorption of different oxime ion-
2
004). Since the aging of tabun-inhibited AChE is rel-
atively slow (t1/2 = 13 h; Heilbronn, 1963), reactivation
seemed to mainly depend on the oxime structure. All
oximes displayed up to 500 times lower tabun-inhibited
AChE reactivation potency than bispyridinium oximes
TMB-4, Hl o¨ -7, obidoxime, K048 or K027 (Worek et al.,
*
ization forms as a result of ␲ → ␲ transitions within the
pyridinium oxime aromatic system (Stern and Timmons,
1970). The first maximum around 280 nm was a result of
the absorption of protonated oxime group and the max-
imum above 340 nm was a result of the absorption of
deprotonated oxime group. The sharp isobestic point
ˇ
2
004; Cali c´ et al., 2006), even if they had the oxime
group in the position four on the pyridinium ring. Their
potency is comparable to that of the monopyridinium
oxime 2-PAM (Worek et al., 2004). It seems that linker

R. Od zˇ ak et al. / Toxicology 233 (2007) 85–96
91
Table 3
Eckert et al., 2006). Therefore, we measured reversible
inhibition of human erythrocyte AChE by the oximes
(Table 4). In the studied concentration range of acetylth-
iocholine, the oxime binding to the catalytic site was
competitive as shown in Fig. 4. The evaluated disso-
ciation constants for reversible inhibition of AChE are
given in Table 4. The catalytic site of AChE showed
Dissociation constants (pKa) for the oximes
Oxime
pKa ± S.D.
%Deprotonated
at pH 7.4
Py-4-H
8.55 ± 0.04
8.60 ± 0.04
8.55 ± 0.04
8.56 ± 0.04
8.48 ± 0.04
7.1
6.3
7.1
6.9
8.3
Py-4-CH3
Py-4-Br
Py-4-Cl
Py-4-NO2
the highest affinity (lowest K ) for oxime Py-4-NO2,
i
5-fold higher than oxime Py-4-Br, which had the low-
est affinity, while affinities of the rest oximes lied in
at 305–308 nm referred to the acid–base equilibrium.
Oxime dissociation constants (Ka) were obtained from
the absorbance maximums, for protonated and deproto-
nated ionic forms, by the non-linear fitting using Eq. (1)
between. Evaluated Ki constants were more similar to
constants published for bispyridinium compounds (Calic´
ˇ
et al., 2006) than for monopyridinium oxime 2-PAM
(Simeon et al., 1981) showing that the second ring,
even without charge, improved stabilization of the oxime
binding. The KM values, derived from the kinetics of
inhibition with oximes were higher, but close to the
determined KM previously (Bosak et al., 2005). This
supports our belief that determined Ki constants corre-
sponded to binding to the catalytic site. However, the
binding affinity of free enzymes for oximes does not
correlate with the oxime efficacy in enzyme reactivation
(cf. Table 2). The phosphorylated AChE had lower affini-
ties than free enzyme for all oximes i.e. KOX constants
were 5–20 times higher than the respective Ki. Neverthe-
less, the relevant enzyme–oxime dissociation constant
should be perhaps determined before doing reactivation
experiments, because they might indicate the binding
affinity of phosphorylated enzyme which is expected to
be lower (Kovarik et al., 2006). On the other hand, effi-
cacy of oxime-mediated reactivation depends mainly on
displacement of the phosphoryl moiety from the active
(Table 3). Our results showed that all oximes had similar
pKa values around 8.50. We calculated the percentage
of deprotonated oxime for our experimental conditions
(physiological pH 7.4). At physiological pH the studied
oximes would be only about 6–8% deprotonated, which
could be one of the reasons for slow reactivation rates.
Such results are in agreement with previous findings on
pyridinium oximes with oxime group in position 4 on
ˇ
the pyridinium ring (Sinko et al., 2006) and pointing out
that pKa values should be taken into consideration while
searching for new oxime-reactivators.
Besides acting as reactivators, oximes are also
reversible inhibitors of AChE and therefore they could
protect the catalytic site against phosphorylation due to
a direct competition between the oxime and the phos-
phorylating agent (Harris et al., 1978; Reiner, 1986;
ˇ
ˇ
Simeon-Rudolf et al., 1998; Skrinjari c´ -Spoljar et al.,
999; Lallement et al., 2002; Hammond et al., 2003;
1
Fig. 4. Reversible inhibition of human erythrocyte AChE by the studied oximes. Points indicate the calculated average Kapp from measured activities
with acetylthiocholine in the presence of oximes using Eq. (5). Lines represent linear regression analysis and y-intercepts represent Ki constants.

9
2
R. Od zˇ ak et al. / Toxicology 233 (2007) 85–96
Table 4
Reversible inhibition of human erythrocyte acetylcholinesterase by the oximes, calculated protective index of tested oximes against phosphylation
of human erythrocyte acetylcholinesterase and non-enzymatic reaction of the acetylthiocholine with tested oximes (constants ± S.E.)
−
1
−1
M )
Oxime
mM
ATCh (mM)
Ki (mM)
KM (mM)
PIcalc
k (min
Py-4-H
0.02–0.3
0.05–0.5
0.05–0.5
0.05–0.4
0.03–0.2
0.1–1.0
0.1–1.0
0.1–1.0
0.1–1.0
0.1–1.0
0.038 ± 0.006
0.063 ± 0.020
0.10 ± 0.01
1.72 ± 0.76
0.36 ± 0.13
1.48 ± 0.33
1.34 ± 0.28
0.58 ± 0.13
3.6
2.6
2.0
2.2
5.0
7.3 ± 0.2
19.3 ± 0.2
15.0 ± 0.2
14.9 ± 0.6
16.1 ± 0.5
Py-4-CH3
Py-4-Br
Py-4-Cl
Py-4-NO2
0.085 ± 0.008
0.025 ± 0.004
ˇ
serine. Due to this fact, the contribution of reversible
binding in reactivation reactions is not the most rele-
vant one. As reversible inhibitors oximes can protect the
catalytic serine against phosphorylation, meaning that,
although oxime-mediated reactivation is poor, oximes
could be used as prophylactic agents (Cali c´ et al., 2006).
The protective index (PI) is an important parameter for
oxime treatment, especially when reactivation is slow.
We calculated tested oximes protective potency against
phosphorylation by Eq. (6) (Table 4). All oximes had
Fig. 5. Orientations of geometries with the lowest docked energies of all ligands in the active site of human AChE represented by some structurally
important amino acids. The green ball and stick model represents Py-4-Br ( NOH) and the blue one Py-4-H ( NOH) with a hydroxyl oxime group
emphasized. Hydrogen atoms, except the polar ones, are omitted. Hydrogen bonds between atoms of the ligands and amino acids are highlighted
with the dotted lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

R. Od zˇ ak et al. / Toxicology 233 (2007) 85–96
Table 5
93
a relatively good protective potency. The highest pro-
The mean of final docked energies (FDEm) and the lowest docked
energies (FDEl) obtained by AutoDock simulations for all oximes for
the protonated ( NOH) and deprotonated form ( NO ) of the oxime
tection potential had oxime Py-4-NO2, which exhibited
the highest binding affinity to native AChE. Therefore,
although oximes Py-4-NO2 and Py-4-H did not show
significant reactivation ability (cf. Table 2), these oximes
might be of interest as pre-treatment drugs due to their
high affinity for the native AChE.
−
group
Oxime
FDEm (kcal mol 1)
−
−1
FDEl (kcal mol )
Py-4-H ( NOH)
−11.13
−10.50
−11.57
−10.83
−
Py-4-H ( NO )
It has been shown earlier that oximes react with
AChE substrate, acetylthiocholine, on a 1:1 molar basis
Py-4-CH3 ( NOH)
−11.21
−11.94
−
Py-4-CH3( NO )
−10.64
−11.58
whereby thiocholine is one of the reaction products
ˇ
(
Simeon et al., 1981; Sinko et al., 2006). All five studied
Py-4-Br ( NOH)
−10.96
−10.36
−11.72
−11.29
−
monopyridinium oximes reacted with acetylthiocholine
inanon-enzymaticreaction. Therateconstantforoxime-
catalysed hydrolysis of acetylthiocholine was calculated
by Eq. (7) (Table 4). All five rate constants were very
similar to rate constant obtained for 2-PAM, monopyri-
dinium oxime (Simeon et al., 1981).
Py-4-Br ( NO )
Py-4-Cl ( NOH)
−11.05
−11.87
−
Py-4-Cl ( NO )
−10.47
−11.49
Py-4-NO2 ( NOH)
−11.33
−10.87
−12.37
−11.93
−
Py-4-NO2 ( NO )
We performed docking simulations for the protonated
and deprotonated oxime group for all ligands in the
human AChE. Final mean docked energies for proto-
nated and deprotonated oxime forms (FDEm), presented
in Table 5, were calculated as average values of ener-
gies for all obtained geometries within the active site.
AutoDock predicted the strongest affinity for Py-4-NO2
and the lowest affinity to the enzyme for Py-4-Br, which
is in good agreement with the experimental data (cf.
Table 4). Affinities for the rest of the ligands fell in
between, and only the value for the Py-4-CH devi-
3
ated, probably because the AutoDock program finds
H and CH groups very similar to observe the dif-
3
Fig. 6. Orientations of all geometries for Py-4-NO2 ( NOH) in the active site of human AChE represented by some structurally important amino
acids obtained by AutoDock simulation. Hydrogen atoms, except the polar ones, are omitted. Hydrogen bonds between the oxime group and amino
acids are highlighted with the dotted lines.

9
4
R. Od zˇ ak et al. / Toxicology 233 (2007) 85–96
ferences in the calculations. So, the predicted inhibi-
tion potency increased in the following order: Py-4-Br
only the oxime group is pointed toward the His447 and
is making a H-bond with its carbonyl group (Fig. 5, blue
model). The green model in Fig. 5 is the geometry of
Py-4-Br ( NOH) with oxime group pointed toward the
rim of the active site gorge and making two H-bonds with
Tyr72 and Asp74. Aromatic rings are stabilized with the
␲–␲ interactions with Trp86 and T-shaped complex with
Phe338.
The results obtained from flexible ligand docking
revealed that all oximes have similar interactions in the
active site. In Fig. 6, all geometries of the Py-4-NO2,
the most potent reversible inhibitor of the enzyme, are
presented. It can be seen that ligand is oriented in such
a way that the hydrogen bond is always formed between
the oxime oxygen and various amino acids. Furthermore
aromatic rings of the ligand are stabilized via the ␲–␲
interactions mainly with Trp86, occupying the choline-
binding site. Some clusters of geometries are found to
be positioned toward the rim of the active site gorge
making H-bonds with the Thr75 and Ser293 and ␲–␲
<
Py-4-Cl < Py-4-H < Py-4-CH3 < Py-4-NO2. In the case
−
of deprotonated form ( NO ) of the oxime group, cal-
culated mean docked energies were slightly higher than
that for the protonated form indicating weaker affinity
for the enzyme (Table 5).
In Fig. 5, the geometries of the most stable oxime-
AChE complexes in the active site of the enzyme were
superimposed. It can be seen that regardless of the sub-
stituent on the benzyl ring and protonated or deproto-
nated oxime groups, there is a region in the choline-
binding site which all ligands favour (except Py-4-Br
(
NOH)). That geometry is stabilized with the H-bond
between the Ala127 amide nitrogen and the oxime oxy-
gen atom. Furthermore, there is ␲–␲ stabilization of the
pyridinium and benzyl ring with the Trp86. The benzyl
ring is also stabilized with Tyr337. The orientation of
the aromatic rings of Py-4-H with the protonated oxime
group is in a way similar to geometries described above;
−
Fig. 7. Selected geometry of the Py-4-Br ( NO ) in the active site of human AChE inhibited with tabun. Oxime oxygen is pointed toward the
phosphorus atom, distance being ∼5.3 A˚ . Hydrogen atoms, except the polar ones, are omitted.

R. Od zˇ ak et al. / Toxicology 233 (2007) 85–96
95
interactions with some aromatic amino acids present in
the active site gorge. The main difference in binding of
Py-4-Br and Py-4-NO2 is in that region; in the case of Py-
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4
-Br there are much more clusters of geometries which
270, 6370–6380.
is probably due to the size of that ligand. For Py-4-H
none of the clusters in that region were found.
Based on the knowledge of the mechanism of reacti-
vation of AChE inhibited by tabun, we have chosen the
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most probable orientation of the best reactivator Py-4-Br
1
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AChE–tabun–oxime interactions. Such visualization
helped us to consider possible solutions for the oxime
structure optimisation that would lead to a greater reac-
tivation potency of oximes to reactivate human AChE
inhibited by tabun.
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M.A., Cheeseman, J.R., Montgomery Jr., J.A., Vreven, T., Kudin,
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V., Mennucci, B., Cossi, M., Scalmani, G., Rega, N., Petersson,
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R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao,
O., Nakai, H., Klene, M., Li, X., Knox, J.E., Hratchian, H.P.,
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R.E., Yazyev, O., Austin, A.J., Cammi, R., Pomelli, C., Ochterski,
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Acknowledgement
–
The authors thank Professor Srdanka Tomi c´ for gen-
eral support and for discussion throughout the study.
We also thank Dr. Elsa Reiner and Dr. Zoran Radi c´ for
critical review of the manuscript and their valuable sug-
gestions. The work was supported by the Ministry of
Science, Education and Sports of the Republic of Croa-
tia (Grant No. 0022014 and 0119610) and by the NATO
Reintegration Grant (EAP.RIG.981791).
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