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Z. Wei et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5743–5748
inhibition of hAChE
inhibition of hAChE
not washed
oxime concentration(1 mM)
oxime concentration(0.1 mM)
120
110
100
90
80
70
60
50
40
30
20
10
0
110
100
90
80
70
60
50
40
30
20
10
0
washed once
Figure 4. Inhibition of hAChE by conjugates 3, 7a–7h and the reference oximes at different concentrations (1 mM and 0.1 mM, left figure) and under different treatments
(1 mM, right figure). Data depicted shows the average and standard deviation (calculated as a fraction of positive control samples without oximes) for AChE activity (n = 3
incubations per oxime) after inhibition. Unless otherwise indicated, the standard deviation bars were smaller than the data marker.
Considering the fact that the inhibition ability would help to pro-
vide an insight into the oximes binding affinity for hAChE, and a
strong or irreversible inhibitor of AChE would lead to toxicity,37,38
we firstly evaluated the AChE inhibition potency of these new
reactivators (7a–7h and 3).
to the poisoned control (85–98% AChE inhibition) and the results
are shown in Table 1. In the case of sarin, the most promising
uncharged reactivators were 7h, 7g and 3. Their reactivation rates
equaled and even exceeded those of MMB-4 and HI-6, while 7d
and 7f showed relatively low reactivation potency. For VX-inhib-
ited hAChE, it is inspiring that most of the novel oximes were
promising reactivators; some of them even exhibited higher reac-
tivation abilities than obidoxime and HI-6 (such as 7c, 7d, 7g, 7h
and 3). In terms of tabun-inhibited hAChE, to our best knowledge,
oxime 3, 7g and 7h were the most promising nonquaternary reac-
tivators reported until now, while HI-6 and oximes 7a–7f only
slightly reactivated tabun-inhibited hAChE. For conjugate 3, our
results are in line with what Mercey et al. had reported, but they
did not evaluate the reactivation ability against sarin-inhibited
hAChE.24,25 In contrast to those quaternary oximes, 7g, 7h and 3
emerged as more broad-spectrum for sarin-, VX- and tuban-
inhibited hAChE, while obidoxime and TMB-4 were less efficient
for sarin; HI-6 and MMB-4 were much less efficient for tabun
poisoning. We also determined the reactivation potency of
imidazolium-2-aldoxime (5b), the results demonstrated that most
conjugates showed improved reactivation potency, especially in
the cases of 7d, 7g and 7h, which justified the concept of dual-site
biding strategy.
In addition, in order to get a complete comprehension of the
reactivation of conjugates 7g and 7h, we measured their reactiva-
tion rate constant Kr, dissociation constant KD and second order
reactivation rate constant Kr2 for sarin-, VX- and tabun-inhibited
hAChE,39 the results were presented in Table 2. As a result of
improved Kr or KD, their reactivation efficiencies, or Kr2 (Kr/KD),
equaled and even exceeded those of obidoxime, HI-6 and TMB-4
in similar conditions, which were in line with the conclusions we
got above. For details, conjugate 7h was more efficient than HI-6
and 3 for sarin poisoning due to a better affinity (lower KD). For
Four quaternary reactivators (obidoxime, HI-6, MMB-4 and
TMB-4, Fig. 1) were used as reference compounds in the inhibition
experiments, the results are depicted in Figure 4. It is apparent that
these new oximes, including 3, were moderate or weak inhibitors
of hAChE, while the reference reactivators just showed slight
inhibitory abilities (Fig. 4). At 1 mM oxime concentration, enzyme
inhibition percentages by 3, 7g, 7h were 51%, 36% and 47% respec-
tively, while the inhibition percentages by all other oximes were
below 40%. In contrast, at 0.1 mM oxime concentration, the
enzyme activity was almost completely released (>85%). We fur-
ther determined the IC50 of 3, 7g and 7h (data show in Table 2)
and confirm that they were weak inhibitors of AChE. In order to
determine whether the inhibition was reversible, the erythrocyte
(AChE bound to it covalently) was washed once with PBS (0.1 M,
pH = 7.4). For most tested oximes, the activities of the hAChE were
greatly restored (>85%) after wash (Fig. 4). For examples, at 1 mM
oxime concentration, hAChE activities increased from 53% to 68%
for 7h, 49% to 73% for 3 and 64% to 95% for 7g, respectively. On
the contrary, it was found that there was no change in the activity
of the nerve agent-inhibited hAChE in an extra experiment. There-
fore, we concluded that the synthesized oximes inhibited hAChE in
a moderate and reversible way, this could not only provide suitable
AChE affinity for reactivation of OP poisoning, but also avoid
toxicology caused by strong inhibition of AChE. These results
encouraged us to proceed to the reactivation experiments.
Sarin, VX and tabun were used to determine the reactivation
potency of oximes 7a–7h,
3 and the reference compounds
mentioned above. The reactivation rates were calculated relatively
Table 2
Reactivation rate constant (Kr), dissociation constant (KD), second order reactivation rate constant (Kr2) and IC50 of obidoxime, HI-6, TMB-4, 3, 7g and 7h
Oxime
Kr/minÀ1
VX
KD/
l
M
Kr2/mMÀ1minÀ1
IC50 (mM)
Sarin
Tabun
Sarin
VX
Tabun
Sarin
VX
Tabun
Obidoxime
0.011 0.002
0.048 0.003
n.d.
0.063 0.022
0.040 0.008
0.033 0.004
0.22 0.02
0.25 0.01
n.d.
0.22 0.02
0.14 0.02
0.21 0.01
0.028 0.002
n.d.a
0.056 0.004
0.027 0.003
0.009 0.001
0.012 0.001
90 39
93 19
n.d.
88 77
143 78
41 16
32
27
n.d.
22
24
25
9
3
66 16
n.d.
79 20
18 10
26 15
0.12
0.52
n.d.
0.72
0.28
0.81
6.95
9.30
n.d.
10.03
5.75
8.43
0.43
n.d.
0.70
1.54
0.36
0.86
2.21
1.36
2.57
0.89
0.93
0.87
HI-6
TMB-4
3
7g
7h
7
8
4
14
8
a
not determined.