Biochemical Pharmacology 83 (2012) 1700–1706

Reactivation kinetics of a series of related bispyridinium oximes withorganophosphate-inhibited human acetylcholinesterase—Structure–activityrelationships

Franz Worek *, Timo Wille, Marianne Koller, Horst Thiermann

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany

A R T I C L E I N F O

Article history:

Received 23 January 2012

Accepted 2 March 2012

Available online 12 March 2012

This article is devoted to Prof. Dr. Peter Eyer

on the occasion of his 70th birthday.

Keywords:

Acetylcholinesterase

Organophosphorus compounds

Oximes

Kinetics

Structure–activity relationship

A B S T R A C T

Despite extensive research in the last six decades, oximes are the only available drugs which enable a

causal treatment of poisoning by organophosphorus compounds (OP). However, numerous in vitro and

in vivo studies demonstrated a limited ability of these oximes to reactivate acetylcholinesterase (AChE)

inhibited by different OP pesticides and nerve agents. New oximes were mostly tested for their

therapeutic efficacy by using different animal models and for their reactivating potency with AChE from

different species. Due to the use of different experimental protocols a comparison of data from the

various studies is hardly possible. Now, we found it tempting to determine the reactivation kinetics of a

series of bispyridinium oximes bearing one or two oxime groups at different positions and having an

oxybismethylene or a trimethylene linker under identical conditions with human AChE inhibited by

structurally different OP. The data indicate that the position of the oxime group(s) is decisive for the

reactivating potency and that different positions of the oxime groups are important for different OP

inhibitors while the nature of the linker, oxybismethylene or trimethylene, is obviously of minor

importance. Hence, these and previous data emphasize the necessity for thorough kinetic investigations

of OP-oxime-AChE interactions and underline the difficulty to develop a broad spectrum oxime

reactivator which is efficient against structurally different OP inhibitors.

� 2012 Elsevier Inc. All rights reserved.

Contents lists available at SciVerse ScienceDirect

Biochemical Pharmacology

jo u rn al h om epag e: ww w.els evier .c o m/lo cat e/bio c hem p har m

1. Introduction

Despite extensive research in the last six decades, oximes arethe only available drugs which enable a causal treatment ofpoisoning by organophosphorus compounds (OP) [1]. The primarymechanism of action of oximes is the reactivation of OP-inhibitedacetylcholinesterase (AChE), the main target of OP inhibitors [2–4].At present, only few oximes, i.e. obidoxime, pralidoxime and TMB-4, are used clinically. However, numerous in vitro and in vivostudies demonstrated a limited ability of these oximes toreactivate AChE inhibited by different OP pesticides and nerveagents [1,5,6].

This situation led to the search for more effective oximes in thepast decades and up to now a countless number of oximes has beensynthetized [7]. A major contribution to this effort was made byProf. I. Hagedorn from the University of Freiburg, Germany, whosynthetized more than 1000 oximes including some of the mosteffective reactivators, e.g. obidoxime, HI-6 and HLo 7 [8].

* Corresponding author. Tel.: +49 89 3168 2930; fax: +49 89 3168 2333.

E-mail address: franzworek@bundeswehr.org (F. Worek).

0006-2952/$ – see front matter � 2012 Elsevier Inc. All rights reserved.

doi:10.1016/j.bcp.2012.03.002

New oximes were mostly tested for their therapeutic efficacy byusing different animal models and for their reactivating potencywith AChE from different species [7,9,10]. In view of the use ofdifferent experimental protocols it is hardly possible to comparedata of the various studies in order to quantify the ability of oximesto reactivate inhibited AChE.

Evaluation of the reactivating potency of oximes requires thedetermination of the reactivation kinetics including the dissocia-tion and reactivity constants [11]. Hereby, the determination ofmeaningful kinetic constants is a major challenge and requires theuse of appropriate experimental procedures. Recently, we estab-lished a protocol which allows the investigation of oximes withlargely different affinities and reactivities [12]. Now, we found ittempting to determine the reactivation kinetics of a series ofbispyridinium oximes with human AChE inhibited by structurallydifferent OP, i.e. the organophosphate paraoxon, the organopho-sphonate cyclosarin and the phosphoramidate tabun (Fig. 1) atidentical experimental conditions. For this attempt oximes bearingone or two oxime functions at different positions and having anoxybismethylene or a trimethylene linker were selected (Table 1),namely the Hagedorn oximes obidoxime, HI-6, HS 3 and HS 4 [13–15], TMB-4 [16], ICD585 [17] and K005 [18] which was firstdescribed by [19]. With these data at hand it should be possible to

Fig. 1. Chemical structure of OP inhibitors used in this study.

F. Worek et al. / Biochemical Pharmacology 83 (2012) 1700–1706 1701

get more insight into structural requirements for reactivation and toprovide a database for investigating structure–activity relationships.

2. Materials and methods

2.1. Materials

Paraoxon-ethyl (paraoxon, Fig. 1) was purchased from Dr.Ehrenstorfer GmbH (Augsburg, Germany), tabun and cyclosarin(>98% by GC–MS, 1H NMR and 31P NMR, Fig. 1) were madeavailable by the German Ministry of Defence. The tested oximes(>95% by 1H NMR; Table 1) were from different sources:obidoxime dichloride was purchased from Merck (Darmstadt,Germany) and TMB-4 from Sigma–Aldrich (Taufkirchen,Germany), HI-6 dichloride monohydrate was provided by Dr.

Table 1Chemical structure and acidity of tested oximes.

Code a R1 Y b

TMB-4 4 CHNOH (CH2)3 4

Obidoxime 4 CHNOH CH2OCH2 4

HS 3 2 CHNOH CH2OCH2 4

HS 4 2 CHNOH CH2OCH2 2

K005 2 CHNOH (CH2)3 2

HI-6 2 CHNOH CH2OCH2 4

ICD585 2 CHNOH (CH2)3 4

pKa values were taken from: a [36]; b,f [31]; c [37]; d [27]; e [18]; g unpublished data.

Clement (Defence Research Establishment Suffield, Ralston,Alberta, Canada), K005 dibromide by Dr. Kuca (Faculty of MilitaryHealth Sciences, Hradec Kralove, Czech Republic), ICD585 by Prof.Taylor (University of California, San Diego, USA) and HS 3 and HS 4were made available by Prof. Eyer (University of Munich, Munich,Germany). 5,50-Dithiobis(2-nitrobenzoic acid) (DTNB) and acet-ylthiocholine iodide (ATCh) were supplied by Sigma–Aldrich. Allother chemicals were from Merck (Darmstadt, Germany).

Tabun (6.2 mM) and cyclosarin stock solutions (5.5 mM) wereprepared in acetonitrile and paraoxon stock solutions (10 mM) in2-propanol and were stored at 20 8C and �80 8C, respectively.Oxime stock solutions (200 mM) were prepared in distilled waterand were stored at �80 8C. Working solutions were appropriatelydiluted in distilled water just before the experiment and were kepton ice until use.

Hemoglobin-free human erythrocyte ghosts were prepared asdescribed from heparinized human blood and served as AChEsource [20]. Aliquots of the erythrocyte ghosts with an AChEactivity adjusted to that found in whole blood were stored at�80 8C and aliquots were homogenized prior to use to achieve ahomogeneous matrix for the kinetic studies.

2.2. AChE assay

AChE activities were measured with a modified Ellman assay[21] at 412 nm (Cary 50, Varian, Darmstadt) using polystyrolcuvettes, 0.45 mM ATCh as substrate and 0.3 mM DTNB as achromogen in 0.1 M phosphate buffer (pH 7.4).

All experiments were performed at 37 8C and pH 7.4. Allconcentrations refer to final concentrations.

2.3. Reactivation of OP-inhibited human AChE by oximes

Human erythrocyte ghosts were incubated for 15 min at 37 8Cwith a small volume (�1%, v/v) of paraoxon (100 nM), tabun(100 nM) or cyclosarin (20 nM final concentration) to achieve anAChE inhibition of >95%. Then, the treated ghosts were dialyzed(phosphate buffer, 0.1 M, pH 7.4) overnight at 4 8C to removeresidual inhibitor followed by incubation of treated and controlghost aliquots (30 min, 37 8C) to verify the absence of inhibitoryactivity. Aliquots were stored at �80 8C until use.

150 ml inhibited AChE were mixed with 150 ml phosphatebuffer containing 0.2% gelatin in order to stabilize AChE activityduring prolonged incubation at 37 8C. At t = 0 5 ml oxime wasadded to initiate reactivation. After specified time intervals (2–60 min) 20 ml aliquots were transferred to tempered cuvettes

R2 X pKa1 pKa2 Refs.

CHNOH Br 7.78 8.61 a

CHNOH Cl 7.54 8.12 b

CHNOH Cl 7.23 8.24 c

CHNOH Cl 7.04 7.95 d

CHNOH Br 7.13 8.55 e

CONH2 Cl 7.28 – f

CONH2 Cl 7.7 – g

Table 2Reactivation constants for oxime-induced reactivation of tabun-inhibited human

AChE.

Oxime KD (mM) kr (min�1) kr2 (mM�1 min�1) Maximum

reactivation

(%)

TMB-4 111 � 1 0.1 � 0.005 0.9 � 0.04 91

Obidoxime 203 � 15 0.046 � 0.002 0.23 � 0.005 88

HS 3 236 � 15 0.006 � 0.0003 0.028 � 0.0004 27

HS 4 229 � 79 0.003 � 0.0003 0.014 � 0.003 14

K005 n.d. n.d. n.d. 9

HI-6 n.d. n.d. n.d. 3

ICD585 n.d. n.d. n.d. 2

The pseudo-first order rate constant kobs was determined by non-linear regression

analysis and these data were used for the calculation of the reactivity rate constant

kr, the dissociation constant KD and the hybrid reactivation rate constant kr2 (from

the ratio of kr/KD). Data are given as means � SD (n = 2). n.d. inadequate reactivation of

tabun-inhibited AChE for the calculation of reactivation constants. Maximum

reactivation gives the highest AChE activity after 60 min incubation of inhibited AChE

with the respective oxime.

Table 3Reactivation constants for oxime-induced reactivation of cyclosarin-inhibited

human AChE.

Oxime KD (mM) kr (min�1) kr2 (mM�1 min�1)

TMB-4 343 � 57 0.21 � 0.02 0.62 � 0.03

Obidoxime 582 � 123 0.39 � 0.03 0.68 � 0.09

HS 3 151 � 14 1.77 � 0.05 11.86 � 1.46

HS 4 274 � 94 0.62 � 0.06 2.47 � 0.62

K005 110 � 7 0.11 � 0.001 1.00 � 0.05

HI-6 123 � 6 2.75 � 0.02 22.41 � 1.27

ICD585 164 � 19 1.71 � 0.17 10.45 � 0.18

The pseudo-first order rate constant kobs was determined by non-linear regression

analysis and these data were used for the calculation of the reactivity rate constant

kr, the dissociation constant KD and the hybrid reactivation rate constant kr2 (from

the ratio of kr/KD). Data are given as means � SD (n = 2). Maximum reactivation was

100% with all oximes.

Table 4Reactivation constants for oxime-induced reactivation of paraoxon-inhibited

human AChE.

Oxime KD (mM) kr (min�1) kr2 (mM�1 min�1) Maximum

reactivation

(%)

TMB-4 62 � 19 0.97 � 0.01 17.44 � 5.3 100

Obidoxime 65 � 17 1.22 � 0.01 19.98 � 5.1 100

HS 3 202 � 27 0.50 � 0.03 2.53 � 0.51 100

HS 4 786 � 53 0.05 � 0.003 0.07 � 0.001 73

K005 130 � 7 0.01 � 0.001 0.08 � 0.001 45

HI-6 478 � 11 0.16 � 0.004 0.34 � 0.002 92

ICD585 514 � 25 0.15 � 0.001 0.29 � 0.01 84

The pseudo-first order rate constant kobs was determined by non-linear regression

analysis using Eq. (2) with HS 4, K005, HI-6 and ICD585 or Eq. (3) with TMB-4,

obidoxime and HS 3. These data were used for the calculation of the reactivity rate

constant kr, the dissociation constant KD and the hybrid reactivation rate constant

kr2 (from the ratio of kr/KD). Data are given as means � SD (n = 2). Maximum

reactivation gives the highest AChE activity after 60 min incubation of inhibited AChE

by the respective oxime.

F. Worek et al. / Biochemical Pharmacology 83 (2012) 1700–17061702

containing 3000 ml phosphate buffer and 100 ml DTNB for themeasurement of AChE activity after addition of 50 ml ATCh.

8–10 different oxime concentrations (10–1000 mM) were usedfor the determination of the reactivation rate constants induplicate experiments (n = 2). For the reactivation of tabun-inhibited AChE higher oxime concentrations (up to 5000 mM) wereused in part, too. OP-inhibited AChE activity as well as AChEactivity after time-dependent reactivation was referred to controlAChE and % reactivation was calculated thereof.

2.4. Reactivation kinetics

The reactivation of OP-inhibited AChE by oximes may bedescribed by a two-step reaction (Scheme 1) and can be quantifiedby the determination of reactivation rate constants. In this scheme[EP] is the OP-inhibited AChE, [OX] the oxime, [EPOX] theMichaelis-type OP-AChE-oxime conjugate, [E] the reactivatedenzyme and [POX] the phosphylated oxime. KD stands for thedissociation constant which is inversely proportional to the affinityof the oxime to [EP], and kr the rate constant for removing the OPresidue from [EPOX] by the oxime, quantifying its reactivity. Thehybrid reactivation rate constant kr2 was calculated from the ratioof kr and KD, resembling a second-order rate constant.

In case of complete reactivation and with [Ox] � [EP]0 apseudo-first-order rate equation can be derived for the reactiva-tion process (Eq. (1)):

kobs ¼kr � ½OX�KD þ ½OX� (1)

kobs was calculated by non-linear regression analysis [12] usingEq. (2)

vt ¼ v0 � ð1 � e�kobs�tÞ (2)

In case of substantial deviation of the reactivation curves, i.e. byre-inhibition of the reactivated enzyme by phosphoryloximes [22],kobs1 and kobs2 were estimated by using Eq. (3)

vt ¼ v01� ð1 � e�kobs1�tÞ þ v02

� ð1 � e�kobs2�tÞ (3)

Finally, kr and KD were obtained by the nonlinear fit of therelationship between kobs versus [OX] and kr2 was calculated fromthe ratio of kr and KD. In case of analysis of the data by Eq. (3) thepseudo-first-order rate constant of the initial rapid phase, kobs1,was used for the calculation of kr, KD and kr2.

2.5. Data analysis

Processing of experimental data for the determination of thedifferent kinetic constants was performed by non-linear regressionanalysis using curve fitting programs provided by PrismTM Vers.4.03 (GraphPad Software, San Diego, USA). Hereby, individual datasets were analyzed to obtain kobs and these constants were used forthe calculation of kr, KD and kr2 which are presented as means � SDin Tables 2–4.

3. Results

3.1. Reactivation of tabun-inhibited AChE

The ability of the tested oximes to reactivate tabun-inhibitedAChE was dependent on the position of the oxime group(s)

[EP ] + [OX] [EP OX] [E] + [POX]

KD kr

Scheme 1. Reaction scheme for the reactivation of OP-inhibited AChE by oximes.

(Table 2). With TMB-4 and obidoxime, bearing an oxime group atposition 4 on both pyridinium rings, an almost completereactivation could be recorded after 60 min with 1000 mM oxime(Fig. 2A). Use of the 2,40-oxime HS 3 resulted only in partialreactivation which was even lower with the 2,20-oximes HS 4 andK005. The 2-oximes HI-6 and ICD585 failed to reactivate theinhibited AChE even when 5000 mM oxime were used.

Due to the moderate affinity and low reactivity of the oximesthe overall reactivating potency was low, i.e. a kr2 of<1 mM�1 min�1 with TMB-4 and obidoxime and substantiallylower with HS 3 and HS 4 (Table 2).

Fig. 2. Time- and concentration-dependent reactivation of tabun- (A), cyclosarin-

(B) and paraoxon-inhibited human AChE (C) by obidoxime. Inhibited AChE was

incubated with 10, 50, 100, 200, 400, 600, 800 and 1000 mM obidoxime and AChE

activity was determined after 2–60 min. Data were analyzed by non-linear

regression (A and B: Eq. (2); C: Eq. (3)). Data are means � SD (n = 2).

F. Worek et al. / Biochemical Pharmacology 83 (2012) 1700–1706 1703

3.2. Reactivation of cyclosarin-inhibited AChE

A different pattern was observed for the reactivation ofcyclosarin-inhibited AChE. Under conditions of, i.e. high oximeconcentrations and long incubation time (60 min), all oximes wereable to reactivate the inhibited enzyme completely. Nevertheless,marked differences of the calculated reactivation constants,depending on the oxime structure, were recorded (Table 3). The4,40-oximes TMB-4 and obidoxime showed a low affinity andreactivity while the 2-oximes HI-6 and ICD585 and the 2,40-oximeHS 3 had a substantially higher affinity and reactivity resulting in a15- to 32-fold higher second order reactivation rate constant of thelatter. In contrast, the 2,20-oximes HS 4 and K005 were character-ized by a moderate affinity and a low reactivity.

3.3. Reactivation of paraoxon-inhibited AChE

The reactivation of paraoxon-inhibited AChE was markedlyaffected by the position of the oxime group(s). Hereby, the increase

of AChE activity was bi-phasic with obidoxime, TMB-4 and HS 3,indicating the formation of phosphoryloxime and subsequentpartial re-inhibition of the reactivated enzyme [22]. Analysis ofthese data by mono-exponential analysis resulted in a R2 of <0.98while a two-phase exponential analysis according to Eq. (3)provided a substantially better fitting of the data (R2 > 0.99) andenabled to provide estimates of kobs (Fig. 2C). Hereby, the pseudo-first-order rate constant of the initial rapid phase (kobs1) was usedfor the further calculation of the constants kr, KD and kr2. The 4,40-oximes obidoxime and TMB-4 were characterized by a high affinityand reactivity while 2,20- and 2-oximes had a markedly loweraffinity and reactivity (Table 4). Especially, 2,20-oximes (HS 4 andK005) had an outstanding low reactivity resulting in only partialreactivation of the enzyme after 60 min incubation. In effect, TMB-4 and obidoxime had a 50- to 280-fold higher kr2 compared tooximes bearing only oxime groups in position 2.

4. Discussion

4.1. Methodological aspects

The ability of oximes to reactivate human AChE inhibited bystructurally different OP was investigated with a uniformexperimental protocol. This enabled reactivation constants to begenerated under identical conditions as a prerequisite for theproper evaluation of the reactivating potency of the tested oximes.In fact, the recorded reactivation curves of oxime-inducedreactivation of tabun- and cyclosarin-inhibited AChE could bebest described by a one-phase exponential association as shown inFig. 2B for the reactivation of cyclosarin-inhibited AChE byobidoxime. In contrast, reactivation of paraoxon-inhibited AChEby obidoxime, TMB-4 and HS 3 was bi-phasic (Fig. 2C). Thisphenomenon may be attributed to the re-inhibition of reactivatedenzyme by stable phosphoryloximes formed during the reactiva-tion process with oximes bearing an oxime group at position 4 ofthe pyridinium ring [23,24]. Therefore, these reactivation curveswere analyzed by two-phase exponential association, an approachwhich enabled the estimation of kobs. Although this procedure doesnot eliminate the impact of phosphoryloxime completely, thegenerated reactivation constants (Table 4) were in good agreementwith results from previous studies using a different protocol[25,26].

4.2. Reactivation kinetics

The reactivation of OP-inhibited human AChE by the testedoximes was strongly dependent on the structure of the inhibitorand reactivator. This fact was expected since previous studiesusing selected oximes showed a relationship between structureand reactivation [1,9,11,17,27–30].

Now, the results of the present study demonstrate that theposition of the oxime group(s) is decisive for the ability of an oximeto reactivate tabun-, cyclosarin- or paraoxon-inhibited AChE andthe type of the linker, oxybismethylene versus trimethylene, is ofless importance (Tables 2–4). In fact, an oxime group at position 4of the pyridinium ring is mandatory for an at least moderatereactivating potency toward tabun-inhibited AChE and results in ahigh reactivity with paraoxon-inhibited AChE. In contrast, anoxime group at position 2 is essential for a high bimolecularreactivation rate constant with cyclosarin-inhibited AChE.

Apart from the position of the oxime group its acidity wasconsidered important for the reactivating effectiveness of oximesby various authors since the removal of the phosphyl moiety fromthe active site is caused by the oximate anion [19,31]. Hence, thereactivity of oximes should be inversely proportional to the pKa. Acomparison of the published pKa values (Table 1) with the

F. Worek et al. / Biochemical Pharmacology 83 (2012) 1700–17061704

reactivation constants (Tables 2–4) does not support the view thatthe acidity is a decisive factor for the reactivity of an oxime. HI-6,pKa 7.28, was an excellent reactivator of cyclosarin-inhibited AChE,weak with paraoxon-inhibited AChE and failed with tabun-inhibited enzyme while TMB-4, pKa 8.2, was excellent withparaoxon-, moderate with tabun- and weak with cyclosarin-inhibited AChE. Hence, these data indicate that other factors, i.e.position of the oxime group(s) and/or other substituents, are moreimportant for the reactivating potency of oximes.

4.3. Structure–activity relationship

The investigation of the reactivation kinetics with structurallydifferent oximes should enable the assessment of potentialstructure–activity relationships. Previous studies attempted toperform such an assessment with selected oximes and inhibitors[9,11,27,29,32–35] but a comparison of results from the variousstudies is hampered by the use of different experimentalprotocols.

The use of oxime pairs with identical oxime groups anddifferent linkers prompted us to calculate the ratios of kr, KD andkr2 between oximes with oxybismethylene and trimethylenelinkers (Fig. 3). With 4,40- and 2-oximes the ratio was close to 1with paraoxon-inhibited AChE and varied only moderately withcyclosarin-inhibited enzyme. A different pattern was recorded for2,20-oximes. With both inhibitors K005 had a several-fold lowerreactivity but higher affinity compared to HS 4 resulting in a

Fig. 3. Reactivation kinetics of cyclosarin- and paraoxon-inhibited human AChE.

The data show the ratio of the reactivation rate constants kr, KD and kr2 of

corresponding oximes with different linkers, i.e. oxybismethylene relative to

trimethylene. The dashed line indicates a ratio of 1. Please note the different scale of

the Y-axis.

2.5-fold higher kr2 with cyclosarin- but equal kr2 with paraoxon-inhibited AChE.

In addition, the impact of the position of the oxime group on thevarious kinetic constants was assessed by forming the ratio ofoxime pairs as shown in Fig. 4. These calculations point to thedecisive effect of the position of the oxime group on thereactivating potency. A comparison of the different constantsbetween 4,40 and 2,40-oximes revealed a substantially loweraffinity and reactivity of 4,40-oximes resulting in a more than 16-fold lower bimolecular reactivation rate constant with cyclosarin-inhibited AChE. The ratio was not as high but still relevant in caseof 4,40- versus 2,20-oximes. In contrast, the comparison of 2,40- and2,20-oximes revealed a higher affinity and reactivity and a kr2 ratioof 5. Hence, it may be assumed that a high reactivating potency ofcyclosarin-inhibited AChE with bispyridinium oximes bearing anoxybismethylene linker requires an oxime group in position 2 anda substitute, i.e. oxime or carbamoyl group, in position 4 of thesecond pyridinium ring.

Different results were obtained with paraoxon-inhibited AChE(Fig. 4). The ratio of 4,40- and 2,40-oximes revealed a moderatelyhigher reactivity and affinity and an almost 8-fold higher kr2 ofobidoxime while this oxime in comparison to the 2,20-oxime HS 4was distinctively more potent having a 300-fold higher bimolecu-lar reactivation rate constant. In consequence, the ratio of 2,40- and2,20-oxime resulted in a 38-fold higher kr2 value. These data

Fig. 4. Reactivation kinetics of cyclosarin- and paraoxon-inhibited human AChE.

The data show the ratio of the reactivation rate constants kr, KD and kr2 of

homologous 2,20 , 2,40 and 4,40 oximes bearing an oxybismethylene linker. The

dashed line indicates a ratio of 1. Please note the different scale of the Y-axis.

F. Worek et al. / Biochemical Pharmacology 83 (2012) 1700–1706 1705

underline the necessity of at least one oxime group in position 4 forthe effective reactivation of paraoxon-inhibited AChE, an assump-tion which is in agreement with previous data generated withbovine AChE [27]. 2,20-Oximes, either with oxybismethylene ortrimethylene linker, were rather ineffective and a carbamoyl groupat position 4 of the second pyridinium ring (HI-6 and ICD585)resulted in an only minor improvement.

The failure of 2-oximes to reactivate tabun-inhibited AChEprevented comparable calculations. However, the determinedreactivation constants indicate that oxime groups in position 4 atboth pyridinium rings are essential for an at least moderatereactivating potency (Table 2). Previous studies showed thatoximes bearing an oxime and a carbamoyl group at position 4 ofthe pyridinium rings are able to reactivate bovine AChE as well [28]and that HLo 7, an oxime with two oxime groups in position 2 and 4and a carbamoyl group in position 4 of the second ring, wasapproximately half as potent as obidoxime [11]. This conclusion issupported by the fact that the 2,40-oxime HS 3 had an almost 10-fold lower kr2 compared to obidoxime (Table 2).

The testing of the asymmetric 2,40-oxime HS 3 may give insightinto the question whether a differential coordination of the oximein the AChE gorge may occur for the reactivation of the enzymeinhibited by different OP inhibitors. In fact, HS 3 had a highreactivating potency toward cyclosarin-inhibited AChE, a reason-able potency toward paraoxon-inhibited AChE and an at leastmeasurable kr2 with tabun-inhibited AChE. Hence, it may behypothesized that there is no exclusive coordination of this oximewith either oxime group 2 or 4 attaching to the phosphyl moietybut that both oxime groups at an unknown ratio contribute to thereactivating effect of this oxime.

4.4. Conclusions

The investigation of the reactivation kinetics of a series ofbispyridinium oximes bearing one or two oxime functions atdifferent positions and having an oxybismethylene or a trimethy-lene linker with tabun-, cyclosarin- and paraoxon-inhibitedhuman AChE gives further insight into structural requirementsfor oxime reactivators. The data indicate that the position of theoxime group(s) is decisive for the reactivating potency and thatdifferent positions of the oxime groups are important for differentOP inhibitors while the nature of the linker, oxybismethylene ortrimethylene, is obviously of minor importance. Hence, these andprevious data emphasize the necessity for thorough kineticinvestigations of OP-oxime–AChE interactions and underline thedifficulty to develop a broad spectrum oxime reactivator which isefficient against structurally different OP inhibitors.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

The study was funded by the German Ministry of Defence. Theauthors are grateful to T. Hannig and E. Wagner for expert technicalassistance and to Prof. Eyer, Prof. Taylor, Dr. Clement and Dr. Kucafor the donation of oximes.

References

[1] Eyer P, Worek F. Oximes. In: Marrs TC, Maynard RL, Sidell FR, editors. Chemicalwarfare agents: toxicology and treatment. Chichester: John Wiley & Sons Ltd.;2007. p. 305–29.

[2] Holmstedt B. Pharmacology of organophosphorus cholinesterase inhibitors.Pharmacol Rev 1959;11:567–688.

[3] Aldridge WN, Reiner E. Enzyme inhibitors as substrates – interactions ofesterases with esters of organophosphorus and carbamic acids. Amsterdam,London: North-Holland Publishing Company; 1972.

[4] Hobbiger F. Reactivation of phosphorylated acetylcholinesterase. In: KoelleGB, editor. Cholinesterases and anticholinesterase agents. Berlin: Springer-Verlag; 1963. p. 921–88.

[5] Jokanovic M. Medical treatment of acute poisoning with organophosphorusand carbamate pesticides. Toxicol Lett 2009;190:107–15.

[6] Zilker T. Medical management of incidents with chemical warfare agents.Toxicology 2005;214:221–31.

[7] Worek F, Eyer P, Aurbek N, Szinicz L, Thiermann H. Recent advances inevaluation of oxime efficacy in nerve agent poisoning by in vitro analysis.Toxicol Appl Pharmacol 2007;219:226–34.

[8] Eyer P. In memory of Ilse Hagedorn. Toxicology 2007;233:3–7.[9] Schoene K. Pyridinium salts as organophosphate antagonists. Monogr Neural

Sci 1980;7:85–98.[10] Dawson RM. Review of oximes available for treatment of nerve agent poison-

ing. J Appl Toxicol 1994;14:317–31.[11] Worek F, Thiermann H, Szinicz L, Eyer P. Kinetic analysis of interactions

between human acetylcholinesterase, structurally different organophospho-rus compounds and oximes. Biochem Pharmacol 2004;68:2237–48.

[12] Worek F, Wille T, Aurbek N, Eyer P, Thiermann H. Reactivation of organophos-phate-inhibited human, Cynomolgus monkey, swine and guinea pig acetyl-cholinesterase by MMB-4: a modified kinetic approach. Toxicol Appl Pharmacol2010;249:231–7.

[13] Luttringhaus A, Hagedorn I. Quartare Hydroxyiminomethylpyridiniumsalze.Drug Res 1964;14:1–5.

[14] Schoene K. Darstellung und Untersuchung neuer Acetylcholinesterase-Reak-tivatoren. Thesis, University of Freiburg; 1967.

[15] Stark I. Versuche zur Darstellung eines LuH 6 (Toxogonin1) uberlegenenAcetylcholinesterase-Reaktivators. Thesis, University of Freiburg; 1968.

[16] Poziomek EJ, Hackley BE, Steinberg GM. Pyridinium aldoximes. J Org Chem1958;23:714–7.

[17] Bharate SP, Guo L, Reeves TE, Cerasoli DM, Thompson CM. Bisquaternarypyridinium oximes: comparison of in vitro reactivation potency of compoundsbearing aliphatic linkers and heteroaromatic linkers for paraoxon-inhibitedelectric eel and recombinant human acetylcholinesterase. Bioorg Med Chem2010;18:787–94.

[18] Kuca K, Cabal J, Patocka J, Kassa J. Synthesis of bisquaternary symmetric -x,d-bis(2-hydroxyiminomethylpyridinium) alkane dibromides and their reacti-vation of cyclosarin-inhibited acetylcholinesterase. Lett Org Chem 2004;1:84–6.

[19] Wilson IB, Ginsburg S. Reactivation of alkylphosphate inhibited acetylcholin-esterase by bis-quaternary derivatives of 2-PAM and 4-PAM. Biochem Phar-macol 1958;1:200–6.

[20] Worek F, Reiter G, Eyer P, Szinicz L. Reactivation kinetics of acetylcholinester-ase from different species inhibited by highly toxic organophosphates. ArchToxicol 2002;76:523–9.

[21] Worek F, Mast U, Kiderlen D, Diepold C, Eyer P. Improved determination ofacetylcholinesterase activity in human whole blood. Clin Chim Acta 1999;288:73–90.

[22] Worek F, Eyer P, Kiderlen D, Thiermann H, Szinicz L. Effect of human plasma onthe reactivation of sarin-inhibited human erythrocyte acetylcholinesterase.Arch Toxicol 2000;74:21–6.

[23] Kiderlen D, Eyer P, Worek F. Formation and disposition of diethylphosphoryl-obidoxime, a potent anticholinesterase that is hydrolysed by human para-oxonase (PON1). Biochem Pharmacol 2005;69:1853–67.

[24] Ashani Y, Bhattacharjee AK, Leader H, Saxena A, Doctor BP. Inhibition ofcholinesterases with cationic phosphonyl oximes highlights distinctive prop-erties of the charged pyridine groups of quaternary oxime reactivators.Biochem Pharmacol 2003;66:191–202.

[25] Mast U. Reaktivierung der Erythrozyten-Acetylcholinesterase durch Oxime.Thesis, University of Munich; 1997.

[26] Worek F, Backer M, Thiermann H, Szinicz L, Mast U, Klimmek R, et al.Reappraisal of indications and limitations of oxime therapy in organophos-phate poisoning. Hum Exp Toxicol 1997;16:466–72.

[27] Schoene K, Strake EM. Reaktivierung von diathylphosphoryl-acetylcholines-terase—affinitat und reaktivitat einiger pyridiniumoxime. Biochem Pharmacol1971;20:1041–51.

[28] Schoene K, Oldiges H. Die Wirkungen von Pyridiniumsalzen gegenuber Tabun-und Sarinvergiftungen in vivo un in vitro. Arch Int Pharmacodyn Ther 1973;204:110–23.

[29] Worek F, Aurbek N, Koller M, Becker C, Eyer P, Thiermann H. Kinetic analysis ofreactivation and aging of human acetylcholinesterase inhibited by differentphosphoramidates. Biochem Pharmacol 2007;73:1807–17.

[30] Luo C, Tong M, Chilukuri N, Brecht K, Maxwell DM, Saxena A. An in vitrocomparative study on the reactivation of nerve agent-inhibited guinea pig andhuman acetylcholinesterases by oximes. Biochemistry 2007;46:11771–79.

[31] Hagedorn I, Stark I, Lorenz HP. Reaktivierung phosphorylierter Acetylcholin-esterase—Abhangigkeit von der Aktivator-Aciditat. Angew Chem 1972;84:354–6.

[32] Maxwell DM, Koplovitz I, Worek F, Sweeney RE. A structure–activity analysisof the variation in oxime efficacy against nerve agents. Toxicol Appl Pharmacol2008;231:157–64.

[33] Su CT, Wang PH, Lui RF, Shih JH, Ma C, Lin CH, et al. Kinetic studies andstructure–activity relationships of bispyridinium oximes as reactivators of

F. Worek et al. / Biochemical Pharmacology 83 (2012) 1700–17061706

acetylcholinesterase inhibited by organophosphorus compounds. FundamAppl Toxicol 1986;6:506–14.

[34] Aurbek N, Herkert NM, Koller M, Thiermann H, Worek F. Kinetic analysis ofinteractions of different sarin and tabun analogues with human acetylcholin-esterase and oximes: is there a structure–activity relationship? Chem BiolInteract 2010;187:215–9.

[35] Wille T, Ekstrom F, Lee JC, Pang YP, Thiermann H, Worek F. Kinetic analysis ofinteractions between alkylene-linked bis-pyridiniumaldoximes and human

acetylcholinesterases inhibited by various organophosphorus compounds.Biochem Pharmacol 2010;80:941–6.

[36] Bieger D, Wassermann O. Ionization constants of cholinesterase-reactivatingbispyridinium aldoximes. J Pharm Pharmacol 1967;19:844–7.

[37] Stark I. Reaktivierung phosphorylierter Acetylcholinesterase mit quater-nierten Pyridinaldoximen: Ermittlung eines Zusammenhangs zwischenOximaciditat und Reaktivierungsvermogen. Thesis, University of Freiburg;1971.