Chemico-Biological Interactions 203 (2013) 125–128

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Chemico-Biological Interactions

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Structural requirements for effective oximes – Evaluation of kinetic in vitrodata with phosphylated human AChE and structurally different oximes

Franz Worek ⇑, Timo Wille, Marianne Koller, Horst ThiermannBundeswehr Institute of Pharmacology and Toxicology, Munich, Germany

a r t i c l e i n f o a b s t r a c t

Article history:Available online 22 July 2012

Keywords:AcetylcholinesteraseOrganophosphorus compoundsOximesKineticsStructure–activity relationship

0009-2797/$ - see front matter � 2012 Elsevier Irelanhttp://dx.doi.org/10.1016/j.cbi.2012.07.003

⇑ Corresponding author. Address: Bundeswehr InsToxicology, Neuherbergstrasse 11, 80937 Munich, G2930; fax: +49 89 3168 2333.

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

Treatment of poisoning by various organophosphorus (OP) nerve agents with established acetylcholines-terase (AChE) reactivators (oximes) is insufficient. In consequence, extensive research programs havebeen undertaken in various countries in the past decades to identify more effective oximes. The efficacyof new compounds has been investigated with different in vitro and in vivo models which hamper thecomparison of results from different laboratories. The crucial mechanism of action of oximes is the reac-tivation of phosphylated AChE. The kinetic properties of these compounds can be quantified in vitro withisolated AChE from different origin. It was tempting to evaluate the reactivation kinetics of a series of oxi-mes with various OP inhibitors performed under identical experimental conditions in order to get insightinto structural requirements for adequate affinity and reactivity towards inhibited AChE. The determina-tion of reactivation rate constants with bispyridinium oximes having different linkers, bearing oximegroup(s) at different positions and having in part additional substituents revealed that (a) the reactivatingpotency was dependent on the position of the oxime groups and of additional substituents, (b) smallmodifications of the oxime structure had an in part marked effect on the kinetic properties and (c) no sin-gle oxime had an adequate reactivating potency with AChE inhibited by structurally different OP. Theseand previous studies underline the necessity to investigate in detail the kinetic properties of novel oximesand that the identification of a single oxime being effective against a broad range of structurally differentOP will remain a major challenge.

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1. Introduction

The investigation of the toxicology of organophosphorus com-pounds (OP) gave the impetus to research on oxime-based reactiva-tors of OP-inhibited acetylcholinesterase (AChE) in the early 1950s[1–3]. In 1958, Namba and Hiraki published an enthusiastic reporton the therapeutic efficacy of pralidoxime (2-PAM) stating ‘‘Hith-erto, alkylphosphate poisoning has been treated mainly by atropine,but now atropine is replaced by PAM’’ [4]. Unfortunately, subse-quent experimental and clinical research showed that oxime treat-ment of OP poisoning is of limited efficacy at various conditions [5].The main reasons for inadequate oxime efficacy are rapid aging ofOP-inhibited AChE, formation of reactivation resistant OP-AChEcomplexes and mega-dose poisoning with high OP body load [6].

Since the introduction of 2-PAM for clinical use a countlessnumber of oximes has been synthesized by research groups indifferent countries [5,7]. These compounds were mainly tested inanimal models and there is still insufficient knowledge on the

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orek).

structural requirements of oximes being effective against a varietyof structurally different OP [7].

The main mechanism of action of oximes is the reactivation ofOP-inhibited AChE [8]. Hence, the reactivation kinetics of oximescan be investigated with isolated human and animal AChEin vitro which enables the quantification of the reactivity (kr), dis-sociation (KD) and bimolecular reactivation rate constants (kr2). Upto now, various research groups investigated the reactivationkinetics of different oximes with OP-inhibited AChE. However,the use of different experimental protocols and enzyme sourceshampers the comparability of the results (Table 1) and preventsthe proper assessment of data from different studies. The presentpaper compiles and evaluates the reactivation kinetics of differentgroups of homologous or related oximes with phosphylated humanAChE determined under identical conditions in order to get insightinto structural determinants and potential structure–activityrelationships.

2. Reactivation kinetics of oximes: methodological aspects

The reactivation of OP-inhibited AChE by oximes may be de-scribed by a two-step reaction and is determined by the affinity

Table 1Reactivation kinetics of oximes with OP-inhibited human AChE – Impact of experimental protocols and AChE sources.

OP Oxime AChE source kr (min�1) KD (lM) kr2 (mM�1 min�1) Reference

Sarin HI-6 Brain 0.08 182 0.44 [15]Recombinant 0.13 22 5.95 [16]Erythrocyte ghosts 0.68 50 13.5 [10]

Cyclosarin HI-6 Brain 0.07 2 35.0 [17]Recombinant 1.04 35 29.7 [16]Erythrocyte ghosts 2.75 123 22.4 [7]

Tabun Obidoxime Brain 0.06 1412 0.042 [18]Erythrocyte ghosts 0.04 97 0.41 [10]

TMB-4 Brain 0.08 1585 0.05 [18]Erythrocytes 0.15 490 0.31 [19]Erythrocyte ghosts 0.11 111 0.92 [13]

126 F. Worek et al. / Chemico-Biological Interactions 203 (2013) 125–128

of the oxime towards phosphylated AChE (1/KD) and by the veloc-ity of removal of the phosphyl moiety from the enzyme (kr). Fromthe ratio of kr and KD the hybrid reactivation rate constant kr2 canbe calculated, its dimension resembles a second-order rate con-stant [9].

In case of complete reactivation and at oxime concentrationsbeing higher than the concentration of the phosphylated AChEthe pseudo-first-order rate constant kobs of reactivation at any gi-ven oxime concentration can be determined by the equation

Kobs ¼ ðkr � ½oxime�=ðKD þ ½oxime�Þ

Hereby, the value of kobs is not proportional to the oxime con-centration but underlies a saturation kinetics and kr and KD followMichaelis–Menten kinetics [10].

In the past, different procedures for the determination of kobs

have been proposed:

1. Discontinuous procedure: OP-inhibited AChE is incubated withoxime, aliquots are taken after specified time intervals to deter-mine the AChE activity and kobs is calculated by linear regres-sion analysis [10,11].

2. Continuous procedure: OP-inhibited AChE and oxime are addedto a cuvet, the substrate hydrolysis is monitored continuouslyfor a defined time and kobs is calculated by non-linear regressionanalysis [10,12].

With both procedures certain limitations have to be considered.The determination of reactivation kinetics with the discontinuousprocedure and highly reactive oximes results in rapid reactivationof OP-inhibited AChE and in a progressive deviation of the data

Table 2Chemical structure of oximes.

Y

R1 (a) R2 (b)

2X -

Code a R1 Y b R2 X

MMB-4 4 CHNOH CH2 4 CHNOH BrTMB-4 4 CHNOH (CH2)3 4 CHNOH BrObidoxime 4 CHNOH CH2OCH2 4 CHNOH ClK075 4 CHNOH CH2–CH = CH–CH2 4 CHNOH BrHS 3 2 CHNOH CH2OCH2 4 CHNOH ClK053 2 CHNOH CH2–CH = CH–CH2 4 CHNOH BrHS 4 2 CHNOH CH2OCH2 2 CHNOH ClK005 2 CHNOH (CH2)3 2 CHNOH BrK068 2 CHNOH CH2–CH = CH–CH2 2 CHNOH BrHI-6 2 CHNOH CH2OCH2 4 CONH2 ClICD585 2 CHNOH (CH2)3 4 CONH2 Cl

from a straight line. The major disadvantage of the continuous pro-cedure is the limited maximum oxime concentration due to con-centration-dependent oxime-induced oximolysis and AChEinhibition.

In order to enable the investigation of reactivation kinetics ofoximes with largely different affinities and reactivities a modifiedapproach was proposed [9]. In this discontinuous procedure kobs

Fig. 1. Reactivation kinetics of oximes with tabun-inhibited human AChE. Thesecond-order reactivation rate constant kr2 of bispyridinium oximes with differentoxime positions and linkers is shown. (A) 4,40-oximes; (B) 2,20-oximes; (C) 2,40-oximes (HI-6 and ICD585 are bearing an carbamoyl group instead of an oximegroup in position 40). Data are from [7,13].

F. Worek et al. / Chemico-Biological Interactions 203 (2013) 125–128 127

is calculated by non-linear regression analysis which allows theuse of oximes with a broad range of affinities and reactivities andenables the precise calculation of kobs even in case of oximes withlow affinity and high reactivity, e.g. MMB-4 [9]. In addition, thisprocedure allows the estimation of kobs in case of re-inhibition ofreactivated AChE by phosphyloximes formed during the reactiva-tion process [7].

3. Reactivation kinetics: structure–activity relationship

This modified procedure was applied to determine the reactiva-tion kinetics of a series of bispyridinium oximes (Table 2) with ta-bun- and cyclosarin-inhibited human AChE under identicalconditions [7,9,13]. Tabun forms reactivation resistant AChE com-plexes and the clinically used oximes pralidoxime and obidoximeare weak reactivators of cyclosarin-inhibited AChE which, how-ever, can be easily reactivated by HI-6 [5].

These experiments revealed that the position of the oximegroup at the bispyridinium ring(s) is decisive for the reactivatingpotency (Figs. 1 and 2). According to de Jong reactivation oftabun-inhibited AChE requires an oxime with an oxime group in

Fig. 2. Reactivation kinetics of oximes with cyclosarin-inhibited human AChE. Thesecond-order reactivation rate constant kr2 of bispyridinium oximes with differentoxime positions and linkers is shown. (A) 4,40-oximes; (B) 2,20-oximes; (C) 2,40-oximes (HI-6 and ICD585 are bearing an carbamoyl group instead of an oximegroup in position 40). Data are from [7,13].

position 4 at the pyridinium ring [14]. Now, it could be shown thattwo oxime groups in position 4 are necessary for an at least mod-erate reactivating potency (Fig. 1). Compounds bearing oximegroups in position 2 and 4 at different pyridinium rings, i.e. HS3and K053, had a substantially lower reactivating potency. Oximeswith two oxime groups in position 2, HS4, K005 and K068, had anegligible potency while oximes with a single oxime group in po-sition 2, HI-6 and ICD585, were unable to reactivate tabun-inhib-ited AChE at all.

A completely different pattern was observed for the reactivationof cyclosarin-inhibited human AChE (Fig. 2). 2-oximes having asecond oxime, HS3 and K053, or carbamoyl group in position 4,HI-6 and ICD585, had a high reactivating potency which was sub-stantially lower with the 2,20-oximes HS4, K005 and K068. Oximeswith two oxime groups in position 4, i.e. obidoxime, TMB-4, MMB-4 and K075, were weak reactivators of cyclosarin-inhibited AChE.

The impact of the linker, either methylene, oxybismethylene, tri-methylene or (E)-but-2-ene, on the reactivating potency of oximeswas not consistent (Figs. 1 and 2). Reactivation of tabun-inhibitedAChE by 4,40-oximes followed the order (E)-but-2-ene (K075) > tri-methylene (TMB-4) > oxybismethylene (obidoxime) >> methylene(MMB-4) while corresponding 2,40- and 2,20-oximes compoundswith trimethylene linker (K053 and K005) were slightly morepotent compared to those with oxybismethylene linker (HS3 andHS4; Fig. 1). With cyclosarin-inhibited AChE the type of the linkerhad only a small effect with 2,20- and 4,40-oximes but with oximesbearing an oxime group in position 2 and a second function in posi-tion 4 an oxybismethylene linker resulted in a higher reactivatingpotency compared to a trimethylene linker (Fig. 2).

4. Conclusions

The determination of the reactivation kinetics of different bispy-ridinium oximes bearing one or two oxime functions and havingdifferent linkers with tabun- and cyclosarin-inhibited human AChEunder identical conditions provides insight into structural require-ments of oximes. It became evident that the position of the oximegroup(s) and of other functions, i.e. carbamoyl group in case of HI-6 and ICD585, is decisive for the reactivating potency. In addition,these findings underline the difficulty to identify oxime structuresfor an effective reactivation of tabun-inhibited AChE. In contrast,the intricacy to reactivate cyclosarin-inhibited AChE can be over-come by selection of appropriate oxime structures. The in part, sub-stantial impact of small structural modifications on the reactivatingpotency of oximes emphasize the requirement of detailed kineticinvestigations in order to enable a proper evaluation of the potentialand limitations of new oxime reactivators.

5. Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgement

The study was funded by the German Ministry of Defence.

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