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Chemico-Biological Interactions 187 (2010) 215–219

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journa l homepage: www.e lsev ier .com/ locate /chembio int

inetic analysis of interactions of different sarin and tabun analogues with humancetylcholinesterase and oximes: Is there a structure–activity relationship?

adine Aurbek, Nadja M. Herkert, Marianne Koller, Horst Thiermann, Franz Worek ∗

undeswehr Institute of Pharmacology and Toxicology, Munich, Germany

r t i c l e i n f o

rticle history:vailable online 25 January 2010

eywords:rganophosphorus compoundsximesChEeactivation

a b s t r a c t

The repeated misuse of highly toxic organophosphorus compound (OP) based chemical warfare agentsin military conflicts and terrorist attacks poses a continuous threat to the military and civilian sector. Thetoxic symptomatology of OP poisoning is mainly caused by inhibition of acetylcholinesterase (AChE, E.C.3.1.1.7) resulting in generalized cholinergic crisis due to accumulation of the neurotransmitter acetyl-choline (ACh) in synaptic clefts. Beside atropine as competitive antagonist of ACh at muscarinic AChreceptors oximes as reactivators of OP-inhibited AChE are a mainstay of standard antidotal treatment.However, human AChE inhibited by certain OP is rather resistant to oxime-induced reactivation. The

tructure–activity relationship development of more effective oxime-based reactivators may fill the gaps. To get more insight into apotential structure–activity relationship between human AChE, OPs and oximes in vitro studies wereconducted to investigate interactions of different tabun and sarin analogues with human AChE and theoximes obidoxime and HI 6 by determination of various kinetic constants. Rate constants for the inhibitionof human AChE by OPs, spontaneous dealkylation and reactivation as well as reactivation by obidoximeand HI 6 of OP-inhibited human AChE were determined. The recorded kinetic data did not allow a general

tructu

statement concerning a s

. Introduction

The high number of fatalities due to poisoning by organophos-horus compound-based (OP) pesticides and the continuing threatf misuse of highly toxic OP-type chemical warfare agents (nervegents) emphasize the necessity for an effective medical treatment.he toxic symptomatology of OP poisoning is mainly caused bynhibition of AChE by phosphylation of its active site serine [1].he failure of inhibited AChE to hydrolyze the neurotransmittercetylcholine results in an endogenous acetylcholine intoxicationollowed by an over-stimulation of muscarinic and nicotinic ACheceptors in the autonomic, peripheral and central nervous sys-em. Consequences are massive disturbances of numerous bodyunctions finally resulting in death due to central and peripheralespiratory arrest.

Presently, standard treatment of OP poisoning includes the

dministration of a muscarinic antagonist, e.g. atropine, and of aeactivator of inhibited AChE (oxime) [2]. Hereby, anti-muscarinicrugs act only symptomatically while oximes may restore thenzyme function. At present, the oximes obidoxime, pralidoxime

∗ Corresponding author at: Bundeswehr Institute of Pharmacology and Toxicol-gy, Neuherbergstrasse 11, 80937 Munich, Germany. Tel.: +49 89 3168 2930;ax: +49 89 3168 2333.

E-mail address: FranzWorek@Bundeswehr.org (F. Worek).

009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2010.01.035

re–activity relationship between human AChE, OP and oximes.© 2010 Elsevier Ireland Ltd. All rights reserved.

and HI 6 are introduced in different countries as antidotes againstnerve agent poisoning. However, human AChE inhibited by certainOP, e.g. soman and tabun, is rather resistant towards reactiva-tion by oximes while AChE inhibited by structurally differentOP, e.g. sarin and paraoxon, can be easily reactivated [3,4]. Thedevelopment of more effective oximes may fill the gaps of theactually used therapeutic countermeasures in OP poisoning. Agrowing number of studies have been performed to give a struc-tural basis for the development of improved oximes [5–9]. Inorder to get more insight into potential structural requirements forthe reactivatability of OP-inhibited AChE by oximes in vitro stud-ies were conducted to investigate interactions of different sarin(Fig. 1) and tabun (Fig. 2) homologues with human AChE andobidoxime as well as HI 6 by determination of various kinetic con-stants.

2. Materials and methods

2.1. Materials

Acetylthiocholine iodide (ATCh) and 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB) were obtained from Sigma. Obidoximedichloride (obidoxime) was purchased from Merck (Darmstadt,Germany), HLö 7 dimethanesulfonate was a custom synthesis byDr. Braxmeier (Chemisches Labor, Döpshofen, Germany) and HI 6

216 N. Aurbek et al. / Chemico-Biological In

Fig. 1. Structure of sarin homologues.

Fig. 2. Structure of tabun homologues.

teractions 187 (2010) 215–219

dichloride was kindly provided by Dr. Clement (Defence ResearchEstablishment Suffield, Ralston, Alberta, Canada).

Methylsarin, ethylsarin, n-propylsarin, n-butylsarin, n-pentylsarin, iso-pentylsarin, sec-pentylsarin and neo-pentylsarinas well as tabun and its analogues (>98% by GC–MS, 1H NMR and31P NMR, Figs. 1 and 2) were made available by the Ministry ofDefence (Bonn, Germany). All other chemicals were purchasedfrom Merck Eurolab (Darmstadt, Germany). OP stock solutions(0.1%, v/v) were prepared weekly in 2-propanol, stored at 4 ◦C,and were appropriately diluted in distilled water just before use.Oximes (200 mM) were prepared in distilled water, stored at−80 ◦C and diluted daily as required in distilled water at the day ofexperiment.

Hemoglobin-free erythrocyte ghosts, serving as AChE source,were prepared as described before [10]. Aliquots of the erythrocyteghosts were stored at −80 ◦C until use. Just before the experimentthawed ghosts were homogenised on ice with a Sonoplus HD 2070ultrasonic homogenator (Bandelin Electronic, Berlin, Germany),three-times for 5 s with 30 s intervals, to achieve a homogeneousmatrix for the kinetic studies.

In order to prevent AChE denaturation during long-term exper-iments at 37 ◦C AChE was stabilized by addition of human plasmathat was incubated with soman (100 nM) for 30 min at 37 ◦C toensure complete inhibition and aging of BChE [11]. The inhibitedplasma was dialyzed (phosphate buffer, 0.1 M, pH 7.4) overnight at4 ◦C to adjust pH and to remove residual inhibitor.

2.2. Enzyme assays

AChE activities were measured spectrophotometrically (Cary3Bio, Varian, Darmstadt) with a modified Ellman assay [12,13]. Theassay mixture (3.15 ml) contained 0.45 mM ATCh as substrate and0.3 mM DTNB as a chromogen in 0.1 M phosphate buffer (pH 7.4).

All experiments were performed at 37 ◦C and pH 7.4. All con-centrations refer to final concentrations.

2.3. Inhibition of human AChE

Human erythrocyte ghosts were incubated with a small volume(1%, v/v) of appropriate OP concentrations for 15 min (sarin andanalogues) and 30 min (tabun and analogues) at 37 ◦C to achieve anAChE inhibition by >80%. The samples treated with tabun and ana-logues were dialyzed (phosphate buffer, 0.1 M, pH 7.4) overnightat 4 ◦C to remove residual inhibitor. Then, the absence of inhibitoryactivity was tested by incubation of treated and control ghosts(30 min, 37 ◦C).

2.4. Determination of inhibition rate constants (ki) of sarin andanalogues

The second-order inhibition rate constants (ki) of sarin and itsanalogues were determined with human AChE in the presence ofsubstrate [14]. In brief, 10 �l erythrocyte ghosts and 5 �l dilutedOP were added to a cuvette containing phosphate buffer, DTNB andATCh (final volume 3.165 ml), the resultant OP concentrations were2–75 nM. ATCh hydrolysis was continuously monitored for up to30 min. The recorded curves were analyzed by non-linear regres-sion analysis and used for the further determination of ki = k2/KD[14,15].

2.5. Determination of rate constants for aging (ka) and

spontaneous reactivation (ks)

OP-treated human ghosts were mixed with equal volumes ofsoman-treated, dialyzed human plasma to prevent denaturationof AChE during long-term experiments at 37 ◦C [3]. Samples of

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N. Aurbek et al. / Chemico-Biolog

P-treated, stabilized AChE were temperature equilibrated (37 ◦C;= 0 min). After various time intervals aliquots were taken for deter-ination of AChE activity (spontaneous reactivation) and for the

ecrease in oxime-induced reactivation (aging) which was testedy incubating OP-inhibited ghosts with 100 �M HLö 7 for 15 minsarin and analogues) and 1 mM obidoxime for 15–30 min (tabunnd analogues), respectively. Data were referred to the maxi-um reactivation and the percentage reactivation (% reac) was

alculated. The pseudo-first-order rate constants ks (spontaneouseactivation) and ka (aging) were calculated by a non-linear regres-ion model [3].

.6. Oxime-induced reactivation of OP-inhibited AChE

The reactivation kinetics (37 ◦C) was determined as describedefore by two different procedures depending on the reactivatingotency of the oximes [3]. In case of expected high reactivat-

ng potency (sarin and analogues) the reactivation kinetics wereetermined with the continuous procedure while in case of

ow reactivating potency (tabun and analogues) a discontinuousrocedure was applied which allowed use of higher oxime concen-rations (up to 5 mM). 8–10 different oxime concentrations weresed for the determination of the reactivation rate constants.

. Results and discussion

.1. Inhibition kinetics of sarin homologues with human AChE

The determination of the inhibition rate constants with sarinnalogues revealed a correlation of chain length of the O--alkyl group and the inhibitory potency (Fig. 3A). Hereby,-pentylsarin showed an outstanding high inhibition rate constantf 9.5 × 108 M−1 min−1 which is the highest inhibition rate constantetermined with human AChE so far [3]. Obviously, an increase inhain length facilitates the covalent binding to the active site serinef AChE. The pentylsarin homologues with O-i-alkyl residues exhib-ted lower inhibition rate constants compared to n-pentylsarin,hese results corroborate previous findings of higher inhibition rateonstants of methylphosphonofluoridothionates bearing O-n-alkylroups compared to O-i-alkyl residues [16].

.2. Aging and spontaneous reactivation of human AChEnhibited by sarin homologues

Aging of inhibited AChE proceeded slowly, giving half-timesetween 42 h (methylsarin) and 314 h (n-pentylsarin). There waso obvious structure–activity relationship between chain lengthnd aging. Interestingly, no aging could be determined with sec-entylsarin (Fig. 3B) though its structure is very close to somannd rapid aging of soman-inhibited AChE is the major limitation ofxime treatment in soman poisoning.

There was a correlation between the chain length of O-n-alkylroups (up to C4) and spontaneous reactivation (Fig. 3C) althoughhe half-times were rather long, i.e. between 57 h (methylsarin)nd 9 h (n-butylsarin). With pentylsarin analogues the spontaneouseactivation velocity decreased again, indicating an improved sta-ilization of the phosphonylated enzyme with these agents.

.3. Reactivation kinetics of obidoxime and HI 6 with humanChE inhibited by sarin homologues

The reactivation rate constants of inhibited AChE were stronglyependent on the inhibitor and the oxime (Fig. 4). Obidoxime

nduced reactivation of C1–C3 O-n-alkyl-sarin inhibited AChEhowed higher reactivity but lower affinity compared to HI 6

tion (ks) (C) of AChE inhibited by sarin analogues. Data for methylsarin, ethylsarin,n-propylsarin and n-butylsarin were taken from Ref. [18] and data for n-pentylsarin,iso-pentylsarin, sec-pentylsarin and neo-pentylsarin were taken from Ref. [19].

resulting in comparable oxime potency. However, the reacti-vation of n-butyl- and pentylsarin-inhibited AChE by oximeswas characterized by a superior potency of HI 6 compared toobidoxime. Thereby, only small differences in KD and kr wererecorded between the pentylsarin homologues reactivated by HI 6.A more pronounced variability was observed with obidoxime(Fig. 4).

Unlike inhibition and spontaneous reactivation kinetics oxime-induced reactivation of AChE inhibited by sarin homologues did notfollow a structure–activity relationship.

3.4. Aging and spontaneous reactivation of human AChEinhibited by tabun homologues

Aging of OP-inhibited human AChE followed pseudo-first-orderkinetics and was strongly dependent on the structure of the used

ing chain length of the N-mono-alkyl residue (Fig. 5). Aging kineticscould not be determined with N-diethyl and N-di-n-propyl tabunsince AChE inhibited by these compounds was resistant towardsreactivation by oximes. AChE inhibited by tabun homologues didnot reactivate spontaneously.

218 N. Aurbek et al. / Chemico-Biological Interactions 187 (2010) 215–219

Fig. 4. Reactivation rate constants (kr, A; KD, B and kr2, C) for obidoxime- (blackcolumn) and HI 6- (grey column) induced reactivation of AChE inhibited by sarinanalogues. Data for methylsarin, ethylsarin, n-propylsarin and n-butylsarin weretaken from Ref. [18], data for n-pentylsarin, iso-pentylsarin, sec-pentylsarin andneo-pentylsarin were taken from Ref. [19].

Fig. 5. Rate constants for aging (ka) of AChE inhibited by N-monoalkyl tabun homo-logues. Abscissa labelling indicates the N-alkyl residues. Data were taken from Ref.[17].

Fig. 6. Reactivation rate constants (kr, A; KD, B and kr2, C) for obidoxime-inducedreactivation of AChE inhibited by N-monoalkyl tabun homologues. Abscissa labellingindicates the N-alkyl residues. Data were taken from Ref. [17].

3.5. Reactivation kinetics of obidoxime with human AChEinhibited by tabun homologues

Obidoxime-induced reactivation of human AChE inhibited bytabun homologues was strongly dependent on the structure ofthe phosphyl moiety. The reactivation by oximes was markedlyaffected by the different residues at the amido group. The reac-tivation kinetics of obidoxime with N-mono-alkyl tabun analoguesrevealed a dependence of the reactivity rate constant kr (Fig. 6A)and the dissociation constant KD (Fig. 6B) on the chain length of theN-alkyl residue. The affinity as well as the reactivity of obidoximedecrease with the chain length of the N-alkyl residue resulting inan enormous difference of the second-order reactivation rate con-stant (Fig. 6C). The relationship between chain length, reactivation(Fig. 6) and aging (Fig. 5) kinetics indicates a stabilization of thephosphyl–AChE complex with increased chain length of the N-alkylgroup.

AChE inhibited by tabun analogues bearing a N,N-diethyl orN,N-di-n-propyl residue could not be reactivated by oximes. Upto now, it is not understood whether this is due to an inability of

oximes to attack the phosphyl–AChE complex or an extremely rapidaging. Hence it is surprising that AChE inhibited by N,N-di-i-propyltabun was shown to be readily accessible towards reactivation byobidoxime (not shown) [17].

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. Conclusion

The determination of inhibition, reactivation and aging con-tants of different sarin homologues with human AChE indicateshat a structure–activity relationship can be derived for inhibitions well as for spontaneous reactivation but not for dealkylation andxime-induced reactivation.

The kinetic analysis of interactions between human AChE andtructurally different tabun homologues did not show a cleartructure–activity relationship concerning the reactivation potencyf obidoxime. With N-mono-alkyl analogues a structure–activityelationship of oxime-induced reactivation and aging of the inhib-ted AChE depending on the chain length of N-alkyl residue wasbserved. No such relationship could be derived with data from,N-dialkyl tabun analogues.

These data in conjunction with kinetic data generated with otherximes and OPs emphasize the need for the determination of reac-ivation rate constants in order to evaluate the ability of oximes toeactivate inhibited AChE. Up to now, it is not obvious that the reac-ivating potency of oximes can be predicted on basis of availableinetic data.

onflict of interests

The authors declare that there are no conflicts of interest.

cknowledgements

The authors are grateful to T. Hannig and L. Windisch for engagedechnical assistance.

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