reactivation kinetics of 31 structurally different bispyridinium oximes with...

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Arch ToxicolDOI 10.1007/s00204-014-1288-5

MOleculAr TOxIcOlOgy

Reactivation kinetics of 31 structurally different bispyridinium oximes with organophosphate‑inhibited human butyrylcholinesterase

Gabriele Horn · Timo Wille · Kamil Musilek · Kamil Kuca · Horst Thiermann · Franz Worek

received: 2 April 2014 / Accepted: 28 May 2014 © Springer-Verlag Berlin Heidelberg 2014

to serve as reactivators of human Bche inhibited by differ-ent OP and it is doubtful whether further modifications of the bispyridinium template will lead to more potent reacti-vators. In the end, novel structures of oxime and non-oxime reactivators are urgently needed for the development of human Bche into an effective pseudo-catalytic scavenger.

Keywords Organophosphorus compounds · Butyrylcholinesterase · Oxime · reactivation · Scavenger · In vitro

Introduction

Toxic organophosphorus compounds (OP), pesticides and nerve agents, act primarily by inhibition of the pivotal enzyme acetylcholinesterase (Ache), resulting in accu-mulation of acetylcholine at cholinergic synapses (Holm-stedt 1959; Aldridge and reiner 1972). Overstimulation of nicotinic and muscarinic receptors leads to disturbance of multiple body functions and finally to respiratory arrest and death (grob 1956; Krieger 2001). For several decades, the standard treatment of OP poisoning has consisted of an antimuscarinic, e.g., atropine, and an Ache reactivator (oxime), e.g., obidoxime, pralidoxime or TMB-4 (eyer and Worek 2007; Jokanovic 2009). However, numerous in vitro and in vivo studies demonstrated the limitations of oxime therapy, depending on the type and incorporated amount of OP and the oxime (Worek and Thiermann 2013).

In contrast to Ache, the inhibition of plasma and tis-sue butyrylcholinesterase (Bche) by OP does not directly translate into a toxic effect (lockridge and Masson 2000; Aurbek et al. 2009). However, Bche serves as an endoge-nous scavenger being able to bind OP and thus reducing the toxic body load to some extent (Nachon et al. 2013). The

Abstract Organophosphorus compounds (OP) are bound to human butyrylcholinesterase (Bche) and endogenous or exogenous Bche may act as a stoichiometric scaven-ger. Adequate amounts of Bche are required to minimize toxic OP effects. Simultaneous administration of Bche and oximes may transfer the enzyme into a pseudo-catalytic scavenger. The present study was initiated to determine the reactivation kinetics of 31 structurally different bispyridin-ium oximes with paraoxon-, tabun- and cyclosarin-inhib-ited human Bche. Human plasma was incubated with OP and the reactivation of inhibited Bche was tested with mul-tiple oxime concentrations followed by nonlinear regres-sion analysis for the determination of reactivity, affinity and overall reactivation constants. The generated data indicate that the tested oximes have a low-to-negligible reactivating potency with paraoxon- and tabun-inhibited human Bche. Several oximes showed a moderate-to-high potency with cyclosarin-inhibited Bche. Thus, the present study indi-cates that bispyridinium oximes are obviously not suitable

g. Horn · T. Wille · H. Thiermann · F. Worek (*) Bundeswehr Institute of Pharmacology and Toxicology, Munich, germanye-mail: [email protected]

K. Musilek Department of chemistry, Faculty of Science, university of Hradec Kralove, Hradec Kralove, czech republic

K. Kuca Faculty of Military Health Sciences, center of Advanced Studies and Faculty Hospital, university of Defence, Hradec Kralove, czech republic

K. Kuca Biomedical research center, university Hospital Hradec Kralove, Hradec Kralove, czech republic

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concept to use human Bche as a stoichiometric bioscav-enger was investigated in numerous studies, and the results underline the principal suitability of Bche to prevent toxic OP effects if given as a pre-treatment and under certain conditions even as a post-exposure treatment (Mumford et al. 2013; Mumford and Troyer 2011; Saxena et al. 2011; lenz et al. 2007).

A major drawback of Bche as a bioscavenger is the need to administer large doses since Bche binds OP at a molar ratio (lenz et al. 2007). In order to overcome this limitation, several research groups proposed the com-bination of human Bche and reactivators which would transfer Bche from a stoichiometric to a ‘pseudo-cat-alytic’ bioscavenger (radic et al. 2013; Kovarik et al. 2010). Human Bche as pseudo-catalytic scavenger would reduce the required enzyme dose resulting in a more convenient administration of the enzyme, lower therapy costs and potentially diminish an immunologic response.

recently, we determined the reactivation rate constants of a number of bispyridinium oximes with human Ache inhibited by structurally different OP under standardized conditions (Worek et al. 2010, 2012a, b). Now, we found it tempting to investigate the reactivation kinetics of more than 30 structurally different bispyridinium oximes with human plasma Bche inhibited by the organophosphate paraoxon, the organophosphonate cyclosarin and the phos-phoramidate tabun. By applying a standardized protocol, the data should give insight into structural requirements of an effective reactivator and may provide a basis for a devel-opment of novel oximes.

Materials and methods

chemicals

The organophosphorus compounds (OP) tabun (gA) and cyclosarin (gF) (>98 % by gc–MS, 1H NMr and 31P NMr, Fig. 1) were supplied by the german Ministry of Defence and paraoxon-ethyl (paraoxon, Pxe, Fig. 1) was obtained from Dr. ehrensdorfer gmbH (Augsburg, ger-many). Tabun (6.2 mM) and cyclosarin stock solutions (5.5 mM) were prepared in acetonitrile and paraoxon stock solution (10 mM) in 2-propanol and stored at 20 °c.

Tested oximes (>95 % by 1H NMr, Table 1) were of dif-ferent origin:

MMB-4 was obtained from Prof. Fusek (Purkyne Mili-tary Medical Academy, Hradec Kralove, czech republic). Obidoxime was purchased from Merck (Darmstadt, ger-many), TMB-4 and pralidoxime (2-PAM) from Sigma-Aldrich (Taufkirchen, germany). Hlö7 was synthesized by J. Braxmeier (chemisches labor, Döpshofen, germany).

HI-6 was made available by Dr. clement (Defence research establishment Suffield, ralston, Alberta, canada) and HS 3 and HS 4 were provided by Prof. eyer (univer-sity of Munich, Munich, germany). All K-oximes were prepared by Assoc. Prof. Musilek and Prof. Kuca (Kim et al. 2005; Kuca et al. 2003a, b; Musilek et al. 2005, 2006, 2007, 2008, 2011).

Oxime stock solutions (100 or 200 mM) of K068, K108, K114, K181, K239 and HS 4 were prepared in dimethyl sulfoxide (DMSO) due to low solubility; all other oximes were dissolved in distilled water and stored at −80 °c. At the day of experiment, oxime stock solutions were diluted appropriately with DMSO and distilled water, respectively.

The chemicals S-butyrylthiocholine iodide (BTch) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were obtained from Sigma-Aldrich. All other substances were purchased from Merck (Darmstadt, germany).

Human plasma

Plasma was obtained from heparinized human whole blood by centrifugation (3,000 rpm, 10 min, 4 °c) and was stored at −80 °c until use. Plasma samples were incubated with small volumes (1 % v/v) of appropriate tabun, paraoxon or cyclosarin to inhibit Bche by >90 %. Inhibited and con-trol plasma were dialyzed (phosphate buffer, 0.1 M, pH 7.4) overnight to remove residual inhibitor and to adjust pH 7.4. Absence of residual inhibitor was verified by incuba-tion of inhibited and control plasma and determination of Bche activity. Aliquots were stored at −80 °c. At the day

Fig. 1 chemical structures of tested OP

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of experiment, plasma was centrifuged (10,000 rpm, 5 min, 4 °c) to remove cryoprecipitates.

Bche assay

Bche activity was determined with an uV–Vis spectro-photometer (cary 50 Bio, Varian, Darmstadt, germany) at a wavelength of 412 nm with a modified ellman assay

(Worek et al. 1999) using 0.3 mM DTNB and 1.0 mM BTch as substrate. All experiments were performed at 37 °c.

Inhibition of human Bche by oximes

20 µl Oximes (1–1,000 µM final concentration) were trans-ferred to tempered cuvettes (37 °c) containing 1,820 µl

Table 1 chemical structure and inhibitory activity of tested oximes

The inhibition of native human Bche by oximes was tested with 10 concentrations (1–1,000 µM) in duplicate with SD < 5 %

n.d. not determined

# The oxime precipitated at higher concentrations

code a r1 y b r2 x Ic50 (µM)

MMB-4 4 cHNOH cH2 4 cHNOH Br >1,000

K191 4 cHNOH (cH2)2 4 cHNOH Br n.d.

TMB-4 4 cHNOH (cH2)3 4 cHNOH Br >1,000

K074 4 cHNOH (cH2)4 4 cHNOH Br >1,000


K027 4 cHNOH (cH2)3 4 cONH2 Br >1,000

K048 4 cHNOH (cH2)4 4 cONH2 Br >1,000

K203 4 cHNOH cH2–cH=cH–cH2 4 cONH2 Br >1,000

K075 4 cHNOH cH2–cH=cH–cH2 4 cHNOH Br >1,000

Obidoxime 4 cHNOH cH2–O–cH2 4 cHNOH cl >1,000

K117 4 cHNOH (cH2)2–O–(cH2)2 4 cHNOH Br >1,000

K127 4 cHNOH (cH2)2–O–(cH2)2 4 cONH2 Br >1,000

Hlö 7 2, 4 cHNOH cH2–O–cH2 4 cONH2 DMS >1,000

K239 2, 4 cHNOH cH2–O–cH2 2, 4 cHNOH cl #

K114 4 cHNOH p-xylene 4 cHNOH Br 544

K208 2 cHNOH (cH2)3 4 cHNOH Br 17

K290 2 cHNOH (cH2)3 4 cONH2 Br 542


K053 2 cHNOH cH2–cH=cH–cH2 4 cHNOH Br 696

HS 3 2 cHNOH cH2–O–cH2 4 cHNOH cl 646

HI-6 2 cHNOH cH2–O–cH2 4 cONH2 cl >1,000




K068 2 cHNOH cH2–cH=cH–cH2 2 cHNOH Br 147

HS 4 2 cHNOH cH2–O–cH2 2 cHNOH J 68

K129 2 cHNOH (cH2)2–O–(cH2)2 2 cHNOH Br 313





2-PAM 2 cHNOH cH3 – – cl >1,000

N+

YN

+ R2 (b)R

1 (a)

2 X-

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phosphate buffer (0.1 M, pH 7.4), 100 µl DTNB (0.3 mM) and 10 µl native human plasma. Then, 50 µl BTch was added (final volume 2,000 µl) and the Bche activity was measured. In addition, concentration-dependent oxime blanks were determined in the absence of plasma. All experiments were performed in duplicate.

The Ic50 values were calculated from semi-logarithmic plots of the oxime concentration versus the Bche activity.

reactivation of OP-inhibited human Bche by oximes

2 µl Oxime was added at t = 0 to 200 µl OP-inhibited Bche to start enzyme reactivation. 10 µl aliquots were transferred to tempered cuvettes at specified time intervals (paraoxon- or tabun-inhibited Bche: 2–60 min, cyclosarin-inhibited Bche: 0.5–60 min) filled with 3,000 µl phosphate buffer and 100 µl DTNB. Measurement of Bche activity was initiated after addition of 50 µl BTch. All experiments ran in dupli-cate with 6–8 different oxime concentrations (1–5,000 µM).

OP-inhibited Bche in the presence and absence of oxi-mes was referred to control Bche activity for the calcula-tion of the time-dependent % reactivation.

reactivation kinetics

OP-inhibited Bche is reactivated by oximes in two steps. First, the Michaelis-type OP–Bche–oxime complex [ePOx] is generated from OP-inhibited Bche [eP] and the oxime [Ox]. Second, reactivated Bche [e] and phos-phylated oxime [POx] are formed from [ePOx]. KD repre-sents the dissociation constant and is inversely proportional to the affinity of the oxime to [eP] and kr the reactivation rate constant for removing the OP residue from [ePOx].

A pseudo-first-order rate equation (eq. 1) can be derived in the event of a complete enzyme reactivation and with [Ox] ≫ [eP]0.

kobs was calculated by nonlinear regression analysis (Worek et al. 2010) using eq. (2):

Finally, oxime concentrations were plotted versus kobs and kr and kD were obtained from nonlinear regression analysis and the second-order rate constant kr2 was calcu-lated from the ratio of kr and kD.

Oxime concentrations necessary to obtain a defined frac-tion of reactivated Bche at a given time in the absence of excess inhibitor were calculated according to (Worek et al. 2011) with eq. (3):

(1)kobs =kr × [OX]

KD + [OX]

(2)vt = v0 ×

(

1 − ekobs×t

)

Table 2 reactivation of paraoxon-inhibited human Bche by 4,4-oximes

The pseudo-first-order rate constant kobs was determined by nonlinear regression analysis using eq. (2). These data were used for the calcu-lation 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 mean ± SD (n = 2). Ø calculation of reactivation constants not possible due to inadequate reactivation of inhibited Bche

Oxime KD (µM) kr (min−1) kr2 (mM−1 min−1)

MMB-4 544 ± 42 0.05 ± 0.001 0.08 ± 0.004

K191 1,402 ± 1 0.12 ± 0.0002 0.09 ± 0.0001

TMB-4 578 ± 12 0.09 ± 0.0005 0.15 ± 0.002

K074 255 ± 22 0.06 ± 0.0005 0.24 ± 0.02

K305 119 ± 2 0.05 ± 0.0002 0.42 ± 0.008

K027 360 ± 26 0.04 ± 0.0008 0.12 ± 0.007

K048 671 ± 23 0.05 ± 0.0002 0.07 ± 0.002

K203 271 ± 9 0.04 ± 0.0005 0.16 ± 0.007

K075 181 ± 22 0.06 ± 0.0001 0.34 ± 0.04

Obidoxime 317 ± 2 0.07 ± 0.0004 0.23 ± 0.002

K117 292 ± 3 0.07 ± 0.0002 0.25 ± 0.002

K127 307 ± 29 0.05 ± 0.001 0.17 ± 0.01

Hlö 7 267 ± 5 0.01 ± 0.0 0.04 ± 0.0005

K239 Ø Ø Ø

K114 266 ± 27 0.06 ± 0.001 0.24 ± 0.02

Table 3 reactivation of tabun-inhibited human Bche by 4,4-oximes



MMB-4 1,317 ± 39 0.008 ± 0.0001 0.006 ± 0.0001

K191 458 ± 12 0.044 ± 0.0005 0.097 ± 0.003

TMB-4 823 ± 18 0.012 ± 0.0 0.015 ± 0.0003

K074 603 ± 27 0.008 ± 0.0 0.013 ± 0.0005

K305 411 ± 18 0.009 ± 0.0001 0.021 ± 0.0008

K027 1,152 ± 52 0.007 ± 0.0002 0.006 ± 0.0001

K048 1,341 ± 25 0.006 ± 0.0001 0.004 ± 0.0

K203 1,929 ± 65 0.007 ± 0.0 0.004 ± 0.0001

K075 421 ± 26 0.006 ± 0.0002 0.013 ± 0.0004

Obidoxime 735 ± 49 0.013 ± 0.0 0.017 ± 0.001

K117 Ø Ø Ø

K127 Ø Ø Ø

Hlö 7 490 ± 36 0.004 ± 0.0 0.009 ± 0.0005

K239 Ø Ø Ø

K114 252 ± 8 0.002 ± 0.0 0.009 ± 0.0004

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Data analysis

Data processing for the calculation of the kinetic constants KD, kr and kr2 was performed by nonlinear regression analy-sis using curve fitting program provided by Prism™ ver-sion 4.03 (graphPad Software, San Diego, cA, uSA). In all cases, individual data sets were analyzed to obtain kobs and the derived constants KD, kr and kr2 are presented as mean ± SD.

Results

The determination of the reactivation kinetics of human Bche inhibited by the organophosphate paraoxon, the organophosphonate cyclosarin or the phosphorami-date tabun with 31 structurally different bispyridinium oximes and the monopyridinium oxime pralidoxime resulted in general in a low reactivating potency with paraoxon- and tabun-inhibited Bche, while some oxi-mes were remarkably potent with cyclosarin-inhibited Bche.

(3)[OX] = −

KD

1 +t×kr

ln

(

v0−vt

v0−vi

)

reactivation kinetics of 4,4-oximes

The reactivation of paraoxon-inhibited Bche with com-pounds bearing two oxime or one oxime and one carba-moyl group at position 4 of the pyridinium rings was char-acterized by a moderate-to-low affinity and a low reactivity, i.e. kr < 0.2 min−1 (Table 2). This resulted in second-order reactivation rate constants of <0.5 mM−1 min−1 throughout.

Tabun-inhibited Bche was even more resistant toward reactivation with these oximes (Table 3). The low reac-tivating potency (kr2 ≤ 0.2 mM−1 min−1) was a result of a low affinity and an exceptionally low reactivity (kr < 0.05 min−1). No reactivation rate constants could be determined with K117, K127 and K239 due to substantial deviation of the reactivation curves being most likely an effect of re-inhibition by phosphyloximes formed during the reactivation reaction.

The ability to reactivate cyclosarin-inhibited Bche was strongly dependent on the linker type (Table 4). Oxi-mes bearing an alkanyl, alkenyl or xylenyl linker had a

Table 4 reactivation of cyclosarin-inhibited human Bche by 4,4-oximes



MMB-4 3,591 ± 27 0.51 ± 0.02 0.14 ± 0.006

K191 1,513 ± 44 0.43 ± 0.0237 0.29 ± 0.02

TMB-4 921 ± 26 0.26 ± 0.0004 0.28 ± 0.008

K074 233 ± 27 0.05 ± 0.0 0.22 ± 0.02

K305 163 ± 12 0.04 ± 0.0004 0.26 ± 0.02

K027 562 ± 2 0.07 ± 0.0 0.13 ± 0.0005

K048 927 ± 33 0.05 ± 0.001 0.06 ± 0.001

K203 1,407 ± 2 0.10 ± 0.001 0.07 ± 0.0007

K075 310 ± 17 0.08 ± 0.002 0.25 ± 0.007

Obidoxime 296 ± 23 0.44 ± 0.004 1.51 ± 0.1

K117 682 ± 72 0.78 ± 0.006 1.15 ± 0.1

K127 1,618 ± 77 0.86 ± 0.02 0.53 ± 0.01

Hlö 7 118 ± 13 0.85 ± 0.004 7.28 ± 0.8

K239 20.9 ± 1 4.54 ± 0.08 217.7 ± 3.27

K114 Ø Ø Ø

Table 5 reactivation of OP-inhibited human Bche by 2,4-oximes


OP Oxime KD (µM) kr (min−1) kr2 (mM−1 min−1)

Pxe K208 Ø Ø Ø

K290 172 ± 3 0.01 ± 0.0001 0.03 ± 0.0002

K308 313 ± 42 0.04 ± 0.0007 0.14 ± 0.02

K053 268 ± 33 0.06 ± 0.001 0.24 ± 0.03

HS 3 350 ± 25 0.02 ± 0.0001 0.07 ± 0.004

HI-6 582 ± 12 0.02 ± 0.0 0.03 ± 0.0006

K181 42 ± 0 0.01 ± 0.0 0.29 ± 0.004

Tabun K208 Ø Ø Ø

K290 466 ± 40 0.001 ± 0.0 0.002 ± 0.0002

K308 579 ± 28 0.005 ± 0.0001 0.009 ± 0.0003

K053 512 ± 18 0.041 ± 0.0004 0.080 ± 0.004

HS 3 287 ± 6 0.007 ± 0.0001 0.024 ± 0.0

HI-6 747 ± 55 0.003 ± 0.0 0.004 ± 0.0003

K181 Ø Ø Ø

cyclosarin K208 Ø Ø Ø

K290 405 ± 20 0.88 ± 0.02 2.18 ± 0.1

K308 455 ± 43 0.12 ± 0.005 0.26 ± 0.01

K053 812 ± 71 3.74 ± 0.003 4.65 ± 0.4

HS 3 541 ± 3 2.20 ± 0.004 4.07 ± 0.03

HI-6 670 ± 0 0.98 ± 0.01 1.46 ± 0.02

K181 154 ± 3 0.46 ± 0.003 2.99 ± 0.07

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low reactivating potency with a kr2 ≤ 0.5 mM−1 min−1. In contrast, oximes bearing an ether bridge were substan-tially more reactive and potent. even more potent was Hlö 7, an oxime bearing two oxime groups in position 2 and

4 on one pyridinium ring, due to a moderate affinity and reactivity. An outstanding effect was recorded with K239, an oxime bearing 4 oxime groups (position 2 and 4 at both pyridinium rings). K239 had an exceptionally high affin-ity and reactivity resulting in an extraordinary high kr2 of 217.7 mM−1 min−1.


Oximes bearing an oxime group at position 2 of one pyri-dinium ring and an oxime or carbamoyl group at position 4 of the second pyridinium ring exhibited a low reactivating potency with paraoxon- and tabun-inhibited Bche which was mainly due to a low reactivity (Table 5). Most 2,4-oxi-mes showed a reasonably high reactivating potency with cyclosarin-inhibited Bche, primarily due to a moderate-to-high reactivity.


The reactivation kinetics of oximes with an oxime group at position 2 of both pyridinium rings was comparable to 2,4-oximes, i.e., low reactivity and reactivating potency with paraoxon- and tabun-inhibited Bche and a moderate-to-high reactivity and reactivating potency with cyclosarin-inhibited Bche (Table 6).

reactivation kinetics of 3-oximes

Oximes bearing an oxime group at position 3 of one pyri-dinium ring and an oxime at position 2, 3 or 4 of the second pyridinium ring showed a negligible ability to reactivate paraoxon-, tabun- and cyclosarin-inhibited Bche (Table 7). With most oximes, it was impossible to determine reli-able reactivation constants despite a reactivation time up to 60 min and use of oxime concentrations up to 5 mM.

reactivation kinetics of pralidoxime

The monopyridinium oxime pralidoxime exhibited a low reactivating potency with all tested OP (Table 8). With

Table 6 reactivation of OP-inhibited human Bche by 2,2-oximes



Pxe K005 Ø Ø Ø

K033 121 ± 4 0.01 ± 0.0002 0.09 ± 0.001

K068 437 ± 14 0.09 ± 0.0001 0.21 ± 0.007

HS 4 395 ± 3 0.03 ± 0.0005 0.06 ± 0.002

K129 191 ± 10 0.01 ± 0.0001 0.07 ± 0.003

K108 23 ± 0 0.01 ± 0.0 0.20 ± 0.004

Tabun K005 Ø Ø Ø

K033 161 ± 0.1 0.007 ± 0.0001 0.044 ± 0.0006

K068 940 ± 31 0.087 ± 0.005 0.092 ± 0.002

HS 4 199 ± 16 0.002 ± 0.0 0.011 ± 0.001

K129 253 ± 0 0.005 ± 0.0 0.020 ± 0.0001

K108 Ø Ø Ø


K033 320 ± 3 1.16 ± 0.0005 3.62 ± 0.04

K068 899 ± 17 3.90 ± 0.04 4.34 ± 0.03

HS 4 677 ± 4 2.18 ± 0.01 3.23 ± 0.002

K129 1,734 ± 46 1.76 ± 0.02 1.02 ± 0.02

K108 179 ± 1 1.50 ± 0.01 8.39 ± 0.12

Table 7 reactivation of OP-inhibited human Bche by 3-oximes



Pxe K207 Ø Ø Ø

K099 Ø Ø Ø

K209 899 ± 88 0.08 ± 0.0001 0.09 ± 0.009

Tabun K207 Ø Ø Ø

K099 1,344 ± 111 0.004 ± 0.0 0.003 ± 0.0002

K209 837 ± 6 0.008 ± 0.0 0.010 ± 0.0001


K099 Ø Ø Ø

K209 Ø Ø Ø

Table 8 reactivation of OP-inhibited human Bche by pralidoxime

The pseudo-first-order rate constant kobs was determined by nonlinear regression analysis using eq. (2). These data were used for the calcu-lation 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 mean ± SD (n = 2)

OP KD (µM) kr (min−1) kr2 (mM−1 min−1)

Pxe 1,603 ± 21 0.13 ± 0.0003 0.08 ± 0.001

Tabun 1,642 ± 40 0.003 ± 0.0 0.002 ± 0.0001

cyclosarin 12,496 ± 40 1.41 ± 0.007 0.11 ± 0.001

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paraoxon- and tabun-inhibited Bche, this was due to low affinity and reactivity, while with cyclosarin-inhib-ited Bche, an outstanding low affinity, i.e., KD of more than 12 mM, combined with a rather high reactivity was recorded.

reactivation rate constants could not be determined with a number of oxime–OP combinations (Tables 2, 3, 4, 5, 6, 7). This was either due to a negligible reactivation or to a high intrinsic inhibitory activity which prevented the use of sufficiently high oxime concentrations.

Inhibition of native Bche by oximes

The inhibition of native Bche was tested with oxime concen-trations ranging from 1 to 1,000 µM. Approximately 50 % of the tested oximes, i.e., 4,4-oximes, 3-oximes and pralidoxime, induced no or only a slight inhibition at the highest oxime con-centration indicating an Ic50 of >1,000 µM (Table 1). eight oximes had Ic50 values between 100 and 700 µM, and only few oximes resulted in Bche inhibition at low concentrations. These were the 2,2-oxime K005, the 2,3-oxime K207 and 2,4-oxime K208. No Ic50 value could be determined with K239 due to precipitation of the oxime at higher concentrations.

calculated oxime concentrations

The determined reactivation constants enabled the calcula-tion of required oxime concentrations for the reactivation of a defined fraction of inhibited Bche within a given time in the absence of excess inhibitor (Table 9). The calculated values demonstrate that rather high oxime concentrations would be needed to reactivate a small portion (20 %) of paraoxon-inhibited Bche within 10 min. With few excep-tions, K053, K068 and K191, more than millimolar oxime concentrations would be needed to reactivate tabun-inhib-ited Bche partially. In contrast, a larger number of oximes would be able to reactivate cyclosarin-inhibited Bche at low, potentially therapeutically relevant, concentrations. With some oximes, even less than 10 µM should be able to reactivate the inhibited enzyme at the selected conditions.

Discussion

The results of the present study demonstrate a low reac-tivating potency of all investigated oximes with human

Table 9 calculated oxime concentrations for the reactivation of OP-inhibited human Bche

The data represent calculated oxime concentrations (µM) necessary to reactivate 20 % inhibited Bche within 10 min in the absence of excess inhibitor. The data were calculated by applying eq. (3), assuming Bche being fully inhibited and being 100 % reactivatable. Ø no calculation due to lack of reactivation rate constants. concentra-tions exceeding the Ic50 are marked in bold italics

Oxime code Paraoxon Tabun cyclosarin

4,4-Oximes MMB-4 528 >1,000 165

K191 323 463 82

TMB-4 197 >1,000 87

K074 153 >1,000 184

K305 97 >1,000 181

K027 367 >1,000 253

K048 569 >1,000 658

K203 302 >1,000 411

K075 105 >1,000 128

Obidoxime 139 >1,000 16

K117 129 Ø 20

K127 234 Ø 43

Hlö 7 >1,000 >1,000 3

K239 Ø Ø <1

K114 146 >1,000 Ø

2,4-Oximes K208 Ø Ø Ø

K290 >1,000 >1,000 11

K308 319 >1,000 109

K053 143 616 5

HS 3 >1,000 >1,000 6

HI-6 >1,000 >1,000 16

K181 >1,000 Ø 8

2,2-Oximes K005 Ø Ø Ø

K033 >1,000 >1,000 6

K068 143 326 5

HS 4 >1,000 >1,000 7

K129 >1,000 >1,000 22

K108 >1,000 Ø 3

2,3-Oxime K207 Ø Ø Ø

3,3-Oxime K099 Ø >1,000 Ø

3,4-Oxime K209 334 >1,000 Ø

2-Oxime 2-PAM 343 >1,000 201

Fig. 2 reactivation of cyclosarin-inhibited human Bche by MMB-4, K191, TMB-4, K074 and K305. The reactivation constants kr, KD and kr2 of 4,4′-oximes with alkane linkers are plotted in dependence to the linker length

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Bche inhibited by paraoxon and tabun. These data are in line with previous studies examining a limited number of oximes with different protocols (Kovarik et al. 2010; calic et al. 2008; Aurbek et al. 2009). Tabun-inhibited Ache and Bche are known to be particularly resistant toward reacti-vation by oximes (carletti et al. 2009; Worek et al. 2007). This was attributed to the tight binding of the tabun to ser-ine at the active site of cholinesterases and may give an explanation for the low reactivity of the tested oximes.

A crucial factor for effective reactivation of OP-inhib-ited cholinesterases by oximes is an adequate affinity. In

contrast to Ache, Bche lacks aromatic residues at the peripheral binding site which are essential for proper bind-ing of an oxime (carletti et al. 2009; Wiesner et al. 2010; Vellom et al. 1993). In fact, most of the tested oximes had a low affinity toward native and OP-inhibited Bche.

In addition, the acyl binding pocket of Bche is more spacious than the corresponding Ache pocket (Vellom et al. 1993). This may affect the proper orientation of an oxime for in-line attack of the phosphorus moiety and may contribute to the weak reactivation potency.

However, the various described mechanisms were obvi-ously of less importance for the reactivation of cyclosarin-inhibited Bche. 14 of 32 tested oximes had a second-order reactivation rate constant kr2 of >1 mM−1 min−1. This reac-tivation potency was not determined by the position of the oxime group(s) which is in contrast to cyclosarin-inhibited Ache (Worek et al. 2012b). The most potent reactivator of cyclosarin-inhibited Bche was K239 having a kr2 of >217.7 mM−1 min−1. This bispyridinium oxime with an ether bridge and two oxime groups at both pyridinium rings showed an exceptionally high affinity and reactivity. Nota-bly, this molecule was most suitable for proper orientation and subsequent attack of the phosphonyl moiety.

The reactivation of OP-inhibited Bche with 3,2′-, 3,3′- and 3,4′-oximes did not follow first-order kinetics. With these oximes, a biphasic reactivation curve was obtained indicating a re-inhibition of the reactivated enzyme by highly potent and stable phosphyloxime generated during reactivation (Ashani et al. 2003).

The evaluation of a structure–activity relationship of the tested oximes was hampered by the lack of reactiva-tion rate constants for a number of OP–oxime–Bche com-binations. reactivation of cyclosarin-inhibited Bche with 4,4′-oximes bearing a c1 to c5 linker resulted in very

Fig. 3 reactivation of OP-inhibited Bche by K075, K053 and K068. The reactivation constants kr (a), KD (b) and kr2 (c) of the oxi-mes bearing an (E)-but-2-ene linker are plotted in dependence to the position of the oxime groups

Fig. 4 reactivation kinetics of OP-inhibited Bche. The data show the ratio of the second-order reactivation constant kr2 of oximes bear-ing two oxime groups and oximes bearing one oxime and one car-bonyl group. The dashed line indicates a ratio of 1

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comparable second-order reactivation constants (Fig. 2). However, increase in affinity was correlated with linker length, while reactivity decreased from c1 to c5. cor-responding phenomena were observed for the reactiva-tion of cyclosarin-inhibited Ache by a homologous series of bispyridinium 2,2′-oximes with c1 to c9 linkers (Wille et al. 2010). The impact of the position of the oxime groups could be assessed with K075, K053 and K068, oximes having E-but-2-ene linkers (Fig. 3). With all tested OP, the reactivity increased in the order 4,4′ < 4,2′ < 2,2′ and the affinity decreased in the inverse order. The differences were most pronounced with cyclosarin-inhibited Bche leading to a dramatic increase of reactivity and reactivating potency from 4,4′ to 4,2′. Finally, the impact of an oxime versus a carbamoyl group at the second pyridinium ring was inves-tigated (Fig. 4). It became apparent that the ratio of the second-order reactivation constant between an oxime bear-ing two oxime moieties and one oxime with one oxime and one carbamoyl moiety was >1 throughout. This indicates that under the tested conditions, oxime groups on both pyri-dinium rings are more beneficial for the reactivation of OP-inhibited Bche.

The investigation of the intrinsic inhibitory activity of the tested oximes with native human Bche revealed a low inhibitory potency with most of the oximes which cor-relates with the aforementioned low affinity. Few oximes showed a remarkably low Ic50 value of <20 µM. The com-mon characteristic of K208, K005 and K207 was a propyl linker with different position of the oxime groups, i.e. 2,4′, 2,2′ and 2,3′. However, other oximes with propyl linkers exhibited a low inhibitory activity indicating that the spe-cific configuration of the substitutes bound to the pyridin-ium rings together with the linker type are determining the inhibitory potency of an oxime.

The determined reactivation constants enabled the cal-culation of necessary oxime concentrations for the reac-tivation of a fraction of inhibited Bche. Hereby, rather moderate conditions, 20 % reactivation within 10 min in the absence of free OP, were applied (Table 9). The data indicate that none of the tested oximes would be adequate to reactivate tabun-inhibited Bche and most of the oxi-mes would fail the goal with paraoxon-inhibited Bche at therapeutically relevant concentrations in humans. At least several oximes should be able to reactivate cyclosarin-inhibited Bche at a rate which could transform endoge-nous or exogenous Bche into a pseudo-catalytic scavenger. None of the investigated oximes showed adequate efficacy against structurally different OP.

Hence, the present study indicates that bispyridinium oximes are obviously not suitable to serve as reactivators of human Bche inhibited by different OP and it is doubt-ful whether further modifications of the bispyridinium tem-plate will lead to more potent reactivators. In the end, novel

structures of oxime and non-oxime reactivators are urgently needed (Bhattacharjee et al. 2012; radic et al. 2013) for the development of human Bche into an effective pseudo-cat-alytic scavenger.

Acknowledgments This study was funded by the german Min-istry of Defence, university Hradec Kralove (long Term Develop-ment Plan, lH13009) and Ministry of Health of the czech republic (NT12062, FNHK00179906). The authors are grateful to T. Hannig for her engaged technical assistance.

Conflict of interest The authors declare that there are no conflicts of interest.

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reactivation kinetics of 31 structurally different bispyridinium oximes with...

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