Theoretical study on the aging and reactivation mechanism of tabun-inhibited acetylcholinesterase by using the quantum mechanical / molecular mechanical method

The organophosphorous compound tabun is highly neurotoxic because of its irreversible inhibition on acetylcholinesterase (AChE). It is wildly used as a warfare agent in the military. In this work, the aging and reactivation mechanism of tabun-inhibited AChE were studied by using the quantum mechanical / molecular mechanical (QM/MM) method. Geometry optimization of the stationary points were performed at the B3LYP/6–31G(d) level. Single-point energies were computed at the B3LYP/6–311++G(d,p) level. On the basis of the QM/MM results, a conclusion that the C–O bond scission is caused by water attack on the ethoxy group in the aging mechanism can be drawn. The reactivation process initialed by the antidotes CH2NO– or HLÖ-7 consists of three elemental steps, the nucleophilic attack on the P atom by the antidote, the dephosphorylation process, and the decomposition of the antidote–tabun complex. The highest energy barriers of the aging reaction, CH2NO–-induced reactivation, and HLÖ-7-induced reactivation are 19.9, 20.0, and 14.8 kcal/mol (1 cal = 4.184 J), respectively. The relative lower overall energy barrier of HLÖ-7-induced reactivation compared with that of the aging reaction indicates that HLÖ-7 is able to reactivate tabun-inhibited AChE. In addition, whether a newly designed antidote is able to reactivate tabun-inhibited AChE can be examined by the inequation X < 19.9 kcal/mol,where X means the highest energy barrier of the reactivation reaction of the newly designed antidote.

Structure–activity analysis of aging and reactivation of human butyrylcholinesterase inhibited by analogues of tabun

hBChE [human BChE (butyrylcholinesterase)] naturally scavenges OPs (organophosphates). This bioscavenger is currently in Clinical Phase I for pretreatment of OP intoxication. Phosphylated ChEs (cholinesterases) can undergo a spontaneous time-dependent process called ‘aging’ during which the conjugate is dealkylated, leading to creation of an enzyme that cannot be reactivated. hBChE inhibited by phosphoramidates such as tabun displays a peculiar resistance to oxime-mediated reactivation. We investigated the basis of oxime resistance of phosphoramidyl–BChE conjugates by determining the kinetics of inhibition, reactivation (obidoxime {1,1′-(oxybis-methylene) bis[4-(hydroxyimino) methyl] pyridinium dichloride}, TMB-4 [1,3-trimethylene-bis(4-hydroxyiminomethylpyridinium) dibromide], HLö 7 {1-[[[4-(aminocarbonyl) pyridinio]methoxy]methyl]-2,4-bis-[(hydroxyimino)methyl] pyridinium dimethanesulfonate)}, HI-6 {1-[[[4-(aminocarbonyl) pyridinio] methoxy] methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride monohydrate} and aging, and the crystal structures of hBChE inhibited by different N-monoalkyl and N,N-dialkyl tabun analogues. The refined structures of aged hBChE conjugates show that aging proceeds through O-dealkylation of the P(R) enantiomer of N,N-diethyl and N-propyl analogues, with subsequent formation of a salt bridge preventing reactivation, similarly to a previous observation made on tabun–ChE conjugates. Interestingly, the N-methyl analogue projects its amino group towards the choline-binding pocket, so that aging proceeds through deamination. This orientation results from a preference of hBChE's acyl-binding pocket for larger than 2-atoms linear substituents. The correlation between the inhibitory potency and the N-monoalkyl chain length is related to increasingly optimized interactions with the acyl-binding pocket as shown by the X-ray structures. These kinetics and X-ray data lead to a structure–activity relationship that highlights steric and electronic effects of the amino substituent of phosphoramidate. This study provides the structural basis to design new oximes capable of reactivating phosphoramidyl-hBChE conjugates after intoxication, notably when hBChE is used as pretreatment, or to design BChE-based catalytic bioscavengers.

A Comparison of the Potency of the Oxime HLö-7 and Currently Used Oximes (HI-6, Pralidoxime, Obidoxime) to Reactivate Nerve Agent-Inhibited Rat Brain Acetylcholinesterase by in vitro Methods

1. The efficacy of the oxime HLö-7 and currently used oximes (pralidoxime, obidoxime, HI-6) to reactivate acetylcholinesterase inhibited by various nerve agents (sarin, tabun, cyclosarin, VX) was tested by in vitro methods. 2. Both H oximes (HLö-7, HI-6) were found to be more efficacious reactivators of sarin and VX-inhibited acetylcholinesterase than pralidoxime and obidoxime. On the other hand, their potency to reactivate tabun-inhibited acetylcholinesterase is very low and does not reach the reactivating efficacy of obidoxime. In the case of cyclosarin, the oxime HI-6 was only found to be able to sufficiently reactivate cyclosarin-inhibited acetylcholinesterase in vitro. 3. Thus, the oxime HLö-7 does not seem to be more efficacious reactivator of nerve agent-inhibited acetylcholinesterase than HI-6 according to in vitro evaluation of their reactivation potency and, therefore, it is not more suitable to be introduced for antidotal treatment of nerve agent-exposed people than HI-6.

A comparison of the efficacy of a new asymmetric bispyridinium oxime BI-6 with presently used oximes and H oximes against sarin by in vitro and in vivo methods

1 The reactivating and therapeutic efficacy of a new acetylcholinesterase reactivator, designated BI-6 (1-/2-hydroxyiminomethylpyridinium/-4-/carbamoylpyridinium/-2-butene dibromide), against organophosphate sarin was compared with presently used oximes (pralidoxime, obidoxime, methoxime) and H oximes (HI-6, HLö-7) by in vitro and in vivo methods. 2 Our results confirm that the new oxime BI-6 is a significantly more efficacious acetylcholinesterase reactivator than currently available pralidoxime and obidoxime but not as effective as H oximes (HI-6, HLö-7) in vitro as well as in vivo. In addition, the oxime BI-6 is able to protect supralethal sarin poisoned rats at human-relevant doses. 3 Our data also suggest that the potency of oximes tested to reactivate sarin-inhibited acetylcholinesterase in vitro closely corresponds to their reactivating efficacy in vivo and their ability to protect rats poisoned with supralethal doses of sarin.