Phosphoryl Oxime Inhibition of Acetylcholinesterase during Oxime Reactivation Is Prevented by Edrophonium

Abstract

Reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) is a key objective in the treatment of OP poisoning. This study with native, wild-type, and mutant recombinant DNA-expressed AChEs, each inhibited by representative OP compounds, establishes a relationship between edrophonium acceleration of oxime-induced reactivation of OP-AChE conjugates and phosphoryl oxime inhibition of the reactivated enzyme that occurs during reactivation by pyridinium oximes LüH6 and TMB4. No such recurring inhibition could be observed with HI-6 as the reactivator due to the extreme lability of the phosphoryl oximes formed by this oxime. Phosphoryl oximes formed during reactivation of the ethoxy methylphosphonyl-AChE conjugate by LüH6 and TMB4 were isolated for the first time and their structures confirmed by (31)P NMR. However, phosphoryl oximes formed during the reactivation of the diethylphosphoryl-AChE conjugate were not sufficiently stable to be detected by (31)P NMR. The purified ethoxy methylphosphonyl oximes formed during the reactivation of ethoxy methylphosphonyl-AChE conjugate with LüH6 and TMB4 are 10- to 22-fold more potent than MEPQ as inhibitors of AChE and stable for several hours at pH 7.2 in HEPES buffer. Reactivation of both ethoxy methylphosphonyl- and diethylphosphoryl-AChE by these two oximes was accelerated in the presence of rabbit serum paraoxonase, suggesting that organophosphorus hydrolase can hydrolyze phosphoryl oxime formed during the reactivation. Our results emphasize that certain oximes, such as LüH6 and TMB4, if used in the treatment of OP pesticide poisoning may cause prolonged inhibition of AChE due to formation of phosphoryl oximes.

Unequal Efficacy of Pyridinium Oximes in Acute Organophosphate Poisoning

“…88 Generally, phosphyloxime accumulation may occur in the many reactivation/ re-inhibition cycles, particularly with the more stable compounds deriving from 4-pyridinium oximes and may alter the efficacy of the 4-pyridinium oximes. [89][90][91] It remains doubtful whether this occurs in humans with the usual dose regimens. It was also demonstrated that human paraoxonase with the prevailing Q192 phenotype 92 is able to hydrolyze effectively O,O-diethylphosphoryl LüH-6.…”

Acetylcholinesterase active centre and gorge conformations analysed by combinatorial mutations and enantiomeric phosphonates

“…Known concentrations of MEPQ or DEPQ were utilized to titrate the number of active sites, according to procedures described previously in [17,18].…”

Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6

“…Treatment of nerve agent-inactivated AChE with an antidote causes the release of the functional enzyme and the formation of a phosphonyl oxime (Fig. 1A) (4,5). Although the process has been studied for decades, detailed mechanistic information is still lacking.…”

“…The addition of HI-6 to the sarin-AChE complex leads to a reaction that cleaves the bond between the oxygen of Ser203 and the phosphorus atom of sarin (Fig. 1A) (4,5,11). Furthermore, the sarin adduct can undergo a dealkylation reaction, termed "aging," that renders the enzyme resistant to reactivation by HI-6 (7).…”