Characterization of O,O-diethylphosphoryl oximes as inhibitors of cholinesterases and substrates of phosphotriesterases

Reactivators of organophosphate (OP)-inhibited cholinesterases (ChEs) are b elieved to give rise to phosphorylated oximes (POX) that reinhibit the enzy me. Diethylphosphoryl oximes (DEP-OX) that were generated In situ were demo nstrated in the past to be unstable, yet were more potent inhibitors of ace tylcholinesterase (AChE) than the parent OPs. In view of the inconsistencie s among reported results, and the potential toxicity of POXs, it seemed imp ortant to characterize authentic DEP-OXs, and to evaluate their interferenc e with reactivation of diethylphosphoryl-ChE (DEP-ChE) conjugates. To this end, the diethylphosphoric acid esters of 1-methyl-2-pyridinium carboxaldeh yde oxime (DEP-2PAM) and 1-methyl-4 pyridinium carboxaldehyde oxime (DEP-4P AM) were synthesized and chemically defined. The half-lives of DEP-2PAM and DEP-4PAM in 10 mM Tris buffer, pH 7.8, at 29 degrees were found to be 10 a nd 980 sec, respectively. The two DEP-OXs inhibited ChEs with the following ranking order: for DEP-2PAM, human butyrylcholinesterase (HuBChE, k(i) = 2 .03 x 10(9) M-1 min(-1)) > mouse AChE (MoAChE) congruent to fetal bovine se rum AChE (FBS-AChE) congruent to equine BChE (EqBChE); for DEP-4PAM, WuBChE (k(i) = 0.71 x 10(9) M-1 min(-1)) > EqBChE > MoAChE > FBS-AChE. A dialkyla rylphosphate hydrolase (phosphotriesterase; PTE) from Pseudomonas sp. catal yzed the hydrolysis of DEP-4PAM with k(cat)/K-m = 3.56 x 10(7) M-1 min(-1) and K-m = 0.78 mM. Reactivation of DEP-ChEs was enhanced by PTE when di-PAM -based oximes were used as reactivators, whereas reactivation with 2-PAM-ba sed oximes was not affected by PTE. This observation is attributed primaril y to the short half-life of DEP-OXs derived from the latter oximes. Relativ ely low doses of PTE can detoxify large quantities of DEP-OXs rapidly, and thereby augment the efficacy of antidotes that contain the oxime function i n position 4 of the pyridine ring. (C) 1999 Elsevier Science Inc.