Discovery of (E)-2-(hydroxyimino)-N-(2 ((4methylpentyl)amino)ethyl)acetamide (KR-27425) as a non-pyridinium oxime reactivator of paraoxon-inhibited acetylcholinesterase.

This study aimed to explore non-pyridinium oxime acetylcholinesterase (AChE) reactivators that could hold the potential to overcome the limitations of the currently available compounds used in the clinic to treat the neurologic manifestations induced by intoxication with organophosphorus agents. Fifteen compounds with various non-pyridinium oxime moieties were evaluated for AChE activity at different concentrations, including aldoximes, ketoximes, and α-ketoaldoximes. The therapeutic potential of the oxime compounds was evaluated by assessing their ability to reactivate AChE inhibited by paraoxon. Among the tested compounds, α-Ketoaldoxime derivative 13 showed the highest reactivation (%) reaching 67 % and 60 % AChE reactivation when evaluated against OP-inhibited electric eel AChE at concentrations of 1,000 and 100 µM, respectively. Compound 13 showed a comparable reactivation ability of AChE (60 %) compared to that of pralidoxime (56 %) at concentrations of 100 µM. Molecular docking simulation of the most active compounds 12 and 13 was conducted to predict the binding mode of the reactivation of electric eel AChE. As a result, a non-pyridinium oxime moiety 13, is a potential reactivator of OP-inhibited AChE and is taken as a lead compound for the development of novel AChE reactivators with enhanced capacity to freely cross the blood-brain barrier.

Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P-S Bonded Organophosphorus as Monitored by Spectrofluorimetry.

Enzyme-catalyzed hydrolysis of echothiophate, a P-S bonded organophosphorus (OP) model, was spectrofluorimetrically monitored, using Calbiochem Probe IV as the thiol reagent. OP hydrolases were: the G117H mutant of human butyrylcholinesterase capable of hydrolyzing OPs, and a multiple mutant of Brevundimonas diminuta phosphotriesterase, GG1, designed to hydrolyze a large spectrum of OPs at high rate, including V agents. Molecular modeling of interaction between Probe IV and OP hydrolases (G117H butyrylcholinesterase, GG1, wild types of Brevundimonas diminuta and Sulfolobus solfataricus phosphotriesterases, and human paraoxonase-1) was performed. The high sensitivity of the method allowed steady-state kinetic analysis of echothiophate hydrolysis by highly purified G117H butyrylcholinesterase concentration as low as 0.85 nM. Hydrolysis was michaelian with Km = 0.20 ± 0.03 mM and kcat = 5.4 ± 1.6 min-1. The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (Km = 2.6 ± 0.2 mM; kcat = 53400 min-1). With a kcat/Km = (2.6 ± 1.6) × 107 M-1min-1, GG1 fulfills the required condition of potential catalytic bioscavengers. quantum mechanics/molecular mechanics (QM/MM) and molecular docking indicate that Probe IV does not interact significantly with the selected phosphotriesterases. Moreover, results on G117H mutant show that Probe IV does not inhibit butyrylcholinesterase. Therefore, Probe IV can be recommended for monitoring hydrolysis of P-S bonded OPs by thiol-free OP hydrolases.

Identification of new binding sites of human transferrin incubated with organophosphorus agents via Q Exactive LC-MS/MS.

Organophosphorus agents (OPs) like sarin, VX, or soman could inhibit acetylcholinesterase activity and cause poisoning. OPs could bind many proteins, such as butyrylcholinesterase and albumin, and the adducts formed could identify the exposure. In this paper, we studied human transferrin, which was one of the proteins that could be labeled by OPs. Pure human transferrin was incubated with an overdose of organophosphorus agents, including sarin, soman, VX, tabun, cyclosarin, ethyl tabun, and propyl tabun, and then additional OPs was removed through dialysis. Trypsin was used to cleave the OP-treated proteins and Q Exactive liquid chromatography tandem mass spectrometry (Q Exactive LC-MS/MS) was used to identify them. The present study set out to accomplish two goals. The first goal was to find a good method for identifying multiple binding sites on a given protein through Q Exactive LC-MS/MS. The second goal was to investigate the labeled peptides when transferrin was incubated with a numerous molar excess of OPs. Results showed that tyrosine, lysine, and serine formed covalent bonds with OPs. Twenty OP-labeled sites were found: ten tyrosine sites (including two reported sites), seven lysine sites, and three serine sites. Characteristic fragments for labeled-tyrosine and labeled-lysine adducts were summarized in detail. In conclusion, the method by Q Exactive LC-MS/MS using in this present work is a good way to diagnose exposure to OPs accurately when the binding sites of OPs are uncertain. Novel modified peptides and the characteristic ions found in this work could help investigators assess exposure to OPs.

[Interest of the cholinesterase assay during organophosphate poisonings]. / Intérêt du dosage des cholinestérases dans le cadre des intoxications aux organophosphorés.

Cholinesterases are the main targets of organophosphorus compounds. The two enzymes present in the blood (butyrylcholinesterase, BChE; acetylcholinesterase, AChE) are biomarkers of their systemic toxicity. Activity of the plasma BChE is very often determined as it allows a rapid diagnostic of poisoning and is a marker of the persistence of the toxicant in the blood. The activity of the red blood cell AChE gives a better picture of the synaptic inhibition in the nervous system but the assay is less commonly available in routine laboratories. Better biomarker of the exposure, it allows a diagnosis of the severity of the poisoning and helps to assess the efficacy of oxime therapy. Besides the practical aspects of blood collection and sample processing, and the interpretation of the assays, this review stresses the complementarity of both enzyme assays and recalls their crucial interest for the confirmation of poisoning with an organophosphorus in a situation of war or terrorist attack and for the monitoring of occupational exposures.