Enhanced Stability of Blood Matrices Using a Dried Sample Spot Assay To Measure Human Butyrylcholinesterase Activity and Nerve Agent Adducts

In the present paper we show a comprehensive in vitro, ex vivo and in vivo study on hydrolytic detoxification of nerve agent and pesticide OPs (organophosphates) catalysed by purified hBChE (human butyrylcholinesterase) in combination with novel non-pyridinium oxime reactivators. We identified TAB2OH (2-trimethylammonio-6-hydroxybenzaldehyde oxime) as an efficient reactivator of OP–hBChE conjugates formed by the nerve agents VX and cyclosarin, and the pesticide paraoxon. It was also functional in reactivation of sarin- and tabun-inhibited hBChE. A 3–5-fold enhancement of in vitro reactivation of VX-, cyclosarin- and paraoxon-inhibited hBChE was observed when compared with the commonly used N-methylpyridinium aldoxime reactivator, 2PAM (2-pyridinealdoxime methiodide). Kinetic analysis showed that the enhancement resulted from improved molecular recognition of corresponding OP–hBChE conjugates by TAB2OH. The unique features of TAB2OH stem from an exocyclic quaternary nitrogen and a hydroxy group, both ortho to an oxime group on a benzene ring. pH-dependences reveal participation of the hydroxy group (pKa=7.6) forming an additional ionizing nucleophile to potentiate the oxime (pKa=10) at physiological pH. The TAB2OH protective indices in therapy of sarin- and paraoxon-exposed mice were enhanced by 30–60% when they were treated with a combination of TAB2OH and sub-stoichiometric hBChE. The results of the present study establish that oxime-assisted catalysis is feasible for OP bioscavenging.

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Non-quaternary oximes detoxify nerve agents and reactivate nerve agent-inhibited human butyrylcholinesterase

Communications Biology ◽

10.1038/s42003-021-02061-w ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Gabriel Amitai ◽

Alexander Plotnikov ◽

Shira Chapman ◽

Shlomi Lazar ◽

Rellie Gez ◽

...

Keyword(s):

Small Molecules ◽

High Throughput Screening ◽

Reaction Rates ◽

Nerve Agents ◽

Nerve Agent ◽

The Novel ◽

Quaternary Compounds ◽

Toxicological Studies ◽

Pyridinium Oximes ◽

Human Butyrylcholinesterase

AbstractGovernment-sanctioned use of nerve agents (NA) has escalated dramatically in recent years. Oxime reactivators of organophosphate (OP)-inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) serve as antidotes toward poisoning by OPNAs. The oximes used as therapeutics are quaternary compounds that cannot penetrate the blood–brain barrier (BBB). There remains an urgent need for the development of next generation OPNA therapeutics. We have developed two high-throughput screening (HTS) assays using a fluorogenic NA surrogate, O-ethyl methylphosphonyl O-4-methyl-3-cyano-coumarin (EMP-MeCyC). EMP-MeCyC detoxification and EMP-BChE reactivation screening campaigns of ~155,000 small molecules resulted in the identification of 33 nucleophile candidates, including non-quaternary oximes. Four of the oximes were reactivators of both Sarin- and VX-inhibited BChE and directly detoxified Sarin. One oxime also detoxified VX. The novel reactivators included a non-quaternary pyridine amidoxime, benzamidoxime, benzaldoxime and a piperidyl-ketoxime. The VX-inhibited BChE reactivation reaction rates by these novel molecules were similar to those observed with known bis-quaternary reactivators and faster than mono-quaternary pyridinium oximes. Notably, we discovered the first ketoxime reactivator of OP-ChEs and detoxifier of OPNAs. Preliminary toxicological studies demonstrated that the newly discovered non-quaternary oximes were relatively non-toxic in mice. The discovery of unique non-quaternary oximes opens the door to the design of novel therapeutics and decontamination agents following OPNA exposure.

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Human butyrylcholinesterase as a general prophylactic antidote for nerve agent toxicity

Biochemical Pharmacology ◽

10.1016/0006-2952(93)90228-o ◽

1993 ◽

Vol 45 (12) ◽

pp. 2465-2474 ◽

Cited By ~ 147

Author(s):

Lily Raveh ◽

Jacob Grunwald ◽

Dino Marcus ◽

Yoel Papier ◽

Ephraim Cohen ◽

...

Keyword(s):

Nerve Agent ◽

Human Butyrylcholinesterase

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High-Confidence Qualitative Identification of Organophosphorus Nerve Agent Adducts to Human Butyrylcholinesterase

Analytical Chemistry ◽

10.1021/acs.analchem.6b04441 ◽

2017 ◽

Vol 89 (3) ◽

pp. 1955-1964 ◽

Cited By ~ 16

Author(s):

Thomas P. Mathews ◽

Melissa D. Carter ◽

Darryl Johnson ◽

Samantha L. Isenberg ◽

Leigh Ann Graham ◽

...

Keyword(s):

Nerve Agent ◽

High Confidence ◽

Human Butyrylcholinesterase

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Evaluation of anticholinesterasic substances in cigarettes manufactured in Brazil through a human butyrylcholinesterase activity inhibition method

Estudos de Biologia ◽

10.7213/reb.v31i73/75.22843 ◽

2009 ◽

Vol 31 (73/75) ◽

Author(s):

Mariana Hiromi Azuma ◽

Marina de Almeida ◽

Sérgio Eduardo Fontoura-da-Silva ◽

Thais Campos Peliky ◽

Rafaela Alexandre Saad

Keyword(s):

Indirect Method ◽

Activity Inhibition ◽

Butyrylcholinesterase Activity ◽

Tobacco Extract ◽

Human Butyrylcholinesterase

The objective of this research was to evaluate, through an indirect method, the presence of anticholinesterasicsubstances in tobacco from cigarettes manufactured in Brazil. Organophosphate and carbamatescompounds, which are used in the crops of the tobacco, are the main substances with this characteristic.From each one of the ten main cigarette labels, the tabacco had been separated and extracted anticholinesterasicsubstances using the method described by Leite (1980) and the n-hexane as solvent. After thepreparation of the extract, of the blood was separated the plasma for the determination of the activityof butyrylcholinesterase (BChE) using the method described by Dietz et al (1973).The enzyme activitywas measured in the presence of the extracts of each tobacco extract. An signifi cative average inhibitionof 20.16%, (p < 0.001).

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