Asymmetric fluorogenic organophosphates for the development of active organophosphate hydrolases with reversed stereoselectivity

Amitai, Gabriel; Adani, R.; Yacov, Guy; Yishay, Shelly; Teitlboim, Shai; Tveria, Liat; Limanovich, Osnat; Kushnir, M.; Meshulam, Haim

doi:10.1016/j.tox.2006.09.020

Cited by 37 publications

(30 citation statements)

References 17 publications

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“…Low toxicity, nonvolatile fluorescent methylphosphonates (Flu-MPs) [11] were used in in vitro experiments as analogues of nerve agents sarin, cyclosarin and VX. The Flu-MPs differ from actual nerve agent OPs only by structure of their respective leaving groups.…”

Section: Methodsmentioning

confidence: 99%

Catalytic detoxification of nerve agent and pesticide organophosphates by butyrylcholinesterase assisted with non-pyridinium oximes

Radić

Dale

Kovarik

et al. 2013

Biochemical Journal

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SYNOPSIS We present here a comprehensive in vitro, ex vivo and in vivo study on hydrolytic detoxification of nerve agent and pesticide organophosphates (OPs) catalyzed by purified human butyrylcholinesterase (hBChE) in combination with novel non-pyridinium oxime reactivators. We identified 2-trimethylammonio-6-hydroxybenzaldehyde oxime (TAB2OH) 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 three to five-fold enhancement of in vitro reactivation of VX, cyclosarin and paraoxon inhibited hBChE was observed, when compared to the commonly used N-methylpyridinium aldoxime reactivator, 2PAM. Kinetic analysis showed 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 hydroxyl, both ortho to an oxime group on a benzene ring. pH dependences reveal participation of the hydroxyl (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. These results establish that oxime-assisted catalysis is feasible for OP bioscavenging.

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Section: Methodsmentioning

confidence: 99%

Catalytic detoxification of nerve agent and pesticide organophosphates by butyrylcholinesterase assisted with non-pyridinium oximes

Radić

Dale

Kovarik

et al. 2013

Biochemical Journal

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“…Herein we report the development of an efficient fluorescent assay that is capable of direct detection of low level hydrolysis of nerve agent model compounds. This approach compliments strategies previously reported, where OP analogs with larger fluorescent leaving groups were used as substrates [6, 7]. The thiocholine leaving group for our analogs is the same as standard cholinesterase substrates acetylthiocholine and butyrylthiocholine (BTCh).…”

Section: Introductionmentioning

confidence: 83%

Direct detection of the hydrolysis of nerve agent model compounds using a fluorescent probe

Zheng

Okolotowicz

Wang

et al. 2010

Chemico-Biological Interactions

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Nerve agents are highly toxic organophosphorus compounds (OP) that are used as chemical warfare agents. Developing a catalytic bioscavenger to efficiently detoxify nerve agents in the bloodstream of affected individuals has been recognized as an attractive approach to prevent nerve agent toxicity. However, the search for nerve agent catalysts has been hindered by the lack of efficient direct assays for nerve agent hydrolysis. In addition, authentic nerve agents are restricted and access to use for experiments by the general research community is prohibited. Herein we report development of a method that combines use of novel nerve agent model compounds possessing a thiocholine leaving group that reacts with the fluorescent thio-detection probe, BES-Thio, to afford detection of sub-micromolar amounts of nerve agent model compounds hydrolysis products. The detection sensitivity of BES-Thio assay was approximately 10 times better than the Ellman assay. This developed method is useful as a direct, sensitive screening method for evaluating OP hydrolysis efficiency from catalytic cholinesterases. When the assay was assembled in the presence of oxime, OP-inhibited cholinesterases that were able to be reactivated by specific oxime showed oxime-assisted enzyme-mediated OP hydrolysis. Therefore, this method is also useful to screen oxime analogs to identify novel agents that can reactivate OP-inhibited cholinesterases or to screen various enzymes to identify pseudo-catalytic bioscavengers that can be readily reactivated by clinically approved oximes.

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“…Mipafox (mipafox, purity >98%) was acquired from Lark Enterprise (Webster, MA, USA). The now-toxicity nonvolatile Flu-MP (fluorescent methylphosphonate) used in the in vitro experiments as an analog of nerve agent sarin (Amitai et al, 2007) were kindly provided by Dr. Gabriel Amitai of the Israel Institute for Biological Research, Ness-Ziona, Israel. This Flu-MP differs from Sarin by only the structure of the respective leaving group.…”

Section: Methodsmentioning

confidence: 99%

Resolving pathways of interaction of mipafox and a sarin analog with human acetylcholinesterase by kinetics, mass spectrometry and molecular modeling approaches

et al. 2015

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The hydroxyl oxygen of the catalytic triad serine in the active center of serine hydrolase acetylcholinesterase (AChE) attacks organophosphorus compounds (OPs) at the phosphorus atom to displace the primary leaving group and to form a covalent bond. Inhibited AChE can be reactivated by cleavage of the Ser-phosphorus bond either spontaneously or through a reaction with nucleophilic agents, such as oximes. At the same time, the inhibited AChE adduct can lose part of the molecule by progressive dealkylation over time in a process called aging. Reactivation of the aged enzyme has not yet been demonstrated. Here our goal was to study oxime reactivation and aging reactions of human AChE inhibited by mipafox or a sarin analog (Flu-MPs, fluorescent methylphosphonate). Progressive reactivation was observed after Flu-MPs inhibition using oxime 2-PAM. However, no reactivation was observed after mipafox inhibition with 2-PAM or the more potent oximes used. A peptide mass fingerprinted mass spectrometry (MS) method, which clearly distinguished the peptide with the active serine (active center peptide, ACP) of the human AChE adducted with OPs, was developed by MALDI-TOF and MALDI-TOF-TOF. The ACP was detected with a diethyl phosphorylated adduct after paraoxon inhibition, and with an isopropylmethyl phosphonylated and a methyl phosphonylated adduct after Flu-MPs inhibition and subsequent aging. Nevertheless, nonaged nonreactivated complexes were seen after mipafox inhibition and incubation with oximes, where MS data showed an ACP with an NN diidopropyl phosphoryl adduct. The kinetic experiments showed no reactivation of activity. The computational molecular model analysis of the mipafox-inhibited hAChE plots of energy versus distance between the atoms separated by dealkylation showed a high energy demand, thus little aging probability. However with Flu-MPs and DFP, where aging was observed in our MS data and in previously published crystal structures, the energy demand calculated in modeling was lower and, consequently, aging appeared as a more likely reaction. We document here direct evidence for a phosphorylated hAChE refractory to oxime reactivation, although we observed no aging.

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Asymmetric fluorogenic organophosphates for the development of active organophosphate hydrolases with reversed stereoselectivity

Cited by 37 publications

References 17 publications

Catalytic detoxification of nerve agent and pesticide organophosphates by butyrylcholinesterase assisted with non-pyridinium oximes

Catalytic detoxification of nerve agent and pesticide organophosphates by butyrylcholinesterase assisted with non-pyridinium oximes

Direct detection of the hydrolysis of nerve agent model compounds using a fluorescent probe

Resolving pathways of interaction of mipafox and a sarin analog with human acetylcholinesterase by kinetics, mass spectrometry and molecular modeling approaches

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