Resolving pathways of interaction of mipafox and a sarin analog with human acetylcholinesterase by kinetics, mass spectrometry and molecular modeling approaches.
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 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 diisopropyl 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.
Unit of Toxicology and Chemical Safety, Institute of Bioengineering, University Miguel Hernandez of Elche, Alicante, Spain. imangas@umh.es.|Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, 92093, USA. imangas@umh.es.|Laboratory of Molecular Modeling Applied to the Chemical and Biological Defense, Military Institute of Engineering, Rio de Janeiro, Brazil. imangas@umh.es.
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Mangas
Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, 92093, USA.
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Taylor
Unit of Toxicology and Chemical Safety, Institute of Bioengineering, University Miguel Hernandez of Elche, Alicante, Spain.
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Vilanova
Unit of Toxicology and Chemical Safety, Institute of Bioengineering, University Miguel Hernandez of Elche, Alicante, Spain.
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Estévez
Laboratory of Molecular Modeling Applied to the Chemical and Biological Defense, Military Institute of Engineering, Rio de Janeiro, Brazil.|Center for Basic and Applied Research, Faculty of Informatics and Management, University of Hradec Králové, Hradec Králové, Czech Republic.
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França
Department of Chemistry-Biochemistry, University of California San Diego, La Jolla, CA, 92093, USA.
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Komives
Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, 92093, USA.
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Radić
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002800:Cholinesterase Inhibitors / Q000493:pharmacokinetics*; D002801:Cholinesterase Reactivators / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007531:Isoflurophate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000493:pharmacokinetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000493:pharmacokinetics; D010091:Oximes / Q000737:chemistry; D010261:Paraoxon / Q000493:pharmacokinetics; D010766:Phosphorylation; D011487:Protein Conformation; D012524:Sarin / Q000031:analogs & derivatives* / Q000737:chemistry; D012694:Serine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002800:Cholinesterase Inhibitors; D002801:Cholinesterase Reactivators; D009943:Organophosphorus Compounds; D010091:Oximes; D007531:Isoflurophate; C005238:mipafox; C032627:methylphosphonic acid; D012694:Serine; D012524:Sarin; D000110:Acetylcholinesterase; D010261:Paraoxon
Acetylcholinesterase; Aging; Mipafox; Organophosphorus compounds; Oximes; Sarin