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

Abstract

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.

Immunopurification of Acetylcholinesterase from Red Blood Cells for Detection of Nerve Agent Exposure

“…Laboratories that routinely assay AChE activity stabilize their dilute AChE solutions with albumin. 30 − 32…”

New insights on molecular interactions of organophosphorus pesticides with esterases

“…Protein MS has been used with some OPs as a valuable tool to identify OP-conjugated proteins employing peptide mass fingerprinting to match the isolated proteins to protein sequences by analyzing the OP-protein conjugates of AChE and BuChE Sun and Lynn, 2009;Grigoryan et al, 2009;Doorn et al, 2001;Jennings et al, 2003;Kropp and Richardson., 2006;. Advances in protein MS have been most useful to identify not only the adduction but also the post inhibition processes such as reactivation or aging reactions (Grigoryan et al, 2009;Doorn et al, 2001;Jennings et al, 2003;Mangas et al, 2016a). Figure 7 shows the active center peptide (ACP) of AChE trypsin digested enzyme detected after inhibition of most of the activity with the OPs paraoxon, sarin and mipafox, The ACP with sarin aged was also detected (Mangas et al, 2016a).…”

“…Advances in protein MS have been most useful to identify not only the adduction but also the post inhibition processes such as reactivation or aging reactions (Grigoryan et al, 2009;Doorn et al, 2001;Jennings et al, 2003;Mangas et al, 2016a). Figure 7 shows the active center peptide (ACP) of AChE trypsin digested enzyme detected after inhibition of most of the activity with the OPs paraoxon, sarin and mipafox, The ACP with sarin aged was also detected (Mangas et al, 2016a). Moreover, the detection of OP-adducted protein biomarkers as a more sensitive and accurate assessment of OP exposure has been highlighted in the last years.…”

The inhibition, reactivation and mechanism of VX-, sarin-, fluoro-VX and fluoro-sarin surrogates following their interaction with HuAChE and HuBuChE

“…To further delineate the mechanism of inhibition, quadrupole-time of flight (Q-TOF) and MALDI-TOF MS analyses were conducted with tryptic active-site peptides obtained following reaction of HuAChE or HuBChE modified by VX-, fluoro-VX-, sarin- and fluoro-sarin surrogates. The use of MALDI-TOF MS analyses in particular has proven valuable in the elucidation of OP-cholinesterase adducts and mechanistic pathways [13, 17–20], as the conditions for Q-TOF ionization can sometimes limit characterization [25].…”