The pyridinium-2-carbaldoximes with quinolinium carboxamide moiety were designed and synthesised as cholinesterase reactivators. The prepared compounds showed intermediate-to-high inhibition of both cholinesterases when compared to standard oximes. Their reactivation ability was evaluated in vitro on human recombinant acetylcholinesterase (hrAChE) and human recombinant butyrylcholinesterase (hrBChE) inhibited by nerve agent surrogates (NIMP, NEMP, and NEDPA) or paraoxon. In the reactivation screening, one compound was able to reactivate hrAChE inhibited by all used organophosphates and two novel compounds were able to reactivate NIMP/NEMP-hrBChE. The reactivation kinetics revealed compound 11 that proved to be excellent reactivator of paraoxon-hrAChE better to obidoxime and showed increased reactivation of NIMP/NEMP-hrBChE, although worse to obidoxime. The molecular interactions of studied reactivators were further identified by in silico calculations. Molecular modelling results revealed the importance of creation of the pre-reactivation complex that could lead to better reactivation of both cholinesterases together with reducing particular interactions for lower intrinsic inhibition by the oxime.

• Klíčová slova
• Organophosphate, acetylcholinesterase, butyrylcholinesterase, oxime, reactivator,
• MeSH
• acetylcholinesterasa metabolismus   MeSH
• butyrylcholinesterasa metabolismus   MeSH
• chinolinové sloučeniny chemická syntéza   chemie   farmakologie   MeSH
• cholinesterasové inhibitory chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• molekulární struktura MeSH
• pyridinové sloučeniny chemická syntéza   chemie   farmakologie   MeSH
• rekombinantní proteiny metabolismus   MeSH
• simulace molekulového dockingu MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetylcholinesterasa MeSH
• butyrylcholinesterasa MeSH
• chinolinové sloučeniny MeSH
• cholinesterasové inhibitory MeSH
• pyridinové sloučeniny MeSH
• rekombinantní proteiny MeSH

The deleterious effects of nerve agents over the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) turned these compounds into the most dangerous chemical weapons known. Among the antidotes in use today against these agents, oximes in combination with other drugs are the only treatment with any action. HI-6 and 2-PAM are cationic oximes proved to be effective for the reactivation of AChE inhibited by the nerve agents VX and sarin (GB). However, when it comes to reactivation of AChE inside the central or peripheral nervous systems, charged molecules present low diffusion due to low penetration through the blood-brain barrier. Uncharged oximes appear as an interesting alternative to solve this problem, but the development and enhancement of more efficient uncharged oximes capable of reactivating human AChE is still necessary. Given the limitations for in vivo and in vitro experimental studies with nerve agents, modeling is an important tool that can contribute to a better understanding of factors that may affect the efficiency of uncharged oximes. In order to investigate the interaction and behavior of cationic and uncharged oximes, we performed here molecular docking, molecular dynamics simulations, and binding energies calculations of the known cationic oximes HI-6 and 2-PAM plus four uncharged oximes found in the literature, complexed with human AChE (HssACHE) conjugated with the nerve agents VX and GB. The uncharged oximes showed different behaviors, especially RS194B, which presented stability inside AChE-VX, but presented free binding energy lower than cationic oximes, suggesting that structural alterations could favor its interactions with these complexes. In contrast, HI-6 and 2-PAM showed higher affinities with more negative binding energy values and larger contribution of the amino acid Asp74, demonstrating the importance of the quaternary nitrogen to the affinity and interaction of oximes with AChE-GB and AChE-VX conjugates.

• Publikační typ
• časopisecké články MeSH

Casualties caused by organophosphorus pesticides are a burden for health systems in developing and poor countries. Such compounds are potent acetylcholinesterase irreversible inhibitors, and share the toxic profile with nerve agents. Pyridinium oximes are the only clinically available antidotes against poisoning by these substances, but their poor penetration into the blood-brain barrier hampers the efficient enzyme reactivation at the central nervous system. In searching for structural factors that may be explored in future SAR studies, we evaluated neutral aryloximes as reactivators for paraoxon-inhibited Electrophorus eel acetylcholinesterase. Our findings may result into lead compounds, useful for development of more active compounds for emergencies and supportive care.

• Klíčová slova
• acetylcholinesterase, antidotes, drug design, neutral oximes, pesticides,
• MeSH
• acetylcholinesterasa metabolismus   MeSH
• Electrophorus metabolismus   MeSH
• enzymové reaktivátory chemie   farmakologie   MeSH
• molekulární struktura MeSH
• oximy chemie   farmakologie   MeSH
• paraoxon toxicita   MeSH
• rybí proteiny metabolismus   MeSH
• techniky in vitro MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetylcholinesterasa MeSH
• enzymové reaktivátory MeSH
• oximy MeSH
• paraoxon MeSH
• rybí proteiny MeSH

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.

• Klíčová slova
• Acetylcholinesterase, Aging, Mipafox, Organophosphorus compounds, Oximes, Sarin,
• MeSH
• acetylcholinesterasa chemie   metabolismus   MeSH
• cholinesterasové inhibitory farmakokinetika   MeSH
• fosforylace MeSH
• isofluorofát analogy a deriváty   chemie   farmakokinetika   MeSH
• katalytická doména MeSH
• kinetika MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• organofosforové sloučeniny chemie   farmakokinetika   MeSH
• oximy chemie   MeSH
• paraoxon farmakokinetika   MeSH
• reaktivátory cholinesterasy chemie   farmakologie   MeSH
• sarin analogy a deriváty   chemie   MeSH
• sekvence aminokyselin MeSH
• serin metabolismus   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• acetylcholinesterasa MeSH
• cholinesterasové inhibitory MeSH
• isofluorofát MeSH
• methylphosphonic acid MeSH Prohlížeč
• mipafox MeSH Prohlížeč
• organofosforové sloučeniny MeSH
• oximy MeSH
• paraoxon MeSH
• reaktivátory cholinesterasy MeSH
• sarin MeSH
• serin MeSH