Recent advances in cholinergic mechanisms as reactions to toxicity, stress, and neuroimmune insults.

This review presents recent studies of the chemical and molecular regulators of acetylcholine (ACh) signaling and the complexity of the small molecule and RNA regulators of those mechanisms that control cholinergic functioning in health and disease. The underlying structural, neurochemical, and transcriptomic concepts, including basic and translational research and clinical studies, shed new light on how these processes inter-change under acute states, age, sex, and COVID-19 infection; all of which modulate ACh-mediated processes and inflammation in women and men and under diverse stresses. The aspect of organophosphorus (OP) compound toxicity is discussed based on the view that despite numerous studies, acetylcholinesterase (AChE) is still a vulnerable target in OP poisoning because of a lack of efficient treatment and the limitations of oxime-assisted reactivation of inhibited AChE. The over-arching purpose of this review is thus to discuss mechanisms of cholinergic signaling dysfunction caused by OP pesticides, OP nerve agents, and anti-cholinergic medications; and to highlight new therapeutic strategies to combat both the acute and chronic effects of these chemicals on the cholinergic and neuroimmune systems. Furthermore, OP toxicity was examined in view of cholinesterase inhibition and beyond in order to highlight improved small molecules and RNA therapeutic strategies and assess their predicted pitfalls to reverse the acute toxicity and long-term deleterious effects of OPs.

Who's in, who's out? Re-evaluation of lipid raft residents.

Lipid rafts, membrane microdomains enriched with (glyco)sphingolipids, cholesterol, and select proteins, act as cellular signalosomes. Various methods have been used to separate lipid rafts from bulk (non-raft) membranes, but most often, non-ionic detergent Triton X-100 has been used in their isolation. However, Triton X-100 is a reported disruptor of lipid rafts. Histological evidence confirmed raft disruption by Triton X-100, but remarkably revealed raft stability to treatment with a related polyethylene oxide detergent, Brij O20. We report isolation of detergent-resistant membranes from mouse brain using Brij O20 and its use to determine the distribution of major mammalian brain gangliosides, GM1, GD1a, GD1b and GT1b. A different distribution of gangliosides-classically used as a raft marker-was discovered using Brij O20 versus Triton X-100. Immunohistochemistry and imaging mass spectrometry confirm the results. Use of Brij O20 results in a distinctive membrane distribution of gangliosides that is not all lipid raft associated, but depends on the ganglioside structure. This is the first report of a significant proportion of gangliosides outside raft domains. We also determined the distribution of proteins functionally related to neuroplasticity and known to be affected by ganglioside environment, glutamate receptor subunit 2, amyloid precursor protein and neuroplastin and report the lipid raft populations of these proteins in mouse brain tissue. This work will enable more accurate lipid raft analysis with respect to glycosphingolipid and membrane protein composition and lead to improved resolution of lipid-protein interactions within biological membranes.

Design, synthesis and cholinesterase inhibitory properties of new oxazole benzylamine derivatives.

The enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are primary targets in attenuating the symptoms of neurodegenerative diseases. Their inhibition results in elevated concentrations of the neurotransmitter acetylcholine which supports communication among nerve cells. It was previously shown for trans-4/5-arylethenyloxazole compounds to have moderate AChE and BChE inhibitory properties. A preliminary docking study showed that elongating oxazole molecules and adding a new NH group could make them more prone to bind to the active site of both enzymes. Therefore, new trans-amino-4-/5-arylethenyl-oxazoles were designed and synthesised by the Buchwald-Hartwig amination of a previously synthesised trans-chloro-arylethenyloxazole derivative. Additionally, naphthoxazole benzylamine photoproducts were obtained by efficient photochemical electrocyclization reaction. Novel compounds were tested as inhibitors of both AChE and BChE. All of the compounds exhibited binding preference for BChE over AChE, especially for trans-amino-4-/5-arylethenyl-oxazole derivatives which inhibited BChE potently (IC50 in µM range) and AChE poorly (IC50≫100 µM). Therefore, due to the selectivity of all of the tested compounds for binding to BChE, these compounds could be applied for further development of cholinesterase selective inhibitors.HIGHLIGHTSSeries of oxazole benzylamines were designed and synthesisedThe tested compounds showed binding selectivity for BChENaphthoxazoles were more potent AChE inhibitors.

Counteracting tabun inhibition by reactivation by pyridinium aldoximes that interact with active center gorge mutants of acetylcholinesterase.

Tabun represents the phosphoramidate class of organophosphates that are covalent inhibitors of acetylcholinesterase (AChE), an essential enzyme in neurotransmission. Currently used therapy in counteracting excessive cholinergic stimulation consists of a muscarinic antagonist (atropine) and an oxime reactivator of inhibited AChE, but the classical oximes are particularly ineffective in counteracting tabun exposure. In a recent publication (Kovarik et al., 2019), we showed that several oximes prepared by the Huisgen 1,3 dipolar cycloaddition and related precursors efficiently reactivate the tabun-AChE conjugate. Herein, we pursue the antidotal question further and examine a series of lead precursor molecules, along with triazole compounds, as reactivators of two AChE mutant enzymes. Such studies should reveal structural subtleties that reside within the architecture of the active center gorge of AChE and uncover intimate mechanisms of reactivation of alkylphosphate conjugates of AChE. The designated mutations appear to minimize steric constraints of the reactivating oximes within the impacted active center gorge. Indeed, after initial screening of the triazole oxime library and its precursors for the reactivation efficacy on Y337A and Y337A/F338A human AChE mutants, we found potentially active oxime-mutant enzyme pairs capable of degrading tabun in cycles of inhibition and reactivation. Surprisingly, the most sensitive ex vivo reactivation of mutant AChEs occurred with the alkylpyridinium aldoximes. Hence, although the use of mutant enzyme bio-scavengers in humans may be limited in practicality, bioscavenging and efficient neutralization of tabun itself or phosphoramidate mixtures of organophosphates might be achieved efficiently in vitro or ex vivo with these mutant AChE combinations.

Pharmacology, Pharmacokinetics, and Tissue Disposition of Zwitterionic Hydroxyiminoacetamido Alkylamines as Reactivating Antidotes for Organophosphate Exposure.

In the development of antidotal therapy for treatment of organophosphate exposure from pesticides used in agriculture and nerve agents insidiously employed in terrorism, the alkylpyridinium aldoximes have received primary attention since their early development by I. B. Wilson in the 1950s. Yet these agents, by virtue of their quaternary structure, are limited in rates of crossing the blood-brain barrier, and they require administration parenterally to achieve full distribution in the body. Oximes lacking cationic charges or presenting a tertiary amine have been considered as alternatives. Herein, we examine the pharmacokinetic properties of a lead ionizable, zwitterionic hydroxyiminoacetamido alkylamine in mice to develop a framework for studying these agents in vivo and generate sufficient data for their consideration as appropriate antidotes for humans. Consequently, in vitro and in vivo efficacies of immediate structural congeners were explored as leads or backups for animal studies. We compared oral and parenteral dosing, and we developed an intramuscular loading and oral maintenance dosing scheme in mice. Steady-state plasma and brain levels of the antidote were achieved with sequential administrations out to 10 hours, with brain levels exceeding plasma levels shortly after administration. Moreover, the zwitterionic oxime showed substantial protection after gavage, whereas the classic methylpyridinium aldoxime (2-pyridinealdoxime methiodide) was without evident protection. Although further studies in other animal species are necessary, ionizing zwitterionic aldoximes present viable alternatives to existing antidotes for prophylaxis and treatment of large numbers of individuals in terrorist-led events with nerve agent organophosphates, such as sarin, and in organophosphate pesticide exposure.

Potent 3-Hydroxy-2-Pyridine Aldoxime Reactivators of Organophosphate-Inhibited Cholinesterases with Predicted Blood-Brain Barrier Penetration.

A new series of 3-hydroxy-2-pyridine aldoxime compounds have been designed, synthesised and tested in vitro, in silico, and ex vivo as reactivators of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibited by organophosphates (OPs), for example, VX, sarin, cyclosarin, tabun, and paraoxon. The reactivation rates of three oximes (16-18) were determined to be greater than that of 2-PAM and comparable to that of HI-6, two pyridinium aldoximes currently used by the armies of several countries. The interactions important for a productive orientation of the oxime group within the OP-inhibited enzyme have been clarified by molecular-modelling studies, and by the resolution of the crystal structure of the complex of oxime 17 with Torpedo californica AChE. Blood-brain barrier penetration was predicted for oximes 15-18 based on their physicochemical properties and an in vitro brain membrane permeation assay. Among the evaluated compounds, two morpholine-3-hydroxypyridine aldoxime conjugates proved to be promising reactivators of OP-inhibited cholinesterases. Moreover, efficient ex vivo reactivation of phosphylated native cholinesterases by selected oximes enabled significant hydrolysis of VX, sarin, paraoxon, and cyclosarin in whole human blood, which indicates that the oximes have scavenging potential.

A comprehensive evaluation of novel oximes in creation of butyrylcholinesterase-based nerve agent bioscavengers.

A well-considered treatment of acute nerve agents poisoning involves the exogenous administration of butyrylcholinesterase (BChE, EC 3.1.1.8) as a stoichiometric bioscavenger efficient in preventing cholinergic crises caused by acetylcholinesterase (AChE, EC 3.1.1.7) inhibition. An additional improvement in medical countermeasures would be to use oximes that could reactivate BChE as well to upgrade bioscavenging from stoichiometric to oxime-assisted catalytic. Therefore, in this paper we investigated the potency of 39 imidazolium and benzimidazolium oximes (36 compounds synthesized for the first time) to be considered as the reactivators specifically designed for reactivation of phosphylated human BChE. Their efficiency in the reactivation of paraoxon-, VX-, and tabun-inhibited human BChE, as well as human AChE was tested and compared with the efficiencies of HI-6 and obidoxime, used in medical practice today. A comprehensive analysis was performed for the most promising oximes defining kinetic parameters of reactivation as well as interactions with uninhibited BChE. Furthermore, experimental data were compared with computational studies (docking, QSAR analysis) as a starting point in future oxime structure refinement. Considering the strict criteria set for in vivo applications, we determined the cytotoxicity of lead oximes on two cell lines. Among the tested oxime library, one imidazolium compound was selected for preliminary in vivo antidotal study in mice. The obtained protection in VX poisoning outlines its potential in development oxime-assisted OP-bioscavenging with BChE.

Catalytic Soman Scavenging by the Y337A/F338A Acetylcholinesterase Mutant Assisted with Novel Site-Directed Aldoximes.

Exposure to the nerve agent soman is difficult to treat due to the rapid dealkylation of the soman-acetylcholinesterase (AChE) conjugate known as aging. Oxime antidotes commonly used to reactivate organophosphate inhibited AChE are ineffective against soman, while the efficacy of the recommended nerve agent bioscavenger butyrylcholinesterase is limited by strictly stoichiometric scavenging. To overcome this limitation, we tested ex vivo, in human blood, and in vivo, in soman exposed mice, the capacity of aging-resistant human AChE mutant Y337A/F338A in combination with oxime HI-6 to act as a catalytic bioscavenger of soman. HI-6 was previously shown in vitro to efficiently reactivate this mutant upon soman, as well as VX, cyclosarin, sarin, and paraoxon, inhibition. We here demonstrate that ex vivo, in whole human blood, 1 µM soman was detoxified within 30 min when supplemented with 0.5 µM Y337A/F338A AChE and 100 µM HI-6. This combination was further tested in vivo. Catalytic scavenging of soman in mice improved the therapeutic outcome and resulted in the delayed onset of toxicity symptoms. Furthermore, in a preliminary in vitro screen we identified an even more efficacious oxime than HI-6, in a series of 42 pyridinium aldoximes, and 5 imidazole 2-aldoxime N-propylpyridinium derivatives. One of the later imidazole aldoximes, RS-170B, was a 2-3-fold more effective reactivator of Y337A/F338A AChE than HI-6 due to the smaller imidazole ring, as indicated by computational molecular models, that affords a more productive angle of nucleophilic attack.

Efficient detoxification of nerve agents by oxime-assisted reactivation of acetylcholinesterase mutants.

The recent advancements in crystallography and kinetics studies involving reactivation mechanism of acetylcholinesterase (AChE) inhibited by nerve agents have enabled a new paradigm in the search for potent medical countermeasures in case of nerve agents exposure. Poisonings by organophosphorus compounds (OP) that lead to life-threatening toxic manifestations require immediate treatment that combines administration of anticholinergic drugs and an aldoxime as a reactivator of AChE. An alternative approach to reduce the in vivo toxicity of OP centers on the use of bioscavengers against the parent organophosphate. Our recent research showed that site-directed mutagenesis of AChE can enable aldoximes to substantially accelerate the reactivation of OP-enzyme conjugates while dramatically slowing down rates of OP-conjugate dealkylation (aging). Therefore, this review focuses on oxime-assisted catalysis by AChE mutants that provides a potential means for degradation of organophosphates in the plasma before reaching the cellular target site. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'.

Evaluation of high-affinity phenyltetrahydroisoquinoline aldoximes, linked through anti-triazoles, as reactivators of phosphylated cholinesterases.

Acetylcholinesterase (AChE) is a pivotal enzyme in neurotransmission. Its inhibition leads to cholinergic crises and could ultimately result in death. A related enzyme, butyrylcholinesterase (BChE), may act in the CNS as a co-regulator in terminating nerve impulses and is a natural plasma scavenger upon exposure to organophosphate (OP) nerve agents that irreversibly inhibit both enzymes. With the aim of improving reactivation of cholinesterases phosphylated by nerve agents sarin, VX, cyclosarin, and tabun, ten phenyltetrahydroisoquinoline (PIQ) aldoximes were synthesized by Huisgen 1,3 dipolar cycloaddition between alkyne- and azide-building blocks. The PIQ moiety may serve as a peripheral site anchor positioning the aldoxime moiety at the AChE active site. In terms of evaluated dissociation inhibition constants, the aldoximes could be characterized as high-affinity ligands. Nevertheless, high binding affinity of these oximes to AChE or its phosphylated conjugates did not assure rapid and selective AChE reactivation. Rather, potential reactivators of phosphylated BChE, with its enlarged acyl pocket, were identified, especially in case of cyclosarin, where the reactivation rates of the lead reactivator was 100- and 6-times that of 2-PAM and HI-6, respectively. Nevertheless, the return of the enzyme activity was affected by the nerve agent conjugated to catalytic serine, which highlights the lack of the universality of reactivators with respect to both the target enzyme and OP structure.

Pharmacokinetic Evaluation of Brain Penetrating Morpholine-3-hydroxy-2-pyridine Oxime as an Antidote for Nerve Agent Poisoning.

Nerve agents, the deadliest chemical warfare agents, are potent inhibitors of acetylcholinesterase (AChE) and cause rapid cholinergic crisis with serious symptoms of poisoning. Oxime reactivators of AChE are used in medical practice in the treatment of nerve agent poisoning, but the search for novel improved reactivators with central activity is an ongoing pursuit. For numerous oximes synthesized, in vitro reactivation is a standard approach in biological evaluation with little attention given to the pharmacokinetic properties of the compounds. This study reports a comprehensive physicochemical, pharmacokinetic, and safety profiling of five lipophilic 3-hydroxy-2-pyridine aldoximes, which were recently shown to be potent AChE reactivators with a potential to be centrally active. The oxime JR595 was singled out as highly metabolically stable in human liver microsomes, noncytotoxic oxime for SH-SY5Y neuroblastoma and 1321N1 astrocytoma cell lines, and its pharmacokinetic profile was determined after intramuscular administration in mice. JR595 was rapidly absorbed into blood after 15 min with simultaneous distribution to the brain at up to about 40% of its blood concentration; however, it was eliminated from both the brain and blood within an hour. In addition, the MDCKII-MDR1 cell line assay showed that oxime JR595 was not a P-glycoprotein efflux pump substrate. Finally, the preliminary antidotal study against multiple LD50 doses of VX and sarin in mice showed the potential of JR595 to provide desirable therapeutic outcomes with future improvements in its circulation time.

The estimation of oxime efficiency is affected by the experimental design of phosphylated acetylcholinesterase reactivation.

Reactivation of acetylcholinesterase (AChE), an essential enzyme in neurotransmission, is a key point in the treatment of acute poisoning by nerve agents and pesticides, which structurally belong to organophosphorus compounds (OP). Due to the high diversity of substituents on the phosphorous atom, there is a variety of OP-AChE conjugates deriving from AChE inhibition, and therefore not only is there no universal reactivator efficient enough for the most toxic OPs, but for some nerve agents there is still a lack of any reactivator at all. The endeavor of many chemists to find more efficient reactivators resulted in thousands of newly-designed and synthesized oximes-potential reactivators of AChE. For an evaluation of the oximés reactivation efficiency, many research groups employ a simple spectrophotometric Ellman method. Since parameters that describe reactivator efficiency are often incomparable among laboratories, we tried to emphasize the critical steps in the determination of reactivation parameters as well as in the experimental design of a reactivation assay. We highlighted the important points in evaluation of reactivation kinetic parameters with an aim to achieve better agreement and comparability between the results obtained by different laboratories and overall, a more efficient evaluation of in vitro reactivation potency.

Oxime-assisted reactivation of tabun-inhibited acetylcholinesterase analysed by active site mutations.

The antidotal property of oximes is attributed to their ability to reactivate acetylcholinesterase (AChE) inhibited by organophosphorus compounds (OP) such as pesticides and nerve warfare agents. Understanding their interactions within the active site of phosphylated AChE is of great significance for the search for more efficient reactivators, especially in the case of the most resistant OP to reactivation, tabun. Therefore, herein we studied the interactions and reactivation of tabun-inhibited AChE by site-directed mutagenesis and a series of bispyridinium oximes. Our results indicated that the replacement of aromatic residues with aliphatic ones at the acyl pocket and choline binding site mostly interfered with the stabilisation of the oxime's pyridinium ring(s) within the active site gorge needed to obtain the proper orientation of the oxime group toward the phosphorylated active site serine. However, in the case of W286A, the mutation in the peripheral binding site by preventing a π-π interaction with one of the oxime's pyridinium rings allowed a more favourable position of the oxime for a nucleophilic attack on the phosphorylated catalytic serine. The mutation resulted in a 2-5 fold increase in the reactivation rates when compared to the AChE wild type. Therefore, it seems that aromatic amino acids at the peripheral binding site presented a limitation in bispyridinium oxime reactivation efficiency of tabun-phosphorylated AChE. Moreover, this is further corroborated by the reactivation by mono-pyridinium oxime 2-PAM, in which mutations at the peripheral site did not influence either the affinity or reactivation of tabun-inhibited AChE.

Pyridinium Oximes with Ortho-Positioned Chlorine Moiety Exhibit Improved Physicochemical Properties and Efficient Reactivation of Human Acetylcholinesterase Inhibited by Several Nerve Agents.

Six chlorinated bispyridinium mono-oximes, analogous to potent charged reactivators K027, K048, and K203, were synthesized with the aim of improving lipophilicity and reducing the p Ka value of the oxime group, thus resulting in a higher oximate concentration at pH 7.4 compared to nonchlorinated analogues. The nucleophilicity was examined and the p Ka was found to be lower than that of analogous nonchlorinated oximes. All the new compounds efficiently reactivated human AChE inhibited by nerve agents cyclosarin, sarin, and VX. The most potent was the dichlorinated analogue of oxime K027 with significantly improved ability to reactivate the conjugated enzyme due to improved binding affinity and molecular recognition. Its overall reactivation of sarin-, VX-, and cyclosarin-inhibited AChE was, respectively, 3-, 7-, and 8-fold higher than by K027. Its universality, PAMPA permeability, favorable acid dissociation constant coupled with its negligible cytotoxic effect, and successful ex vivo scavenging of nerve agents in whole human blood warrant further analysis of this compound as an antidote for organophosphorus poisoning.

HI-6 assisted catalytic scavenging of VX by acetylcholinesterase choline binding site mutants.

The high toxicity of organophosphorus compounds originates from covalent inhibition of acetylcholinesterase (AChE), an essential enzyme in cholinergic neurotransmission. Poisonings that lead to life-threatening toxic manifestations require immediate treatment that combines administration of anticholinergic drugs and an aldoxime as a reactivator of AChE. An alternative approach to reduce the in vivo toxicity of OPs focuses on the use of bioscavengers against the parent organophosphate. Our previous research showed that AChE mutagenesis can enable aldoximes to substantially accelerate the reactivation of OP-enzyme conjugates, while dramatically slowing down rates of OP-conjugate dealkylation (aging). Herein, we demonstrate an efficient HI-6-assisted VX detoxification, both ex vivo in human blood and in vivo in mice by hAChE mutants modified at the choline binding site (Y337A and Y337A/F338A). The catalytic scavenging of VX in mice improved therapeutic outcomes preventing lethality and resulted in a delayed onset of toxicity symptoms.

Translation of in vitro to in vivo pyridinium oxime potential in tabun poisoning.

Even if organophosphorus (OP) nerve agents were banned entirely, their presence would remain a problem as weapons of terror (like in Syria). Oxime antidotes currently used in medical practice still fall short of their therapeutic purpose, as they fail to fully restore the activity of cholinesterases, the main target for OPs. As orphan drugs, these antidotes are tested too seldom for anybody's benefit. Over the last few decades, search for improved reactivators has reached new levels, but the translation of data obtained in vitro to in vivo application is still a problem that hinders efficient therapy. In this study, we tested the strengths and weaknesses of extrapolating pyridinium oxime antidotes reactivation efficiency from in vitro to in vivo application. Our results show that this extrapolation is possible with well-determined kinetic constants, but that it also largely depends on oxime circulation time and its tissue-specific distribution. This suggests that pharmacokinetic studies should be planned at the early stages of antidote development. Special attention should also be given to improving oxime distribution throughout the organism to overcome this major constraint in improving overall OP therapy.