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.
We reviewed the current advances in our understanding of the effects of a variety of stressors in order to connect acute poisoning with organophosphates, neurotoxicity, changes in microRNAs, and transfer RNA fragments with cholinergic signaling dysfunction. We highlighted recent studies of the chemical and molecular regulators of acetylcholine (ACh) signaling and the complexity of the mechanisms controlling cholinergic functioning in health and disease.
Phosphylation of the pivotal enzyme acetylcholinesterase (AChE) by nerve agents (NAs) leads to irreversible inhibition of the enzyme and accumulation of neurotransmitter acetylcholine, which induces ...cholinergic crisis, that is, overstimulation of muscarinic and nicotinic membrane receptors in the central and peripheral nervous system. In severe cases, subsequent desensitisation of the receptors results in hypoxia, vasodepression, and respiratory arrest, followed by death. Prompt action is therefore critical to improve the chances of victim’s survival and recovery. Standard therapy of NA poisoning generally involves administration of anticholinergic atropine and an oxime reactivator of phosphylated AChE. Anticholinesterase compounds or NA bioscavengers can also be applied to preserve native AChE from inhibition. With this review of 70 years of research we aim to present current and potential approaches to counteracting NA poisoning.
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.
Nerve‐agent poisoning. A new class of uncharged oximes tested as reactivators of human acetylcholinesterase and butyrylcholinesterase inhibited by nerve agents have been found more efficient than those oximes already in use. The crystal structure of an example oxime–enzyme complex displays a productive conformation for the reactivation of the phosphylated AChE. Furthermore, the oximes are predicted to efficiently cross the blood–brain barrier and therefore, could achieve significant concentrations both at the neuromuscular junction and in the brain. This could lead to improved therapeutic outcome after nerve‐agent poisoning.
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’.
Display omitted
•Detoxification of nerve agents by human AChE mutants is proved ex vivo and in vivo.•Y337A/F338A mutant combined with HI-6 improves therapy in VX and soman exposed mice.•AChE mutant reactivation and re-inhibition cycles attenuate symptoms of poisoning.
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 pK a 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 pK a 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.
Display omitted
•A wide oxime concentration range is needed for an accurate evaluation of reactivation constants.•Reactivation efficiency depends on the enzyme concentration in the assay.•Oxime ...side-reactions could affect the 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.
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.
•Phenyltetrahydroisoquinoline moiety acts as peripheral anionic site ligand.•Potential reactivators of phosphylated BChE were identified.•High binding affinity of AChE for oximes does not ensure ...efficient reactivation in situ.•Oxime efficacy depends on the organophosphate conjugated to the cholinesterase.
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.
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.
Using a data‐driven partitioning strategy, we report that a significant proportion of gangliosides, long believed to be lipid raft resident, reside outside raft domains. Immunohistochemistry and imaging mass spectrometry revealed that the distribution of gangliosides remains largely unchanged by treatment with Brij O20 detergent compared to the widely used Triton X‐100, which induces ganglioside redistribution. Lipid raft analyses of Brij O20 brain extracts revealed that gangliosides are not all lipid raft depending on the ganglioside structures. Likewise, different neuroplasticity protein raft distributions were revealed. This work enables more accurate lipid raft analyses for resolution of lipid–protein interactions in biological membranes.
Acetylcholinesterase (AChE), an enzyme that degrades the neurotransmitter acetylcholine, when covalently inhibited by organophosphorus compounds (OPs), such as nerve agents and pesticides, can be ...reactivated by oximes. However, tabun remains among the most dangerous nerve agents due to the low reactivation efficacy of standard pyridinium aldoxime antidotes. Therefore, finding an optimal reactivator for prophylaxis against tabun toxicity and for post‐exposure treatment is a continued challenge. In this study, we analyzed the reactivation potency of 111 novel nucleophilic oximes mostly synthesized using the CuAAC triazole ligation between alkyne and azide building blocks. We identified several oximes with significantly improved in vitro reactivating potential for tabun‐inhibited human AChE, and in vivo antidotal efficacies in tabun‐exposed mice. Our findings offer a significantly improved platform for further development of antidotes and scavengers directed against tabun and related phosphoramidate exposures, such as the Novichok compounds.
Developing antidotes: A substantial number of N‐methylpyridinium and 2‐methylimidazolium oximes that show significantly improved in vitro reactivating efficacies for tabun‐inhibited AChE have been synthesized and pharmacologically characterized. These findings offer a valuable and comprehensive platform for further development of antidotes and scavengers against tabun and related phosphoramidate exposures.
PDF
