The Cyclic Imine Core Common to the Marine Macrocyclic Toxins Is Sufficient to Dictate Nicotinic Acetylcholine Receptor Antagonism.

Macrocyclic imine phycotoxins are an emerging class of chemical compounds associated with harmful algal blooms and shellfish toxicity. Earlier binding and electrophysiology experiments on nAChR subtypes and their soluble AChBP surrogates evidenced common trends for substantial antagonism, binding affinities, and receptor-subtype selectivity. Earlier, complementary crystal structures of AChBP complexes showed that common determinants within the binding nest at each subunit interface confer high-affinity toxin binding, while distinctive determinants from the flexible loop C, and either capping the nest or extending toward peripheral subsites, dictate broad versus narrow receptor subtype selectivity.

A single post-exposure oxime RS194B treatment rapidly reactivates acetylcholinesterase and reverses acute symptoms in macaques exposed to diethylphosphorothioate parathion and chlorpyrifos insecticides.

Millions of individuals globally suffer from inadvertent, occupational or self-harm exposures from organophosphate (OP) insecticides, significantly impacting human health. Similar to nerve agents, insecticides are neurotoxins that target and inhibit acetylcholinesterase (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with an oxime to reactivate the OP-inhibited AChE.

Differential Activation and Desensitization States Promoted by Noncanonical 7 Nicotinic Acetylcholine Receptor Agonists.

A series of dipicolyl amine pyrimidines (DPPs) were previously identified as potential 7 agonists by means of a calcium influx assay in the presence of the positive allosteric modulator (PAM) 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596). The compounds lack the quaternary or strongly basic nitrogens of typical nicotinic agonists. Although differing in structure from typical nicotinic agonists, based on crystallographic data with the acetylcholine binding protein, they appeared to engage the site shared by such typical orthosteric agonists.

Interactions of Nereistoxin and Its Analogs with Vertebrate Nicotinic Acetylcholine Receptors and Molluscan ACh Binding Proteins.

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond.

Covalent inhibition of hAChE by organophosphates causes homodimer dissociation through long-range allosteric effects.

Acetylcholinesterase (EC 3.1.1.

Enhancing Target Tissue Levels and Diminishing Plasma Clearance of Ionizing Zwitterionic Antidotes in Organophosphate Exposures.

Inhibition of acetylcholinesterase (AChE) by certain organophosphates (OPs) can be life-threatening and requires reactivating antidote accessibility to the peripheral and central nervous systems to reverse symptoms and enhance survival parameters. In considering dosing requirements for oxime antidotes in OP exposures that inactivate AChE, clearance of proton ionizable, zwitterionic antidotes is rapid and proceeds with largely the parent antidotal compound being cleared by renal transporters. Such transporters may also control disposition between target tissues and plasma as well as overall elimination from the body.

Ligand design for human acetylcholinesterase and nicotinic acetylcholine receptors, extending beyond the conventional and canonical.

We detail here distinctive departures from lead classical cholinesterase re-activators, the pyridinium aldoximes, to achieve rapid CNS penetration and reactivation of AChE in the CNS (brain and spinal cord). Such reactivation is consistent with these non-canonical re-activators enhancing survival parameters in both mice and macaques following exposure to organophosphates. Thus, the ideal cholinesterase re-activator should show minimal toxicity, limited inhibitory activity in the absence of an organophosphate, and rapid CNS penetration, in addition to its nucleophilic potential at the target, the conjugated AChE active center.

Cholinergic Capsules and Academic Admonitions.

Herein, I intend to capture highlights shared with my academic and research colleagues over the 60 years I devoted initially to my graduate and postdoctoral training and then to academic endeavors starting as an assistant professor in a new medical school at the University of California, San Diego (UCSD). During this period, the Department of Pharmacology emerged from a division within the Department of Medicine to become the first basic science department, solely within the School of Medicine at UCSD in 1979. As part of the school's plans to reorganize and to retain me at UCSD, I was appointed as founding chair.

Drysdalin, a snake neurotoxin with higher affinity for soluble acetylcholine binding protein from Aplysia californica than from Lymnaea stagnalis.

Acetylcholine binding proteins (AChBPs), structural and functional surrogates of the extracellular binding domain of nicotinic acetylcholine receptor (nAChRs), in complex with various antagonists and agonists have provided detailed insights into the neurotransmitter binding site of nAChRs. The classical long-chain α-neurotoxins bungarotoxin (44-fold) and cobratoxin (7-fold) bind to Lymnaea stagnalis (Ls)-AChBP with higher affinity compared to Aplysia californica (Ac)-AChBP. In this study, we describe a novel long chain α-neurotoxin Drysdalin, which has higher binding affinity (7-fold) to Ac-AChBP when compared to Ls-AChBP.

Positron emission tomography evaluation of oxime countermeasures in live rats using the tracer O-(2-[ F]fluoroethyl)-O-(p-nitrophenyl)methylphosphonate [ F]-VXS.

Oxime antidotes regenerate organophosphate-inhibited acetylcholinesterase (AChE). Although they share a common mechanism of AChE reactivation, the rate and amount of oxime that enters the brain are critical to the efficacy, a process linked to the oxime structure and charge. Using a platform based on the organophosphate [ F]-VXS as a positron emission tomography tracer for active AChE, the in vivo distribution of [ F]-VXS was evaluated after an LD dose (250 μg/kg) of the organophosphate paraoxon (POX) and following oximes as antidotes.

Lessons from nature: Structural studies and drug design driven by a homologous surrogate from invertebrates, AChBP.

It has been almost 20 years since the discovery and crystallization of a structural surrogate, the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP), comprising the extracellular domain of the nicotinic acetylcholine receptors (nAChRs). Structural characterization of this soluble protein has increased our understanding of the requirements for agonist and antagonist interactions at the ligand recognition site of the nAChRs. Application can be extended to orthologs in the pentameric ligand-gated ion channel superfamily, encompassing receptors that depolarize or hyperpolarize upon neurotransmitter association.

Rational design, synthesis, and evaluation of uncharged, "smart" bis-oxime antidotes of organophosphate-inhibited human acetylcholinesterase.

Organophosphate (OP) intoxications from nerve agent and OP pesticide exposures are managed with pyridinium aldoxime-based therapies whose success rates are currently limited. The pyridinium cation hampers uptake of OPs into the central nervous system (CNS). Furthermore, it frequently binds to aromatic residues of OP-inhibited acetylcholinesterase (AChE) in orientations that are nonproductive for AChE reactivation, and the structural diversity of OPs impedes efficient reactivation.

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.

Synthesis, Pharmacological Characterization, and Structure-Activity Relationships of Noncanonical Selective Agonists for α7 nAChRs.

A lack of selectivity of classical agonists for the nicotinic acetylcholine receptors (nAChR) has prompted us to identify and develop a distinct scaffold of α7 nAChR-selective ligands. Noncanonical 2,4,6-substituted pyrimidine analogues were framed around compound 40 for a structure-activity relationship study. The new lead compounds activate selectively the α7 nAChRs with EC's between 30 and 140 nM in a PNU-120596-dependent, cell-based calcium influx assay.

Adhesion, Catalysis and Signaling: A Commonality of Association Followed by Distinctive Events Driving Function.

In this issue of Structure,Ranaivoson et al. (2019) highlight neuronal adhesion molecules with an extracellular exposure in synapses that have the potential to open new avenues in the study of the development and organization of the nervous system and their role in health and disease.

A new crystal form of human acetylcholinesterase for exploratory room-temperature crystallography studies.

Structure-guided design of novel pharmacologically active molecules relies at least in part on functionally relevant accuracy of macromolecular structures for template based drug design. Currently, about 95% of all macromolecular X-ray structures available in the PDB (Protein Data Bank) were obtained from diffraction experiments at low, cryogenic temperatures. However, it is known that functionally relevant conformations of both macromolecules and pharmacological ligands can differ at higher, physiological temperatures.

Productive reorientation of a bound oxime reactivator revealed in room temperature X-ray structures of native and VX-inhibited human acetylcholinesterase.

Exposure to organophosphorus compounds (OPs) may be fatal if untreated, and a clear and present danger posed by nerve agent OPs has become palpable in recent years. OPs inactivate acetylcholinesterase (AChE) by covalently modifying its catalytic serine. Inhibited AChE cannot hydrolyze the neurotransmitter acetylcholine leading to its build-up at the cholinergic synapses and creating an acute cholinergic crisis.

Assessment of ionizable, zwitterionic oximes as reactivating antidotal agents for organophosphate exposure.

Since the development in the 1950's of 2-PAM (Pralidoxime), an antidote that reactivates organophosphate conjugated acetylcholinesterase in target tissues upon pesticide or nerve agent exposure, improvements in antidotal therapy have largely involved congeneric pyridinium aldoximes. Despite seminal advances in detailing the structures of the cholinesterases as the primary target site, progress with small molecule antidotes has yet to define a superior agent. Two major limitations are immediately apparent.

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.

Reversal of Tabun Toxicity Enabled by a Triazole-Annulated Oxime Library-Reactivators of Acetylcholinesterase.

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.

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.

Planarian cholinesterase: molecular and functional characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity.

The asexual freshwater planarian Dugesia japonica has emerged as a medium-throughput alternative animal model for neurotoxicology. We have previously shown that D. japonica are sensitive to organophosphorus pesticides (OPs) and characterized the in vitro inhibition profile of planarian cholinesterase (DjChE) activity using irreversible and reversible inhibitors.

Post-exposure treatment with the oxime RS194B rapidly reactivates and reverses advanced symptoms of lethal inhaled paraoxon in macaques.

Fatalities from organophosphate (OP) insecticide result from both occupational and deliberate exposure; significantly impacting human health. Like nerve agents, insecticides are neurotoxins which target and inhibit acetylcholinesterases (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with a pyridinium aldoxime e.