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. From these data, small spiroimine enantiomers mimicking the functional core motif of phycotoxins were chemically synthesized and characterized. Voltage-clamp analyses involving three nAChR subtypes revealed preserved antagonism for both enantiomers, despite lower subtype specificity and binding affinities associated with faster reversibility compared with their macrocyclic relatives. Binding and structural analyses involving two AChBPs pointed to modest affinities and positional variability of the spiroimines, along with a range of AChBP loop-C conformations denoting a prevalence of antagonistic properties. These data highlight the major contribution of the spiroimine core to binding within the nAChR nest and confirm the need for an extended interaction network as established by the macrocyclic toxins to define high affinities and marked subtype specificity. This study identifies a minimal set of functional pharmacophores and binding determinants as templates for designing new antagonists targeting disease-associated nAChR subtypes.

Connectivity between surface and interior in catalytic subunits of acetylcholinesterases inferred from their X-ray structures.

Catalytic activity and function of acetylcholinesterase (AChE; EC 3.1.1.7) have been recognized and studied for over a century and its quaternary and primary structures for about half a century, and its tertiary structure has been known for about 33 years. Clear understanding of relationships between the structure and the function is still pending for this enzyme. Hundreds of crystallographic, static snapshots of AChEs from different sources reveal largely one general backbone conformation with narrow entry into the active center gorge, tightly fit to accept one acetylcholine (ACh) molecule, in contrast to its high catalytic turnover. This short review of available X-ray structures of AChEs from electric ray Torpedo californica, mouse and human, finds some limited, yet consistent deviations in conformations of selected secondary structure elements of AChE relevant for its function. The observed conformational diversity of the acyl pocket loop of AChE, unlike the large Ω-loop, appears consistent with structurally dynamic INS data and solution-based SAXS experiments to explain its dominant role in controlling the size of the active center gorge opening, as well as connectivity between the immediate surroundings of the buried active Ser, and catalytically relevant sites on the AChE surface.

Structural and dynamic effects of paraoxon binding to human acetylcholinesterase by X-ray crystallography and inelastic neutron scattering.

Organophosphorus (OP) compounds, including nerve agents and some pesticides, covalently bind to the catalytic serine of human acetylcholinesterase (hAChE), thereby inhibiting acetylcholine hydrolysis necessary for efficient neurotransmission. Oxime antidotes can reactivate the OP-conjugated hAChE, but reactivation efficiency can be low for pesticides, such as paraoxon (POX). Understanding structural and dynamic determinants of OP inhibition and reactivation can provide insights to design improved reactivators. Here, X-ray structures of hAChE with unaged POX, with POX and oximes MMB4 and RS170B, and with MMB4 are reported. A significant conformational distortion of the acyl loop was observed upon POX binding, being partially restored to the native conformation by oximes. Neutron vibrational spectroscopy combined with molecular dynamics simulations showed that picosecond vibrational dynamics of the acyl loop soften in the ∼20-50 cm-1 frequency range. The acyl loop structural perturbations may be correlated with its picosecond vibrational dynamics to yield more comprehensive template for structure-based reactivator design.

Cobinamide is a strong and versatile antioxidant that overcomes oxidative stress in cells, flies, and diabetic mice.

Increased oxidative stress underlies a variety of diseases, including diabetes. Here, we show that the cobalamin/vitamin B12 analog cobinamide is a strong and multifaceted antioxidant, neutralizing superoxide, hydrogen peroxide, and peroxynitrite, with apparent rate constants of 1.9 × 108, 3.7 × 104, and 6.3 × 106 M-1 s-1, respectively, for cobinamide with the cobalt in the +2 oxidation state. Cobinamide with the cobalt in the +3 oxidation state yielded apparent rate constants of 1.1 × 108 and 8.0 × 102 M-1 s-1 for superoxide and hydrogen peroxide, respectively. In mammalian cells and Drosophila melanogaster, cobinamide outperformed cobalamin and two well-known antioxidants, imisopasem manganese and manganese(III)tetrakis(4-benzoic acid)porphyrin, in reducing oxidative stress as evidenced by: (i) decreased mitochondrial superoxide and return of the mitochondrial membrane potential in rotenone- and antimycin A-exposed H9c2 rat cardiomyocytes; (ii) reduced JNK phosphorylation in hydrogen-peroxide-treated H9c2 cells; (iii) increased growth in paraquat-exposed COS-7 fibroblasts; and (iv) improved survival in paraquat-treated flies. In diabetic mice, cobinamide administered in the animals' drinking water completely prevented an increase in lipid and protein oxidation, DNA damage, and fibrosis in the heart. Cobinamide is a promising new antioxidant that has potential use in diseases with heightened oxidative stress.

Room temperature crystallography of human acetylcholinesterase bound to a substrate analogue 4K-TMA: Towards a neutron structure.

Acetylcholinesterase (AChE) catalyzes hydrolysis of acetylcholine thereby terminating cholinergic nerve impulses for efficient neurotransmission. Human AChE (hAChE) is a target of nerve agent and pesticide organophosphorus compounds that covalently attach to the catalytic Ser203 residue. Reactivation of inhibited hAChE can be achieved with nucleophilic antidotes, such as oximes. Understanding structural and electrostatic (i.e. protonation states) determinants of the catalytic and reactivation processes is crucial to improve design of oxime reactivators. Here we report X-ray structures of hAChE conjugated with a reversible covalent inhibitor 4K-TMA (4K-TMA:hAChE) at 2.8 â€‹Å resolution and of 4K-TMA:hAChE conjugate with oxime reactivator methoxime, MMB4 (4K-TMA:hAChE:MMB4) at 2.6 â€‹Å resolution, both at physiologically relevant room temperature, as well as cryo-crystallographic structure of 4K-TMA:hAChE at 2.4 â€‹Å resolution. 4K-TMA acts as a substrate analogue reacting with the hydroxyl of Ser203 and generating a reversible tetrahedral hemiketal intermediate that closely resembles the first tetrahedral intermediate state during hAChE-catalyzed acetylcholine hydrolysis. Structural comparisons of room temperature with cryo-crystallographic structures of 4K-TMA:hAChE and published mAChE complexes with 4K-TMA, as well as the effect of MMB4 binding to the peripheral anionic site (PAS) of the 4K-TMA:hAChE complex, revealed only discrete, minor differences. The active center geometry of AChE, already highly evolved for the efficient catalysis, was thus indicative of only minor conformational adjustments to accommodate the tetrahedral intermediate in the hydrolysis of the neurotransmitter acetylcholine (ACh). To map protonation states in the hAChE active site gorge we collected 3.5 â€‹Å neutron diffraction data paving the way for obtaining higher resolution datasets that will be needed to determine locations of individual hydrogen atoms.

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

Acetylcholinesterase (EC 3.1.1.7), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human acetylcholinesterase (hAChE) in solution occurs through a C-terminal four-helix bundle at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the RP enantiomer of sarin promotes a 10-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6, or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of an SP-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wildtype hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket toward the four-helix bundle dimerization interface 25 Å away.

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. An ideal small-molecule antidote should access and be retained in primary target tissues-central nervous system (brain), skeletal muscle, and peripheral autonomic sites-for sufficient periods to reactivate AChE and prevent acute toxicity. We show here that we can markedly prolong the antidotal activity of zwitterionic antidotes by inhibiting P-glycoprotein (P-gp) transporters in the brain capillary and renal systems. We employ the P-gp inhibitor tariquidar as a reference compound and show that tissue and plasma levels of RS194B, a hydroxyl-imino acetamido alkylamine reactivator, are elevated and that plasma clearances are reduced. To examine the mechanism, identify the transporter, and establish the actions of a transport inhibitor, we compare the pharmacokinetic parameters in a P-glycoprotein knockout mouse strain and see dramatic enhancements of short-term plasma and tissue levels. Hence, repurposed transport inhibitors that are candidate or Food and Drug Administration-approved drugs, should enhance target tissue concentrations of the zwitterionic antidote through inhibition of both renal elimination and brain capillary extrusion. SIGNIFICANCE STATEMENT: We examine renal and brain capillary transporter inhibition as means for lowering dose and frequency of dosing of a blood-brain barrier permanent reactivating antidote, RS194B, an ionizable zwitterion. Through a small molecule, tariquidar, and gene knockout mice, CNS antidote concentrations are enhanced, and total body clearances are concomitantly diminished. RS194B with repurposed transport inhibitors should enhance reactivation of central and peripheral OP-inhibited acetylcholinesterase. Activities at both disposition sites are a desired features for replacing the antidote, pralidoxime, for acute OP exposure.

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. These are structural properties directed to reactivity profiles at the conjugated AChE active center, reinforced by the pharmacokinetic and tissue disposition properties of the re-activator leads. In the case of nicotinic acetylcholine receptor (nAChR) agonists and antagonists, with the many existing receptor subtypes in mammals, we prioritize subtype selectivity in their design. In contrast to nicotine and its analogues that react with panoply of AChR subtypes, the substituted di-2-picolyl amine pyrimidines possess distinctive ionization characteristics reflecting in selectivity for the orthosteric site at the α7 subtypes of receptor. Here, entry to the CNS should be prioritized for the therapeutic objectives of the nicotinic agent influencing aberrant CNS activity in development or in the sequence of CNS ageing (longevity) in mammals, along with general peripheral activities controlling inflammation.

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. Improvements of OP antidotes need to include much better access of AChE reactivators to the CNS and optimized orientation of the antidotes' nucleophile within the AChE active-center gorge. On the basis of X-ray structures of a CNS-penetrating reactivator, monoxime RS194B, reversibly bound to native and venomous agent X (VX)-inhibited human AChE, here we created seven uncharged acetamido bis-oximes as candidate antidotes. Both oxime groups in these bis-oximes were attached to the same central, saturated heterocyclic core. Diverse protonation of the heterocyclic amines and oxime groups of the bis-oximes resulted in equilibration among up to 16 distinct ionization forms, including uncharged forms capable of diffusing into the CNS and multiple zwitterionic forms optimal for reactivation reactions. Conformationally diverse zwitterions that could act as structural antidote variants significantly improved in vitro reactivation of diverse OP-human AChE conjugates. Oxime group reorientation of one of the bis-oximes, forcing it to point into the active center for reactivation, was confirmed by X-ray structural analysis. Our findings provide detailed structure-activity properties of several CNS-directed, uncharged aliphatic bis-oximes holding promise for use as protonation-dependent, conformationally adaptive, "smart" accelerated antidotes against OP toxicity.

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.

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. We describe in this article development and properties of new human acetylcholinesterase (AChE) crystals of space group P31 and a new unit cell, amenable for room-temperature X-ray diffraction studies. We co-crystallized hAChE in P31 unit cell with the reversible inhibitor 9-aminoacridine that binds at the base of the active center gorge in addition to inhibitors that span the full length of the gorge, donepezil (Aricept, E2020) and AChE specific inhibitor BW284c51. Their new low temperature P31 space group structures appear similar to those previously obtained in the different P3121 unit cell. Successful solution of the new room temperature 3.2 Å resolution structure of BW284c51*hAChE complex from large P31 crystals enables us to proceed with studying room temperature structures of lower affinity complexes, such as oxime reactivators bound to hAChE, where temperature-related conformational diversity could be expected in both oxime and hAChE, which could lead to better informed structure-based design under conditions approaching physiological temperature.

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. Current antidotes, including oxime reactivators that attack the OP-AChE conjugate to free the active enzyme, are inefficient. Better reactivators are sought, but their design is hampered by a conformationally rigid portrait of AChE extracted exclusively from 100K X-ray crystallography and scarcity of structural knowledge on human AChE (hAChE). Here, we present room temperature X-ray structures of native and VX-phosphonylated hAChE with an imidazole-based oxime reactivator, RS-170B. We discovered that inhibition with VX triggers substantial conformational changes in bound RS-170B from a "nonproductive" pose (the reactive aldoxime group points away from the VX-bound serine) in the reactivator-only complex to a "semi-productive" orientation in the VX-modified complex. This observation, supported by concurrent molecular simulations, suggested that the narrow active-site gorge of hAChE may be significantly more dynamic than previously thought, allowing RS-170B to reorient inside the gorge. Furthermore, we found that small molecules can bind in the choline-binding site hindering approach to the phosphorous of VX-bound serine. Our results provide structural and mechanistic perspectives on the reactivation of OP-inhibited hAChE and demonstrate that structural studies at physiologically relevant temperatures can deliver previously overlooked insights applicable for designing next-generation antidotes.

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. The first is the impacted space within the active center gorge, particularly when the active center serine at its base is conjugated with an organophosphate. The reactivating nucleophile will have to negotiate the tortuous gorge terrain to access the phosphorus atom with its most nucleophilic form or ionization state, the oximate anion. A second limitation stems from the antidote crossing the blood-brain barrier sufficiently rapidly, since it is well documented that central acetylcholinesterase inhibition gives rise to cardiovascular and respiratory compromise. The associated hypoxia then leads to a sequelae of events, including poor perfusion of the brain and periphery, along with muscle fasciculation, tremors and eventually seizures. We consider both the barriers confronting and further achievements necessary to enhance efficacy of antidotes.

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.

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. 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.

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.

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. We found that DjChE has intermediate features of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Here, we identify two candidate genes (Djche1 and Djche2) responsible for DjChE activity. Sequence alignment and structural homology modeling with representative vertebrate AChE and BChE sequences confirmed our structural predictions, and show that both DjChE enzymes have intermediate sized catalytic gorges and disrupted peripheral binding sites. Djche1 and Djche2 were both expressed in the planarian nervous system, as anticipated from previous activity staining, but with distinct expression profiles. To dissect how DjChE inhibition affects planarian behavior, we acutely inhibited DjChE activity by exposing animals to either an OP (diazinon) or carbamate (physostigmine) at 1 µM for 4 days. Both inhibitors delayed the reaction of planarians to heat stress. Simultaneous knockdown of both Djche genes by RNAi similarly resulted in a delayed heat stress response. Furthermore, chemical inhibition of DjChE activity increased the worms' ability to adhere to a substrate. However, increased substrate adhesion was not observed in Djche1/Djche2 (RNAi) animals or in inhibitor-treated day 11 regenerates, suggesting this phenotype may be modulated by other mechanisms besides ChE inhibition. Together, our study characterizes DjChE expression and function, providing the basis for future studies in this system to dissect alternative mechanisms of OP toxicity.

Post-exposure treatment with the oxime RS194B rapidly reverses early and advanced symptoms in macaques exposed to sarin vapor.

Organophosphate (OP) nerve agents and pesticides trigger a common mechanism of neurotoxicity resulting from critical targeting and inhibition of acetylcholinesterases (AChE) in central and peripheral synapses in the cholinergic nervous system. Therapeutic countermeasures have thus focused on either administering an oxime post-exposure, that can rapidly reactivate OP-inhibited AChE, or by preventing OP poisoning through administering pre-exposure treatments that scavenge OPs before they inhibit their physiological AChE targets. While several pyridinium aldoxime antidotes are currently approved, their utility is impaired due to their inability to cross the blood-brain barrier (BBB) efficiently. The present study utilized a macaque (Ma) model to demonstrate the efficacy of a novel zwitterionic and centrally acting oxime RS194B to reactivate sarin- and paraoxon-inhibited macaque AChE and butyrylcholinesterase (BChE) in vitro and to further assess the capacity of RS194B to effect a reversal of clinical symptoms following sarin inhalation in vivo. In vitro, oxime reactivation of MaAChE and MaBChE was shown to be comparable to their human orthologs, while the macaque studies indicated that IM administration of 62.5 mg/kg of RS194B and 0.28 mg/kg atropine after continuous exposure to 49.6 µg/kg sarin vapor, rapidly reactivated the inhibited AChE and BChE in blood and reversed both early and advanced clinical symptoms of sarin-induced toxicity following pulmonary exposure within 1 h. The rapid cessation of autonomic and central symptoms, including convulsions, observed in macaques bodes well for the use of RS194B as an intra- or post-exposure human treatment and validates the macaque model in generating efficacy and toxicology data required for approval under the FDA Animal rule.

Butyrylcholinesterase identification in a phenylvalerate esterase-enriched fraction sensitive to low mipafox concentrations in chicken brain.

Multiple epidemiological and experimental studies have demonstrated that exposure to organophosphorus compounds (OPs) is associated with a variety of neurological disorders. Some of these exposure symptoms cannot be precisely correlated with known molecular targets and mechanisms of toxicity. Most of the known molecular targets of OPs fall in the protein family of serine esterases. We have shown that three esterase components in the soluble fraction of chicken brain (an animal model frequently used in OP neurotoxicity assays) can be kinetically distinguished using paraoxon, mipafox and phenylmethyl sulfonyl fluoride as inhibitors, and phenyl valerate as a substrate; we termed them Eα, Eß and Eγ. The Eα-component, which is highly sensitive to paraoxon and mipafox and resistant to PMSF, has shown sensitivity to the substrate acetylthiocholine, and to ethopropazine and iso-OMPA (specific inhibitors of butyrylcholinesterase; BChE) but not to BW 284C51 (a specific inhibitor of acetylcholinesterase; AChE). In this work, we employed a large-scale proteomic analysis B with a LC/MS/MS TripleTOF system; 259 proteins were identified in a chromatographic fractionated sample enriched in Eα activity of the chicken brain soluble fraction. Bioinformatics analysis revealed that BChE is the only candidate protein identified to be responsible for almost all the Eα activity. This study demonstrates the potential information to be gained from combining kinetic dissection with large-scale proteomics and bioinformatics analyses for identification of proteins that are targets of OP toxicity and may be involved in detoxification of phosphoryl and carbonyl esters.