Search

Nerve agents have posed a huge threat to national and human security, and their sensitive detection is crucial. Herein, based on the oxidation of Ce 4+ and the aggregation-induced emission (AIE) of glutathione-protected gold nanoclusters (GSH-Au NCs), a cascade reaction was designed to prepare oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB) and GSH-Au NCs crosslinked by Ce 3+ (Ce 3+ -GSH-Au NCs). oxTMB had a broad UV-visible absorption range (500-700 nm) and was capable of quenching the fluorescence of Ce 3+ -GSH-Au NCs at 590 nm through the internal filtration effect (IFE). Thiocholine (TCh), the hydrolysis product of acetylthiocholine chloride (ATCl) catalyzed by acetylcholinesterase (AChE), reduced oxTMB completely, resulting in a decrease in the absorption of oxTMB and the recovery of IFE-quenched fluorescence of Ce 3+ -GSH-Au NCs. Nerve agent sarin (GB) hindered the production of TCh and the reduction of oxTMB by inhibiting the AChE activity, leading to the fluorescence of Ce 3+ -GSH-Au NCs being quenched again. The dual-output sensing system (AChE + ATCl + oxTMB + Ce 3+ -GSH-Au NCs) exhibited a low limit of detection to GB (2.46 nM for colorimetry and 1.18 nM for fluorimetry) and excellent selectivity toward common interferences being unable to inhibit AChE. Moreover, the intelligent logic gate constructed based on the sensing system showed promising applications in the field of smart sensing of nerve agents.

This study addresses a comprehensive assessment of the interaction between chemical warfare agents (CWA) and acetylcholinesterase (AChE) systems, focus on the intriguing pnictogen-bond interaction (PnB). Utilizing the crystallographic data from the Protein Data Bank pertaining to the AChE-CWA complex involving Sarin (GB), Cyclosarin (GF), 2-[fluoro(methyl)phosphoryl]oxy-1,1-dimethylcyclopentane (GP) and venomous agent X (VX) agents, the CWA is systematically displaced by increments of 0.1 Å along the PO bond axis, extending its distance by 4 Å from the original position. The AIM analysis was carried out and consistently revealed the presence of a significant interaction along the PO bond. Investigating the intrinsic nature of the PnB, the NBO and the EDA analysis unearthed the contribution of orbital factors to the overall energy of the system. Strikingly, this observation challenges the conventional σ-hole explanation commonly associated with such interactions. This finding adds a layer of complexity to understanding of PnB, encouraging further exploration into the underlying mechanisms governing these intriguing chemical phenomena., (© 2024 The Authors. Journal of Computational Chemistry published by Wiley Periodicals LLC.)

Olesoxime, a cholesterol derivative with an oxime group, possesses the ability to cross the blood-brain barrier, and has demonstrated excellent safety and tolerability properties in clinical research. These characteristics indicate it may serve as a centrally active ligand of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), whose disruption of activity with organophosphate compounds (OP) leads to uncontrolled excitation and potentially life-threatening symptoms. To evaluate olesoxime as a binding ligand and reactivator of human AChE and BChE, we conducted in vitro kinetic studies with the active metabolite of insecticide parathion, paraoxon, and the warfare nerve agents sarin, cyclosarin, tabun, and VX. Our results showed that both enzymes possessed a binding affinity for olesoxime in the mid-micromolar range, higher than the antidotes in use (i.e., 2-PAM, HI-6, etc.). While olesoxime showed a weak ability to reactivate AChE, cyclosarin-inhibited BChE was reactivated with an overall reactivation rate constant comparable to that of standard oxime HI-6. Moreover, in combination with the oxime 2-PAM, the reactivation maximum increased by 10-30% for cyclosarin- and sarin-inhibited BChE. Molecular modeling revealed productive interactions between olesoxime and BChE, highlighting olesoxime as a potentially BChE-targeted therapy. Moreover, it might be added to OP poisoning treatment to increase the efficacy of BChE reactivation, and its cholesterol scaffold could provide a basis for the development of novel oxime antidotes.

Poisoning with organophosphorus compounds used as pesticides or misused as chemical weapons remains a serious threat to human health and life. Their toxic effects result from irreversible blockade of the enzymes acetylcholinesterase and butyrylcholinesterase, which causes overstimulation of the cholinergic system and often leads to serious injury or death. Treatment of organophosphorus poisoning involves, among other strategies, the administration of oxime compounds. Oximes reactivate cholinesterases by breaking the covalent bond between the serine residue from the enzyme active site and the phosphorus atom of the organophosphorus compound. Although the general mechanism of reactivation has been known for years, the exact molecular aspects determining the efficiency and selectivity of individual oximes are still not clear. This hinders the development of new active compounds. In our research, using relatively simple and widely available molecular docking methods, we investigated the reactivation of acetyl- and butyrylcholinesterase blocked by sarin and tabun. For the selected oximes, their binding modes at each step of the reactivation process were identified. Amino acids essential for effective reactivation and those responsible for the selectivity of individual oximes against inhibited acetyl- and butyrylcholinesterase were identified. This research broadens the knowledge about cholinesterase reactivation and demonstrates the usefulness of molecular docking in the study of this process. The presented observations and methods can be used in the future to support the search for new effective reactivators.

The oximes 4-carbamoyl-1-[({2-[(E)-(hydroxyimino) methyl] pyridinium-1-yl} methoxy) methyl] pyridinium (known as HI-6) and 3-carbamoyl-1-[({2-[(E)-(hydroxyimino) methyl] pyridinium-1-yl} methoxy) methyl] pyridinium (known as HS-6) are isomers differing from each other only by the position of the carbamoyl group on the pyridine ring. However, this slight difference was verified to be responsible for big differences in the percentual of reactivation of acetylcholinesterase (AChE) inhibited by the nerve agents tabun, sarin, cyclosarin, and VX. In order to try to find out the reason for this, a computational study involving molecular docking, molecular dynamics, and binding energies calculations, was performed on the binding modes of HI-6 and HS-6 on human AChE (HssAChE) inhibited by those nerve agents.

Organophosphorus nerve agents (NAs) irreversibly inhibit acetylcholinesterase (AChE), the enzyme responsible for breaking down the neurotransmitter acetylcholine (ACh). The over accumulation of ACh after NA exposure leads to cholinergic toxicity, seizure, and death. Current medical countermeasures effectively mitigate peripheral symptoms, however; the brain is often unprotected. Alternative acute treatment with the adenosine A 1 receptor agonist N 6 -cyclopentyladensosine (CPA) has previously been demonstrated to prevent AChE inhibition as well as to suppress neuronal activity. The mechanism of AChE protection is unknown. To elucidate the feasibility of potential CPA-AChE interaction mechanisms, we applied a truncated molecular model approach and density functional theory. The candidate mechanisms studied are reversible enzyme inhibition, enzyme reactivation, and NA blocking prior to enzyme conjugation. Our thermodynamic data suggest that CPA can compete with the NAs sarin and soman for the active site of AChE, but may, in contrast to NAs, undergo back-reaction. We found a strong interaction between CPA and NA conjugated AChE, making enzyme reactivation unlikely but possibly allowing for CPA protection through the prevention of NA aging. The data also indicates that there is an affinity between CPA and unbound NAs. The results from this study support the hypothesis that CPA counters NA toxicity via multiple mechanisms and is a promising therapeutic strategy that warrants further development., (Published by Elsevier Ltd.)

In the present work, we performed docking and molecular dynamics simulations studies on two groups of long-tailored oximes designed as peripheral site binders of acetylcholinesterase (AChE) and potential penetrators on the blood brain barrier. Our studies permitted to determine how the tails anchor in the peripheral site of sarin-inhibited human AChE, and which aminoacids are important to their stabilization. Also the energy values obtained in the docking studies corroborated quite well with the experimental results obtained before for these oximes.

Currently fielded treatments for nerve agent intoxication include atropine, an acetylcholine receptor antagonist, and pralidoxime (2PAM), a small molecule reactivator of acetylcholinesterase (AChE). 2PAM reactivates nerve agent-inhibited AChE via direct nucleophilic attack by the oxime moiety on the phosphorus center of the bound nerve agent. Due to a permanently charged pyridinium motif, 2PAM is not thought to cross the blood brain barrier and therefore cannot act directly in the neuronal junctions of the brain. In this study, ADOC, a non-permanently charged, non-oxime molecule initially identified using pesticide-inhibited AChE, was characterized in vitro against nerve agent-inhibited recombinant human AChE. The inhibitory and reactivation potentials of ADOC were determined with native AChE and AChE inhibited with tabun, sarin, soman, cyclosarin, VX, or VR and then compared to those of 2PAM. Several structural analogs of ADOC were used to probe the reactivation mechanism of the molecule. Finally, guinea pigs were used to examine the protective efficacy of the compound after exposure to sarin. The results of both in vitro and in vivo testing will be useful in the design of future small molecule reactivators., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The terroristic availability of highly toxic nerve agents (NAs) highlights the necessity for a deep understanding of their toxicities and effective medical treatments. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for a characterization of the NAs poisoning and an evaluation on the efficacy of reactivators in in vitro was developed for the first time. After exposure to sarin or VX and pepsin digestion, the specific peptides of acetylcholinesterase (AChE) in a purified status, i.e. undecapeptide "GESAGAASVGM" in free, unaged, or aged status was identified and quantified. A key termination procedure is focused to make the reaction system "frozen" and precisely "capture" the poisoning, aging and spontaneous reactivation status of AChE, and the abundance of such specific peptides can thus be simultaneously measured. In our established method, as low as 0.72% and 0.84% inhibition level of AChE induced by 0.5nM sarin and VX can be detected from the measurement of peptide adducts, which benefits a confirmation of NAs exposure, especially at extremely low levels. Comparing with conventional colorimetric Ellman assays, our method provides not only enzyme activity and inhibition rate, but also the precise poisoning status of NAs exposed AChE. Based on the full information provided by this method, the efficacy of reactivators, such as HI-6, obidoxime and pralidoxime, in the typical treatment of NAs poisoned AChE in in vitro was further evaluated. Our results showed that this method is a promising tool for the characterization of NAs poisoning and the evaluation of reactivator efficacy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Organophosphorus nerve agents interfere with cholinergic signaling by covalently binding to the active site of the enzyme acetylcholinesterase (AChE). This inhibition causes an accumulation of the neurotransmitter acetylcholine, potentially leading to overstimulation of the nervous system and death. Current treatments include the use of antidotes that promote the release of functional AChE by an unknown reactivation mechanism. We have used diffusion trap cryocrystallography and density functional theory (DFT) calculations to determine and analyze prereaction conformers of the nerve agent antidote HI-6 in complex with Mus musculus AChE covalently inhibited by the nerve agent sarin. These analyses reveal previously unknown conformations of the system and suggest that the cleavage of the covalent enzyme-sarin bond is preceded by a conformational change in the sarin adduct itself. Together with data from the reactivation kinetics, this alternate conformation suggests a key interaction between Glu202 and the O-isopropyl moiety of sarin. Moreover, solvent kinetic isotope effect experiments using deuterium oxide reveal that the reactivation mechanism features an isotope-sensitive step. These findings provide insights into the reactivation mechanism and provide a starting point for the development of improved antidotes. The work also illustrates how DFT calculations can guide the interpretation, analysis, and validation of crystallographic data for challenging reactive systems with complex conformational dynamics.

The main treatment for organophosphorus (OP) compound poisoning in clinics is to restore the activity of acetylcholinesterase (AChE) through oxime-induced reactivation of the phosphorylated OP-AChE adduct. It suffers from a competitive and irreversible aging reaction of the phosphorylated OP-AChE adduct, resulting in permanent inactivity of AChE. However, it was recently reported that N-methyl-2-methoxypyridinium species can act as methylating agents to methylate the methyl methane-phosphonate monoanion, in which the reaction mimics the reverse of the aging reaction of the phosphorylated OP-AChE adduct. If the aging reaction could be really reversed, the efficiency for the OP detoxification should be significantly improved, bringing up the possibility to develop an agent to reverse the aging process of the phosphorylated OP-AChE adduct. However, such a reaction with the N-methyl-2-methoxypyridinium species in the enzyme is still not reported so far. It is of great interest to know whether or not this reaction is observable in the enzyme, and more importantly, if it turns out to be not observable in the enzyme, why such a reaction proceeds quickly in aqueous solution but not in the enzyme. In the present study, we performed DFT calculations and quantum mechanical/molecular mechanical (QM/MM) calculations to reveal the fundamental mechanism for the methylation of both the methyl methane-phosphonate monoanion and the aged sarin-AChE adduct by N-methyl-2-methoxypyridinium species, respectively. The obtained results support the SN2 reaction mechanism, not the stepwise mechanism, for the methylation of the methyl methane-phosphonate monoanion by 9 reported N-methyl-2-methoxypyridinium compounds. The calculated free energy barriers are in good agreement with the experimental data. The methylation of the aged sarin-AChE adduct by one N-methyl-2-methoxypyridinium compound (labeled as compound 2) also employs the SN2 reaction mechanism with an extremely high free energy barrier of 30.4 ± 3.5 (or 26.6) kcal mol(-1), implying that this reaction in the enzyme hardly occurs. Our results clearly show that compound 2 forms a strong π-π stacking interaction with the aromatic ring of the W86 residue of AChE, making itself unable to approach sarin for the reverse of the aging process. On the basis of the structure and mechanism, several possible strategies have been suggested for designing methylating agents with higher activity against the aged sarin-AChE adduct.

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.

Presently available medications for treatment of organiphosphorus poisoning are not sufficiently effective due to various pharmacological and toxicological reasons. In this regard, herein we report the synthesis of a series of N-thiazolylacetamide monoquaternary pyridinium oximes and its analogs (1a-1b to 6a-6b) with diversely substituted thiazole ring and evaluation of their in vitro reactivation efficacies against nerve agent (sarin, O-ethylsarin and VX) inhibited human erythrocyte acetylcholinesterase (hAChE). Reactivation kinetics was performed to determine dissociation constant (KD), reactivity rate constant (kr) and the second order rate constant (kr2) for all the compounds and compared their efficacies with commercial antidotes viz. 2-PAM and obidoxime. All the newly synthesized oximes were evaluated for their physicochemical parameters (pKa) and correlated with their respective reactivation efficacies to assess the capability of the oxime reactivator. Three of these novel compounds showed promising reactivation efficacies toward OP inhibited hAChE. Molecular docking studies were performed in order to correlate the reactivation efficacies with their interactions in the active site of the AChE., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A series of bis-quaternary pyridinium derivatives 3a-3i of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide (2) have been synthesized. The synthesized pyridinium compounds have an amide group in conjugation to the oxime moiety. These compounds were evaluated in vitro for their reactivation efficacy against organophosphorus (OP) nerve agents (NAs) (sarin and VX) inhibited human erythrocyte ghost acetylcholinesterase (hAChE) and compared with the reactivation efficacy of 2-PAM and obidoxime. The pKa values of the synthesized compounds were found closer to the pKa values of 2- and 4-pyridinium oxime reactivators such as 2-PAM and obidoxime. Some of the compounds have shown better reactivation efficacy than 2-PAM, and obidoxime against sarin and VX inhibited AChE., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The inactivation of acetylcholinesterase (AChE) by organophosphorus agent (OP) compounds is a serious problem regardless of how the individual was exposed. The reactivation of OP-inactivated AChE is dependent on the OP conjugate, and commonly a specific oxime is better at reactivating a specific OP conjugate than several diverse OP conjugates. The presented research explores the physicochemical properties needed for the reactivation of OP-inactivated AChE. Four different OPs, cyclosarin, sarin, tabun, and VX, were analyzed using the same set of oxime reactivators. A trial descriptor pool of semiempirical, traditional, and molecular interaction field descriptors was used to construct an ensemble of QSAR models for each OP-conjugate pair. Based on the molecular information and the cross-validation ability, individual QSAR models were selected to be part of an OP-conjugate consensus model. The OP-conjugate specific models provide important insight into the physicochemical properties required to reactivate the OP conjugates of interest. The reactivation of AChE inactivated with either cyclosarin or tabun requires the oxime therapeutic to possess an overall polar-positive surface area. Oxime therapeutics for the reactivation of sarin-inactivated AChE are conformationally dependent while oxime reverse therapeutics for VX require a compact region with a highly hydrophilic region and two positively charged pyridine rings.

Administration of oxime therapy is currently the standard approach used to reverse the acute toxicity of organophosphorus (OP) compounds, which is usually attributed to OP inhibition of acetylcholinesterase (AChE). Rate constants for reactivation of OP-inhibited AChE by even the best oximes, such as HI-6 and obidoxime, can vary >100-fold between OP-AChE conjugates that are easily reactivated and those that are difficult to reactivate. To gain a better understanding of this oxime specificity problem for future design of improved reactivators, we conducted a QSAR analysis for oxime reactivation of AChE inhibited by OP agents and their analogues. Our objective was to identify common mechanism(s) among OP-AChE conjugates of phosphates, phosphonates and phosphoramidates that result in resistance to oxime reactivation. Our evaluation of oxime reactivation of AChE inhibited by a sarin analogue, O-methyl isopropylphosphonofluoridate, or a cyclosarin analogue, O-methyl cyclohexylphosphonofluoridate, indicated that AChE inhibited by these analogues was at least 70-fold more difficult to reactivate than AChE inhibited by sarin or cyclosarin. In addition, AChE inhibited by an analogue of tabun (i.e., O-ethyl isopropylphosphonofluoridate) was nearly as resistant to reactivation as tabun-inhibited AChE. QSAR analysis of oxime reactivation of AChE inhibited by these OP compounds and others suggested that the presence of both a large substituent (i.e., ≥ the size of dimethylamine) and an alkoxy substituent in the structure of OP compounds is the common feature that results in resistance to oxime reactivation of OP-AChE conjugates whether the OP is a phosphate, phosphonate or phosphoramidate., (Published by Elsevier Ireland Ltd.)

Inactivation of acetylcholinesterase (AChE) due to inhibition by organophosphorus (OP) compounds is a major threat to human since AChE is a key enzyme in neurotransmission process. Oximes are used as potential reactivators of OP-inhibited AChE due to their α-effect nucleophilic reactivity. In search of more effective reactivating agents, model studies have shown that α-effect is not so important for dephosphylation reactions. We report the importance of α-effect of nucleophilic reactivity towards the reactivation of OP-inhibited AChE with hydroxylamine anion. We have demonstrated with DFT [B3LYP/6-311G(d,p)] calculations that the reactivation process of sarin-serine adduct 2 with hydroxylamine anion is more efficient than the other nucleophiles reported. The superiority of hydroxylamine anion to reactivate the sarin-inhibited AChE with sarin-serine adducts 3 and 4 compared to formoximate anion was observed in the presence and absence of hydrogen bonding interactions of Gly121 and Gly122. The calculated results show that the rates of reactivation process of adduct 4 with hydroxylamine anion are 261 and 223 times faster than the formoximate anion in the absence and presence of such hydrogen bonding interactions. The DFT calculated results shed light on the importance of the adjacent carbonyl group of Glu202 for the reactivation of sarin-serine adduct, in particular with formoximate anion. The reverse reactivation reaction between hydroxylamine anion and sarin-serine adduct was found to be higher in energy compared to the other nucleophiles, which suggests that this α-nucleophile can be a good antidote agent for the reactivation process., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The repeated misuse of highly toxic organophosphorus compound (OP) based chemical warfare agents in military conflicts and terrorist attacks poses a continuous threat to the military and civilian sector. The toxic symptomatology of OP poisoning is mainly caused by inhibition of acetylcholinesterase (AChE, E.C. 3.1.1.7) resulting in generalized cholinergic crisis due to accumulation of the neurotransmitter acetylcholine (ACh) in synaptic clefts. Beside atropine as competitive antagonist of ACh at muscarinic ACh receptors oximes as reactivators of OP-inhibited AChE are a mainstay of standard antidotal treatment. However, human AChE inhibited by certain OP is rather resistant to oxime-induced reactivation. The development of more effective oxime-based reactivators may fill the gaps. To get more insight into a potential structure-activity relationship between human AChE, OPs and oximes in vitro studies were conducted to investigate interactions of different tabun and sarin analogues with human AChE and the oximes obidoxime and HI 6 by determination of various kinetic constants. Rate constants for the inhibition of human AChE by OPs, spontaneous dealkylation and reactivation as well as reactivation by obidoxime and HI 6 of OP-inhibited human AChE were determined. The recorded kinetic data did not allow a general statement concerning a structure-activity relationship between human AChE, OP and oximes., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

The phosphonylation mechanism of AChE and the S203C mutation by sarin (GB) is evaluated using two reaction schemes: a small model nucleophile (ethoxide, CH(3)CH(2)O(-)) and quantum mechanical/molecular mechanical (QM/MM) simulations. Calculations utilizing small model nucleophiles indicate that the reaction barrier for addition to GB is the rate-limiting step for both ethoxide and ethyl thiolate (CH(3)CH(2)S(-)); moreover, the activation barrier for addition to the phosphorus center of GB by ethyl thiolate is significantly larger (13.2 kcal/mol) than for ethoxide (8.3 kcal/mol). The decomposition transition state for both nucleophiles was determined to be approximately 1 kcal/mol. QM/MM simulations for AChE suggest a similar reaction mechanism for phosphonylation of the catalytic S203; however, the relative energetics are altered significantly compared to the isolated system. QM/MM results indicate that formation of the penta-coordinate intermediate is the rate-limiting step in the enzymatic system, with an activation barrier of 3.6 kcal/mol. Hydrogen-bonding interactions between the fluoride leaving group of GB with Y124 in AChE are observed throughout the reaction profile. The S203C mutation alters the relative energetics of the reaction, increasing the energy barrier for formation of the penta-coordinate intermediate to a value of 4.5 kcal/mol; moreover, the penta-coordinate intermediate (as product) is stabilized by an additional 6 kcal/mol when compared to wild-type AChE., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Both G and V type nerve agents possess a center of chirality about phosphorus. The S(p) enantiomers are generally more potent inhibitors than their R(p) counterparts toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). To develop model compounds with defined centers of chirality that mimic the target nerve agent structures, we synthesized both the S(p) and the R(p) stereoisomers of two series of G type nerve agent model compounds in enantiomerically enriched form. The two series of model compounds contained identical substituents on the phosphorus as the G type agents, except that thiomethyl (CH(3)-S-) and thiocholine [(CH(3))(3)NCH(2)CH(2)-S-] groups were used to replace the traditional nerve agent leaving groups (i.e., fluoro for GB, GF, and GD and cyano for GA). Inhibition kinetic studies of the thiomethyl- and thiocholine-substituted series of nerve agent model compounds revealed that the S(p) enantiomers of both series of compounds showed greater inhibition potency toward AChE and BChE. The level of stereoselectivity, as indicated by the ratio of the bimolecular inhibition rate constants between S(p) and R(p) enantiomers, was greatest for the GF model compounds in both series. The thiocholine analogues were much more potent than the corresponding thiomethyl analogues. With the exception of the GA model compounds, both series showed greater potency against AChE than BChE. The stereoselectivity (i.e., S(p) > R(p)), enzyme selectivity, and dynamic range of inhibition potency contributed from these two series of compounds suggest that the combined application of these model compounds will provide useful research tools for understanding interactions of nerve agents with cholinesterase and other enzymes involved in nerve agent and organophosphate pharmacology. The potential of and limitations for using these model compounds in the development of biological therapeutics against nerve agent toxicity are also discussed.

Oximes have been used as reactivators for organophosphorus-inhibited acetylcholinesterase (AChE). However, it is still not clear why oximes are more efficient than other nucleophiles, since the reactivation consists of a simple nucleophilic substitution. In an attempt to answer this question, we have modeled the sarin-inhibited AChE reactivation by other nucleophiles (with and without the so-called alpha-effect) by applying the B3LYP/6-311G(d,p) level of theory. We have concluded that oximes reactivate AChE by a three-step mechanism in opposition to the four-step processes of the other modeled nucleophiles. In addition, our model suggests that oximes react with AChE in the deprotonated form (oximate). Our results also indicate that other nucleophiles may be used as AChE reactivators. We propose the use of hydrazones and hydrazonates for further evaluation as antidotes for intoxication by chemical warfare agents.

Organophosphonates such as isopropyl metylphosphonofluoridate (sarin) are extremely toxic as they phosphonylate the catalytic serine residue of acetylcholinesterase (AChE), an enzyme essential to humans and other species. Design of effective AChE reactivators as antidotes to various organophosphonates requires information on how the reactivators interact with the phosphonylated AChEs. However, such information has not been available hitherto because of three main challenges. First, reactivators are generally flexible in order to change from the ground state to the transition state for reactivation; this flexibility discourages determination of crystal structures of AChE in complex with effective reactivators that are intrinsically disordered. Second, reactivation occurs upon binding of a reactivator to the phosphonylated AChE. Third, the phosphorous conjugate can develop resistance to reactivation. We have identified crystallographic conditions that led to the determination of a crystal structure of the sarin(nonaged)-conjugated mouse AChE in complex with [(E)-[1-[(4-carbamoylpyridin-1-ium-1-yl)methoxymethyl]pyridin-2-ylidene]methyl]-oxoazanium dichloride (HI-6) at a resolution of 2.2 A. In this structure, the carboxyamino-pyridinium ring of HI-6 is sandwiched by Tyr124 and Trp286, however, the oxime-pyridinium ring is disordered. By combining crystallography with microsecond molecular dynamics simulation, we determined the oxime-pyridinium ring structure, which shows that the oxime group of HI-6 can form a hydrogen-bond network to the sarin isopropyl ether oxygen, and a water molecule is able to form a hydrogen bond to the catalytic histidine residue and subsequently deprotonates the oxime for reactivation. These results offer insights into the reactivation mechanism of HI-6 and design of better reactivators.

Previous kinetic studies investigating the interactions between human acetylcholinesterase (AChE), structurally different organophosphorus compounds (OP) and oximes did not reveal a conclusive structure-activity relationship of the different reactions. The only exception was for a homologous series of methylphosphonofluoridates bearing C1-C4 O-n- or O-i-alkyl residues. Hence, it was tempting to investigate the kinetic interactions between different pentylsarin analogues, human AChE and two oximes, obidoxime and HI 6, in order to increase the understanding of structure-activity relationship between highly toxic OP and human AChE. The rate constants for the inhibition of human erythrocyte AChE by four pentylsarin compounds (k(i)), for the spontaneous dealkylation (aging, k(a)) and reactivation (k(s)) of inhibited AChE as well as for the oxime-induced reactivation of inhibited AChE by obidoxime and HI 6 reflected by the dissociation constant (K(D)) and the reactivity constant (k(r)) were determined. All pentylsarin analogues had a high inhibitory potency towards AChE. Inhibited AChE was subject to spontaneous reactivation which outweighed aging substantially. Pentylsarin-inhibited AChE could be reactivated by oximes, HI 6 being more potent than obidoxime. The determination of inhibition, reactivation and aging kinetics of pentylsarin analogues with human AChE extends the database on interactions between AChE and methylphosphonofluoridate homologues with C1-C4 n- and i-alkyl residues demonstrating a structure-activity relationship depending on the chain length with certain differences regarding inhibition and post-inhibitory reactions. Unfortunately, no structure-activity relationship could be observed for the oxime-induced reactivation of inhibited AChE. In view of previous results with numerous structurally different organophosphates, organophosphonates and phosphoramidates it has to be concluded that up to now kinetic studies did not provide decisive information for the development of more effective oxime-based reactivators.

Enzyme-catalysed degradation of the nerve agents tabun, sarin, ethyl sarin and soman by three variants of an organophosphate-degrading enzyme was studied at low concentrations of nerve agent. The concentration of nerve agent at a given time was determined by its ability to inhibit the enzyme acetylcholinesterase. Experiments were conducted in 96-well microtitre plates. Values of the ratio of k(cat) (turnover number) to K(m) (Michaelis-Menten constant) were calculated. For tabun, this value (for the most effective OpdA variant) exceeded any value published to date for other enzymes. The value was within an order of magnitude for the highest value reported for sarin, but there appears to be no published value for ethyl sarin for comparison. The OpdA enzymes were relatively inefficient in degrading soman.

Organophosphorus compounds (OPs), such as nerve agents and a group of insecticides, irreversibly inhibit the enzyme acetylcholinesterase (AChE) by a rapid phosphorylation of the catalytic Ser203 residue. The formed AChE-OP conjugate subsequently undergoes an elimination reaction, termed aging, that results in an enzyme completely resistant to oxime-mediated reactivation by medical antidotes. In this study, we present crystal structures of the non-aged and aged complexes between Mus musculus AChE (mAChE) and the nerve agents sarin, VX, and diisopropyl fluorophosphate (DFP) and the OP-based insecticides methamidophos (MeP) and fenamiphos (FeP). Non-aged conjugates of MeP, sarin, and FeP and aged conjugates of MeP, sarin, and VX are very similar to the noninhibited apo conformation of AChE. A minor structural change in the side chain of His447 is observed in the non-aged conjugate of VX. In contrast, an extensive rearrangement of the acyl loop region (residues 287-299) is observed in the non-aged structure of DFP and in the aged structures of DFP and FeP. In the case of FeP, the relatively large substituents of the phosphorus atom are reorganized during aging, providing a structural support of an aging reaction that proceeds through a nucleophilic attack on the phosphorus atom. The FeP aging rate constant is 14 times lower than the corresponding constant for the structurally related OP insecticide MeP, suggesting that tight steric constraints of the acyl pocket loop preclude the formation of a trigonal bipyramidal intermediate.

The pertinent threat of using organophosphorus compound (OP)-type chemical warfare agents (nerve agents) during military conflicts and by non-state actors requires the continuous search for more effective medical countermeasures. OP inhibit acetylcholinesterase (AChE) and therefore standard treatment of respective poisoning includes AChE reactivators (oximes) in combination with antimuscarinic agents. Hereby, standard oximes, 2-PAM and obidoxime, are considered to be rather insufficient against various nerve agents. Numerous experimental oximes have been investigated in the last decades by in vitro and in vivo models. Recently, we studied the reactivating potency of several oximes with human AChE inhibited by structurally different OP and observed remarkable differences depending on the OP and oxime. In order to investigate structure-activity relationships we determined the various kinetic constants (inhibition, reactivation, aging) for a series of sarin analogues bearing a methyl, ethyl, n-propyl, n-butyl, i-propyl, i-butyl, cyclohexyl or pinacolyl group with human AChE and BChE. The rate constants for the inhibition of human erythrocyte AChE and plasma BChE by these OP (k(i)), for the spontaneous dealkylation (k(a)) and reactivation (k(s)) of OP-inhibited AChE and BChE as well as for the oxime-induced reactivation of OP-inhibited AChE and BChE by the oximes obidoxime, 2-PAM, HI 6, HLö 7 and MMB-4 were determined. With compounds bearing a n-alkyl group the inhibition rate constant increased with chain length. A relation between chain length and spontaneous reactivation velocity was also observed. In contrast, no structure-activity dependence could be observed for the oxime-induced reactivation of AChE and BChE inhibited by the compounds tested. In general, OP-inhibited AChE and BChE were susceptible towards reactivation by oximes. HLö 7 was the most potent reactivator followed by HI 6 and obidoxime while 2-PAM and MMB-4 were rather weak reactivators. These data indicate a potential structure-activity relationship concerning inhibition and spontaneous reactivation but not for oxime-induced reactivation.

Potential energy surfaces for the phosphonylation of sarin and acetylcholinesterase (AChE) have been theoretically studied at the B3LYP/6-311G(d,p) level of theory. The obtained results show that the phosphonylation process involves a two-step addition-elimination mechanism, with the first step (addition process) being the rate-determining step, while by comparison, the ensuing steps are very rapid. Stable trigonal bipyramidal intermediates are formed in the studied pathways. It is also revealed that the catalytic triad of acetylcholinesterase plays the catalytic role in the reaction by speeding up the phosphonylation process, as it does in the acylation reaction of ACh and AChE. The effect of aqueous solvation was accounted for via the polarizable continuum model. It is concluded that the enzymatic reaction here is influenced strongly by the solvent environment.

Organophosphorus acid anhydride (OP) nerve agents are potent inhibitors which rapidly phosphonylate acetylcholinesterase (AChE) and then may undergo an internal dealkylation reaction (called "aging") to produce an OP-enzyme conjugate that cannot be reactivated. To understand the basis for irreversible inhibition, we solved the structures of aged conjugates obtained by reaction of Torpedo californica AChE (TcAChE) with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman) by X-ray crystallography to 2.3, 2.6, or 2.2 A resolution, respectively. The highest positive difference density peak corresponded to the OP phosphorus and was located within covalent bonding distance of the active-site serine (S200) in each structure. The OP-oxygen atoms were within hydrogen-bonding distance of four potential donors from catalytic subsites of the enzyme, suggesting that electrostatic forces significantly stabilize the aged enzyme. The active sites of aged sarin- and soman-TcAChE were essentially identical and provided structural models for the negatively charged, tetrahedral intermediate that occurs during deacylation with the natural substrate, acetylcholine. Phosphorylation with DFP caused an unexpected movement in the main chain of a loop that includes residues F288 and F290 of the TcAChE acyl pocket. This is the first major conformational change reported in the active site of any AChE-ligand complex, and it offers a structural explanation for the substrate selectivity of AChE.