Crystal structures of aged phosphonylated acetylcholinesterase: nerve agent reaction products at the atomic level.

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.     

Refined crystal structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin

"Aged" organophosphoryl conjugates of serine hydrolases differ from the corresponding "non-aged" conjugates in their striking resistance to nucleophilic reactivation. The refined X-ray structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin were compared in order to understand the molecular basis for this resistance of "aged" conjugates. "Aged" and "non-aged" crystalline organophosphoryl-gamma-chymotrypsin conjugates were obtained by prolonged soaking of native gamma-chymotrypsin crystals with appropriate organophosphates. Thus, a representative "non-aged" conjugate, diethylphosphoryl-gamma-chymotrypsin, was obtained by soaking native crystals with paraoxon (diethyl-p-nitrophenyl phosphate), and a closely related "aged" conjugate, monoisopropyl-gamma-chymotrypsin, was obtained by soaking with diisopropylphosphorofluoridate. In both crystalline conjugates, the refined structures clearly reveal a high occupancy of the active site by the appropriate organophosphoryl moiety within covalent bonding distance of Ser195 O gamma. Whereas in the "non-aged" conjugate both ethyl groups can be visualized clearly, in the putative "aged" conjugate, as expected, only one isopropyl group is present. There is virtually no difference between the "aged" and "non-aged" conjugates either with respect to the conformation of the polypeptide backbone as a whole or with respect to the positioning of the side-chains within the active site. In the "aged" conjugate, however, close proximity (2.6 A) of the negatively charged phosphate oxygen atom of the dealkylated organophosphoryl group to His57 N epsilon 2 indicates the presence of a salt bridge between these two moieties. In contrast, in the "non-aged" conjugate the DEP moiety retains its two alkyl groups; thus, lacking a negative oxygen atom, it does not enter into such a charge-charge interaction and its nearest oxygen atom is 3.6 A away from His57 N epsilon 2. It is suggested that steric constraints imposed by the salt bridge in the "aged" conjugate lie at the basis of its resistance to reactivation.     

An attempt to assess functionally minimal acetylcholinesterase activity necessary for survival of rats intoxicated with nerve agents

Acetylcholinesterase (AChE, EC 3.1.1.7) is an important enzyme for cholinergic nerve transmission. The action of toxic organophosphates such as nerve agents is based on AChE inhibition. The death following acute nerve agent poisoning is due to central or peripheral respiratory/cardiac failure. Therefore, the changes in AChE activity following nerve agents acting predominantly on the central (sarin, soman) or peripheral (VX) level were studied. It is known that AChE activity in different structures exists in relative excess. Female Wistar rats intoxicated with sarin, soman, and VX in different doses (0.5-2.0xLD(50)) were divided into groups of survived and died animals. AChE activities in diaphragm, brain parts (pontomedullar area, frontal cortex, basal ganglia, in some cases other parts of the brain) were determined and the rest of activity (in %) was correlated with survival/death of animals. More precise elucidation of action of nerve agents and the assessment of minimal AChE activity in different organs compatible with the survival of organism poisoned with nerve agents were the aims of this study.     

Synthesis and in vitro reactivation study of isonicotinamide derivatives of 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide as reactivators of Sarin and VX inhibited human acetylcholinesterase (hAChE)

Previously (Karade et al., 2014), we have reported the synthesis and in vitro evaluation of bis-pyridinium derivatives of pyridine-3-yl-(2-hydroxyimino acetamide), as reactivators of sarin and VX inhibited hAChE. Few of the molecules showed superior in vivo protection efficacy (mice model) (Kumar et al., 2014; Swami et al., 2016) in comparison to 2-PAM against DFP and sarin poisoning. Encouraged by these results, herein we report the synthesis and in vitro evaluation of isonicotinamide derivatives of pyridine-3-yl-(2-hydroxyimino acetamide) (4a–4d) against sarin and VX inhibited erythrocyte ghost hAChE. Reactivation kinetics of these compounds was studied and the determined kinetic parameters were compared with that of commercial reactivators viz. 2-PAM and obidoxime. In comparison to 2-PAM and obidoxime, oxime 4a and 4b exhibited enhanced reactivation efficacy toward sarin inhibited hAChE while oxime 4c showed far greater reactivation efficacy toward VX inhibited hAChE. The acid dissociation constant and IC₅₀ values of these oximes were determined and correlated with the observed reactivation potential.     

Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging

Organophosphorus poisons (OP) bind covalently to the active-site serine of cholinesterases. The inhibited enzyme can usually be reactivated with powerful nucleophiles such as oximes. However, the covalently bound OP can undergo a suicide reaction (termed aging) yielding nonreactivatable enzyme. In human butyrylcholinesterase (hBChE), aging involves the residues His438 and Glu197 that are proximal to the active-site serine (Ser198). The mechanism of aging is known in detail for the nerve gases soman, sarin, and tabun as well as the pesticide metabolite isomalathion. Aging of soman- and sarin-inhibited acetylcholinesterase occurs by C-O bond cleavage, whereas that of tabun- and isomalathion-inhibited acetylcholinesterase occurs by P-N and P-S bond cleavage, respectively. In this work, the crystal structures of hBChE inhibited by the ophthalmic reagents echothiophate (nonaged and aged) and diisopropylfluorophosphate (aged) were solved and refined to 2.1, 2.25, and 2.2 A resolution, respectively. No appreciable shift in the position of the catalytic triad histidine was observed between the aged and nonaged conjugates of hBChE. This absence of shift contrasts with the aged and nonaged crystal structures of Torpedo californica acetylcholinesterase inhibited by the nerve agent VX. The nonaged hBChE structure shows one water molecule interacting with Glu197 and the catalytic triad histidine (His438). Interestingly, this water molecule is ideally positioned to promote aging by two mechanisms: breaking either a C-O bond or a P-O bond. Pesticides and certain stereoisomers of nerve agents are expected to undergo aging by breaking the P-O bond.     

Oxime K027: novel low-toxic candidate for the universal reactivator of nerve agent- and pesticide-inhibited acetylcholinesterase

Oxime K027 is a low-toxic bisquaternary compound originally developed as a reactivator of acetylcholinesterase (AChE) inhibited by nerve agents. The reactivation potency of K027 has been tested as a potential reactivator of AChE inhibited by tabun, sarin, cyclosarin, soman, VX, Russian VX, paraoxon, methylchlorpyrifos, and DDVP. The results show that oxime K027 reactivated AChE inhibited by almost all tested inhibitors to more than 10%, which is believed to be enough for saving the lives of intoxicated organisms. In the case of cyclosarin- and soman-inhibited AChE, oxime K027 did not reach sufficient reactivation potency.     

Choline derivatives and sodium fluoride protect acetylcholinesterase against irreversible inhibition and aging by DFP and paraoxon

A light addressable potentiometric sensor was used to measure acetylcholinesterase (AChE) activity in order to evaluate the protective effects of quaternary compounds and NaF against enzyme phosphorylation and aging by two organophosphates. The use of the immobilized AChE made possible the quick removal of reagents (i.e., organophosphate, 2-pralidoxime, and protectant), thereby permitting accurate determination of AChE activity before and after phosphorylation and aging. Paraoxon was 15-fold more potent in inhibiting AChE than DFP, while the percent aging following phosphorylation by diiso-propylfluorophosphate (DFP) was much higher. Sodium fluoride (NaF), the most effective protectant against phosphorylation and aging, and the quaternary ammonium compounds reduced significantly AChE inhibition by DFP and paraoxon, to similar degrees. Even though the percent AChE activity that was lost to aging was reduced by these agents, aging as a percent of phosphorylated AChE was not reduced. Thus, their major effect was in reducing the percent AChE phosphorylation, which consequently resulted in reduction of total aged AChE. The finding that quaternary ammonium compounds protect against phosphorylation is consonant with the proposed presence of the active site of AChE in an aromatic gorge.     

Structural changes of phenylalanine 338 and histidine 447 revealed by the crystal structures of tabun-inhibited murine acetylcholinesterase

Organophosphorus compounds (OPs) interfere with the catalytic mechanism of acetylcholinesterase (AChE) by rapidly phosphorylating the catalytic serine residue. The inhibited enzyme can at least partly be reactivated with nucleophilic reactivators such as oximes. The covalently attached OP conjugate may undergo further intramolecular dealkylation or deamidation reactions, a process termed "aging" that results in an enzyme considered completely resistant to reactivation. Of particular interest is the inhibition and aging reaction of the OP compound tabun since tabun conjugates display an extraordinary resistance toward most reactivators of today. To investigate the structural basis for this resistance, we determined the crystal structures of Mus musculus AChE (mAChE) inhibited by tabun prior to and after the aging reaction. The nonaged tabun conjugate induces a structural change of the side chain of His447 that uncouples the catalytic triad and positions the imidazole ring of His447 in a conformation where it may form a hydrogen bond to a water molecule. Moreover, an unexpected displacement of the side chain of Phe338 narrows the active site gorge. In the crystal structure of the aged tabun conjugate, the side chains of His447 and Phe338 are reversed to the conformation found in the apo structure of mAChE. A hydrogen bond between the imidazole ring of His447 and the ethoxy oxygen of the aged tabun conjugate stabilizes the side chain of His447. The displacement of the side chain of Phe338 into the active site gorge of the nonaged tabun conjugate may interfere with the accessibility of reactivators and thereby contribute to the high resistance of tabun conjugates toward reactivation.     

Structural study of the complex stereoselectivity of human butyrylcholinesterase for the neurotoxic V-agents

Nerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can be reactivated using oximes, but a spontaneous time-dependent process called aging alters the adduct, leading to resistance toward oxime reactivation. Human butyrylcholinesterase (BChE) functions as a bioscavenger, protecting the cholinergic system against OPs. The stereoselectivity of BChE is an important parameter for its efficiency at scavenging the most toxic OPs enantiomer for AChE. Crystals of BChE inhibited in solution or in cristallo with racemic V-agents (VX, Russian VX, and Chinese VX) systematically show the formation of the P(S) adduct. In this configuration, no catalysis of aging seems possible as confirmed by the three-dimensional structures of the three conjugates incubated over a period exceeding a week. Crystals of BChE soaked in optically pure VX(R)-(+) and VX(S)-(-) solutions lead to the formation of the P(S) and P(R) adduct, respectively. These structural data support an in-line phosphonylation mechanism. Additionally, they show that BChE reacts with VX(R)-(+) in the presence of racemic mixture of V-agents, at odds with earlier kinetic results showing a moderate higher inhibition rate for VX(S)-(-). These combined results suggest that the simultaneous presence of both enantiomers alters the enzyme stereoselectivity. In summary, the three-dimensional data show that BChE reacts preferentially with P(R) enantiomer of V-agents and does not age, in complete contrast to AChE, which is selectively inhibited by the P(S) enantiomer and ages.     

不同有机磷酸酯磷酰化乙酰胆碱酯酶活性中心的构象差异

通过观察2位肟化合物HI-6和HGG-42及它们的4位胎异构体对不同有机磷毒剂抑制的AChE的重活化作用发现塔崩、梭曼、沙林等有机磷毒剂磷酰化的AChE活性中心的构象可能存在着明显差异;又从变构剂C₁₀和丙吡啶对TMB₄重活化这几种毒剂磷酰化AChE的影响中证实塔崩磷酰化AChE活性中心构象与沙林、梭曼和VX3种毒剂磷酰化的AChE明显不同.     

Synthesis and in vitro evaluation of bis-quaternary 2-(hydroxyimino)-N-(pyridin-3-yl)acetamide derivatives as reactivators against sarin and VX inhibited human acetylcholinesterase (hAChE)

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 pK_a values of the synthesized compounds were found closer to the pK_a 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.     

New quaternary pyridine aldoximes as casual antidotes against nerve agents intoxications

In this work, the ability of four newly synthesized oximes--K005 (1,3-bis(2-hydroxyiminomethylpyridinium) propane dibromide), K027 (1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide), K033 (1,4-bis(2-hydroxyiminomethylpyridinium) butane dibromide) and K048 (1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide) to reactivate acetylcholinesterase (AChE, EC 3.1.1.7) inhibited by nerve agents is summarized. Reactivation potency of these compounds was tested using standard in vitro reactivation test. Tabun, sarin, cyclosarin and VX agent were used as appropriate testing nerve agents. Rat brain AChE was used as a source of the enzyme. Efficacies of new reactivators to reactivate tabun-, sarin-, cyclosarin- and VX-inhibited AChE were compared with the currently used AChE reactivators (pralidoxime, obidoxime and HI-6). Oxime K048 seems to be promising reactivator of tabun-inhibited AChE. Its reactivation potency is significantly higher than that of HI-6 and pralidoxime and comparable with the potency of obidoxime. The best reactivator of sarin-inhibited AChE seems to be oxime HI-6. None of the new AChE reactivators reached comparable reactivation potency. The same results were obtained for cyclosarin-inhibited AChE. However, oxime K033 is also potent reactivator of AChE inhibited by this nerve agent. In the case of VX inhibition, obidoxime and new oximes K027 and K048 seem to be the best AChE reactivators. None from the currently tested AChE reactivators is able to reactivate AChE inhibited by all nerve agents used and, therefore, the search for new potential broad spectrum AChE reactivators is needed.     

Refinement of structural leads for centrally acting oxime reactivators of phosphylated cholinesterases

We present a systematic structural optimization of uncharged but ionizable N-substituted 2-hydroxyiminoacetamido alkylamine reactivators of phosphylated human acetylcholinesterase (hAChE) intended to catalyze the hydrolysis of organophosphate (OP)-inhibited hAChE in the CNS. Starting with the initial lead oxime RS41A identified in our earlier study and extending to the azepine analog RS194B, reactivation rates for OP-hAChE conjugates formed by sarin, cyclosarin, VX, paraoxon, and tabun are enhanced severalfold in vitro. To analyze the mechanism of intrinsic reactivation of the OP-AChE conjugate and penetration of the blood-brain barrier, the pH dependence of the oxime and amine ionizing groups of the compounds and their nucleophilic potential were examined by UV-visible spectroscopy, (1)H NMR, and oximolysis rates for acetylthiocholine and phosphoester hydrolysis. Oximolysis rates were compared in solution and on AChE conjugates and analyzed in terms of the ionization states for reactivation of the OP-conjugated AChE. In addition, toxicity and pharmacokinetic studies in mice show significantly improved CNS penetration and retention for RS194B when compared with RS41A. The enhanced intrinsic reactivity against the OP-AChE target combined with favorable pharmacokinetic properties resulted in great improvement of antidotal properties of RS194B compared with RS41A and the standard peripherally active oxime, 2-pyridinealdoxime methiodide. Improvement was particularly noticeable when pretreatment of mice with RS194B before OP exposure was combined with RS194B reactivation therapy after the OP insult.     

Synthesis,in vitro screening and molecular docking of isoquinolinium-5-carbaldoximes as acetylcholinesterase and butyrylcholinesterase reactivators

Abstract

The series of symmetrical and unsymmetrical isoquinolinium-5-carbaldoximes was designed and prepared for cholinesterase reactivation purposes. The novel compounds were evaluated for intrinsic acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) inhibition, when the majority of novel compounds resulted with high inhibition of both enzymes and only weak inhibitors were selected for reactivation experiments on human AChE or BChE inhibited by sarin, VX, or paraoxon. The AChE reactivation for all used organophosphates was found negligible if compared to the reactivation ability of obidoxime. Importantly, two compounds were found to reactivate BChE inhibited by sarin or VX better to obidoxime at human attainable concentration. One compound resulted as better reactivator of NEMP (VX surrogate)-inhibited BChE than obidoxime. The in vitro results were further rationalized by molecular docking studies showing future directions on designing potent BChE reactivators.     

A comparison of the ability of a new bispyridinium oxime--1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane dibromide and currently used oximes to reactivate nerve agent-inhibited rat brain acetylcholinesterase by in vitro methods

The efficacy of a new bispyridinium oxime 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane dibromide, called K048, and currently used oximes (pralidoxime, obidoxime, the oxime HI-6) to reactivate acetylcholinesterase inhibited by various nerve agents (sarin, tabun, cyclosarin, VX) was tested by in vitro methods. The new oxime K048 was found to be a more efficacious reactivator of nerve agent-inhibited acetylcholinesterase than pralidoxime (in the case of VX, tabun and cyclosarin), obidoxime (cyclosarin and tabun) and HI-6 (tabun) but it did not reach the efficacy of currently used oximes for the reactivation of acetylcholinesterase inhibited by sarin. Thus, the oxime K048 seems to be a relatively efficacious broad spectrum acetylcholinesterase reactivator and, therefore, it could be useful for the treatment of a nerve agent-exposed population if information about detection of the type of nerve agent is not available.     

The role of the oximes HI-6 and HS-6 inside human acetylcholinesterase inhibited with nerve agents: a computational study

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.     

New group of xylene linker-containing acetylcholinesterase reactivators as antidotes against the nerve agent cyclosarin

Nerve agents such as sarin, cyclosarin and tabun are organophosphorus substances able to inhibit the enzyme acetylcholinesterase (AChE; EC 3.1.1.7). AChE reactivators and anticholinergics are generally used as antidotes in the case of intoxication with these agents. None of the known AChE reactivators is able to reactivate AChE inhibited by all nerve agents used. In this work, reactivation potency of nine newly developed AChE reactivators with an incorporated xylene ring in their structure was measured in vitro. Cyclosarin was chosen as an appropriate member of the nerve agent family. Reactivation potency of the tested AChE reactivators was compared with the gold standard of AChE reactivators--pralidoxime. Two oximes (K107 and K108) surpassed the reactivation potency of pralidoxime. Moreover, from the obtained results it could be deduced that AChE reactivators with a functional oxime group in position-2 are the most potent AChE reactivators in the case of cyclosarin intoxications.     

Reactivation of DFP- and paraoxon-inhibited acetylcholinesterases by pyridinium oximes

Exposure to the organophosphorus nerve agents such as sarin, soman, cyclosarin, and VX causes acute intoxication by inhibiting acetylcholinesterase (AChE), where the serine residue of the active site can attack the phosphorous atom of the organophosphorus agents to form a strong P–O bond. The purpose of the present study was to evaluate new oxime antidotes to reactivate the inhibited AChE. We have designed and synthesized several new oximes, and have evaluated the substances that differ from the currently used oximes in linker between the two pyridinium rings. The potency of newly synthesized oximes was compared with two currently used AChE reactivators (2-PAM, HI-6). The reactivation potencies of the bis-pyridinium oximes connected with a (CH₂)_n linker between the two quaternary nitrogen atoms were evaluated with housefly (HF) AChE inhibited by diisopropyl fluorophosphates (DFP) and by paraoxon. The bis-pyridinium oximes showed stronger activity compared with mono-pyridinium oxime, and the magnitude of reactivation potency depended on the length of the methylene linker. The potency order was (CH₂) < (CH₂)₂ < (CH₂)₃ > (CH₂)₄ > (CH₂)₇. A (CH₂)₃ linker was optimal in HF AChE inhibited by either DFP or paraoxon. Thus, bis-pyridinium oxime 5 which has (CH₂)₃ linker showed the highest activity in this series of compounds. Interestingly, 5 was not as active as 2-PAM, showing that the position of the oxime group on the pyridinium ring is also very important for the reactivation potency.     

Advantages of the WRAIR whole blood cholinesterase assay: comparative analysis to the micro-Ellman, Test-mate ChE, and Michel (DeltapH) assays

Red blood cell AChE (RBC-AChE) and plasma BChE can be used as sensitive biomarkers to detect exposure to OP nerve agents, pesticides, and cholinergic drugs. In a comparative study, RBC-AChE and serum BChE activities in whole blood was obtained from forty seven healthy male and female human volunteers, and then exposed separately ex vivo to three OP nerve agents (soman (GD), sarin (GB) and VX) to generate a wide range of inhibition of AChE and BChE activity (up to 90% of control). These samples were measured using four different ChE assays: (i) colorimetric microEllman (using DTNB at 412 nm), (ii) Test-mate ChE field kit (also based on the Ellman assay), (iii) Michel (delta pH), and (iv) the Walter Reed Army Institute of Research Whole Blood (WRAIR WB) cholinesterase assay. The WRAIR assay is a modified Ellman method using DTP at 324 nm (which minimizes hemoglobin interference and improves sensitivity), and determines AChE and BChE in a small whole blood sample simultaneously. Scatter plots of RBC-AChE activities were determined using the WRAIR ChE assay versus the micro-Ellman, Test-mateTM and Michel after exposure to varying concentrations of soman, sarin and VX. Regression analyses yielded mostly linear relationships with high correlations (r2 = 0.83-0.93) for RBC-AChE values in the WRAIR assay compared to the alternate methods. For the plasma BChE measurements, individual human values were significantly more variable (as expected), resulting in lower correlations using WRAIR ChE versus the alternate assays (r2 values 0.5 - 0.6). To circumvent the limitations of simple correlation analysis, Bland and Altman analysis for comparing two independent measurement techniques was performed. For example, a Bland and Altman plot of the ratio of the WRAIR whole blood AChE and Michel AChE (plotted on the y-axis) vs. the average of the two methods (x-axis) shows that the majority of the individual AChE values are within +/- 1.96 S.D. of the mean difference, indicating that the two methods may be used interchangeably with a high degree of confidence. The WRAIR ChE assay can be thus be used as a reliable inter-conversion assay when comparing results from laboratory-based (Michel) and field-based (Test-mateTM ChE kit), which use different methodology and report in different units of AChE activity.     

Efficient hydrolysis of the chemical warfare nerve agent tabun by recombinant and purified human and rabbit serum paraoxonase 1

Paraoxonase 1 (PON1) has been described as an efficient catalytic bioscavenger due to its ability to hydrolyze organophosphates (OPs) and chemical warfare nerve agents (CWNAs). It is the future most promising candidate as prophylactic medical countermeasure against highly toxic OPs and CWNAs. Most of the studies conducted so far have been focused on the hydrolyzing potential of PON1 against nerve agents, sarin, soman, and VX. Here, we investigated the hydrolysis of tabun by PON1 with the objective of comparing the hydrolysis potential of human and rabbit serum purified and recombinant human PON1. The hydrolysis potential of PON1 against tabun, sarin, and soman was evaluated by using an acetylcholinesterase (AChE) back-titration Ellman method. Efficient hydrolysis of tabun (100 nM) was observed with ∼25-40 mU of PON1, while higher concentration (80-250 mU) of the enzyme was required for the complete hydrolysis of sarin (11 nM) and soman (3 nM). Our data indicate that tabun hydrolysis with PON1 was ∼30-60 times and ∼200-260 times more efficient than that with sarin and soman, respectively. Moreover, the catalytic activity of PON1 varies from source to source, which also reflects their efficiency of hydrolyzing different types of nerve agents. Thus, efficient hydrolysis of tabun by PON1 suggests its promising potential as a prophylactic treatment against tabun exposure.