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Current medical countermeasures (MCMs) for nerve agent poisoning have limited efficacy, and can cause serious adverse effects, prompting the requirement for new broad-spectrum therapeutics. Human plasma-derived butyrylcholinseterase (huBChE) is a promising novel bioscavenger MCM which has shown potential in animal studies, however, is economically prohibitive to manufacture at scale. This study addresses current challenges for the economical production of a bioactive and long-acting recombinant huBChE (rBChE) in mammalian cells by being the first to directly compare novel rBChE design strategies. These include co-expression of a proline rich attachment domain (PRAD) and fusion of BChE with a protein partner. Additionally, a pre-purification screening method developed in this study enables parallel comparison of the expression efficiency, activity and broad-spectrum binding to nerve agents for ten novel rBChE molecular designs. All designed rBChE demonstrated functionality to act as broad-spectrum MCMs to G, V and A series nerve agents. Expression using the ExpiCHO™ Max protocol provided greatest expression levels and activity for all constructs, with most rBChE expressing poorly in Expi293™. Fc- or hSA-fused rBChE significantly outperformed constructs designed to mimic huBChE, including PRAD-BChE, and proved an effective strategy to significantly improve enzyme activity and expression. Choice of protein partner, directionality and the addition of a linker also impacted fusion rBChE activity and expression. Overall, hSA fused rBChE provided greatest expression yield and activity, with BChE-hSA the best performing construct. The purified and characterised BChE-hSA demonstrated similar functionality to huBChE to be inhibited by GD, VX and A-234, supporting the findings of the pre-screening study and validating its capacity to assess and streamline the selection process for rBChE constructs in a cost-effective manner. Collectively, these outcomes contribute to risk mitigation in early-stage development, providing a systematic method to compare rBChE designs and a focus for future development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Given the promising prospect of aggregation-induced emission luminogens (AIEgens) in fluorescence assays, it is interesting and significant to endow AIEgens with molecular recognition capability (such as enzyme-like activity). Here, an AIE nanomaterial with intrinsic enzyme-like activity (named as "AIEzyme") is designed and synthesized via a facile coordination polymerization of Zr 4+ and AIE ligands. AIEzyme possesses enhanced and stable fluorescence in different solvents because of the AIE effect of ligands in the rigid structure of a coordination polymer. On the other hand, the organophosphorus hydrolase (OPH)-mimicking activity of AIEzyme exhibits excellent affinity and specific activity. Interestingly, the OPH-like activity can quench the inherent fluorescence of AIEzyme by the hydrolysate of a typical organophosphorus nerve agent (OPNA), diethyl-4-nitrophenylphosphate. Due to the sensitive activity-induced quenching effect for AIE, the self-reporting fluorescence assay method based on AIEzyme was established, which shows ultrahigh sensitivity, high selectivity, good storage stability, and acceptable reliability for a real sample assay. Moreover, the simultaneous colorimetric method broadens the detection range and the application scenarios. The proposed assay method avoided the interference of O 2 during detection because the OPH-like activity does not derive from the generation of ROS. As a bonus, AIEzyme can also be used for the degradation of OPNAs by OPH-like activity, and the process can be self-monitored by AIE quenching. This work would provide a new opportunity for expanding the application of AIEgens and artificial enzymes by endowing AIEgens with enzyme-like activity.

The biocatalyzed oxidative detoxification of the V-series simulant PhX, by mean of the microperoxidase AcMP11, affords the corresponding phosphonothioate as the prominent product instead of the classical P-S and P-O bond cleavage. While PhX is structurally very close to the live agent VX (the methyl group is replaced by a phenyl), assessment with other surrogates missing the nucleophilic amino function displayed more resistance under the same conditions with no phosphonothioate observed. These encouraging results highlight 1) the efficacy of AcMP11 microperoxidase to efficiently detoxify V-series organophosphorus nerve agents (OPNA), and 2) the necessity to use representative alkyl or aryl phosphonothioates simulants such as PhX bearing the appropriate side chain as well as the P-O and P-S cleavable bond to mimic accurately the V-series OPNA to prevent false positive or false negative results., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

VX, a well-known organophosphorus nerve agent (OPNA), poses a significant threat to public safety if employed by terrorists. Obtaining complete metabolites is critical to unequivocally confirm its alleged use/exposure and elucidate its whole-molecular metabolism. However, the nitrogenous VX metabolites containing 2-diisopropylaminoethyl moiety from urinary excretion remain unknown. Therefore, this study applied a newly developed untargeted workflow platform to discover and identify them using VX-exposed guinea pigs as animal models. 2-(N,N-diisopropylamino)ethanesulfonic acid (DiPSA) was revealed as a novel nitrogenous VX metabolite in urine, and 2-(Diisopropylaminoethyl) methyl sulfide (DAEMS) was confirmed as another in plasma, indicating that VX metabolism differed between urine and plasma. It is the first report of a nitrogenous VX metabolite in urine and a complete elucidation of the VX metabolic pathway. DiPSA was evaluated as an excellent VX exposure biomarker. The whole-molecule VX metabolism in urine was characterized entirely for the first time via the simultaneous quantification of DiPSA and two known P-based biomarkers. About 52.1% and 32.4% of VX were excreted in urine as P-based and nitrogenous biomarkers within 24 h. These findings provide valuable insights into the unambiguous detection of OPNA exposure/intoxication and human and environmental exposure risk assessment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nerve agents pose significant threats to civilian and military populations. The reactivation of acetylcholinesterase (AChE) is critical in treating acute poisoning, but there is still lacking broad-spectrum reactivators, which presents a big challenge. Therefore, insights gained from the reactivation kinetic analysis and molecular docking are essential for understanding the behavior of reactivators towards intoxicated AChE. In this research, we present a systematic determination of the reactivation kinetics of three V agents-inhibited four human ChEs [(AChE and butyrylcholinesterase (BChE)) from either native or recombinant resources, namely, red blood cell (RBC) AChE, rhAChE, hBChE, rhBChE) reactivated by five standard oximes. We unveiled the effect of native and recombinant ChEs on the reactivation kinetics of V agents ex vitro, where the reactivation kinetics characteristic of Vs-inhibited BChE was reported for the first time. In terms of the inhibition type, all of the five oxime reactivators exhibited noncompetitive inhibition. The inhibition potency of these reactivators would not lead to the difference in the reactivation kinetics between native and recombinant ChE. Despite the significant differences between the native and recombinant ChEs observed in the inhibition, aging, and spontaneous reactivation kinetics, the reactivation kinetics of V agent-inhibited ChEs by oximes were less differentiated, which were supported by the ligand docking results. We also found differences in the reactivation efficiency between five reactivators and the phosphorylated enzyme, and molecular dynamic simulations can further explain from the perspectives of conformational stability, hydrogen bonding, binding free energies, and amino acid contributions. By Poisson-Boltzmann surface area (MM-PBSA) calculations, the total binding free energy trends aligned well with the experimental k r2 values., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We herein present for the first time a micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS) procedure to detect phosphonylated tyrosine (Tyr) and lysine (Lys) residues obtained from human hair exposed to organophosphorus nerve agents (OPNA). In general, toxic OPNA react with endogenous blood proteins causing the formation of adducts representing well-known targets for biomedical analysis to prove exposure. In contrast, no protein-derived biomarker has been introduced so far to document local exposure of hair. Accordingly, we developed and characterized a μLC-ESI MS/HR MS method for the analysis of scalp hair exposed to OPNA in vitro. Type I and Type II keratin from hair was dissolved during lysis, precipitated and subjected to pronase-catalyzed hydrolysis yielding single adducted Lys and in a much higher amount Tyr residues. Exposure to sarin caused the adduction of an isopropyl methylphosphonic acid moiety and exposure to VX yielded adducts of ethyl methylphosphonic acid, well suited as biomarkers of exposure. These were of appropriate stability in the autosampler for 24 h. The biomarker yield obtained from hair of six individuals as well as from hair of six different parts of the body of one individual (armpit, beard, leg, arm, scalp, and pubic) differed reasonably indicating the variable individual protein composition and structure of hair. Exposed hair stored at ambient temperature for 9 weeks with contact to air and daylight showed stability of all adducts and therefore their suitability for verification of exposure., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Organophosphorus nerve agents (OPNAs) that damage the central nervous system by inhibiting acetylcholinesterase activity, pose severe threats to human health and life security. Reliable biomarkers that quickly and accurately detect OPNAs exposure are urgently needed to help diagnose quickly and treat in time. Albumins that covalently bind to OPNAs could serve as important targets for retrospective verification of OPNAs exposure. The goal of this study is to explore the potential biomarkers in albumins with high reactivity and good stability and expand the group of potential biomarkers in different species for detecting the exposure of V-type OPNAs including O-ethyl S-(2-(diisopropylamino)ethyl) methylphosphonothioate (VX), O-isobutyl S-(2(diethylamino)ethyl) methylphosphonothioate (VR), and O-butyl S-(2-(diethylamino)ethyl) methylphosphonothioate (Vs). Taking human serum albumin (HSA), bovine serum albumin (BSA) and rabbit serum albumin (RSA) as the research objectives, multiple active sites including phosphonylation and disulfide adduct sites were observed in albumins from different species. Numerous phosphonylation sites labeled by all agents in one type of albumin were found. Among the different species, four shared phosphonylation sites with high reactivity include K499, K549, K249, and Y108. In addition, Y108 on ETY*GEMADCCAK, Y287 on Y*ICENQDSISSK, Y377 on TY*ETTLEK and Y164 on YLY*EIAR in HSA were stably phosphonylated by all agents in gradient concentration, making them stable and suitable potential biomarkers for V-type OPNAs exposure. Notably, Y108 on ETY*GEMADCCAK in HSA, on DTY*GDVADCCEK in RSA, and on ETY*GDMADCCEK in BSA were highly reactive to all V-type agents, regardless of species. It was also successfully labeled in HSA exposed to class V agents in gradient concentration. Y108 is expected to be used to screen and identify the exposure of V-type agents in the retrospective research. Disulfide adducts sites, consisted of four sites in HSA and two sites in BSA were also successfully labeled by V-type agents, and characteristic ion fragments from these disulfide adducts were also identified by secondary mass spectrometry. Molecular simulation of the stably modified sites were conducted to discover the promoting factors of covalent adduct formation, which help further clarify formation mechanism of albumin adducts at active sites., (© 2022. The Author(s).)

Cascade catalysis that combines chemical catalysis and biocatalysis has received extensive attention in recent years, especially the integration of metal nanoparticles (MNPs) with enzymes. However, the compatibility between MNPs and enzymes, and the stability of the integrated nanocatalyst should be improved to promote the application. Therefore, in this study, we proposed a strategy to space-separately co-immobilize MNPs and enzymes to the pores and surface of a highly stable covalent organic framework (COF), respectively. Typically, Pd NPs that were prepared by in situ reduction with triazinyl as the nucleation site were distributed in COF (Tz-Da), and organophosphorus hydrolase (OPH) was immobilized on the surface of Tz-Da by a covalent method to improve its stability. The obtained integrated nanocatalyst Pd@Tz-Da@OPH showed high catalytic efficiency and reusability in the cascade degradation of organophosphate nerve agents. Furthermore, the versatility of the preparation strategy of COF-based integrated nanocatalyst has been preliminarily expanded: (1) Pd NPs and OPH were immobilized in the triazinyl COF (TTB-DHBD) with different pore sizes for cascade degradation of organophosphate nerve agent and the particle size of MNPs can be regulated. (2) Pt NPs and glucose oxidase were immobilized in COF (Tz-Da) to obtain an integrated nanocatalyst for efficient colorimetric detection of phenol.

Novichok (NV) nerve agents were recently added to the list of Schedule 1 chemicals of the Chemical Weapons Convention. There is a well-accepted method for assessment of nerve agent exposure based on mass spectrometric analysis of a nonapeptide with the serine-198 residue modified by the nerve agent, but this approach has not yet been reported for the class of NV agents and requires the availability of reference standards, which may be a limitation for NV agent exposure assessment. Thus, a goal of this study was to first verify the utility of the nonapeptide method for the characterization of human plasma samples exposed in vitro to the NV agents A-230, A-232, and A-234. A second aim was to evaluate the possibility of identifying unknown exposures by applying precursor ion scanning in combination with high resolution mass spectrometry (HRMS). Thus, precursor ion scanning, with a generic fragment ion ( m / z 778) of the nonapeptide, was used to pinpoint any modified nonapeptide, while HRMS was used for structural elucidation of the adduct moiety. By this approach, use of HRMS enabled differentiation between adducts of agents with similar molecular masses. A new unique feature that could be exploited for NV nonapeptide analysis was that the modification was released from the peptide during fragmentation in the mass spectrometer and was detected in the low-mass region of the mass spectrum. This low-mass region was extremely informative and contributed to the assignment of the structure of the particular agent used, which is especially important in case no reference materials are available. The presented method is important for verification purposes by the Organisation for Prohibition of Chemical Weapons (OPCW), e.g., in case of investigations of alleged use of NV agents, and for regular forensic investigations.

Organophosphorus (OP) compounds are typically a broad class of compounds that possess various uses such as insecticides, pesticides, etc. One of the most evil utilizations of these compounds is as chemical warfare agents, which pose a greater threat than biological weapons because of their ease of access. OP compounds are highly toxic compounds that cause irreversible inhibition of enzyme acetylcholinesterase, which is essential for hydrolysis of neurotransmitter acetylcholine, leading to series of neurological disorders and even death. Due to the extensive use of these organophosphorus compounds in agriculture, there is an increase in the environmental burden of these toxic chemicals, with severe environmental consequences. Hence, the rapid and sensitive, selective, real-time detection of OP compounds is very much required in terms of environmental protection, health, and survival. Several techniques have been developed over a few decades to easily detect them, but still, numerous challenges and problems remain to be solved. Major advancement has been observed in the development of sensors using the spectroscopic technique over recent years because of the advantages offered over other techniques, which we focus on in the presented review., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

In analogy to the fluoride-induced regeneration of butyrylcholinesterase (BChE) inhibited by nerve agents a method was developed and optimized for whole blood samples. Compared to the plasma method, regeneration grade was found to be higher for cyclosarin (GF), i-butylsarin from VR, and n-butylsarin from CVX, but lower for sarin (GB), fluorotabun from tabun (GA), and ethylsarin from VX. Regeneration grade of soman (GD) is the same for both matrices because it is released from serum albumin and not from cholinesterases. The method was fully validated for GB and GF to prove selectivity, linearity (n = 6), limit of determination (LOD1), reproducibility (within day (n = 8) and from day to day (n = 8)), effectiveness of extraction, matrix effect, and sample stability (after sample preparation and during three freeze/thaw cycles). The other agents were tested for selectivity, linearity (n = 2), limit of determination, and stability after sample preparation. The method showed high selectivity, good linearity up to the protein's saturation concentration (GB: R 2  = 0.9995, GF: 0.9968), and high reproducibility (GB: C.V. 5.9-13.7%, GF: 4.9-10.3%). The limits of determination (calculated from the spiked amount of the original agent) were found with 0.3 ng/mL VX, 0.5 ng/mL GB, 1 ng/mL VR, 0.5 ng/mL GA, 1 ng/mL CVX, and 8 ng/mL GD. In the case of GF, it was found with 4 ng/mL using Isolute ENV + SPE cartridges as for the other analytes and with 2.5 ng/mL using Isolute C8 EC SPE cartridges instead. This method was then applied to a denatured whole blood sample obtained from an individual exposed to GB. While previously only the GB metabolite isopropyl methylphosphonic acid (IMPA) could be detected in this blood sample it was now possible to successfully release GB from the blood proteins by excess fluoride., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Highly toxic organophosphorus nerve agents, especially the extremely stable and persistent V-type agents such as VX, still pose a threat to the human population and require effective medical countermeasures. Engineered mutants of the Brevundimonas diminuta phosphotriesterase (BdPTE) exhibit enhanced catalytic activities and have demonstrated detoxification in animal models, however, substrate specificity and fast plasma clearance limit their medical applicability. To allow better assessment of their substrate profiles, we have thoroughly investigated the catalytic efficacies of five BdPTE mutants with 17 different nerve agents using an AChE inhibition assay. In addition, we studied one BdPTE version that was fused with structurally disordered PAS polypeptides to enable delayed plasma clearance and one bispecific BdPTE with broadened substrate spectrum composed of two functionally distinct subunits connected by a PAS linker. Measured k cat /K M values were as high as 6.5 and 1.5 × 10 8  M -1  min -1 with G- and V-agents, respectively. Furthermore, the stereoselective degradation of VX enantiomers by the PASylated BdPTE-4 and the bispecific BdPTE-7 were investigated by chiral LC-MS/MS, resulting in a several fold faster hydrolysis of the more toxic P(-) VX stereoisomer compared to P(+) VX. In conclusion, the newly developed enzymes BdPTE-4 and BdPTE-7 have shown high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(-) VX enantiomer in vitro and offer promise for use as bioscavengers in vivo., (© 2021. The Author(s).)

Intramolecular charge transfer (ICT) is a fundamental mechanism that enables the development of numerous fluorophores and probes for bioimaging and sensing. However, the electron-withdrawing targets (EWTs)-induced fluorescence quenching is a long-standing and unsolved issue in ICT fluorophores, and significantly limits the widespread applicability. Here we report a simple and generalizable structural-modification for completely overturning the intramolecular rotation driving energy, and thus fully reversing the ICT fluorophores' quenching mode into light-up mode. Specifically, the insertion of an indazole unit into ICT scaffold can fully amplify the intramolecular rotation in donor-indazole-π-acceptor fluorophores (fluorescence OFF), whereas efficiently suppressing the rotation in their EWT-substituted system (fluorescence ON). This molecular strategy is generalizable, yielding a palette of chromophores with fluorescence umpolung that spans visible and near-infrared range. This strategy expands the bio-analytical toolboxes and allows exploiting ICT fluorophores for light-up sensing of EWTs including N-acetyltransferases and nerve agents.

Metal-organic frameworks (MOFs) have shown promising properties for removal of chemical warfare agents, in particular for material decontamination and functionalized fabrics. The MOF-properties could also be beneficial for skin decontamination, especially when exposed to highly toxic and low volatile nerve agents. In such exposures, efficient decontamination is crucial for adequate medical management. In the present study, seven zirconium-based MOFs were evaluated for their ability to degrade VX and subsequently tested in vitro for decontamination of VX on human dermatomed skin. Of the MOFs evaluated, MOF-808 showed the greatest ability to degrade VX in an alkaline buffer with complete degradation of VX within 5 min. PCN-777, Zr-NDC and NU-1000 displayed degradation half-lives of approximately 10 min. When including MOF-808 in a skin friendly carrier with slightly acidic pH, a decreased agent degradation rate was observed, requiring over 24 h to reach complete degradation. In skin decontamination experiments, MOF-808 enhanced the efficacy compared to the carrier alone, essentially by improved agent absorption. Adding MOF-808 to Reactive Skin Decontamination Lotion (RSDL) did not improve the high effectiveness of RSDL alone. The present study showed that including MOF in skin decontamination lotions could be beneficial. Further studies should include optimizing the particulates and formulations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We present a simple method for chiral separation and analysis of organophosphorus nerve agents and apply it to monitor the enantioselective blood elimination kinetics of sarin in-vitro. The method is implemented in standard reverse phase LC-MS operating conditions, relieving the user of the dedicated operating conditions frequently demanded in chiral LC-MS analysis. The method consists of formation of diastereomers by a rapid derivatization with (R)-2-(1 aminoethyl) phenol, followed by LC-MS/MS analysis. Derivatization enantioselectivity was studied by comparing the reaction of optically pure sarin and racemic sarin, proving no substantial enantiomeric preference in the reaction and demonstrating the enantiomeric discrimination abilities of the technique. Enantioselective sarin elimination pathways were probed in-vitro by following the fast elimination kinetics of the two sarin enantiomers as well as its hydrolysis metabolite (isopropyl methyl-phosphonic acid, IMPA) in whole blood and plasma compared to water. Sarin enantiomers showed the known marked differences in elimination kinetics with rapid elimination of the (+) enantiomer and slower elimination of the (-) enantiomer in whole blood and plasma as well as dose-dependent kinetics (faster elimination at lower concentrations). We found that small amounts of acetonitrile in plasma prevent the rapid elimination of the (+) enantiomer, resulting in similar, slower elimination kinetics for both enantiomers.

Highly toxic organophosphorous nerve agents (OPAs) have been used in several armed conflicts and terror attacks in the last few decades. A new method for retrospective determination of alkyl methylphosphonic acid (AMPA) metabolites in urine after exposure to VX, GB and GF nerve agents was developed. This method enables a rapid, sensitive and selective determination of trace levels of the nerve agent biomarkers ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA) in urine. The new technique involves a unique combination of two solid phase extraction (SPE) cartridges: a Ba/Ag/H cartridge for urine interference removal, and a ZrO 2 cartridge for selective reconstitution and enrichment of the AMPAs. Extraction of AMPAs from the ZrO 2 cartridge was accomplished with a 1% ammonium hydroxide (NH 4 OH) solution and was followed by analysis via liquid chromatography-mass spectrometry (LC-MS). The limits of quantitation (LOQs) were in the range of 10-100 pg/mL with recoveries of 64-71% (± 5-19%) after fast sample preparation and a total LC-MS analysis cycle time of 15 min and 13 min, respectively. This method was successfully applied in vivo in a rabbit that was exposed to 0.5 LD 50 (7.5 µg/kg, i.v.) sarin for retrospective monitoring of the IMPA metabolite in urine. For the first time, IMPA was determined in rabbit urine samples for 15 days post-exposure, which is longer than any reported post-exposure method for AMPAs. To the best of our knowledge, this new method is the most sensitive and rapid for AMPA determination in urine by LC-MS/MS analysis.

Early initiated decontamination is demonstrated to be crucial to avoid systemic effects of highly toxic and low volatile agents exposed on the skin. Skin decontamination can be performed by simple procedures, such as washing with soap and water, or by using advanced decontamination products containing absorption and agent degradation properties. Reactive Skin Decontamination Lotion (RSDL) has demonstrated high efficacy to remove nerve agents from the skin. However, contrary to the current operational recommendations, experimental studies have shown that prolonged skin contact time of RSDL is important for efficient decontamination of VX. In the present study, several RSDL-protocols were evaluated for the efficacy to remove neat VX from human skin in vitro. The decontamination efficacies of the RSDL-procedures were compared with the efficacy of the simple procedure of washing off the skin with soapy water. The RSDL-protocols containing repeated swabbing with the sponge and a 10 min skin contact time of RSDL-lotion demonstrated the greatest decontamination efficacy of all procedures evaluated. Repeating the protocol 2 h after the initial decontamination step resulted in a transient increased skin penetration of remaining intact agent on skin and was followed by rapidly declined agent penetration rate. Decontamination performed with soapy water significantly increased agent amounts penetrating skin, most likely caused by skin hydration and agent dilution. In conclusion, a slightly extended procedure for RSDL-decontamination showed improved efficacy and is therefore recommended for removal of nerve agents from the skin. In addition, it is of highest importance that skin decontamination of nerve agents should consist of procedures using low water content., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Kinetic enhancement of organophosphate hydrolysis is a long-standing challenge in catalysis. For prophylactic treatment against organophosphate exposure, enzymatic hydrolysis needs to occur at high rates in the presence of low substrate concentrations and enzymatic activity should persist over days and weeks. Here, the conjugation of small DNA scaffolds was used to introduce substrate binding sites with micromolar affinity to VX, paraoxon, and methyl-parathion in close proximity to the enzyme phosphotriesterase (PTE). The result was a decrease in K M and increase in the rate at low substrate concentrations. An optimized system for paraoxon hydrolysis decreased K M by 11-fold, with a corresponding increase in second-order rate constant. The initial rates of VX and methyl-parathion hydrolysis were also increased by 3.1- and 6.7-fold, respectively. The designed scaffolds not only increased the local substrate concentration, but they also resulted in increased stability and PTE-DNA particle size tuning between 25 and ~150 nm. The scaffold engineering approach taken here is focused on altering the local chemical and physical microenvironment around the enzyme and is therefore compatible with active site engineering via combinatorial and computational approaches., (© 2020 Wiley Periodicals, Inc.)

The molecular toxicology of the chemical warfare agent sulfur mustard (SM) is still not completely understood. It has been suggested that in addition to SM itself also biotransformation products thereof mediate cytotoxicity. In the current study, we assessed this aspect by exposing a human hepatocyte cell line (HepG2) to SM or to its oxidation products sulfur mustard sulfoxide (SMO), sulfur mustard sulfone (SMO 2 ), and divinyl sulfone (DVS). Cytotoxicity, determined with the XTT assay, revealed a significant higher toxicity of SMO 2 and DVS compared to SM while SMO had no effect at any concentration. The exact biotransformation of SM leading to SMO, SMO 2 and finally DVS is unknown so far. Involvement of the CYP450 system is discussed and was also investigated in the presented study. Modulation of CYP1A2 activity, taken as a model enzyme for CYP450, affected cytotoxicity of SM, SMO 2 or DVS significantly. Induction of CYP1A2 with omeprazole led to decreased cytotoxicity for all compounds whereas inhibition with cimetidine resulted in an increased cytotoxicity for SM, but not for SMO 2 and DVS. Our results indicate a distinctive role of the CYP450 system in SM poisoning. Future studies should address the metabolic conversion of SM in more detail. Our data may suggest the well-tolerated drug omeprazole as a potential co-treatment after contact to SM., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Inhibitors of cholinesterases are a wide group of low molecular weight compounds with a significant role in the current pharmacology. Besides the pharmacological importance, they are also known as toxic compounds like military nerve agents. In the pharmacology, drugs for Alzheimer disease, myasthenia gravis and prophylaxis of poisoning by nerve agents can be mentioned as the relevant applications. Besides this, anti-inflammation and antiphrastic drugs are other pharmacological applications of these inhibitors. This review is focused on a survey of cholinesterase inhibitors with known or expected pharmacological impact and indications of their use. Recent literature with comments is provided here as well., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Nerve agents are organophosphates (OPs) that potently inhibit acetylcholinesterase, and their enzymatic detoxification has been a long-standing goal. Nerve agents vary widely in size, charge, hydrophobicity and the cleavable ester bond. A single enzyme is therefore unlikely to efficiently hydrolyze all agents. Here, we describe a mixture of three previously developed variants of the bacterial phosphotriesterase (Bd-PTE) that are highly stable and nearly sequence identical. This mixture enables effective detoxification of a broad spectrum of known threat agents-GA (tabun), GB (sarin), GD (soman), GF (cyclosarin), VX and Russian-VX. The potential for dimer dissociation and exchange that could inactivate Bd-PTE has minimal impact, and the three enzyme variants are as active in a mixture as they are individually. To our knowledge, this engineered enzyme 'cocktail' comprises the first solution for enzymatic detoxification of the entire range of threat nerve agents., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Nerve agent metabolites (NAMs) derived from alkyl methyl phosphonic acids, such as ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA), and pinacolyl methylphosphonic acid (PMPA), were extracted from human urine using diethyl ether as an extractant. After exchanging the diethyl ether solvent to acetonitrile, the analytes were derivatized with 2-(bromomethyl)naphthalene (BMN). The reaction products of the BMN and NAMs, i.e., MN-EMPA, MN-IMPA, and MN-PMPA, were separated by gas chromatography (GC) and measured by mass spectrometry (MS) using a femtosecond laser emitting at 267 nm as the ionization source for resonance-enhanced two-photon ionization (RE2PI). The limits of detection (LOD) were <1 ng/mL for these analytes. The use of BMN increased the volatility of the analytes for separation by GC and also increased the ionization efficiency via the RE2PI process as the result of presence of a naphthalene functional group. A two-dimensional GC-MS display can be used for comprehensive analysis of NAMs, by-products, and impurities in the sample. Then, this approach could be used to confirm the use of chemical weapons and for forensic identification., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Studies with oximes have been extensively developed to design new reactivators with better efficiency, and greater spectrum of action. In this study, we aimed to analyze the influence of the Carbamoyl group position change in two isomeric oximes, K203 and K206, on the reactivation percentage of Mus musculus Acetylcholinesterase (MmAChE), inhibited by different nerve agents. Theoretical calculations were performed to assess the difference for the oxime activity with inhibited AChE-complexes and the factors that govern this difference. Comparing theoretical and experimental data, it is possible to observe that this change between the oximes results in different reactivation percentage for the same nerve agent, due to the different interaction modes and activation energy for the studied systems., (Copyright © 2019. Published by Elsevier B.V.)

Organophosphorus compounds have been widely employed to the development of warfare nerve agents and pesticides, resulting in a huge number of people intoxicated annually, being a serious problem of public health. Efforts worldwide have been done in order to design new technologies that are capable of combating or even reversing the poisoning caused by these OP nerve agents. In this line, the bioremediation arises as a promising and efficient alternative for this purpose. As an example of degrading enzymes, there is the organophosphate-degrading (OpdA) enzyme from Agrobacterium radiobacter, which has been quite investigated experimentally due to its high performance in the degradation of neurotoxic nerve agents. This work aims to look into the structural and electronic details that govern the interaction modes of these compounds in the OpdA active site, with the posterior hydrolysis reaction prediction. Our findings have brought about data about the OpdA performance towards different nerve agents, and among them, we may realize that the degradation efficiency strongly depends on the nerve agent structure and its stereochemistry, being in this case the compound Tabun the one more effectively hydrolyzed. By means of the chemical bonds (AIM) and orbitals (FERMO) analysis, it is suggested that the initial reactivity of the OP nerve agents in the OpdA active site does not necessarily dictate the reactivity and interaction modes over the reaction coordinate., (Copyright © 2019. Published by Elsevier B.V.)

In March 2018 the term Novichok (Hoвичoκ) became publically known following an attempted murder of a former Russian spy in Salisbury, UK. Novichok is the name of a group of nerve agents secretly produced by Russia in the later stages of the Cold War. These compounds were never declared under the Chemical Weapons Convention and very little is known about the actual identity and characteristics of these compounds. Structures of some of the Novichoks have been reported by a former Russian chemist, Vil Mirzayanov, previously working at the Russian State Scientific Research Institute of Organic Chemistry and Technology (GOSNIIOKhT). It was in this context claimed that at least two compounds of the Novichok family, known as Novichok-5 and Novichok-7 were 5-8 times more potent than the hitherto most toxic nerve agent, VX. The present study elucidates, applying a series of QSAR models toxicity, skin permeation, pharmacokinetic aspects as well as the environmental fate of a series of Novichoks. Virtually the results from the different studies related to human health point in the same direction, i. e., the Novichoks are significantly less toxic than VX and the skin permeation much lower and less efficient than observed for VX. Hence, the claim by Mirzayanov could not be substantiated., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this research an accumulation of nerve agent markers in garden cress (Lepidium sativum) as a model plant object was studied using LC-QTOF hybrid system. For the determination of methylphosphonic acid and alkyl methylphosphonates, which are specific markers of sarin, soman, VR and VX, simple and sensitive approach was developed. Direct analysis of aqueous extracts on the reversed phase column with polar endcapping allowed to achieve satisfactory retention factor for methylphosphonic acid, which has high polarity and is usually very weakly retained on the ordinary reversed phase columns. Application of the QTOF mass spectrometer with high mass resolution led to the increase in the accuracy of the conducted measurements. The HPLC-HRMS technique developed exclusively for this study has been validated for linearity, limit of detection, limit of quantification, precision, accuracy and matrix effect prior to the analysis of plant extract samples. Hydroponic growth model was employed to examine accumulation of nerve agent markers in garden cress. It was found that after elimination of nerve agent markers from the plant growth medium, garden cress was able to store these substances for at least 5 weeks providing high retrospectivity of the analysis. Moreover, during the cress growth, no metabolization of alkyl methylphosphonates was observed. This allows not only to reveal the fact of nerve agents release into environment, but also to define its type after a long period of time., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The effective detoxification of chemical warfare agents, specifically nerve agents, is a pressing issue in the modern world. Due to the high toxicity of these molecules, simulants are often used in experiments as substitutes for the agents. However, there is little reason to believe that the current simulants used in the literature are optimal predictors of nerve agent reactivity. Density functional theory calculations were performed on the alkaline hydrolysis of over 100 organophosphate molecules to identify improved simulants for the G-series nerve agents soman and sarin, based on low toxicity and similarity to nerve agent hydrolysis energetics and degradation mechanism. This screening highlighted 5 molecules that have nearly identical reaction barriers to the actual agents, while being far less toxic. Quantitative structure-activity relationship (QSAR) models were also derived to determine the most significant molecular descriptors for describing the hydrolysis free energy barriers of these reactions. The optimal QSAR model was subjected to a thorough statistical analysis and validation procedure to confirm its predictive capacity, showing excellent quantitative and ranking accuracy. It was further shown that the model trained on G-series agents can reliably predict energetics for other organophosphate classes as well, including VX. Through these computational insights, experimentalists may be aided in accurately and safely studying these reactions with less toxic simulants., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A major challenge in organophosphate compound (OP) and OP nerve agent (OPNA) research has been in the identification and utilization of reliable biomarkers for rapid, sensitive, and efficient detection of OP exposure. Albumin has been widely studied as a biomarker for retrospective verification of exposure to OPNAs, including soman (GD), by detecting the phosphonylation of specific amino acid residues. The aim of the present study was to identify binding sites between GD and rabbit serum albumin in vitro and in vivo. A nano-liquid chromatography coupled with a quadrupole-orbitrap mass spectrometry (nLC-Q-Orbitrap-MS) was used to examine the GD-modified adducts of rabbit albumin. A total of 11 GD-modified sites were found in rabbit serum albumin across three experimental models. The following five GD-modified rabbit albumin sites, which were all lysine residues, were established in vivo: K188, K329, K162, K233, and K525. Two of these five lysine residues, K188 in peptide EK*ALISAAQER and K162 in peptide YK*AILTECCEAADK, were stable for at least 7 days in vivo. Molecular simulation of the GD-albumin interaction provided theoretical evidence for reactivity of the identified lysine residues. The findings suggest that these modifiable lysine residues are potential biomarkers of GD exposure for retrospective analysis by Q-Orbitrap-MS.

Bispyridinium oximes with one (K865, K866, K867) or two (K868, K869, K870) ortho-positioned chlorine moiety, analogous to previously known K027, K048 and K203 oximes, and potent reactivators of human acetylcholinesterase (AChE) inhibited by nerve agents, were tested in the reactivation of human butyrylcholinesterase (BChE) inhibited by sarin, cyclosarin, VX, and tabun. A previously highlighted AChE reactivator, dichlorinated bispyridinium oxime with propyl linker (K868), was tested in more detail for reactivation of four nerve agent-BChE conjugates. Its BChE reactivation potency was showed to be promising when compared to the standard oximes used in medical practice, asoxime (HI-6) and pralidoxime (2-PAM), especially in case of sarin and tabun. This finding could be used in the pseudo-catalytic scavenging of the most nerve agents due to its cumulative capacity to reactivate both AChE and BChE., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Organophosphorus compounds (OP) nerve agents are among the most toxic chemical substances known. Their toxicity is due to their ability to bind to acetylcholinesterase. Currently, some enzymes, such as phosphotriesterase, human serum paraoxonase 1 and diisopropyl fluorophosphatase, capable of degrading OP, have been characterized. Regarding the importance of bioremediation methods for detoxication of OP, this work aims to study the interaction modes between the human human deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and Sarin and VX, considering their R p and S p enantiomers, to evaluate the asymmetric catalysis of those compounds. In previous work, this enzyme has shown good potential to degrade phosphotriesters, and based on this characteristic, we have applied the human dUTPase to the OP degradation. Molecular docking, chemometrics and mixed quantum and molecular mechanics calculations have been employed, showing a good interaction between dUTPase and OP. Two possible reaction mechanisms were tested, and according to our theoretical results, the catalytic degradation of OP by dUTPase can take place via both mechanisms, beyond being stereoselective, that is, dUTPase cleaves one enantiomer preferentially in relation to other. Chemometric techniques provided excellent assistance for performing this theoretical investigation. The dUTPase study shows importance by the fact of it being a human enzyme. Communicated by Ramaswamy H. Sarma.

Organophosphorus (OP) nerve agents and pesticides present significant threats to civilian and military populations. OP compounds include the nefarious G and V chemical nerve agents, but more commonly, civilians are exposed to less toxic OP pesticides, resulting in the same negative toxicological effects and thousands of deaths on an annual basis. After decades of research, no new therapeutics have been realized since the mid-1900s. Upon phosphylation of the catalytic serine residue, a process known as inhibition, there is an accumulation of acetylcholine (ACh) in the brain synapses and neuromuscular junctions, leading to a cholinergic crisis and eventually death. Oxime nucleophiles can reactivate select OP-inhibited acetylcholinesterase (AChE). Yet, the fields of reactivation of AChE and butyrylcholinesterase encounter additional challenges as broad-spectrum reactivation of either enzyme is difficult. Additional problems include the ability to cross the blood brain barrier (BBB) and to provide therapy in the central nervous system. Yet another complication arises in a competitive reaction, known as aging, whereby OP-inhibited AChE is converted to an inactive form, which until very recently, had been impossible to reverse to an active, functional form. Evaluations of uncharged oximes and other neutral nucleophiles have been made. Non-oxime reactivators, such as aromatic general bases and Mannich bases, have been developed. The issue of aging, which generates an anionic phosphylated serine residue, has been historically recalcitrant to recovery by any therapeutic approach-that is, until earlier this year. Mannich bases not only serve as reactivators of OP-inhibited AChE, but this class of compounds can also recover activity from the aged form of AChE, a process referred to as resurrection. This review covers the modern efforts to address all of these issues and notes the complexities of therapeutic development along these different lines of research., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

As a countermeasure against terrorism involving highly toxic chemical warfare agents, the rapid identification of the causative toxic substances is extremely important. This symposium review describes analytical methods the author's group has developed for detecting nerve gases after either high level or low level exposure. As a method for assessing human exposure to high levels of nerve gases, a technology that detects nerve gas hydrolysis products, i.e., strong anion exchange extraction-tert-butyldimethylsilyl derivatization-selectable one-dimensional or two-dimensional GC-MS, is explained. As a method for assessing human exposure to low levels of nerve gases, two technologies that detect adducts of nerve gas with blood cholinesterase, i.e., adduct purification-enzymatic digestion-LC/MS and fluoride-mediated regeneration-solid phase extraction-large volume introduction GC-MS, are explained.

The in vitro percutaneous penetration of the nerve agent VX through pig skin was measured in the absence and presence of water or artificial sweat to determine the impact such conditions might have on penetration rates. Experiments were performed with Franz-type glass diffusion cells containing dermatomed pig skin as a surrogate for human skin. The receptor fluid used was 50% aqueous ethanol. 14 C labelled VX was applied to the skin surface under infinite or finite dosing conditions with regular receptor fluid sampling up to 24 h. Penetration from aqueous solutions of VX showed the maximal steady state penetration rate (Jss) was from the 50% (v:v) solution (366 ± 149 μg·cm -2 ·h -1 ), this being ~2.2 fold greater than Jss measured for neat VX (169 ± 89 μg·cm -2 ·h -1 ). Application of neat VX to water-wetted skin was also found to result in an increased penetration rate, compared to dry skin, shortly after contamination. These studies have shown that, in vitro, aqueous solutions of VX in contact with the skin can result in increased penetration rates when compared to neat VX applied to dry skin., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

A method was developed to detect and quantify organophosphate nerve agent (OPNA) metabolites in dried blood samples. Dried blood spots (DBS) and microsampling devices are alternatives to traditional blood draws, allowing for safe handling, extended stability, reduced shipping costs, and potential self-sampling. DBS and microsamplers were evaluated for precision, accuracy, sensitivity, matrix effects, and extraction recovery following collection of whole blood containing five OPNA metabolites. The metabolites of VX, Sarin (GB), Soman (GD), Cyclosarin (GF), and Russian VX (VR) were quantitated from 5.0 to 500 ng mL -1 with precision of ≤16% and accuracy between 93 and 108% for QC samples with controlled volumes. For unknown spot volumes, OPNA metabolite concentrations were normalized to total blood protein to improve interpretation of nerve agent exposures. This study provides data to support the use of DBS and microsamplers to collect critical exposure samples quickly, safely, and efficiently following large-scale chemical exposure events., (Published by Elsevier B.V.)

Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.

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.)

The NMR-observable nuclei of the acidic and basic compounds experience pH dependence in chemical shift. This phenomenon can be exploited in NMR titrations to determine p K a values of compounds, or in pH measurement of solutions using dedicated pH reference compounds. On the other hand, this sensitivity can also cause problems in, for example, metabolomics, where slight changes in pH result in significant difficulties for peak alignment between spectra of set of samples for comparative analysis. In worst case, the pH sensitivity of chemical shifts can prevent unambiguous identification of compounds. Here, we propose an alternative approach for NMR identification of pH-sensitive analytes. The 1 H and X ( 13 C, 15 N, 31 P, ...) chemical shifts in close proximity to the acidic or basic functional group should, when presented as ordered pairs, express piecewise linear correlation with distinct slope, intercept, and range. We have studied the pH dependence of 1 H and 31 P chemical shifts of the CH 3 -P moiety in urinary metabolites of nerve agents sarin, soman and VX using 2D 1 H- 31 P fast-HMQC spectroscopy. The 1 H and 31 P chemical shifts of these chemicals appear in very narrow range, and due to subtle changes in sample pH the identification on either 1 H or 31 P chemical shift alone is uncertain. However, if the observed 1 H and 31 P chemical shifts of the CH 3 -P moiety of individual compounds are presented as ordered pairs, they fall into distinct linear spaces, thus, facilitating identification with high confidence.

The wild-type OPAA enzyme has relatively high levels of catalytic activity against several organophosphate G-type nerve agents. A series of mutants containing replacement amino acids at the OPAA Y212, V342, and I215 sites showed several fold enhanced catalytic efficiency on sarin, soman, and GP. One mutant, Y212F/V342L, showed enhanced stereospecificity on sarin and that enzyme along with a phosphotriesterase mutant, GWT, which had the opposite stereospecificity, were used to generate enriched preparations of each sarin enantiomer. Inhibition of acetylcholinesterase by the respective enantioenriched sarin solutions subsequently provided identification of the sarin enantiomers as separated by normal phase enantioselective liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometry., (Published by Elsevier Inc.)

O-ethyl S-2-diisopropylaminoethyl methyl phosphonothiolate (VX) is a highly toxic chemical warfare agent because it inhibits cholinesterase (ChE) activity in the nervous system. Inhibition of butyrylcholinesterase (BChE) activity by VX is due to formation of a phosphorylated BChE adduct; this adduct in human plasma can serve as a biomarker of exposure to nerve agents. We compared purification efficiency between the procainamide affinity gel method and immunomagnetic separation (IMS) for the nerve agent adduct of BChE in plasma and then optimized the sample preparation by purifying BChE to measure biomarkers of human exposure to organophosphorus nerve agents. The purification efficiency of IMS was 5-fold greater than that of the procainamide affinity gel method because the antibody conjugate with protein G magnetic beads ensured highly selective capture and high recovery of VX-inhibited BChE from plasma. Protein isolation and extraction of the adduct of VX-inhibited BChE from plasma were made more specific by IMS. A 50 µL of the IMS solution was enough to bind VX-inhibited BChE in up to 0.5 mL of plasma. Nonetheless, the IMS method has a limitation in terms of reutilization of the complexes antibody-magnetic beads. We expect that this approach can be used to quantify other types of organophosphorus adducts in human plasma, thus serving as a possible general assay for biomarkers of exposure to nerve agents.

Chemical warfare agents represent a continuous and considerable threat to military personnel and the civilian population. Such compounds are prohibited by the Chemical Weapons Convention, to which adherence by the member states is strictly controlled. Therefore, reliable analytical methods for verification of an alleged use of banned substances are required. Accordingly, current research focuses on long-term biomarkers derived from covalent adducts with biomolecules such as proteins. Recently, we have introduced a microbore liquid chromatography/electrospray ionization high-resolution tandem mass spectrometry method allowing for the investigation of two different classes of adducts of the nerve agent VX with human serum albumin (HSA). Phosphonylated tyrosine residues and novel disulfide adducts at cysteine residues of HSA were produced by enzymatic cleavage with pronase and detected simultaneously. Notably, the thiol containing leaving group of VX (2-(diisopropylamino)ethanethiol, DPAET) formed disulfide adducts that were released as cysteine and proline containing dipeptides originating from at least two different sites of HSA. Aim of this study was to identify assumed and novel adducts of DPAET with HSA using synthetic peptide reference compounds. Two novel tripeptides were identified representing disulfide adducts with DPAET (Met-Pro-Cys-DPAET, MPC-DPAET and Asp-Ile-Cys-DPAET, DIC-DPAET). MPC-DPAET was shown to undergo partial in-source decay during electrospray ionization for MS detection thereby losing the N-terminal Met residue. This results in the more stable Pro-Cys-DPAET (PC-DPAET) dipeptide detectable as protonated ion. The limit of detection for MPC-DPAET was evaluated, revealing toxicologically relevant VX plasma concentrations. The results provide novel insights into the reactivity of VX and its endogenous targets. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

A direct approach for the determination of a specific hydrolysis product of organophosphorus nerve agents such as methylphosphonic acid (MPA) in urine by ion chromatography and tandem mass spectrometry (IC-MS/MS) has been developed. The first advantage of the proposed approach is a rapid and simple sample preparation, which does not require a large sample volume, complicated and laborious preconcentration and derivatization steps, and takes less than 7min per sample. The second advantage is the fast and selective IC determination of MPA carried out on a noncommercial anion exchanger based on a poly(styrene-co-divinylbenzene) (PS-DVB) substrate with a high degree of crosslinking and a covalently-bonded branched functional layer, which enables complete resolution of MPA from major urine matrix components and allows one to overcome matrix effects. Hyphenation of IC with tandem mass spectrometry results in highly sensitive and reliable MPA determination with the lowest detection limit (4ngmL -1 ) reported so far for HPLC determination of MPA in urine. The proposed approach is successfully applied for the analysis of urine from rats exposed to nonlethal doses of organophosphorus nerve agents such as sarin, soman, and VR in up to 13days after initial exposure, which shows the possibility to verify the nerve agent exposure after a long period of time., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Nerve agents (NAs) are extremely neurotoxic synthetic organophosphate (OP) compounds exploited as weapons of mass destruction in terrorist attacks and chemical warfare. Considering the current world scenario, there is a persistent threat of NA-exposure to military personals and civilians. Various prophylactic and post-exposure treatments (such as atropine and oximes) available currently for NA-poisoning are inadequate and unsatisfactory and suffer from severe limitations. Hence, developing safe and effective treatment(s) against NA-poisoning is a critical necessity. With regards to counteracting NA-toxicity, the OP-hydrolyzing enzymes (OPHEs), which can hydrolyze and inactivate a variety of NAs, have emerged as promising candidates for the development of prophylactic therapy against NA-poisoning. However, there are many hurdles to be crossed before these enzymes can be brought to therapeutic use in humans. In this article, we have reviewed the various advancements in the field of development of OPHEs as prophylactic against NA-poisoning. The article majorly focuses on the toxic effects of NAs, various available therapies to counteract NA poisoning, the current status of OPHEs and attempts made to improve the various properties of these enzymes. Further, we have also briefly discussed about the prospective work that is needed to be undertaken for developing these OPHEs into those suitable for use in humans.

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 nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k cat /K M ) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23's ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence-activity relationships along the pathway of PTE's laboratory evolution, we examined k cat /K M values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k cat /K M values >10 7  M -1  min -1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence-activity relationships.

A well-considered treatment of acute nerve agents poisoning involves the exogenous administration of butyrylcholinesterase (BChE, EC 3.1.1.8) as a stoichiometric bioscavenger efficient in preventing cholinergic crises caused by acetylcholinesterase (AChE, EC 3.1.1.7) inhibition. An additional improvement in medical countermeasures would be to use oximes that could reactivate BChE as well to upgrade bioscavenging from stoichiometric to oxime-assisted catalytic. Therefore, in this paper we investigated the potency of 39 imidazolium and benzimidazolium oximes (36 compounds synthesized for the first time) to be considered as the reactivators specifically designed for reactivation of phosphylated human BChE. Their efficiency in the reactivation of paraoxon-, VX-, and tabun-inhibited human BChE, as well as human AChE was tested and compared with the efficiencies of HI-6 and obidoxime, used in medical practice today. A comprehensive analysis was performed for the most promising oximes defining kinetic parameters of reactivation as well as interactions with uninhibited BChE. Furthermore, experimental data were compared with computational studies (docking, QSAR analysis) as a starting point in future oxime structure refinement. Considering the strict criteria set for in vivo applications, we determined the cytotoxicity of lead oximes on two cell lines. Among the tested oxime library, one imidazolium compound was selected for preliminary in vivo antidotal study in mice. The obtained protection in VX poisoning outlines its potential in development oxime-assisted OP-bioscavenging with BChE., (Copyright © 2016 Elsevier Inc. All rights reserved.)

An October 14, 2014 article in The New York Times reported that the US Department of Defense (DoD) concealed, for nearly a decade, circumstances surrounding service members' exposure to chemical warfare agents (CWA) while deployed to Iraq in support of Operations Iraqi Freedom and New Dawn from March 13, 2003, to December 31, 2011, and alleged failure of the DoD to provide expedient and adequate medical care. This report prompted the DoD to devise a public health investigation, with the Army Public Health Center (Provisional) as the lead agency to identify, evaluate, document, and track CWA casualties of the Iraq war. Further, the DoD revisited and revised clinical guidelines and health policies concerning CWA exposure based on current evidence-based guidelines and best practices.

While several methods for the bioanalysis of nerve agents or their metabolites have been developed for the verification of nerve agent exposure, these methods are generally limited in the amount of time after an exposure that markers of exposure can be detected (due to rapid metabolism from biological matrices). In this study, a method for the analysis of nerve agent hydrolysis products from nail clippings was developed to allow evaluation of nails as a long-term repository of these markers. Pinacolyl methylphosphonic acid (PMPA) and isopropyl methylphosphonic acid (IMPA) were extracted from nail samples with N,N-dimethylformamide and subsequently analyzed by liquid chromatography-tandem mass spectrometry. Limits of detection for PMPA and IMPA were 0.3μg/kg and 7.5μg/kg and linear ranges were 0.75-300μg/kg and 30-1500μg/kg, respectively. Precision was within 10% and 8% for PMPA and IMPA, respectively, and accuracy was 100±12% for both analytes. The approach presented here is complementary to current methods for nerve agent exposure verification, and should allow for long-term determination of nerve agent poisoning., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The recent attacks with the nerve agent sarin in Syria reveal the necessity of effective countermeasures against highly toxic organophosphorus compounds. Multiple studies provide evidence that a rapid onset of antidotal therapy might be life-saving but current standard antidotal protocols comprising reactivators and competitive muscarinic antagonists show a limited efficacy for several nerve agents. We here set out to test the newly developed phosphotriesterase (PTE) mutant C23AL by intravenous (i.v.), intramuscular (i.m.; model for autoinjector) and intraosseous (i.o.; model for intraosseous insertion device) application in an in vivo guinea pig model after VX challenge (∼2LD50). C23AL showed a Cmax of 0.63μmolL(-1) after i.o. and i.v. administration of 2mgkg(-1) providing a stable plasma profile up to 180min experimental duration with 0.41 and 0.37μmolL(-1) respectively. The i.m. application of C23AL did not result in detectable plasma levels. All animals challenged with VX and subsequent i.o. or i.v. C23AL therapy survived although an in part substantial inhibition of erythrocyte, brain and diaphragm AChE was detected. Theoretical calculation of the time required to hydrolyze in vivo 96.75% of the toxic VX enantiomer is consistent with previous studies wherein similar activity of plasma containing catalytic scavengers of OPs resulted in non-lethal protection although accompanied with a variable severity of cholinergic symptoms. The relatively low C23AL plasma level observed immediately after its i.v. or i.o load, point at a possible volume of distribution greater than the guinea pig plasma content, and thus underlines the necessity of in vivo experiments in antidote research. In conclusion the i.o. application of PTE is efficient and resulted in comparable plasma levels to the i.v. application at a given time. Thus, i.o. vascular access systems could improve the post-exposure PTE therapy of nerve agent poisoning., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Several methods for the bioanalysis of nerve agents or their metabolites have been developed for the verification of nerve agent exposure. However, parent nerve agents and known metabolites are generally rapidly excreted from biological matrixes typically used for analysis (i.e., blood, urine, and tissues), limiting the amount of time after an exposure that verification is feasible. In this study, hair was evaluated as a long-term repository of nerve agent hydrolysis products. Pinacolyl methylphosphonic acid (PMPA; hydrolysis product of soman) and isopropyl methylphosphonic acid (IMPA; hydrolysis product of sarin) were extracted from hair samples with N,N-dimethylformamide and subsequently analyzed by liquid chromatography-tandem mass spectrometry. Limits of detection for PMPA and IMPA were 0.15 μg/kg and 7.5 μg/kg and linear ranges were 0.3-150 μg/kg and 7.5-750 μg/kg, respectively. To evaluate the applicability of the method to verify nerve agent exposure well after the exposure event, rats were exposed to soman, hair was collected after approximately 30 days, and stored for up to 3.5 years prior to initial analysis. PMPA was positively identified in 100% of the soman-exposed rats (N = 8) and was not detected in any of the saline treated animals (N = 6). The hair was reanalyzed 5.5 years after exposure and PMPA was detected in 6 of the 7 (one of the soman-exposed hair samples was completely consumed in the analysis at 3.5 years) rat hair samples (with no PMPA detected in the saline exposed animals). Although analysis of CWA metabolites from hair via this technique is not appropriate as a universal method to determine exposure (i.e., it takes time for the hair to grow above the surface of the skin and typical analysis times are >24 h), it complements existing methods and could become the preferred method for verification of exposure if 10 or more days have elapsed after a suspected exposure.