Novichok Nerve Agents as Inhibitors of Acetylcholinesterase-In Silico Study of Their Non-Covalent Binding Affinity.

In silico studies were performed to assess the binding affinity of selected organophosphorus compounds toward the acetylcholinesterase enzyme (AChE). Quantum mechanical calculations, molecular docking, and molecular dynamics (MD) with molecular mechanics Generalized-Born surface area (MM/GBSA) were applied to assess quantitatively differences between the binding energies of acetylcholine (ACh; the natural agonist of AChE) and neurotoxic, synthetic correlatives (so-called "Novichoks", and selected compounds from the G- and V-series). Several additional quantitative descriptors like root-mean-square fluctuation (RMSF) and the solvent accessible surface area (SASA) were briefly discussed to give-to the best of our knowledge-the first quantitative in silico description of AChE-Novichok non-covalent binding process and thus facilitate the search for an efficient and effective treatment for Novichok intoxication and in a broader sense-intoxication with other warfare nerve agents as well.

Looking after each other in a crisis-Lessons from Novichok and the parallels with Covid-19.

BACKGROUND: In March 2018, three patients were admitted to the Emergency Department of a District General Hospital. Originally suspected of having suffered an opiate overdose, it became clear that they were the victims of anti-cholinesterase poisoning-the Soviet era poison Novichok. Twenty-five days later, two further patients were admitted with the same symptoms. One of these patients died 8 days later and the second remained in hospital for 3 weeks. A Clinical Psychologist was present on the unit throughout the major incident and all staff directly involved received psycho-educational support regarding self-care. AIMS: To examine the psychological impact of the longest running major incident in NHS history on the staff directly involved. DESIGN: A cross-sectional design was used, with structured questionnaires administered retrospectively. METHODS: A link to an electronic survey was emailed to every member of staff in the organization. The survey included the Hospital Anxiety and Depression Scale, the Maslach Burnout Inventory, the Impact of Events Scale-Revised (to both the March and June events). RESULTS: 540/4000 hospital staff responded (13.5% response rate) with a 29/59 (49%) response rate in intensive care staff. Frontline staff had significantly lower scores on anxiety (P < .05 for the June incident), depressive symptoms (P < .05 March and June) and subscales of burnout than managers (depersonalization P < .05). On the remaining two burnout subscales and on anxiety scores for those involved in March, results trended towards significance (P < .1). CONCLUSIONS: Staff in management roles during major incidents may experience higher levels of psychological distress than staff in front line clinical roles and should be encouraged to seek psychological support. RELEVANCE TO CLINICAL PRACTICE: This article informs teams of the psychological impact of major incidents on staff in intensive care settings.

Unambiguous identification and determination of A234-Novichok nerve agent biomarkers in biological fluids using GC-MS/MS and LC-MS/MS.

The present study was intended to develop suitable methods for unambiguous identification and determination of ethyl (1-(diethylamino)ethylidene) phosphoramidofluoridate (known as A234-Novichok) biomarkers in urine and plasma samples. Multiple biomarkers were investigated for the first time, to verify intoxication by the A234-Novichok agent, using sensitive and accurate techniques including gas and liquid chromatography-tandem mass spectrometry (GC-MS/MS and LC-MS/MS). Like other nerve agents, in biological matrices, the A234-Novichok agent reacts with several proteins to form related adducts. Considering this, two different protein adduct biomarkers in blood samples were analyzed, and the regenerated A234 was determined. Two-dimensional chromatography and solid-phase extraction techniques were employed for blood sample preparation. Limits of detection for butyrylcholinesterase (BChE) adduct, the regenerated A234, and albumin covalent adduct were determined and reported as 1, 1, and 10 ng mL-1, while the related calibration curves were linear within the range of 2-100, 2-100, and 15-100 ng mL-1, respectively. The detection limit and linear range for the intact agent in the urine sample were determined as 0.1 and 1-100 ng mL-1, respectively. Since A234 and some other Novichok chemicals have been added to the Schedule 1 of the Chemical Weapons Convention (CWC), Annex on Chemicals, after UK incidents, the analytical methods developed in this work might be used for verification purposes, as well as OPCW Biomedical Proficiency Tests.

Chemical, biological, radiological, and nuclear mass casualty medicine: a review of lessons from the Salisbury and Amesbury Novichok nerve agent incidents.

On March 4, 2018, two casualties collapsed on a park bench in Salisbury, Wiltshire, UK. They were later discovered to have been the victims of an attempted murder using the Soviet-era Novichok class of nerve agent. The casualties, along with three further critically ill patients, were cared for in Salisbury District Hospital's Intensive Care Unit. Before the COVID-19 pandemic, the Salisbury and Amesbury incidents were the longest-running major incidents in the history of the UK National Health Service. This narrative review seeks to reflect on the lessons learned from these chemical incidents, with a particular focus on hospital and local organisational responses.

Enzymatic Decontamination of G-Type, V-Type and Novichok Nerve Agents.

Organophosphorus nerve agents (OPNAs) are highly toxic compounds inhibiting cholinergic enzymes in the central and autonomic nervous systems and neuromuscular junctions, causing severe intoxications in humans. Medical countermeasures and efficient decontamination solutions are needed to counteract the toxicity of a wide spectrum of harmful OPNAs including G, V and Novichok agents. Here, we describe the use of engineered OPNA-degrading enzymes for the degradation of various toxic agents including insecticides, a series of OPNA surrogates, as well as real chemical warfare agents (cyclosarin, sarin, soman, tabun, VX, A230, A232, A234). We demonstrate that only two enzymes can degrade most of these molecules at high concentrations (25 mM) in less than 5 min. Using surface assays adapted from NATO AEP-65 guidelines, we further show that enzyme-based solutions can decontaminate 97.6% and 99.4% of 10 g∙m-2 of soman- and VX-contaminated surfaces, respectively. Finally, we demonstrate that these enzymes can degrade ethyl-paraoxon down to sub-inhibitory concentrations of acetylcholinesterase, confirming their efficacy from high to micromolar doses.

Characterization and Study on Fragmentation Pathways of a Novel Nerve Agent, 'Novichok (A234)', in Aqueous Solution by Liquid Chromatography-Tandem Mass Spectrometry.

As a first step toward studying the properties of Novichok (ethyl (1-(diethylamino)ethylidene)phosphoramidofluoridate (A234)), we investigated its degradation products and fragmentation pathways in aqueous solution at different pH levels by liquid chromatography-tandem mass spectrometry. A234 was synthesized in our laboratory and characterized by nuclear magnetic resonance spectroscopy. Three sets of aqueous samples were prepared at different pH levels. A stock solution of A234 was prepared in acetonitrile at a concentration of 1 mg/mL and stored at -20 °C until use. Aqueous samples (0.1 mg/mL) were prepared by diluting the stock solution with deionized water. The acidic aqueous sample (pH = 3.5) and basic aqueous sample (pH = 9.4) were prepared using 0.01 M acetic acid and 0.01 M potassium carbonate, respectively. The analysis of the fragmentation patterns and degradation pathways of A234 showed that the same degradation products were formed at all pH levels. However, the hydrolysis rate of A234 was fastest under acidic conditions. In all three conditions, the fragmentation pattern and the major degradation product of A234 were determined. This information will be applicable to studies regarding the decontamination of Novichok and the trace analysis of its degradation products in various environmental matrices.

Identification and Study of Biomarkers from Novichok-Inhibited Butyrylcholinesterase in Human Plasma.

To identify biomarkers of ethyl (1-(diethylamino)ethylidene)phosphoramidofluoridate (A234)- or methyl (1-(diethylamino)ethylidene)phosphoramidofluoridate (A232)-inhibited butyrylcholinesterase (BChE), we investigated nonapeptide adducts containing the active site serine, which plays a key role in enzyme activity, using LC-MS/HRMS. Biomarkers were acquired as expected, and they exhibited a significant amount of fragment ions from the inhibiting agent itself, in contrast to the MS2 spectra of conventional nerve agents. These biomarkers had a higher abundance of [M+2H]2+ ions than [M+H]+ ions, making doubly charged ions more suitable for trace analysis.

Organophosphorus nerve agent poisoning: managing the poisoned patient.

Organophosphorus (OP) nerve agent poisoning made the headlines in 2018 with the nerve agent 'Novichok' poisonings in Salisbury, England. This event highlighted a gap in the knowledge of most clinicians in the UK. In response, this special article aims to enlighten and signpost anaesthetists and intensivists towards the general management of OP nerve agent poisoned patients. Drawing on a broad range of sources, we will discuss what OP nerve agents are, how they work, and how to recognise and treat OP nerve agent poisoning. OP nerve agents primarily act by inhibiting the enzyme acetylcholinesterase, causing an acute cholinergic crisis; death usually occurs through respiratory failure. The antimuscarinic agent atropine, oximes (to reactivate acetylcholinesterase), neuroprotective drugs, and critical care remain the mainstays of treatment. The risk to medical staff from OP poisoned patients appears low, especially if there is a thorough decontamination of the poisoned patient and staff wear appropriate personal protective equipment. The events in Salisbury in the past year were shocking, and the staff at Salisbury District General Hospital performed admirably in treating those affected by Novichok nerve agent poisoning. We eagerly anticipate their future clinical publications so that the medical community might learn from their valuable experiences.

Effective skin decontamination with RSDL® (reactive skin decontamination lotion kit) following dermal exposure to a Novichok class nerve agent.

In recent years, various poisoning incidents have been reported, involving the alleged use of the so-called Novichok agents, resulting in their addition to the Schedule I list of the Organisation for the Prohibition of Chemical Warfare (OPCW). As the physicochemical properties of these agents are different from the 'classical' nerve agents, such as VX, research is needed to evaluate whether and to what extent existing countermeasures are effective. Here, we evaluated the therapeutic potential of RSDL® (Reactive Skin Decontamination Lotion Kit) for the neutralization of percutaneous toxicity caused by Novichok agents, both in vitro and in vivo. Experiments showed the three selected Novichok agents (A230, A232, A234) could be degraded by RSDL lotion, but at a different rate. The half-life of A234, in the presence of an excess of RSDL lotion, was 36 min, as compared to A230 (<5 min) and A232 (18 min). Following dermal exposure of guinea pigs to A234, application of the RSDL kit was highly effective in preventing intoxication, even when applied up until 30 min following exposure. Delayed use of the RSDL kit until the appearance of clinical signs of intoxication (3-4 h) was not able to prevent intoxication progression and deaths. This study determines RSDL decontamination as an effective treatment strategy for dermal exposure to the Novichok agent A234 and underscores the importance of early, forward use of skin decontamination, as rapidly as possible.

Persistence of A-234 nerve agent on indoor surfaces.

Understanding the fundamental physical characteristics of extremely toxic compounds and their behavior across different environments plays a crucial role in assessing their danger. Additionally, this knowledge informs the development of protocols for gathering forensic evidence related to harmful chemicals misuse. In 2018, former Russian spy Sergei Skripal and his daughter were poisoned in Salisbury, England, with a substance later identified as the unconventional nerve agent A-234. Contamination with the compound was found on items inside Skripal's home. The aim of this paper was to determine the persistence of A-234 on selected indoor surfaces. Ceramics, aluminum can, laminated chipboard, polyvinyl chloride (PVC) floor tile, polyethylene terephthalate (PET) bottle, acrylic paint and computer keyboard were used as matrices. The decrease in surface contamination and further fate of the compound was monitored for 12 weeks. Persistence determination involved optimizing the wipe sampling method. Simultaneously, evaporation from the surface and permeation of the contaminant into the matrix were closely monitored. The experimental findings indicate that the nerve agent exhibits remarkable persistence, particularly on impermeable surfaces. Notably, the process of A-234 evaporation plays a minor role in determining its fate, with detectable concentrations observed solely above solid, non-porous surfaces such as ceramics and aluminum can. The surface persistence half-life varied significantly, ranging from 12 min to 478 days, depending on the material. The article has implications for emergency response protocols, decontamination strategies, public health and crime scene investigations.

Simultaneous analysis of degradation products of Novichok agents and conventional nerve agents in human urine by ion chromatography-tandem mass spectrometry using ammonium regeneration solution.

An ion chromatography (IC)-tandem mass spectrometry (MS/MS) method to analyze nerve agent degradation products in human urine was developed. Six degradation products of conventional nerve agents and six Novichok agent degradation products were analyzed simultaneously despite their differences in hydrophilicity and acidity. Using ammonium regeneration solution improved the peak shapes greatly compared with the results obtained with the ordinary IC-MS/MS configuration. For urine samples, a simple pretreatment method of dilution with water and ultrafiltration was used. The detection limits of the nerve agent degradation products were sufficiently low (10-250 ng/mL) and the calibration curves showed acceptable linearity. Due to the absence of a derivatization step, throughput was higher than for our previous derivatization-liquid chromatography-MS/MS method.

Molecular modeling of Mannich phenols as reactivators of human acetylcholinesterase inhibited by A-series nerve agents.

The A-series is the most recent generation of chemical warfare nerve agents (CWA) which act directly on the inhibition of the human acetylcholinesterase (HssAChE) enzyme. These compounds lack accurate experimental data on their physicochemical properties, and there is no evidence that traditional antidotes effectively reactivate HssAChE inhibited by them. In the search for potential antidotes, we employed virtual screening, molecular docking, and molecular dynamics (MD) simulations for the theoretical assessment of the performance of a library of Mannich phenols as potential reactivators of HssAChE inhibited by the Novichok agents A-230, A-232, and A-234, in comparison with the commercial oximes pralidoxime (2-PAM), asoxime (HI-6), trimedoxime (TMB-4), and obidoxime. Following the near-attack conformation (NAC) approach, our results suggest that the compounds assessed would face difficulties in triggering the proposed nucleophilic in-line displacement mechanism. Despite this, it was observed that certain Mannich phenols presented similar or superior results to those obtained by reference oximes against A-232 and A-234 model, suggesting that these compounds can adopt more favourable conformations. Additional binding energy calculations confirmed the stability of the model/ligands complexes and the reactivating potential observed in the molecular docking and MD studies. Our findings indicate that the Mannich phenols could be alternative antidotes and that their efficacy should be evaluated experimentally against the A-series CWA.

Analysis of degradation products of Novichok agents in human urine by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

PURPOSE: The detection of hydrolysis products of Novichok agents in biological samples from victims is important for confirming exposure to these agents. However, Novichok agents are new class of nerve agent and there have been only few reports on analyses of Novichok agent degradation products. Here, we developed hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry (MS/MS) methods to detect Novichok agent degradation products in human urine with simple pretreatment and high sensitivity. METHODS: A Poroshell 120 HILIC-Z column was used to analyze six Novichok agent degradation products. For urine samples, we used a simple pretreatment method, which consisted of deproteinization with acetonitrile and microfiltration. We calculated the pKa values of the OH groups, the log P values, and the molecular weights to investigate the difference in chromatographic behaviors of the Novichok agent degradation products and the degradation products of conventional nerve agents. RESULTS: Six Novichok agent degradation products, including N-(bis-(diethylamino)methylidene)-methylphosphonamidic acid (MPGA), which could not be detected by our previous method, could be analyzed with sufficient peak shape and mutual separation. The detection limits of six Novichok agent degradation products were sufficiently low (1-50 ng/mL) and the calibration curves showed sufficient linearity. The physicochemical parameters of Novichok agent degradation products were different from those of conventional nerve agent degradation products, and this explains the difference in chromatographic behaviors. CONCLUSION: Six Novichok agent degradation products were successfully analyzed by HILIC-MS/MS. Due to the absence of a derivatization step, throughput performance was higher than our previous derivatization-liquid chromatography-MS/MS method.

Selective Colorimetric Detection of Novichok Agents with Hydrazone Chemosensors.

Novichok is a recently identified class of neurotoxic organophosphorus compounds that have subsequently been banned by the Organization for Prohibition of Chemical Weapons (OPCW) as chemical warfare agents. Selective, rapid detection of Novichok remains a challenge. Several colorimetric paper-based detection devices have recently been developed for the rapid and selective detection of sarin, VX, and sulfur mustard. In this article, we present a similar colorimetric device designed specifically for the detection of Novichok, based on hydrazone derivatives from 2,4-dinitrophenylhydrazine (2,4-DNPH) impregnated on a glass fiber substrate. Results show that these compounds rapidly and selectively reveal the presence of Novichok agents to the naked eye. The low cost, ease of use, portability, and high selectivity to Novichok of this device complete the detection range of colorimetric paper-based sensors for chemical warfare agents.

A complete, evidence-based review on novichok poisoning based on epidemiological aspects and clinical management.

Background: The whole world has learned about the existence of a highly toxic neuro-paralytic substance called Novichok. A wide range of neuro-paralytic toxins were used during the wars of decades ago, which also had harmful and irreversible effects. Fortunately, the establishment of conventions prohibiting the use of these weapons prevented the adverse clinical consequences of these compounds. What we did in the present study was to evaluate the clinical features of Novichok, how to manage exposure to it, and to evaluate the prognostic aspects associated with this poisoning agent. Methods: The manuscript especial databases including Medline, Web of knowledge, Google scholar, and Scopus were deeply searched by the two blinded investigators for all eligible studies based on the considered keywords. Initially 98 articles were initially collected by database searching that considering eligibility criteria, 83 articles were finally eligible for the final assessment. There is a lack of clinical trials and case-cohort studies on general population about treatment and side effects when it comes to human nerve agents and most of the data in our search is based on animal studies. Results: In evaluating various clinical, auto physiological and prognostic aspects of exposure to these substances, special attention was necessary to the following points. First, Novichok agents are considered more potent than other toxic agents. Pathophysiologically, these agents irreversibly bind acetylcholinesterase and produce a rapid cholinergic toxidrome which is responsible for the clinical manifestations as well as the potential dangerous and life threatening side effects caused by these agents. Uniquely, these agents are thought to also target every neuron in the central and peripheral nervous system. As a managerial and therapeutic approach, early and timely treatment of its related complication along with prevents massive exposure and decontamination in addition to rapid resuscitation can prohibit debilitating neuropathy and death due to facing it. Conclusion: The present review highlights the importance of recognizing the potential acute toxic effects of Novichok agents, diagnostic and therapeutic approaches (life-saving antidotal therapy) to complications and ultimately the application of guidelines to improve the prognosis of exposure to these agents for both victims and medical community.

Selected Political Criminal Poisonings in the Years 1978-2020: Detection and Treatment.

Criminal poisonings are among the least frequently detected crimes in the world. Lack of suspicion of this type of event by police officers and prosecutors, clinical symptoms imitating many somatic diseases and technical difficulties in diagnostics, as well as high research costs make the actual frequency of these events difficult to estimate. The substance used for criminal poisoning is often characterized by: lack of taste, color and smell, delayed action, easy availability and difficulty to detect. The aim of the study was to analyze selected cases of political poisoning that took place in the years 1978-2020, to describe the mechanisms of action of the substances used and to evaluate the diagnosis and treatment. The analyzed cases of criminal poisoning concerned: Georgi Markov (ricin), Khalid Maszal (fentanyl), Wiktor Yushchenko (TCDD dioxin), Jasir Arafat (polonium 210Po isotope), Alexander Litvinenko (polonium 210Po isotope), Kim Jong-Nam (VX), Sergei Skripal (Novichok) and Alexei Navalny (Novichok). Contemporary poisons, to a greater extent than in the past, are based on the use of synthetic substances from the group of organophosphorus compounds and radioactive substances. The possibility of taking appropriate and effective treatment in such cases is the result of many factors, including the possibility of quick and competent rescue intervention, quick and reliable detection of the toxic substance and the possibility of using an antidote.

Simulation of the electron ionization mass spectra of the Novichok nerve agent.

Novichok is one of the most feared and controversial nerve agents, which existence was confirmed only after the Salisbury attack in 2018. A new attack on August 2020, in Russia, was confirmed. After the 2018 attack, the agent was included in the list of the most dangerous chemicals of the Chemical Weapons Convention (CWC). However, information related to its electron ionization mass spectrometry (EI/MS), essential for unambiguous identification, is scarce. Therefore, investigations about Novichok EI/MS are urgent. In this work, we employed Born-Oppenheimer molecular dynamics through the Quantum Chemistry Electron Ionization Mass Spectrometry (QCEIMS) method to simulate and rationalize the EI/MS spectra and fragmentation pathways of 32 Novichok molecules recently incorporated into the CWC. The comparison of additional simulations with the measured EI spectrum of another Novichok analog is very favorable. A general scheme of the fragmentation pathways derived from simulation results was presented. The present results will be useful for elucidation and prediction of the EI spectra and fragmentation pathways of the dangerous Novichok nerve agent.

Hydrolysis and enzymatic degradation of Novichok nerve agents.

Several parameters of the Novichok nerve agents A230, A232 and A234 were determined. Hydrolysis rates were approximately one to three orders of magnitude slower than G-type nerve agents and approximately zero to two orders of magnitude slower than V-type nerve agents. A230 was the most labile Novichok compound followed by A232 then A234. Activation energies (Ea) and frequency factors (A) were determined for all three compounds. The organophosphorus acid anhydrolase (OPAA) enzyme had catalytic efficiencies on the Novichok compounds ranging between 104 and 105 M-1 min-1 with the highest k cat/Km value for A230, then A232 and lastly, A234.

Theoretical study on the toxicity of 'Novichok' agent candidates.

The identities and properties of 'Novichok' (Russian for 'newcomer' or 'novice') agents allegedly used in the recent terrorist attack in the UK have not been well documented. Although several people previously involved in the synthesis of these materials claimed Novichok agents to be five to eight times more potent than VX, a deadly nerve agent, no open data on these species are currently available. To bridge this gap, we herein performed a theoretical study on several Novichok agent candidates and conducted natural population charge analysis to evaluate the possible mechanisms of their toxicity, suggesting that these agents might promote the ageing and deformation of acetylcholinesterase. Moreover, the reaction of Novichok agents with acetylcholinesterase serine was calculated to be most thermodynamically favoured for Novichok candidate A234. Thus, this work is believed to provide a basis for finding Novichok antidotes and should inspire further detoxification studies to prepare for possible terrorist attacks in the future.

Theoretical study of the molecular aspect of the suspected novichok agent A234 of the Skripal poisoning.

Novichoks are the suspected nerve agents in the March 2018 Skripal poisoning. In this context, the novichok agent A234 (chemical structure proposed by Mirzayanov) was studied using computational methods to shed light on its molecular, electronic, spectroscopic, thermodynamic and toxicity parameters as well as on potential thermal and hydrolysis degradation pathways. The poisoning action and antidote of A234 were also investigated. Some of these parameters were compared to three common G- and V-series nerve agents, namely GB, VR and VX. The research findings should be useful towards the detection, development of antidotes and destruction of A234.