Backbone Conformation Shifts in X-ray Structures of Human Acetylcholinesterase upon Covalent Organophosphate Inhibition

Conformations of Cα backbones in X-ray structures of most organophosphate (OP)-inhibited human acetylcholinesterases (hAChEs) have been previously shown to be similar to that of the native hAChE. One of the exceptions is the structure of the diethylphosphoryl-hAChE conjugate, where stabilization of a large ethoxy group into the acyl pocket (AP) of hAChE-triggered notable loop distortions and consequential dissociation of the hAChE homodimer. Recently, six X-ray structures of hAChE conjugated with large OP nerve agents of the A-type, Novichoks, have been deposited to PDB. In this study we analyzed backbone conformation shifts in those structures, as well as in OP-hAChE conjugates formed by Paraoxon, Soman, Tabun, and VX. A Java-based pairwise alpha carbon comparison tool (PACCT 3) was used for analysis. Surprisingly, despite the snug fit of large substituents on phosphorus, inside Novichok-conjugated hAChEs only minor conformational changes were detected in their backbones. Small magnitudes of observed changes were due to a 1.2–2.4 Å shift of the entire conjugated OP away from the AP. It thus appears that the small AP of AChEs can accommodate, without distortion, substituents of the size of ethoxy or butyryl groups, provided that conjugated OP is “pulled” away from the AP. This observation has practical consequences in the structure-based design of nucleophilic reactivation antidotes as well as in the definition of the AChE specificity that relies on the size of its AP.

In Vitro Interaction of Organophosphono- and Organophosphorothioates with Human Acetylcholinesterase

The implementation of the Chemical Weapons Convention (CWC) in 1997 was a milestone in the prohibition of chemical warfare agents (CWA). Yet, the repeated use of CWA underlines the ongoing threat to the population. Organophosphorus (OP) nerve agents still represent the most toxic CWA subgroup. Defensive research on nerve agents is mainly focused on the “classical five”, namely tabun, sarin, soman, cyclosarin and VX, although Schedule 1 of the CWC covers an unforeseeable number of homologues. Likewise, an uncounted number of OP pesticides have been produced in previous decades. Our aim was to determine the in vitro inhibition kinetics of selected organophosphono- and organophosphorothioates with human AChE, as well as hydrolysis of the agents in human plasma and reactivation of inhibited AChE, in order to derive potential structure–activity relationships. The investigation of the interactions of selected OP compounds belonging to schedule 1 (V-agents) and schedule 2 (amiton) of the CWC with human AChE revealed distinct structural effects of the P-alkyl, P-O-alkyl and N,N-dialkyl residues on the inhibitory potency of the agents. Irrespective of structural modifications, all tested V-agents presented as highly potent AChE inhibitors. The high stability of the tested agents in human plasma will most likely result in long-lasting poisoning in vivo, having relevant consequences for the treatment regimen. In conclusion, the results of this study emphasize the need to investigate the biological effects of nerve agent analogues in order to assess the efficacy of available medical countermeasures.

Trends in the Recent Patent Literature on Cholinesterase Reactivators (2016–2019)

Acetylcholinesterase (AChE) is the key enzyme responsible for deactivating the ACh neurotransmitter. Irreversible or prolonged inhibition of AChE, therefore, elevates synaptic ACh leading to serious central and peripheral adverse effects which fall under the cholinergic syndrome spectra. To combat the toxic effects of some AChEI, such as organophosphorus (OP) nerve agents, many compounds with reactivator effects have been developed. Within the most outstanding reactivators, the substances denominated oximes stand out, showing good performance for reactivating AChE and restoring the normal synaptic acetylcholine (ACh) levels. This review was developed with the purpose of covering the new advances in AChE reactivation. Over the past years, researchers worldwide have made efforts to identify and develop novel active molecules. These researches have been moving farther into the search for novel agents that possess better effectiveness of reactivation and broad-spectrum reactivation against diverse OP agents. In addition, the discovery of ways to restore AChE in the aged form is also of great importance. This review will allow us to evaluate the major advances made in the discovery of new acetylcholinesterase reactivators by reviewing all patents published between 2016 and 2019. This is an important step in continuing this remarkable research so that new studies can begin.

Review of Biomarkers and Analytical Methods for Organophosphate Pesticides and Applicability to Nerve Agents

Introduction Recent malicious use of chemical warfare agents (CWAs) is a reminder of their severity and ongoing threat. One of the main categories of CWAs is the organophosphate (OP) nerve agents. Presently, there is an urgent need to identify and evaluate OP nerve agent biomarkers that can facilitate identification of exposed individuals post-CWA incident. While exposures to OP nerve agents may be scenario-specific, the public is commonly exposed to OP compounds through the ubiquitous use of OP pesticides, which are chemically related to nerve agents. Therefore, a systematic literature review and methodological quality assessment were conducted for OP pesticide biomarker studies to serve as a baseline to assess if these approaches may be adapted to OP nerve agent exposures. Materials and Methods We conducted a systematic literature review to identify biomarkers of OP pesticide exposures. English language studies of any design that reported primary data on biomarkers for exposures in nonhuman primates or adult human study participants were eligible for inclusion. Using standard criteria for assessing the completeness of reported analytical methods, the quality of study methods was critically evaluated. Results A total of 1,044 studies of biomarkers of OP pesticide exposure were identified, of which 75 articles satisfied the inclusion and exclusion criteria. These studies described 143 different analyte/sample matrix combinations: 99 host-based biomarkers, 28 metabolites, 12 pesticides, and 4 adducts. The most commonly reported biomarkers were dialkyl phosphate urinary metabolites (22 studies), blood acetylcholinesterase, and plasma butyrylcholinesterase (26 studies each). None of the assessed quality review criteria were fully addressed by all identified studies, with almost all criteria scoring less than 50%. Conclusion Cholinesterase activity may have utility for identifying individuals with exposures surpassing a given threshold of OP nerve agent, but further investigation of how acetylcholinesterase and butyrylcholinesterase levels correlate with observed patient symptoms may be required to ensure accuracy of results. As CWAs and nerve agents are more readily used, more standardized reporting of biomarker measurements are needed to develop new approaches for OP nerve agent biomarkers.

A microneedle biosensor for minimally-invasive transdermal detection of nerve agents

A microneedle array based biosensor for minimally invasive electrochemical monitoring of organophosphate (OP) nerve agents under the skin.

Translation of in vitro to in vivo pyridinium oxime potential in tabun poisoning / Translacija učinkovitosti piridinijevih oksima kod trovanja tabunom iz in vitro sustava u in vivo primjenu

Even if organophosphorus (OP) nerve agents were banned entirely, their presence would remain a problem as weapons of terror (like in Syria). Oxime antidotes currently used in medical practice still fall short of their therapeutic purpose, as they fail to fully restore the activity of cholinesterases, the main target for OPs. As orphan drugs, these antidotes are tested too seldom for anybody’s benefit. Over the last few decades, search for improved reactivators has reached new levels, but the translation of data obtained in vitro to in vivo application is still a problem that hinders efficient therapy. In this study, we tested the strengths and weaknesses of extrapolating pyridinium oxime antidotes reactivation efficiency from in vitro to in vivo application. Our results show that this extrapolation is possible with well-determined kinetic constants, but that it also largely depends on oxime circulation time and its tissue-specific distribution. This suggests that pharmacokinetic studies should be planned at the early stages of antidote development. Special attention should also be given to improving oxime distribution throughout the organism to overcome this major constraint in improving overall OP therapy.

Pharmacokinetics of MMB4 DMS in Rats, Rabbits, and Dogs Following a Single IV Administration

Organophosphorus (OP) nerve agents pose tremendous threats to both military and civilian populations. The substance 1,1′-methylenebis[4-[(hydroxyimino)methyl]-pyridinium] (MMB4) is being developed as a replacement for the currently fielded 2-pyridine aldoxime, or pralidoxime (2-PAM) as a treatment for OP nerve agent–induced toxicity. The present study characterized pharmacokinetic (PK) profiles of MMB4 in male and female Sprague-Dawley rats, New Zealand White rabbits, and beagle dogs given a single intravenous (IV) administration of MMB4 dimethanesulfonate (DMS) at 55, 25, and 15 mg/kg dose, respectively. The plasma MMB4 concentration versus time profiles were biphasic for all species tested and fit a 2-compartment model with first-order elimination. There were no overt sex-related differences in the calculated PK parameters. For the rat, rabbit, and dog, the average systemic exposure parameters predicted Cmax (µg/mL) and AUC∞ (µg·h/mL) were 273 and 71.0, 115 and 48.1, and 87.4 and 39.6; the average volume of distribution (mL/kg) values to the central and peripheral compartments were 207 and 143, 242 and 172, and 198 and 213; and the average elimination half-life (hour) and clearance (mL/h/kg) values were 0.18 and 778, 0.29 and 577, and 0.32 and 430, respectively, when the PK parameters for males and females were combined. The current study revealed a similarity in the volume of distribution to the central compartment for MMB4 among the 3 species tested while demonstrating species-related differences in the elimination half-life and clearance of MMB4.

Improving the Catalytic Activity of HyperthermophilicPyrococcus horikoshiiProlidase for Detoxification of Organophosphorus Nerve Agents over a Broad Range of Temperatures

Prolidases hydrolyze Xaa-Pro dipeptides and can also cleave the P-F and P-O bonds found in organophosphorus (OP) compounds, including the nerve agents soman and sarin.Ph1prol (PH0974) has previously been isolated and characterized fromPyrococcus horikoshiiand was shown to have higher catalytic activity over a broader pH range, higher affinity for metal, and increased thermostability compared toP. furiosusprolidase,Pfprol (PF1343). To obtain a better enzyme for OP nerve agent decontamination and to investigate the structural factors that may influence protein thermostability and thermoactivity, randomly mutatedPh1prol enzymes were prepared. FourPh1prol mutants (A195T/G306S-, Y301C/K342N-, E127G/E252D-, and E36V-Ph1prol) were isolated which had greater thermostability and improved activity over a broader range of temperatures against Xaa-Pro dipeptides and OP nerve agents compared to wild typePyrococcusprolidases.