Synthesis and in vitro assessment of the reactivation profile of clinically available oximes on the acetylcholinesterase model inhibited by A-230 nerve agent surrogate.

The risk of the use of toxic chemicals for unlawful acts has been a matter of concern for different governments and multilateral agencies. The Organisation for the Prohibition of Chemical Weapons (OPCW), which oversees the implementation of the Chemical Weapons Convention (CWC), considering recent events employing chemical warfare agents as means of assassination, has recently included in the CWC "Annex on Chemicals" some organophosphorus compounds that are regarded as acting in a similar fashion to the classical G- and V-series of nerve agents, inhibiting the pivotal enzyme acetylcholinesterase. Therefore, knowledge of the activity of the pyridinium oximes, the sole class of clinically available acetylcholinesterase reactivators to date, is plainly justified. In this paper, continuing our research efforts in medicinal chemistry on this class of toxic chemicals, we synthesized an A-230 nerve agent surrogate and applied a modified Ellman's assay to evaluate its ability to inhibit our enzymatic model, acetylcholinesterase from Electrophorus eel, and if the clinically available antidotes are able to rescue the enzyme activity for the purpose of relating the findings to the previously disclosed in silico data for the authentic nerve agent and other studies with similar A-series surrogates. Our experimental data indicates that pralidoxime is the most efficient compound for reactivating acetylcholinesterase inhibited by A-230 surrogate, which is the opposite of the in silico data previously disclosed.

Are the current commercially available oximes capable of reactivating acetylcholinesterase inhibited by the nerve agents of the A-series?

The misuse of novichok agents in assassination attempts has been reported in the international media since 2018. These relatively new class of neurotoxic agents is claimed to be more toxic than the agents of the G and V series and so far, there is no report yet in literature about potential antidotes against them. To shed some light into this issue, we report here the design and synthesis of NTMGMP, a surrogate of A-242 and also the first surrogate of a novichok agent useful for experimental evaluation of antidotes. Furthermore, the efficiency of the current commercial oximes to reactivate NTMGMP-inhibited acetylcholinesterase (AChE) was evaluated. The Ellman test was used to confirm the complete inhibition of AChE, and to compare the subsequent rates of reactivation in vitro as well as to evaluate aging. In parallel, molecular docking, molecular dynamics and MM-PBSA studies were performed on a computational model of the human AChE (HssAChE)/NTMGMP complex to assess the reactivation performances of the commercial oximes in silico. Experimental and theoretical studies matched the exact hierarchy of efficiency and pointed to trimedoxime as the most promising commercial oxime for reactivation of AChE inhibited by A-242.

Theoretical assessment of the performances of commercial oximes on the reactivation of acetylcholinesterase inhibited by the nerve agent A-242 (novichok).

The nerve agents of the A-series are relatively recent chemical weapons with no antidote available yet. Once inside the human body, those chemicals act similarly to the classic nerve agents, by binding to the catalytic residue Serine 203 (Ser203) of human acetylcholinesterase (HssAChE) and thus preventing the proper function of this enzyme. However, there is no experimental evidence yet if the current antidotes for intoxication by nerve agents are also capable of restoring AChE inhibited by the nerve agents of the A-series. In order to launch some light on this issue, we used computational techniques (molecular docking, molecular dynamics and MM-PBSA interaction energy calculations) to assess the performances of the four currently available commercial oximes (2-PAM, HI-6, obidoxime and trimedoxime) when in contact with HssAChE inhibited by the agent A-242. Based on the near-attack conformation (NAC) criterion, our results suggest that the commercial oximes would have limited efficacy to reactivate the enzyme since they are not able to properly approach the adduct Ser203-A-242. Among those oximes, trimedoxime seems to be the most promising, since it showed lower values of energy in the MM-PBSA calculations, a higher stability inside the catalytic anionic center (CAS) of HssAChE, and was able to adopt a position closer to the NAC that could enable the reactivation mechanism.

A New Method for Extraction and Analysis of Ricin Samples through MALDI-TOF-MS/MS.

We report for the first time the efficient use of accelerated solvent extraction (ASE) for extraction of ricin to analytical purposes, followed by the combined use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and MALDI-TOF MS/MS method. That has provided a fast and unambiguous method of ricin identification for in real cases of forensic investigation of suspected samples. Additionally, MALDI-TOF MS was applied to characterize the presence and the toxic activity of ricin in irradiated samples. Samples containing ricin were subjected to ASE, irradiated with different dosages of gamma radiation, and analyzed by MALDI-TOF MS/MS for verification of the intact protein signal. For identification purposes, samples were previously subjected to SDS-PAGE, for purification and separation of the chains, followed by digestion with trypsin, and analysis by MALDI-TOF MS/MS. The results were confirmed by verification of the amino acid sequences of some selected peptides by MALDI-TOF MS/MS. The samples residual toxic activity was evaluated through incubation with a DNA substrate, to simulate the attack by ricin, followed by MALDI-TOF MS/MS analyses.