A-series agent A-234: initial in vitro and in vivo characterization.

A-series agent A-234 belongs to a new generation of nerve agents. The poisoning of a former Russian spy Sergei Skripal and his daughter in Salisbury, England, in March 2018 led to the inclusion of A-234 and other A-series agents into the Chemical Weapons Convention. Even though five years have already passed, there is still very little information on its chemical properties, biological activities, and treatment options with established antidotes. In this article, we first assessed A-234 stability in neutral pH for subsequent experiments. Then, we determined its inhibitory potential towards human recombinant acetylcholinesterase (HssAChE; EC 3.1.1.7) and butyrylcholinesterase (HssBChE; EC 3.1.1.8), the ability of HI-6, obidoxime, pralidoxime, methoxime, and trimedoxime to reactivate inhibited cholinesterases (ChEs), its toxicity in rats and therapeutic effects of different antidotal approaches. Finally, we utilized molecular dynamics to explain our findings. The results of spontaneous A-234 hydrolysis showed a slow process with a reaction rate displaying a triphasic course during the first 72 h (the residual concentration 86.2%). A-234 was found to be a potent inhibitor of both human ChEs (HssAChE IC50 = 0.101 ± 0.003 µM and HssBChE IC50 = 0.036 ± 0.002 µM), whereas the five marketed oximes have negligible reactivation ability toward A-234-inhibited HssAChE and HssBChE. The acute toxicity of A-234 is comparable to that of VX and in the context of therapy, atropine and diazepam effectively mitigate A-234 lethality. Even though oxime administration may induce minor improvements, selected oximes (HI-6 and methoxime) do not reactivate ChEs in vivo. Molecular dynamics implies that all marketed oximes are weak nucleophiles, which may explain the failure to reactivate the A-234 phosphorus-serine oxygen bond characterized by low partial charge, in particular, HI-6 and trimedoxime oxime oxygen may not be able to effectively approach the A-234 phosphorus, while pralidoxime displayed low interaction energy. This study is the first to provide essential experimental preclinical data on the A-234 compound.

Exploring Structure-Activity Relationship in Tacrine-Squaramide Derivatives as Potent Cholinesterase Inhibitors.

Tacrine was the first drug to be approved for Alzheimer's disease (AD) treatment, acting as a cholinesterase inhibitor. The neuropathological hallmarks of AD are amyloid-rich senile plaques, neurofibrillary tangles, and neuronal degeneration. The portfolio of currently approved drugs for AD includes acetylcholinesterase inhibitors (AChEIs) and N-methyl-d-aspartate (NMDA) receptor antagonist. Squaric acid is a versatile structural scaffold capable to be easily transformed into amide-bearing compounds that feature both hydrogen bond donor and acceptor groups with the possibility to create multiple interactions with complementary sites. Considering the relatively simple synthesis approach and other interesting properties (rigidity, aromatic character, H-bond formation) of squaramide motif, we combined this scaffold with different tacrine-based derivatives. In this study, we developed 21 novel dimers amalgamating squaric acid with either tacrine, 6-chlorotacrine or 7-methoxytacrine representing various AChEIs. All new derivatives were evaluated for their anti-cholinesterase activities, cytotoxicity using HepG2 cell line and screened to predict their ability to cross the blood-brain barrier. In this contribution, we also report in silico studies of the most potent AChE and BChE inhibitors in the active site of these enzymes.

Cytotoxicity of acetylcholinesterase reactivators evaluated in vitro and its relation to their structure.

The development of acetylcholinesterase reactivators, i.e., antidotes against organophosphorus poisoning, is an important goal of defense research. The aim of this study was to compare cytotoxicity and chemical structure of five currently available oximes (pralidoxime, trimedoxime, obidoxime, methoxime, and asoxime) together with four perspective oximes from K-series (K027, K074, K075, and K203). The cytotoxicity of tested substances was measured using two methods - colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and impedance based real-time cytotoxicity assay - in three different cell lines (HepG2, ACHN, and NHLF). Toxicity was subsequently expressed as toxicological index IC50. The tested compounds showed different cytotoxicity ranging from 0.92 to 40.06 mM. In HepG2 cells, K027 was the least and asoxime was the most toxic reactivator. In ACHN and NHLF cell lines, trimedoxime was the compound with the lowest adverse effects, whereas the highest toxicity was found in methoxime-treated cells. The results show that at least five structural features affect the reactivators' toxicity such as the number of oxime groups in the molecule, their position on pyridinium ring, the length of carbon linker, and the oxygen substitution or insertion of the double bond into the connection chain. Newly synthetized oximes with IC50 ≥ 1 mM evaluated in this three cell lines model might appear suitable for further testing.