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Inhibition of the physiological function of acetylcholinesterase (AChE) by organophosphate (OP) molecules that readily cross the blood- brain barrier (BBB) promotes hypercholinergic activity which induces seizures leading to brain damage and neuroinflammation. OP-induced overstimulation of nicotinic and muscarinic membrane receptors in severe cases can lead to hypoxia, vasodepression, and respiratory arrest, followed by death. Currently approved therapy by permanently charged oxime compounds that don’t cross BBB readily is inadequate for restoring the activity of synaptic AChE, leaving the brain vulnerable to long-term damage. We anticipate that the treatment with uncharged, but ionizable oximes that cross the BBB and reactivate OP-inhibited synaptic AChE will act protectively on the brain of mice exposed to an organophosphorus nerve agent. For that purpose, a microglial response, detected with IBA-1 protein, glial cells, detected with glial fibrillary acidic protein (GFAP), and neuronal cell viability, detected by the neuronal nuclei antigen NeuN, were all monitored in order to indicate the survival of neurons and neuroprotection by RS194B oxime in the brain of mice exposed to a nerve agent.

The main action mechanism of organophosphorus compounds (OP) is the inhibition of acetylcholinesterase (AChE) that causes the accumulation of the neurotransmitter acetylcholine and excessive stimulation of nicotinic and muscarinic receptors in the central and peripheral nervous system, leading to the paralysis of cholinergic synaptic transmission. Although BChE is generally considered as having no natural physiological function, the most likely function for BChE is as backup for AChE and protection of synaptic AChE from man-made and naturally occurring poisons. Both enzymes should be reactivated by strong nucleophiles such as oximes to avoid severe health effects after exposure to OP. However, inhibition and reactivation of both enzymes are fine-tuning chemical processes that depend on the structure of all reactants. Therefore, we evaluated the inhibition of cholinesterase activity with selected OP-herbicides, insecticides and newly scheduled nerve agents as well as reactivation of OP-inhibited AChE and BChE with click chemistry-synthetized oximes. Although several oximes showed reasonable potency in reactivating AChE and BChE conjugated with methylphosphonates, phosphorates and phosphoramidates, a universally superior antidote was not identified. However, our results showed that toxicity of various OP can be reduced by efficient reactivation of phosphylated AChE and BChE. Our findings also offer a valuable and comprehensive platform for further development of antidotes and scavengers against tabun and related phosphoramidate exposures, such as the Novichok series of compounds. This research was supported by the Croatian Science Foundation (IP-2018-01-7683).

Organophosphorus (OP) compounds were developed in Germany in the 1930s as pesticides and as warfare nerve agents (NA) since they were found to have a lethal effect on insects and mammals. Although 193 states signed the Chemical Weapons Convention in the 1997, which prohibits the production and use of NA, and they are slowly being replaced as pesticides by newer less hazardous formulations, OP compounds still pose a threat in terrorist attacks and they account for more than three million accidental or deliberate cases of poisoning a year worldwide [1]. OP exert their toxic effect primarily by inhibiting acetylcholinesterase (AChE), an essential enzyme for neurotransmitter acetylcholine (ACh) hydrolysis, and related enzyme butyrylcholinesterase (BChE). AChE inhibition leads to accumulation of ACh in the nerve system and overstimulation of cholinergic receptors in the entire body, which induces symptoms like nausea, convulsions, loss of consciousness, respiratory failure and ultimately death, while poisoning survivors additionally suffer from considerable irreversible neurological complications [2]. Emergency treatment of acute OP poisoning aims to recover the activity of the inhibited enzyme by pyridinium oxime reactivators that displace the phosphorus moiety bound within the enzyme catalytic site. Their disadvantages are that they generally weakly reactivate BChE, do not reactivate AChE in the brain due to their positive charge and the subsequent inability to cross the blood-brain barrier, and they are not universal reactivators of all NA and pesticides [3]. The goal of our study is to identify and kinetically characterize novel oxime antidotes which would efficiently reactivate BChE and potentially establish a pseudocatalytic scavenger system which could detoxify OP in the bloodstream before they reach target tissues [3]. Structurally various novel oximes were tested as reactivators of NA sarin-, cyclosarin-, VX- and tabun-inhibited human AChE and BChE, as well as chosen OP pesticides. For several oximes BChE reactivation potency was showed to be superior when compared to the standard oximes used in medical practice, and binding affinities of phosphylated BChE for some oximes increased up to 1900-fold compared to pyridinium oximes, establishing them as promising candidates for future treatment development.

The cholinesterase reactivators (so-called "oximes”) are used as causal antidotes in case of organophosphorus intoxications. The effectiveness of the reactivator is strongly dependent on the formation of active nucleophile, the oximate anion [1]. Its formation can be supported by optimizing the physical-chemical properties (i.e. pKa) of the oxime and the decreased pKa should lead to enhanced reactivation of phosphylated cholinesterases. For this reason, the charged chlorinated oximes were introduced and proved to highly effective in vitro for reactivation of acetylcholinesterase [2] and butyrylcholinesterase [3] inhibited by multiple organophosphorus agents. The chlorinated oximes were also proved to be not cytotoxic and effective after in vivo administration. More recently, the charged fluorinated oximes were prepared and thoroughly evaluated with promising reactivation results against multiple organophosphorus agents, although their particular lower stability was found [5]. Finally, further modified halogenated oximes with optimized oximate formation were designed and evaluated on cholinesterases inhibited by nerve agent surrogates. Some modified reactivators were proved to be in vitro effective for reactivation of NEMP, NIMP or NEDPA-inhibited AChE and surprisingly one oxime resulted as excellent reactivator of NEMP and NIMP-inhibited BChE. These promising results make prospects for further detailed investigation of modified oximes nucleophiles. This work was supported by Czech Science Foundation (no. 21- 03000S).

The widespread and frequent use of herbicides has led to the emergence of two major global issues: 1) increasing weed resistance to herbicides and 2) detrimental impact on health. A set of 509 herbicides collected from the literature and online databases was classified based on their modes of action and screened for toxic effects on human health based on predicted toxicities for in vitro and animal models and impact on environment. The analyses enabled us to estimate the toxicity profiles for herbicides of different herbicidal modes of action, including also their physicochemical properties (MW, TPSA, log P, log D, HBA, HBD, log BB, BBB penetration, etc.). We selected 11 compounds with potential neurotoxic effect for biological tests focusing on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), enzymes involved in neurotoxicity and detoxification mechanisms, respectively. Out of organophosphate herbicides (anilophos, piperophos, bensulide and butamiphos) and carbamates (flufenacet, desmedipham and phenmedipham), all of which exhibited an inhibition of both cholinesterases, anilophos was the most potent inhibitor of AChE (IC50=26 M) and BChE (8 M). Moreover, the inhibition of all of the listed herbicides was with a slight preference to bind BChE, which is known as a natural endogenous bioscavenger of various xenobiotics. The selected glyphosate, oxadiazon, tembotrione and terbuthylazine were poor inhibitors with an estimated IC50 above 1 mM. Our results given by in silico and in vitro analyses could give insight into the toxic effects of herbicides in use and may be the origin of new herbicides with less hazardous effects to humans and the environment. This research was supported by the Croatian Science Foundation (HrZZ-IP-2018-01-7683).

Carbamates are a structural part of many drugs for the treatment of various diseases, including neurodegenerative disorders like Alzheimer’s disease (AD). AD is one of the most common causes of mental deterioration in elderly people caused by a loss of cholinergic innervation in the cerebral cortex and characterized by decreased levels of the neurotransmitter acetylcholine in neurons, which has led to the development of AD drugs that inhibit the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The mechanism of action of carbamates and cholinesterases is similar to that of the physiological substrate of AChE, acetylcholine ; the only difference lies in the turnover rate of the enzyme’s activity where the decarbamylation rate is much slower than the deacetylation. We synthesized 17 new heterocyclic bi scarbamates with aliphatic carbamate groups in meta position on the benzene ring and different substituents in the amino part of the molecule. All biscarbamates were potent inhibitors of both cholinesterases with inhibition rate constants within 10^3-10^6 M^-1 min^-1. The most potent BChE inhibitors were ethyl -methyl carbamates with piperidine or adamantylamine in the amino part of the molecule, while the most potent AChE inhibitor was diethyl carbamate with tert- pentylamine. The inhibition potential and selectivity were analysed by molecular docking studies. Nine biscarbamates were determined to possess the ability to penetrate the blood-brain barrier by passive transport, while seven biscarbamates exhibited one deviation from the recommended values for CNS active drugs. Twelve biscarbamates didn’t exhibited hepatotoxicity, nephrotoxicity not neurotoxicity, while five biscarbamates were toxic to at least one of the cell lines at concentrations in which they showed inhibition activity. The ethyl - methyl biscarbamate with piperidine in the amine moiety could be pointed out as the most promising compound for further evaluation and structural modifications for development as a potential AD drug.

Pyridinium oximes are an essential part of antidotal therapy used in organophosphorous (OP) compounds poisoning. Through reactivation of phosphorylated acetylcholinesterase (AChE) oximes ameliorate symptoms arising from acute OP poisoning. However, survivors of OP exposures often have delayed neurologic symptoms of memory loss and cognitive dysfunction. It was shown that oxidative stress mediates secondary OP injury so long-term neurological consequences may be amendable to antioxidant therapy. Pyridiunium oximes have incorporated iminium and oxime moieties that are electron-affinic which enables them various physiological properties and might contribute to their overall therapeutic efficacy. The antioxidant activity of novel pyridinium oximes with the addition of chlorine or fluorine atoms was evaluated using DPPH (2, 2-diphenyl-1- picrylhydrazyl) and FRAP (ferric reducing antioxidant power) assays and compared to their non-halogenated analogues, standard pyridinium oximes and standard antioxidants. According to calculated EC50 values (the concentration of a compound necessary to decrease the initial concentration of DPPH radicals by 50 %), di- fluorinated oximes exhibited the best radical scavenging activity, with the strongest effect being observed for oxime 7b (EC50 250 µM). In case of chlorinated oximes, mono-chlorinated oximes showed better DPPH radical scavenging activity than di-chlorinated. Standard oximes and nonhalogenated analogues showed weak scavenging capacity (EC50 1.05 – 2.99 mM). In addition, FRAP assay highlighted di-fluorinated analogue 7b as the oxime with the highest antioxidant activity in terms of its reducing capacity (FRAP value 1.6 mM). Generally, a trend of higher reduction capacity of fluorinated oximes compared to chlorinated and non-halogenated analogues was observed. Both assays clearly indicated that 7b possesses an antioxidant potential probably due to the radical stabilizing effect of fluorine substituents associated with the resonance delocalization of the unpaired electron. Therefore, a multi-purpose (reactivation and antioxidant) activity of studied oximes could contribute to the entire therapeutic outcome in OP poisoning.

Toxicity of organophosphorus compounds remains a major public health concern due to their widespread use as pesticides and the existence of nerve agents. Their common mechanism of action involves inhibition of enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) which are crucial for neurotransmission. Both chronic and acute poisoning by organophosphates can leave long- lasting health effects even when the patients are treated with standard medical therapy. Therefore, an increasing urgency exists to find more effective oxime reactivators for compounds which are resistant to reactivation, especially phosphoramidates. Here, we investigated in silico and in vitro interactions and kinetics of inhibition for human cholinesterases with four organophosphate pesticides - ethoprophos, fenamiphos, methamidophos and phosalone. Overall, ethoprophos and fenamiphos displayed higher potency as inhibitors for tested cholinesterases. Our results show that methamidophos-inhibited AChE was more susceptible to reactivation than AChE inhibited by fenamiphos by selected oximes. Molecular modelling enabled an evaluation of interactions important for specificity and selectivity of both inhibition and reactivation of cholinesterases. Two newly developed reactivators – bispyridinium triazole oxime 14A and zwitterionic oxime RS194B possess remarkable potential for further development of antidotes directed against pesticides and related phosphoramidate exposures, such as nerve agents tabun or Novichoks.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are enzymes crucial for neurotransmitter acetylcholine metabolism in both central and peripheral nervous system. The progressive inhibition of these enzymes by nerve agents (NA) during poisoning disrupts acetylcholine breakdown, leading to excessive impulse transmission and cholinergic crisis. Since BChE circulates freely in the bloodstream, it has been considered as a possible exogenous bioscavenging enzyme which would act as an oxime- assisted catalytic scavenger of NAs, neutralising them before they reach target tissues. Oximes are used in standard clinical therapy to recover activity of inhibited cholinesterases, but reactivating BChE with standard pyridinium oximes proved to be ineffective as they are mainly AChE reactivators. To approach this issue, based on previous research we synthesized eight novel N- benzyl supstituted imidazolium oximes to be considered as reactivators of nerve agent sarin-, cyclosarin- and tabun-inhibited human AChE and BChE. Two oximes emerged as effective reactivators of all three NA-BChE conjugates, possessing superior overall reactivation rates (kr) than standard oxime HI-6 which was up to 3000-times higher for cyclosarin conjugate and 210-times for sarin conjugate, or 100-times higher than oxime TMB-4 in case of tabun conjugate. Binding affinities of phosphylated BChE for novel oximes in terms of 1/KOX increased up to 1900-fold compared to standard oximes and they achieved their reactivation maximum in a shorter time frame. However, standard pyridinium oximes remain better reactivators of phosphylated AChE which indicates that imidazolium oximes with 1, 3- aromatic substituents do not position optimally into the AChE active site gorge. Novel oximes' low toxicity on hepatic (HepG2) and neuronal (SH-SY5Y) cell lines and high capacity for reactivation of BChE in the studied concentration range establishes them as promising candidates for future treatment development.

The covalent inhibition of cholinesterases (ChE) with organophosphates (OPs) requires immediate treatment mostly based on oximes as reactivators of acetylcholinesterase (AChE). A downfall of this well-known treatment is its inapplicability for the reactivation of butyrylcholesterase (BChE) or targeted AChE in brain due to the poor blood brain barrier (BBB) penetration of the oximes. Thus new oximes were synthesized, referring to standard oxime pralidoxime (2-PAM), with permanently charged pyridinium rings, but with longer aliphatic linkers which improved their ability to penetrate the BBB regarding in silico results. We singled out three analogs of 2-PAM for which both enzymes had moderate affinity, with a 2- to 14- fold higher binding proficiency of AChE than BChE. In a previous study, certain pyridinium oximes were identified with a significantly improved in vitro reactivation potential for tabun inhibited- AChE. So in this research we wanted to test the reactivation ability, not only for ChE inhibited by nerve agents but also by organophosphorus pesticides that are analogs of tabun, metamidophos and fenamiphos. From several combinations of an oxime and the OP-ChE conjugate, 2-PAM analogs have shown the highest reactivation efficiency for cyclosarin, with a 300-fold higher overall kinetic reactivation rate for BChE than standard oxime. Moreover, the reactivation potency of cyclosarin- inhibited AChE was up to 90% for one oxime. With regard to ChE inhibited with pesticides, the acquired results were slightly better than for 2- PAM, with the highest reactivation of 60% recorded for fenamifos-inhibited BChE. Taking into account the reactivation efficacy for cyclosarin exposure, oximes were also evaluated in ex vivo conditions together with exogenous BChE. The obtained results demonstrated up to 80% of restored phosphonylated cholinesterase activity within a short time. Considering our results, we identified three efficient reactivators of phosphylated BChE that, due to a cumulative capacity to reactivate both AChE and BChE, possess the potential for further evaluation in OP bioscavenging.

The inhibition of AChE and BChE by organophosphates (OPs) as nerve agents and pesticides compromises normal cholinergic nerve signal transduction in the peripheral and central nervous systems (CNS) leading to cholinergic crisis. The treatment comprises an antimuscarinic drug and an oxime reactivator of the inhibited enzyme. Oximes in use have quaternary nitrogens, and therefore poorly cross the brain–blood barrier. In this work, we synthesized novel uncharged thienostilbene oximes by sequence of three reactions in very high yields. The expected targeted products were pure cis- and trans-isomers of syn- and anti-oximes containing different substituents bound in the para- position of the benzene ring.1 Eight trans, anti- and trans, syn-isomers of oximes were tested as reactivators of nerve-agent- inhibited AChE and BChE. Four derivatives reactivated cyclosarin- inhibited BChE up to 70% in two hours of reactivation, and docking studies confirmed their productive interactions with the active site of cyclosarin- inhibited BChE. Based on the moderate binding affinity of both AChE and BChE for all selected oximes, these compounds present a new class of oximes with the potential for further development of CNS- active therapeutics in OP poisoning. This is the first study to show the potential of thienostilbene oximes as therapeutics in OP poisoning, and it seems that further design of the compounds e.g., with amide, OH, mono- and dimethylamino groups, or triazole ring, could provide a new platform for further antidote and scavenger development for exposure to organophosphates.

Acutely toxic, small lipophilic organophosphate (OP) molecules readily cross the blood-brain barrier and inhibit the physiological function of acetylcholinesterase (AChE) - hydrolysis of the neurotransmitter acetylcholine. Consequently, hypercholinergic activity will induce seizures which lead to brain damage and trigger neuroinflammation. Also, overstimulation of nicotinic and muscarinic membrane receptors will induce symptoms of cholinergic crises, which in severe cases can lead to hypoxia, vasodepression, and respiratory arrest, followed by death. Therapy against OP poisoning includes oxime compounds that can restore AChE activity preventing the cholinergic crisis and an anti-muscarinic drug, such as atropine. Nevertheless, oximes currently approved for therapy do not cross the blood-brain barrier readily, due to the permanent positive charge, and cannot restore the activity of synaptic AChE, leaving the brain vulnerable to long-term damage. We anticipate that the treatment with uncharged, but ionizable oximes that cross the blood-brain barrier and reactivate OP- inhibited synaptic AChE will act protectively on the brain of mice exposed to a nerve agent. For that purpose, we have investigated the effect of centrally acting oxime RS194B in the brain of mice exposed to a nerve agent and compared it to poisoned mice by monitoring effects on neuronal and glial cells. A microglial response, detected with IBA-1 protein, glial cells, detected with glial fibrillary acidic protein (GFAP), and neuronal cell viability, detected by the neuronal nuclei antigen NeuN were all monitored to track the level of GFAP and IBA-1 proteins, and the preservation of NeuN expression in order to indicate the survival of neurons and neuroprotection by uncharged, ionizable oxime in mice exposed to a nerve agent. This research was supported by the HDTRA-19- 1-006-UCSD-113020, and Croatian Science Foundation (IP-2018-01-7683, and IP- 2016-06-8636).