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Notwithstanding clinical effectiveness evidences continue to suggest benefit from the acetylcholinesterase inhibitors (AChEIs) in alleviating Alzheimer’s disease (AD) symptoms, these drugs do not appear to delay or prevent the underlying neurodegeneration. In this context, novel prospects are offered by the strategy of developing single chemical entities able to modulate multiple targets, i.e. the multi-target-directed ligands (MTDLs). On this basis, several multifunctional AChEIs have been rationally designed with the deliberate aim of enlarging their biological profiles, beyond the ability to inhibit cholinesterases. This is because it has been recognized that a balanced simultaneous modulation of multiple targets critically intertwined in AD pathological cascade can provide a superior therapeutic and toxicological profile compared to the action of a selective AChEI.[1] Building on this founding principle, we and others have developed several series of anti-AD MTDL compounds that combine cholinesterase inhibition with anti-aggregating, anti-oxidant, and anti-neuroinflammatory properties.[2] As a further step, to explore the possibility to discover new MTDLs based on inexpensive resources, we have developed a series of MTDLs obtained by properly modifying constituents from the cashew nut shell liquid (CNSL), a waste from cashew nut processing factories.[3] Such hybrid compounds, obtained from renewable and inexpensive material, might be promising bio-based, sustainable MTDLs for AD drug discovery. Working in the field for almost 20 years, we should draw lessons from the past and try our best to chart innovative directions and hopefully address the scientific and societal challenges of neurodegenerative diseases.

Alzheimer’s disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD. Potent, selective and reversible human BChE inhibitors were developed. First, a hierarchical virtual screening was performed followed by biochemical evaluation of highest scoring hit compounds. Three compounds showed significant inhibitory activities against BChE and the best inhibitor was selected for further SAR studies. More than 100 different analogues were synthesized and among them, two compounds were found to be promising lead compounds as they were not cytotoxic, they crossed the blood-brain barrier and improved memory, cognitive functions and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic adverse effects. The solved crystal structures of human BChE in complex with the most potent inhibitors revealed their binding modes and provided the structural basis for their further development into multi-target-directed ligands, which in addition to good inhibition of BChE possess good antioxidant, metal chelating, neuroprotective and other properties beneficial for AD.

Combination of tacrine/huprine, connected through a different linker tether length, with tryptophan led to the generation of a novel, highly-potent family of multi-target directed ligands targeting key molecular mechanisms of Alzheimer’s disease. Based on in vitro biological profile, the 6-chloro-tacrine-(CH₂)₆-L-tryptophan heterodimer S-K1035 was found to be the most potent inhibitor of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE) within the series, with nanomolar IC₅₀ values (6.31 and 9.07 nM, respectively). Moreover, S‑K1035 showed good ability to inhibit Aβ₄₂ self-aggregation and hAChE-induced Aβ₄₀ aggregation. The X-ray crystallographic analysis of TcAChE in complex with S-K1035 highlighted the utility of the hybridization approach used in the structure based drug design. S‑K1035 also exerted moderate inhibition against neuronal nitric oxide synthase (nNOS). In vivo studies displayed low toxicity profile compared to parent tacrine. S-K1035 also significantly ameliorated performances of scopolamine-treated animals.

Neurodegenerative diseases are multifactorial. Therefore, their treatment requires drugs that can act simultaneously on multiple pathogenic targets. We synthesized several series of hybrid structures combining certain pharmacophores essential for neurodegenerative disease treatment: γ-carbolines, carbazoles, phenothiazines, and aminoadamantanes [1-3]. Inhibitory activity of these conjugates against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CaE) was studied along with their ability to competitively displace propidium iodide from the peripheral anionic site of electric eel AChE to assess their potential effect on AChE-induced aggregation of β-amyloid. Antioxidant properties were examined computationally with density functional theory and measured experimentally using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and oxygen radical absorbance capacity (ORAC-FL) assays. Binding modes of conjugates to AChE and BChE were studied using quantum mechanical-assisted molecular docking. Results revealed structures that were selective inhibitors of BChE [1,2] or that combined high potency and selectivity toward BChE with high radical-scavenging activity, e.g., conjugates of γ-carbolines and tetrahydrocarbazoles [3]. Conjugates of γ-carbolines and cycloalcaneindoles with the phenothiazine derivative Methylene Blue demonstrated high potency against AChE and BChE combined with effective displacement of propidium from the peripheral anionic site of AChE. Additionally, the conjugates were extremely active in both antioxidant tests. All conjugates were poor CaE inhibitors and therefore expected to lack drug-drug interactions by this pathway. Good agreement was found between experimental and computational results. Lead compounds were identified for future optimization and development of new multi-target drugs against neurodegenerative diseases that combined cognition enhancement with neuroprotective potential.

Today, treatment of Alzheimer's Disease (AD) mainly involves acetylcholinesterase inhibitors (AChEIs). AChEIs display solely a symptomatic benefit, alleviating the cognitive disorders associated to AD through a temporary restoration of the cholinergic neurotransmission impaired by the neurodegeneration. The gradual loss of efficiency for AChEIs led to associate them to drugs exhibiting potential disease-modifying properties. The “Multi-Target-Directed Ligands” (MTDLs) was used in the recent years with a great potential benefit towards multiple targets implicated in the complex AD,¹ as well as other neurodegenerative syndromes, which involve multiple pathogenic factors. Our contribution to the field led recently to the discovery of Donecopride, the first 5-HT₄R partial agonist, which possesses important acetylcholinesterase (AChE) inhibition properties currently under preclinical development.^2,3 Based on this experience, we have recently developed a novel pleiotropic prodrugs approach to generate promising in vivo active compounds. Based on the structure of rivastigmine, novel MTDLs were designed, acting as prodrugs, able to temporarily covalently bind and inhibit AChE (for a symptomatic effect). and to secondarily release a drug able to selectively reach another AD target (for a potential disease-modifying effect) This concept was applied to several secondary targets, including different 5-HT receptors of interest⁴ for the treatment of AD. The concept, the synthetic development, in vitro and in vivo evaluation of these candidates and our undisclosed results will be presented for the first time in this communication.

Alzheimer’s disease (AD) is a complex and progressive neurodegenerative disorder. The available therapy is limited to the symptomatic treatment and its efficacy remains unsatisfactory ^[1]. In view of the prevalence and expected increase in the incidence of AD, the development of an effective therapy is crucial for public health. Due to the multifactorial etiology of this disease, the multi-target-directed ligand (MTDL) approach is a promising method in search for new drugs for AD. Aiming at developing new MTDLs, this project consists on the development of new multifunctional agents, which will act simultaneously on the different players in AD pathology. The project aims at developing MTDLs by combining an AChE inhibitory activity with an alpha-7 nAChR activation ^[2].

In response to the complex and still not fully understood pathomechanism of Alzheimer's disease, many researchers have turned towards the promising paradigm of designing ligands with a multi-target nature¹. One of the possible benefits of this approach in Alzheimer's disease is an opportunity to merge activity against cholinesterases, which are used in the current symptomatic therapies, with disease-modifying targets associated with β-amyloid and tau protein pathways². Optimization of ligand with respect to several biological targets while maintaining good physicochemical parameters is not an easy task.  Computer modeling can be a huge help in this task. Computer modeling in the design of biologically active substances can be used to effectively search through the huge, available chemical space, or provide support for drawing conclusions of results obtained during the study³. In the work presented here, we would like to describe how the molecular modeling methods were used to design and obtain new series of 1-benzylamino-2-hydroxyalkyl derivatives that are effective against both acetyl- and butyrylcholinesterase as valid, symptomatic targets with an anti-aggregating properties against Tau protein, β-amyloid and inhibition properties against β-secretase (BACE-1) as disease-modified targets⁴.

Cocaine abuse is a major medical and health problem. There is no FDA-approved medication for treatment of cocaine overdose and addiction. Statistical data show that 92% of cocaine users also consume alcohol. The risk of immediate death is 18 - 25 times greater for cocaine co-ingested with alcohol than for cocaine alone. Alcohol can react with cocaine to get a series of toxious compounds in body including cocaine, cocaethylene, norcocaine, norcocaethylene and benzoylecgonine. In combination of our “virtual screening of transition states” computational protocol and artificial intelligence, a novel approach was used to design BChE mutants as multiple functional cocaine hydrolases (mfCocHs) for treatment of toxicity caused by concurrent use of cocaine and alcohol. Comparing the kinetic parameters of native human BChE and mfCocH against cocaine as well as its four toxic/harmful metabolites (i.e. norcocaine, cocaethylene, norcocaethylene and benzoylecgonine) determined by us, the most effective mfCocH has at least a ~1000-fold improved catalytic efficiency against three of the substrates (cocaine, norcocaine, and cocaethylene), ~100-fold and ~10-fold improved catalytic efficiency against norcocaethylene and benzoylecgonine, respectively. In vivo studies have revealed that the mfCocH can effectively hydrolyze cocaine and its four metabolites in rats produced from the concurrent abuse of cocaine and alcohol in both addiction and overdose models. The mfCocHs was powerful antidote to treat cocaine (w/ or w/o alcohol) induced toxicity, even from the lethal toxicity after co-administrated 1 g/kg alcohol (IP) and 180 mg/kg cocaine (IP), at any time point as long as the subject is alive before treatment.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the mammalian central nervous system (CNS), but their dysregulation results in the aetiology of many human CNS disorders. Several NMDAR modulators including memantine have been used successfully in clinical trials. Indeed, 1,2,3,4-tetrahydro-9-aminoacridine (tacrine; THA) was the first approved drug for Alzheimer’s disease (AD) treatment. 7-methoxyderivative of THA (7-MEOTA) is less toxic and showed promising results in patients with tardive dyskinesia. Here, we employed electrophysiological recordings in HEK293 cells and rat neurones to examine the mechanism of action of THA and 7-MEOTA at the NMDAR. We showed that both THA and 7-MEOTA are “foot-in-the-door” open-channel blockers of GluN1/GluN2 and GluN1/GluN3 NMDARs and that 7-MEOTA is a more potent but slower blocker than THA. Furthermore, the inhibitory potency of 7-MEOTA at synaptic and extrasynaptic hippocampal NMDARs was similar, and 7-MEOTA exhibited better neuroprotective activity in rats exposed NMDA-induced lesions in hippocampus when compared with THA and memantine. Finally, intraperitoneal administration of 7-MEOTA attenuated MK-801-induced hyperlocomotion in rats. We conclude that 7-MEOTA is a promising candidate for the treatment of diseases associated with the dysfunction of NMDARs.

C. elegans is a free-living worm widely used as model to study neurotoxicology. Despite its simplicity, C. elegans has a high level of genetic and molecular conservation with vertebrates. Similar to mammals, intoxication with anti-cholinestereses triggers the accumulation of synaptic acetylcholine causing continuous stimulation of both nicotinic and muscarinic receptors, hypercontracting the muscles of the worm¹. The pharynx, the nematode feeding organ, depends on cholinergic function. Pharyngeal movements, readily observed in whole organism, are disrupted by impairments in cholinergic transmission. Therefore, quantitative analysis of pharyngeal structure and function has excellent potential to probe anti-cholinesterase mode of action that may translate to human toxicology. We establish the IC₅₀ values for the carbamate aldicarb and the organophosphates paraoxon-ethyl, paraoxon-methyl and DFP, highlighting a distinct dose-time dependence inhibition of pharyngeal activity. In recovery experiments, aldicarb and paraoxon-ethyl but not paraoxon-methyl or DFP intoxicated worms recover the pharyngeal function onto empty and oxime plates. A cycle of aldicarb intoxication-recovery-intoxication revealed aldicarb-induced plasticity as a reduced sensitivity of pre-conditioned worms to a subsequent drug exposure. We investigated molecular determinants of this plasticity by using uncoordinated locomotion and reduced pharyngeal movement mutant worms due to impairments in cholinergic transmission. Interestingly, preconditioned mutant worms exhibits a switch in the aldicarb-induced plasticity observed in wild type, becoming more sensitive to post-exposure of aldicarb. Defining the molecular identity of this mutant will reveal pathways that mediate cholinesterase induced structural reorganization at the pharyngeal NMJ. Thus, the drug and genetic tractability of C. elegans offers a new route to anti-cholinesterase poisoning antidotes.

Bis-pyridinium mono-aldoxime (BPMA) compounds are potential antidotes against organophosphorus inhibitors of either acetylcholinesterase or these of butyrylcholinesterase. From the points of drug distribution and pharmacokinetics essential characteristics were determined (concentration versus time curves). Experimental results of pharmacokinetics of BPMA will be detailed with special focus on drug distribution and HPLC analysis of oxime K117. The concentration of BPMAs decreases fast in the body of rats, and thus they fulfil the basic requirement for antidotes: elimination should be as fast as possible. Their elimination curve should be characterized by the term „tenth-life” rather than half-life. BPMA compounds penetrate into the brain in considerable amounts of their concentration in the serum. As blood-brain penetration can have vital importance, time of the maximum extent of blood-brain barrier should also be conceived as a novel pharmacokinetic parameter.

Human butyrylcholinesterase (BChE) is a stoichiometric bioscavenger of toxic organophosphates. It can be used as an antidote to protect acetylcholinesterase, and is a protein of choice for development of detoxification biocatalysts for clinical applications. Despite the number of different monomeric structures of recombinant human BChE obtained to date, all attempts to obtain an atomic structure of the natural glycosylated tetrameric BChE were unsuccessful. Here, we present for the first time the 3D structure of the natural tetrameric form of human butyrylcholinesterase, obtained by Cryo-EM technique at a final resolution of 8.8Å. The tetramer has a C2 symmetry, with all subunits arranged as a “propeller-like” tetramer. This is in contrast with previous “flat” model of subunits arrangement in tetramer. Cryo-EM structure shows that the two opposite BChE subunits are placed higher (or lower) the plane of the other two subunits. Despite glycan chains were obscured in the electron density due to their relative disoder, they could be modeled based on the positions of the residues anchoring these glycans. The electron density allowed to distinguish that C-terminal tails of all the subunits interact with each other and form a helix around the PRAD-peptide, supporting rigidity of the tail. The tail is situated in the center of the tetramer and is oriented nearly perpendicular to the tetramer “plane”.It was also observed that the subunits in the tetramer have different contacts with neighbouring subunits. This allows to consider the tetramer as a dimer of dimers which is additionally strengthened by the C-terminal tail interactions.

Blood serum proteins serve various functions, including transport of lipids, hormones, vitamins. They are responsible for maintaining acid-base balance, oncotic pressure, plasma viscosity, and functioning of the immune system. There are several hundred different proteins in the blood serum, which total concentration varies within the limits of 6.6-8.7 g/dl, but only a small amount is determined for laboratory diagnostics. One of the serum protein is butyrylcholinesterase  (BChE, EC 3.1.1.8), which exists predominantly in the form of a glycosylated tetramer (G4) with a mass of 340 kDa. Four identical subunits assemble into a tetramer by the interaction of a proline-rich peptide with the BChE tetramerization domain at the C-terminus. Our results suggest that BChE interacts with plasma proteins and form much larger complexes than predicted from mass of tetramer. In order to investigate and isolate such complexes we developed  a strategy to find protein-protein interactions by combined native size-exclusion chromatography (SEC) with affinity chromatography using resin that binds BChE. Moreover, to confirm specificity of protein complexes we performed also fractionation of blood serum proteins by density gradient ultracentrifugation followed by co-immunoprecipitation using anti-BCHE monoclonal antibodies. The proteins isolated in complexes with BChE were identified by mass spectroscopy.

For acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), several oligomeric forms are known. In vivo, the monomers in dimers are covalently bound by a C-terminal disulfide bond formed after association of the monomers. Available crystal structures were obtained for truncated forms without disulfide bonds and serve as good models for describing the role of non-covalent interactions in the dimerization of cholinesterases before linking by disulfide bonds. Here, we analyze the formation of the four-alpha-helix bundle in cholinesterases and differences between AChE and BChE dimers. To identify interactions stabilizing the four-alpha-helix bundle, we counted hydrophobic interactions, solvent accessible surface (SASA), and hydrogen bonds between monomers and estimated electrostatic contributions to dimerization. To reveal the contribution of amino acids in the area of contact to dimerization, we performed free energy perturbation (FEP) alanine screening. Potential of mean force (PMF) calculations of dimerization revealed a difference between acetylcholinesterase and butyrylcholinesterase in the dimerization process and stability of non-covalent dimers. According to replica exchange molecular dynamics umbrella sampling (REMD-US) calculations, the free energy of BChE dimerization is 20 kcal/mol, which is 15 kcal/mol less than the free energy of hAChE dimerization. BChE has less hydrophobic contacts than hAChE. Electrostatic contribution to oligomerization energy is almost the same for hAChE, mAChE, tcAChE, and BChE. In the case of BChE, contribution from the loops surrounding the helices forming bundles is less significant than that from the helices, whereas in all AChEs, vice versa. The in silico alanine screening showed that hydrophobic interactions between the helices are most important for dimerization with stabilization by charged amino acids, mostly lying on surrounding loops.

We synthesised eight derivatives of 4-aminoquinolines differing in the substituents attached to the C(4)-amino group and C(7) carbon of 4-aminoquinoline, and tested their potency to inhibit human AChE and BChE. All of the compounds reversibly inhibited both enzymes with dissociation inhibition (K_i) constants from 0.50 to 50 µM exhibiting selectivity. In other words, for all compounds, AChE exhibited higher affinity than BChE. The most potent inhibitors of AChE were compounds with an octyl chain or adamantane, regardless of the group in position C(7). The shortening of the chain length caused the AChE inhibition decrease by 5-20 times. Docking studies made it clear that the high AChE affinity resulted from simultaneous interactions of the quinoline group with aromatic residues of both the catalytic active site and the peripheral site. In conclusion, the inhibition potency and selectivity classify several novel compounds as leads for further modification and optimization towards the development of new inhibitors of AChE and potential drugs for treatment of neurodegenerative diseases.

Nerve Agents are toxic organophosphorus compounds which inhibit cholinesterases, pivotal enzyme in Parasympathetic Neurotransmission. As they are Schedule 1 compounds in accordance to Chemical Weapons Convention, strict controls are applied and some research groups may have their work hampered due to requirements for synthesis and manipulation. Nerve Agents’ surrogates have emerged as affordable substitutes for more realistic approach for development of antidotes and biochemical and toxicity studies, as they are structurally related to Nerve Agents and considered as CWC Schedule 2 compounds, yielding similar enzyme adducts. As Laboratório de Análises Químicas – LAQ (ISO 17025) at IDQBRN have been participated in OPCW Proficiency Tests, striven to obtain the “OPCW Designated Laboratory” status, we have synthesized different dialkyl alkylphosphonates for verification purposes. Therefore, we have proposed synthesis of surrogates for our research on Medicinal Chemistry using them as starting materials. They have proven to be very useful compounds in our research and our syntheses have delivered good yields and purity of final compounds.

According to the World Malaria Report, there were 216 million cases of malaria with 445000 causalties in 2016. Current anticholinesterase insecticides, such as carbamates and organophosphates, act via covalent modification of serine at the bottom of the active site. Traditional chemical insecticides are highly toxic to insect but similarly to mammals. The cysteine-targeting concept of new insecticides is focused on cysteine 447 located in the peripheral site of mosquito acetylcholinesterase. In mammalian enzyme, the cysteine residue is replaced by phenylalanine, whereas honeybees or bumble-bees have this cysteine residue protected. This approach has been proposed to overcome insecticide resistance and to develop promising environmental-friendly insecticides. The eight cysteine-targeted insecticides (succimides or maleinimides) were prepared via optimised synthetic route. The inhibitory activity of novel compounds and standards (paraoxon, bendiocarb and carbofuran) towards human acetylcholinesterase, human butyrylcholinesterase and mosquito acetylcholinesterase from Anopheles gambiae were determined using the modified spectrophotometric Ellman’s method. The potentiometric titration using acetylcholine as a substrate was used for validation of Ellman’s method. All data showed that the IC₅₀ values obtained from both methods were almost similar. Human butyrylcholinesterase was used as common off-target for acetylcholinesterase inhibitors, and no inhibitory effect was determined. The binding mode of the inhibitors was determined using the rapid dilution assay. Pyridinium maleimides were found with excellent efficacy towards mosquito acetylcholinesterase in contrast to the human enzyme and with significantly improved selectivity index compared to paraoxon. Despite some limitations, we believe that specific optimisation of the structure of molecule connected to maleimide moiety may lead to the development of novel promising insecticides. This work was supported by Ministry of Health of the Czech Republic (no. 16-34390A) and University of Defense (Long-term organization development plan Medical Aspect of Weapons of Mass Destruction).

Acetylcholinesterase (AChE) is the target of pesticides like organophosphates (OP). OP exert their toxic effect by irreversible phosphorylation of the AChE leading to cholinergic crisis and neurotoxicity. Erythrocyte AChE is the surrogate biomarker for the detection of inhibition by OP. There are numerous methods for the detection of AChE activity.¹ Unfortunately, the method popularly used for AChE detection has inherent limitations.¹ To overcome such a problem, we have explored 1-Naphthyl acetate (1-NA) as an alternative substrate for the assessment of AChE activity using in silico tools and in vitro experiments. The in silico results have shown that 1-NA is a better substrate for AChE. The fluorescence and chromogenic properties of 1-naphthol were studied. The results proved that 1-NA has specificity for AChE similar to Acetylthiocholine. Moreover, it was observed that in terms of Michaelis constant (Km) 1-NA is a better substrate than Acetylthiocholine. We believe that 1-NA is a candidate substrate for development of a method for screening of OP poisoning.

Progress in the development of biodegradable ionic liquids (ILs) [1] allowed finding sustainable fragments to assist the synthesis of sustainable molecules by means of “benign by design” approach. Based on our recent experience in creating micellar catalytic systems for decomposition of organophosphates [2, 3] we have elaborated the following oxime-functionalized low-toxic biodegradable ILs as potential AcChE reactivators: amide/ester linked (amino acid free) IL (I) as well as L-alanine (II) and L-phenylalanine (III) containing compounds with pyridinium aldoxime moiety in cationic part. Variation of amino acid variation (e.g. Me for I and phenyl for II) can help us to analyze a role of hydrophobicity of IL’s cation in AcChE reactivation. The reactivation capacity of novel ILs were evaluated towards AcChE inhibited by typical toxic organophosphate agents. The regularities of antidotal activity of studied compounds are to use in the further improvement of their structures.

Several years, there are ideas how to use reactivators of BChE in prophylaxis of OP-poisoning. They could be applied in combination with human BChE as a pseudo-catalytic scavenging system. However, the effective hBChE reactivator is still missing. The aim of this project is to find highly active and plausibly universal reactivator of hBChE. In the first phase, a database of about 6 mil. structures (ZINC Lead Like) was screened by rigid molecular docking. The receptor (hBChE) was found in the PDB database (pdb code 3DJY, hBChE inhibited by tabun) and prepared for docking. For the second phase, over one hundred molecules were selected. These structures were docked to hBChE with flexible residues within the active site. After manual inspection, over twenty molecules were chosen. Such molecules were modified (e.g. addition of oxime moiety, pK_a optimization) and redocked to hBChE with flexible residues. Finally, two novel compounds were recommended for synthesis. The newly designed compounds will be further synthesized and evaluated on the model of OP-inhibited hBChE and hAChE. They could be used for development of new series of hBChE reactivators.

Ten phenyltetrahydroisoquinoline-based compounds synthesized using alkyne+azide [3+2] building block cycloaddition were tested as potential reactivators of human acetylcholinesterase (hAChE) inhibited by different organophosphates. Computational docking indicated molecule phenyltetrahydroisoquinoline moiety association with the hAChE peripheral anionic binding site (Trp286, Tyr337 and Tyr341). Therefore, stabilization near the gorge opening seemed to control the general orientation of the pyridinium ring with its attached aldoxime group inserted into the internal gorge of the hAChE active center. All of the oximes were tested in vitro as potential reactivators of sarin-, cyclosarin-, tabun- and VX-conjugated hAChE and potent reactivators were identified, especially with the cyclosarin-hAChE conjugate. Nevertheless, in order to acquire results applicable to reactivation in vivo, compounds should be tested at concentrations higher than 10µM, which proved limiting due to the concomitant reversible inhibition of unconjugated hAChE. High oxime affinity was observed for hAChE, but not for human butyrylcholinesterase, where an aromatic peripheral site is absent. Therefore, we tested the oximes as reversible inhibitors of hAChE. All of the compounds potently inhibited hAChE with dissociation inhibition constants in nM range. To further explore potential for safe antidotal activity, we tested oxime cytotoxicity on the human neuroblastoma SH-SY5Y cell line. No cytotoxicity was observed at studied concentrations. In conclusion our study has shown that likely binding poses of an oxime in the hAChE active center do not always ensure enhanced enzyme activity for in vivo reactivation. Very high affinity of a candidate oxime for unconjugated hAChE may prove counterproductive for reactivation in tissue.

Organophosphorus compounds (OPs) irreversibly inhibit cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). There is wide variety of applications of OP compounds including warfare chemicals and pesticides. Oxime-type reactivators are used to reactivate the OP inhibited AChE and BChE. Present study was aimed to evaluate the reactivation potency of two novel oximes K456 and K733 against organophosphate inhibited AChE and BChE. Efficacy was compared with K27 and pralidoxime (2-PAM). Molecular mechanism of reactivation by the oximes is predicted by In silico method. Intrinsic toxicity of novel oximes in term of IC₅₀ and 50 % reactivation of inhibited enzymes (R₅₀) were evaluated by in vitro methods using human RBC-AChE and plasma BChE. In silico study revealed lower free binding energies, but novel oximes did not bind with catalytic anionic site of enzymes. In vitro studies showed higher intrinsic toxicity by K456 and K733 than K27 and pralidoxime. R₅₀ for human RBC-AChE were K456=203.59µM±66.96; K733= 405.55µM±67.36; K27=2.68µM ±0.98 and pralidoxime 30.71µM±5.10 (mean±SEM) respectively. No substantial reactivation in BChE was noted by tested concentration of novel oximes.  The study concludes that oximes with peripheral binding/far from catalytic anionic site are ineffective reactivators. K27 with central (inside the active gorge) binding was superior to all tested oximes.

Malaria is annually responsible for more than 400 thousands causalties. The disease is transmitted via infected female Anopheles mosquitoes. Spread of the malaria can be prevented by using either chemical compounds known as insecticides or by genetically engineered plants.^[1,2] Mechanism of action of currently deployed insecticide involves inactivating acetylcholinesterase (AChE, EC 3.1.1.7) enzyme by binding to Ser360 (Anopheles gambiae numbering). More recently, Cys447 located close to active site entrance was emerged as an alternative target to overcome insecticide resistance and also improving selectivity towards insect AChE over mammalian one.^[3] In our contribution, we have developed novel, straightforward and facile synthesis for Cys-targeted insecticides containing either maleimide or succinimide scaffolds. Employment of Grubbs olefin metathesis allowed us to obtain the final compounds in multistep synthesis in relatively high yields. We propose that the described synthetic route might be used in large scale-up for further studies.

Cholinesterases are divided into two classes according to differences in substrate specificity, behaviour in high substrate concentrations, inhibitor sensitivity and tissue distribution: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The both enzymes are sensitive to broad spectrum of molecules and may be inhibited by several compounds, such as organophosphate and carbamate pesticides or nerve agents. In a previous study, a phenazine-derived natural product, geranyl-phenazine-diol was shown to inhibit human AChE with IC50 value of 2.62 mM. Phenazines which are naturally produced by bacteria and archaeal Methanosarcina species are nitrogen containing tricyclic molecules with antibiotic, antitumor, and antiparasitic activities. Phenazines are used as electron acceptors-donors in wide range of fields including environmental biosensors. In this study, the inhibitory effect of a synthetic phenazine dye, methylene violet 3RAX (also known as diethyl safranine) was tested on human erythrocyte AChE and human plasma BChE and its inhibitory mechanism on both enzymes was studied in detail. AChE and BChE activities were assayed spectrophotometrically at 25 ^oC in 50 mM MOPS buffer pH 8, using 0.05-0.4 mM butyrylthiocholine or  0.025-0.4 mM acetylthiocholine as substrate, 0.125 mM DTNB and 0-80 µM dye. Kinetic analyses showed that methylene violet 3RAX acts as a hyperbolic noncompetitive inhibitor of AChE with K_i value of 1.42±0.09 µM; α=1 β=0.11. On the other hand, it caused linear competitive inhibition of BChE with K_i value of 0.46±0.02 µM; α=∞. In conclusion, methylene violet 3RAX with K_i value in the low micromolar range may be a promising lead candidate for the treatment of Alzheimer’s disease.

Aflatoxins are secondary metabolites of the fungi Aspergillus flavus and A. parasiticus. Among them Aflatoxin B1 (AFB1) is the most frequent type in nature and the most carcinogenic and hepatotoxic for mammals. AFB1 is also inhibitor of the enzyme acetylcholinesterase (AChE) and, therefore, a potential chemical and biological warfare agent, as well as its metabolites. In order to investigate this, we performed inedited theoretical studies on the interactions of AFB1 and its metabolites inside the catalytic and the peripheral anionic sites (CAS and PAS) of human acetylcholinesterase (HssAChE), to verify their stability, suggest the preferential ways of inhibition, and compare their behavior to each other. Molecular docking, molecular dynamics and MM-PBSA calculations for the systems HssAChE/AFB1-metabolites, on both sites were performed. All the metabolites presented negative values of interaction energies in comparison to AFB1. This suggests that they can be better inhibitors of HssAChE. Also, the energy values obtained for the CAS were lower than for the PAS for all metabolites, suggesting that they may preferentially bind in the CAS and come closer to the active site. This behavior is different from the experimentally observed for AFB1, pointing to a different way of inhibition for its metabolites.

Chlorinated bispyridinium aldoximes (Cl-oximes) analogous to previously reported efficient reactivators of inhibited AChE (K027, K048, K203) were designed and synthesized with the premise that the addition of a chlorine atom increases their lipophilicity in comparison to the reference oximes and that they could therefore achieve higher brain concentrations than the ones reported for non-chlorinated analogues. The affinity of hAChE for Cl-oximes was moderate, but higher than for analogous non-chlorinated oximes, as well as higher than the affinity of hBChE for Cl-oximes. Their reactivation efficacy for nerve agent-inhibited AChE was in the following order: cyclosarin>VX>sarin>tabun. Predictably, the electron-withdrawing effect of the chlorine atom led to a lower pK_a value of the oxime groups as confirmed by UV/VIS measurements. Finally, using the molecular modelling approach we attempted to attribute the differences in the predicted binding modes of the tested oximes to their observed reactivity. As molecular docking results suggested, the non-bonding interactions between the chlorine atoms and neighbouring amino acid residues play a significant role in the stabilization of Cl-oximes in a productive conformation in the case of cyclosarin-inhibited AChE.

An endogenous tetrameric wild-type human BChE expressed in Expi293 cells hydrolyzes the neutral substrate N-methylindoxyl acetate (NMIA) with the same K_m as wild-type huBChE (0.14 mM) [1]. For this enzyme, the steady state is preceded by a pre-steady state phase of several minutes in 10 mM Bis-Tris, pH 7 at 25°C. Thermal inactivation of this BChE is biphasic. Kinetic constants (k₁ and k₂) for thermal inactivation shows differences between this mutant and plasma derived wtBChE: the Expi293 is more stable at 55°C and less stable at 60°C than natural wtBChE [2]. At 55°C half-life times of the first and the second phases are 11 min and 43 min for plasma wtBChE; 14 min and 36 min for the Expi293 wtBChE, respectively. At 60°C, the corresponding values are 6 min and 14 min for natural wtBChE; 3 min and 11 min for Expi293 wtBChE. The endogenous enzyme is more stable in urea: urea-induced denaturation is 10 % slower than for the wtBChE and the urea concentration at the mid-point of denaturation is 4.1M for wtBChE and 4.6M for the endogenous enzyme. A bistable dynamic behavior of the endogenous BChE is also observed from pre-steady state behavior for hydrolysis of 1 mM NMIA, showing either long lags or bursts under the same conditions while plasma BChE shows only lags. Molecular mechanic simulations have been undertaken to determine the molecular basis of bistability of this wild-typeBChE.

A human embryonic kidney cell line (Expi293), adapted for suspension growth in serum-free medium, secretes a tetrameric butyrylcholinesterase (BChE). Expression levels are very low, but are increased 10-fold upon treatment with polyethylenimine. DNA sequencing shows that this enzyme is wild-type BCHE. This endogenous BChE displays catalytic properties very close to that of natural huBChE with butyrylthiocholine and N-methylindoxyl acetate as substrates [1]. Several endogenous co-secreted esterases self-reactivate after inhibition by echothiophate, paraoxon, cresyl saligenin phosphate (CBDP), racemic coumarin(CM)-soman, CM-tabun and CM-VX. Overall reactivation rate constants, k_r, of diethylphosphorylated enzymes after inhibition by echothiophate and paraoxon are 0.171 min^-1 and 0.059 min^-1, respectively, suggesting multiple OP-hydrolyzing enzymes. After phosphonylation by CM-soman, CM-tabun and CM-VX, k_r values range from 0.0375 min^-1 to 0.0078 min^-1. k_r of CBDP-inhibited enzyme is 0.028 min^-1. Interestingly, an apparent aging rate is observed after phosphylation. The aging rate of the soman-phosphonylated enzyme(s) is approximately 2-fold slower than for wtBChE (half-time =16 min against 9 min for wtBChE [2]). The half-time for aging after inhibition by CBDP is 31 min whereas aging of wtBChE-CBDP is almost instantaneous [3]. Diethylphosphorylated enzyme(s) inhibited by paraoxon and echiothiophate age(s) with apparent k_a =0.162 min^-1 and 0.057 min^-1, respectively. This difference also supports the multiple enzyme hypothesis.  Further studies are in progress to indentify the different OP-reacting enzymes produced by this Expi293 cell line.

In efficacy testing of experimental oximes, traditionally reactivation of OP-inhibited acethylcholinesterase (AChE) has been analysed by comparing the obtained effects of the single dose with the control [1]. However, quantitative analysis of in vivo dose-response data by benchmark dose (BMD) approach would improve both identification and quantification of the effect and it will allow more rigorous comparison of different oximes efficacies [2]. Thus, we have evaluated in vivo dose-response relationship for two promising experimental oximes, K203 and K027, concerning reactivation of diaphragmal AChE inhibited by dichlorvos (DDVP). To compare the oximes effects, BMD-covariate method was used to estimate oxime dose (with 90% confidence intervals) that elicits a pre-specified effect size of 50% (1.5-fold increase in AChE activity compared to DDVP-treated group). Wistar rats (5/group) were treated with oxime (0/1.25/2.5/5/25/50% LD₅₀ im) immediately after DDVP challenge (75% LD₅₀ sc). Activity of AChE was measured in rat diaphragm homogenates by modified Ellman´s method 60 min after the treatment. Dose-response modeling was done by PROAST software (version 65.5, RIVM, Nederlands). Exponential model m5-ab (y=a[c-(c-1)exp(-bx^d)]) was selected as best estimate with parameters: a_K203=0.1525, a_K027=0.1498, b_K203=0.008472, b_K027=0.03941, c=2.117 and d=0.8916. Derived BMD₅₀ were K203=117 (56, 209) and K027=21 (10, 37) µmol/kg bw, indicating that oxime K027 induces the same effect size with 5.5-times lower dose compared to oxime K203. Moreover, obtained confidence intervals of BMDs did not overlap allowing the conclusion that more potent dose-response relationship belongs to experimental oxime K027.

Female Wistar rats were percutaneously (p.c.) intoxicated (1xLD₅₀) with VX and its two derivatives differing in their substitution on nitrogen (diethyl- and dibutyl- derivatives). Blood cholinesterase activity was continuously monitored; 100 min after the intoxication (or after death), acetylcholinesterase (AChE)  activity was determined in diaphragm and brain parts (pontomedullar area - PM, frontal cortex - FC and basal ganglia – BG). Blood ChE activity remains unchanged at very short interval (5 min) after VX administration; this interval was prolonged for diethyl- and dibutyl derivatives. AChE activity was decreased to 20-30% of control values in diaphragm, then in FC (60-70%) and PM (54-74%). AChE activity in BG was relatively resistant (cca 80%). When the AChE activity was compared for all three agents in relationship to survival (11 animals) or death (7 animals), significant differences between the activities in survived (32%) and died (13%) rats were demonstrated in diaphragm but not in the blood. This tendency (higher AChE activity in survived animals) was also observed in PM and FC, however, not statistically significant. It is concluded that substitution on nitrogen atom probably influences penetration through the skin; the rest of agent molecule (phosphorus head) probably influences AChE inhibition. As hypothesis, AChE activity in diaphragm could be important for survival  or death in case of p.c. intoxication with these types of V agents.