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The novel 7-methoxytacrine-4-pyridinealdoxime agent, named hybrid 5C, is a hybrid compound comprised of a linkage between 7-methoxytacrine (7-MEOTA-4-PA) and reactivator 4-pyridinealdoxime (4-PA) moieties through a 5-carbon length-spacer. This compound was formerly designed as a prophylactic agent for intoxication by organophosphates (OP), able to form a complex with acetylcholinesterase (AChE) and reactivate this enzyme in case of OP inhibition. In order to check if the 5 carbons spacer is the ideal to maximize the interactions of this compound inside AChE, we performed in this work docking, molecular dynamics and mmpbsa studies on a series of analogues of hybrid 5C, varying the spacer-length from 1 to 10 carbons long. Our results helped to elucidate the interactions of these compounds with the different binding sites inside human AChE (HssAChE) and pointed to the 4 and 5 carbons long as the best spacers for optimizing these interactions.

Acetylcholinesterase (AChE) is a key enzyme of the Central Nervous System (CNS) hydrolyzing the neurotransmitter acetylcholine. By targeting AChE, OPNA and organophosphorus pesticides irreversibly inhibit the cholinergic transmission leading to a certain death if untreated. The current treatment available in the French army consists of an auto-injector containing a methanesulfonate salt of 2-PAM for AChE reactivation, an anticholinergic drug, atropine and avizafone, a prodrug of diazepam for limiting convulsions. However, this treatment displays major drawbacks in terms of CNS bioavailability, restricted spectrum action and effectiveness. The aim of this project is to develop a new class of more efficient human nerve agent-inhibited acetylcholinesterase. We designed, synthesized and evaluated a new class of bifunctional uncharged hybrid reactivators composed of a 3-hydroxypyridinaldoxime linked to a tacrine derivative. The in vitro efficacy of this reactivators has been assessed. We show that this new class of reactivators outperform HI-6 in restoring the human AChE activity inhibited by VX, sarin, tabun and paraoxon. By X-ray crystallography, we have been able to observe some of these new hybrids inside of the catalytic site of hAChE and TcAChE.

Newly developed oximes, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the different organs. In this work, we focused our attention on an investigation of morphological lesions produced by increasing doses of asoxime, obidoxime, K027, K048, and K075 were selected as experimental reactivators. The whole experiment was conducted on Wistar rats. All rats were sacrificed 24 hrs and 7 days after single im application of 0.1 LD₅₀, 0.5 LD₅₀ and 1.0 LD₅₀ of each reactivator. Tissue alterations were carefully quantified by semiquantitative grading scales - cardiac, diaphragm, muscular, pulmonary, gastric, hepatic and splenic damage score, respectively. Morfological structure of different tissues treated with of 0.1 LD₅₀ of all reactivators were similar to those evaluated in the control groups. Focal and reversible degenerative and vascular changes, were established in tissue samples after treatment with 0.5 LD₅₀ of asoxime, obidoxime and K027 (p < 0.01 vs. control group). Acute alterations were developed in tissue samples within 7 days following treatment with 0.5 LD₅₀ and 1.0 LD₅₀ of all reactivators. The most severe tissue alterations were found in rats treated with .0 LD₅₀ of K048 and K075 (p < 0.001 vs. control and asoxime group, respectively). Our results showed that all AChE reactivators given by a single, high, unitary dose regimen, have adverse effect not only on the main visceral and muscular tissues, but on the whole rat as well, but the exact cause-effect relationship causing cellular injury remains to be established in further investigation.

Acetylcholinesterase (AChE, E.C. 3.1.1.7) reactivators (also known as oximes) represent a class of antidotes that are used as therapeutics in cases of organophosphate (pesticide or nerve agent) poisoning. The AChE reactivators are highly hydrophilic compounds due to their quaternary nitrogen/s and hydrophilic oxime groups included in the structure. The absorption and distribution of such antidotes is limited by these structural factors. Their delivery may be improved through their encapsulation into macrocycles. Use of these vehicles may provide some retention effect or better targeting into the central nervous system via enhanced biological barriers´ permeability. Cucurbit[n]urils are a family of rigid macrocycles provided by the acid condensation of glycoluril and formaldehyde. Encapsulation of oximes K048 and asoxime by cucurbit[7]uril (CB[7]) might provide controlled/delayed drug release from a depot or enhanced biological barriers permeability. In our work we compared the cytotoxicity of oximes K048 and asoxime with their encapsulated forms using 3-(4, 5- dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Panel of five different cell lines was used . The cytotoxicity was calculated for 24 h interval after the treatment. Our results show, that oxime@CB7 complexes decrease the cytotoxic effect of oximes used individually.

A number of strategies through structural modification of pyridinium oximes have been developed to circumvent the Blood-Brain Barrier (BBB). Some of the attempted examples are (1) enhancement of lipophilicity by introduction of a fluorine atom into pyridinium ring, (2) facilitation of glucose transporters introduction of glucose moiety on the pyridinium nitrogen, (3) use of a prodrug by uncharged dihydropyridyl moiety, etc One of the strategies that our group tried was the introduction of fluorine atoms into the heterocyclic ring of pyridinium oximes to increase their lipophilicity.¹ In our continuing effort towards the development of new oxime reactivators, we were interested in monoquaternary pyridinium oximes with N-alkyl side chains, because oximes with hydrophobic side chains may penetrate the BBB more easily than 2-PAM with an N-methyl side chain. We also investigated bis-pyridinium oximes with diethyl ether linker between two pyridine rings. The synthesized pyridinium oximes were evaluated their inhibitory activities on AChE, as well as their potency to reactivate AChE inhibited by paraoxon organophosphorus agent. The plasma and brain disposition of oximes were evaluated in male ICR mice, and the oximes concentrations in the plasma and brain were measured by LC-MSMS analysis. Therefore, KR-26256 which is a bis-pyridinium oxime showed higher brain concentration as well as better brain/plasma ratio compared with HI-6.

Bis-pyridinium mono-aldoximes (BPMA) are established first-line antidotes against anticholinergic toxicants. Several novel BPMA substances are even more promising candidates, yet their pharmacological properties have not been elucidated. The most prominent candidate compounds are K-117, K-127  and K-269. A bioanalytical method employing high-performance liquid chromatography with ultraviolet absorbance detection is presented for the quantitative assay of K-117, K-127 and K-269. Following brief sample preparation consisting of extraction or dilution with 0.3 mol/L perchloric acid, the substances were determined in serum, cerebrospinal fluid, kidney, liver, eye and cochlea. The analytes were baseline-separated on a Phenomenex Kinetex EVO-C18 100x3mm (5 µm) column using reversed phase ion-pair chromatography in an isocratic run lasting 4 min and were detected at 275 nm. The employed internal standard was K-117 and K-127 for the evaluation of K-127, and K-117 and K-269, respectively. The method was validated according to the effective guideline of the European Medicines Agency. The approach has been applied for determining the pharmacokinetic properties of K-117, K-127 and K-269 in rats following intramuscular administration.

The ability of a novel bispyridinium oxime K870 and currently available oximes (pralidoxime, HI-6) to reactivate sarin-inhibited acetylcholinesterase and to reduce acute toxicity of sarin was evaluated. In vivo determined percentage of reactivation of sarin-inhibited rat blood, diaphragm and brain acetylcholinesterase showed that the potency of newly developed oxime K870 to reactivate sarin-inhibited acetylcholinesterase roughly corresponds to the reactivating efficacy of pralidoxime with the exception of diaphragm where the oxime K870 was more effective than pralidoxime. However, the oxime HI-6 was found to be the most efficient reactivator of sarin-inhibited acetylcholinesterase. While the oxime HI-6 was able to reduce the acute toxicity of sarin more than five times, the novel oxime K870 and pralidoxime decreased the acute toxicity of sarin less than three times. Based on the results, we can conclude that the reactivating and therapeutic efficacy of newly developed oxime K870 is significantly lower compared to the oxime HI-6 and, therefore, it is not suitable for the replacement of the oxime HI-6 for the antidotal treatment of acute sarin poisoning.

Mono- and bis-pyridinium aldoximes are the only causal antidotes that are designated for the treatment of organophosphate (OP) poisoning. Intoxication by OPs is caused either by pesticides or by the nerve agents, the latter belong to group of chemical warfare agents. These compounds irreversibly inhibit enzyme acetylcholinesterase (AChE) that is no more able to fulfill its physiological function. Mono- and bis-pyridinium aldoximes are able to restore catalytic function of AChE. The reactivating ability of aldoximes is limited by several drawbacks like low blood-brain barrier permeation, low reactivation potency against specific nerve agents etc. In order to obtain efficient treatment of OP, the introduction of novel AChE reactivators raised as an important issue. For over 60 years of intensive research, none of the reactivators reached sufficient activity. Herein, we present novel mono quaternary reactivators that possess excellent in vitro activity to restore AChE activity after intoxication with different nerve agents as well as pesticides. The molecular docking simulations, total synthesis and biological evaluation will be discussed.

Project is aimed at the development of new combined micellar decontamination systems based on quaternary nitrogen compounds having detergent and active decontamination properties, which will cause faster hydrolysis of chemical warfare agents. In the case of biological agents, these molecules are strong disinfectants, able to destabilize pathogen membrane structures. Several decontamination mixtures will be prepared and tested both in vitro and in vivo for their decontamination and disinfection properties against selected chemical and biological agents. The expected result of the project is efficient decontamination solution for personal skin decontamination with good tolerability.

Acetylcholinesterase (AChE; 3.1.1.7) reactivators play a key role in the treatment of organophosphate poisoning. The main mechanism of reactivators is disruption of the covalent bond between organophosphorus compounds and AChE and restore the physiological function of this enzyme. On the other hand, there are some evidence, other mechanisms not related to reactivation, which may lead to survival. Thus, their effect on muscarinic (M₁ subtype), nicotinic (α₇ subtype) and N-methyl-D-aspartat (NMDA; 2B subtype) receptor was studied. They are able to significantly modulate the receptors at higher concentration (100 µM) and for this reason, their toxicities were tested. Cytotoxicity of standard oximes was evaluated using neuroblastoma cell line SH-SY5Y. MTT assay and real-time cell viability assay were used to measure cytotoxicity of selected compounds. The tested reactivators showed different cytotoxicity. Methoxime was the most and K027 was the least toxic. Reactivators had no influence on NMDA receptor in tested concertation. The nicotinic receptor was the most inhibit by K027. However, trimedoxime and obidoxime showed the highest inhibition of muscarinic receptor.

The acute toxicity of OPNA results from irreversible inhibition of AChE (EC 3.1.1.7), a key enzyme in neurotransmission, via the formation of a covalent P–O bond at the catalytic serine. Inhibition of AChE leads to the accumulation of acetylcholine neurotransmitter (ACh) in the synaptic cleft causing among other symptoms, seizures and respiratory arrest leading to death.  The current urgency treatment of OPNA poisoning is based on the administration of a cocktail of three components: an antimuscarinic agent (e.g. atropine), an anticonvulsant drug (e.g. diazepam) and mono or bispyridinium AChE reactivator (e.g. pralidoxime, obidoxime, trimedoxime). The high nucleophilicity of these alpha-nucleophiles allows the displacement of the phosphyl group from the catalytic serine, yielding to the restoration of AChE activity.  However, reactivation of central AChE is inefficient due to the fact that positively charged pyridiniums poorly cross the brain blood barrier (BBB). Moreover pyridinium(s) oximes exhibit a quite narrow spectrum of reactivation. Despite decades of research in this field, there are no efficient and general broad-spectrum reactivators for OP-inhibited AChE.  In this context, we have developed families of new uncharged reactivators of OP-inhibited acetylcholinesterase and/or OP-inhibited butyrylcholinesterase with the potential to cross the BBB. Three new families of uncharged reactivators display in vitro reactivation potencies towards VX-, tabun- and paraoxon-inhibited human AChE that are superior to those of the mono- and bis-pyridinium aldoximes (e.g. 2-PAM, HI-6, obidoxime, HLö-7, TMB-4) which include those currently used in the armed forces.

Even though reactive oxygen/nitrogen species (ROS/RNS) are physiologically generated in biological systems, their excessive production may cause severe damage of cellular components. Excessive production of ROS/RNS can occur in response to various stressors such as xenobiotics, radiation or pathological processes. Oxidative stress has also been reported to cause adverse effects of some therapeutic drugs including acetylcholinesterase (AChE) oxime reactivators which are used in therapy of organophosphate poisoning.  In this study, we determined the effect of obidoxime, methoxime, asoxime, pralidoxime and trimedoxime on redox homeostasis in cultured human hepatoma (HepG2) cells. The cells were incubated with oximes at concentration corresponding with their IC₅₀ for 1, 4 and 24 hours. Intracellular ROS levels were determined using two fluorescent probes (2',7'‑dichlorodihydrofluorescein diacetate and dihydroethidium). Malondialdehyde and 3‑nitrotyrosine were measured using LC-MS/MS. Additionally, non-protein thiols and non-protein disulfides were evaluated to reflect antioxidant capacity. Individual reactivators displayed distinct quantitative and/or qualitative changes in redox homeostasis reflecting different role of oxidative stress in their intrinsic toxicity. Future perspectives are to test new AChE reactivators synthetized at Department of Toxicology and Military Pharmacy in order to minimalize their unwanted side effect related to oxidative stress.

This study focused on the evaluation of the use of quinuclidinium oximes as potential antidotes in organophosphorus compound (OPs) poisoning. We determined the reversible inhibition of human red blood cell acetylcholinesterase (AChE) and human plasma butyrylcholinesterase (BChE) by 14 quinuclidinium oximes as well as the reactivation of tabun-, VX-, paraoxon-, sarin- and cyclosarin-inhibited enzymes. Reversible inhibition constants were within 3 μM to 4 mM, depending on the oxime structure. The highest inhibition was observed for Q5, which has a long aliphatic chain on the quinuclidinium ring quaternary nitrogen. It seems that AChE is selective toward oximes that have groups in meta position on the benzene ring and BChE to those with a group in para position. Quinuclidinium potency to reactivate organophosphorus-inhibited cholinesterases in vitro proved promising in restoring cholinesterase activity. VX- and paraoxon-inhibited AChE was reactivated by several candidates at up to 90 - 100 % within 1-4 hours. Oximes with a group in para position showed reactivation potency for cyclosarin-inhibited BChE with reactivation up to 90-100 %. Furthermore, at the very beginning of antidote development, we investigated if quinuclidinium oximes are cytotoxic to selected cell lines. As results indicate, quinuclidinium oximes did not show cytotoxic profiles up to 800 μM. An exception was observed only for Q5, an oxime with a long aliphatic chain in the structure, influencing cell vitality at concentrations significant for reactivation of cholinesterases.

Phosphonylated butyrylcholiesterase (BChE) is a marker of exposure to organophosphorus compounds, including nerve agents and pesticides. In cases of poisoning with nerve agents, it is important not only to establish the fact of poisoning, but also to give a quantitative estimate. The most common quantitative characteristic is BChE inhibition. We developed a highly sensitive method for the quantification of BChE inhibition by Russian Vx (VR) by mass spectrometry. For model experiments we used donor human blood plasma exposed to VR at concentrations of 1‒100 ng/ml. Butyrylcholinesterase was selectively isolated from plasma by immunoprecipitation and then subjected to enzymatic hydrolysis with pepsin. The hydrolysate was analyzed by HPLC-MS/MS using MRM mode, which allowed determination of the VR-modified nonapeptide  FGESAGAAS (m/z 930 Da) at a very low level of VR (1 ng/mL). To measure the inhibition of BChE, an excess of VR is added to one sample, and the nonapeptide peak area is considered to correspond to 100% inhibition. The inhibition of BChE in samples containing different concentrations of VR are determined by ratio of the nonapeptide peak area in each specific  sample to that at 100% inhibition. BChE inhibition,% = (S₉₃₀/S₉₃₀100%)*100 It was found that the VR-modified nonapeptide peak area is linearly related to VR concentration. The BChE inhibition measured by mass spectrometry was consistent with the results of Ellman’s assay (R²≥0.98). The advantages of the proposed approach over Ellman’s assay include the possibility of quantification of inhibition at low doses of nerve agent and lack of necessity to construct calibration plots.

BACKGROUND: Managing burden of Coronary Artery Disease (CAD) is a battle for researchers over the globe as disease seems to be multifactorial. Duet concert of genetics and environmental factors over oxidative stress and inflammation accounts for disease progression. Human Paraoxonase 3 an HDL associated endogenous antioxidant enzyme, has been identified as antiatherogenic entity. Modifiable risk factors like diet and lifestyle play a supreme role in regulating paraoxonase activity. RATIONALE: To understand how the activity of Paraoxonase 3 can be modulated by using various pharmacological agents to derive the therapeutic benefit in CAD patients. METHODOLOGY: After approval of Institutional review board (No.55/IAEC/293), Hepatoma derived cell line (HepG2) was exposed to resveratrol, tempol, quercetin, simvastatin and nicotine in varying doses. MTT based optimum dose was selected and measured the PON3 enzymatic activity (Spectrophotometry/ HPLC), concentration (ELISA), cellular ROS (using H₂DCFH-DA), NOS (Griess assay) and protein expression (western blot) in cell lysates and supernatants. RESULTS: Resveratrol treatment led to significant increase in PON3 activity (p≤0.001) in HepG2 cells whereas other pharmacological agents had no major significant effect on PON3 activity, expression and concentration. CONCLUSION: PON3 induction by resveratrol translates new avenues in cardio-therapeutics.

CHEMFORASE¹ is a french innovative biotechnological start-up, built in 2016, and specialized in the manufacturing and marketing of affinity resins for purification of cholinesterases. The lead product, Hupresin®, makes it possible to purify efficiently both plasmatic and recombinant human butyrylcholinesterase (BChE). This innovative Hupresin® technology has perfect characteristics to specifically bind cholinesterases, providing the best performances on the market. CHEMFORASE also developed a fast flow affinity resin, Hupresin^® AC, efficient for the purification of plasmatic BChE. A new affinity resin with better capacity is under development. These new chromatographic supports should facilitate the large-scale production of BChE and reduce the costs associated for the production of BChE-based drugs such as nerve agents bioscavengers. Hupresin^® Magnet is the magnetic version of Hupresin^® that is compatible with the efficient extraction of BChE from small samples. This technology might facilitate the development of diagnosis tool useful for proving exposure to nerve agents and for identifying the type of poison involved. Based on its know-how, CHEMFORASE offers his knowledge for your research. The company has expertise in organic chemistry synthesis and has laboratory equipment to manage gram scale synthesis: organophosphorus nerve agents simulants, organic fluorophores, fluorescent probes, heterocyclic molecules. As part of its research and development program, CHEMFORASE is continuously seeking for new academic and industrial collaborations in order to develop innovative tools for cholinesterases applications.

Bioscavengers are considered to be a promising approach in the prophylaxis or treatment of poisoning by organophosphorus inhibitors (OPI; nerve agents and organophosphate pesticides). They can efficiently neutralize diverse OPIs in the bloodstream before they reach their natural targets - cholinesterases. Antidotal efficacy of administered butyrylcholinesterase (BChE; EC 3.1.1.8), one of the possible bioscavengers, could be further increased when it is co-administered with an oxime reactivator of a sufficient reactivation potency. Therefore, the activity of BChE, inhibited by OPI, could be continuously renewed (pseudo-catalytic bioscavenger). In this study, we evaluated the ability of standard reactivators (pralidoxime, obidoxime, HI-6, methoxime and  trimedoxime) and newly developed ones (K027, K048 and K203) to reactivate human plasma BChE inhibited by nerve agents (sarin, cyclosarin, VX and tabun) and dimethoxy and diethoxy pesticide (dichlorvos and paraoxon). Overall reactivation potency was decreased as follows: cyclosarin > sarin > VX > paraoxon > dichlorvos > tabun. HI-6 was the most efficient reactivator of cyclosarin- and sarin-inhibited BChE, whereas pralidoxime achieved highest potency for VX. Obidoxime was the most active in the case of pesticide inhibited enzyme. Reactivation of tabun-inhibited BChE was negligible for all tested compounds. Generally, reactivation ability of examined standard reactivators was deficient and uneven as they were designed for the reactivation of acetylcholinesterase. Therefore, there is a need for development of both more balanced and potent reactivators, suitable for pseudo-catalytic bioscavengers. Assayed oximes will serve for further standardization of our in vitro testing method and subsequent evaluation of newly synthesized BChE reactivators.

Human butyrylcholinesterase (hBChE) is well-known stoichiometric scavenger in case of organophosphorus (OP) intoxication. However, its major limitation lies in binding of only one OP moiety per hBChE molecule and thus necessity of its very high dosage prior or post intoxication. This issue might be resolved by use of hBChE reactivators that could cleave irreversibly bound OP moiety from the enzyme active site and restore its scavenging function. This concept has been called pseudo-catalytic scavenger. Within our contribution, we would like to present bisquaternary heteroaromatic compounds that are butyrylcholinesterase reactivators and might act as potential pseudo-catalytic bioscavengers. Recently, we have prepared and evaluated over 20 novel compounds that displayed better hBChE reactivation activity than clinically used reactivators.

Major Depressive Disorder (MDD) is a psychiatric condition. Globally, it is known to be the fourth leading source of ill health. Butyrylcholinesterase is a cholinergic enzyme with diversified reported functions. Objectives of the present study was to find the status of BChE in depressive individuals and to investigate the association of two SNPs of BCHE (rs3495; c.*189G<A) and (rs1803274; c.*1699G>A). Study was conducted with the approval from Ethical Review Board of the Department of Biosciences and consents from participants. Seventy six MDD patients and fifty four healthy controls were recruited for the study. Depressive individuals were diagnosed by the consultant psychiatrist. BChE activity was measured using plasma by Ellman’s method. The blood samples were genotyped for rs3495 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and rs1803274 by allele refractory mutation system-polymerase chain reaction (tetra-primer ARMS-PCR). Biochemical estimation of BChE showed a significant decrease activity in MDD patients (0.020 µmol/L/min; n=54) than healthy controls (0.028 µmol/L/min; n=76). Genetic analysis revealed no significant association for rs3495. However, the statistical analysis of the genotyped data of rs1803274 showed statistically significant association under dominant model (OR: 2.32; 95% CI: 1.09-4.96; p-value =0.025). Homozygous GG genotype was higher in control (p-value=0.01) as compared to the cases. Significant result was also noted in allele frequency distribution (p-value =0.01). The study concludes that BChE may have a tentative role in pathophysiology of MDD. Genetic association of rs1803274 with the disease is also evident. A further study with different ethnic groups is suggested.

The inhibitors for acetylcholinesterase (AChE), an enzyme hydrolyzing acetylcholine in cholinergic synapses, have been used for the treatment of Alzheimer's disease (AD). Alkaloids inhibiting AChE activity are commonly found in traditional Chinese medicine (TCM), e.g. gelantamine from Lycoris radiata, berberine from Coptis chinensis, huperzine A from Huperzia serrata. Many of these alkaloids also show regulatory role on inflammation, including the suppression on neuro-inflammation. Here, we aimed to reveal the possible relationship of these alkaloids in having both anti-inflammation and anti-AChE properties, in particular the role of which in “cholinergic anti-inflammatory pathway (CAP)”. A compound database containing 1,500 alkaloids from 113 kinds of TCM was developed. By molecular docking, the database was probed for AChE-inhibitory effect. Over 200 alkaloids showing AChE binding effect were further tested by its activities in inhibition of AChE, as well as in LPS-induced inflammatory responses. Thus, the current results could provide a good foundation for further research and development of TCM alkaloids on AD treatment.

Acetylcholine (ACh), the primary neurotransmitter released by vagus nerve, suppresses the levels of pro-inflammatory cytokines and tissue damage via the α7-nicotinic ACh receptor (α7-nAChR); this connection is being known as “cholinergic anti-inflammatory pathway (CAP)”, a communication between immune and nervous systems. Acetylcholinesterase (AChE) is responsible for rapid elimination of ACh in vertebrate. In the treatment of Alzheimer's disease (AD), AChE inhibitors are commonly employed. The modulatory role of AChE inhibitors in inflammation have been reported. Here, the expression profile of AChE was determined in cultured macrophages. The tetramic form of PRiMA-linked AChE was found to be the predominant form, and its glycosylation pattern was similar to that of brain AChE. The challenge of LPS induced the rate of transcription of AChE, and this induction was shown to be triggered by NFκB, a key transcription factor in regulating immune responses. In LPS-treated macrophages, the release of cytokines was inhibited by co-applied galantamine, or other AChE inhibitors, in a dose-dependent manner: this LPS-induced inflammation was also altered by over expression of PRiMA-linked AChE. In cultured macrophages, the LPS-induced cell migration, confirmed by Transwell^® motility assay, was suppressed by applied ACh, and this suppression was further enhanced by the co-applied galantamine, or other AChE inhibitors. In parallel, the levels of MMP2 and CDC42, two pro-migratory genes, were suppressed in the present of galantamine. Thus, the role of AChE in CAP needs to be elucidated.

Several flavonoids have been identified to induce the expression of AChE in PC12 cells, e.g. daidzin, irisflorentin, cardamonin and genistein. Among them, genistein is the most robust inducer for AChE activity. Genistein, 4',5,7-trihydroxyisoflavone, is a major isoflavone in soybean, which is known as phytoestrogen having known benefit to brain functions. Being a common phytoestrogen, the possible role of genistein in the brain protection needs to be further explored. In PC12 cells, application of genistein significantly induced the expression of neurofilaments, markers for neuronal differentiation. In parallel, the expression of tetrameric form of proline-rich membrane anchor (PRiMA)-linked acetyl-cholinesterase (G4 AChE), a key enzyme to hydrolyze acetylcholine in cholinergic synapses, was induced in a dose-dependent manner: this induction included the associated protein PRiMA. Genistein-induced AChE expression was fully blocked by the pre-treatment of H89 (an inhibitor of protein kinase A) and G15 (a selective G protein-coupled receptor 30 (GPR30) antagonist), which suggested a direct involvement of a membrane-bound estrogen receptor-GPR30-in the cultures. In parallel, the estrogen-induced activation of GPR30 induced AChE expression in a dose-dependent manner. The genistein/estrogen-induced AChE expression was triggered by a cyclic AMP responding element (CRE) located on the ACHE gene promoter. The binding of this CRE site by cAMP response element-binding protein (CREB) induced ACHE gene transcription. We have shown for the first time the activation of GPR30 could be one way for estrogen or flavonoids, possessing estrogenic properties, to enhance cholinergic functions in the brain, which could be a good candidate for possible treatment of neurodegenerative diseases.

Drug addiction is strongly influenced by biochemical, neuromodulator and genetics. It has been established that cholinergic system acts as neuromodulator with dopaminergic system, a major player in addiction. Putative role of cholinergic enzymes other than cocaine is hardly addressed. Present study was designed to evaluate the status of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in heroin, hashish (cannabis) and polydrug users. Study was conducted with the approval from Ethical Review Board of the Department of Biosciences and consent from participants. Twenty healthy non-addict age and sex matched individuals and eighteen male substance abusers from each group were included who fulfilled the inclusion criteria. Exclusions criteria include no chronic diseases of any kinds, no other neuronal disorders and used drugs for three or more months. Age group of non-addicts was 29.50±2.17. Age groups of the addicts were; heroin, 28.44±1.32, hashish 27.00±1.38 and polydrug users 26.06±2.27. Cholinergic enzymes were measured by Worek et al.1999 method based on Ellman’s principal. AChE was measured from whole blood and BChE from plasma. Results are expressed as (µmol/L/min; Mean±SEM). Results showed statistically significant increased activity of AChE in heroin addicts (0.029 ±0.003) than non-addicts (0.021±0.002). AChE activity in hashish and polydrug users were 0.017±0.001 and 0.016±0.033 respectively and were not statistically significant. BChE measurement showed higher enzyme activity in all three groups; 0.031±0.007, 0.027±0.006, 0.027±0.006 for heroin, hashish and polydrug users respectively. The study concluded that butyrylcholinesterase have tentative physiological roles in addiction. Further studies in this direction may lead to novel approaches in therapy.

Acetylcholinesterase (EC3.1.1.7; AChE) is one of the most important enzymes in the cholinergic system. Our previous works showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a notorious persistent organic pollutants, suppressed neuronal AChE activity by both transcriptional and post-transcriptional regulations via aryl hydrocarbon receptor pathway in SK-N-SH human neuroblastoma cells [1, 2]. In the nervous system, the most abundant cell type, astrocyte is regarded to play vital roles in protecting neurons from various kinds of insults, including environmental pollutants. Astrocytes have been considered as one of the target cells of dioxin in the nervous system. However, whether astrocytes are able to mediate indirect effect of dioxin on neuronal AChE is still unknown. In the present study, we aimed to reveal the potential indirect effect by using conditional medium derived from dioxin-treated astrocytes. Rat primary astrocytes were employed which were exposed to TCDD at 0.01 to 1 nM directly for 4 days. After the treatment, the astrocyte conditioned medium (ACM) was collected and administrated to the primary neurons on DIV (day in vitro) 2 for 4 days. Meanwhile, the primary neurons (DIV 2) from the same bench were exposed to TCDD directly at same concentrations for 4 days. The results showed that the enzymatic activity and mRNA expression of AChE was suppressed in TCDD-ACM-treated neurons compared to those of solvent-ACM-treated neurons. The effective concentrations of TCDD were 0.01 and 0.03 nM, which are close to the average serum TCDD concentration in exposed population from different areas of the world. However, AChE was less sensitive in the primary neurons directly exposed to TCDD. These results suggested that astrocytes play roles in mediating the indirect effect of TCDD on neuronal AChE expression.

We have showed before that both cholinesterases are present in rat aorta, while inhibition of butyrylcholinesterase impairs physiology of the isolated organ. The endothelium-dependent vasodilatory effect of acetylcholine (ACh) on vessels is well known, but physiological or pathological importance of this effect in live animals is questionable, and origin of possibly acting ACh unclear. Hypothesizing that aorta is a non-neuronal cholinergic tissue, the main aim of this project was to examine the presence of the proteins involved in the synthesis, storage, release, and degradation of ACh. Target-specific primers were used in RT-qPCR for determination of relative expressions and proteins were visualized by immunohistochemistry using commercially available antibodies. We confirmed the presence of high-affinity transporter and vesicular acetylcholine transporter in aorta, but no choline acetyltransferase was detected. Instead, relatively high levels of carnitine acetyltransferase were observed thus we assume this enzyme to be responsible for ACh synthesis in aorta. Additionally, present organic cation transporters OCT2 and OCT3 (but not OCT1) suggest possible involvement in ACh transmembrane transport. We confirmed the presence of both cholinesterases in rat aorta, more precisely in the smooth muscle, while no protein or activity was detected in the endothelium. Our results confirm aorta to be a non-neuronal cholinergic tissue carrying a full machinery for synthesis, storage and release and degradation of ACh.

Acetylcholinesterase (AChE) hydrolyses acetylcholine that functions as a neurotransmitter in neurons. The non-neuronal functions of AChE have been proposed in different cell types. Here, we revealed the expression of AChE in melanocyte and melanoma, in which the tetrameric (G4) form was the major isoform. In the melanogenesis of cultured B16F10 murine melanoma, the amount of AChE was markedly decreased. The differentiation of melanoma led to: (i) increase of melanin and its synthesis enzyme tyrosinase; (ii) change of intracellular cAMP level; and (iii) decrease of microphthalmia-associated transcription factor (MITF). The regulation of AChE during melanogenesis was hypothesized to be mediated by two transcriptional factors: cAMP responsive element binding protein (CREB) and MITF. In cultured melanoma, application of cAMP suppressed the expression of AChE, as well as the promoter activity of human ACHE gene. This suppression was shown to be mediated by a cAMP responsive element (CRE) located on the ACHE promoter, and mutation of this site eliminated the suppression. In melanoma, over expression of MITF induced the transcription of ACHE gene, and mutation of E-box site of the promoter blocked the induction. In parallel, application of an AChE inhibitor in vitro greatly enhanced acetylcholine-mediated responses of melanogenic gene expressions; but the enhancement was not revealed in the present of agonists of muscarinic acetylcholine receptor. Therefore, our results indicated that AChE transcription is specifically regulated by cAMP-dependent signaling pathway during melanogenesis of B16F10 cells, suggesting a potential role of AChE being played in this differentiation process.

Alzheimer’s disease (AD) is a broadly spread neurodegenerative disorder of ageing population manifesting itself in progressing loss of cognitive functions down to total demolition of intellect and disability. Profound synaptic dysfunction contributes to early loss of short-term memory in Alzheimer’s disease. Here we show the protective effects against amyloid-induced synaptic toxicity of C-35, a potent reversible inhibitor of acetylcholinesterase (AChE). Crystal structure of the complex between human AChE and C-35 revealed tight contacts of ligand along the enzyme active site gorge. Molecular dynamics simulations indicated that the external flexible part of the ligand establishes multiple transient interactions with the enzyme peripheral anionic site. Thus, C-35 is a dual binding site inhibitor of AChE. In amyloid-transgenic mice, C-35, when administered after disease onset, reversed synapse loss, decreased the number of amyloid plaques and restored learning and memory. When administration of C-35 and the clinically relevant AChE dual inhibitor donepezil was terminated three weeks after the trial started, animals, that were receiving C-35 showed a much better ability to learn than those who received physiological saline or donepezil. Our results provide evidence that C-35 has a more pronounced Alzheimer’s disease-modifying action than donepezil.

Drug candidate 1-EN-142 was designed and synthesized as a multipotent therapeutic agent to treat Alzheimer´s disease. In its molecule it combines tacrine moiety with naphthoquinone scaffold. For the study of centrally-active molecules in biological samples it is necessary to develop appropriate detection methodology that would determine such compounds in low concentration. Spectroscopy based on Surface-Enhanced Raman Scattering (SERS) was chosen as a comparative method for the electronic detection of compound 1-EN-142 by interdigitated impedance sensor decorated with gold nanoparticles. Since spectroscopic data were not available for this new drug candidate, it was necessary, as well, to acquire its classical Raman spectra in the solution and the solid state. SERS-active substrates were prepared by straightforward procedure so that 20 nm thick layer of gold was deposited by fast magnetron sputtering on silicon wafer. The substrates with roughened gold surface were immersed in solution of 1-EN-142 in methanol for 30 min and dried in the stream of nitrogen. SERS spectra of 1-EN-142 were obtained as an average of 100 spectra measured from an array of 20 x 5 points with 2 mm spacing. Subsequently, the reference spectrum, obtained by the same procedure from a SERS substrate unexposed to 1-EN-142, was subtracted, and the spectrum baseline was corrected using cubic splines. SERS spectra were recorded with a Raman microspectrometer using excitation wavelengths of 633 nm and 785 nm, respectively. Raman spectrum of 1-EN-142 solution in methanol in the range of 390 – 1741 cm^-1 was collected with laser excitation of 532 nm. SERS has proved to be a suitable method of detecting compound 1-EN-142.

Current symptomatic treatment has only limited clinical efficacy and minute effect on progression of Alzheimer’s disease. The research focus has thus shifted from single targets towards multifunctional ligands targeting several pathological processes of the disease [1, 2]. A potent picomolar selective inhibitor of human butyrylcholinesterase [3] was used as the starting point to develop a new series of multifunctional ligands. A focused library of derivatives was designed and synthesized that showed both butyrylcholinesterase inhibition and good antioxidant activity comparable to natural antioxidants. The crystal structure of compound 11 in complex with butyrylcholinesterase revealed the molecular basis for its low nanomolar inhibition of butyrylcholinesterase (K_i = 1.09 ±0.12 nM). In addition, compounds 8 and 11 show metal-chelating properties as determined by the UV-Vis titrations, and reduce the redox activity of chelated Cu²⁺ ions in a Cu-ascorbate redox system. Compounds 8 and 11 decrease intracellular levels of reactive oxygen species, and are not substrates of the active efflux transport system, as determined in Caco2 cells. Compound 11 also protects neuroblastoma SH-SY5Y cells from toxic Aβ_1–42 species. These data indicate that compounds 8 and 11 are promising multifunctional lead ligands for treatment of Alzheimer’s disease.

Alzheimerʼs disease (AD) is a devastating neurodegenerative disorder characterized by a severe, progressive loss of memory. Currently, AD therapy is limited on the administration of cholinesterase inhibitors (ChEIs) and the N-methyl-D-aspartate (NMDA) antagonist, memantine. Tacrine as the first registered acetylcholinesterase (AChE, E.C. 3.1.1.7) inhibitor was withdraw due to its adverse effects. 7-Methoxytacrine (7-MEOTA) was prepared as a pharmacologically equal active compound with lower toxicity compared to THA. Donepezil as a highly selective inhibitor for AChE was connected with 7-MEOTA scaffold due to the ability to interact within calatytic anionic site (CAS) as well as peripheral anionic site (PAS) regions of AChE [1]. Recent research has been focused on studying the association between the intracellular amyloid beta (Aβ) cascade and the dysfunction of subcellular organelles, especially mitochondria. Mitochondrial enzyme amyloid beta binding alcohol dehydrogenase (ABAD) might contribute to the neuronal dysfunction associated with AD by interacting with intracellular Aβ [2]. These derivatives embodying 7-MEOTA and donepezil moieties [3] could be effective in the treatment of AD with the respect of their ability to interact with the multiple targets. Within our contribution, synthesis, in vitro biological evaluation including cholinesterase inhibitory activity and effects on mitochondrial function of 7-MEOTA-donepezil series will be reported.