Human AChE Research Articles

Synthesis and kinetic analysis of some phosphonate analogs of cyclophostin as inhibitors of human acetylcholinesterase

Two new monocyclic analogs of the natural AChE inhibitor cyclophostin and two exocyclic enol phosphates were synthesized. The potencies and mechanisms of inhibition of the bicyclic and monocyclic enol phosphonates and the exocyclic enol phosphates toward human AChE are examined. One diastereoisomer of the bicyclic phosphonate exhibits an IC 50 of 3 μM. Potency is only preserved when the cyclic enol phosphonate is intact and conjugated to an ester. Kinetic analysis indicates both a binding and a slow inactivation step for all active compounds. Mass spectrometric analysis indicates that the active site Ser is indeed phosphorylated by the bicyclic phosphonate.

Kinetic analysis of interactions of different sarin and tabun analogues with human acetylcholinesterase and oximes: Is there a structure–activity relationship?

The repeated misuse of highly toxic organophosphorus compound (OP) based chemical warfare agents in military conflicts and terrorist attacks poses a continuous threat to the military and civilian sector. The toxic symptomatology of OP poisoning is mainly caused by inhibition of acetylcholinesterase (AChE, E.C. 3.1.1.7) resulting in generalized cholinergic crisis due to accumulation of the neurotransmitter acetylcholine (ACh) in synaptic clefts. Beside atropine as competitive antagonist of ACh at muscarinic ACh receptors oximes as reactivators of OP-inhibited AChE are a mainstay of standard antidotal treatment. However, human AChE inhibited by certain OP is rather resistant to oxime-induced reactivation. The development of more effective oxime-based reactivators may fill the gaps. To get more insight into a potential structure–activity relationship between human AChE, OPs and oximes in vitro studies were conducted to investigate interactions of different tabun and sarin analogues with human AChE and the oximes obidoxime and HI 6 by determination of various kinetic constants. Rate constants for the inhibition of human AChE by OPs, spontaneous dealkylation and reactivation as well as reactivation by obidoxime and HI 6 of OP-inhibited human AChE were determined. The recorded kinetic data did not allow a general statement concerning a structure–activity relationship between human AChE, OP and oximes.

Next generation OP-bioscavengers: A circulatory long-lived 4-PEG hypolysine mutant of F338A-HuAChE with optimal pharmacokinetics and pseudo-catalytic characteristics

We have shown previously that conjugation of polyethylene glycol (PEG) chains to recombinant human acetylcholinesterase (rHuAChE) results in the extension of its residence time in the circulation of mice and monkeys [1,2]. By profiling the pharmacokinetic behavior of an array of well-defined hypolysine human mutant AChE molecules following PEGylation, we now determine that the duration of these enzyme forms in the circulation of rhesus macaques correlates with their number of appended PEG moieties, and is influenced by the actual location of the PEG chains at the molecule surface, as well. These findings, which concur with those we have previously established in mice, indicate that a common set of rules dictates the circulatory fate of PEGylated HuAChEs in rodents and non-human primates. In addition to its effect on circulatory residence, PEGylation reduces the ability of the rHuAChE bioscavenger to elicit an immune response in the heterologous mouse animal system. Thus, an inverse relationship between anti-AChE antibody production and PEG loading was observed following repeated administration of the different PEGylated hypolysine human AChEs to mice. We note however, that in rhesus macaques, the essentially homologous (human) AChE does not induce specific anti-AChE antibodies after repeated administration of high doses of the enzyme in its PEGylated form, and even in its non-PEGylated form. Taken together, these findings indicate that PEG acts by veiling enzyme-related epitopes, which would otherwise interact with host circulatory elimination pathways and immune system. The barring of such interactions by obstructive PEGs, confers the enzyme molecule with both extended circulatory residence and mitigated immunogenic properties. Further modulation by incorporation of the F338A mutation into the PEGylated hypolysine rHuAChE enzyme mold, resulted in the generation of an OP-bioscavenger that displayed reduced aging rates and could effectively protect mice against repeated exposure to CW agents. This selected 4-PEG F338A-AChE can serve as a paradigm for new generation OP-bioscavengers, specifically tailored for prophylactic treatment against toxic OP-agents.

Respective roles of the catalytic domains and C‐terminal tail peptides in the oligomerization and secretory trafficking of human acetylcholinesterase and butyrylcholinesterase

Butyrylcholinesterase (BChE) and the T splice variant of acetylcholinesterase that is predominant in mammalian brain and muscles (AChE(T)) possess a characteristic C-terminal tail (t) peptide. This t peptide allows their assembly into tetramers associated with the anchoring proteins ColQ and PRiMA. Although the t peptides of all vertebrate cholinesterases are remarkably similar and, in particular, contain seven strictly conserved aromatic residues, these enzymes differ in some of their oligomerization properties. To explore these differences, we studied human AChE (Aa) and BChE (Bb), and chimeras in which the t peptides (a and b) were exchanged (Ab and Ba). We found that secretion was increased by deletion of the t peptides, and that it was more efficient with a than with b. The patterns of oligomers were similar for Aa and Ab, as well as for Ba and Bb, indicating a predominant influence of the catalytic domains. However, addition of a cysteine within the aromatic-rich segment of the t peptides modified the oligomeric patterns: with a cysteine at position 19, the proportion of tetramers was markedly increased for Aa(S19C) and Ba(S19C), and to a lesser extent for Bb(N19C); the Ab(N19C) mutant produced all oligomeric forms, from monomers to hexamers. These results indicate that both the catalytic domains and the C-terminal t peptides contribute to the capacity of cholinesterases to form and secrete various oligomers. Sequence comparisons show that the differences between the t peptides of AChE and BChE are remarkably conserved among all vertebrates, suggesting that they reflect distinct functional adaptations.

Hysteresis of insect acetylcholinesterase

Pre-steady-state catalytic properties of insect acetylcholinesterase (AChE, EC 3.1.1.7) were studied with the neutral substrate N-methylindoxylacetate. Kinetics of soluble Apis mellifera and Drosophila melanogaster AChE forms showed lags ( v i = 0 ) before reaching the steady-state. Results were interpreted in terms of slow equilibrium between two conformational states E and E′ of insect AChE. Hysteresis of insect AChE has been pointed out for the first time. The hysteretic behaviour was found to depend on the NMIA concentration and the nature of the enzyme. The maximum induction times ( τ max) to reach the steady-state were 800 and 1000 s with soluble AChE from A. mellifera and D. melanogaster, respectively. The orders of magnitude of the τ max were high and similar to human AChE and BuChE.

Antisense Treatment for Myasthenia Gravis

Acetylcholinesterase pre-mRNA is susceptible to alternative splicing. Myasthenia gravis has been shown to be associated with the expression of the readthrough transcript (AChE-R), which, unlike the normal "synaptic" transcript (AChE-S) is not tethered to the post-synaptic membrane, but is a soluble monomer in the synaptic cleft. In rats with experimental autoimmune myasthenia gravis (EAMG), inhibition of production of AChE-R using antisense is associated with a significant reduction in synaptic expression of AChE-R mRNA and protein, with improved muscle strength and stamina and increased survival. Synaptic AChE does not appear to be significantly affected by the induction of EAMG or treatment with antisense to AChE-R. Monarsen (EN101) is a synthetic 20-base antisense oligodeoxynucleotide directed against the human AChE gene. It is modified to achieve stability for oral administration. Sixteen patients with seropositive myasthenia gravis who were responsive to pyridostigmine were withdrawn from it and treated with Monarsen. Fourteen patients experienced a clinically significant response. In some, the improvement was dramatic. Although the dose of pyridostigmine was not optimized before the study, the majority of responders achieved better Quantitative Myasthenia Gravis scores than on pyridostigmine. The response of an individual muscle group to Monarsen was related to the degree of deterioration following the withdrawal of pyridostigmine. Cholinergic side effects were conspicuous by their absence. Monarsen is now being investigated in a phase II study.

Potency of several oximes to reactivate human acetylcholinesterase and butyrylcholinesterase inhibited by paraoxon in vitro

Organophosphorus pesticides (e.g. chlorpyrifos, malathion, and parathion) and nerve agents (sarin, tabun, and VX) are highly toxic organophosphorus compounds with strong inhibition potency against two key enzymes in the human body—acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8). Subsequent accumulation of acetylcholine at synaptic clefts can result in cholinergic crisis and possible death of intoxicated organism. For the recovery of inhibited AChE, derivatives from the group of pyridinium or bispyridinium aldoximes (called oximes) are used. Their efficacy depends on their chemical structure and also type of organophosphorus inhibitor. In this study, we have tested potency of selected cholinesterase reactivators (pralidoxime, obidoxime, trimedoxime, methoxime and H-oxime HI-6) to reactivate human erythrocyte AChE and human plasma BuChE inhibited by pesticide paraoxon. For this purpose, modified Ellman's method was used and two different concentrations of oximes (10 and 100 μM), attainable in the plasma within antidotal treatment of pesticide intoxication were tested. Results demonstrated that obidoxime (96.8%) and trimedoxime (86%) only reached sufficient reactivation efficacy in case of paraoxon-inhibited AChE. Other oximes evaluated did not surpassed more than 25% of reactivation. In the case of BuChE reactivation, none of tested oximes surpassed 12.5% of reactivation. The highest reactivation efficacy was achieved for trimedoxime (12.4%) at the concentration 100 μM. From the data obtained, it is clear that only two from currently available oximes (obidoxime and trimedoxime) are good reactivators of paraoxon-inhibited AChE. In the case of BuChE, none of these reactivators could be used for its reactivation.

Oximes: Reactivators of phosphorylated acetylcholinesterase and antidotes in therapy against tabun poisoning

One of the therapeutic approaches to organophosphate poisoning is to reactivate AChE with site-directed nucleophiles such as oximes. However, pyridinium oximes 2-PAM, HI-6, TMB-4 and obidoxime, found as the most effective reactivators, have limiting reactivating potency in tabun poisoning. We tested oximes varying in the type of ring (pyridinium and/or imidazolium), the length and type of the linker between rings, and in the position of the oxime group on the ring to find more effective oximes to reactivate tabun-inhibited human erythrocyte AChE. Three of our tested pyridinium oximes K027, K048, K074, along with TMB-4, were the most promising for AChE reactivation. Promising oximes were further tested in vivo on tabun poisoned mice not only as antidotes in combination with atropine but also as pretreatment drug. Herein, we showed that a promising treatment in tabun poisoning by selected oximes and atropine could be improved if oximes are also used in pretreatment. Since the reactivating efficacy of the oximes in vitro corresponded to their therapeutic efficacy in vivo, it seems that pharmacological effect of these oximes is indeed primarily related to the reactivation of tabun-phosphorylated AChE.

In vitro screening of acetylcholinesterase reactivating potency and oxime assisted organophosphate hydrolysis for a library of novel oxime reactivators synthesized by “click‐chemistry”.

Organophosphorus (OP) pesticides and nerve warfare agents stoichiometrically inactivate acetylcholinesterase (AChE; EC ) by phosphylation of the active serine 203. Catalytic activity of the covalently inhibited AChE can be effectively reclaimed only by nucleophiles stronger than water. In this study we analyzed reactivating potency of close to 100 novel oximes synthesized using [3+2] cycloaddition between alkyne and azide building blocks, the “Click reaction”. Human AChE (hAChE) covalent adducts resulting from inhibition by nerve agents soman, sarin, cyclosarin, VX and tabun and pesticide derived paraoxon were used for reactivation experiments. We were able to identify several oximes with significantly improved reactivating efficacy against larger spectrum of offending OPs. We furthermore measured rates of hydrolysis of fluorescent derivatives of nerve agents soman, sarin, cyclosarin and VX assisted by oxime reactivators, for the wt AChE and Tyr337Ala mutant of hAChE, and found that in the presence of selected oximes rates of OP hydrolysis increased up to forty times with wt AChE and more with the Tyr337Ala hAChE mutant.

Viral vector-medicated expression of human collagen Q in cultured cells

Congenital myasthenic syndromes are caused by mutations in molecules expressed at the neuromuscular junction. Collagen Q (ColQ) makes a triple helical structure and anchors the catalytic subunit of acetylcholinesterase (AChE) to the synaptic basal lamina in the form of asymmetric AChE. Mutations in the collagen Q gene ( COLQ) cause endplate AChE deficiency. As an initial step to develop a novel therapeutic strategy for endplate acetylcholinesterase deficiency, we expressed AChE species in cultured cells using retrovirus and adeno-associated virus (AAV). The retroviral vectors carried human ACHE and COLQ either in a single construct (EF1α- ACHE-IRES- COLQ) or in two separate constructs (EF1α- ACHE and EF1α- COLQ). We produced high-titer retroviruses using the PLAT-E retrovirus packaging cells. We also confirmed expression of asymmetric AChE in the PLAT-E cells. We infected NIH3T3 and confirmed expression of the transgenes by RT-PCR. The AAV vector carried human COLQ-IRES- EGFP downstream of the CMV promoter (pAAV-CMV- COLQ-IRES- EGFP). We produced recombinant AAV using HEK293 cells carrying pDF6 encoding the AAV6 capsid gene. We infected AAVHT1080 cells and confirmed expression of COLQ by RT-PCR and EGFP by flow cytometry. We are currently trying to achieve further higher expression levels of transgenes in cultured cells to make the current strategy applicable to an animal model.

Structure-activity study of phosphoramido acid esters as acetylcholinesterasf inhibitors

Phosphoramido acid esters (CH3)2NP(O)X(p-OC6H4-CH3) (containing P-Cl (1), P-O (2), P-F (3), P-CN (5), and P-N (4,6) bonds, X for 2, 4 and 6 is OCH3, (C2H5)2N and morpholin) have been synthesized to investigate the structure-activity study of AChE enzyme inhibition, through the parameters logP, δ31P and IC50. After their characterization by 31P, 31P{1H}, 13C, 1H NMR, IR and mass spectroscopy, the parameters logP and δ31P (31P chemical shift in NMR) were used to evaluated the lipophilicity and electronical properties. The ability of compounds to inhibit human AChE was predicted by PASS software (version 1.193), and experimentally evaluated by a modified Ellman's assay.

Multi-Target-Directed Drug Design Strategy: From a Dual Binding Site Acetylcholinesterase Inhibitor to a Trifunctional Compound against Alzheimer’s Disease

A design strategy to convert a dual-binding site AChE inhibitor into triple functional compounds with promising in vitro profile against multifactorial syndromes, such as Alzheimer's disease, is proposed. The lead compound bis(7)-tacrine (2) was properly modified to confer to the new molecules the ability of chelating metals, involved in the neurodegenerative process. The multifunctional compounds show activity against human AChE, are able to inhibit the AChE-induced amyloid-beta aggregation, and chelate metals, such as iron and copper.

Pathogenic point mutations in a transmembrane domain of the ε subunit increase the Ca2+ permeability of the human endplate ACh receptor

The epsilon subunit of the human endplate ACh receptor (AChR) is a key determinant of the large fraction of the ACh-evoked current carried by Ca2+ ions (P(f)). Consequently, missense mutations in the epsilon subunit are potential targets for altering the P(f) of human AChR. In this paper we investigate the effects of two pathogenic point mutations in the M2 transmembrane segment AChR epsilon subunit, epsilonT264P and epsilonV259F, that cause slow-channel syndromes (SCS). When expressed in GH4C1 cells, the mutant receptors subunits raise Ca2+ permeability of the receptors approximately 1.5 and approximately 2-fold above that of wild-type, to attain P(f) values of 11.8% (epsilonT264P) and 15.4% (epsilonV259F). The latter value exceeds most P(f) values reported to date for ligand-gated ion channels. Consistent with these findings, the biionic Ca2+ permeability ratio (P(Ca)/P(Cs)) of the mutant AChRs is also increased. Upon repetitive stimulation with ACh, the mutant receptors show an enhanced current run-down compared with wild-type, leading to a strong reduction of their function. We propose that the enhanced Ca2+ permeability of the mutant receptors overrides the protective effect of desensitization and, together with the prolonged opening events of the AChR channel, is an important determinant of the excitotoxic endplate damage in the SCS.

The Kinetics of Inhibition of Human Acetylcholinesterase and Butyrylcholinesterase by Two Series of Novel Carbamates

Controlled inhibition of brain acetyl- and butyrylcholinesterases (AChE and BChE, respectively) and of monoamine oxidase-B (MAO-B) may slow neurodegeneration in Alzheimer's and Parkinson's diseases. It was postulated that certain carbamate esters would inhibit AChE and BChE with the concomitant release in the brain of the OH-derivatives of rasagiline or selegiline that can serve as inhibitors of MAO-B and as antioxidants. We conducted a detailed in vitro kinetic study on two series of novel N-methyl, N-alkyl carbamates and compared them with rivastigmine, a known anti-Alzheimer drug. The rates of carbamylation (k(i)) and decarbamylation (k(r)) of recombinant human AChE were mainly determined by the size of the N-alkyl substituent and to a lesser extent by the nature of the leaving group. k(i) was highest when the alkyl was methyl, hexyl, cyclohexyl, or an aromatic substituent and lowest when it was ethyl. This suggested that k(i) depends on a delicate balance between the length of the residue and its degree of freedom of rotation. By contrast, presumably because of its wider gorge, inhibition of human BChE was less influenced by the size of the alkyl group and more dependent on the structure of the leaving group. The data show how the degree of enzyme inhibition can be manipulated by structural changes in the N-methyl, N-alkyl carbamates and the corresponding leaving group to achieve therapeutic levels of brain AChE, BChE, and MAO-B inhibition.

Kinetic analysis of reactivation and aging of human acetylcholinesterase inhibited by different phosphoramidates

The high number of fatalities due to poisoning by organophosphorus compound-based (OP) pesticides and the availability of highly toxic OP-type chemical warfare agents (nerve agents) emphasize the necessity for an effective medical treatment. Acute OP toxicity is mainly caused by inhibition of acetylcholinesterase (AChE, EC 3.1.1.7). Reactivators (oximes) of inhibited AChE are a mainstay of treatment. However, human AChE inhibited by certain OP, e.g. the phosphoramidates tabun and fenamiphos, is rather resistant towards reactivation by oximes while AChE inhibited by others, e.g. the phosphoramidate methamidophos is easily reactivated by oximes. To get more insight into a potential structure-activity relationship human AChE was inhibited by 16 different tabun analogues and the time-dependent reactivation by 1 mM obidoxime, TMB-4, MMB-4, HI 6 or HLö 7, the reactivation kinetics of obidoxime and the kinetics of aging and spontaneous reactivation were investigated. A clear structure-activity relationship of aging, spontaneous and oxime-induced reactivation kinetics could be determined with AChE inhibited by N-monoalkyl tabun analogues depending on the chain length of the N-alkyl residue. N, N-dialkyl analogues bearing ethyl and n-propyl residues were completely resistant towards reactivation while N, N-di-i-propyl tabun was highly susceptible towards reactivation by oximes. AChE inhibited by phosphonoamidate analogues of tabun, bearing a N, N-dimethyl and N, N-diethyl group, could be reactivated and had comparable reactivation kinetics with obidoxime. These results in conjunction with previous data with organophosphates and organophosphonates emphasizes the necessity for kinetic studies as basis for future work on structural analysis with human AChE and for the development of effective broad-spectrum oximes.

Characterization of reversible and pseudo-irreversible acetylcholinesterase inhibitors by means of an immobilized enzyme reactor

The aim of the present study was the application of a human AChE-CIM-IMER (enzyme reactor containing acetylcholinesterase immobilized on a monolithic disk) for the rapid evaluation of the thermodynamic and kinetic constants, and the mechanism of action of new selected inhibitors. For this application, human recombinant AChE was covalently immobilized onto an ethylenediamine (EDA) monolithic Convective Interaction Media (CIM) disk and on-line studies were performed by inserting this IMER into a HPLC system. Short analysis time, absence of backpressure, low nonspecific matrix interactions and immediate recovery of enzyme activity were the best characteristics of this AChE-CIM-IMER. Mechanisms of action of selected reversible inhibitors (tacrine, donepezil, edrophonium, ambenonium) were evaluated by means of Lineweaver–Burk plot analysis. Analyses were performed on-line by injecting increasing concentrations of the tested inhibitor and substrate and by monitoring the product peak area. AChE-CIM-IMER kinetic parameters ( K m app and v max app ) were derived as well as inhibitory constants ( K i app ) of selected compounds. Moreover, noteworthy results were obtained in the application of the AChE-CIM-IMER to the characterization of the carbamoylation and decarbamoylation steps in pseudo-irreversible binding of carbamate derivatives (physostigmine and rivastigmine). AChE-CIM-IMER appeared to be a valid tool to determine simultaneously the kinetic constants in a reliable and fast mode. The obtained values were found in agreement with those obtained with the classical methods with the free enzyme. Furthermore, after inactivation by carbamates, activity could be fully recovered and the AChE-CIM-IMER could be reused for further studies. Results showed that the AChE-CIM-IMER is a valid tool not only for automated fast screening in the first phase of the drug discovery process but also for the finest characterization of the mode of action of new hit compounds with increased accuracy and reproducibility and with saving of time and materials.

Asymmetric fluorogenic organophosphates for the development of active organophosphate hydrolases with reversed stereoselectivity

In order to enhance the enzymatic detoxification rate of organophosphorus (OP) nerve agents we have searched for more active variants of recombinant mammalian paraoxonase (PON1). We have previously identified three key positions in PON1 that affect OP hydrolysis: Leu69, Val346 and His115, that significantly enhance the hydrolysis of cyclosarin (GF), soman, chlorpyrifos-oxon (ChPo), O-isopropyl- O-( p-nitrophenyl)methylphosphonate (IMP- pNP) and diisopropyl fluorophosphate (DFP). GC/FPD analysis compared to residual AChE inhibition assay displayed stereoselective hydrolysis of GF, soman and IMP- pNP, indicating that wild type PON1 and its variant V346A are more active toward the less toxic P(+) optical isomer. In order to obtain new PON1 variants with reversed stereoselectivity, displaying augmented activity toward the more toxic isomer P(−) of nerve agents, we synthesized new asymmetric fluorogenic OPs (Flu-OPs). Six Flu-OPs were prepared containing either ethyl (E), cyclohexyl (C) or pinacolyl (P) alkyl radicals attached to methyl-phosphonyl (MP) moiety analogous to the structure of VX, GF and soman, respectively. The fluorescent moieties are either 3-cyano-4-methyl-7-hydroxy coumarin (MeCyC) or 1,3-dichloro-7-hydroxy-9,9-dimethyl-9 H-acridin-2-one (DDAO). The kinetics of AChE and BChE inhibition by these new Flu-OPs display k i values 8.5 × 10 4 to 8.5 × 10 7 and 5 × 10 4 to 2 × 10 6 M −1 min −1, respectively. EMP–MeCyC and EMP–DDAO are the most active inhibitors of AChE whereas CMP–MeCyC and CMP–DDAO are better inhibitors of BChE than AChE, indicating accommodation of bulky cyclohexyl group inside the active site of BChE. PMP–MeCyC and PMP–DDAO are the least active inhibitors of both AChE and BChE. CMP–MeCyC and CMP–DDAO were significantly detoxified only by the five-site mutations PON1 variant L69V/S138L/S193P/N287D/V346A. Degradation kinetics of Flu-OPs measured by increase in absorbance of the released fluorogenic group was fit by a two exponential function, indicating faster hydrolysis of the less toxic optical isomer. Interestingly, wt PON1 caused only 50% degradation of racemic EMP–MeCyC, CMP–MeCyC and CMP–DDAO indicating complete hydrolysis of P(+) isomer. This remarkable stereoselectivity was used for the enzymatic separation of the P(−) isomer of CMP–MeCyC. The bimolecular rate constant k i for human AChE inhibition by the isolated P(−) isomer of CMP–MeCyC is five-fold larger than that of its P(+) isomer. The marked preference of wt PON1 toward P(+) stereo-isomer of CMP–MeCyC and CMP–DDAO renders their P(−) stereo-isomers suitable for the selection of new OP hydrolase variants with reversed stereoselectivity.

Evaluation of monoquaternary pyridinium oximes potency to reactivate tabun-inhibited human acetylcholinesterase

Monoquaternary N-benzyl-4-hydroxyiminomethylpyridinium bromide (Py-4-H) and its analogous with diverse substituents introduced into the phenyl ring (Py-4-CH 3, Py-4-Br, Py-4-Cl and Py-4-NO 2) were synthesized in order to examine their potency as reactivators of tabun-inhibited human erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7). Within 24 h, the reactivation of tabun-inhibited AChE reached 80% with Py-4-CH 3, Py-4-Br and Py-4-Cl, 40% with Py-4-NO 2, and 30% with Py-4-H. The overall reactivation rate constants were up to 5.0 min −1 M −1. All oximes inhibited human AChE reversibly, and the inhibition potency increased in the following order Py-4-Br < Py-4-Cl < Py-4-CH 3 < Py-4-H < Py-4-NO 2. Although oximes Py-4-H and Py-4-NO 2 did not show significant reactivation ability, these oximes might be of interest as pre-treatment drugs due to their high affinity for the native AChE. Docking studies were carried out to elucidate the differences in oximes potency. The orientations of all studied oximes in the active site of human AChE have been proposed by flexible ligand docking with AutoDock 3.0. Analyses of the obtained complexes revealed the presence of numerous hydrogen bonds and close contacts between the oximes and the residues in the active site. Final docked energies predicted correctly the relative order of the inhibition potency of compounds (except in the case of Py-4-CH 3) as well as the most probable orientation of the best reactivator, Py-4-Br, which can result in an attack on the phosphorus atom of the tabun-phosphorylated human AChE.

In vitro interactions between pyridinium aldoximes and endogenous bio-scavenger butyrylcholinesterase in tabun poisoning

Kinetic parameters were evaluated for inhibition and reactivation of tabun-inhibited human plasma butyrylcholinesterase (BChE ; EC 3.1.1.8) by five pyridinium aldoximes: K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide], K033 [1, 4-bis(2-hydroxyiminomethylpyridinium) butane dibromide], HI-6 [(1-(2’ -hydroxyimi-nomethyl-1'-pyridinium)-3-(4''-carbamoyl-1''-pyridinium)-2-oxapropane dichloride)] and TMB-4 [1, 3-bis(4-hydroxyiminomethylpyridinium) propane dibromide]. Selected aldoximes have been recently evaluated as protectors/reactivators of tabun-inhibited human erythrocyte AChE [1]. Since BChE can be considered an endogenous bio-scavenger of anticholinesterase compounds, in order to discuss the possible use of BChE as scavenging agent, and BChE prophylactic properties, we examined aldoxime interactions with BChE. Progressive inhibition by tabun was faster for BChE than for AChE, indicating that BChE would scavenge tabun prior to AChE inhibition [1]. The reactivation of tabun-inhibited BChE by selected aldoximes was very slow, reaching maximum of 70%, which means that BChE in combination with the aldoximes investigated cannot be considered as catalytic scavenger of tabun. The overall reactivation rate constants were between 2.2 and 53.6 min-1M-1. Interestingly, the fastest reactivation was achieved by K033, aldoxime with the lowest efficacy for reactivation of tabun-inhibited AChE. The phosphorylated BChE had lower affinities for two potent AChE reactivators, K027 and K048, than phosphorylated AChE, indicating that fast AChE reactivation by these aldoximes would not be obstructed by interactions between BChE and aldoxime [1]. All aldoximes reversibly inhibited BChE and the enzyme-aldoxime dissociation constants ranged between 0.04 and 0.70 mM. However, BChE had lower affinities for aldoximes than AChE (except for TMB-4 for which both enzymes had similar affinity) indicating aldoxime preference for binding to the native AChE [1]. These results imply possible use of the aldoximes as protectors of AChE against phosphorylation by tabun. [1] Calic, M. et al (2006) Toxicology 219, 85-96.

Expression of the α7, α4 and α3 nicotinic receptor subtype in the brain and adrenal medulla of transgenic mice carrying genes coding for human AChE and β-amyloid

Human AChE-enzyme (hAChE) enhances the over-expression of β-amyloid (Aβ) containing plaques in the brain of transgenic mice (APPSWE/hAChE-Tg) carrying mutated genes for human amyloid precursor protein (APPSWE) and hAChE. In this study, we showed that interaction of hAChE with Aβ affects the plasticity of the α7 nicotinic acetylcholine receptors (nAChRs) both in the brain and adrenal medulla. An age-related increase in the 125I-αbungarotoxin (125I-αBTX) binding (specific to α7 nAChRs) was observed in the adrenal medulla of 3, 7 and 10 months old control mice. In contrast, a significant decrease in 125I-αBTX binding was detected in the adrenal medulla of 10 months old APPSWE/hAChE-Tg.A significantly higher α7 nAChR mRNA level was observed in the brain of APPSWE/hAChE-Tg at 3 and 7 months of age and in the adrenal medulla at 3 and 10 months of age compared to those of the control mice. The α3 nAChR mRNA level was significantly higher in the brain of APPSWE/hAChE-Tg at 3 months of age and in the adrenal medulla at 10 months of age. The α4 nAChR mRNA level remained unchanged in the brain and adrenal medulla of APPSWE/hAChE-Tg for all age groups.Based on these observations, we conclude that a high load of Aβ and an over-expression of hAChE induce differences in the expression of the nAChR subtypes at various ages in the brain and in the adrenal medulla of hAChE/APPSWETg mice. The findings may have implications for a better understanding the underlying mechanism for AD-related pathogenesis.