Horse Serum Butyrylcholinesterase Research Articles

Structural Insights into the Marine Alkaloid Discorhabdin G as a Scaffold towards New Acetylcholinesterase Inhibitors.

In this study, Antarctic Latrunculia sponge-derived discorhabdin G was considered a hit for developing potential lead compounds acting as cholinesterase inhibitors. The hypothesis on the pharmacophore moiety suggested through molecular docking allowed us to simplify the structure of the metabolite. ADME prediction and drug-likeness consideration provided valuable support in selecting 5-methyl-2H-benzo[h]imidazo[1,5,4-de]quinoxalin-7(3H)-one as a candidate molecule. It was synthesized in a four-step sequence starting from 2,3-dichloronaphthalene-1,4-dione and evaluated as an inhibitor of electric eel acetylcholinesterase (eeAChE), human recombinant AChE (hAChE), and horse serum butyrylcholinesterase (BChE), together with other analogs obtained by the same synthesis. The candidate molecule showed a slightly lower inhibitory potential against eeAChE but better inhibitory activity against hAChE than discorhabdin G, with a higher selectivity for AChEs than for BChE. It acted as a reversible competitive inhibitor, as previously observed for the natural alkaloid. The findings from the in vitro assay were relatively consistent with the data available from the AutoDock Vina and Protein-Ligand ANTSystem (PLANTS) calculations.

Rethinking common solvents in butyrylcholinesterase activity assays

• The effect of solvents on enzyme activity is quite diverse. Even two enzymes, which are members of the same family, may have different responses to the same solvent. • Acetone has the highest potential to inhibit the butyrylcholinesterase enzyme. • Butyrylcholinesterase is slightly inhibited by ethanol and methanol, making these solvents comparatively preferable in activity measurement assays. • Owing to their inhibitory potentials, the final concentrations of common solvents in the assay medium must be considered carefully. Cholinesterases are a group of enzymes that prevent the message of the neurotransmitter acetylcholine from being transmitted in an undesirable manner. The primary classes of this family are acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). In neurodegenerative disorders, including Alzheimer’s disease (AD) and Parkinson’s disease, the levels of these enzymes are altered. The level of AChE declines in Alzheimer’s disease, while the level of BChE increases or remains unchanged. There have been a number of licenced medications developed to combat the negative health impacts of these disorders. However, synthetic medications have a number of unfavorable side effects. The search for alternative BChE inhibitors in the treatment of AD is also on the rise. Some of the compounds examined are not soluble in water and must be dissolved in organic solvents. However, these organic solvents may have a negative or detrimental influence on enzymes. This study examined the inhibitory effects of acetone, dimethyl sulfoxide (DMSO), acetonitrile, ethanol, and methanol on horse serum BChE (hsBChE). Competitive inhibition was observed with acetone, DMSO and ethanol, non-competitive inhibition was observed with methanol, and acetonitrile exhibited linear mixed-type competitive inhibition. Acetone, DMSO, acetonitrile, ethanol, and methanol had IC 50 values of 707 mM, 1719 mM, 1848 mM, 7741 mM, and 12199 mM, respectively, and K i values of 186 ± 15 mM; 529 ± 35 mM, 725 ± 67 mM, 3281 ± 29 mM, and 22991 ± 6535 mM, respectively. These IC 50 and K i values show that methanol is the best solvent, with almost minimal interaction with hsBChE.

Structural Isomerism and Enhanced Lipophilicity of Pyrithione Ligands of Organoruthenium(II) Complexes Increase Inhibition on AChE and BuChE.

The increasing number of Alzheimer’s disease (AD) cases requires the development of new improved drug candidates, possessing the ability of more efficient treatment as well as less unwanted side effects. Cholinesterase enzymes are highly associated with the development of AD and thus represent important druggable targets. Therefore, we have synthesized eight organoruthenium(II) chlorido complexes 1a–h with pyrithione-type ligands (pyrithione = 1-hydroxypyridine-2(1H)-thione, a), bearing either pyrithione a, its methyl (b-e) or bicyclic aromatic analogues (f–h) and tested them for their inhibition towards electric eel acetylcholinesterase (eeAChE) and horse serum butyrylcholinesterase (hsBuChE). The experimental results have shown that the novel complex 1g with the ligand 1-hydroxyquinoline-2-(1H)-thione (g) improves the inhibition towards eeAChE (IC50 = 4.9 μM) and even more potently towards hsBuChE (IC50 = 0.2 μM) in comparison with the referenced 1a. Moreover, computational studies on Torpedo californica AChE have supported the experimental outcomes for 1g, possessing the lowest energy value among all tested complexes and have also predicted several interactions of 1g with the target protein. Consequently, we have shown that the aromatic ring extension of the ligand a, though only at the appropriate position, is a viable strategy to enhance the activity against cholinesterases.

Structural and functional characterization of an organometallic ruthenium complex as a potential myorelaxant drug

In addition to antibacterial and antitumor effects, synthetic ruthenium complexes have been reported to inhibit several medicinally important enzymes, including acetylcholinesterase (AChE). They may also interact with muscle-type nicotinic acetylcholine receptors (nAChRs) and thus affect the neuromuscular transmission and muscle function. In the present study, the effects of the organometallic ruthenium complex of 5-nitro-1,10-phenanthroline (nitrophen) were evaluated on these systems. The organoruthenium-nitrophen complex [(η6-p-cymene)Ru(nitrophen)Cl]Cl; C22H21Cl2N3O2Ru (C1-Cl) was synthesized, structurally characterized and evaluated in vitro for its inhibitory activity against electric eel acetylcholinesterase (eeAChE), human recombinant acetylcholinesterase (hrAChE), horse serum butyrylcholinesterase (hsBChE) and horse liver glutathione-S-transferase. The physiological effects of C1-Cl were then studied on isolated mouse phrenic nerve-hemidiaphragm muscle preparations, by means of single twitch measurements and electrophysiological recordings. The compound C1-Cl acted as a competitive inhibitor of eeAChE, hrAChE and hsBChE with concentrations producing 50 % inhibition (IC50) of enzyme activity ranging from 16 to 26 μM. Moreover, C1-Cl inhibited the nerve-evoked isometric muscle contraction (IC50 = 19.44 μM), without affecting the directly-evoked muscle single twitch up to 40 μM. The blocking effect of C1-Cl was rapid and almost completely reversed by neostigmine, a reversible cholinesterase inhibitor. The endplate potentials were also inhibited by C1-Cl in a concentration-dependent manner (IC50 = 7.6 μM) without any significant change in the resting membrane potential of muscle fibers up to 40 μM. Finally, C1-Cl (5–40 μM) decreased (i) the amplitude of miniature endplate potentials until a complete block by concentrations higher than 25 μM and (ii) their frequency at 10 μM or higher concentrations. The compound C1-Cl reversibly blocked the neuromuscular transmission in vitro by a non-depolarizing mechanism and mainly through an action on postsynaptic nAChRs. The compound C1-Cl may be therefore interesting for further preclinical testing as a new competitive neuromuscular blocking, and thus myorelaxant, drug.

Arachidonoylcholine and Other Unsaturated Long-Chain Acylcholines Are Endogenous Modulators of the Acetylcholine Signaling System.

Cholines acylated with unsaturated fatty acids are a recently discovered family of endogenous lipids. However, the data on the biological activity of acylcholines remain very limited. We hypothesized that acylcholines containing residues of arachidonic (AA-CHOL), oleic (Ol-CHOL), linoleic (Ln-CHOL), and docosahexaenoic (DHA-CHOL) acids act as modulators of the acetylcholine signaling system. In the radioligand binding assay, acylcholines showed inhibition in the micromolar range of both α7 neuronal nAChR overexpressed in GH4C1 cells and muscle type nAChR from Torpedo californica, as well as Lymnaea stagnalis acetylcholine binding protein. Functional response was checked in two cell lines endogenously expressing α7 nAChR. In SH-SY5Y cells, these compounds did not induce Ca2+ rise, but inhibited the acetylcholine-evoked Ca2+ rise with IC50 9 to 12 μM. In the A549 lung cancer cells, where α7 nAChR activation stimulates proliferation, Ol-CHOL, Ln-CHOL, and AA-CHOL dose-dependently decreased cell viability by up to 45%. AA-CHOL inhibited human erythrocyte acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by a mixed type mechanism with Ki= 16.7 ± 1.5 μM and αKi= 51.4 ± 4.1 μM for AChE and Ki= 70.5 ± 6.3 μM and αKi= 214 ± 17 μM for BChE, being a weak substrate of the last enzyme only, agrees with molecular docking results. Thus, long-chain unsaturated acylcholines could be viewed as endogenous modulators of the acetylcholine signaling system.

Mass spectrometry based proteomic approach for the screening of butyrylcholinesterase adduct formation with organophosphates

Organophosphates’ toxic effect causes covalent binding to serine-198 in the active site of human plasma butyrylcholinesterase (BChE) with loss of enzymatic function (covalent inhibition). Mass spectrometric detection of modified FGESAGAAS peptide at the active site is a powerful exposure biomarker tool. The aim of this study was to develop mass spectrometry-based method for BChE adduct formation screening, avoiding the use of standard peptides. Immunomagnetic separation of proteins from plasma was optimized. Commercially available anti-butyrylcholinesterase monoclonal antibodies, immobilized on magnetic beads, resulted in stable and reusable affinity sorbent. The method was tested on horse serum BChE and real human plasma from healthy donors, treated with Russian VX (VR). The BChE isolated from blood plasma was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The method was evaluated by using synthetic peptides and by comparison to the enzymatic activity Ellman's assay. The minimum concentration of VR exposure, resulting in detectable VR-adduct, was 0.2 ng/mL, which corresponded to the relative BChE inhibition of less than 2%. Adduct formation assessment was performed via monitoring of decrease in non-modified peptide LC-MS/MS signal and increase in VR-modified peptide signal. The designed approach was tested in a pilot study with 5 blood samples from healthy volunteers. Mass spectrometry-based method for BChE adduct formation was found to be in agreement with Ellman's inhibition assay, so the method is applicable for direct BChE inhibition assessment.

Evaluation of the inhibitory effect of abamectin on mammalian butyrylcholinesterase: Enzyme kinetic and molecular docking studies

ABSTRACTAbamectin, a blend of the natural avermectins B1a and B1b, is a widely-used insecticide/miticide with relatively low toxicity to mammals. Exposure to high doses of it, however, leads to cholinergic-like neurotoxic effects. Butyrylcholinesterase, which is best known for its abundant presence in plasma, is a serine hydrolase loosely coupled with the cholinergic system. It protects and supports the neurotransmitter function of its sister enzyme acetylcholinesterase. Here, using experimental and computational studies, we provide evidence demonstrating that abamectin is a potent (IC50 = 10.6 μM; Ki = 2.26 ± 0.35 μM) inhibitor of horse serum butyrylcholinesterase and that it interacts with the enzyme in a reversible, competitive manner predictively to block the mouth of the active-site gorge of the enzyme and to bind to several critical residues that normally bind/hydrolyze choline esters.

Design, synthesis and biological evaluation of tacrine-1,2,3-triazole derivatives as potent cholinesterase inhibitors.

We report herein the design and synthesis of a series of 11 novel tacrine-1,2,3-triazole derivatives via a Cu(i)-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction. The newly synthesized compounds were evaluated for their inhibition activity against Electrophorus electricus acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) as potential drug targets for Alzheimer's disease (AD). Among the designed compounds, compound 8a2 exhibited potent inhibition against AChE and BChE with IC50 values of 4.89 μM and 3.61 μM, respectively. Further structure-activity relationship (SAR) and molecular modeling studies may provide valuable insights into the design of better tacrine-triazole analogues with potential therapeutic applications for AD.

Discorhabdin alkaloids from Antarctic Latrunculia spp. sponges as a new class of cholinesterase inhibitors

The brominated pyrroloiminoquinone alkaloids discorhabdins B, L and G and 3-dihydro-7,8- dehydrodiscorhabdin C, isolated from methanol extracts of two specimens of Latrunculia sp. sponges collected near the Antarctic Peninsula, are here demonstrated for the first time to be reversible competitive inhibitors of cholinesterases. They showed Ki for electric eel acetylcholinesterase of 1.6–15.0 μM, for recombinant human acetylcholinesterase of 22.8–98.0 μM, and for horse serum butyrylcholinesterase of 5.0–76.0 μM. These values are promising when compared to the current cholinesterase inhibitors used for treatment of patients with Alzheimer's disease, to counteract the acetylcholine deficiency in the brain. Good correlation was obtained between IC50 data and results by molecular docking calculation on the binding interactions within the acetylcholinesterase active site, which also indicated the moieties in discorhabdin structures involved. To avoid unwanted peripheral side effects that can appear in patients using some acetylcholinesterase inhibitors, electrophysiological experiments were carried out on one of the most active of these compounds, discorhabdin G, which confirmed that it had no detectable undesirable effects on neuromuscular transmission and skeletal muscle function. These findings are promising for development of cholinesterase inhibitors based on the scaffold of discorhabdins, as potential new agents for treatment of patients with Alzheimer's disease.

Synthesis and studies of biological activity of N-(4-tert-butylbenzyl)-N-(pyridin-3-ylmethyl)-2-aminothiazolines as potential multifunctional agents for treatment of neurodegenerative diseases

A number of N-(4-tert-butylbenzyl)-N-(pyridin-3-ylmethyl)-2-aminothiazolines with different substituents at position 5 of thiazoline ring was synthesized and their inhibitory activity against human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7), horse serum butyrylcholinesterase (BChE, EC 3.1.1.8), and porcine liver carboxylesterase (CaE, EC 3.1.1.1) was studied, as well as their antioxidant properties in DPPH and ABTS tests (DPPH is the 2,2-diphenyl-1-picrylhydrazyl, ABTS is the 2,2´-azinobis(3-ethylbenzthiazolino-6-sulfonic acid)). All the studied compounds virtually do not inhibit AChE, whereas the inhibition degree of BChE and CaE depends on the substituent structure. The presence of radical-binding activity comparable with the standard antioxidant ascorbic acid was shown for compounds containing a iodomethyl substituent. The found lead compound (R1 = H, R2 = CH2I) combines the antioxidant properties and the ability to efficiently inhibit BChE, simultaneously possessing an optimal esterase profile. The performed studies showed that N-(4-tert-butylbenzyl)-N-(pyridin-3-ylmethyl)-2-aminothiazolines are a new promising class of compounds, which can be used for the development of multifunctional agents for treatment of neurodegenerative diseases.

Alkyl 2-arylhydrazinylidene-3-oxo-3-polyfluoroalkylpropionates as new effective and selective inhibitors of carboxylesterase.

A series of alkyl 2-Arylhydrazinylidene-3-oxo-3-polyfluoroalkylpropionates was synthesized and their inhibitory activity with respect to porcine liver carboxylesterase (CaE, EC 3.1.1.1), human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7), and horse serum butyrylcholinesterase (BChE, EC 3.1.1.8) was studied. The molecular docking method was used to study the binding mode of the compounds in the active site of CaE. It was found that compounds containing the trifluoromethyl group in the third position of carbonyl chain are highly effective and selective inhibitors of CaE with nanomolar IC50 values, which agrees well with the results of molecular docking.

Multitarget Therapeutic Leads for Alzheimer's Disease: Quinolizidinyl Derivatives of Bi- and Tricyclic Systems as Dual Inhibitors of Cholinesterases and β-Amyloid (Aβ) Aggregation.

Multitarget therapeutic leads for Alzheimer's disease were designed on the models of compounds capable of maintaining or restoring cell protein homeostasis and of inhibiting β-amyloid (Aβ) oligomerization. Thirty-seven thioxanthen-9-one, xanthen-9-one, naphto- and anthraquinone derivatives were tested for the direct inhibition of Aβ(1-40) aggregation and for the inhibition of electric eel acetylcholinesterase (eeAChE) and horse serum butyrylcholinesterase (hsBChE). These compounds are characterized by basic side chains, mainly quinolizidinylalkyl moieties, linked to various bi- and tri-cyclic (hetero)aromatic systems. With very few exceptions, these compounds displayed inhibitory activity on both AChE and BChE and on the spontaneous aggregation of β-amyloid. In most cases, IC50 values were in the low micromolar and sub-micromolar range, but some compounds even reached nanomolar potency. The time course of amyloid aggregation in the presence of the most active derivative (IC50 =0.84 μM) revealed that these compounds might act as destabilizers of mature fibrils rather than mere inhibitors of fibrillization. Many compounds inhibited one or both cholinesterases and Aβ aggregation with similar potency, a fundamental requisite for the possible development of therapeutics exhibiting a multitarget mechanism of action. The described compounds thus represent interesting leads for the development of multitarget AD therapeutics.

Active compounds from a diverse library of triazolothiadiazole and triazolothiadiazine scaffolds: Synthesis, crystal structure determination, cytotoxicity, cholinesterase inhibitory activity, and binding mode analysis

In an effort to identify novel cholinesterase candidates for the treatment of Alzheimer’s disease (AD), a diverse array of potentially bioactive compounds including triazolothiadiazoles (4a–h and 5a–f) and triazolothiadiazines (6a–h) was obtained in good yields through the cyclocondensation reaction of 4-amino-5-(pyridin-3-yl)-4H-1,2,4-triazole-3-thiol (3) with various substituted aryl/heteroaryl/aryloxy acids and phenacyl bromides, respectively. The structures of newly prepared compounds were confirmed by IR, 1H and 13C NMR spectroscopy and, in case of 4a, by single crystal X-ray diffraction analysis. The purity of the synthesized compounds was ascertained by elemental analysis. The newly synthesized conjugated heterocycles were screened for cholinesterase inhibitory activity against electric eel acetylcholinesterase (EeAChE) and horse serum butyrylcholinesterase (hBChE). Among the evaluated hybrids, several compounds were identified as potent inhibitors. Compounds 5b and 5d were most active with an IC50 value of 3.09±0.154 and 11.3±0.267μM, respectively, against acetylcholinesterase, whereas 5b, 6a and 6g were most potent against butyrylcholinesterase, with an IC50 of 0.585±0.154, 0.781±0.213, and 1.09±0.156μM, respectively, compared to neostigmine and donepezil as standard drugs. The synthesized heteroaromatic compounds were also tested for their cytotoxic potential against lung carcinoma (H157) and vero cell lines. Among them, compound 6h exhibited highest antiproliferative activity against H157 cell lines, with IC50 value of 0.96±0.43μM at 1mM concentration as compared to vincristine (IC50=1.03±0.04μM), standard drug used in this study.

Synthesis, cytotoxicity and molecular modelling studies of new phenylcinnamide derivatives as potent inhibitors of cholinesterases

The present study reports the synthesis of cinnamide derivatives and their biological activity as inhibitors of both cholinesterases and anticancer agents. Controlled inhibition of brain acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) may slow neurodegeneration in Alzheimer's diseases (AD). The anticholinesterase activity of phenylcinnamide derivatives was determined against Electric Eel acetylcholinesterase (EeAChE) and horse serum butyrylcholinesterase (hBChE) and some of the compounds appeared as moderately potent inhibitors of EeAChE and hBChE. The compound 3-(2-(Benzyloxy)phenyl)-N-(3,4,5-trimethoxyphenyl)acrylamide (3i) showed maximum activity against EeAChE with an IC50 0.29 ± 0.21 μM whereas 3-(2-chloro-6-nitrophenyl)-N-(3,4,5-trimethoxyphenyl)acrylamide (3k) was proved to be the most potent inhibitor of hBChE having IC50 1.18 ± 1.31 μM. To better understand the enzyme–inhibitor interaction of the most active compounds toward cholinesterases, molecular modelling studies were carried out on high-resolution crystallographic structures. The anticancer effects of synthesized compounds were also evaluated against cancer cell line (lung carcinoma). The compounds may be useful leads for the design of a new class of anticancer drugs for the treatment of cancer and cholinesterase inhibitors for Alzheimer's disease (AD).

Microcalorimetric study of the inhibition of butyrylcholinesterase by sodium arsenite and zinc chloride: Use of a fractal kinetic model

The inhibition of horse serum butyrylcholinesterase (EC 3.1.1.8) by sodium arsenite (NaAsO2) and zinc chloride (ZnCl2) was studied at 37°C in a TRIS buffer, pH 7.5, with a flow microcalorimeter operating in the mixing mode. The dose–response curves were analyzed with the Hill equation, as deduced from the “fractal kinetic” model of Savageau by considering an exponential distribution of reaction rate constants. The C50 values were in the millimolar range and zinc chloride was a better inhibitor than sodium arsenite. This was confirmed by the values of the apparent rate constants. The Hill exponent, interpreted as a fractional reaction order, was also higher for zinc chloride. According to the statistical interpretation, the heterogeneity of binding reactions was slightly higher for Zn, in agreement with a speciation study which showed the existence of several Zn species in solution, with the free Zn2+ ion accounting for about 10% of the total.

Synthesis and evaluation of 4-substituted coumarins as novel acetylcholinesterase inhibitors

A series of 4-hydroxycoumarin derivatives were designed and synthesized as new acetylcholinesterase (AChE) inhibitors which could be considered for Alzheimer's disease therapeutics. Among the 19 coumarin-derived compounds tested toward Electrophorus electricus acetylcholinesterase (eelAChE) and horse serum butyrylcholinesterase (eqBChE), N-(1-benzylpiperidin-4-yl)acetamide derivative 4m displayed highest AChE inhibitory activity (IC50 = 1.2 μM) and good selectivity (37 times). The docking study of the most potent compound 4m, indicated that Phe330 is responsible for ligand recognition and trafficking by forming π-cation interaction with benzylpiperidine moiety. Furthermore, the formation of an additional π–π interaction between coumarin moiety and Trp279 of peripheral anionic site could stabilize the ligand in the active site resulting in more potent inhibition of the enzyme.

Synthesis and biological activity of O-carbamoylated 1,1,1,3,3,3-hexafluoroisopropanols as new specific inhibitors of carboxylesterase

A series of O-carbamoylated 1,1,1,3,3,3-hexafluoroisopropanols of general formula RNHC(O)OCH(CF3)2, where R = CH3, n-C3H7, tert-C4H9, cyclo-C6H11, C6H5–CH2, C6H5, 4-Cl-C6H4, 3-Cl-C6H4, 3,4-Cl2-C6H3, and naphthylen-2-yl were synthesized. The reaction kinetics of the synthesized carbamates with human erythrocyte acetylcholinesterase (EC 3.1.1.7), horse serum butyrylcholinesterase (EC 3.1.1.8), and porcine liver carboxylesterase (EC 3.1.1.1) were studied. It was shown that the synthesized carbamates did not inhibit acetylcholinesterase, inhibited weakly butyrylcholinesterase, and inhibited selectively the activity of carboxylesterase. A new selective irreversible inhibitor of carboxylesterase, 2,2,2-trifluoro-1-trifluoromethylethyl cyclohexylcarbamate, which had low acute toxicity, was obtained.

Reversible inhibition of cholinesterases by opioids: possible pharmacological consequences.

The inhibitory potency of opioids belonging to different structural categories on electric eel and rat brain acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BuChE) was investigated. The phenylazepine meptazinol, the pyrrolo-[2,3-b]-indole derivative eseroline and the benzomorphan normetazocine were the most potent inhibitors of AChE among the compounds tested. These were followed by (-)-metazocine, N-allylnorcyclazocine, 3-(1,3-dimethyl-3-pyrrodinyl)-phenol, levallorphan, levorphanol and pentazocine. The opioids which inhibited horse serum BuChE were in order of potency: meptazinol, methadone, profadol, levallorphan and 1,2,3-trimethyl-3-(3-hydroxyphenyl)-piperidine. The results of this work appear consistent with the fact that the anticholinesterase activity of the opioids is not confined to specific structural categories, although conformationally constrained molecules, like those of morphinans, benzomorphans or pyrrolo-[2,3-b]-indoles, appear to favour affinity for AChE, whereas highly flexible molecules, like those of acyclic opioids, inhibit BuChE in a rather selective way. In all cases, the inhibitory action of opioids markedly differed from that of carbamates or organophosphorous compounds, in that it was time-independent and immediately reversible on dilution. In general the anticholinesterase action of opioids does not seem to influence appreciably the pharmacological properties of the drugs since it is evidenced at drug doses higher than those which are analgesic. However, in the case of mixed agonist/antagonist opioids with rather weak analgesic activity, the enzyme inhibition caused by the levels of circulating drugs can be so marked as to exert also a cholinergic component of action.

In-vitro and in-vivo protection of acetylcholinesterase by eseroline against inactivation by diisopropyl fluorophosphate and carbamates

The protective action of eseroline--(3aS,8aR)-1,2,3,3a,8,8a-hexahydro-1,3 a, 8-trimethyl-pyrrolo[2,3-b]indol-5-ol--salicylate against (DFP) diisopropyl fluorophosphate and carbamate poisoning of cholinesterases (ChEs) has been examined in-vitro with human erythrocytes and purified preparations of electric eel acetylcholinesterase (AChE) and of horse serum butyrylcholinesterase (BuChE), and in-vivo using mice. Eseroline afforded 50% protection (ED 50) of erythrocyte AChE against inactivation by 1 microM DFP, physostigmine or neostigmine, at concentrations of 4.3, 22 and 23.5 microM, respectively, while for eel AChE protection against 10 and 30 microM DFP, 0.3 and 1 microM physostigmine and 1 microM neostigmine the eseroline ED 50 values were 0.3, 0.4, 0.7, 1.9 and 5.6 microM, respectively. On the other hand, up to 0.3 mM eseroline did not appreciably affect the inhibitory action of the same drugs on horse serum BuChE. Eseroline concentrations in the range 0.1-1 mM were able to reactivate 20-42% of erythrocyte AChE previously inhibited by 100 microM physostigmine, but failed to reactivate the DFP (10 microM)-pretreated enzyme to any extent. Finally, eseroline salicylate injected into mice (10 mg kg-1 s.c.) protected an average of 82 and 26% of the animals against lethal doses of DFP (7 mg kg-1 s.c.) and physostigmine sulphate (1 mg kg-1 i.p.) respectively, which were administered 15 min later. These results indicate that the protective activity of eseroline correlates well with its own anti-ChE profile, and that the effectiveness of the protection depends largely on the rate of AChE inhibition by the agents used to inactivate the enzyme.

Influence of the Acyl Moiety on the Hydrolysis of Quinuclidinium Esters Catalyzed by Butyrylcholinesterase

Eight chiral esters of quinuclidin-3-ol and butyric, acetic, pivalic and benzoic acid were synthe- sized as well as their racemic and chiral, quaternary N-benzyl derivatives. All racemic and chiral quater- nary compounds were studied as substrates and/or inhibitors of horse serum butyrylcholinesterase (BChE). The best substrate for the enzyme was (R)-N-benzyl butyrate. The rates of hydrolysis decreased in order (R)-butyrate >> (R)-acetate (7-fold slower) > (R)-pivalate (8-fold slower) > (R)-benzoate (9-fold slower reaction), while (S)-N-benzyl esters were much poorer substrates (320 (butyrate) - 4360-fold slow- er (pivalate) than the appropriate (R)-enantiomer). For all (S)-N-benzyl esters excluding (S)-N-benzyl ace- tate inhibition constants were determined (Ka = 3.3−60 μmol dm −3 ). The hydrolysis of racemic mixtures of N-benzyl esters proceeded 1.4 (for acetate) − 5.1 (for benzoate) times slower than that of pure (R)- enantiomers of the corresponding concentrations due to the inhibition with (S)-enantiomers. Change of the acyl moiety of the substrate effected both activity and stereoselectivity of the BChE.(doi: 10.5562/cca1829)