Effect of cetyltrimethylammonium on the human blood cholinesterases activity

The influence of cationic detergent cetyltrimethylammonium on the human blood cholinesterases activity (erythrocyte acetylcholinesterase and plasma butyrylcholinesterase) in reactions of hydrolysis of alpha-thionaphthylacetat and acetylthiocholine is studied. It is shown, that cetyltrimethylammonium is reversible effector for both cholinesterases. This compound competitively inhibited enzymatic hydrolysis of acetylthiocholine by both cholinesterases, and in the reactions of enzymatic hydrolysis alpha-thionaphthylacetat display as the synergistic activator--in experiments with butyrylcholinesterase, and as the reversible inhibitor--in experiments with acetylcholinesterase. Kinetic constants in reaction of acetylcholinesterase inhibition by cetyltrimethylammonium defined by means of different substrates--alpha-thionaphthylacetat and acetylthiocholin. They are close among themselves and amount (2.5 +/- 0.3) x 10(-5) and (2.8 +/- 0.3) x 10(-5) M, accordingly. Butyrylcholinesterase was more sensitive to influence of cetyltrimethylammonium. The kinetic constants defined for this enzyme by the effect of inhibition of acetylthiocholin hydrolysis or activation of alpha-thionaphthylatcetat hydrolysis, are also close among themselves and amount (3.9 +/- 0.4) x 10(-6) and (4.4 +/- 0.4) x 10(-6) M, accordingly.     

Synthesis,molecular modeling and evaluation of novel N′-2-(4-benzylpiperidin-/piperazin-1-yl)acylhydrazone derivatives as dual inhibitors for cholinesterases and Aβ aggregation

To develop new drugs for treatment of Alzheimer’s disease, a group of N′-2-(4-Benzylpiperidin-/piperazin-1-yl)acylhydrazones was designed, synthesized and tested for their ability to inhibit acetylcholinesterase, butyrylcholinesterase and aggregation of amyloid beta peptides (1–40, 1–42 and 1–40_1–42). The enzyme inhibition assay results indicated that compounds moderately inhibit both acetylcholinesterase and butyrylcholinesterase. β-Amyloid aggregation results showed that all compounds exhibited remarkable Aβ fibril aggregation inhibition activity with a nearly similar potential as the reference compound rifampicin, which makes them promising anti-Alzheimer drug candidates. Docking experiments were carried out with the aim to understand the interactions of the most active compounds with the active site of the cholinesterase enzymes.     

Comparison of active sites of butyrylcholinesterase and acetylcholinesterase based on inhibition by geometric isomers of benzene‐di‐N‐substituted carbamates

We have reported that benzene‐1,2‐, 1,3‐, and 1,4‐di‐N‐substituted carbamates ( 1–15 ) are characterized as the conformationally constrained inhibitors of acetylcholinesterase and mimic gauche, eclipsed, and anti‐conformations of acetylcholine, respectively (J Biochem Mol Toxicol 2007;21:348–353). We further report the inhibition of butyrylcholinesterase by these inhibitors. Carbamates 1–15 are also characterized as the pseudosubstrate inhibitors of butyrylcholinesterase as in the acetylcholinesterase catalysis. Benzene‐1,4‐di‐N‐n‐hexylcarbamate ( 12 ) and benzene‐1,4‐di‐N‐n‐octylcarbamate ( 13 ) are the two most potent inhibitors of butyrylcholinesterase among inhibitors 1–15 . These two para compounds, with the angle of 180° between two C(benzene)? O bonds, mimic the preferable anti C? O/C? N conformers for the choline ethylene backbone of butyrylcholine during the butyrylcholinesterase catalysis. The second n‐hexylcarbamyl or n‐octylcarbamyl moiety of inhibitors 12 and 13 is proposed to bind tightly to the peripheral anionic site of butyrylcholinesterase from molecular modeling. Butyrylcholinesterase prefers para‐carbamates to ortho‐ and meta‐carbamates, whereas acetylcholinesterase prefers para‐ and meta‐carbamates to ortho‐carbamates. This result implies that the anionic site of butyrylcholinesterase is relatively smaller than that of acetylcholinesterase because meta‐carbamates, which may bind to the anionic sites of both enzymes, are not potent inhibitors of butyrylcholinesterase. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:303–308, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20286     

Spontaneous reactivation of phosphinylated human erythrocyte acetylcholinesterase and human serum butyrylcholinesterase

This report documents studies on the spontaneous reactivation of human erythrocyte acetylcholinesterase and human serum butyrylcholinesterase following inhibition by organophosphinate esters. The spontaneous reactivation reactions were carried out at 26.0 degrees C in 0.10 M phosphate buffer of pH 7.6. Based upon results at 24 h, human serum butyrylcholinesterase inhibited with 4-nitrophenyl methyl (4-methoxyphenyl) phosphinate was the most responsive (92.5% recovery) of the nine esters studied. Using the same criteria, the most active compound in the human erythrocyte acetylcholinesterase studies was 4-nitrophenyl methyl(phenyl)phosphinate (74.2% recovery). With seven of the nine compounds examined the response was greater from the serum enzyme than from the erythrocyte enzyme.     

Novel isosorbide di-ester compounds as inhibitors of acetylcholinesterase

We report herein that a variety of isosorbide di-esters, previously reported to be novel substrates for butyrylcholinesterase (BuChE, EC 3.1.1.8), are in fact inhibitors of the homologous enzyme acetylcholinesterase (AChE), with IC(50) values in the micromolar range. In vitro studies show that they are mixed inhibitors of the enzyme, and thus the ternary enzyme-inhibitor-substrate complex can form in acetylcholinesterase. This is rationalised by molecular modelling which shows that the compounds bind in the mid-gorge area. In this position, simultaneous substrate binding might be possible, but the hydrolysis of this substrate is prevented. The di-esters dock within the butyrylcholinesterase gorge in a very different manner, with the ester sidechain at the 5-position occupying the acyl pocket at residues Leu286 and Val288, and the 2-ester binding to Trp82. The carbonyl group of the 2-ester is susceptible to nucleophilic attack by Ser198 of the catalytic triad. The larger residues of the acyl pocket in acetylcholinesterase prevent binding in this manner. The results complement each other and explain the differing behaviours of the esters in the cholinesterase enzymes. These findings may prove very significant for future work.     

A subunit-sized butyrylcholinesterase present in high concentrations in pooled rabbit serum

A butyrylcholinesterase of mol.wt. approx. 83000 was observed in pooled rabbit serum. The enzyme was named monomeric butyrylcholinesterase to distinguish it from the larger oligomeric butyrylcholinesterase of horse and human serum whose subunits are the same size as the monomeric enzyme. The active-site concentration of monomeric butyrylcholinesterase in the pooled serum was 0.18mum, which is five times the concentration of butyrylcholinesterase in pooled horse serum. This was surprising, since the horse serum is regarded as a rich source of butyrylcholinesterase, whereas rabbit serum is not generally thought to contain significant amounts of any butyrylcholinesterase. The explanation, in large part, was the relatively low k(cat.) of the monomeric enzyme, which was approx. 57s(-1) with butyrylthiocholine as substrate and is one-thirtieth of the comparable k(cat.) of horse butyrylcholinesterase. The substrate specificity of monomeric butyrylcholinesterase also differed significantly from that of horse and human butyrylcholinesterase. For example, with the monomeric enzyme, the hydrolysis of 1mm-acetylthiocholine was only 4% the rate for 1mm-butyrylthiocholine, whereas human and horse butyrylcholinesterases hydrolysed 1mm-acetylthiocholine at 50% of the rate for 1mm-butyrylthiocholine. Moreover, monomeric butyrylcholinesterase generally hydrolysed aromatic esters more rapidly than choline esters, whereas the reverse is true of the butyrylcholinesterases. To facilitate the study of monomeric butyrylcholinesterase, it was separated from the larger butyrylcholinesterase and acetylcholinesterase, also present in rabbit serum, and purified 89-fold by fractionation with (NH(4))(2)SO(4) and ion-exchange chromatography.     

Design and synthesis of tacrine-ferulic acid hybrids as multi-potent anti-Alzheimer drug candidates

Five tacrine–ferulic acid hybrids (6a–e) were designed and synthesized as multi-potent anti-Alzheimer drug candidates. All target compounds have better acetylcholinesterase inhibitory activity and comparable butyrylcholinesterase inhibitory activity in relation to tacrine. Interestingly, 6d showed a reversible and non-competitive inhibitory action for acetylcholinesterase indicating interaction with the peripheral anionic site, whereas a reversible but competitive inhibitory action for butyrylcholinesterase. The antioxidant study revealed that four target compounds have, compared to Trolox, high ability to absorb reactive oxygen species.     

Acetylcholinesterase and butyrylcholinesterase released from normal and dystrophic muscles by treatment with proteolytic enzymes

1. Skeletal muscle from C57BL dystrophic mice demonstrated decreased activities of acetylcholinesterase with increased activities of butyrylcholinesterase. These changes were less distinct when compared to those observed with 129 ReJ mice. 2. Collagenase or trypsin treatment solubilized less acetylcholinesterase activity but more butyrylcholinesterase activity from muscle of C57BL dystrophic mice than from muscle of control mice. 3. These treatments resulted in similar pattern of release of acetylcholinesterase activity from muscle of 129 ReJ mice, except that more acetylcholinesterase activity was released from dystrophic muscle (129 ReJ) than from control by pepsin treatment. 4. The acetylcholinesterase activities released by proteolytic enzymes were characterized by sucrose density gradient centrifugation.     

Synthesis and acetylcholinesterase/butyrylcholinesterase inhibition activity of new tacrine-like analogues

The synthesis and preliminary results for acetylcholinesterase and butyrylcholinesterase inhibition activity of a series of pyrano[2,3-b]quinolines (2, 3) and benzonaphthyridines (5, 6) derivatives are described. These molecules are tacrine-like analogues which have been prepared from readily available polyfunctionalized ethyl [6-amino-5-cyano-4H-pyrans and 6-amino-5-cyanopyridines]-3-carboxylates via Friedlander condensation with selected ketones. These compounds showed moderate acetylcholinesterase inhibition activity, the more potent (2e, 5b) being 6 times less active than tacrine. The butyrylcholinesterase activity of some of these molecules is also discussed.     

Preparation,in vitro evaluation and molecular modelling of pyridinium–quinolinium/isoquinolinium non-symmetrical bisquaternary cholinesterase inhibitors

Two series of non-symmetrical bisquaternary pyridinium–quinolinium and pyridinium–isoquinolinium compounds were prepared as molecules potentially applicable in myasthenia gravis treatment. Their inhibitory ability towards human recombinant acetylcholinesterase and human plasmatic butyrylcholinesterase was determined and the results were compared to the known effective inhibitors such as ambenonium dichloride, edrophonium bromide and experimental compound BW284C51.Two compounds, 1-(10-(pyridinium-1-yl)decyl)quinolinium dibromide and 1-(12-(pyridinium-1-yl)dodecyl)quinolinium dibromide, showed very promising affinity for acetylcholinesterase with their IC₅₀ values reaching nM inhibition of acetylcholinesterase. These most active compounds also showed satisfactory selectivity towards acetylcholinesterase and they seem to be very promising as leading structures for further modifications and optimization. Two of the most promising compounds were examined in the molecular modelling study in order to find the possible interactions between the ligand and tested enzyme.     

Inhibition and protection of cholinesterases by methanol and ethanol

The cholinesterases have been investigated in terms of the effects of methanol and ethanol on substrate and carbamate turnover, and on their phosphorylation. It was found: 1) that at low substrate concentrations the two alcohols inhibit all three tested cholinesterases and that the optimum activities are shifted towards higher substrate concentrations, but with a weak effect on horse butyrylcholinesterase; 2) that methanol slows down carbamoylation by eserine and does not influence decarbamoylation of vertebrate and insect acetylcholinesterase and 3) that ethanol decreases the rate of phosphorylation of vertebrate acetylcholinesterase by DFP. Our results are in line with the so-called 'approach-and-exit' hypothesis. By hindering the approach of substrate and the exit of products, methanol and ethanol decrease cholinesterase activity at low substrate concentrations and allow for the substrate inhibition only at higher substrate concentrations. Both effects appears to be a consequence of the lower ability of substrate to substitute alcohol rather than water. It also seems that during substrate turnover in the presence of alcohol the transacetylation is negligible.     

Expression of cholinesterases in human kidney and its variation in renal cell carcinoma types

Despite the aberrant expression of cholinesterases in tumours, the question of their possible contribution to tumorigenesis remains unsolved. The identification in kidney of a cholinergic system has paved the way to functional studies, but details on renal cholinesterases are still lacking. To fill the gap and to determine whether cholinesterases are abnormally expressed in renal tumours, paired pieces of normal kidney and renal cell carcinomas (RCCs) were compared for cholinesterase activity and mRNA levels. In studies with papillary RCC (pRCC), conventional RCC, chromophobe RCC, and renal oncocytoma, acetylcholinesterase activity increased in pRCC (3.92 ± 3.01 mU·mg(-1), P = 0.031) and conventional RCC (2.64 ± 1.49 mU·mg(-1), P = 0.047) with respect to their controls (1.52 ± 0.92 and 1.57 ± 0.44 mU·mg(-1)). Butyrylcholinesterase activity increased in pRCC (5.12 ± 2.61 versus 2.73 ± 1.15 mU·mg(-1), P = 0.031). Glycosylphosphatidylinositol-linked acetylcholinesterase dimers and hydrophilic butyrylcholinesterase tetramers predominated in control and cancerous kidney. Acetylcholinesterase mRNAs with exons E1c and E1e, 3'-alternative T, H and R acetylcholinesterase mRNAs and butyrylcholinesterase mRNA were identified in kidney. The levels of acetylcholinesterase and butyrylcholinesterase mRNAs were nearly 1000-fold lower in human kidney than in colon. Whereas kidney and renal tumours showed comparable levels of acetylcholinesterase mRNA, the content of butyrylcholinesterase mRNA was increased 10-fold in pRCC. The presence of acetylcholinesterase and butyrylcholinesterase mRNAs in kidney supports their synthesis in the organ itself, and the prevalence of glycosylphosphatidylinositol-anchored acetylcholinesterase explains the splicing to acetylcholinesterase-H mRNA. The consequences of butyrylcholinesterase upregulation for pRCC growth are discussed.     

Ursolic and oleanolic acid derivatives with cholinesterase inhibiting potential

Triterpenoids are in the focus of scientific interest, and they were evaluated for many pharmacological applications among them their ability to act as inhibitors of cholinesterases. These inhibitors are still of interest as drugs that improve the life quality of patients suffering from age-related dementia illnesses especially of Alzheimer’s disease. Herein, we prepared several derivatives of ursolic and oleanolic acid and screened them in Ellman’s assays for their ability to inhibit acetylcholinesterase and/or butyrylcholinesterase, and for each of the active compounds the type of inhibition was determined. As a result, several compounds were shown as good inhibitors for acetylcholinesterase and butyrylcholinesterase even in a micromolar range. An ursolic acid derived hydroxyl-propinyl derivative 10 was a competitive inhibitor for butyrylcholinesterase with an inhibition constant of K_i = 4.29 μM, and therefore being twice as active as gold standard galantamine hydrobromide. The best inhibitor for acetylcholinesterase, however, was 2-methyl-3-oxo-methyl-ursoloate (18), acting as a mixed-type inhibitor showing K_i = 1.72 µM and K_i′ = 1.28 μM, respectively.     

Investigation of potent inhibitors of cholinesterase based on thiourea and pyrazoline derivatives: Synthesis,inhibition assay and molecular modeling studies

Owing to the desperate need of new drugs development to treat Alzheimer's ailment the synthesis of 1-aroyl-3-(5-(4-chlorophenyl)-1,2,4-triazole-3-thioneaminylthioureas (2–6) starting from (4-amino-5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol) (1) and synthesis of 1-(3-(4-aminophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-(4-isobutylphenyl)propan-1-one (7–9) starting from 2-(4-isobutylphenyl)propanehydrazide (a) with the cyclization with substituted chalcones (c-e) was carried out. To check the biological potential of the synthesized compounds, all were subjected to acetylcholinesterase (AChE) and butrylcholinesterase (BChE) inhibition assays. The most potent and selective inhibitor for the acetylcholinesterase was compound 7 having an inhibitory concentration of 123 ± 51 nM, whereas, compound 6 was found as selective inhibitor of butyrylcholinesterase (BChE) with an IC₅₀ value of 201 ± 80 nM. However, the compounds 1 and 2 were found as dual inhibitors i.e. active against both acetylcholinesterase as well as butyrylcholinesterase.     

Inhibition and protection of cholinesterases by methanol and ethanol

The cholinesterases have been investigated in terms of the effects of methanol and ethanol on substrate and carbamate turnover, and on their phosphorylation. It was found: 1) that at low substrate concentrations the two alcohols inhibit all three tested cholinesterases and that the optimum activities are shifted towards higher substrate concentrations, but with a weak effect on horse butyrylcholinesterase; 2) that methanol slows down carbamoylation by eserine and does not influence decarbamoylation of vertebrate and insect acetylcholinesterase and 3) that ethanol decreases the rate of phosphorylation of vertebrate acetylcholinesterase by DFP. Our results are in line with the so-called ‘approach-and-exit’ hypothesis. By hindering the approach of substrate and the exit of products, methanol and ethanol decrease cholinesterase activity at low substrate concentrations and allow for the substrate inhibition only at higher substrate concentrations. Both effects appears to be a consequence of the lower ability of substrate to substitute alcohol rather than water. It also seems that during substrate turnover in the presence of alcohol the transacetylation is negligible.     

Ammonium Compounds with Localized and Delocalized Charge as Reversible Inhibitors of Cholinesterases of Different Origin

Five derivatives of benzimidazole, compounds with delocalized charge in cationic group, are studied and turned out to be reversible inhibitors of hydrolysis of acetylthiocholine under action of acetylcholinesterase from human erythrocytes, butyrylcholinesterase from horse blood serum, and cholinesterases from brain of the brown frog Rana temporaria and from optical ganglion of the Pacific squid Todarodes pacificus. It was only for acetylcholinesterase from erythrocyte as well as (with propyonylthiocholine as substrate) from squid that sensitivity to the studied benzimidazole derivatives correlated with degree of localization of the charge in the cationic group; this confirms the current concepts of functioning of the enzyme active center. A comparative study of 9 ammonium inhibitors with localized cation in their molecules, including the complete sterical analogue of the benzimidazole derivatives, benzimidazolinium iodide, has revealed both quantitative and qualitative differences.     

Selective cholinesterase inhibition by lanostane triterpenes from fruiting bodies of Ganoderma lucidum

Two new lanostane triterpenes, named methyl ganoderate A acetonide (1) and n-butyl ganoderate H (2), were isolated from the fruiting bodies of Ganoderma lucidum together with 16 known compounds (3-18). Extensive spectroscopic and chemical studies established the structures of these compounds as methyl 7β,15α-isopropylidenedioxy-3,11,23-trioxo-5α-lanost-8-en-26-oate (1) and n-butyl 12β-acetoxy-3β-hydroxy-7,11,15,23-tetraoxo-5α-lanost-8-en-26-oate (2). Because new compounds exhibiting specific anti-acetylcholinesterase activity are being sought as possible drug candidates for the treatment of Alzheimer's and related neurodegenerative diseases, compounds 1-18 were examined for their inhibitory activities against acetylcholinesterase and butyrylcholinesterase. All of the compounds exhibited moderate acetylcholinesterase-inhibitory activity, with IC(50) values ranging from 9.40 to 31.03μM. In contrast, none of the compounds except lucidadiol (13) and lucidenic acid N (14) exhibited butyrylcholinesterase-inhibitory activity at concentrations up to 200μM. These results indicate that these lanostane triterpenes are preferential inhibitors of acetylcholinesterase and may be suitable drug candidates.     

Cholinesterase hydrolysis of acetylcholine derivatives with different structures of the ammonium grouping]

15 acetoxyethylenammonium compounds are studied as substrates for acetylcholinesterase (ACE) from bovine erythrocytes and for butyrylcholinesterase (BCE) from horse serum. Substitution of methyl groups of the ammonium grouping with other radicals and incorporation of onium nitrogen in the cycle resulted in the decrease of the hydrolysis rate under the action of BCE and ACE, the effect of BCE being more pronounced. The rate of the hydrolysis of N-acetoxyethylene-N-methylpiperidine iodide in the presence of ACE was 65 times as much as in the presence of BCE. This compound is a new specific substrate of ACE. Dipropylmethyl derivative turned not to be a good substrate for both enzymes. Dibutylmethyl and pyridinic derivatives were not attacked by ACE and BCE. Kinetic analysis of the compounds listed is performed, taking account of non-productive sorbtion. Possible role of hydrofobic regions in the orientation of substrates on the active surface of ACE and BCE is discussed.     

Synthesis, biological evaluation and molecular modelling of diversely functionalized heterocyclic derivatives as inhibitors of acetylcholinesterase/butyrylcholinesterase and modulators of Ca2+ channels and nicotinic receptors

The synthesis and the biological activity of compounds 5-40 as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), as well as modulators of voltage-dependent Ca(2+) channels and nicotinic receptors, are described. These molecules are tacrine analogues, which have been prepared from polyfunctionalized 6-amino-5-cyano-4H-pyrans, 6-amino-5-cyano-pyridines and 5-amino-2-aryl-3-cyano-1,3-oxazoles via Friedl?nder reaction with selected cycloalkanones. These compounds are moderate acetylcholinesterase and butyrylcholinesterase inhibitors, the BuChE/AChE selectivity of the most active molecules ranges from 10.0 (compound 29) to 76.9 (compound 16). Interestingly, the 'oxazolo-tacrine' derivatives are devoid of any activity. All compounds showed an important inhibitory effect on the nicotinic acetylcholine receptor. Most of them also blocked L-type Ca(2+) channels, and three of them, 64, 19 and 67, the non-L type of Ca(2+) channels. Molecular modelling studies suggest that these compounds might bind at the peripheral binding site of AChE, which opens the possibility to design inhibitors able to bind at both, the catalytic and peripheral binding sites of the enzyme.