Novel N-benzylpyridinium moiety linked to arylisoxazole derivatives as selective butyrylcholinesterase inhibitors: Synthesis,biological evaluation,and docking study

A novel series of N-benzylpyridinium moiety linked to arylisoxazole ring were designed, synthesized, and evaluated for their in vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. Synthesized compounds were classified into two series of 5a-i and 5j-q considering the position of positively charged nitrogen of pyridinium moiety (3- or 4- position, respectively) connected to isoxazole carboxamide group. Among the synthesized compounds, compound 5n from the second series of compounds possessing 2,4-dichloroaryl group connected to isoxazole ring was found to be the most potent AChE inhibitor (IC₅₀ = 5.96 µM) and compound 5j also from the same series of compounds containing phenyl group connected to isoxazole ring demonstrated the most promising inhibitory activity against BChE (IC₅₀ = 0.32 µM). Also, kinetic study demonstrated competitive inhibition mode for both AChE and BChE inhibitory activity. Docking study was also performed for those compounds and desired interactions with those active site amino acid residues were confirmed through hydrogen bonding as well as π-π and π-anion interactions. In addition, the most potent compounds were tested against BACE1 and their neuroprotectivity on Aβ-treated neurotoxicity in PC12 cells which depicted negligible activity. It should be noted that most of the synthesized compounds from both categories 5a-i and 5j-q showed a significant selectivity toward BChE. However, series 5j-q were more active toward AChE than series 5a-i.     

Cholinesterase inhibitory and spasmolytic potential of steroidal alkaloids

A new steroidal alkaloid, isosarcodine (1) along with four known bases, sarcorine (2), sarcodine (3), sarcocine (4) and alkaloid-C (5) were isolated from the MeOH extract of Sarcococca saligna. The structures of these alkaloids were identified by spectral data interpretation. These compounds were subjected to acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition studies, and were found to be noncompetitive inhibitors of AChE (K_i = 21.8, 90.3, 32.2, 16.0 and 50.0 μM, respectively) and uncompetitive or noncompetitive inhibitors of BChE (K_i = 8.3, 7.5, 15.6, 5.0 and 12.0 μM, respectively).

The compounds (2–5) also showed dose-dependent spasmolytic activity in the rabbit jejunum intestinal preparations and also relaxed the high K⁺ (80 mM)-induced contraction, indicative of a calcium channel-blocking mechanism.

Structure–activity relationship suggested that the nitrogen substituents at C-3 and/or C-20 of steroidal skeleton and the hydrophobic properties of the pregnane skeleton are the key structural features contributed to the inhibitory potency of these steroidal alkaloids against AChE and BChE.     

Effect of polyethylene glycol modification on the circulatory stability and immunogenicity of recombinant human butyrylcholinesterase

The therapeutic value of human serum butyrylcholinesterase (Hu BChE) as a bioscavenger of chemical warfare agents is due to its high reactivity with organophosphorus compounds and prolonged circulatory stability. Native Hu BChE is mostly tetrameric in form while the enzyme produced using molecular cloning technology is a mixture of tetramers, dimers, and monomers. Previous studies revealed that monomers and dimers of recombinant human (rHu) BChE cleared rapidly from the circulation of mice compared to tetrameric rHu BChE and native Hu BChE, which have mean residence times (MRTs) of 18h and 45h, respectively. It was also shown that polyethylene glycol-20K (PEG) modification of tetrameric rHu BChE prolonged its circulatory stability and bioavailability in vivo. The goal of this study was to determine if modification with PEG could prolong the circulatory stability and eliminate the immunogenicity of monomeric rHu BChE. Monomeric rHu BChE was expressed in human 293A cells using a cDNA lacking the 45 amino acid tetramerization domain from the carboxyl terminus and the adenovirus expression system. The catalytic and inhibitory properties of purified monomeric rHu BChE were similar to those for native Hu BChE and were not affected by PEG modification. As expected, monomeric rHu BChE rapidly cleared from the circulation of mice (MRT=3.2+/-0.3h) while monomeric PEG-rHu BChE demonstrated significant improvement in its bioavailability and circulatory stability in blood (MRT=31.4+/-5.4h). However, a second injection of monomeric PEG-rHu BChE, 28 days after the first, displayed a much shorter MRT=11.6+/-0.4h, and circulating anti-monomeric PEG-rHu BChE antibodies were detected in the blood of mice. These results suggest that PEG modification increased the circulatory stability of monomeric rHu BChE but failed to reduce or eliminate its immunogenicity.     

Interaction of human butyrylcholinesterase variants with bambuterol and terbutaline

Bambuterol, a dimethylcarbamate, carbamoylates butyrylcholinesterase (BChE; EC 3.1.1.8). The carbamoylated enzyme is not very stable and the final product of the two-step hydrolysis is a bronchodilator drug, terbutaline (1-(3,5-dihydroxyphenyl)-2-t-butylamino-ethanol sulphate). Both bambuterol and terbutaline inhibit BChE, but their affinities differ in human serum BChE variants (U, A, F, K and S) due to their positive charge. Bambuterol inhibition rate constants for the homozygous usual (UU), Kalow (KK), fluoride-resistant (FF) or atypical (AA) variant ranged from 4.4 to 0.085min (-1)microM(-1). Terbutaline showed competitive reversible inhibition for all BChE variants. The dissociation constants for UU, FF and AA homozygotes were 0.18, 0.31 and 3.3 mM, respectively. The inhibition rate or dissociation constants for heterozygotes were distributed between the respective constants for the corresponding homozygotes. A 50-fold difference in inhibition between the UU and AA enzyme might affect terbutaline release in humans. The affinity of all studied BChE variants for terbutaline was low, which suggests that terbutaline originating from bambuterol hydrolysis should not affect the hydrolysis of bambuterol by BChE.     

Predicted Michaelis-Menten complexes of cocaine-butyrylcholinesterase. Engineering effective butyrylcholinesterase mutants for cocaine detoxication

Butyrylcholinesterase (BChE) is important in cocaine metabolism, but it hydrolyzes (-)-cocaine only one-two thousandth as fast as the unnatural (+)-stereoisomer. A starting point in engineering BChE mutants that rapidly clear cocaine from the bloodstream, for overdose treatment, is to elucidate structural factors underlying the stereochemical difference in catalysis. Here, we report two three-dimensional Michaelis-Menten complexes of BChE liganded with natural and unnatural cocaine molecules, respectively, that were derived from molecular modeling and supported by experimental studies. Such complexes revealed that the benzoic ester group of both cocaine stereoisomers must rotate toward the catalytic Ser(198) for hydrolysis. Rotation of (-)-cocaine appears to be hindered by interactions of its phenyl ring with Phe(329) and Trp(430). These interactions do not occur with (+)-cocaine. Because the rate of (-)-cocaine hydrolysis is predicted to be determined mainly by the re-orientation step, it should not be greatly influenced by pH. In fact, measured rates of this reaction were nearly constant over the pH range from 5.5 to 8.5, despite large rate changes in hydrolysis of (+)-cocaine. Our models can explain why BChE hydrolyzes (+)-cocaine faster than (-)-cocaine, and they suggest that mutations of certain residues in the catalytic site could greatly improve catalytic efficiency and the potential for detoxication.     

Synthesis of readily available fluorophenylalanine derivatives and investigation of their biological activity

A series of thirty novel N-acetylated fluorophenylalanine-based aromatic amides and esters was synthesized using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide or phosphorus trichloride in pyridine. They were characterized by spectral methods and screened against various microbes (Mycobacterium tuberculosis, non-tuberculous mycobacteria, other bacteria, fungi), for their inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and cytotoxicity. All amino acids derivatives revealed a moderate inhibition of both cholinesterases with IC₅₀ values for AChE and BChE of 57.88–130.75 µM and 8.25–289.0 µM, respectively. Some derivatives were comparable or superior to rivastigmine, an established drug. Phenyl 2-acetamido-3-(4-fluorophenyl)propanoate was identified as the selective and most potent inhibitor of BChE. The esterification and amidation of parent acids led to an improved BChE inhibition. The esters are better inhibitors of BChE than the amides. The introduction of NO₂ and CH₃ groups into aniline ring and CF₃ moiety in phenol is translated into lower IC₅₀ values. Seven compounds showed selectivity index higher than 10 for at least one cholinesterase. Especially the esters exhibited a mild activity against Gram-positive bacteria, mycobacteria and several fungal strains with minimum inhibitory concentrations starting from 125 µM. The highest susceptibility was recorded for Trichophyton mentagrophytes fungus.     

Probing the mid-gorge of cholinesterases with spacer-modified bivalent quinazolinimines leads to highly potent and selective butyrylcholinesterase inhibitors

The spacer structure of homobivalent quinazolinimes acting as potent acetyl-(AChE)- and butyrylcholinesterase (BChE) inhibitors was chemically modified introducing tertiary amine and acyl-amide moieties, and the activities at both ChEs were evaluated. Molecular docking was applied to explain the data and probe the capacity of the mid-gorge site of both ChEs. The novel spacer structures considerably alter the biological profile of bivalent quinazolinimines with regard to both inhibitory activity and selectivity. Mutual interaction of binding to the various sites of the enzymes was further investigated by applying also different spacer lengths and ring sizes of the alicycle of the tricyclic quinazolinimines. In order to achieve selectivity toward BChE and to improve inhibitory activities, the spacer structure was optimized and identified a highly potent and selective BChE inhibitor.     

Comparison of methods used for the determination of cholinesterase activity in whole blood

Cholinesterases (ChEs) are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) based on their substrate and inhibitor specificity. Organophosphate and carbamate compounds commonly represented by herbicides, pesticides, and nerve gases irreversibly inhibit ChEs. Therefore, exposure to organophosphates and carbamates is normally assessed by measuring ChE activity in blood. There are two approaches for measuring AChE and BChE activity present in whole blood: (1) separating blood into erythrocytes, which contain only AChE, and plasma which contains only BChE, to measure their activity individually, or (2) use a BChE-specific inhibitor to measure the activity of AChE in whole blood. A number of studies have reported the use of different inhibitors for the simultaneous measurement of AChE and BChE activities. However, the inhibitors used for completely inhibiting BChE activity also inhibited AChE activity leading to errors in reported values. The goal of this study was to find the most accurate and simple method for the simultaneous determination of AChE and BChE activity in animal whole blood. Solutions containing human AChE and BChE in various proportions were prepared and AChE and BChE activities were measured using three reported methods. Results demonstrate that ethopropazine and (-) huperzine A appear to be the most specific ChE inhibitors. Preliminary results with human and animal whole blood suggest that 20muM ethopropazine and 500nM (-) huperzine A can be used for measuring AChE and BChE activities across species.     

Rational design and synthesis of highly potent anti-acetylcholinesterase activity huperzine A derivatives

By targeting multi-active sites of acetylcholinesterase (AChE), a series of huperzine A (Hup A) derivatives with various aromatic ring groups were designed and synthesized by Schiff reaction. They were evaluated as AChE and butyrylcholinesterase (BChE) inhibitors. Results showed very significant specificity that the group of imine derivatives could inhibit TcAChE and hAChE, but no inhibitory effect on hBChE was detected. The experiment was explained by a docking study. In the docking model, we confirmed that aromatic ring of Hup A derivatives played the π–π stacking against aminophenol residues of AChE, and the structure–activity relationship (SAR) was discussed.     

Evolution of acetylcholinesterase and butyrylcholinesterase in the vertebrates: an atypical butyrylcholinesterase from the Medaka Oryzias latipes

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are thought to be the result of a gene duplication event early in vertebrate evolution. To learn more about the evolution of these enzymes, we expressed in vitro, characterized, and modeled a recombinant cholinesterase (ChE) from a teleost, the medaka Oryzias latipes. In addition to AChE, O. latipes has a ChE that is different from either vertebrate AChE or BChE, which we are classifying as an atypical BChE, and which may resemble a transitional form between the two. Of the fourteen aromatic amino acids in the catalytic gorge of vertebrate AChE, ten are conserved in the atypical BChE of O. latipes; by contrast, only eight are conserved in vertebrate BChE. Notably, the atypical BChE has one phenylalanine in its acyl pocket, while AChE has two and BChE none. These substitutions could account for the intermediate nature of this atypical BChE. Molecular modeling supports this proposal. The atypical BChE hydrolyzes acetylthiocholine (ATCh) and propionylthiocholine (PTCh) preferentially but butyrylthiocholine (BTCh) to a considerable extent, which is different from the substrate specificity of AChE or BChE. The enzyme shows substrate inhibition with the two smaller substrates but not with the larger substrate BTCh. In comparison, AChE exhibits substrate inhibition, while BChE does not, but may instead show substrate activation. The atypical BChE from O. latipes also shows a mixed pattern of inhibition. It is effectively inhibited by physostigmine, typical of all ChEs. However, although the atypical BChE is efficiently inhibited by the BChE-specific inhibitor ethopropazine, it is not by another BChE inhibitor, iso-OMPA, nor by the AChE-specific inhibitor BW284c51. The atypical BChE is found as a glycophosphatidylinositol-anchored (GPI-anchored) amphiphilic dimer (G(2) (a)), which is unusual for any BChE. We classify the enzyme as an atypical BChE and discuss its implications for the evolution of AChE and BChE and for ecotoxicology.     

Synthesis and biological evaluation of aminomethyl and alkoxymethyl derivatives as carbonic anhydrase,acetylcholinesterase and butyrylcholinesterase inhibitors

Compounds containing nitrogen and sulfur atoms can be widely used in various fields such as industry, medicine, biotechnology and chemical technology. Therefore, the reactions of aminomethylation and alkoxymethylation of mercaptobenzothiazole, mercaptobenzoxazole and 2-aminothiazole were developed. Additionally, the alkoxymethyl derivatives of mercaptobenzoxazole and 2-aminothiazole were synthesized by a reaction with hemiformals, which are prepared by the reaction of alcohols and formaldehyde. In this study, the inhibitory effects of these molecules were investigated against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) enzymes and carbonic anhydrase I, and II isoenzymes (hCA I and II). Both hCA isoenzymes were significantly inhibited by the recently synthesized molecules, with K_i values in the range of 58–157?nM for hCA I, and 81–215?nM for hCA II. Additionally, the K_i parameters of these molecules for BChE and AChE were calculated in the ranges 23–88 and 18–78?nM, respectively.     

Adenovirus-mediated gene transfer of human butyrylcholinesterase results in persistent high-level transgene expression in vivo

Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents, pesticide intoxication, and cocaine overdose. However, its widespread application is hampered by difficulties in large-scale production of the native protein from human plasma and/or availability as a recombinant protein suitable for use in vivo. This limitation may be resolved by in vivo delivery and expression of the Hu BChE gene. In this study, recombinant (r) adenoviruses (Ads) encoding full-length and truncated rHu BChEs were tested for in vivo expression in mice. Mice injected with these rAds intraperitoneally failed to express rHu BChE. However, a single tail vein injection of both rAds resulted in persistent high serum levels of rHu BChE in BChE knockout mice, which peaked on days 4/5 at 377+/-162U/ml for full-length rHu BChE and 574+/-143U/ml for truncated rHu BChE. These activity levels are orders of magnitude higher than 1.9U/ml of mouse BChE present in wild-type mouse serum. Thereafter, rHu BChE levels dropped rapidly and very little or no activity was detected in the serum 10 days post-virus administration. In conclusion, the present study demonstrates the potential of rAd-mediated Hu BChE gene therapy to counteract multiple lethal doses of chemical warfare nerve agent toxicity.     

The role of Phe329 in binding of cationic triarylmethane dyes to human butyrylcholinesterase

Cationic triarylmethane dyes (TAM(+))s which are used as colorants in industry and as frequent tools and reagents in analytical, cell biological and biomedical research have been recently characterized as reversible inhibitors of human butyrylcholinesterase. In this study, the inhibitory effects of two TAM(+)s, malachite green (MG) and methyl green (MeG) on five human BChE mutants (A277V, P285L, H77L, A328F and F329A) were studied spectrophotometrically at 25°C in 50mM MOPS buffer pH 8, using butyrylthiocholine as substrate. The kinetic results obtained with mutant enzymes were compared to those obtained with recombinant wild type BChE. MG and MeG were found to act as competitive/linear mixed inhibitors of recombinant wild type BChE and all BChE mutants except the F329A mutant. Both dyes caused complex nonlinear inhibition of F329A mutant, pointing to multisite binding. K(i) values for MG and MeG, estimated by nonlinear regression analysis, were 3.8 and 27 μM, respectively, as compared to the 50- to 150-fold lower values observed with recombinant wild type BChE. The observed significant differences in kinetic pattern and K(i) values between recombinant wild type BChE and F329A mutant suggest that phenylalanine at position 329 in human BChE is a critical residue in MG and MeG binding to enzyme.     

Inhibition effects of benactyzine and drofenine on human serum butyrylcholinesterase

Benactyzine and drofenine are widely used anticholinergic drugs. Benactyzine is used to treat organophosphate poisoning and drofenine acts on smooth muscle to stop muscle spasms. Both of these drugs are esters. After they enter the bloodstream, they will interact with butyrylcholinesterase (BChE; acylcholine acyl hydrolase: EC 3.1.1.8), which has an ability to hydrolyze a wide variety of esters. Therefore, the kinetic analysis of their inhibitory effects on human serum BChE was examined using butyrylthiocholine as substrate. Both drugs were competitive inhibitors of BChE and the Ki values of benactyzine and drofenine were calculated to be 0.010 +/- 0.001 and 0.003 +/- 0.000 mM, respectively, using the Systat (version 5.03, 1991) nonlinear regression analysis software package. According to these parameters, drofenine is a more potent competitive inhibitor of BChE than benactyzine.     

Resorcinol-, catechol- and saligenin-based bronchodilating β2-agonists as inhibitors of human cholinesterase activity

We investigated the influence of bronchodilating β2-agonists on the activity of human acetylcholinesterase (AChE) and usual, atypical and fluoride-resistant butyrylcholinesterase (BChE). We determined the inhibition potency of racemate and enantiomers of fenoterol as a resorcinol derivative, isoetharine and epinephrine as catechol derivatives and salbutamol and salmeterol as saligenin derivatives. All of the tested compounds reversibly inhibited cholinesterases with K_i constants ranging from 9.4?μM to 6.4?mM and had the highest inhibition potency towards usual BChE, but generally none of the cholinesterases displayed any stereoselectivity. Kinetic and docking results revealed that the inhibition potency of the studied compounds could be related to the size of the hydroxyaminoethyl chain on the benzene ring. The additional π–π interaction of salmeterol’s benzene ring and Trp286 and hydrogen bond with His447 probably enhanced inhibition by salmeterol which was singled out as the most potent inhibitor of all the cholinesterases.     

Discovery of potent carbonic anhydrase,acetylcholinesterase, and butyrylcholinesterase enzymes inhibitors: The new amides and thiazolidine‐4‐ones synthesized on an acetophenone base

Compounds containing nitrogen and sulfur atoms can be widely used in various fields, including industry, medicine, biotechnology, and chemical technology. Among them, amides of acids and heterocyclic compounds have an important place. These amides and thiazolidine‐4‐ones showed good inhibitory action against butyrylcholinesterase (BChE), acetylcholinesterase (AChE), and human carbonic anhydrase isoforms. AChE exists at high concentrations in the brain and red blood cells. BChE is an important enzyme that is plentiful in the liver, and it is released into the blood in a soluble form. They were demonstrated to have effective inhibition profiles with K_i values of 23.76–102.75 nM against hCA I, 58.92–136.64 nM against hCA II, 1.40–12.86 nM against AChE, and 9.82–52.77 nM against BChE. On the other hand, acetazolamide showed K_i value of 482.63 ± 56.20 nM against hCA I, and 1019.60 ± 163.70 nM against hCA II. Additionally, Tacrine inhibited AChE and BChE, showing K_i values of 397.03 ± 31.66 and 210.21 ± 15.98 nM, respectively.     

Acetylcholinesterase and butyrylcholinesterase activities in brain and plasma of freshwater teleosts: cross-species and cross-family differences

Brain and plasma acetylcholinesterase (AChE; EC 3.1.1.7) and plasma butyrylcholinesterase (BChE; EC 3.1.1.8) specific activities were assayed in 16 freshwater teleosts belonging to four families: Cyprinidae, Percidae, Esocidae and Lotidae. Brain AChE activity varied among fish species approximately 15-fold, ranging from 138 to 2011 micromol/g per h. All cyprinids had higher brain AChE activity than other fish families. Plasma AChE activity was on average 100-fold lower than that in brain, varying from 1.2 to 18.6 micromol/ml per h. Plasma BChE activity was found only in cyprinids with the exception of the common and crucian carp, and sabrefish. It varied from 26 to 1083 micromol/ml per h. In bream (Abramis brama) only 30% of specimens studied had BChE activity. The correlation coefficient values between activities of brain and plasma AChE, brain AChE and plasma BChE, plasma AChE and BChE were 0.67, 0.68 and 0.84, respectively. Cross-species and also cross-family differences in AChE and BChE activities among fish were demonstrated. Possible reasons for the differences are discussed.     

Adenovirus-mediated gene transfer of human butyrylcholinesterase results in persistent high-level transgene expression in vivo

Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents, pesticide intoxication, and cocaine overdose. However, its widespread application is hampered by difficulties in large-scale production of the native protein from human plasma and/or availability as a recombinant protein suitable for use in vivo. This limitation may be resolved by in vivo delivery and expression of the Hu BChE gene. In this study, recombinant (r) adenoviruses (Ads) encoding full-length and truncated rHu BChEs were tested for in vivo expression in mice. Mice injected with these rAds intraperitoneally failed to express rHu BChE. However, a single tail vein injection of both rAds resulted in persistent high serum levels of rHu BChE in BChE knockout mice, which peaked on days 4/5 at 377 ± 162 U/ml for full-length rHu BChE and 574 ± 143 U/ml for truncated rHu BChE. These activity levels are orders of magnitude higher than 1.9 U/ml of mouse BChE present in wild-type mouse serum. Thereafter, rHu BChE levels dropped rapidly and very little or no activity was detected in the serum 10 days post-virus administration. In conclusion, the present study demonstrates the potential of rAd-mediated Hu BChE gene therapy to counteract multiple lethal doses of chemical warfare nerve agent toxicity.     

Synthesis,in vitro screening and molecular docking of isoquinolinium-5-carbaldoximes as acetylcholinesterase and butyrylcholinesterase reactivators

Abstract

The series of symmetrical and unsymmetrical isoquinolinium-5-carbaldoximes was designed and prepared for cholinesterase reactivation purposes. The novel compounds were evaluated for intrinsic acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) inhibition, when the majority of novel compounds resulted with high inhibition of both enzymes and only weak inhibitors were selected for reactivation experiments on human AChE or BChE inhibited by sarin, VX, or paraoxon. The AChE reactivation for all used organophosphates was found negligible if compared to the reactivation ability of obidoxime. Importantly, two compounds were found to reactivate BChE inhibited by sarin or VX better to obidoxime at human attainable concentration. One compound resulted as better reactivator of NEMP (VX surrogate)-inhibited BChE than obidoxime. The in vitro results were further rationalized by molecular docking studies showing future directions on designing potent BChE reactivators.     

Screening assays for cholinesterases resistant to inhibition by organophosphorus toxicants

Methods to measure resistance to inhibition by organophosphorus toxicants (OP) for mutants of butyrylcholinesterase (EC 3.1.1.8; BChE) and acetylcholinesterase (EC 3.1.1.7; AChE) enzymes were devised. Wild-type cholinesterases were completely inhibited by 0.1 mM echothiophate or 0.001 mM diisopropylfluorophosphate, but human BChE mutants G117H, G117D, L286H, and W231H and snake AChE mutant HFQT retained activity. Tissues containing a mixture of cholinesterases could be assayed for amount of G117H BChE. For example, the serum of transgenic mice expressing human G117H BChE contained 0.5 microg/ml human G117H BChE, 2 microg/ml wild-type mouse BChE, and 0.06 microg/ml wild-type mouse AChE. The oligomeric structure of G117H BChE in the serum of transgenic mice was determined by nondenaturing gel electrophoresis followed by staining for butyrylthiocholine hydrolysis activity in the presence of 0.1 mM echothiophate. Greater than 95% of the human G117H BChE in transgenic mouse serum was a tetramer. To visualize the distribution of G117H BChE in tissues of transgenic mice, sections of small intestine were treated with echothiophate and then stained for BChE activity. Both wild-type and G117H BChE were in the epithelial cells of the villi. These assays can be used to identify OP-resistant cholinesterases in culture medium and in animal tissues.