Molecularly imprinted polymers (MIPs) mimic the binding sites of antibodies by substituting the amino acid-scaffold of proteins by synthetic polymers. In this work, the first MIP for the recognition of the diagnostically relevant enzyme butyrylcholinesterase (BuChE) is presented. The MIP was prepared using electropolymerization of the functional monomer o-phenylenediamine and was deposited as a thin film on a glassy carbon electrode by oxidative potentiodynamic polymerization. Rebinding and removal of the template were detected by cyclic voltammetry using ferricyanide as a redox marker. Furthermore, the enzymatic activity of BuChE rebound to the MIP was measured via the anodic oxidation of thiocholine, the reaction product of butyrylthiocholine. The response was linear between 50 pM and 2 nM concentrations of BuChE with a detection limit of 14.7 pM. In addition to the high sensitivity for BuChE, the sensor responded towards pseudo-irreversible inhibitors in the lower mM range.

Measuring various biochemical and cellular components in the blood is a routine procedure in clinical practice. Human serum contains hundreds of diverse proteins secreted from all cells and tissues in healthy and diseased states. Moreover, some serum proteins have specific strong interactions with other blood components, but most interactions are probably weak and transient. One of the serum proteins is butyrylcholinesterase (BChE), an enzyme existing mainly as a glycosylated soluble tetramer that plays an important role in the metabolism of many drugs. Our results suggest that BChE interacts with plasma proteins and forms much larger complexes than predicted from the molecular weight of the BChE tetramer. To investigate and isolate such complexes, we developed a two-step strategy to find specific protein-protein interactions by combining native size-exclusion chromatography (SEC) with affinity chromatography with the resin that specifically binds BChE. Second, to confirm protein complexes' specificity, we fractionated blood serum proteins by density gradient ultracentrifugation followed by co-immunoprecipitation with anti-BChE monoclonal antibodies. The proteins coisolated in complexes with BChE were identified by mass spectroscopy. These binding studies revealed that BChE interacts with a number of proteins in the human serum. Some of these interactions seem to be more stable than transient. BChE copurification with ApoA-I and the density of some fractions containing BChE corresponding to high-density lipoprotein cholesterol (HDL) during ultracentrifugation suggest its interactions with HDL. Moreover, we observed lower BChE plasma activity in individuals with severely reduced HDL levels (≤20 mg/dL). The presented two-step methodology for determination of the BChE interactions can facilitate further analysis of such complexes, especially from the brain tissue, where BChE could be involved in the pathogenesis and progression of AD.

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.

Caffeine is an alkaloid with a stimulant effect in the body. It can interfere in transmissions based on acetylcholine, epinephrine, norepinephrine, serotonin, dopamine and glutamate. Clinical studies indicate that it can be involved in the slowing of Alzheimer disease pathology and some other effects. The effects are not well understood. In the present work, we focused on the question whether caffeine can inhibit acetylcholinesterase (AChE) and/or, butyrylcholinesterase (BChE), the two enzymes participating in cholinergic neurotransmission. A standard Ellman test with human AChE and BChE was done for altering concentrations of caffeine. The test was supported by an in silico examination as well. Donepezil and tacrine were used as standards. In compliance with Dixon's plot, caffeine was proved to be a non-competitive inhibitor of AChE and BChE. However, inhibition of BChE was quite weak, as the inhibition constant, Ki, was 13.9 ± 7.4 mol/L. Inhibition of AChE was more relevant, as Ki was found to be 175 ± 9 µmol/L. The predicted free energy of binding was -6.7 kcal/mol. The proposed binding orientation of caffeine can interact with Trp86, and it can be stabilize by Tyr337 in comparison to the smaller Ala328 in the case of human BChE; thus, it can explain the lower binding affinity of caffeine for BChE with reference to AChE. The biological relevance of the findings is discussed.

Butyrylcholinesterase (BChE) is a nonspecific cholinesterase enzyme that hydrolyzes choline-based esters. BChE plays a critical role in maintaining normal cholinergic function like acetylcholinesterase (AChE) through hydrolyzing acetylcholine (ACh). Selective BChE inhibition has been regarded as a viable therapeutic approach in Alzheimer's disease. As of now, a limited number of selective BChE inhibitors are available. To identify BChE inhibitors rapidly and efficiently, we have screened 8998 compounds from several annotated libraries against an enzyme-based BChE inhibition assay in a quantitative high-throughput screening (qHTS) format. From the primary screening, we identified a group of 125 compounds that were further confirmed to inhibit BChE activity, including previously reported BChE inhibitors (e.g., bambuterol and rivastigmine) and potential novel BChE inhibitors (e.g., pancuronium bromide and NNC 756), representing diverse structural classes. These BChE inhibitors were also tested for their selectivity by comparing their IC50 values in BChE and AChE inhibition assays. The binding modes of these compounds were further studied using molecular docking analyses to identify the differences between the interactions of these BChE inhibitors within the active sites of AChE and BChE. Our qHTS approach allowed us to establish a robust and reliable process to screen large compound collections for potential BChE inhibitors.

Human butyrylcholinesterase (hBChE) is currently being developed as a detoxication enzyme for stoichiometric binding and/or catalytic hydrolysis of organophosphates. Herein, we describe the use of a molecular evolution method to develop novel hBChE variants with increased resistance to stereochemically defined nerve agent model compounds of soman, sarin, and cyclosarin. Novel hBChE variants (Y332S, D340H, and Y332S/D340H) were identified with an increased resistance to nerve agent model compounds that retained robust intrinsic catalytic efficiency. Molecular dynamics simulations of these variants revealed insights into the mechanism by which these structural changes conferred nerve agent model compound resistance.

Selective butyrylcholinesterase inhibitors are considered promising drug candidates for the treatment of Alzheimer's disease. In this work, one rivastigmine-bambuterol hybrid (MTR-1) and fourteen of its analogues were synthesized, purified, and characterized. In vitro cholinesterase assays showed that all the compounds were more potent inhibitors of BChE when compared to AChE. Further investigations indicated that MTR-3 (IC50(AChE) > 100,000 nM, IC50(BChE) = 78 nM) was the best compound in the series, showing high butyrylcholinesterase selectivity and inhibition potency, the potential to permeate the blood-brain barrier, and longer-lasting BChE inhibition than bambuterol. These compounds could be used to discover novel specific BChE inhibitors for the treatment of Alzheimer's disease.

Alzheimer's disease is a topic of deep research interest across the global scientific community. Butyrylcholinesterase (BuChE) is an important enzyme, and an interesting anti-Alzheimer's target. Identification or fresh design of promising BuChE-inhibitors is warranted. Virtual screening supported by molecular dynamics simulations has emerged as a key component of present drug-discovery cascades. The research piece aimed at identification of a putative BuChE-inhibitor as a fresh molecular frame that might aid drug design in the context of Alzheimer's disease. The study utilized 'MCULE' to screen a set of 5 million ligands to test their ability to bind to human BuChE. Pharmacokinetic profiling was achieved by the 'SWISS ADME' program. Toxicities were duly assessed. YASARA STRUCTURE version 20.10.4.W.64 was employed to run 133 ns molecular dynamics (MD) simulation for the complex of 'the top screened out inhibitor' and 'the human BuChE enzyme'. The simulation was executed for approx. 4 days (~93 hrs) on an HP ZR30w workstation. YANACONDA, a special language contained in YASARA STRUCTURE was employed to perform complex tasks. Fine resolution figures (notably the RMSD vs time plot) were created. Snapshots were extracted at every 250 ps. The selected ligand, (3-Bromophenyl)[5-(4-chlorophenyl)-5-hydroxy-3-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl]methanone, exhibited the best overall binding with human BuChE. It interacted with human BuChE through 19 residues. Markedly, 9 of the 19 residues were confirmed to be matching to those of the reference complex (PDB ID 5DYW). Trajectory analysis returned 533 snapshots. The RMSD versus time plot indicated that around 22 ns, equilibrium was achieved and, from then on, the 'BuChE-Top inhibitor' complex remained predominantly stable. From 22 ns and onwards till 133 ns, the backbone RMSD fluctuations were observed to remain limited within a range of 1.2-1.9 Å. The molecule, (3-Bromophenyl)[5-(4-chlorophenyl)-5-hydroxy-3-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl]methanone, satisfied ADMET requirements. Additionally, the feasibility of the proposed enzyme-inhibitor complex was supported by an adequately extended MD simulation of 133 ns. Hence, the proposed molecule could be a likely lead for designing inhibitor(s) against human BuChE. Scope remains for validatory wet laboratory investigation.

Human plasma to be analyzed for exposure to cholinesterase inhibitors is stored at 4 °C or lower to prevent denaturation of human butyrylcholinesterase (HuBChE), the biomarker of exposure. Currently published protocols immunopurify HuBChE using antibodies that bind native HuBChE before analysis by mass spectrometry. It is anticipated that the plasma collected from human casualties may be stored nonideally at elevated temperatures of up to 45 °C for days or maybe weeks. At 45 °C, the plasma loses 50% of its HuBChE activity in 8 days and 95% in 40 days. Our goal was to identify a set of monoclonal antibodies that could be used to immunopurify HuBChE from plasma stored at 45 °C. The folding states of pure human HuBChE stored at 4 and 45 °C and boiled at 100 °C were visualized on nondenaturing gels stained with Coomassie blue. Fully active pure HuBChE tetramers had a single band, but pure HuBChE stored at 45 °C had four bands, representing native, partly unfolded, aggregated, and completely denatured, boiled tetramers. The previously described monoclonal B2 18-5 captured native, partly unfolded, and aggregated HuBChE tetramers, whereas a new monoclonal, C191 developed in our laboratory, was found to selectively capture completely denatured, boiled HuBChE. The highest quantity of HuBChE protein was extracted from 45 °C heat-denatured human plasma when HuBChE was immunopurified with a combination of monoclonals B2 18-5 and C191. Using a mixture of these two antibodies in future emergency response assays may increase the capability to confirm exposure to cholinesterase inhibitors.

Cultured SH-SY5Y human neuroblastoma cells are used in neurotoxicity assays. These cells express markers of the cholinergic and dopaminergic systems. Acetylcholinesterase (AChE) activity has been reported in these cells. Neurotoxic organophosphate compounds that inhibit AChE, also inhibit butyrylcholinesterase (BChE). We confirmed the presence of AChE in the cell lysate by activity assays, Western blot, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of immunopurified AChE. A nondenaturing gel stained for AChE activity identified the catalytically active AChE in SH-SY5Y cells as the unstable monomer. We also identified immature BChE in the cell lysate. The concentration of active BChE protein was similar to that of active AChE protein. The rate of substrate hydrolysis by AChE was 10-fold higher than substrate hydrolysis by BChE. The higher rate was due to the 10-fold higher specific activity of AChE over BChE (5000 units/mg for AChE; 500 units/mg for BChE). Neither cholinesterase was secreted. Tryptic peptides of immunopurified AChE and BChE were identified by LC-MS/MS on an Orbitrap Lumos Fusion mass spectrometer. The unfolded protein chaperone, binding immunoglobulin protein BiP/GRP78, was identified in the mass spectral data from all cholinesterase samples, suggesting that BiP was co-extracted with cholinesterase. This suggests that the cytoplasmic cholinesterases are immature forms of AChE and BChE that bind to BiP. It was concluded that SH-SY5Y cells express active AChE and active BChE, but the proteins do not mature to glycosylated tetramers.

Alzheimer's disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD. Potent, selective and reversible human BChE inhibitors were developed. The solved crystal structure of human BChE in complex with the most potent inhibitor reveals its binding mode and provides the molecular basis of its low nanomolar potency. Additionally, this compound is noncytotoxic and has neuroprotective properties. Furthermore, this inhibitor moderately crosses the blood-brain barrier and improves memory, cognitive functions and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic adverse effects. Our study provides an advanced lead compound for developing drugs for alleviating symptoms caused by cholinergic hypofunction in advanced AD.

Metal phosphides are widely used as a rodenticide and insecticide and poisoning with these substances has a very high mortality. The aim of this study was to evaluate the butyrylcholinesterase (BuCh) level in poisoning with metal phosphides.

The ghrelin hormone is a 28 amino acid peptide esterified on serine 3 with octanoic acid. Ghrelin is inactivated by hydrolysis of the ester bond. Previous studies have relied on inhibitors to identify human butyrylcholinesterase (BChE) as the hydrolase in human plasma that converts ghrelin to desacyl ghrelin. The reaction of BChE with ghrelin is unusual because the rate of hydrolysis is very slow and the substrate is ten times larger than standard BChE substrates. These unusual features prompted us to re-examine the reaction, using human BChE preparations that were more than 98% pure. Conversion of ghrelin to desacyl ghrelin was monitored by MALDI TOF mass spectrometry. It was found that 5 different preparations of pure human BChE all hydrolyzed ghrelin, including BChE purified from human plasma, from Cohn fraction IV-4, BChE immunopurified by binding to monoclonals mAb2 and B2 18-5, and recombinant human BChE purified from culture medium. We reasoned that it was unlikely that a common contaminant that could be responsible for ghrelin hydrolysis would appear in all of these preparations. km was <1 μM, and kcat was ~1.4 min(-1). A Michaelis-Menten analysis employing these kinetic values together with serum concentrations of ghrelin and BChE demonstrated that BChE could hydrolyze all of the ghrelin in serum in ~1 h. It was concluded that BChE is physiologically relevant for the hydrolysis of ghrelin.

A series of tryptophan-based selective nanomolar butyrylcholinesterase (BChE) inhibitors was designed and synthesized. Compounds were optimized in terms of potency, selectivity, and synthetic accessibility. The crystal structure of the inhibitor 18 in complex with BChE revealed the molecular basis for its low nanomolar inhibition (IC50 = 2.8 nM). The favourable in vitro results enabled a first-in-animal in vivo efficacy and safety trial, which demonstrated a positive impact on fear-motivated and spatial long-term memory retrieval without any concomitant adverse motor effects. Altogether, this research culminated in a handful of new lead compounds with promising potential for symptomatic treatment of patients with Alzheimer's disease.

A newly recognized action of organophosphates (OP) is the ability to crosslink proteins through an isopeptide bond. The first step in the mechanism is covalent addition of the OP to the side chain of lysine. This activates OP-lysine for reaction with a nearby glutamic or aspartic acid to make a gamma glutamyl epsilon lysine bond. Crosslinked proteins are high molecular weight aggregates. Our goal was to identify the residues in the human butyrylcholinesterase (HuBChE) tetramer that were crosslinked following treatment with 1.5 mM chlorpyrifos oxon. High molecular weight bands were visualized on an SDS gel. Proteins in the gel bands were digested with trypsin, separated by liquid chromatography and analyzed in an Orbitrap mass spectrometer. MSMS files were searched for crosslinked peptides using the Batch-Tag program in Protein Prospector. MSMS spectra were manually evaluated for the presence of ions that supported the crosslinks. The crosslink between Lys544 in VLEMTGNIDEAEWEWK544AGFHR and Glu542 in VLEMTGNIDEAEWE542WK satisfied our criteria including that of spatial proximity. Distances between Lys544 and Glu542 were 7.4 and 9.5 Å, calculated from the cryo-EM (electron microscopy) structure of the HuBChE tetramer. Paraoxon ethyl, diazoxon, and dichlorvos had less pronounced effects as visualized on SDS gels. Our proof-of-principle study provides evidence that OP have the ability to crosslink proteins. If OP-induced protein crosslinking occurs in the brain, OP exposure could be responsible for some cases of neurodegenerative disease.

Butyrylcholinesterase (BChE) is a plasma enzyme that hydrolyzes ghrelin and bioactive esters, suggesting a role in modulating metabolism. Serum BChE is reduced in cancer patients. In prostate cancer (PC), the down-regulation is associated with disease recurrence. Nonetheless, how BChE is expressed in PC and its impact on PC remain unclear. We report here the biphasic changes of BChE expression in PC. In vitro, BChE expression was decreased in more tumorigenic PC stem-like cells (PCSLCs), DU145, and PC3 cells compared to less tumorigenic non-stem PCs and LNCaP cells. On the other hand, BChE was expressed at a higher level in LNCaP cells than immortalized but non-tumorigenic prostate epithelial BPH-1 cells. In vivo, BChE expression was up-regulated in DU145 xenografts compared to LNCaP xenografts; DU145 cell-derived lung metastases displayed comparable levels of BChE as subcutaneous tumors. Furthermore, LNCaP xenografts produced in castrated mice exhibited a significant increase of BChE expression compared to xenografts generated in intact mice. In patients, BChE expression was down-regulated in PCs (n = 340) compared to prostate tissues (n = 86). In two independent PC populations MSKCC (n = 130) and TCGA Provisional (n = 490), BChE mRNA levels were reduced from World Health Organization grade group 1 (WHOGG 1) PCs to WHOGG 3 PCs, followed by a significant increase in WHOGG 5 PCs. The up-regulation was associated with a reduction in disease-free survival (P = .008). Collectively, we demonstrated for the first time a biphasic alteration of BChE, its down-regulation at early stage of PC and its up-regulation at advanced PC.

The search for genetic vulnerability factors in cocaine dependence has focused on the role that neuroplasticity plays in addiction. However, like many other drugs, the ability of an individual to metabolize cocaine can also influence susceptibility to dependence. Butyrylcholinesterase (BChE) metabolizes cocaine, and genetic variants of the BChE gene (BCHE) alter its catalytic activity. Therefore, we hypothesize that cocaine users with polymorphisms in BCHE can show diverse addictive behaviors due to differences in effective plasma concentrations of cocaine. Those polymorphisms might also influence users to prefer one of the two main preparations (crack or powder cocaine), despite having equal access to both. The present work investigates polymorphisms in BCHE and if those genetic variants constitute risk factors for cocaine dependence and for crack cocaine use.

Butyrylcholinesterase (BChE), a hydrolytic enzyme, is responsible for the termination of the action of acetylcholine besides acetylcholinesterase (AChE) in the synaptic cleft of the brain. The alteration in the enzyme level, in patients with the progression of Alzheimer's disease, makes it a therapeutic target. In the present study, we developed BChE inhibitors through scaffold hopping by exploring two previously reported compounds, i.e., 1,4-bis((4-chlorophenyl) sulfonyl)-3,6-diphenylpiperazine-2,5-dione and N-(2-chlorophenyl)-4-(phenylsulfonamido)benzamide, to afford scaffold and pharmacophore fragments, respectively. The N,2-diphenyl-2-(phenylsulfonamido)acetamide derivatives, thus designed, were synthesised and screened for the inhibition of AChE and BChE enzymes. Compounds 30 and 33 were found to be most active against BChE among the derivatives, with IC50 values of 7.331 ± 0.946 and 10.964 ± 0.936 μM, respectively. The compounds displayed a non-competitive mode of inhibition along with BBB permeability and good cell viability on SH-SY5Y cell line. The molecular docking analysis of the compounds with BChE showed interactions with Trp82, Trp231, Leu286, and His438. The molecular dynamics study revealed the stability of the protein-ligand complexes.

Butyrylcholinesterase (BChE) is a hydrolytic enzyme that together with acetylcholinesterase (AChE) belongs to the cholinesterase family. Whereas AChE has a well-established role in regulating cholinergic neurotransmission in central and peripheral synapses, the physiological role of BChE remains elusive. In this morphological immunohistochemical and double-label confocal microscopy study we investigated the distribution of BChE in the mouse gastrointestinal tract. BChE-positive cells were detected in the liver (both in hepatocytes and cholangiocytes), in the keratinised layers of the squamous epithelium of the oesophagus and forestomach, in the oxyntic mucosa of the stomach, in the mucus-secreting cells of duodenal Brunner glands and the small and large intestinal mucosa. Interestingly, BChE-positive cells were often detected close to gastrointestinal proliferative niches. In the oxyntic mucosa, the close proximity of ghrelin-producing and BChE-positive parietal cells suggests that BChE may be involved in ghrelin hydrolysation through paracrine action. To our knowledge, this is the first comprehensive morphological study performed to gain insight into the physiological role of BChE in the gastrointestinal tract.

Neurodegenerative diseases, e.g., Alzheimer's disease (AD), are a key health problem in the aging population. The lack of effective therapy and diagnostics does not help to improve this situation. It is thought that ligands influencing multiple but interconnected targets can contribute to a desired pharmacological effect in these complex illnesses. Histamine H3 receptors (H3Rs) play an important role in the brain, influencing the release of important neurotransmitters, such as acetylcholine. Compounds blocking their activity can increase the level of these neurotransmitters. Cholinesterases (acetyl- and butyrylcholinesterase) are responsible for the hydrolysis of acetylcholine and inactivation of the neurotransmitter. Increased activity of these enzymes, especially butyrylcholinesterase (BuChE), is observed in neurodegenerative diseases. Currently, cholinesterase inhibitors: donepezil, rivastigmine and galantamine are used in the symptomatic treatment of AD. Thus, compounds simultaneously blocking H3R and inhibiting cholinesterases could be a promising treatment for AD. Herein, we describe the BuChE inhibitory activity of H3R ligands. Most of these compounds show high affinity for human H3R (Ki < 150 nM) and submicromolar inhibition of BuChE (IC50 < 1 µM). Among all the tested compounds, 19 (E153, 1-(5-([1,1'-biphenyl]-4-yloxy)pentyl)azepane) exhibited the most promising in vitro affinity for human H3R, with a Ki value of 33.9 nM, and for equine serum BuChE, with an IC50 of 590 nM. Moreover, 19 (E153) showed inhibitory activity towards human MAO B with an IC50 of 243 nM. Furthermore, in vivo studies using the Passive Avoidance Task showed that compound 19 (E153) effectively alleviated memory deficits caused by scopolamine. Taken together, these findings suggest that compound 19 can be a lead structure for developing new anti-AD agents.