Selective Butyrylcholinesterase Inhibitors Research Articles

Evaluating Fmoc-amino acids as selective inhibitors of butyrylcholinesterase.

Cholinesterases are involved in neuronal signal transduction, and perturbation of function has been implicated in diseases, such as Alzheimer's and Huntington's disease. For the two major classes of cholinesterases, such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), previous studies reported BChE activity is elevated in patients with Alzheimer's disease, while AChE levels remain the same or decrease. Thus, the development of potent and specific inhibitors of BChE have received much attention as a potential therapeutic in the alleviation of neurodegenerative diseases. In this study, we evaluated amino acid analogs as selective inhibitors of BChE. Amino acid analogs bearing a 9-fluorenylmethyloxycarbonyl (Fmoc) group were tested, as the Fmoc group has structural resemblance to previously described inhibitors. We identified leucine, lysine, and tryptophan analogs bearing the Fmoc group as selective inhibitors of BChE. The Fmoc group contributed to inhibition, as analogs bearing a carboxybenzyl group showed ~tenfold higher values for the inhibition constant (K I value). Inclusion of a t-butoxycarbonyl on the side chain of Fmoc tryptophan led to an eightfold lower K I value compared to Fmoc tryptophan alone suggesting that modifications of the amino acid side chains may be designed to create inhibitors with higher affinity. Our results identify Fmoc-amino acids as a scaffold upon which to design BChE-specific inhibitors and provide the foundation for further experimental and computational studies to dissect the interactions that contribute to inhibitor binding.

Discovery and Structure-Activity Relationships of a Highly Selective Butyrylcholinesterase Inhibitor by Structure-Based Virtual Screening.

Structure-based virtual screening of two libraries containing 567 981 molecules was used to discover novel, selective BuChE inhibitors, which are potentially superior symptomatic treatments in late-stage Alzheimer's disease. Compound 16 was identified as a highly selective submicromolar inhibitor of BuChE (huBuChE IC50 = 0.443 μM) with high permeability in the PAMPA-BBB model. The X-ray crystal structure of huBuChE in complex with 16 revealed the atomic-level interactions and offers opportunities for further development of the series.

1H-benzimidazole derivatives as butyrylcholinesterase inhibitors: synthesis and molecular modeling studies

A series of N-{2-[2-(1H-benzimidazole-2-yl)phenoxy]ethyl} substituted amine derivatives were synthesized and tested for their cholinesterase inhibitor activity. Acetylcholinesterase and butyrylcholinesterase inhibitor activities were evaluated in vitro by using Ellman’s method. According to the activity results, all of the compounds displayed moderate acetylcholinesterase inhibitory activity and most of the compounds displayed remarkable butyrylcholinesterase inhibitory activity. Compound 3d was the most active compound in the series and also a selective butyrylcholinesterase inhibitor. Molecular docking studies and molecular dynamic simulations were also carried out.

1,2,4-Thiadiazoles as promising multifunctional agents for treatment of neurodegenerative diseases

Detailed studies of properties of new 3-substituted 5-anilino-1,2,4-thiadiazoles containing different substituents at position 3 of the thiadiazole ring were carried out, in particular, their esterase profile and antioxidant properties. It was found that the presence in the molecule of 2-aminopropyl fragment determines an efficient and selective inhibition of butyrylcholinesterase as compared to acetylcholinesterase and carboxylesterase, with radical-scavenging activity being weak. The compounds containing a 2-aminopropenyl fragment possess a high radicalscavenging activity, weakly inhibit cholinesterases, and exhibit anticarboxylesterase activity. A wide spectrum of activity of 3-substituted 5-anilino-1,2,4-thiadiazoles as inhibitors of cholinesterases and highly efficient scavengers of free radicals gives a basis for the optimization of structure and development in this series of original agents for therapy of neurodegenerative diseases.

Synthesis of glucopyranos-6′-yl purine and pyrimidine isonucleosides as potential cholinesterase inhibitors. Access to pyrimidine-linked pseudodisaccharides through Mitsunobu reaction

Abstract The synthesis of new isonucleosides comprising purine and pyrimidine-derived systems linked to methyl glucopyranosidyl units at C-6 and evaluation of their cholinesterase inhibitory profiles is reported. Their access was based on the Mitsunobu coupling of partially acetylated and benzylated methyl glucopyranosides with purine and pyrimidine derivatives. While the reactions with purines and theobromine proceeded with complete regioselectivity, affording exclusively N9- or N1-linked 6′-isonucleosides, respectively, the use of pyrimidine nucleobases led to N1 and/or N3-glucopyranosid-6′-yl pyrimidines and/or to N1,N3/2-O,4-O-pyrimidine-linked pseudodisaccharides through bis-coupling, depending on the substitution pattern of the sugar precursor and on the nature of the nucleobase. From this series of compounds, four were shown to be effective and selective inhibitors of acetylcholinesterase with inhibition constants in the micromolar concentration range. A tri-O-acetylated N1-glucopyranosid-6′-yl theobromine and a benzylated N1,N3-bis-glucopyranosid-6-yl thymine were the most active molecules with K i values of 4 μM. A tri-O-benzylated glucopyranosid-6′-yl uracil displayed good and selective inhibition of butyrylcholinesterase (K i=8.4±1.0 μM), similar to that exhibited by the standard galantamine. Molecular docking simulations, performed with the two most effective acetylcholinesterase inhibitors, showed interactions with key amino acid residues located at the enzyme’s active site gorge, which explain the competitive component of their inhibitory activities.

Novel Selective Butyrylcholinesterase Inhibitors Incorporating Antioxidant Functionalities as Potential Bimodal Therapeutics for Alzheimer's Disease.

Isosorbide-2-carbamates-5-aryl esters are highly potent and very selective butyrylcholinesterase inhibitors. The objective of the present work was to address the hypothesis that the isosorbide-aryl-5-ester group could be replaced with an antioxidant functionality while maintaining inhibitor effects and selectivity. We successfully incorporated ferulic acid or lipoic acid groups producing potent selective inhibitors of butyrylcholinesterase (BuChE). The hybrid compounds were non-toxic to the murine hippocampal cell line HT-22 and lipoate esters were neuroprotective at 10 and 25 µM when the cells were challenged with glutamate (5 mM) in a similar manner to the positive control quercetin. The benzyl carbamate 7a was a potent inhibitor of BuChE (IC50 150 nM) and it was effective in reducing glutamate toxicity to neuronal cells at >5 µM. Representative compounds exhibited an antioxidant effect in the oxygen radical absorbance capacity assay as the lipoate 7d was not active, whereas the ferulate 8a showed a weak, but significant, activity with 0.635 ± 0.020 Trolox Equivalent.

Conjugates of γ-Carbolines and Phenothiazine as new selective inhibitors of butyrylcholinesterase and blockers of NMDA receptors for Alzheimer Disease.

Alzheimer disease is a multifactorial pathology and the development of new multitarget neuroprotective drugs is promising and attractive. We synthesized a group of original compounds, which combine in one molecule γ-carboline fragment of dimebon and phenothiazine core of methylene blue (MB) linked by 1-oxo- and 2-hydroxypropylene spacers. Inhibitory activity of the conjugates toward acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and structurally close to them carboxylesterase (CaE), as well their binding to NMDA-receptors were evaluated in vitro and in silico. These newly synthesized compounds showed significantly higher inhibitory activity toward BChE with IC50 values in submicromolar and micromolar range and exhibited selective inhibitory action against BChE over AChE and CaE. Kinetic studies for the 9 most active compounds indicated that majority of them were mixed-type BChE inhibitors. The main specific protein-ligand interaction is π-π stacking of phenothiazine ring with indole group of Trp82. These compounds emerge as promising safe multitarget ligands for the further development of a therapeutic approach against aging-related neurodegenerative disorders such as Alzheimer and/or other pathological conditions.

Structure-based development of nitroxoline derivatives as potential multifunctional anti-Alzheimer agents

Tremendous efforts have been dedicated to the development of effective therapeutics against Alzheimer’s disease, which represents the most common debilitating neurodegenerative disease. Multifunctional agents are molecules designed to have simultaneous effects on different pathological processes. Such compounds represent an emerging strategy for the development of effective treatments against Alzheimer’s disease. Here, we report on the synthesis and biological evaluation of a series of nitroxoline-based analogs that were designed by merging the scaffold of 8-hydroxyquinoline with that of a known selective butyrylcholinesterase inhibitor that has promising anti-Alzheimer properties. Most strikingly, compound 8g inhibits self-induced aggregation of the amyloid beta peptide (Aβ1–42), inhibits with sub-micromolar potency butyrylcholinesterase (IC50=215nM), and also selectively complexes Cu2+. Our study thus designates this compound as a promising multifunctional agent for therapeutic treatment of Alzheimer’s disease. The crystal structure of human butyrylcholinesterase in complex with compound 8g is also solved, which suggests ways to further optimize compounds featuring the 8-hydroxyquinoline scaffold.

A color-reaction-based rapid screening for null activity of butyrylcholinesterase: a step toward point-of-care screening for succinylcholine apnea.

Succinylcholine apnea happens in cases of null butyrylcholinesterase activity after administration of preintubation succinylcholine. So far, there is no such popular test that can rapidly screen null butyrylcholinesterase activity from plasma. Development of a novel method for rapid screening of null butyrylcholinesterase activity of plasma samples was the objective of the current work. Dichromate reagent was added to 1-naphthol, 2-naphthol, phenol, and para-nitrophenol in separate aliquots and watched for the color formation. Plasma samples preincubated with and without selective butyrylcholinesterase inhibitor were mixed with 1-naphthylacetate and watched for color development after addition of dichromate reagent. Fitting of 1-naphthylacetate at the active site of butyrylcholinesterase was analyzed by using tools of computational biology. It was seen that 1-naphthol formed color with dichromate reagent in a concentration-dependent manner. Other phenols did not form color with dichromate reagent even at 500-µm concentrations. Plasma sample with and without selective butyrylcholinesterase inhibitor (tetra isopropyl pyrophosphoramide) was distinguishable by color formation when incubated with 1-naphthylacetate, followed by the addition of dichromate reagent. In silico analysis also showed that 1-naphthylacetate fitted well at the active site of butyrylcholinesterase. The developed method may be used for rapid screening for null butyrylcholinesterase activity at point of care.

Isoxazolotacrines as non-toxic and selective butyrylcholinesterase inhibitors for Alzheimer's disease.

Owing to the complex nature of Alzheimer's disease, there is a renewed and growing search for multitarget non-toxic tacrines as simple, easily available drugs in order to stop the progress and development of the disease. This paper describes our preliminary results on the synthesis, in vitro biochemical evaluation and molecular modeling of isoxazolotacrines as potential drugs for the treatment of Alzheimer's disease. Novel 3-phenyl-5,6,7,8-tetrahydroisoxazolo[5,4-b]quinolin-4-amine (OC41) is a promising, 31% less toxic than tacrine in HepG2 cells, and selective reversible human butyrylcholinesterase inhibitor (IC50 = 5.08 ± 1.12 µM), also showing good drug-like properties according to the absorption, Distribution, Metabolism, Excretion, Toxicity analysis. A new family of non-hepatotoxic permeable tacrine analogs, showing selective butyrylcholinesterase inhibition, have been discovered for the potential treatment of Alzheimer's disease.

Discovery, biological evaluation, and crystal structure of a novel nanomolar selective butyrylcholinesterase inhibitor.

Butyrylcholinesterase (BChE) is regarded as a promising drug target as its levels and activity significantly increase in the late stages of Alzheimer's disease. To discover novel BChE inhibitors, we used a hierarchical virtual screening protocol followed by biochemical evaluation of 40 highest scoring hit compounds. Three of the compounds identified showed significant inhibitory activities against BChE. The most potent, compound 1 (IC50 = 21.3 nM), was resynthesized and resolved into its pure enantiomers. A high degree of stereoselective activity was revealed, and a dissociation constant of 2.7 nM was determined for the most potent stereoisomer (+)-1. The crystal structure of human BChE in complex with compound (+)-1 was solved, revealing the binding mode and providing clues for potential optimization. Additionally, compound 1 inhibited amyloid β(1-42) peptide self-induced aggregation into fibrils (by 61.7% at 10 μM) and protected cultured SH-SY5Y cells against amyloid-β-induced toxicity. These data suggest that compound 1 represents a promising candidate for hit-to-lead follow-up in the drug-discovery process against Alzheimer's disease.

Quinoxaline derivatives: novel and selective butyrylcholinesterase inhibitors.

Alzheimer's disease (AD) is a progressive brain disorder which occurs due to lower levels of acetylcholine (ACh) neurotransmitters, and results in a gradual decline in memory and other cognitive processes. Acetycholinesterase (AChE) and butyrylcholinesterase (BChE) are considered to be primary regulators of the ACh levels in the brain. Evidence shows that AChE activity decreases in AD, while activity of BChE does not change or even elevate in advanced AD, which suggests a key involvement of BChE in ACh hydrolysis during AD symptoms. Therefore, inhibiting the activity of BChE may be an effective way to control AD associated disorders. In this regard, a series of quinoxaline derivatives 1-17 was synthesized and biologically evaluated against cholinesterases (AChE and BChE) and as well as against α- chymotrypsin and urease. The compounds 1-17 were found to be selective inhibitors for BChE, as no activity was found against other enzymes. Among the series, compounds 6 (IC50 = 7.7 ± 1.0 µM) and 7 (IC50 = 9.7 ± 0.9 µM) were found to be the most active inhibitors against BChE. Their IC50 values are comparable to the standard, galantamine (IC50 = 6.6 ± 0.38 µM). Their considerable BChE inhibitory activity makes them selective candidates for the development of BChE inhibitors. Structure-activity relationship (SAR) of this new class of selective BChE inhibitors has been discussed.

Identification of a neuroprotective and selective butyrylcholinesterase inhibitor derived from the natural alkaloid evodiamine

Two sets of carbamates based on the natural alkaloid evodiamine were designed, synthesized and evaluated as potential butyrylcholinesterase inhibitors. Although a set of carbamates of 3-hydroxyevodiamine (10a–f) is inactive both at AChE and BChE, carbamates of 5-deoxo-3-hydroxyevodiamine (11a–f) exhibit much better potency with selectivity toward BChE. The heptyl carbamate of 5-deoxo-3-hydroxyevodiamine (11c) shows the best potency with an IC50 value of 77 nM and very good selectivity over AChE. ORAC and cell-based assays indicate 11c owns pronounced antioxidant properties with 1.75 Trolox equivalents and strong neuroprotection even from 1 μM onwards. These combined activities might enable compound 11c to be a potential candidate for treatment of Alzheimer's disease.

Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli

Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1–14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1–14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1–14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1–14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis.

Long-Term Reduction of Cocaine Self-Administration in Rats Treated with Adenoviral Vector-Delivered Cocaine Hydrolase: Evidence for Enzymatic Activity

A new pharmacokinetic approach treating cocaine addiction involves rapidly metabolizing cocaine before it reaches brain reward centers using mutated human butyrylcholinesterase (BChE) or cocaine hydrolase (CocH). Recent work has shown that helper-dependent adenoviral (hdAD) vector-mediated plasma CocH reduced the locomotor-activating effects of cocaine and prevented reinstatement of cocaine-seeking behavior up to 6 months in rats. The present study investigated whether hdAD-CocH could decrease ongoing intravenous cocaine (0.4 mg/kg) self-administration. The hdAD-CocH vector was injected into self-administering rats, and after accumulation of plasma CocH, there was a dramatic reduction in cocaine infusions earned under a fixed ratio 1 schedule of reinforcement that lasted for the length of the study (>2 months). Pretreatment with the selective BChE and CocH inhibitor iso-OMPA (1.5 mg/kg) restored cocaine intake; therefore, the decline in self-administration was likely due to rapid CocH-mediated cocaine metabolism. Direct measurements of cocaine levels in plasma and brain samples taken after the conclusion of behavioral studies provided strong support for this conclusion. Further, rats injected with hdAD-CocH did not experience a deficit in operant responding for drug reinforcement and self-administered methamphetamine (0.05 mg/kg) at control levels. Overall, these outcomes suggest that viral gene transfer can yield plasma CocH levels that effectively diminish long-term cocaine intake and may have potential treatment implications for cocaine-dependent individuals seeking to become and remain abstinent.

Microwave-assisted synthesis of novel purine nucleosides as selective cholinesterase inhibitors

Alzheimer's disease (AD), the most common form of senile dementia, is characterized by high butyrylcholinesterase (BChE) levels in the brain in later AD stages, for which no treatment is available. Pursuing our studies on selective BChE inhibitors, that may contribute to understand the role of this enzyme in disease progression, we present now microwave-assisted synthesis and anticholinesterase activity of a new nucleoside series embodying 6-chloropurine or 2-acetamido-6-chloropurine linked to D-glucosyl, D-galactosyl and D-mannosyl residues. It was designed to assess the contribution of sugar stereochemistry, purine structure and linkage to the sugar for cholinesterase inhibition efficiency and selectivity. Compounds were subjected to Ellman's assay and their inhibition constants determined. The α-anomers were the most active compounds, while selectivity for BChE or acetylcholinesterase (AChE) inhibition could be tuned by the purine base, by the glycosyl moiety and by N(7)-ligation. Some of the nucleosides were far more potent than the drug galantamine, and the most promising competitive and selective BChE inhibitor, the N(7)-linked 2-acetamido-α-D-mannosylpurine, showed a Ki of 50 nM and a selectivity factor of 340 fold for BChE over AChE.

Synthesis of Selective Butyrylcholinesterase Inhibitors Coupled between α-Lipoic Acid and Polyphenols by Using 2-(Piperazin-1-yl)ethanol Linker

In the previous paper (Bull. Korean Chem. Soc., 2011, 32, 2997), the hybrid molecules between α-lipoic acid (ALA) and polyphenols (PPs) connected with neutral 2-(2-aminoethoxy)ethanol linker (linker-1) showed new biological activity such as butyrylcholinesterase (BuChE) inhibition. In order to increase the binding affinity of the hybrid compounds to cholinesterase (ChE), the neutral 2-(2-aminoethoxy)ethanol (linker 1) was switched to the cationic 2-(piperazin-1-yl)ethanol linker (linker 2). The IC50 values of the linker-2 hybrid molecules for BuChE inhibition were lower than those of linker-1 hybrid molecules (except 9-2) and they also had the same great selectivity for BuChE over AChE (> 800 fold) as linker-1 hybrid molecules. ALA-acetyl caffeic acid (102, ALA-AcCA) was shown as an effective inhibitor of BuChE (IC50 = 0.44 ± 0.24 μM). A kinetic study using 72 showed that it is the same mixed type inhibition as 7-1. Its inhibition constant (Ki) to BuChE is 4.3 ± 0.09 μM.

Synthesis and biological evaluation of lycorine derivatives as dual inhibitors of humanacetylcholinesterase and butyrylcholinesterase

BackgroundAlzheimer’s disease (AD) is a neurologically degenerative disorder that affects more than 20 million people worldwide. The selective butyrylcholinesterase (BChE) inhibitors and bivalent cholinesterase (ChE) inhibitors represent new treatments for AD.FindingsA series of lycorine derivatives (1–10) were synthesized and evaluated for anti-cholinesterase activity. Result showed that the novel compound 2-O-tert-butyldimethylsilyl-1-O-(methylthio)methyllycorine (7) was a dual inhibitor of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) with IC50 values of 11.40 ± 0.66 μM and 4.17 ± 0.29 μM, respectively. The structure-activity relationships indicated that (i) the 1-O-(methylthio)methyl substituent in lycorine was better than the 1-O-acetyl group for the inhibition of cholinesterase; (ii) the acylated or etherified derivatives of lycorine and lycorin-2-one were more potent against hBChE than hAChE; and (iii) the oxidation of lycorine at C-2 decreases the activity.ConclusionAcylated or etherified derivatives of lycorine are potential dual inhibitors of hBChE and hAChE. Hence, further study on the modification of lycorine for ChE inhibition is necessary.

Combined Cocaine Hydrolase Gene Transfer and Anti-Cocaine Vaccine Synergistically Block Cocaine-Induced Locomotion

Mice and rats were tested for reduced sensitivity to cocaine-induced hyper-locomotion after pretreatment with anti-cocaine antibody or cocaine hydrolase (CocH) derived from human butyrylcholinesterase (BChE). In Balb/c mice, direct i.p. injection of CocH protein (1 mg/kg) had no effect on spontaneous locomotion, but it suppressed responses to i.p. cocaine up to 80 mg/kg. When CocH was injected i.p. along with a murine cocaine antiserum that also did not affect spontaneous locomotion, there was no response to any cocaine dose. This suppression of locomotor activity required active enzyme, as it was lost after pretreatment with iso-OMPA, a selective BChE inhibitor. Comparable results were obtained in rats that developed high levels of CocH by gene transfer with helper-dependent adenoviral vector, and/or high levels of anti-cocaine antibody by vaccination with norcocaine hapten conjugated to keyhole limpet hemocyanin (KLH). After these treatments, rats were subjected to a locomotor sensitization paradigm involving a “training phase" with an initial i.p. saline injection on day 1 followed by 8 days of repeated cocaine injections (10 mg/kg, i.p.). A 15-day rest period then ensued, followed by a final “challenge" cocaine injection. As in mice, the individual treatment interventions reduced cocaine-stimulated hyperactivity to a modest extent, while combined treatment produced a greater reduction during all phases of testing compared to control rats (with only saline pretreatment). Overall, the present results strongly support the view that anti-cocaine vaccine and cocaine hydrolase vector treatments together provide enhanced protection against the stimulatory actions of cocaine in rodents. A similar combination therapy in human cocaine users might provide a robust therapy to help maintain abstinence.

Metaproterenol, Isoproterenol, and Their Bisdimethylcarbamate Derivatives as Human Cholinesterase Inhibitors

Metaproterenol and isoproterenol are bronchodilators that provide a structural basis for many other bronchodilators currently in use. One of these structurally related bronchodilators is terbutaline; it is administered as a prodrug, bambuterol, and is metabolized (bioconverted) into terbutaline by butyrylcholinesterase (BChE). The metabolism rate can be affected by BChE gene polymorphism in the human population and BChE stereoselectivity. The aim of our study was to investigate inhibition of human BChE and acetylcholinesterase (AChE) with metaproterenol, isoproterenol, and newly synthesized racemic bisdimethylcarbamate derivatives of metaproterenol (metacarb) and isoproterenol (isocarb) and their (R)-enantiomers to see if their bioconversion is affected by BChE inhibition in the same way as that for bambuterol. Metacarb and isocarb proved to be selective BChE inhibitors, as they progressively inhibited AChE 960 to 80 times more slowly than BChE(UU). All studied cholinesterases displayed poor affinity for metaproterenol and isoproterenol, yet BChE(UU) had an affinity about five times higher than that of AChE.