Quantum-Chemical Multiligand Simultaneous Docking of Three-Membered Rings in the Active Site of Butyrylcholinesterase.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by dementia, particularly in older adults. It is a process that is increasing significantly with the aging population worldwide, has yet to be cured, and therefore challenges healthcare systems. The ability of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors to modulate neurotransmitter levels has made AChE/BChE inhibitors central therapeutic targets in drug development studies for the treatment of AD. Previous studies have demonstrated the beneficial effects of pyrimidine derivatives on cognitive functions and highlighted their high therapeutic potential against neurodegenerative diseases. Considering the pharmacological importance of AChE/BChE inhibitors and pyrimidine derivatives, this study investigated the inhibitory potential of seven different pyrimidine derivatives (1-7) on AChE and BChE using both in vitro and in silico approaches. Analysis of IC50 values indicated that compounds 1-7 (IC50: 14.89-77.70nM) exhibited strong inhibitory effect. Compound 6 (IC50:14.89nM) had the strongest inhibitory effect on AChE, while it showed a much weaker inhibitory effect against BChE (IC50: 357nM), corresponding to an approximately 24-fold selectivity for AChE. Molecular modeling results indicate that compounds 6 and 7 exhibit favorable interactions within the active site of the enzyme. In addition, compounds 1 and 3, which exhibited the strongest inhibitory effects on BChE, appear to display a multiple binding profile with the active site of BChE. Correlation and regression analyses indicated that compounds 1-7 display a structure-activity relationship (SAR) consistent with strong inhibitory potency toward AChE, while showing comparatively weaker inhibition toward BChE.

Cholinesterase (ChE) inhibition represents the most efficacious treatment approach for Alzheimer's disease (AD) to date. This multiple-dose study has examined the relationship between inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities in the cerebrospinal fluid (CSF) and cognitive change (measured by the Computerised Neuropsychological Test Battery [CNTB]) following administration of the ChE inhibitor, rivastigmine (Exelon). In 18 patients with mild to moderate AD, CNTB scores, activities of AChE and BuChE in the CSF, and plasma BuChE activity were determined prior to treatment with rivastigmine. Doses of rivastigmine were then titrated (1 mg b.i.d./week) to final doses of 1, 2, 3, 4, 5 or 6 mg b.i.d. (n = 3 per dose). Following treatment with the target dose of rivastigmine for at least 3 days, CNTB scores were re-determined. CSF samples were continuously collected together with plasma samples prior to and for 12 hours after the final dose of rivastigmine, and AChE and BuChE activities determined.AChE in CSF and BuChE in plasma were dose-dependently inhibited by rivastigmine treatment. The inhibition of BuChE in CSF was not clearly dose-dependent. A statistically significant correlation was observed between the change in CNTB summary score and inhibition of AChE activity (r = -0.56, p < 0.05) and BuChE activity (r = -0.65, p < 0.01) in CSF. Improvement in speed-, attention- and memory-related subtests of the CNTB correlated significantly with inhibition of BuChE but not AChE activity in CSF. Weak or absent correlation with change in cognitive performance was noted for inhibition of plasma BuChE. These results indicate that cognitive improvement with rivastigmine in AD is associated with central inhibition of ChEs and support a role for central BuChE in addition to AChE inhibition in modulating cholinergic function in AD.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors are used to increase acetylcholine levels in current symptomatic treatments for Alzheimer's disease (AD), which is still the most common neurodegenerative illness. Through the synthesis and thorough assessment of a novel series of Imidazo-pyrazinone derivatives (8a-8l), this study sought to identify and create highly effective cholinesterase inhibitors. Using donepezil as the standard reference medication, the produced compounds were tested in vitro for their inhibitory activity against both AChE and BuChE (AChE IC50 (μM) 2.16 ± 0.12, BuChE IC50 (μM) 4.50 ± 0.11). The findings showed that most of the recently created derivatives had better inhibitory activity than the reference medication. Compound 8h was the most effective dual inhibitor in the series, outperforming Donepezil with an AChE IC50 of 1.22 ± 0.30 and a BuChE of 3.75 ± 1.20. Derivatives with electron-withdrawing groups, such as the fluoro substituent, particularly at the meta-position, and these strengthens interactions around the enzyme's active site, demonstrated the greatest levels of inhibition, according to the structure-activity relationship (SAR) studies. The ortho-position hydroxyl (-OH) phenolic group in compound 8h was also found to be an important structural feature that increased potency by encouraging helpful hydrogen bonding with catalytic residues. In order to investigate the binding mode, reliability, and electronic characteristics of the most active compounds, molecular docking and Density Functional Theory (DFT) calculations have been carried out in addition to the studies. The results of the experiment were supported by the docking analysis, which verified the advantageous penetration of compound 8h through the active site. Additionally, the most promising candidates' acceptable pharmacokinetic profiles and drug-likeness were predicted through ADME (Absorption, Distribution, Metabolism, and Excretion) evaluation. To sum up, compound 8h, a novel imidazo-pyrazinone derivative, is a potent dual AChE and BuChE inhibitor that deserves more in vivo investigation as a potential lead compound for Alzheimer's disease treatment.

In the present work, a novel series of eleven sulfonate derivatives with potent inhibitory activity against butyrylcholinesterase (BChE) is reported. Of these, compounds 2-[(E)-(2-Benzoylhydrazinylidene)methyl]phenyl 5-(dimethylamino)naphthalene-1-sulfonate (5c, IC50 = 1.11 µM) and tert-butyl (2E)-2-[(2-{[5-(dimethylamino)naphthalene-1-sulfonyl]oxy}phenyl)methylidene]hydrazine-1-carboxylate (5b, IC50 = 11.51 µM) exhibit stronger inhibitory activity than rivastigmine, the reference compound, and exhibit high selectivity for BChE over AChE (e.g., selectivity index 57 for 5c). Interestingly, compound 5c also exhibited anti-inflammatory effects, which is important for potential therapeutic applications, especially in Alzheimer's disease. These new compounds were designed through a structure-based approach using molecular modeling techniques (docking, molecular dynamic (MD) simulations, and QTAIM (quantum theory of atoms in molecules) calculations). The most promising compounds show no detectable toxic effects and satisfy Lipinski's rule of five, indicating that they represent attractive starting structures for the design of new derivatives acting as specific BChE inhibitors. In addition, our results indicate that relatively simple computational techniques such as docking calculations and toxicity prediction programs can be valuable when properly used in the search of new candidates for this particular target. Docking calculations show that the more active compounds of this series reach the bottom region of the gorge interacting with residues within the active site of BChE. However, our data further suggest that the use of more precise techniques, such as MD simulations and QTAIM analysis, is necessary to obtain detailed insight into ligand-enzyme interactions. Regarding QTAIM calculations, they demonstrate that such computations are very useful to evaluate the molecular interactions of the different molecular complexes. In summary, we report a new series of sulfonate derivatives as promising starting structures for the development of new selective BChE inhibitors.

Four types of extracts were prepared from Melia azedarach L. (Meliaceae) leaves: Aqueous, potassium phosphate buffer (pH 7.2), hydroethanolic solution 70:30 and hydroethanolic solution 50:50. Different concentrations of these extracts were investigated for the effect on butyrylcholinesterase (BuChE, EC 3.1.1.8) activity in homogenates rat livers. The introduction of M. azedarach extracts in the reaction mixture produced a variety of inhibitions (> 45 to 100%), independent on its concentration (0.5 to 2.0 mg.ml-1) and extract type. A clear explanation for this interaction between M. azedarach and active site of butyrylcholinesterase is still missing. The UV-VIS (200 to 400 nm) absorption spectrum and the phytochemicals tests of the extracts show diversity of compounds including flavonoids, a known inhibitor of butyrylcholinesterase. On the other hand, decreased butyrylcholinesterase activity in homogenates rat livers was not correlated with flavonoids content, suggesting that other compounds may have contributed to the inhibitory capacity of this plant. Key words: Enzymatic activity, flavonoids, medicinal plants.

In this article, we present the design and synthesis of amino-7,8-dihydro-4H-chromenone derivatives as possible inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) for the management of Alzheimer’s disease (AD). The target compounds were evaluated against AChE and BChE in vitro, and 4k exhibited good potency against BChE (IC50 = 0.65 ± 0.13 µM) compared with donepezil used as a positive control. Kinetic studies revealed that compound 4k exhibited a competitive-type inhibition with a Ki value of 0.55 µM. Molecular docking and molecular dynamics simulations further supported the rationality of our design strategy, as 4k showed promising binding interactions with the active sites of BChE. Overall, our findings highlight the potential of amino-7,8-dihydro-4H-chromenone derivatives as promising candidates for developing novel therapeutics targeting cholinesterase in managing AD.

The serine hydrolase butyrylcholinesterase (BChE), like the related enzyme acetylcholinesterase (AChE), co-regulates metabolism of the neurotransmitter acetylcholine. In the human brain BChE is mainly expressed in white matter and glia and in distinct populations of neurons in regions that are important in cognition and behavior, functions compromised in Alzheimer's disease (AD). AD is a neurodegenerative disorder causing dementia with no cure nor means for definitive diagnosis during life. In AD, BChE is found in association with pathology, such as β-amyloid (Aβ) plaques, particularly in the cerebral cortex where BChE is not normally found in quantity. Up to 30% of cognitively normal older adults have abundant Aβ deposition in the brain. We have designed an imaging agent that can detect, through autoradiography, BChE-associated Aβ plaques in the cerebral cortex of AD brains, but does not visualize Aβ plaques in brains of cognitively normal individuals. Furthermore, in an AD mouse model with BChE gene knocked out, there are up to 70% fewer fibrillar Aβ brain plaques, suggesting diminished BChE activity could prove beneficial as a curative approach to AD. To that end, we have examined numerous N-10-carbonyl phenothiazines that are specific inhibitors of human BChE, revealing important details of the enzyme's active site gorge. These phenothiazines can be designed without potential side effects caused by neurotransmitter receptor interactions. In conclusion, BChE is potentially an important target for diagnosis and treatment of AD.

Selective human butyrylcholinesterase (BChE) inhibitors such as cymserine have shown considerable promise for restoring cognition in Alzheimer's disease. Recently, (-)-debromoflustramine B, 1, a hexahydropyrrolo-[2,3-b]indole natural product isolated from the marine bryozoan Flustra foliacea, has demonstrated micromolar potency as a selective BChE inhibitor. Since (±)-demethyldebromoflustramine B, (±)-2, has an even lower IC(50), and the active enantiomer is (-)-2, derivatives of (-)-2 were constructed in silico and docked into the active site of BChE. Several compounds exhibited improved inhibitor potency and could be candidates for future synthesis and in vitro enzyme inhibition study.

We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/- mice had 50% of normal AChE activity and AChE -/- mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well.

Exploring the selective butyrylcholinesterase inhibition potential of phenol carbamates: Experimental and computational study.

A novel near-infrared fluorescent probe for butyrylcholinesterase: Research for screening of natural anti-AD inhibitors

Butyrylcholinesterase (BuChE) is an enzyme that is highly upregulated in the pathogenesis of Alzheimer's Disease (AD) and contributes to the symptomatic cognitive decline through the hydrolysis of the neurotransmitter, acetylcholine. We have recently developed BuChE-specific, reversible organophosphorus inhibitors in which two phosphates are linked by an alkyl chain. Subsequent changes in drug design led to a better and smaller inhibition constant (Ki) by replacing the two oxygen atoms in the linker with sulfur atoms. Therefore, it is hypothesized that the position as well as an increase in the number of sulfur substitutions around the phosphorus atom will lead to a more potent inhibitor design. To evaluate the effects of these sulfur atoms on inhibition, dibutyl hexyl phosphate will be used as a scaffold for making a library of potential compounds, each with different number and location of substitutions. The reversibility of inhibition and the specificity is determined through an in vitro competition assay by incubating BuChE with inhibitor for various time intervals and then doping in butyrylthiocholine, a molecule that can be hydrolyzed by BuChE; the hydrolyzed product will react with Ellman's reagent to give an absorbance reading at 412 nanometers. Preliminary data for a single sulfur atom substitution on the hexyl chain shows about a 50-fold decrease in Ki compared to the control that has all oxygen atoms (1.3 versus 50 μM, respectively). When assayed against acetylcholinesterase, no inhibition was observed. Analogs bearing a thiophosphoryl group (P=S) show decreased inhibition compared to the phosphoryl (P=O) compounds (1.3 versus 3.4 μM). Despite the difference in location, a single substitution has led to an irreversible, yet selective inhibition of BuChE. The insight from this study will give guidance towards the rational design of better inhibitors of BuChE that can potentially decelerate the symptomatic progression of AD. Support or Funding Information This project is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R25GM071638. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health. Library of phosphate and phosphorothioate compounds that will be evaluated for selective inhibition of butyrylcholinesterase This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alzheimer's disease (AD) is a neurodegenerative disorder with a gradual increase in severity. The underlying cause of the disease is the dysfunction of cholinergic neurotransmission affecting mainly the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Within the context of the present research, a new group of 3,5-disubstituted rhodanine derivatives containing tertiary amine groups has been prepared and their potency in the inhibition of AChE and BChE was assessed. Enzymatic assays demonstrated that compounds 6 and 11 exhibited exceptional inhibitory potency, with Ki values of 13.61 nM and 12.70 nM against AChE, and 10.44 nM and 25.11 nM against BChE, respectively, surpassing the reference inhibitors tacrine (145.21 nM for AChE and 169.54 nM for BChE) and donepezil (67.41 nM for AChE and 62.44 nM for BChE). Cytotoxicity studies confirmed minimal toxicity in human umbilical vein endothelial cells (HUVEC) at concentrations several times higher than the effective inhibitory doses (IC50 = 79.13 µM for 6 and 69.14 µM for 11). The results from molecular docking and MM-GBSA calculations supported this presumption by foretelling strong binding affinities, where compound 11 was the one to show a free energy of −103.26 kcal mol−1 for AChE and compound 6 −86.75 kcal mol−1 for BChE. Moreover, the 250 ns molecular dynamics simulations gave a confirmation of the structural stability and the prolonged existence of the key interactions in the enzyme active sites during the entire time. The findings of this research emphasize compounds 6 and 11 as potential candidates for the creation of strong cholinesterase inhibitors for the treatment of Alzheimer's disease, thus encouraging additional studies.

Inhibitory activities of major anthraquinones and other constituents from Cassia obtusifolia against β-secretase and cholinesterases

Synthesis, molecular docking and biological evaluation of N,N-disubstituted 2-aminothiazolines as a new class of butyrylcholinesterase and carboxylesterase inhibitors