Acetylcholinesterase: structure and use as a model for specific cation—protein interactions (2023)

Multi-pronged spectroscopic techniques were employed to provide quantitative inputs on the modulated photophysics of nine coumarinyl derivatives in an array of solvents. Significant solvatochromic effect on the spectral behavior of the investigated systems was quantified using multiparametric regression analysis along with Density Functional Theory (DFT) calculations. The optical properties of the compounds were seen to be contingent on solvent polarity/polarizability and hydrogen bonding to varying degrees. Assessment of in-vitro anti-acetylcholinesterase (AChE) activity of the investigated systems indicates their efficacy to be even better than FDA approved drug donepezil to treat Alzheimer's disease (AD) and shed novel insights into the biomedical applications of coumarinyl compounds. Interestingly, the AChE inhibition capacity of these compounds showed significant diminution in presence of human serum albumin (HSA), the principal transport protein in blood serum and therefore, reinstated its ability to modulate the therapeutic efficacies of potential drugs. The observed results were validated by quantifying the interaction of the investigated systems with HSA and also investigated through steady state and time-resolved fluorescence spectroscopy along with several other bio-analytical tools. A combination of static and dynamic mechanisms was responsible for the quenching of intrinsic protein fluorescence due to the strong sequestration ability of these coumarin derivatives into the protein binding domain. The observed results emphasized on the need of screening the pharmacological effectiveness of proposed drugs in biologically relevant media rather than in pure buffer.

Phenyl valerate (PV) is a neutral substrate for measuring the PVase activity of neuropathy target esterase (NTE), a key molecular event of organophosphorus-induced delayed neuropathy. This substrate has been used to discriminate and identify other proteins with esterase activity and potential targets of organophosphorus (OP) binding. A protein with PVase activity in chicken (model for delayed neurotoxicity) was identified as butyrylcholinesterase (BChE). Further studies in human BChE suggest that other sites might be involved in PVase activity. From the theoretical docking analysis, other more favorable sites for binding PV related to the Asn289 residue located far from the catalytic site (“PVsite”) were deduced.In this paper, we demonstrate that acetylcholinesterase is also able to hydrolyze PV. Robust kinetic studies of interactions between substrates PV and acetylthiocholine (AtCh) were performed. The kinetics did not fit the classic competition models among substrates. While PV interacts as a competitive inhibitor in AChE activity, AtCh at low concentrations enhances PVase activity and inhibits this activity at high concentrations. Kinetic behavior suggests that the potentiation effect is caused by thiocholine released at the active site, where AtCh could act as a Trojan Horse. We conclude that the products released at the active site could play an important role in the hydrolysis reactions of different substrates in biological systems.

In the field of Chemical Defense toward Nerve Agents, the development of analytical methodologies for analysis of different sample matrices, training of human resources in handling, detection of such toxic chemicals, and design of more effective medical countermeasures may be hampered by the lack of infrastructure and well-prepared human resources. Therefore Nerve Agents Surrogates may be an invaluable alternative to accomplish such tasks, as these compounds present similar behavior and are easier to handle in comparison to actual nerve agents, being useful analytical and toxicological tools for technical purposes.

In the present study, the inhibitory mechanisms and effects of a synthetic phenazine dye, safranin O (SO) on human plasma butyrylcholinesterase (BChE), human erythrocyte acetylcholinesterase (AChE) and recombinant BChE mutants were investigated. Kinetic studies showed the following information: SO leaded to linear competitive inhibition of human plasma BChE with K_i=0.44±0.085μM; α=∞. It acted as a hyperbolic noncompetitive inhibitor of human erythrocyte AChE with K_i=0.69±0.13; α=1; β=0.08±0.02. On the other hand, the inhibitory effects of SO on two BChE mutants, where A328 was modified to either F or Y, revealed differences in terms of inhibitory patterns and K_i values, compared to the obtained results with recombinant wild type BChE. SO was found to act as a linear competitive inhibitor of A328F and A328Y BChE mutants. Compared to recombinant wild type BChE, A328Y and A328F BChE mutants caused a 4- and 10-fold decrease in K_i value for SO, respectively. These findings were supported by molecular modelling studies. In conclusion, SO is a potent inhibitor of human cholinesterases and may be useful in the design and development of new drugs for the treatment of AD.

Acetylcholinesterase inhibitors are the mainstay of Alzheimer's disease treatments, despite having only short-term symptomatic benefits and severe side effects. Selective butyrylcholinesterase inhibitors (BuChEIs) may be more effective treatments in late-stage Alzheimer’s disease with fewer side effects. Virtual screening is a powerful tool for identifying potential inhibitors in large digital compound databases. This study used structure-based virtual screening combined with physicochemical filtering to screen the InterBioScreen and Maybridge databases for novel selective BuChEIs. The workflow rapidly identified 22 potential hits in silico, resulting in the discovery of a human BuChEI with low-micromolar potency in vitro (IC₅₀ 2.4µM) and high selectivity for butyrylcholinesterase over acetylcholinesterase. The compound was a rapidly reversible BuChEI with mixed-model in vitro inhibition kinetics. The binding interactions were investigated using in silico molecular dynamics and by developing structure-activity relationships using nine analogues. The compound also displayed high permeability in an in vitro model of the blood-brain barrier.

Phenazines, naturally produced by bacteria and archaeal Methanosarcina species are nitrogen-containing tricyclic molecules with antibiotic, antitumoral, and antiparasitic activities. Phenazines are used as electron acceptors-donors in wide range of fields including environmental biosensors. In this study, the inhibitory effects of a synthetic phenazine dye, methylene violet 3RAX (also known as diethyl safranine) on human erythrocyte AChE and human plasma BChE were tested and also its inhibitory mechanisms for both enzymes were studied in detail. Kinetic analyses showed that methylene violet 3RAX acts as a hyperbolic noncompetitive inhibitor of AChE with K_i value of 1.58 ± 0.36 μM; α = 1; β = 0.12 ± 0.0003. On the other hand, it caused linear competitive inhibition of BChE with K_i value of 0.51 ± 0.006 μM; α = ∞. In conclusion, methylene violet 3RAX which is a highly effective inhibitor of both human AChE and human BChE with K_i values in low micromolar range may be a promising candidate for the treatment of Alzheimer's disease.

Bioorganic Chemistry, Volume 110, 2021, Article 104792

Twenty novel 7-benzyloxycoumarin based compounds were synthesized with a variety of bioactive chemical fragments. The synthesized compounds showed remarkable acetylcholinesterase (AChE) inhibitory activity. In vitro assay revealed that compounds 7-benzyloxy-4-{[(4-phenylthiazol-2(3H)-ylidene)hydrazono]methyl}-2H-chromen-2-one (5b, IC₅₀=0.451$\mu$M), 7-benzyloxy-4-({[4-(4-methoxyphenyl)thiazol-2(3H)-ylidene]hydrazono}methyl)-2H-chromen-2-one (5d, IC₅₀=0.625$\mu$M), 5-amino-1-[2-(7-benzyloxy-2-oxo-2H-chromen-4-yl)acetyl]-1H-pyrazole-4-carbonitrile (13c, IC₅₀=0.466$\mu$M), 2-(7-benzyloxy-2-oxo-2H-chromen-4-yl)-N-(2-methylimino-4-phenylthiazol-3(2H)-yl)acetamide (16a, IC₅₀=0.500$\mu$M) and 2-(7-benzyloxy-2-oxo-2H-chromen-4-yl)-N-[4-(4-methoxyphenyl)-2-methyliminothiazol-3(2H)-yl]acetamide (16b, IC₅₀=0.590$\mu$M) exhibited promising AChE inhibitory activity even better than donepezil (IC₅₀=0.711$\mu$M). Kinetic study for compound 5b implied mixed type inhibitor which could bind peripheral anionic site (PAS) and catalytic active site (CAS) of AChE enzyme. In addition, in vivo evaluation of compounds 5b, 13c and 16a confirmed significant memory improvement in scopolamine-induced impairment model in tested mice. Furthermore, in silico studies were performed on the synthesized compounds which included molecular docking study at the active site of recombinant human acetylcholinesterase enzyme (rhAChE) as well as prediction of ADMET and other physicochemical parameters. A correlation between the docking results and IC₅₀ of tested compounds was routinely observed and shared similar binding pattern to the co-crystallized ligand donepezil.

Bioorganic Chemistry, Volume 93, 2019, Article 103312

Novel series of pyrrolizine based compounds (4–6 and 9–11) were designed, synthesized and evaluated as potential anti-Alzheimer agents. Most of the tested compounds showed selectivity to hAChE over hBChE and effectively inhibited self–induced amyloid beta aggregation in vitro. Among these derivatives, compound 10 displayed high selectivity towards hAChE (Ki = 1.47 ± 0.63 μM for hAChE and Ki = 40.15 ± 3.31 μM for hBChE). However, compound 11 displayed dual inhibitory effect against hAChE and hBChE at submicromolar range (Ki = 0.40 ± 0.03 and 0.129 ± 0.009 μM, respectively). Kinetic studies of the new ligands showed competitive type inhibition for both hAChE and hBChE. Moreover, compounds 10 and 11 showed lower or comparable cytotoxicity to donepezil against human neuroblastoma (SH-SY5Y) and normal human hepatic (THLE2) cell lines. In vivo studies confirmed that both compounds were able to improve cognitive dysfunction of scopolamine-induced AD mice. Finally, molecular docking simulation of compounds 10 and 11 in hAChE active site showed good agreement with the obtained pharmaco-biological results.

Bioorganic Chemistry, Volume 84, 2019, pp. 511-517

4-(3-Substitutedphenyl-5-polymethoxyphenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamides (9–16) were synthesized and their chemical structures were elucidated by ¹H NMR, ¹³C NMR, and HRMS. The compounds designed include pyrazoline and sulfonamide pharmacophores in a single molecule by hibrit molecule approach which is a useful technique in medicinal chemistry in designing new compounds with potent activity for the desired several bioactivities. Inhibition potency of the sulfonamides were evaluated against human CA isoenzymes (hCA IandhCA II) and acetylcholinesterase (AChE) enzyme and also their cytotoxicities were investigated towards oral squamous cancer cell carcinoma (OSCC) cell lines (Ca9-22, HSC-2, HSC-3, and HSC-4) and non-tumor cells (HGF, HPLF, and HPC). Cytosolic hCA I and hCA II isoenzymes were inhibited by the sulfonamide derivatives (9–16) and Ki values were found in the range of 27.9 ± 3.2–74.3 ± 28.9 nM and 27.4 ± 1.4–54.5 ± 11.6 nM, respectively. AChE enzyme was strongly inhibited by the sulfonamide derivatives with Ki values in the range of 37.7 ± 14.4–89.2 ± 30.2 nM The CC₅₀ values of the compounds were found between 15 and 200 µM towards OSCC malign cell lines. Their tumor selectivities were also calculated with two ways. Compound’s selectivities towards cancer cell line were found generally low, except compounds bearing 3,4-dimethoxyphenyl 14 (TS1 = 1.3, TS2 = 1.4) and 10 (TS2 = 1.4). All sulfonamide derivatives studied here can be considered as good candidates to develop novel CAs or AChE inhibitor candidates based on the enzyme inhibition potencies with their low cytotoxicity and tumor selectivity.

Journal of Molecular Graphics and Modelling, Volume 68, 2016, pp. 176-183

Rational design of active molecules through structure-based methods has been gaining adepts during the last decades due to the wider availability of protein structures, most of them conjugated with relevant ligands. Acetylcholinesterase (AChE) is a molecular target with a considerable amount of data related to its sequence and 3-dimensional structure. In addition, there are structural insights about the mechanism of action of the natural substrate and drugs used in Alzheimer’s disease, organophosphorus compounds, among others. We looked for AChE structural data useful for in silico design of potential interacting molecules. In particular, we focused on information regarding the design of ligands aimed to reactivate AChE catalytic activity. The structures of 178 AChE were annotated and categorized on different subsets according to the nature of the ligand, source organisms and experimental details. We compared sequence homology among the active site from Torpedo californica, Mus musculus and Homo sapiens with the latter two species having the closest relationship (88.9% identity). In addition, the mechanism of organophosphorus binding and the design of effective reactivators are reviewed. A curated data collection obtained with information from several sources was included for researchers working on the field. Finally, a molecular dynamics simulation with human AChE indicated that the catalytic pocket volume stabilizes around 600 ų, providing additional clues for drug design.

Bioorganic Chemistry, Volume 100, 2020, Article 103897

Some metabolic enzyme inhibitors can be used in the treatment of many diseases. Therefore, synthesis and determination of alternative inhibitors are essential. In this study, the inhibition effect of newly synthesized compounds on carbonic anhydrase (cytosolic isoforms, hCA I and hCA II), α-glycosidase (α-GLY), and acetylcholinesterase (AChE) were investigated. The possible binding mechanism of the compounds with a high inhibitory effect on the active site of the enzyme was demonstrated by molecular docking method. We investigated the inhibition effects of novel synthesized compounds (MZ1-MZ11) on metabolic enzymes such as α-GLY, AChE, and hCA I and II. The compound MZ6 for AChE, MZ8 for CA I and CA II and MZ7 for α-GLY showed a very active inhibition profile (K_Is 51.67±4.76 for hCA I, 40.35±5.74nM for hCA II, 41.74±8.08nM for α-GLY and 335.76±46.91nM for AChE). The novel synthesized compounds (MZ1-MZ11) have a higher enzyme (α-GLY, AChE, hCA I, and II) inhibitory potential than ACR, TAC, and AZA, respectively. The compounds may have the potential to be used as alternative medicines after further research in the treatment of many diseases such as diabetes, Alzheimer’s disease, heart failure, ulcer, and epilepsy.

Journal of Molecular Structure, Volume 1249, 2022, Article 131591

A new set of hybrid derivatives bearing pyrazole and coumarin scaffold3a-fwas synthesized and confirmed by different spectroscopic techniques (¹H NMR, ¹³C NMR, FT-IR) and mass spectrometry. This study aimed to evaluate the inhibitory activity of new derivatives against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) using Galanthamine as standard drugs. In vitro studies showed that compounds 3e and 3f were the most effective showing high potential AChE inhibitory activities with respective IC50 values of 4.41±0.53 and 5.04±0.96 µg/ml. Compounds 3a and 3b were found to be the best butyrylcholinesterase inhibitor with an IC50 value comparable to that of the standard drugs. All the newly synthesized derivatives 3a–f are evaluated for their antioxidant activity and showed good activity. Molecular docking study was also carried to get insights into binding interactions of synthesized compounds to act as AChE and BChE inhibitors. The docking study of compound 3e with AChE enzyme showed that PAS are occupied by the ligand. In silico predictions of toxicity analysis indicated that these compounds should have good oral bioavailability.