Molecular docking studies of a phytocompound kanzonol B as a potential acetylcholine esterase inhibitor for epilepsy

Acetylcholinesterase (AChE) is one of the key targets of drugs for treating Alzheimer's disease (AD). Tacrine is an approved drug with AChE-inhibitory activity. In this paper, 3D-QSAR, molecular docking, and molecular dynamics were carried out in order to study 60 tacrine derivatives and their AChE-inhibitory activities. 3D-QSAR modeling resulted in an optimal CoMFA model with q(2) = 0.552 and r(2) = 0.983 and an optimal CoMSIA model with q(2) = 0.581 and r(2) = 0.989. These QSAR models also showed that the steric and H-bond fields of these compounds are important influences on their activities. The interactions between these inhibitors and AChE were further explored through molecular docking and molecular dynamics simulation. A few key residues (Tyr70, Trp84, Tyr121, Trp279, and Phe330) at the binding site of AChE were identified. The results of this study improve our understanding of the mechanisms of AChE inhibitors and afford valuable information that should aid the design of novel potential AChE inhibitors. Graphical Abstract Superposition of backbone atoms of the lowest-energy structure obtained from MD simulation (magenta) onto those of the structure of the initial molecular docking model (green).

Alzheimer’s disease, the leading cause of dementia in older adults, involves memory loss and cognitive decline, with β-amyloid plaques and neurofibrillary tangles (NFTs) as key features. Acetylcholinesterase (AChE), an enzyme that breaks down acetylcholine, plays a role in the formation of these plaques and tangles. AChE is a promising target for the development of small molecule inhibitors in Alzheimer’s disease (AD) treatment. Indonesia’s native red betel (Piper crocatum) contains bioactive compounds that inhibit AChE activity, as shown in previous research. This study aims to evaluate the AChE inhibitory potential of bioactive compounds from P. crocatum extracts (water, n-hexane and ethyl acetate) using an in silico approach (molecular docking) with 3 different docking software programs. Donepezil served as the reference compound, and the results were compared with in vitro AChE inhibition assays. The potential AChE inhibitors, based on molecular docking using PyRx, Autodock Vina and YASARA Structure, from each extract are SM05 (n-hexane extract), SM15 (water extract) and SM18 (ethyl acetate extract), with the most negative ΔGbind values, measuring −8.8, −9.2 and −11.2 kcal/mol (more negative than Donepezil’s ΔGbind values). SM15 and SM18 Compounds show promise, based on its ΔGbind values, interactions with AChE, favorable pharmacokinetic properties, bioavailability, bioactivity and toxicity positioning both compounds as strong candidates for AD therapy. Both compounds were docked to the AChE substrate binding pockets (6O4W), forming hydrogen bonds with His447 and Phe297 at the “gorge” active site, and hydrophobic interactions with key amino acids in the peripheral anionic site (PAS) and substrate-binding sites (Tyr124, Phe297 and Phe338). This is consistent with in vitro assay results, which show that the ethyl acetate extract has strong inhibition, with an IC50 of 16.7908 ppm, while the water infusion extract yields a 26.621 % inhibition of AChE enzyme activity. In addition, the DIY extract exhibited the strongest AChE inhibitory activity with an IC50 of 40.799 ppm. HIGHLIGHTS Piper crocatum (red betel leaf) shows potential as a natural acetylcholinesterase (AChE) inhibitor, with compounds SM05 (2-(3,4-Dimethoxyphenyl)-6-ethoxy-7-methoxy-1-naphthol), SM15 (Columbin) and SM18 (Flemiphilippinin A) identified as promising candidates through in silico molecular modeling, as indicated by the most negative binding free energy values generated through 3 molecular docking methods (Virtual Screening with PyRx, Autodock Vina and YASARA Structure), especially compound SM18 (Flemiphilippinin A) from ethyl acetate extract (−11.2 kcal/mol). This aligns with the in vitro assay results, which show that the ethyl acetate extract has the strong inhibition, with an IC50 of 16.7908 ppm, as provided by Nurinsani et al. [10]. The red betel leaves water extract from DIY exhibited the most potent acetylcholinesterase (AChE) inhibitory activity with the lowest IC50 value (40.799 ppm), highlighting the significant inhibitory activity of the extract. Flemiphilippinin A (SM18) showed the strongest binding affinity to AChE (ΔGbind −2 kcal/mol), supported by favorable pharmacokinetic properties and bioavailability, as indicated by the fulfillment of Lipinski’s Rule of 5, ADME and bioactivity scores. It acts as a Nuclear receptor ligand (0.53), while Columbin (SM15) exhibits bioactivity as a Nuclear receptor ligand [0.66] and as an Enzyme inhibitor (0.57), suggesting that both compounds have potential as therapeutic candidates for Alzheimer’s disease (AD). Virtual screening and molecular docking revealed robust interactions of SM05, SM15 and SM18 with critical AChE active site residues, indicating their capability to inhibit AChE activity and potentially prevent AD progression. The 3 compounds from red betel leaf fractions are safe and have potential as oral drugs for Alzheimer’s therapy, with oral toxicity similar to donepezil (toxicity class IV, LD50 505 mg/kg) based on ProTox3 Prediction (with toxicity class IV for SM15, toxicity class V for SM05 and SM18). Pharmacokinetic evaluation confirmed the 3 compounds met Lipinski’s rule of 5 and showed high gastrointestinal absorption, with SM15 excelling in drug-likeness and bioactivity parameters critical for AD therapy. GRAPHICAL ABSTRACT

Bioactive natural products have been extensively investigated for the discovery of alternative Alzheimer’s disease (AD) treatments. Recently, our structure-based virtual screening (SBVS) campaigns on natural products served in the LOTUS database exhibited the potency of isoliquiritigenin as an acetylcholinesterase (AChE) inhibitor, which propelled us to investigate it further. This study aimed to evaluate the acetylcholinesterase (AChE) inhibitory activity of isoliquiritigenin compared to commonly known inhibitors i.e., donepezil through in vitro and in silico studies. The AChE inhibitory activity of isoliquiritigenin and donepezil was evaluated using the improved Ellman method and the molecular mechanism in inhibiting AChE was identified using molecular docking and dynamics simulations. Our findings verified the AChE inhibitory activity of isoliquiritigenin, possessing an IC50 value of 126.22 µM compared to donepezil with an IC50 value of 36.98 µM. In silico studies revealed that five best-docked poses from 100 redocking and molecular docking simulations established interactions in the AChE active site in 5 ns after 5-ns equilibration run. Further dynamics interactions were explored to 50 ns, showing interactions of isoliquiritigenin and donepezil which were still in the AChE active site. These simulations also revealed the pivotality of the aromatic ring and hydroxyl moiety of isoliquiritigenin reinforcing the receptor-ligand stabilization. Our studies hence exhibited the potency of isoliquiritigenin acting as an AChE inhibitor and might be explored in isoliquiritigenin-containing bioactive natural products as AD alternative treatments.

Acetylcholinesterase (AChE) inhibitors are widely used in medicine for pharmacological correction of cholinergic neurotransmission pathologies such as myasthenia gravis (MG) and Alzheimer's disease [1, 2]. The efficacy of anti-AChE drugs is based on their ability to potentiate the effects of acetylcholine (ACh) due to a decrease in the rate of AChE-catalyzed hydrolysis of ACh. Crystallographic studies showed that the active site of AChE is located at the bottom of a deep gorge [3]. It was shown that, in addition to its catalytic center, AChE has other sites that are crucial for the proper functioning of the enzyme. In particular, the so-called peripheral anionic site (PAS) located at the entrance of the active site gorge is responsible for: 1) allosteric modulation of the catalytic center; 2) enzyme inhibition at high substrate concentration; 3) and non-catalytic functions such as enhancement of cell adhesion and neurite outgrowth. Especially interesting is the relationship between the PAS and pathological beta-amyloid deposition. This led to a new hypothesis for rational design of more effective anti-Alzheimer drugs [4]. Concentration of drug producing 50% of AChE activity inhibition (IC50) was measured using the method of Ellman et al. [5]. Toxicological experiments were performed using IP injection of the different compounds in mice. LD50, dose (in mg/kg) causing lethal effects in 50% of animals was taken as a criterion of toxicity [6]. Molecular docking was performed with Autodock 4.2.6 software. We described previously a new class of selective mammalian AChE vs. butyrylcholinesterase (BChE) inhibitors based on alkylammonium derivatives of 6-methyluracil of acyclic topology [7]. In the present study, taking acyclic derivatives of 6-methyluracil as a model AChE inhibitor, we attempted to develop AChE inhibitors that specifically bind to the PAS with weak binding to the active site of AChE. We attempted to increase the size of AChE ligands to restrict specific binding to the PAS of AChE. To this aim we synthesized pyrimidinophanes bearing two o-nitrobenzylethyldialkylammonium heads. Almost all of synthesized pyrimidinophanes inhibited AChE in the nanomolar range. Based on molecular docking simulations, it was suggested that compounds bind AChE to the active center as well as to the PAS or only to the PAS. Thus, we found that introduction of the spacer, flexible or rigid, between [5-(o-nitrobenzylethylammonium)pentyl] units at N atoms of the 6-methyluracil moiety allows tuning the binding of 6-methyluracil derivatives with AChE. In conclusion, it can be stated that pyrimidinophanes are promising lead scaffold structures for further design of specific ligands for the PAS of AChE. Also AChE inhibitors with a 6-methyluracil moiety may be considered as potential drugs for the treatment of pathological muscle weakness syndromes.

The neuroprotective properties of bis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate-induced excitotoxicity were investigated in primary cultured cerebellar granule neurons (CGNs). Exposure of CGNs to 75 mum glutamate resulted in neuronal apoptosis as demonstrated by Hoechst staining, TUNEL, and DNA fragmentation assays. The bis(7)-tacrine treatment (0.01-1 mum) on CGNs markedly reduced glutamate-induced apoptosis in dose- and time-dependent manners. However, donepezil and other AChE inhibitors, even at concentrations of inhibiting AChE to the similar extents as 1 mum bis(7)-tacrine, failed to prevent glutamate-induced excitotoxicity in CGNs; moreover, both atropine and dihydro-beta-erythroidine, the cholinoreceptor antagonists, did not affect the anti-apoptotic properties of bis(7)-tacrine, suggesting that the neuroprotection of bis(7)-tacrine appears to be independent of inhibiting AChE and cholinergic transmission. In addition, ERK1/2 and p38 pathways, downstream signals of N-methyl-d-aspartate (NMDA) receptors, were rapidly activated after the exposure of glutamate to CGNs. Bis(7)-tacrine inhibited the apoptosis and the activation of these two signals with the same efficacy as the coapplication of PD98059 and SB203580. Furthermore, using fluorescence Ca(2+) imaging, patch clamp, and receptor-ligand binding techniques, bis(7)-tacrine was found effectively to buffer the intracellular Ca(2+) increase triggered by glutamate, to reduce NMDA-activated currents and to compete with [(3)H]MK-801 with an IC(50) value of 0.763 mum in rat cerebellar cortex membranes. These findings strongly suggest that bis(7)-tacrine prevents glutamate-induced neuronal apoptosis through directly blocking NMDA receptors at the MK-801-binding site, which offers a new and clinically significant modality as to how the agent exerts neuroprotective effects.

Comprehensive evaluation of AChE inhibition by Eulophia ochreata extract utilizing in silico, ex vivo, and in vivo zebrafish models.

Part 1 Characterization of acetylcholinesterase inhibitory constituents from Annona glabra assisted by HPLC micro-fractionation Acetylcholinesterase (AChE) inhibitors are used as an effective approach to treat Alzheimer’s disease (AD). Up to now, only a few AChE inhibitors have been approved for such purpose due to the side effects. Thus, the search for safer and effective AChE inhibitors is still a demand for the treatment of AD. Since natural products are versatile and often serve as drug lead for further development, a simple assay system was applied to explore the potential AChE inhibitors from higher plants. The EtOH extract of the stem of Annona glabra L. (Annonaceae) was found to be active against AChE. Thus the characterization of the anti-AChE constituents from this plant extract was the aim of this study. The active fraction was analyzed by combining HPLC micro-fractionation with a bioassay. The analytical-scale sample was fractionated by HPLC-DAD into 96-well microplates, which, after condentration using centrifugal vacuum evaporator, were assayed against AChE. The active subfractions were separated over semi-preparative HPLC to give twenty compounds. Four of these, (7S,14S)-10-O-demethylxylopinine N-metho salt (3), S-(-)-7,8-didehydro-10-O-demethylxylopininium salt (10), S-(-)-7,8-didehydrocorydalminium salt (11), and 5-methoxymarcanine A (17), were assigned as new natural products. In addition, compounds 10 and 11 represent the first natural occurrence of 7,8-didehydroprotoberberines. Compound 3, pseudocolumbamine (12), palmatine (15), and pseudopalmatine (16) showed anti-AChE IC50 values of 8.4, 5.0, 0.4 and 1.8 μM, respectively. Part 2 Flavonoid composition in the leaves of 14 Neolitsea and Litsea plants This study was aimed to study the chemodiversity of flavonoids in the Formosan Litsea and Neolitsea plants. Applications of LC-SPE-NMR and LC/MS hyphenated techniques in analyzing polar constituents from the leaves of L. acuminata, L. akoensis var. Chitouchiaoensis, L. krukovii, N. acuminatissima, and N. konishi led to the identification of 13 known flavonoids and one new flavonol dioside, quercetin 3-O-(2-O-β-D-apiofuranosyl)-α-L-rhamnopyranoside. The quantity and variety of flavonoid composition in the leaves of 14 Litsea and Neolitsea plants were examined to enable more effective utilization of such bioactive ingredients. Of these, N. acuminatissima was found to contain the most quantity of flavonoids (ca. 0.24%, w/w, leaves). Part 3 ALZ-1 attenuates scopolamine-induced amnesia in rats The deficiency in cholinergic neurotransmitter is an important feature of Alzheimer's disease (AD). Acetylcholinesterase (AChE) inhibitor is one of the rational therapeutic targets to improve AD. From in vitro study, IC50 of ALZ-1 for AChE inhibitory activity is 1.56 uM. The effects of ALZ-1 on the scopolamine-induced memory deficits in rats were examined in Morris water maze and active shuttle avoidance. Scopolamine (1 mg/kg, i.p.), the antagonist of cholinergic receptors, caused significant impairments on a wide range of learning and memory tasks. We investigated the effects of ALZ-1 on memory disturbances induced by scopolamine, since ALZ-1 (1 and 10 mg/kg, p.o.) had greater efficacy than scopolamine group on the improvement of scopolamine-induced learning and memory errors, respectively. These in vivo findings by two behavioural paradigms suggest ALZ-1, as a therapeutic potential of orally active AChE inhibitor, for improving learning and memory in AD.

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive memory loss and cognitive impairment due in part to a severe loss of cholinergic neurons in specific brain areas. AD is the most common type of dementia in the aging population. Although several acetylcholinesterase (AChE) inhibitors are currently available, their performance sometimes yields unexpected results. Thus, research is ongoing to find potentially therapeutic AChE inhibitory agents, both from natural and synthetic sources. Here, we synthesized 13 new lupinine triazole derivatives and evaluated them, along with 50 commercial lupinine-based esters of different carboxylic acids, for AChE inhibitory activity. The triazole derivative 15 [1S,9aR)-1-((4-(4-(benzyloxy)-3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)octahydro-2H-quinolizine)] exhibited the most potent AChE inhibitory activity among all 63 lupinine derivatives, and kinetic analysis demonstrated that compound 15 was a mixed-type AChE inhibitor. Molecular docking studies were performed to visualize interaction between this triazole derivative and AChE. In addition, a structure-activity relationship (SAR) model developed using linear discriminant analysis (LDA) of 11 SwissADME descriptors from the 50 lupinine esters revealed 5 key physicochemical features that allowed us to distinguish active versus non-active compounds. Thus, this SAR model could be applied for design of more potent lupinine ester-based AChE inhibitors.

To provide hints for the design of novel acetylcholinesterase (AChE) inhibitors with higher potency and specificity, the binding modes of the (RS, S)-17b and (RS, R)-17b enantiomers on AChE were chosen to investigate by molecular docking and molecular dynamics simulation. The results show that the binding modes of (RS, S)-17b and (RS, R)-17b are clearly different from each other. In particular, the (RS, S)-17b and (RS, R)-17b enantiomers tend to be planar and bend conformations to interact with AChE, respectively. Furthermore, based on the binding mode on AChE and structure modification of (RS, S)-17b, two novel inhibitors (1 and 2) with higher inhibitory activity were designed. Our design strategy suggests that the number of N and O atoms should be increased, the 5, 6-dimethoxy should be transformed into ring and the indanone moiety should be ring-opening, which would result in generating potent and selective AChE inhibitors.

Acetylcholinesterase inhibitors from Evodia rutaecarpa were screened, prepared, and evaluated. To screen the lipophilic alkaloid active constituents in E. rutaecarpa, we improved and optimized an ultrafiltration system. Three acetylcholinesterase (AChE) inhibitors, dehydroevodiamine, evodiamine, and rutecarpine, were screened. Addressing the limitations of the traditional response surface methodology (RSM) for multiobjective screening, we integrated RSM with the Non-dominated Sorting Genetic Algorithm III to achieve the optimal extraction of these active ingredients. High-speed countercurrent chromatography was used to isolate the active components using a two-phase solvent system: n-hexane/ethyl acetate/methanol/water (3.0:2.5:3.5:2.0, v/v/v/v) and ethyl acetate/methanol/water (3.0:1.0:4.0, v/v/v). The nuclear magnetic resonance spectroscopy confirmed the structures of the compounds, and molecular docking and dynamics simulations assessed the inhibitory effects of the chemical components on AChE, which were consistent with the findings of the ultrafiltration experiments. We also confirmed the neuroprotective properties of these compounds against glutamate-induced apoptosis in PC12 cells. Overall, we achieved the systematic optimization of multitarget compound extraction and lipophilic alkaloid ultrafiltration screening, as well as preparation and activity validation, laying the groundwork for the development of AChE inhibitors from lipophilic alkaloids.

Carbamate derivatives of N-propargylaminoindans (Series I) and N-propargylphenethylamines (Series II) were synthesized via multistep procedures from the corresponding hydroxy precursors. The respective rasagiline- and selegiline-related series were designed to combine inhibitory activities of both acetylcholine esterase (AChE) and monoamine oxidase (MAO) by virtue of their carbamoyl and propargylamine pharmacophores. Each compound was tested for these activities in vitro in order to find molecules with similar potencies against each enzyme. Compounds with such dual AChE and MAO inhibitory activities are expected to have potential for the treatment of Alzheimer's disease. The observed SAR also offers insight into the requirements of the active sites on these enzymes. A carbamate moiety was found to be essential for AChE inhibition, which was absent in the corresponding hydroxy precursors. The propargyl group caused 2-70-fold decrease in AChE inhibitory activity (depending on the position of the carbamoyl group) of Series I, but had little or no effect in Series II. Thus, the 6- and 7-carbamyloxyphenyls in Series I were either equipotent to, or slightly (2- to 5-fold) less active as AChE inhibitors than, the corresponding compounds in Series II, while the 4-carbamyloxyphenyls were more potent. The presence of the carbamate moiety in 6- and 7-carbamyloxyphenyls of Series I, considerably decreased MAO-A and -B inhibitory activity, compared to that of the parent hydroxy analogues, while the opposite was true for Series II. Thus, the 6- and 7-carbamyloxyphenyls in Series I were 2-3 orders of magnitude weaker MAO inhibitors while the 4- carbamyloxyphenyls were equipotent with the corresponding compounds in Series II. In both series, N-methylation of the propargylamine enhanced the MAO (A and B equally) inhibitory activities and decreased the AChE inhibitory activity. Two candidates belonging to the indan and tetralin ring systems (24c, 27b) and one phenethylamine (53d) were identified as possible leads for further development based on the following criteria: (a) comparable AChE and MAO-B inhibitory activities, (b) good to moderate AChE inhibitory activity, and (c) lack of strong MAO-A selectivity. However, it is likely that these compounds will be metabolized to the corresponding phenols, with inhibitory activities against AChE and/or MAO-A or -B, different from those of the parent carbamates. Thus, the apparent enzyme inhibition will be a result of the combined inhibition of all of these individual metabolites. The results of our ongoing in vivo screening programs will be published elsewhere.

ABSTRACT: Alzheimer’s disease is a progressive neurodegenerative disorder of the centralnervous system. Acetylcholinesterase inhibition is one of the proposed mechanisms fortreatment of Alzheimer’s disease. Currently, acetylcholinesterase inhibitors such as tacrine,donepezil, rivastigmine and galantamine are applied in different stages of Alzheimer’s diseaseteratment. In recent years, various heterocyclic systems have been used as a skeletonto discover new acetylcholinesterase inhibitors. On the other hand, it is known that the benzoxazoloneheterocyclic structure exhibited a wide range of biological activities. In thisstudy, a series N-substituted-5-chloro-2(3H)-benzoxazolone derivatives were synthesizedand evaluated their acetylcholinesterase inhibitory activity. These compounds were synthesizedby Mannich reaction of 5-chloro-2(3H)-benzoxazolone with the appropriated amines.The acetylcholinesterase inhibitory activity of the title compounds was determined by colorimetricEllman’s method. The preliminary screening results indicated that 5-chloro-2-(3H)-benzoxazolone scaffold demonstrated different inhibition range against acetylcholinesteraseenzyme depending on the structural differences.KEY WORDS: acetylcholinesterase Inhibitory activity, 2(3H)-benzoxazolone, mannich reaction,Ellman’s method, synthesis.

The mixture of semi-synthetic derivatives (-)-3-O-acetyl-cassine hydrochloride and (-)-3-O-acetyl-spectaline hydrochloride, prepared from the mixture of natural alkaloids (-)-cassine and (-)-spectaline (4:1) isolated from Senna spectabilis, has been shown to be a potent acetylcholinesterase (AChE) inhibitor, thereby prompting further molecular studies. In this sense, docking and dynamic molecular studies were carried out in this work, aiming to acquire a deeper understanding about all the structural aspects of molecules (-)-3-O-acetyl-cassine and (-)-3-O-acetyl-spectaline hydrochlorides, which differ with respect to their AChE inhibitory potentials. Both molecules establish important interactions with the peripheral anionic site within the catalytic gorge of Torpedo californica AChE. However, only the major compound (-)-3-O-acetyl-cassine hydrochloride significantly interacts with the catalytic triad. Explicit-solvent molecular dynamic simulations were conducted in order to gain better understanding about the hypothetical interactions taking place between the semi-synthetic alkaloid molecules (-)-3-O-acetyl-cassine and (-)-3-O-acetyl-spectaline hydrochlorides and AChE. The data obtained in this study indicated that (-)-3-O-acetyl-cassine hydrochloride is the most potent inhibitor of AChE possibly due to the favorable interactions of this molecule with the target protein, with lower desolvation cost. These results suggested that the size of the side chain has an effect on the inhibitory potential of the evaluated molecules and may represent the starting point for the development of new derivatives of (-)-3-O-acetyl-cassine hydrochloride, with a view to the discovery of new effective AChE inhibitors.

Naringin, as a component universal existing in the peel of some fruits or medicinal plants, was usually selected as the material to synthesise bioactive derivates since it was easy to gain with low cost. In present investigation, eight new acacetin-7-O-methyl ether Mannich base derivatives (1–8) were synthesised from naringin. The bioactivity evaluation revealed that most of them exhibited moderate or potent acetylcholinesterase (AChE) inhibitory activity. Among them, compound 7 (IC50 for AChE = 0.82 ± 0.08 μmol•L−1, IC50 for BuChE = 46.30 ± 3.26 μmol•L−1) showed a potent activity and high selectivity compared with the positive control Rivastigmine (IC50 for AChE = 10.54 ± 0.86 μmol•L−1, IC50 for BuChE = 0.26 ± 0.08 μmol•L−1). The kinetic study suggested that compound 7 bind to AChE with mix-type inhibitory profile. Molecular docking study revealed that compound 7 could combine both catalytic active site (CAS) and peripheral active site (PAS) of AChE with four points (Trp84, Trp279, Tyr70 and Phe330), while it could bind with BuChE via only His 20.

Dispiropyrrolidinyl-piperidone embedded indeno[1,2-b]quinoxaline heterocyclic hybrids: Synthesis, cholinesterase inhibitory activity and their molecular docking simulation.