Phytochemical analysis, antioxidant, acetylcholinesterase, and α-amylase inhibitors from extract.

OBJECTIVE: To investigate the effects of Hippeastrum hybridum (HH) as a free radical scavenger, and an inhibitor of the two enzymes i-e Alpha-amylase (α-amylase) and acetylcholinesterase (AChE). METHODS: In this study, HH plant was preliminary analyzed for phytochemical screening and then tested for its antioxidant, anti-α-amylase, and anti-AChE efficiency via standard procedures. RESULTS: Phytochemical analysis shows the existence of different compounds; while Coumarins and quinones were absent. The total phenolic, flavonoid, and tannins content were found to be (78.52 ± 0.69) mg GAE/g, (2.01 ± 0.04) mg RUE/g, and (58.12 ± 0.23) mg TAE/g of plant extract respectively. 28.02% ± 0.02% alkaloid and 2.02% ± 0.05% saponins were present in the HH extract. The HH extract showed the anti-oxidant property with IC50 (50% inhibition) of (151.01 ± 0.13) (HH), (79.01 ± 0.04) (Ascorbic acid) for ferric reducing, (91.48 ± 0.13) (HH), (48.02 ± 0.11) (Ascorbic acid) against Ammonium molybdenum, (156.02 ± 0.31) (HH), (52.38 ± 0.21) (Ascorbic acid) against DPPH, 136.01 ± 0.21 (HH), 52.02± 0.31 (Ascorbic acid) against H2O2, and 154.12 ± 0.03 (HH), (40.05 ± 0.15) (Ascorbic acid) µg/mL against ABTS respectively. Statistical analysis indicated that HH caused a competitive type of inhibition of α-amylase (Vmax remained constant and Km increases from 10.65 to 84.37%) while Glucophage caused the un-competitive type of inhibition i-e both Km and Vmax decreased from 40.49 to 69.15% and 38.86 to 69.61% respectively. The Ki, (inhibition constant); KI, (dissociation constant), Km, (Michaelis-Menten constant), and IC50 were found to be 62, 364, 68.1, and 38.08 ± 0.22 for HH and 12, 101.05, 195, 34.01 ± 0.21 for Glucophage. Similarly, HH causes an anon-competitive type of inhibition of AChE i-e Km remains constant while Vmax decreases from 60.5% to 74.1%. The calculated Ki, KI, Km, and IC50 were found to be 32, 36.2, 0.05, and 18.117 ± 0.018. CONCLUSION: From the current results, it is concluded that HH extract contains bioactive compounds, and could be a good alternative to controlling oxidants, Alzheimer's and Type-II diabetic diseases.

Two Fluorescent Probes for Recognition of Acetylcholinesterase: Design, Synthesis, and Comparative Evaluation.

In this study, two "on-off" probes (BF2-cur-Ben and BF2-cur-But) recognizing acetylcholinesterase (AChE) were designed and synthesized. The obtained probes can achieve recognition of AChE with good selectivity and pH-independence with a linear range of 0.5~7 U/mL and 0.5~25 U/mL respectively. BF2-cur-Ben has a lower limit of detection (LOD) (0.031 U/mL), higher enzyme affinity (Km = 16 ± 1.6 µM), and higher inhibitor sensitivity. A responsive mechanism of the probes for AChE was proposed based on HPLC and mass spectra (MS) experiments, as well as calculations. In molecular simulation, BF2-cur-Ben forms more hydrogen bonds (seven, while BF2-cur-But has only four) and thus has a more stable enzyme affinity, which is mirrored by the results of the comparison of Km values. These two probes could enable recognition of intracellular AChE and probe BF2-cur-Ben has superior cell membrane penetration due to its higher log p value. These probes can monitor the overexpression of AChE during apoptosis of lung cancer cells. The ability of BF2-cur-Ben to monitor AChE in vivo was confirmed by a zebrafish experiment.

Cholesterol Oxime Olesoxime Assessed as a Potential Ligand of Human Cholinesterases.

Olesoxime, a cholesterol derivative with an oxime group, possesses the ability to cross the blood-brain barrier, and has demonstrated excellent safety and tolerability properties in clinical research. These characteristics indicate it may serve as a centrally active ligand of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), whose disruption of activity with organophosphate compounds (OP) leads to uncontrolled excitation and potentially life-threatening symptoms. To evaluate olesoxime as a binding ligand and reactivator of human AChE and BChE, we conducted in vitro kinetic studies with the active metabolite of insecticide parathion, paraoxon, and the warfare nerve agents sarin, cyclosarin, tabun, and VX. Our results showed that both enzymes possessed a binding affinity for olesoxime in the mid-micromolar range, higher than the antidotes in use (i.e., 2-PAM, HI-6, etc.). While olesoxime showed a weak ability to reactivate AChE, cyclosarin-inhibited BChE was reactivated with an overall reactivation rate constant comparable to that of standard oxime HI-6. Moreover, in combination with the oxime 2-PAM, the reactivation maximum increased by 10-30% for cyclosarin- and sarin-inhibited BChE. Molecular modeling revealed productive interactions between olesoxime and BChE, highlighting olesoxime as a potentially BChE-targeted therapy. Moreover, it might be added to OP poisoning treatment to increase the efficacy of BChE reactivation, and its cholesterol scaffold could provide a basis for the development of novel oxime antidotes.

The Cholinergic Selectivity of FDA-Approved and Metabolite Compounds Examined with Molecular-Docking-Based Virtual Screening.

The search for selective anticholinergic agents stems from varying cholinesterase levels as Alzheimer's Disease progresses from the mid to late stage. In this computational study, we probed the selectivity of FDA-approved and metabolite compounds against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with molecular-docking-based virtual screening. The results were evaluated using locally developed codes for the statistical methods. The docking-predicted selectivity for AChE and BChE was predominantly the consequence of differences in the volume of the active site and the narrower entrance to the bottom of the active site gorge of AChE.

Acetylcholinesterase purification from human erythrocytes using magnetic nanoparticles containing procainamide.

This work presents a magnetic purification method of human erythrocyte Acetylcholinesterase (EC 3.1.1.7; AChE) based on affinity binding to procainamide (Proca) as ligand. Acetylcholinesterase is an acetylcholine-regulating enzyme found in different areas of the body and associated with various neurological disorders, such as Parkinson, Alzheymer and Amyotrophic Lateral Sclerosis. AChE from human erythrocyte purification has been attempted in recent years with low degree of purity. Here, magnetic nanoparticles (MNP) were synthesized and coated with polyaniline (PANI) and procainamide (PROCA) was covalently linked to the PANI. The extracted human erythrocyte AChE formed a complex with the MNP@PANI-PROCA and an external magnet separated it from the undesired proteins. Finally, the enzyme was collected by increasing the ionic strength. Experimental Box-Behnken design was developed to optimize this process of human erythrocyte AChE purification protocol. The enzyme was purified in all fifteen experiments. However, the best AChE purification result was achieved, about 2000 times purified, when 100 mg of MNP@PANI-PROCA was incubated for one hour with 4 ml hemolysate extract. The SDS-PAGE of this preparation presented a molecular weight of approximately 70 kDa, corroborating with few previous studies of AChE from erythrocyte purification.

Preferential Door for Ligand Binding and Unbinding Pathways in Inhibited Human Acetylcholinesterase.

Rising global population and increased food demands have resulted in the increased use of organophosphate pesticides (OPs), leading to toxin accumulation and transmission to humans. Pralidoxime (2-PAM), an FDA-approved drug, serves as an antidote for OP therapy. However, the atomic-level detoxification mechanisms regarding the design of novel antidotes remain unclear. This is the first study to examine the binding and unbinding pathways of 2-PAM to human acetylcholinesterase (HuAChE) through three identified doors using an enhanced sampling method called ligand-binding parallel cascade selection molecular dynamics (LB-PaCS-MD). Remarkably, LB-PaCS-MD could identify a predominant in-line binding mechanism through the acyl door at 63.79% ± 6.83%, also implicating it in a potential unbinding route (90.14% ± 4.22%). Interestingly, crucial conformational shifts in key residues, W86, Y341, and Y449, and the Ω loop significantly affect door dynamics and ligand binding modes. The LB-PaCS-MD technique can study ligand-binding pathways, thereby contributing to the design of antidotes and covalent drugs.

Disentangling the formation, mechanism, and evolvement of the covalent methanesulfonyl fluoride acetylcholinesterase adduct: Insights into an aged-like inactive complex susceptible to reactivation by a combination of nucleophiles.

Chemical warfare nerve agents and pesticides, known as organophosphorus compounds inactivate cholinesterases (ChEs) by phosphorylating the serine hydroxyl group located at the active site of ChEs. Over the course of time, phosphorylation is followed by loss of an organophosphate-leaving group and the bond with ChEs becomes irreversible, a process known as aging. Differently, structurally related irreversible catalytic poisons bearing sulfur instead of phosphorus convert ChEs in its aged form only by covalently binding to the key catalytic serine. Kinetic and crystallographic studies of the interaction between Torpedo californica acetylcholinesterase (TcAChE) and a small organosulfonate, methanesulfonyl fluoride (MSF), indeed revealed irreversibly methylsulfonylated serine 200, to be isosteric with the bound aged sarin/soman analogues. The potent bulky reversible inhibitor 7-bis-tacrine (BTA) adopts, in the active site of the crystal structure of the MSF-enzyme adduct, a location and an orientation that closely resemble the one being found in the crystal structure of the BTA-enzyme complex. Remarkably, the presence of BTA accelerates the rate of methanesulfonylation by a factor of two. This unexpected result can be explained on the basis of two facts: i) the steric hindrance exerted by BTA to MSF in accessing the active site and ii) the acceleration of the MSF-enzyme adduct formation as a consequence of the lowering of the rotational and translational degrees of freedom in the proximity of the catalytic serine. It is well known that pralidoxime (2-Pyridine Aldoxime Methyl chloride, 2-PAM) alone or in the presence of the substrate acetylcholine cannot reactivate the active site serine of the TcAChE-MSF adduct. We show that the simultaneous presence of 2-PAM and the additional neutral oxime, 2-[(hydroxyimino)methyl]-l-methylimidazol (2-HAM), triggers the reactivation process of TcAChE within the hour timescale. Overall, our results pave the way toward the likely use of a cocktail of distinctive oximes as a promising recipe for an effective and fast reactivation of aged cholinesterases.

Design, Synthesis, and Biological Evaluation of Novel Tetrahydroacridin Hybrids with Sulfur-Inserted Linkers as Potential Multitarget Agents for Alzheimer's Disease.

Alzheimer's disease (AD) is a complex neurodegenerative disease that can lead to the loss of cognitive function. The progression of AD is regulated by multiple signaling pathways and their associated targets. Therefore, multitarget strategies theoretically have greater potential for treating AD. In this work, a series of new hybrids were designed and synthesized by the hybridization of tacrine (4, AChE: IC50 = 0.223 µM) with pyrimidone compound 5 (GSK-3ß: IC50 = 3 µM) using the cysteamine or cystamine group as the connector. The biological evaluation results demonstrated that most of the compounds exhibited moderate to good inhibitory activities against acetylcholinesterase (AChE) and glycogen synthase kinase 3ß (GSK-3ß). The optimal compound 18a possessed potent dual AChE/GSK-3ß inhibition (AChE: IC50 = 0.047 ± 0.002 µM, GSK-3ß: IC50 = 0.930 ± 0.080 µM). Further molecular docking and enzymatic kinetic studies revealed that this compound could occupy both the catalytic anionic site and the peripheral anionic site of AChE. The results also showed a lack of toxicity to SH-SY5Y neuroblastoma cells at concentrations of up to 25 µM. Collectively, this work explored the structure-activity relationships of novel tetrahydroacridin hybrids with sulfur-inserted linkers, providing a reference for the further research and development of new multitarget anti-AD drugs.

Unleashing new MTDL AChE and BuChE inhibitors as potential anti-AD therapeutic agents: In vitro, in vivo and in silico studies.

Alzheimer's disease (AD) is challenging due to its irreversible declining cognitive symptoms and multifactorial nature. This work tackles targeting both acetylcholinesterase (AChE) and BuChE with a multitarget-directed ligand (MTDL) through design, synthesis, and biological and in silico evaluation of a series of twenty eight new 5-substituted-2-anilino-1,3,4-oxadiazole derivatives 4a-g, 5a-g, 9a-g and 13a-g dual inhibitors of the target biomolecules. In vitro cholinesterases inhibition and selectivity assay of the synthesized derivatives showed excellent nanomolar level inhibitory activities. Compound 5a, the most potent inhibitor, elicited IC50s of 46.9 and 3.5 nM against AChE and BuChE, respectively (SI = 0.07), 5 folds better than the known dual inhibitor Rivastagmine. In vivo and ex vivo investigation showed that 5a significantly inhibited MDA levels and increased GSH contents, thus, attenuating the brain tissue oxidative stress. Additionally, 5a significantly decreased AChE and BuChE levels and inhibited self-mediated ß-amyloid aggregation in brains of treated rats. Histopathological and immunohistochemical evaluation demonstrated lessened damage and decreased caspase-3 and VEGF expression levels. In silico prediction of 5a's pharmacokinetics and toxicity profiles reflected promising results. Finally, 5a demonstrated tight binding interactions with the two target biomolecules upon docking along with stable complex formation with its bio-targets throughout the 100 ns MD trajectories.

Bifunctional enzyme-mimicking metal-organic frameworks for sensitive acetylcholine analysis.

The development of nanomaterials with multi-enzyme-like activity is crucial for addressing challenges in multi-enzyme-based biosensing systems, including cross-talk between different enzymes and the complexities and costs associated with detection. In this study, Pt nanoparticles (Pt NPs) were successfully supported on a Zr-based metal-organic framework (MOF-808) to create a composite catalyst named MOF-808/Pt NPs. This composite catalyst effectively mimics the functions of acetylcholinesterase (AChE) and peroxidase (POD). Leveraging this capability, we replaced AChE and POD with MOF-808/Pt NPs and constructed a biosensor for sensitive detection of acetylcholine (ACh). The MOF-808/Pt NPs catalyze the hydrolysis of ACh, resulting in the production of acetic acid. The subsequent reduction in pH value further enhances the POD-like activity of the MOFs, enabling signal amplification through the oxidation of a colorimetric substrate. This biosensor capitalizes on pH variations during the reaction to modulate the different enzyme-like activities of the MOFs, simplifying the detection process and eliminating cross-talk between different enzymes. The developed biosensor holds great promise for clinical diagnostic analysis and offers significant application value in the field.

Direct detection of acetylcholinesterase by Fe(HCOO)2.6(OH)0.3. H2O nanosheets with oxidase-like activity on a smartphone platform.

Monitoring acetylcholinesterase (AChE) is crucial in clinical diagnosis and drug screening. Traditional methods for detecting AChE usually require the addition of intermediates like acetylthiocholine, which complicates the detection process and introduces interference risks. Herein, we develop a direct colorimetric assay based on alkaline iron formate nanosheets (Fe(HCOO)2.6(OH)0.3·H2O NSs, Fef NSs) for the detection of AChE without any intermediates. The as-prepared Fef NSs exhibit oxidase-like activity, catalyzing the generation of O2·-, 1O2 and ·OH, which leads to a color change from colorless to blue when exposed to 3,3',5,5'-tetramethylbenzidine. AChE directly inhibits the oxidase-like activity of Fef NSs, resulting in a hindered color reaction, enabling the detection of AChE. The biosensor has a linear detection range of 0.1-30 mU/mL, with a minimum detection limit of 0.0083 mU/mL (S/N = 3), representing a 100-fold improvement in detection sensitivity over the traditional Ellman's method. Satisfactory results were obtained when analyzing real AChE samples. Attractively, a method for the quantitative detection of AChE by a smartphone is established based on the Fef NSs. This method enables instant acquisition of AChE concentrations, achieving real-time visualized detection.

Acetylcholine triggered enzymatic cascade reaction based on Fe7S8 nanoflakes catalysis for organophosphorus pesticides visual detection.

BACKGROUND: Organophosphorus pesticides (OPs) play important roles in the natural environment, agricultural fields, and biological prevention. The development of OPs detection has gradually become an effective strategy to avoid the dangers of pesticides abuse and solve the severe environmental and health problems in humans. Although conventional assays for OPs analysis such as the bulky instrument required analytical methods have been well-developed, it still remains the limitation of inconvenient, inefficient and lab-dependence analysis in real samples. Hence, there is an urgent demand to develop efficient detection methods for OPs analysis in real scenarios. RESULTS: Here, by virtue of the highly efficient catalytic performance in Fe7S8 nanoflakes (Fe7S8 NFs), we propose an OPs detection method that rationally integrated Fe7S8 NFs into the acetylcholine (ACh) triggered enzymatic cascade reaction (ATECR) for proceeding better detection performances. In this method, OPs serve as the enzyme inhibitors for inhibiting ATECR among ACh, acetylcholinesterase (AChE), and choline oxidase (CHO), then reduce the generation of H2O2 to suppress the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) that catalyzed by Fe7S8 NFs. Benefiting from the integration of Fe7S8 NFs and ATECR, it enables a sensitive detection for OPs (e.g. dimethoate). The proposed method has presented good linear ranges of OPs detection ranging from 0.1 to 10 µg mL-1. Compared to the other methods, the comparable limits of detection (LOD) of OPs are as low as 0.05 µg mL-1. SIGNIFICANCE: Furthermore, the proposed method has also achieved a favorable visual detection performance of revealing OPs analysis in real samples. The visual signals of OPs can be transformed into RGB values and gathered by using smartphones, indicating the great potential in simple, sensitive, instrument-free and on-site analysis of pesticide residues in environmental monitoring and biosecurity research.

Environmental exposure to glyphosate does not inhibit human acetylcholinesterase and butyrylcholinesterase.

Glyphosate has remained the leading herbicide on the global market to date, despite the continuous debate between consumers, scientific community, and regulatory agencies over its carcinogenicity, genotoxicity, environmental persistence, and the role in the development of neurodegenerative disorders. Chemically, glyphosate belongs to a large family of organophosphorus pesticides, which exert a neurotoxic effect by inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), enzymes of the cholinergic system essential for maintaining neurotransmission. Although research shows that glyphosate is a weak cholinesterase inhibitor in fish and mammals compared to other OP compounds, no conclusive data exist concerning the inhibition of human AChE and BChE. In our study we analysed its inhibitory potency on human AChE and BChE, by establishing its IC50 and reversible inhibition in terms of dissociation inhibition constants. Glyphosate concentration of 40 mmol/L caused near total inhibition of enzyme activity (approx. 10 % activity remaining). Inhibition dissociation constants (K i) of glyphosate-AChE and -BChE complexes were 28.4±2.7 mmol/L and 19.3±1.8 mmol/L, respectively. In conclusion, glyphosate shows a slight binding preference for BChE but exhibits inhibition only in a high concentration range. Our results are in line with studies reporting that its neurotoxic effect is not primarily linked to the cholinergic system.

Interactions between forsythoside E and two cholinesterases at the different conditions: fluorescence sections.

Forsythoside E is one secondary metabolite ofForsythia suspensa(Thunb.) Vahl. In the study, the interactions between forsythoside E and two types of cholinesterases, acetylcholinesterase and butyrylcholinesterase were investigated in the different conditions. Forsythoside E increased the fluorescence intensity of acetylcholinesterase but quenched the fluorescence of butyrylcholinesterase. Aß25-35used in the study may not form complexes with cholinesterases, and did not affect the interaction between forsythoside E and cholinesterases. The charged quaternary group of AsCh interacted with the 'anionic' subsite in acetylcholinesterase, which did not affect the interaction between forsythoside E and acetylcholinesterase. The enhancement rate of forsythoside E to acetylcholinesterase fluorescence from high to low was acid solution (pH 6.4), neutral solution (pH 7.4) and alkaline solution (pH 8.0), while the reduction rate of forsythoside E to butyrylcholinesterase fluorescence was in reverse order. Metal ions may interact with cholinesterases, and increased the effects of forsythoside E to cholinesterases fluorescence, in order that Fe3+was the highest, followed by Cu2+, and Mg2+. A forsythoside E-butyrylcholinesterase complex at stoichiometric ratio of 1:1 was spontaneously formed, and the static quenching was the main quenching mode in the process of forsythoside E binding with butyrylcholinesterase. TheKvalues of two complexes were pretty much the same, suggesting that the interaction between cholinesterases and forsythoside E was almost unaffected by acid-base environment and metal ions. Thennumbers of two cholinesterases approximately equaled to one, indicating that there was only one site on each cholinesterase applicable for forsythoside E to bind to.

New amide and diterpene alkaloids with anticholinesterase activity from Delphinium cyphoplectrum roots.

BACKGROUND: The cholinergic hypothesis posits a robust correlation between the onset of Alzheimer's disease and a pronounced deficit in acetylcholine, a pivotal neurotransmitter crucial for the central cholinergic nervous system's function, pivotal for memory and learning. Diterpene alkaloids exhibit intricate and distinctive chemical structures that facilitate their passage through the blood-brain barrier. Moreover, their potent pharmacological attributes render them promising candidates for addressing central nervous system disorders. OBJECTIVES: This investigation aims to scrutinize the alkaloidal composition of Delphinium cyphoplectrum (Ranunculaceae) roots, further exploring their anticholinesterase inhibitory activity and mode of inhibition. METHOD: Innovative chromatography techniques were repetitively employed to purify the alkaloids. Acetylcholinesterase (AChE) inhibition assays were conducted using Ellman's tests. The mode of inhibition was meticulously characterized through Michaelis-Menten, and Lineweaver-Burk plots. Conducting molecular docking studies, we employed the AUTO DOCK 4.2 software package. RESULTS: Eight alkaloids were identified including five C19-diterpene alkaloids (6,14,16,18-tetramethoxy-1,7,8-trihydroxy-4-methylaconitane (1), 6,16,18-trimethoxy-1,7,8,14-tetrahydroxy-4-methylaconitane (2), 6,8,16,18-tetramethoxy-1,7,14-trihydroxy-4-methylaconitane (3), 6,14,16-trimethoxy-1,7,8,18-tetrahydroxy-4-methylaconitane (4), and 14-O-acetyl-8,16-dimethoxy-1,6,7,18-tetrahydroxy-4-methylaconitane (5)), an epoxy C18-diterpene alkaloid (6,8,16-trimethoxy-1,7,14-trihydroxy-3,4-epoxyaconitane (6)), a known (pyrrolidin-2-one (7) and an undescribed amide alkaloid (1-(2'-hydroxylethylamine)-3,5,5,-trimethyl-1,5-dihydro-2H-pyrrol-2-one (8). All diterpene alkaloids underwent assessment for acetylcholinesterase (AChE) inhibition assay and displayed noteworthy AChE activity, surpassing that of the reference drug (with IC50 values of 13.7, 21.8, 23.4, 28.2, 40.4, and 23.9 for compounds 1-6, respectively, in comparison to 98.4 for Rivastigmine). Analysis of Michaelis-Menten and Lineweaver-Burk plots represents an uncompetitive mode of inhibition for compound 1 on AChE. Notably, computational docking simulations indicated that all diterpene alkaloids were accommodated within the same enzymatic cleft as the reference ligand, and displaying superior free binding energy values (from - 10.32 to -8.59 Kcal.mol-1) in contrast to Rivastigmine (-6.31 Kcal.mol-1). CONCLUSION: The phytochemical analysis conducted on the roots of Delphinium cyphoplectrum yielded the identification of eight alkaloidal compounds including one C18-diterpene, five C19-diterpene, one pyrrolidine and one amide alkaloids. AChE inhibition assay and molecular simulations unveiled remarkable significant potency attributed to the C19-diterpene alkaloids by the order of 1 > 2 > 3,6 > 4 > 5. Presence of hydroxyl group on C-1, C-7, C-8, C-14, and C-18 increased the effect. The best in vitro activity was recorded for compound 1 able to bind to Asp72 in the narrow region of PAS, while interacting by pi-sigma with Phe330 at the hydrophobic region of the gorge involving the acyl and choline binding site. This observation underscores the substantial promise of this category of natural products in the realm of drug discovery for Alzheimer's Disease, offering a compelling avenue for further research and therapeutic development.

Sequential Contrastive and Deep Learning Models to Identify Selective Butyrylcholinesterase Inhibitors.

Butyrylcholinesterase (BChE) is a target of interest in late-stage Alzheimer's Disease (AD) where selective BChE inhibitors (BIs) may offer symptomatic treatment without the harsh side effects of acetylcholinesterase (AChE) inhibitors. In this study, we explore multiple machine learning strategies to identify BIs in silico, optimizing for precision over all other metrics. We compare state-of-the-art supervised contrastive learning (CL) with deep learning (DL) and Random Forest (RF) machine learning, across single and sequential modeling configurations, to identify the best models for BChE selectivity. We used these models to virtually screen a vendor library of 5 million compounds for BIs and tested 20 of these compounds in vitro. Seven of the 20 compounds displayed selectivity for BChE over AChE, reflecting a hit rate of 35% for our model predictions, suggesting a highly efficient strategy for modeling selective inhibition.

Lycopodium Alkaloids from Huperzia goebelii.

One new fawcettimine-type Lycopodium alkaloid, hupertimine F (1), together with five known (2-6) Lycopodium alkaloids were isolated from Huperzia goebelii. The structure of 1 was elucidated by 1D and 2D NMR spectra, HRESIMS, and X-ray diffraction. Structurally, 1 represents the fourth example of Lycopodium alkaloids characterized by a 5/5/5/5/6 pentacyclic ring system with a 1-aza-7-oxabicyclo[2.2.1]heptane moiety. These known compounds 2, 3, 5, and 6 were isolated from H. goebelii for the first time. Compounds 1-6 were evaluated for acetylcholinesterase, butyrylcholinesterase and monoamine oxidase B inhibitory activities in vitro.

Design, synthesis and biological evaluation of bakuchiol derivatives as multi-target agents for the treatment of Alzheimer's disease.

The concept of multi-target-directed ligands offers fresh perspectives for the creation of brand-new Alzheimer's disease medications. To explore their potential as multi-targeted anti-Alzheimer's drugs, eighteen new bakuchiol derivatives were designed, synthesized, and evaluated. The structures of the new compounds were elucidated by IR, NMR, and HRMS. Eighteen compounds were assayed for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in vitro using Ellman's method. It was shown that most of the compounds inhibited AChE and BuChE to varying degrees, but the inhibitory effect on AChE was relatively strong, with fourteen compounds showing inhibition of >50% at the concentration of 200 µM. Among them, compound 3g (IC50 = 32.07 ± 2.00 µM) and compound 3n (IC50 = 34.78 ± 0.34 µM) showed potent AChE inhibitory activities. Molecular docking studies and molecular dynamics simulation showed that compound 3g interacts with key amino acids at the catalytically active site (CAS) and peripheral anionic site (PAS) of acetylcholinesterase and binds stably to acetylcholinesterase. On the other hand, compounds 3n and 3q significantly reduced the pro-inflammatory cytokines TNF-α and IL-6 released from LPS-induced RAW 264.7 macrophages. Compound 3n possessed both anti-acetylcholinesterase activity and anti-inflammatory properties. Therefore, an in-depth study of compound 3n is expected to be a multi-targeted anti-AD drug.

Pnictogen bond-driven control of the molecular interaction between organophosphorus and acetylcholinesterase enzyme.

This study addresses a comprehensive assessment of the interaction between chemical warfare agents (CWA) and acetylcholinesterase (AChE) systems, focus on the intriguing pnictogen-bond interaction (PnB). Utilizing the crystallographic data from the Protein Data Bank pertaining to the AChE-CWA complex involving Sarin (GB), Cyclosarin (GF), 2-[fluoro(methyl)phosphoryl]oxy-1,1-dimethylcyclopentane (GP) and venomous agent X (VX) agents, the CWA is systematically displaced by increments of 0.1 Å along the PO bond axis, extending its distance by 4 Å from the original position. The AIM analysis was carried out and consistently revealed the presence of a significant interaction along the PO bond. Investigating the intrinsic nature of the PnB, the NBO and the EDA analysis unearthed the contribution of orbital factors to the overall energy of the system. Strikingly, this observation challenges the conventional σ-hole explanation commonly associated with such interactions. This finding adds a layer of complexity to understanding of PnB, encouraging further exploration into the underlying mechanisms governing these intriguing chemical phenomena.

Binding interactions and in silico ADME prediction of isoconessimine derivatives as potent acetylcholinesterase inhibitors.

In pursuit of new acetylcholinesterase (AChE) inhibitors for treating Alzheimer's disease (AD), a series of ten previously synthesized isoconessimine compounds (7a-7j) was in silico investigated for their binding interactions with AChE and pharmacokinetics based on absorption, distribution, metabolism, and excretion (ADME) properties using molecular docking, ONIOM (Our own N-layered Integrated molecular Orbital and molecular Mechanics) method and SwissADME tools. Docking experiments showed that all compounds bind within the active site gorge of AChE (PDB entry 1C2B), posing its aryloxy-substitutional ethyl group to catalytic site and conessine skeleton to peripheral anionic site. ONIOM interaction energy was used as an ONIOM score to improve docking score, and it ranked 7b as the most potent AChE inhibitor, in agreement with previous experiment. Residues, ASP74, TRP86, GLY122, GLU202, TRP286, GLU292, SER293, ILE294, TYR337, TYR341, and HIS447 were identified as important for the binding of the AChE-isoconessimine complex. The SwissADME investigation suggested that four compounds (7a, 7c, 7d and 7f) agree with the rules of drug-likeness. The steric and electronic effects on the aryloxy-substitutional ethyl group as important factors in the AChE inhibition were also discussed, which brings a better understanding of Alzheimer's disease drug development.