Synthesis, biological evaluation, molecular docking, and MD simulation of novel 2,4-disubstituted quinazoline derivatives as selective butyrylcholinesterase inhibitors and antioxidant agents.

Alzheimer's disease is the most prevalent neurodegenerative disorder characterized by significant memory loss and cognitive impairments. Studies have shown that the expression level and activity of the butyrylcholinesterase enzyme increases significantly in the late stages of Alzheimer's disease, so butyrylcholinesterase can be considered as a promising therapeutic target for potential Alzheimer's treatments. In the present study, a novel series of 2,4-disubstituted quinazoline derivatives (6a-j) were synthesized and evaluated for their inhibitory activities against acetylcholinesterase (AChE) and butyrylcholinestrase (BuChE) enzymes, as well as for their antioxidant activities. The biological evaluation revealed that compounds 6f, 6h, and 6j showed potent inhibitory activities against eqBuChE, with IC50 values of 0.52, 6.74, and 3.65 µM, respectively. These potent compounds showed high selectivity for eqBuChE over eelAChE. The kinetic study demonstrated a mixed-type inhibition pattern for both enzymes, which revealed that the potent compounds might be able to bind to both the catalytic active site and peripheral anionic site of eelAChE and eqBuChE. In addition, molecular docking studies and molecular dynamic simulations indicated that potent compounds have favorable interactions with the active sites of BuChE. The antioxidant screening showed that compounds 6b, 6c, and 6j displayed superior scavenging capabilities compared to the other compounds. The obtained results suggest that compounds 6f, 6h, and 6j are promising lead compounds for the further development of new potent and selective BuChE inhibitors.

New Heterostilbene and Triazole Oximes as Potential CNS-Active and Cholinesterase-Targeted Therapeutics.

New furan, thiophene, and triazole oximes were synthesized through several-step reaction paths to investigate their potential for the development of central nervous systems (CNS)-active and cholinesterase-targeted therapeutics in organophosphorus compound (OP) poisonings. Treating patients with acute OP poisoning is still a challenge despite the development of a large number of oxime compounds that should have the capacity to reactivate acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The activity of these two enzymes, crucial for neurotransmission, is blocked by OP, which has the consequence of disturbing normal cholinergic nerve signal transduction in the peripheral and CNS, leading to a cholinergic crisis. The oximes in use have one or two pyridinium rings and cross the brain-blood barrier poorly due to the quaternary nitrogen. Following our recent study on 2-thienostilbene oximes, in this paper, we described the synthesis of 63 heterostilbene derivatives, of which 26 oximes were tested as inhibitors and reactivators of AChE and BChE inhibited by OP nerve agents-sarin and cyclosarin. While the majority of oximes were potent inhibitors of both enzymes in the micromolar range, we identified several oximes as BChE or AChE selective inhibitors with the potential for drug development. Furthermore, the oximes were poor reactivators of AChE; four heterocyclic derivatives reactivated cyclosarin-inhibited BChE up to 70%, and cis,trans-5 [2-((Z)-2-(5-((E)-(hydroxyimino)methyl)thiophen-2-yl)vinyl)benzonitrile] had a reactivation efficacy comparable to the standard oxime HI-6. In silico analysis and molecular docking studies, including molecular dynamics simulation, connected kinetic data to the structural features of these oximes and confirmed their productive interactions with the active site of cyclosarin-inhibited BChE. Based on inhibition and reactivation and their ADMET properties regarding lipophilicity, CNS activity, and hepatotoxicity, these compounds could be considered for further development of CNS-active reactivators in OP poisoning as well as cholinesterase-targeted therapeutics in neurodegenerative diseases such as Alzheimer's and Parkinson's.

Evaluation of Anticholinesterase Activity of the Fungicides Mefentrifluconazole and Pyraclostrobin.

Triazoles are compounds with various biological activities, including fungicidal action. They became popular through cholinesterase studies after the successful synthesis of the dual binding femtomolar triazole inhibitor of acetylcholinesterase (AChE, EC 3.1.1.7) by Sharpless et al. via in situ click chemistry. Here, we evaluate the anticholinesterase effect of the first isopropanol triazole fungicide mefentrifluconazole (Ravystar®), developed to overcome fungus resistance in plant disease management. Mefentrifluconazole is commercially available individually or in a binary fungicidal mixture, i.e., with pyraclostrobin (Ravycare®). Pyraclostrobin is a carbamate that contains a pyrazole ring. Carbamates are known inhibitors of cholinesterases and the carbamate rivastigmine is already in use for the treatment of Alzheimer's disease. We tested the type and potency of anticholinesterase activity of mefentrifluconazole and pyraclostrobin. Mefentrifluconazole reversibly inhibited human AChE and BChE with a seven-fold higher potency toward AChE (Ki = 101 ± 19 µM). Pyraclostrobin (50 µM) inhibited AChE and BChE progressively with rate constants of (t1/2 = 2.1 min; ki = 6.6 × 103 M-1 min-1) and (t1/2 = 1.5 min; ki = 9.2 × 103 M-1 min-1), respectively. A molecular docking study indicated key interactions between the tested fungicides and residues of the lipophilic active site of AChE and BChE. Additionally, the physicochemical properties of the tested fungicides were compared to values for CNS-active drugs to estimate the blood-brain barrier permeability. Our results can be applied in the design of new molecules with a lesser impact on humans and the environment.

Benzorhodol derived far-red/near-infrared fluorescent probes for selective and sensitive detection of butyrylcholinesterase activity in living cells and the non-alcoholic fatty liver of zebrafish.

Liver diseases are a growing public health concern and the development of non-alcoholic fatty liver disease (NAFLD) has a significant impact on human metabolism. Butyrylcholinesterase (BChE) is a vital biomarker for NAFLD, making it crucial to monitor BChE activity with high sensitivity and selectivity. In this study, we designed and synthesized a range of benzorhodol-derived far-red/near-infrared fluorescent probes, FRBN-B, NF-SB, and NF-B, for the quantitative detection and imaging of BChE. These probes differed in the size of their conjugated systems and in the number of incorporated cyclopropanecarboxylates, acting as the recognition site for BChE. Comprehensive characterization showed that FRBN-B and NF-SB fluorescence was triggered by BChE-mediated hydrolysis, while an additional cyclopropanecarboxylate in NF-B impeded the fluorescence release. High selectivity towards BChE was observed for FRBN-B and NF-SB, with a detection limit of 7.2 × 10-3 U mL-1 for FRBN-B and 1.9 × 10-3 U mL-1 for NF-SB. The probes were further employed in the evaluation of BChE inhibitor efficacy and imaging of intracellular BChE activity. Additionally, FRBN-B was utilized for imaging the BChE activity level in liver tissues in zebrafish, demonstrating its potential as a diagnostic tool for NAFLD.

Regulating Second Coordination Shell of Ce Atom Site and Reshaping of Carrier Enable Single-Atom Nanozyme to Efficiently Express Oxidase-like Activity.

Single-atom nanozymes (SANs) are considered to be ideal substitutes for natural enzymes due to their high atom utilization. This work reported a strategy to manipulate the second coordination shell of the Ce atom and reshape the carbon carrier to improve the oxidase-like activity of SANs. Internally, S atoms were symmetrically embedded into the second coordination layer to form a Ce-N4S2-C structure, which reduced the energy barrier for O2 reduction, promoted the electron transfer from the Ce atom to O atoms, and enhanced the interaction between the d orbital of the Ce atom and p orbital of O atoms. Externally, in situ polymerization of mussel-inspired polydopamine on the precursor helps capture metal sources and protects the 3D structure of the carrier during pyrolysis. On the other hand, polyethylene glycol (PEG) modulated the interface of the material to enhance water dispersion and mass transfer efficiency. As a proof of concept, the constructed PEG@P@Ce-N/S-C was applied to the multimodal assay of butyrylcholinesterase activity.

Synthesis of novel aryl-substituted 2-aminopyridine derivatives by the cascade reaction of 1,1-enediamines with vinamidinium salts to develop novel anti-Alzheimer agents.

Alzheimer's disease (AD), a severe neurodegenerative disorder, imposes socioeconomic burdens and necessitates innovative therapeutic strategies. Current therapeutic interventions are limited and underscore the need for novel inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), enzymes implicated in the pathogenesis of AD. In this study, we report a novel synthetic strategy for the generation of 2-aminopyridine derivatives via a two-component reaction converging aryl vinamidinium salts with 1,1-enediamines (EDAMs) in a dimethyl sulfoxide (DMSO) solvent system, catalyzed by triethylamine (Et3N). The protocol introduces a rapid, efficient, and scalable synthetic pathway, achieving good to excellent yields while maintaining simplistic workup procedures. Seventeen derivatives were synthesized and subsequently screened for their inhibitory activity against AChE and BChE. The most potent derivative, 3m, exhibited an IC50 value of 34.81 ± 3.71 µM against AChE and 20.66 ± 1.01 µM against BChE compared to positive control donepezil with an IC50 value of 0.079 ± 0.05 µM against AChE and 10.6 ± 2.1 µM against BChE. Also, detailed kinetic studies were undertaken to elucidate their modes of enzymatic inhibition of the most potent compounds against both AChE and BChE. The promising compound was then subjected to molecular docking and dynamics simulations, revealing significant binding affinities and favorable interaction profiles against AChE and BChE. The in silico ADMET assessments further determined the drug-like properties of 3m, suggesting it as a promising candidate for further pre-clinical development.

Ratiometric fluorescence and colorimetric strategies for assessing activity of butyrylcholinesterase in human serum using g-C3N4 nanosheets, silver ion and o-phenylenediamine.

Ratiometric fluorescence and colorimetric strategies for detecting activity of butyrylcholinesterase (BChE) in human serum were developed by using g-C3N4 nanosheets, silver ion (Ag+) and o-phenylenediamine (OPD) as chromogenic agents. The oxidation-reduction reaction of OPD and Ag+ generates 2,3-diaminophenazine (oxOPD). Under exciation at 370 nm, g-C3N4 nanosheets and oxOPD emit fluorescence at 440 nm (F440) and 560 nm (F560), respectively. Additionally, oxOPD exhibits quenching ability towards g-C3N4 nanosheets via photoinduced electron transfer (PET) process. Thiocholine (TCh), as a product of BChE-catalyzed hydrolysis reaction of butylthiocholine iodide (BTCh), can coordinate with Ag+ intensively, and consequently diminish the amount of free Ag+ in the testing system. Less amount of free Ag+ leads to less production of oxOPD, resulting in less fluorescence quenching towards g-C3N4 nanosheets as well as less fluorescence emission of oxOPD. Therefore, by using g-C3N4 nanosheets and oxOPD as fluorescence indicators, the intensity ratio of their fluorescence (F440/F560) was calculated and employed to evaluate the activity of BChE. Similarly, the color variation of oxOPD indicated by the absorbance at 420 nm (ΔA420) was monitored for the same purpose. These strategies were validated to be sensitive and selective for detecting BChE activity in human serum, with limits of detection (LODs) of 0.1 U L-1 for ratiometric fluorescence mode and 0.7 U L-1 for colorimetric mode.

Human Plasma Butyrylcholinesterase Hydrolyzes Atropine: Kinetic and Molecular Modeling Studies.

The participation of butyrylcholinesterase (BChE) in the degradation of atropine has been recurrently addressed for more than 70 years. However, no conclusive answer has been provided for the human enzyme so far. In the present work, a steady-state kinetic analysis performed by spectrophotometry showed that highly purified human plasma BChE tetramer slowly hydrolyzes atropine at pH 7.0 and 25 °C. The affinity of atropine for the enzyme is weak, and the observed kinetic rates versus the atropine concentration was of the first order: the maximum atropine concentration in essays was much less than Km. Thus, the bimolecular rate constant was found to be kcat/Km = 7.7 × 104 M-1 min-1. Rough estimates of catalytic parameters provided slow kcat < 40 min-1 and high Km = 0.3-3.3 mM. Then, using a specific organophosphoryl agent, echothiophate, the time-dependent irreversible inhibition profiles of BChE for hydrolysis of atropine and the standard substrate butyrylthiocholine (BTC) were investigated. This established that both substrates are hydrolyzed at the same site, i.e., S198, as for all substrates of this enzyme. Lastly, molecular docking provided evidence that both atropine isomers bind to the active center of BChE. However, free energy perturbations yielded by the Bennett Acceptance Ratio method suggest that the L-atropine isomer is the most reactive enantiomer. In conclusion, the results provided evidence that plasma BChE slowly hydrolyzes atropine but should have no significant role in its metabolism under current conditions of medical use and even under administration of the highest possible doses of this antimuscarinic drug.

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 reactivation kinetic analysis, molecular docking, and dynamics of oximes against three V-type nerve agents inhibited four human cholinesterases.

Nerve agents pose significant threats to civilian and military populations. The reactivation of acetylcholinesterase (AChE) is critical in treating acute poisoning, but there is still lacking broad-spectrum reactivators, which presents a big challenge. Therefore, insights gained from the reactivation kinetic analysis and molecular docking are essential for understanding the behavior of reactivators towards intoxicated AChE. In this research, we present a systematic determination of the reactivation kinetics of three V agents-inhibited four human ChEs [(AChE and butyrylcholinesterase (BChE)) from either native or recombinant resources, namely, red blood cell (RBC) AChE, rhAChE, hBChE, rhBChE) reactivated by five standard oximes. We unveiled the effect of native and recombinant ChEs on the reactivation kinetics of V agents ex vitro, where the reactivation kinetics characteristic of Vs-inhibited BChE was reported for the first time. In terms of the inhibition type, all of the five oxime reactivators exhibited noncompetitive inhibition. The inhibition potency of these reactivators would not lead to the difference in the reactivation kinetics between native and recombinant ChE. Despite the significant differences between the native and recombinant ChEs observed in the inhibition, aging, and spontaneous reactivation kinetics, the reactivation kinetics of V agent-inhibited ChEs by oximes were less differentiated, which were supported by the ligand docking results. We also found differences in the reactivation efficiency between five reactivators and the phosphorylated enzyme, and molecular dynamic simulations can further explain from the perspectives of conformational stability, hydrogen bonding, binding free energies, and amino acid contributions. By Poisson-Boltzmann surface area (MM-PBSA) calculations, the total binding free energy trends aligned well with the experimental kr2 values.

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.

Pre-Steady-State and Steady-State Kinetic Analysis of Butyrylcholinesterase-Catalyzed Hydrolysis of Mirabegron, an Arylacylamide Drug.

The ß-adrenergic drug Mirabegron, a drug initially used for the treatment of an overactive bladder, has new potential indications and is hydrolyzed by butyrylcholinesterase (BChE). This compound is one of the only arylacylamide substrates to be catabolized by BChE. A steady-state kinetic analysis at 25 °C and pH 7.0 showed that the enzyme behavior is Michaelian with this substrate and displays a long pre-steady-state phase characterized by a burst. The induction time, τ, increased with substrate concentration (τ ≈ 18 min at maximum velocity). The kinetic behavior was interpreted in terms of hysteretic behavior, resulting from a slow equilibrium between two enzyme active forms, E and E'. The pre-steady-state phase with the highest activity corresponds to action of the E form, and the steady state corresponds to action of the E' form. The catalytic parameters were determined as kcat = 7.3 min-1 and Km = 23.5 µM for the initial (burst) form E, and kcat = 1.6 min-1 and Km = 3.9 µM for the final form E'. Thus, the higher affinity of E' for Mirabegron triggers the slow enzyme state equilibrium toward a slow steady state. Despite the complexity of the reaction mechanism of Mirabegron with BChE, slow BChE-catalyzed degradation of Mirabegron in blood should have no impact on the pharmacological activities of this drug.

Potential acetylcholinesterase inhibitors to treat Alzheimer's disease.

OBJECTIVE: Alzheimer's disease (AD) is identified by neuropathological symptoms, and there is now no effective treatment for the condition. A lack of the brain neurotransmitter acetylcholine has been related to the etiology of Alzheimer's disease. Acetylcholinesterase is an enzyme that breaks down acetylcholine to an inactive form and causes the death of cholinergic neurons. Conventional treatments were used but had less effectiveness. Therefore, there is a crucial need to identify alternative compounds with potential anti-cholinesterase agents and minimal undesirable effects. MATERIALS AND METHODS: Fluoroquinolones and benzimidazole-benzothiazole derivatives offer antimicrobial, anti-inflammatory, anti-oxidant, anti-diabetic, and anti-Alzheimer activities. To enhance the chemical portfolio of cholinesterase inhibitors, a variety of fluoroquinolones and benzimidazole-benzothiazole compounds were evaluated against acetylcholinesterase (AChE) butyrylcholinesterase (BChE) enzymes. For this purpose, molecular docking and adsorption, distribution, metabolism, excretion, and toxicology ADMET models were used for in-silico studies for both AChE and BChE enzymes to investigate possible binding mechanisms and drug-likeness of the compounds. The inhibitory effect of docked heterocyclic compounds was also verified in vitro against AChE and BChE enzymes. Fluoroquinolones (Z, Z3, Z4, Z6, Z8, Z12, Z15, and Z9) and benzimidazole-benzothiazole compounds (TBIS-16, TBAF-1 to 9) passed through the AChE inhibition assay and their IC50 values were calculated. RESULTS: The compound 1-ethyl-6-fluoro-7-(4-(2-(4-nitrophenylamino)-2-oxoethyl)piperazin-1-yl) -4-oxo-1,4 di-hydroquinoline-3-carboxylic acid and 2-((1H-benzo[d]imidazol-2-yl)methyl)-N'-(3-bromobenzyl)-4-hydroxy-2H-thiochromene-3-carbohydrazide 1,1-dioxide (Z-9 and TBAF-6) showed the lowest IC50 values against AChE/BChE (0.37±0.02/2.93±0.03 µM and 0.638±0.001/1.31±0.01 µM, respectively) than the standard drug, donepezil (3.9±0.01/4.9±0.05 µM). During the in-vivo investigation, behavioral trials were performed to analyze the neuroprotective impact of Z-9 and TBAF-6 compounds on AD mouse models. The groups treated with Z-9 and TBAF-6 compounds had better cognitive behavior than the standard drug. CONCLUSIONS: This study found that Z-9 (Fluoroquinolones) and TBAF-6 (benzimidazole-benzothiazole) compounds improve behavioral and biochemical parameters, thus treating neurodegenerative disorders effectively.

Interaction of exogenous acetylcholinesterase and butyrylcholinesterase with amyloid-ß plaques in human brain tissue.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are associated with amyloid-ß (Aß) plaques and exhibit altered biochemical properties in human Alzheimer's disease (AD), as well as in the transgenic 5XFAD mouse model of AD amyloidosis. In the brains of the 5XFAD mouse model devoid of BChE enzyme (5XFAD/BChE-KO), incubation of tissue sections with exogenous BChE purified from human plasma (pl-BChE) leads to its association with Aß plaques and its biochemical properties are comparable to those reported for endogenous BChE associated with plaques in both human AD and in 5XFAD mouse brain tissue. We sought to determine whether these observations in 5XFAD/BChE-KO mice also apply to human brain tissues. To do so, endogenous ChE activity in human AD brain tissue sections was quenched with 50 % aqueous acetonitrile (MeCNaq) leaving the tissue suitable for further studies. Quenched sections were then incubated with recombinant AChE (r-AChE) or pl-BChE and stained for each enzymes' activity. Exogenous r-AChE or pl-BChE became associated with Aß plaques, and when bound, had properties that were comparable to the endogenous ChE enzymes associated with plaques in AD brain tissues without acetonitrile treatment. These findings in human AD brain tissue extend previous observations in the 5XFAD/BChE-KO mouse model and demonstrate that exogenously applied r-AChE and pl-BChE have high affinity for Aß plaques in human brain tissues. This association alters the biochemical properties of these enzymes, most likely due a conformational change. If incorporation of AChE and BChE in Aß plaques facilitates AD pathogenesis, blocking this association could lead to disease-modifying approaches to AD. This work provides a method to study the mechanism of AChE and BChE interaction with Aß plaque pathology in post-mortem human brain tissue.

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.

Uncharged mono- and bisoximes: In search of a zwitterion to countermeasure organophosphorus intoxication.

The current study imposes a new class of organophosphorus (OP)-inhibited cholinesterase reactivators by conceptualizing a family of asymmetric bisoximes with various reactivating scaffolds. Several novel nucleophilic warheads were investigated, putting forward 29 novel reactivating options, by evaluating their nucleophilicity and ability to directly decompose OP compounds. Adopting the so-called zwitterionic strategy, 17 mono-oxime and nine bisoxime reactivators were discovered with major emphasis on the bifunctional-moiety approach. Compounds were compared with clinically used standards and other known experimentally highlighted reactivators. Our results clearly favor the concept of asymmetric bisoximes as leading reactivators in terms of efficacy and versatility. These top-ranked compounds were characterized in detail by reactivation kinetics parameters and evaluated for potential CNS availability. The highlighted molecules 55, 57, and 58 with various reactivating warheads, surpassed the reactivating potency of pralidoxime and several notable uncharged reactivators. The versatility of lead drug candidate 55 was also inspected on OP-inhibited butyrylcholinesterase, revealing a much higher rate compared to existing clinical antidotes.

Diethoxyphosphoryloxy-oleanolic acid is a nanomolar-inhibitor of butyrylcholinesterase.

A series of new betulin, lupeol, erythrodiol, and oleanolic acid phosphoryloxy- and furoyloxy-derivatives has been synthesized and their structure was confirmed by NMR spectroscopy. Synthesized compounds were subjected to Ellman's assays to determine their ability to inhibit the enzymes AChE and BChE. Among them, diethoxyphosphoryloxy-oleanolic acid inhibited BChE with a value of 99%, thereby acting as a mixed-type inhibitor holding very low Ki values of Ki = 6.59 nM and Ki ' = 1.97 nM, respectively.

Insight into antioxidant-like activity and computational exploration of identified bioactive compounds in Talinum triangulare (Jacq.) aqueous extract as potential cholinesterase inhibitors.

BACKGROUND: Recent reports have highlighted the significance of plant bioactive components in drug development targeting neurodegenerative disorders such as Alzheimer's disease (AD). Thus, the current study assessed antioxidant activity and enzyme inhibitory activity of the aqueous extract of Talinum triangulare leave (AETt) as well as molecular docking/simulation of the identified phytonutrients against human cholinesterase activities. METHODS: In vitro assays were carried out to assess the 2,2- azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) cation radicals and cholinesterase inhibitory activities of AETt using standard protocols. High performance liquid chromatography coupled with diode-array detection (HPLC-DAD) was employed to identify compounds in AETt. Also, for computational analysis, identified bioactive compounds from AETt were docked using Schrodinger's GLIDE against human cholinesterase obtained from the protein data bank ( https://www.rcsb.org/ ). RESULTS: The results revealed that AETt exhibited a significant concentration-dependent inhibition against ABTS cation radicals (IC50 = 308.26 ± 4.36 µg/ml) with butylated hydroxytoluene (BHT) as the reference. Similarly, AETt demonstrated a significant inhibition against acetylcholinesterase (AChE, IC50 = 326.49 ± 2.01 µg/ml) and butyrylcholinesterase (BChE, IC50 = 219.86 ± 4.13 µg/ml) activities with galanthamine as the control. Molecular docking and simulation analyses revealed rutin and quercetin as potential hits from AETt, having showed strong binding energies for both the AChE and BChE. In addition, these findings were substantiated by analyses, including radius of gyration, root mean square fluctuation, root mean square deviation, as well as mode similarity and principal component analyses. CONCLUSION: Overall, this study offers valuable insights into the interactions and dynamics of protein-ligand complexes, offering a basis for further drug development targeting these proteins in AD.

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.

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.