In silico studies of bis-spiro- and Furano-Labdane diterpenoids from Rydingia persica Scheen (Otostegia persica) as α-glucosidase enzyme inhibitor.

The inhibition potential of α-glucosidase enzyme by crude- dichloromethane, methanol, and ethanol -extracts of Rydingia persica were evaluated using colorimetric method. We have isolated four labdane diterpenoids: 15, 16- epoxy-3α, 7ß, 9α -trihydroxylabdan-13- (16), 14-dien-6-one (1), 15, 16- epoxy-3α, 7α, 9α -trihydroxylabdan-13- (16), 14-dien-6-one (2), 9, 13, 15, 16-diepoxy- 3α, 7ß, 15α (ß)- trihydroxy-labdan- 6 one (3, 4) from the most potent enzyme inhibitor fraction; the ethyl acetate soluble part of ethanol extract of the aerial parts of R. persica. The structures of the compounds were elucidated by their 1H and13C NMR and ESIMS spectral data analyses. The enzyme inhibition potential of the compounds was evaluated against acetylcholine esterase (AChE) and α-glucosidase by simulation studies. The predicted binding energy of most diterpenes towards mouse AChE enzyme was low, while the binding energy of diterpenes towards α-glucosidase enzyme was moderate to potent.

Multiple-biomarker approach in the assessment of bisphenol A effect on the grooved carpet clam Ruditapes decussatus (Linnaeus, 1758).

BACKGROUND: Bisphenol A (BPA), a plastic additive monomer, is among the most highly produced chemicals worldwide, and is broadly used in many industries, such as food and beverage containers, milk bottles, and paper products. Previous studies demonstrated that BPA has potential toxicity to aquatic organisms, causing endocrine disturbance and behavioural disorders. The current work aimed to determine the toxic impacts of BPA on the edible marine clam Ruditapes decussatus considering a multi-biomarker approach (mortality, biochemical studies, DNA strand breaks using comet assay, and histopathological examinations with semi-quantitative and quantitative histopathological analyses). The clams were exposed under laboratory conditions to three concentrations of BPA (0 "control", 1, and 5 µg/L) for a period of 21 days. After the exposure period, BPA impacts were assessed in the digestive gland as a versatile and environmentally relevant organ for ecotoxicological studies. RESULTS: In BPA-treated clams, mortality (10%) occurred only at the highest BPA concentration (5 µg/L). Biochemical impairments were detected in a concentration-dependent manner as a consequence of BPA exposure. There were significant increases in malondialdehyde (MDA) and glutathione (GSH) levels, while catalase (CAT) activity was significantly reduced. Our results revealed that BPA induced neurotoxicity in R. decussatus, as evidenced by the inhibition of acetylcholinesterase (AChE) activity in a dose-dependent manner. Furthermore, DNA damage was strongly induced as BPA levels increased. Additionally, our results have been affirmed by alterations in digestive gland tissues at BPA treatments, which consequently can impair the clam's ability for food absorption; these alterations included mainly atrophic and necrotic digestive tubules, epithelial cell vacuolization, hemocyte infiltration, and intertubular fibrosis. Based on the data obtained from the semi-quantitative and quantitative histopathological analyses, the exposure of the clam's digestive gland to BPA with concentrations of 1 and 5 µg/L for 21 days showed significant histopathological alterations compared with the control clams. CONCLUSION: The multi-biomarker approach used in the current study proved to be a useful tool for assessing the impact of diphenylmethane compounds, such as BPA. Water-borne BPA causes oxidative stress, neurotoxicity, genotoxicity, and deleterious effects on the clam digestive gland; all of these could deteriorate clam performance and health, causing tissue dysfunction.

Cholinergic Inhibition and Antioxidant Potential of Gongronema latifolium Benth Leaf in Neurodegeneration: Experimental and In Silico Study.

The use of Gongronema latifolium for the management of various forms of neurological disorders has generated a lot of interest in the need to further investigate its neurotherapeutic constituents. This work, therefore, focused on assessing the inhibitory potential of selected bioactive components derived from G. latifolium against key neurotherapeutic targets and oxidant species associated with neurodegeneration using in vitro analysis and biomolecular modelling. G. latifolium methanol extract (GLME), solvent partition, chromatographic fractions (A-F) of GLME and pregnane compounds (Iloneoside and marsectohexol) derived from fraction-B with the highest activity were investigated for in vitro acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and monoamine oxidase (MAO) inhibition in addition to their in vitro antioxidant activities. The interactions of iloneoside, marsectohexol, and reference drugs with human acetylcholinesterase, butyrylcholinesterase, and ß-secretase (BACE-1) were further assessed using molecular docking, binding free energy calculations, cluster analysis, and molecular dynamics simulations. The GLME and fractions inhibited the activities of both acetylcholinesterase and butyrylcholinesterase in a dose-dependent manner. Iloneoside and marsectohexol exhibited in vitro concentration-dependent inhibitory activities against acetylcholinesterase (IC50 = 19.28, 184.9 µM, respectively) and butyrylcholinesterase (IC50 = 30.75, 43.4 µM, respectively). These compounds also possess ferric ion-reducing, hydroxyl, and superoxide radical-scavenging activities. Iloneoside had the highest docking scores of -9.8, -9.9 -9.4 Kcal for AChE, BChE, and BACE1, respectively. The stability of the interaction of the bioactive compounds with the catalytic residues of the protein targets was preserved in a 100 ns molecular dynamics simulation. Iloneoside, a rare pregnane glycoside, was identified as a neurotherapeutic constituent of G. latifolium leaf. Further studies are suggested to investigate the neurotherapeutic potential in animal models.

Scutellarein derivatives with histamine H3 receptor antagonism and cholinesterase inhibitory potency as multi target-directed ligands for possible Alzheimer's disease therapy.

A series of scutellarein 7-l-amino acid carbamate-4'-cycloalkylamine propyl ether conjugates were designed and synthesized for the first time as multifunctional agents for Alzheimer's disease (AD) therapy. The designed compounds exhibited more balanced and effective multi-target potency. Among them, compound 11l, l-Valine carbamate derivative of scutellarein cycloheptylamine ether, exhibited the most potent inhibition of electric eel AChE enzymes and human AChE enzymes, with an IC50 values of 7.04 µM and 9.73 µM, respectively. Moreover, 11l exhibited more potent H3R antagonistic activities than clobenpropit, with an IC50 value of 1.09 nM. Compound 11l not only displayed excellent inhibition of self- and Cu2+-induced Aß1-42 aggregation (95.48 % and 88.63 % inhibition, respectively) but also induced the disassembly of self- and Cu2+-induced Aß fibrils (80.16 % and 89.30 % disaggregation, respectively). Moreover, 11l significantly reduced tau protein hyperphosphorylation induced by Aß25-35. It exhibited effective antioxidant activity and neuroprotective potency, and inhibited RSL3-induced PC12 cell ferroptosis. Assays of hCMEC/D3 and hPepT1-MDCK cell line permeability indicated that 11l would have optimal blood-brain barrier permeability and intestinal absorption characteristics. In addition, in vivo studies revealed that compound 11l significantly attenuated learning and memory impairment in an AD mouse model. Finally, a pharmacokinetic characterization of 11l indicated favorable druggability and pharmacokinetic properties. Taken together, our results suggest that 11l is a potential candidate for AD treatment and merits further investigation.

Atomistic Origins of Resurrection of Aged Acetylcholinesterase by Quinone Methide Precursors.

Nerve agents are organophosphates (OPs) that act as potent inhibitors of acetylcholinesterase (AChE), the enzyme responsible for the hydrolysis of acetylcholine. After inhibition, a dealkylation reaction of the phosphorylated serine, known as the aging of AChE, can occur. When aged, reactivators of OP-inhibited AChE are no longer effective. Therefore, the realkylation of aged AChE may offer a pathway to reverse AChE aging. In this study, molecular modeling was conducted to propose new ligands as realkylators of aged AChE. We applied a methodology involving docking and quantum mechanics/molecular mechanics (QM/MM) calculations to evaluate the resurrection kinetic constants and ligand interactions with OP-aged AChE, comparing them to data found in the literature. The results obtained confirm that this method is suitable for predicting kinetic and thermodynamic parameters of ligands, which can be useful in the design and selection of new and more effective ligands for AChE realkylation.

A Series of Novel 1-H-isoindole-1,3(2H)-dione Derivatives as Acetylcholinesterase and Butyrylcholinesterase Inhibitors: In Silico, Synthesis and In Vitro Studies.

The derivatives of isoindoline-1,3-dione are interesting due to their biological activities, such as anti-inflammatory and antibacterial effects. Several series have been designed and evaluated for Alzheimer's therapy candidates. They showed promising activity. In this work, six new derivatives were first tested in in silico studies for their inhibitory ability against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. Molecular docking and molecular dynamic simulation were applied. Next, these compounds were synthesized and characterized by 1H NMR, 13C NMR, FT-IR, and ESI-MS techniques. For all imides, the inhibitory activity against AChE and BuChE was tested using Ellaman's method. IC50 values were determined. The best results were obtained for the derivative I, with a phenyl substituent at position 4 of piperazine, IC50 = 1.12 µM (AChE) and for the derivative III, with a diphenylmethyl moiety, with IC50 = 21.24 µM (BuChE). The compounds tested in this work provide a solid basis for further structural modifications, leading to the effective design of potential inhibitors of both cholinesterases.

Factors associated with cognitive function in patient with Alzheimer's disease with newly prescribed acetylcholinesterase inhibitors: A 1-year retrospective cohort study.

Objective: We aimed to examine the factors associated with treatment outcomes in patients with Alzheimer's disease (AD) after 1 year of acetylcholinesterase inhibitors (AChEI) treatment. Method: We obtained electronic medical records from a medical center in Southern Taiwan between January 2015 and September 2021. Participants aged ≥60 who were newly diagnosed with AD and had been prescribed AChEIs were included. Cognitive assessments were performed before the AChEIs were prescribed and at the 1 year follow-up. Cognition progressors were defined as a Mini-Mental State Examination decline of >3 or a Clinical Dementia Rating decline of ≥1 after 1 year of AChEI treatment. The relationship between the baseline characteristics and cognitive status after follow-up was investigated using logistic regression analysis after adjusting for potential confounders. Results: A total of 1370 patients were included in our study (mean age, 79.86 ± 8.14 years). After adjustment, the body mass index (BMI) was found to be significantly lower in the progressor group [adjusted odds ratio (AOR): 0.970, 95% confidence intervals (95% CIs): 0.943 to 0.997, P = 0.033]. The usage of antipsychotics was significantly higher in the progressor group (AOR: 1.599, 95% CIs: 1.202 to 2.202, P = 0.001). The usage of benzodiazepine receptor agonists also tended to be significantly higher in the progressor group (AOR: 1.290, 95% CIs: 0.996 to 1.697, p = 0.054). Conclusion: These results suggest that patients with AD who receive 1 year of AChEI treatment and have a lower BMI or concurrent treatment with antipsychotics and benzodiazepine receptor agonists are more likely to suffer from cognitive decline.

Design, synthesis, biological evaluation, and molecular modeling simulations of new phthalazine-1,4-dione derivatives as anti-Alzheimer's agents.

The development of targeted phthalazine-1,4-dione acetylcholinesterase (AChE) inhibitors for treating Alzheimer's disease involved the synthesis of 32 compounds via a multistage process. Various analytical techniques confirmed the compounds' identities. Thirteen compounds were found to inhibit AChE by more than 50% without affecting butyrylcholinesterase (BChE). Among these, three compounds, 8m, 8n, and 8p, exhibited extraordinary activity similar to donepezil, a reference AChE inhibitor. During enzyme kinetic studies, compound 8n, displaying the highest AChE inhibitory activity, underwent evaluation at three concentrations (2 × IC50, IC50, and IC50/2). Lineweaver-Burk plots indicated mixed inhibition activity for compound 8n against AChE, suggesting a combination of competitive and noncompetitive characteristics. Additionally, effective derivatives 8m, 8n, and 8p exhibited high blood-brain barrier (BBB) permeability in in vitro parallel artificial membrane permeability assay tests. Molecular docking studies revealed that these compounds bind to the enzyme's active site residues in a position similar to donepezil. Molecular dynamic simulations confirmed the stability of the protein-ligand system, and the chemical reactivity characteristics of the compounds were investigated using density functional theory. The compounds' wide energy gaps suggest stability and therapeutic potential. This research represents a significant step toward finding a potential cure for Alzheimer's disease. However, further research and testing are required to determine the compounds' safety and efficacy. The unique structure of phthalazine derivatives makes them suitable for various biological activities, and these compounds show promise for developing effective drugs for treating Alzheimer's disease. Overall, the development of these targeted compounds is a crucial advancement in the search for an effective treatment for Alzheimer's disease.

Unveiling piperazine-quinoline hybrids as potential multi-target directed anti-Alzheimer's agents: design, synthesis and biological evaluation.

Multi-target directed ligands (MTDLs) have recently been popularized due to their outstanding efficacy in combating the complicated features of Alzheimer's disease. This study details the synthesis of piperazine-quinoline-based MTDLs through a multicomponent Petasis reaction, targeting multiple factors such as AChE, BuChE, metal chelation to restore metal dyshomeostasis, and antioxidant activity. Some of the synthesized compounds exhibited notable inhibitory activity against AChE and BuChE enzymes at specific concentrations. Among the synthesized compounds compound (95) containing a 4-chloroaniline moiety and a 4-methoxybenzyl group displayed the most promising inhibitory activities against AChE (IC50 3.013 µM) and BuChE (IC50 = 3.144 µM). Compound (83) featuring 2-methoxyaniline and 4-fluorobenzyl substituents, exhibited the highest BuChE inhibition (IC50 1.888 µM). Notably, compound (79) demonstrated 93-times higher selectivity for BuChE over AChE. Molecular docking and molecular dynamics simulations were also performed to explore the binding modes and stability of these compounds with the AChE amd BuChE proteins. Further, kinetics study was performed against AChE for comounds (83 and 95) which indicated mixed inhibition of the enzyme by these compounds, Amongs the synthesized compounds, nine compounds were assessed for their antioxidant activity, displaying significant antioxidant properties with IC50 values ranging from 156 µM to 310 µM. Moreover, all the compounds demonstrated metal chelating tendency with Cu+2, Zn+2, Fe+2, Fe+3 and Al+3. This study provides insights into the design of novel MTDLs, highlighting compound (95) as a potential candidate for combating Alzheimer's disease.

Multi-generation effects of lead (Pb) on two Daphnia species.

Two monophyletic Daphnia species (Daphnia magna and D. similis) were exposed to a sub-lethal concentration of Pb (50 µg/L) for nine generations under two food regimes (usual and restricted) and analyzed for acetylcholinesterase (AChE) activity, first reproduction delay, lifespan, and net reproductive rate (R0) at the subcellular, individual, and population levels, respectively. In the sixth generation, Pb-acclimated neonates were moved to clean media for three more generations to check for recovery. The net reproductive rate (R0) of D. magna was not affected by Pb. However, Pb stimulated reproduction, reduced lifespan, and decreased AChE activity. First reproduction delay and lifespan did not improve during the recovery process, suggesting a possible genetic adaptation. Food restriction reduced R0, lifespan, delayed hatching, and increased AChE activity; the opposite outcomes were observed for D. similis. The full recovery shown by R0 suggests the physiological acclimation of D. similis. Under food restriction, the animals exhibited a reduction of R0 and lifespan, delayed first reproduction, and increased AChE activity; however, there was no effect of Pb. The recovery process under food restriction showed that D. similis might not cope with Pb exposure, indicating a failed recovery. Such outcomes indicate that one model species' sensitivity may not represent another's sensitivity.

Electrochemical sensor for effective detection of methyl parathion applying multidimensional MXene/CNHs/PPy nanocomposite to synergistically immobilize acetylcholinesterase.

In this study, a novel acetylcholinesterase (AChE)-based electrochemical sensor was successfully constructed using two-dimensional MXene, carbon nanohorns (CNHs) and polypyrrole (PPy) as the substrate material for the detection of methyl parathion (MP) residue. The multidimensional MXene/CNHs composite, formed through electrostatic self-assembly, provided a high specific surface area and excellent conductivity. With an active surface area of 0.1062 cm2, the composite provided numerous electroactive sites for AChE immobilization and facilitated electron diffusion at the sensing interface, amplifying the electrochemical signals. Additionally, polypyrrole (PPy) improved AChE adhesion on the electrode surface, further enhancing the stability of the sensor. The proposed sensor exhibited a wide linear range (0.002-346 ng mL-1) and low detection limit (0.00021 ng mL-1) for MP. This study offers an innovative strategy to detect MP, showcasing the potential of two-dimensional materials in electrochemical sensing.

Rapid identification of chemical components and screening of acetylcholinesterase inhibitors from Dalbergia odorifera based on mass defect and diagnostic ion filtering strategy, affinity ultrafiltration, and liquid chromatography-tandem mass spectrometry.

Dalbergia odorifera is a natural product rich in pharmacological ingredients, but the comprehensive characterization and rapid profiling of active components remain a challenge. Thus, an integrated data mining and identification strategy was exploited to efficiently identify the chemical constituents and screen acetylcholinesterase inhibitors (AChEIs) through affinity ultrafiltration and ultra-high-performance liquid chromatography-mass spectrometry (AUF-UHPLC-MS). As a result, polygonal mass defect filtering, diagnostic product ions, and neutral loss rules were created for rapid structural classification and component identification. A total of 140 flavonoids were tentatively characterized, including 41 isoflavonoids, 23 flavanones, 21 isoflavans, 19 flavones and flavonols, 13 neoflavonoids, 11 isoflavanones, seven flavone glycosides, and five chalcones. Subsequently, six natural AChEIs including tectorigenin, fisetin, dalbergin, pterostilbene, isoliquiritigenin, and biochanin A were screened out using AUF-UHPLC-MS and molecular docking. Meanwhile, the AChE inhibitory activities of the six compounds were assessed in vitro, tectorigenin, fisetinand, and dalbergin have moderate inhibitory activity. In conclusion, a novel strategy for systematic characterization and further screening of active compounds in natural products was established, which provides a material basis for quality control of Dalbergia odorifera.

Synthesis and in vitro assessment of the reactivation profile of clinically available oximes on the acetylcholinesterase model inhibited by A-230 nerve agent surrogate.

The risk of the use of toxic chemicals for unlawful acts has been a matter of concern for different governments and multilateral agencies. The Organisation for the Prohibition of Chemical Weapons (OPCW), which oversees the implementation of the Chemical Weapons Convention (CWC), considering recent events employing chemical warfare agents as means of assassination, has recently included in the CWC "Annex on Chemicals" some organophosphorus compounds that are regarded as acting in a similar fashion to the classical G- and V-series of nerve agents, inhibiting the pivotal enzyme acetylcholinesterase. Therefore, knowledge of the activity of the pyridinium oximes, the sole class of clinically available acetylcholinesterase reactivators to date, is plainly justified. In this paper, continuing our research efforts in medicinal chemistry on this class of toxic chemicals, we synthesized an A-230 nerve agent surrogate and applied a modified Ellman's assay to evaluate its ability to inhibit our enzymatic model, acetylcholinesterase from Electrophorus eel, and if the clinically available antidotes are able to rescue the enzyme activity for the purpose of relating the findings to the previously disclosed in silico data for the authentic nerve agent and other studies with similar A-series surrogates. Our experimental data indicates that pralidoxime is the most efficient compound for reactivating acetylcholinesterase inhibited by A-230 surrogate, which is the opposite of the in silico data previously disclosed.

An In Silico Study Based on QSAR and Molecular Docking and Molecular Dynamics Simulation for the Discovery of Novel Potent Inhibitor against AChE.

Acetylcholinesterase (AChE) is one of the main drug targets for treating Alzheimer's disease. This current study relies on multiple molecular modeling approaches to develop new potent inhibitors of AChE. We explored a 2D QSAR study using the statistical method of multiple linear regression based on a set of substituted 5-phenyl-1,3,4-oxadiazole and N-benzylpiperidine analogs, which were recently synthesized and proved their inhibitory activities against acetylcholinesterase (AChE). The molecular descriptors, polar surface area, dipole moment, and molecular weight are the key structural properties governing AChE inhibition activity. The MLR model was selected based on its statistical parameters: R2 = 0.701, R2test = 0.76, Q2CV = 0.638, and RMSE = 0.336, demonstrating its predictive reliability. Randomization tests, VIF tests, and applicability domain tests were adopted to verify the model's robustness. As a result, 11 new molecules were designed with higher anti-Alzheimer's activities than the model molecule. We demonstrated their improved pharmacokinetic properties through an in silico ADMET study. A molecular docking study was conducted to explore their AChE inhibition mechanisms and binding affinities in the active site. The binding scores of compounds M1, M2, and M6 were (-12.6 kcal/mol), (-13 kcal/mol), and (-12.4 kcal/mol), respectively, which are higher than the standard inhibitor Donepezil with a binding score of (-10.8 kcal/mol). Molecular dynamics simulations over 100 ns were used to validate the molecular docking results, indicating that compounds M1 and M2 remain stable in the active site, confirming their potential as promising anti-AChE inhibitors.

Exploring the therapeutic potential of steroidal alkaloids in managing Alzheimer's disease.

Steroidal alkaloids are secondary metabolites that are often found in plants, fungi and sponges. These compounds are considered as a source of bioactive compounds for the treatment of chronic diseases, such as neurological disorder like Alzheimer's disease (AD). Some examples of alkaloid derivatives currently used to treat AD symptoms include galantamine, huperzine A, and other alkaloids. AD is a multifactorial disease caused by multiple factors such as inflammation, oxidative stress, and protein aggregation. Based on the various important neuroprotective activities and different pharmacological effects of steroidal alkaloids with polypharmacological modulatory effects, they can lead to the development of new drugs for the treatment of AD. There are limited studies on the involvement of steroidal alkaloids in AD. Therefore, the mechanisms and neuroprotective abilities of these compounds are still poorly understood. The purpose of this review article is to provide an overview of the mechanism, toxicity and neuroprotective benefits of steroidal alkaloids and to discuss future possibilities to improve the application of steroidal alkaloids as anti-AD agents. The therapeutic value and limitations of the steroidal alkaloid are investigated to provide new perspectives for future clinical development studies.

Chiral pyrrolidines as multipotent agents in Alzheimer and neurodegenerative diseases.

In pharmaceutical science and drug design the versatility of the pyrrolidine scaffold relating to spatial arrangement, synthetic accessibility and pharmacological profile is a largely explored and most likely interesting one. Nonetheless, few evidences suggest the pivotal role of pyrrolidine as scaffold for multipotent agents in neurodegenerative diseases. We then challenged the enrolling in the field of Alzheimer disease of so far not ravelled targets of this chemical cliché with a structure based and computer-aided design strategy focusing on multi-target action, versatile synthesis as well as pharmacological safeness. To achieve these hits, ten enantiomeric pairs of compounds were obtained and tested, and the biological data will be here presented and discussed. Among the novel compounds, coumarin and sesamol scaffolds containing analogues resulted promising perspectives.

Oral administration of encapsulated catechin in chitosan-alginate nanoparticles improves cognitive function and neurodegeneration in an aluminum chloride-induced rat model of Alzheimer's disease.

The present study aimed to investigate the effect of catechin-loaded Chitosan-Alginate nanoparticles (NPs) on cognitive function in an aluminum chloride (AlCl3)-induced rat model of Alzheimer's disease (AD). The Catechin-loaded Chitosan-Alginate nanocarriers were synthesized through ionotropic gelation (IG) method. Physio-chemical characterization was conducted with the Zetasizer Nano system, the scanning electron microscope, and the Fourier transform infrared spectroscopy. The experiments were performed over 21 days on six groups of male Wistar rats. The control group, AlCl3 treated group, Catechin group, nanocarrier group, treatment group 1 (AlCl3 + Catechin), and treatment group 2 (AlCl3 + nanocarrier). A behavioral study was done by the Morris water maze (MWM) test. In addition, the level of oxidative indices and acetylcholine esterase (AChE) activity was determined by standard procedures at the end of the study. AlCl3 induced a significant increase in AChE activity, along with a significant decrease in the level of Catalase (CAT) and total antioxidant capacity (TAC) in the hippocampus. Moreover, the significant effect of AlCl3 was observed on the behavioral parameters of the MWM test. Both forms of Catechin markedly improved AChE activity, oxidative biomarkers, spatial memory, and learning. The present study indicated that the administration of Catechin-loaded Chitosan-Alginate NPs is a beneficial therapeutic option against behavioral and chemical alteration of AD in male Wistar rats.

Reactivation by novel pyridinium oximes of rat serum and skeletal muscle acetylcholinesterase inhibited by organophosphates.

The treatment of organophosphate (OP) anticholinesterases currently lacks an effective oxime reactivator of OP-inhibited acetylcholinesterase (AChE) which can penetrate the blood-brain barrier (BBB). Our laboratories have synthesized novel substituted phenoxyalkyl pyridinium oximes and tested them for their ability to promote survival of rats challenged with lethal doses of nerve agent surrogates. These previous studies demonstrated the ability of some of these oximes to promote 24-h survival to rats challenged with a lethal level of highly relevant surrogates for sarin and VX. The reactivation of OP-inhibited AChE in peripheral tissues was likely to be a major contributor to their efficacy in survival of lethal OP challenges. In the present study, twenty of these novel oximes were screened in vitro for reactivation ability for AChE in rat skeletal muscle and serum using two nerve agent surrogates: phthalimidyl isopropyl methylphosphonate (PIMP, a sarin surrogate) and 4-nitrophenyl ethyl methylphosphonate (NEMP, a VX surrogate). The oximes demonstrated a range of 23%-102% reactivation of AChE in vitro across both tissue types. Some of the novel oximes tested in the present study demonstrated the ability to more effectively reactivate AChE in serum than the currently approved oxime, 2-PAM. Therefore, some of these novel oximes have the potential to reverse AChE inhibition in peripheral target tissues and contribute to survival efficacy.

Quaternity method for integrated screening, separation, extraction optimization, and bioactivity evaluation of acetylcholinesterase inhibitors from Sophora flavescens Aiton.

INTRODUCTION: Sophora flavescens Aiton (Fabaceae), a ubiquitous plant species in Asia, contains a wide range of pharmacologically active compounds, such as flavonoids, with potential anti-Alzheimer's disease (anti-AD) effects. OBJECTIVES: The objective of the study is to develop a quaternity method for the screening, isolation, extraction optimization, and activity evaluation of acetylcholinesterase (AChE)-inhibiting compounds from S. flavescens to realize high-throughput screening of active substances in traditional Chinese medicine and to provide experimental data for the development of anti-AD drugs. METHODS: With AChE as the target molecule, affinity ultrafiltration and liquid chromatography-mass spectrometry were applied to screen for potential inhibitors of the enzyme in S. flavescens. Orthogonal array experiments combined with the multi-objective Non-Dominated Sorting Genetic Algorithm III was used for the first time to optimize the process for extracting the active substances. Enzyme inhibition kinetics and molecular docking studies were performed to verify the potential anti-AD effects of the active compounds. RESULTS: Five AChE-inhibiting compounds were identified: kushenol I, kurarinone, sophoraflavanone G, isokurarinone, and kushenol E. These were successfully separated at purities of 72.88%, 98.55%, 96.86%, 96.74%, and 95.84%, respectively, using the n-hexane/ethyl acetate/methanol/water (4.0/5.0/4.0/5.0, v/v/v/v), n-hexane/ethyl acetate/methanol/water (5.0/5.0/6.0/4.0, v/v/v/v), and n-hexane/ethyl acetate/methanol/water (4.9/5.1/5.7/4.3, v/v/v/v) mobile phase systems. Enzyme inhibition kinetics revealed that kushenol E had the best inhibitory effect. CONCLUSION: This study elucidates the mechanism of action of five active AChE inhibitors in S. flavescens and provides a theoretical basis for the screening and development of anti-AD and other therapeutic drugs.

Acetylcholinesterase and butyrylcholinesterase co-immobilized on a copper containing Prussian blue modified electrode for the broad screening of insecticides.

We have developed a bienzymatic biosensor that contains acetylcholinesterase together with butyrylcholinesterase co-immobilized on the same electrode modified with a stabilized copper containing Prussian blue electrodeposited on electrodes coated with 4-aminothiophenol monolayer using diazonium chemistry and copper nanoparticles for improved sensitivity. There are organophosphorus and carbamate neurotoxic insecticides that inhibit only one of the two enzymes, e.g., pirimicarb inhibits butyrylcholinesterase at much lower concentrations than acetylcholinesterase while methomyl inhibits only acetylcholinesterase. Our system is simple and in a single measurement provides a sensitive signal for insecticides' presence based on the inhibition of the enzyme with the highest affinity for each toxic compound. The limits of detection are 50 ng/mL pirimicarb for the bienzymatic biosensor in comparison with 400 ng/mL pirimicarb for the acetylcholinesterase biosensor and 6 ng/mL methomyl for the bienzymatic biosensor, while inhibition is obtained for the butyrylcholinesterase biosensor at 700 ng/mL.