Lead optimization based design, synthesis, and pharmacological evaluation of quinazoline derivatives as multi-targeting agents for Alzheimer's disease treatment.

The complexity and multifaceted nature of Alzheimer's disease (AD) have driven us to further explore quinazoline scaffolds as multi-targeting agents for AD treatment. The lead optimization strategy was utilized in designing of new series of derivatives (AK-1 to AK-14) followed by synthesis, characterization, and pharmacological evaluation against human cholinesterase's (hChE) and ß-secretase (hBACE-1) enzymes. Amongst them, compounds AK-1, AK-2, and AK-3 showed good and significant inhibitory activity against both hAChE and hBACE-1 enzymes with favorable permeation across the blood-brain barrier. The most active compound AK-2 revealed significant propidium iodide (PI) displacement from the AChE-PAS region and was non-neurotoxic against SH-SY5Y cell lines. The lead molecule (AK-2) also showed Aß aggregation inhibition in a self- and AChE-induced Aß aggregation, Thioflavin-T assay. Further, compound AK-2 significantly ameliorated Aß-induced cognitive deficits in the Aß-induced Morris water maze rat model and demonstrated a significant rescue in eye phenotype in the Aꞵ-phenotypic drosophila model of AD. Ex-vivo immunohistochemistry (IHC) analysis on hippocampal rat brains showed reduced Aß and BACE-1 protein levels. Compound AK-2 suggested good oral absorption via pharmacokinetic studies and displayed a good and stable ligand-protein interaction in in-silico molecular modeling analysis. Thus, the compound AK-2 can be regarded as a lead molecule and should be investigated further for the treatment of AD.

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.

Structural Insights into the Marine Alkaloid Discorhabdin G as a Scaffold towards New Acetylcholinesterase Inhibitors.

In this study, Antarctic Latrunculia sponge-derived discorhabdin G was considered a hit for developing potential lead compounds acting as cholinesterase inhibitors. The hypothesis on the pharmacophore moiety suggested through molecular docking allowed us to simplify the structure of the metabolite. ADME prediction and drug-likeness consideration provided valuable support in selecting 5-methyl-2H-benzo[h]imidazo[1,5,4-de]quinoxalin-7(3H)-one as a candidate molecule. It was synthesized in a four-step sequence starting from 2,3-dichloronaphthalene-1,4-dione and evaluated as an inhibitor of electric eel acetylcholinesterase (eeAChE), human recombinant AChE (hAChE), and horse serum butyrylcholinesterase (BChE), together with other analogs obtained by the same synthesis. The candidate molecule showed a slightly lower inhibitory potential against eeAChE but better inhibitory activity against hAChE than discorhabdin G, with a higher selectivity for AChEs than for BChE. It acted as a reversible competitive inhibitor, as previously observed for the natural alkaloid. The findings from the in vitro assay were relatively consistent with the data available from the AutoDock Vina and Protein-Ligand ANTSystem (PLANTS) calculations.

Design, synthesis, and biological evaluation of some 2-(3-oxo-5,6-diphenyl-1,2,4-triazin-2(3H)-yl)-N-phenylacetamide hybrids as MTDLs for Alzheimer's disease therapy.

Inspite of established symptomatic relief drug targets, a multi targeting approach is highly in demand to cure Alzheimer's disease (AD). Simultaneous inhibition of cholinesterase (ChE), ß secretase-1 (BACE-1) and Dyrk1A could be promising in complete cure of AD. A series of 18 diaryl triazine based molecular hybrids were successfully designed, synthesized, and tested for their hChE, hBACE-1, Dyrk1A and Aß aggregation inhibitory potentials. Compounds S-11 and S-12 were the representative molecules amongst the series with multi-targeted inhibitory effects. Compound S-12 showed hAChE inhibition (IC50 value = 0.486 ± 0.047 µM), BACE-1 inhibition (IC50 value = 0.542 ± 0.099 µM) along with good anti-Aß aggregation effects in thioflavin-T assay. Only compound S-02 of the series has shown Dyrk1A inhibition (IC50 value = 2.000 ± 0.360 µM). Compound S-12 has also demonstrated no neurotoxic liabilities against SH-SY5Y as compared to donepezil. The in vivo behavioral studies of the compound S-12 in the scopolamine- and Aß-induced animal models also demonstrated attanuation of learning and memory functions in rats models having AD-like characteristics. The ex vivo studies, on the rat hippocampal brain demonstrated reduction in certain biochemical markers of the AD brain with a significant increase in ACh level. The Western blot and Immunohistochemistry further revealed lower tau, APP and BACE-1 molecular levels. The drosophilla AD model also revealed improved eyephenotype after treatment with compound S-12. The molecular docking studies of the compounds suggested that compound S-12 was interacting with the ChE-PAS & CAS residues and catalytic dyad residues of the BACE-1 enzymes. The 100 ns molecular dynamics simulation studies of the ligand-protein complexed with hAChE and hBACE-1 also suggested stable ligand-protein confirmation throughout the simulation run.

New Charged Cholinesterase Inhibitors: Design, Synthesis, and Characterization.

Triazoles and triazolium salts are very common subunits in the structures of various drugs. Medicaments with a characteristic 1,2,3-triazole core are also being developed to treat neurodegenerative disorders associated with cholinesterase enzyme activity. Several naphtho- and thienobenzo-triazoles from our previous research emerged as being particularly promising in that sense. For this reason, in this research, new naphtho- and thienobenzo-triazoles 23-34, as well as 1,2,3-triazolium salts 44-51, were synthesized and tested. Triazolium salts 44-46 showed excellent activity while salts 47 and 49 showed very good inhibition toward both butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) enzymes. In contrast, neutral photoproducts were shown to be selective towards BChE but with very good inhibition potential as molecules 24-27. The representative of newly prepared compounds, 45 and 50, were stable in aqueous solution and revealed intriguing fluorimetric properties, characterized by a strong Stokes shift of >160 nm. Despite their condensed polycyclic structure shaped similarly to well-known DNA-intercalator ethidium bromide, the studied compounds did not show any interaction with ds-DNA, likely due to the unfavorable steric hindrance of substituents. However, the studied dyes bind proteins, particularly showing very diverse inhibition properties toward AChE and BChE. In contrast, neutral photoproducts were shown to be selective towards a certain enzyme but with moderate inhibition potential. The molecular docking of the best-performing candidates to cholinesterases' active sites identified cation-π interactions as the most responsible for the stability of the enzyme-ligand complexes. As genotoxicity studies are crucial when developing new active substances and finished drug forms, in silico studies for all the compounds synthesized have been performed.

Mechanochemical Approach to Obtaining a Multicomponent Fisetin Delivery System Improving Its Solubility and Biological Activity.

In this study, binary amorphous solid dispersions (ASDs, fisetin-Eudragit®) and ternary amorphous solid inclusions (ASIs, fisetin-Eudragit®-HP-ß-cyclodextrin) of fisetin (FIS) were prepared by the mechanochemical method without solvent. The amorphous nature of FIS in ASDs and ASIs was confirmed using XRPD (X-ray powder diffraction). DSC (Differential scanning calorimetry) confirmed full miscibility of multicomponent delivery systems. FT-IR (Fourier-transform infrared analysis) confirmed interactions that stabilize FIS's amorphous state and identified the functional groups involved. The study culminated in evaluating the impact of amorphization on water solubility and conducting in vitro antioxidant assays: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-ABTS, 2,2-diphenyl-1-picrylhydrazyl-DPPH, Cupric Reducing Antioxidant Capacity-CUPRAC, and Ferric Reducing Antioxidant Power-FRAP and in vitro neuroprotective assays: inhibition of acetylcholinesterase-AChE and butyrylcholinesterase-BChE. In addition, molecular docking allowed for the determination of possible bonds and interactions between FIS and the mentioned above enzymes. The best preparation turned out to be ASI_30_EPO (ASD fisetin-Eudragit® containing 30% FIS in combination with HP-ß-cyclodextrin), which showed an improvement in apparent solubility (126.5 ± 0.1 µg∙mL-1) and antioxidant properties (ABTS: IC50 = 10.25 µg∙mL-1, DPPH: IC50 = 27.69 µg∙mL-1, CUPRAC: IC0.5 = 9.52 µg∙mL-1, FRAP: IC0.5 = 8.56 µg∙mL-1) and neuroprotective properties (inhibition AChE: 39.91%, and BChE: 42.62%).

Diosmetin derivatives as multifunctional anti-AD ligands: Design, synthesis, and biological evaluation.

With the increasing aging population, rational design of drugs for Alzheimer's disease (AD) treatment has become an important research area. Based on the multifunctional design strategy, four diosmetin derivatives (1-4) were designed, synthesized, and characterized by 1H NMR, 13C NMR, and MS. Docking study was firstly applied to substantiate the design strategies and then the biological activities including cholinesterase inhibition, metal chelation, antioxidation and ß-amyloid (Aß) aggregation inhibition in vitro were evaluated. The results showed that 1-4 had good acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition, metal chelation (selective chelation of Cu2+ ions), antioxidation, self-induced, Cu2+-induced, and AChE-induced Aß aggregation inhibition activities, and suitable blood-brain barrier (BBB) permeability. Especially, compound 3 had the strongest inhibitory effect on AChE (10-8 M magnitude) and BuChE (10-7 M magnitude) and showed the best inhibition on AChE-induced Aß aggregation with 66.14% inhibition ratio. Furthermore, compound 3 could also reduce intracellular reactive oxygen species (ROS) levels in Caenorhabditis elegans and had lower cytotoxicity. In summary, 3 might be considered as a potential multifunctional anti-AD ligand.

Onset of Alzheimer disease in apolipoprotein ɛ4 carriers is earlier in butyrylcholinesterase K variant carriers.

BACKGROUND: The authors sought to examine the impact of the K-variant of butyrylcholinesterase (BCHE-K) carrier status on age-at-diagnosis of Alzheimer disease (AD) in APOE4 carriers. METHODS: Patients aged 50-74 years with cerebrospinal fluid (CSF) biomarker-confirmed AD, were recruited to clinical trial (NCT03186989 since June 14, 2017). Baseline demographics, disease characteristics, and biomarkers were evaluated in 45 patients according to BCHE-K and APOE4 allelic status in this post-hoc study. RESULTS: In APOE4 carriers (N = 33), the mean age-at-diagnosis of AD in BCHE-K carriers (n = 11) was 6.4 years earlier than in BCHE-K noncarriers (n = 22, P < .001, ANOVA). In APOE4 noncarriers (N = 12) there was no observed influence of BCHE-K. APOE4 carriers with BCHE-K also exhibited slightly higher amyloid and tau accumulations compared to BCHE-K noncarriers. A predominantly amyloid, limited tau, and limbic-amnestic phenotype was exemplified by APOE4 homozygotes with BCHE-K. In the overall population, multiple regression analyses demonstrated an association of amyloid accumulation with APOE4 carrier status (P < .029), larger total brain ventricle volume (P < .021), less synaptic injury (Ng, P < .001), and less tau pathophysiology (p-tau181, P < .005). In contrast, tau pathophysiology was associated with more neuroaxonal damage (NfL, P = .002), more synaptic injury (Ng, P < .001), and higher levels of glial activation (YKL-40, P = .01). CONCLUSION: These findings have implications for the genetic architecture of prognosis in early AD, not the genetics of susceptibility to AD. In patients with early AD aged less than 75 years, the mean age-at-diagnosis of AD in APOE4 carriers was reduced by over 6 years in BCHE-K carriers versus noncarriers. The functional status of glia may explain many of the effects of APOE4 and BCHE-K on the early AD phenotype. TRIAL REGISTRATION: NCT03186989 since June 14, 2017.

Novel 6-alkyl-bridged 4-arylalkylpiperazin-1-yl derivatives of azepino[4,3-b]indol-1(2H)-one as potent BChE-selective inhibitors showing protective effects against neurodegenerative insults.

Due to the putative role of butyrylcholinesterase (BChE) in regulation of acetylcholine levels and functions in the late stages of the Alzheimer's disease (AD), the potential of selective inhibitors (BChEIs) has been envisaged as an alternative to administration of acetylcholinesterase inhibitors (AChEIs). Starting from our recent findings, herein the synthesis and in vitro evaluation of cholinesterase (ChE) inhibition of a novel series of some twenty 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one derivatives, bearing at the indole nitrogen diverse alkyl-bridged 4-arylalkylpiperazin-1-yl chains, are reported. The length of the spacers, as well as the type of arylalkyl group affected the enzyme inhibition potency and BChE/AChE selectivity. Two compounds, namely 14c (IC50 = 163 nM) and 14d (IC50 = 65 nM), bearing at the nitrogen atom in position 6 a n-pentyl- or n-heptyl-bridged 4-phenethylpiperazin-1-yl chains, respectively, proved to be highly potent mixed-type inhibitors of both equine and human BChE isoforms, showing more than two order magnitude of selectivity over AChE. The study of binding kinetics through surface plasmon resonance (SPR) highlighted differences in their BChE residence times (8 and 47 s for 14c and 14d, respectively). Moreover, 14c and 14d proved to hit other mechanisms known to trigger neurodegeneration underlying AD and other CNS disorders. Unlike 14c, compound 14d proved also capable of inhibiting by more than 60% the in vitro self-induced aggregation of neurotoxic amyloid-ß (Aß) peptide at 100 µM concentration. On the other hand, 14c was slightly better than 14d in counteracting, at 1 and 10 µM concentration, glutamate excitotoxicity, due to over-excitation of NMDA receptors, and hydrogen peroxide-induced oxidative stress assessed in neuroblastoma cell line SH-SY5Y. This paper is dedicated to Prof. Marcello Ferappi, former dean of the Faculty of Pharmacy of the University of Bari, in the occasion of his 90th birthday.

Synthesis and investigation of the cholinesterase inhibitory and antioxidant capacities of some novel N'-(quinolin-4-ylmethylene)propanehydrazides against Alzheimer's disease.

One of the worst long-term health issues of the past few decades is Alzheimer's disease (AD). Unfortunately, there are currently insufficient choices for treating and caring for AD, which makes it a popular subject for drug development research. Studies on the development of drugs for AD have primarily concentrated on the use of multitarget directed ligands. Following this strategy, we designed new ChE inhibitors with additional antioxidant and metal chelator effects. In this research, eight novel N'-(quinolin-4-ylmethylene)propanehydrazide derivatives were synthesized and characterized. We then evaluated the inhibition potency of all the final compounds for cholinesterase enzymes. Among them, 4e (IC50 acetylcholinesterase [AChE] = 0.69 µM and butyrylcholinesterase [BChE]= 26.00 µM) and 4h (IC50's AChE= 7.04 µM and BChE= 16.06 µM) were found to be the most potent AChE and BChE inhibitors, respectively.

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.

Phytochemical Profiling and Biological Activities of Pericarps and Seeds Reveal the Controversy on "Enucleation" or "Nucleus-Retaining" of Cornus officinalis Fruits.

The fruits of Cornus officinalis are used not only as a popular health food to tonify the liver and kidney, but also as staple materials to treat dementia and other age-related diseases. The pharmacological function of C. officinalis fruits with or without seeds is controversial for treating some symptoms in a few herbal prescriptions. However, the related metabolite and pharmacological information between its pericarps and seeds are largely deficient. Here, comparative metabolomics analysis between C. officinalis pericarps and seeds were conducted using an ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, and therapeutic effects were also evaluated using several in vitro bioactivity arrays (antioxidant activity, α-glucosidase and cholinesterase inhibitory activities, and cell inhibitory properties). A total of 499 secondary metabolites were identified. Thereinto, 77 metabolites were determined as key differential metabolites between C. officinalis pericarps and seeds, and the flavonoid biosynthesis pathway was identified as the most significantly different pathway. Further, 47 metabolites were determined as potential bioactive constituents. In summary, C. officinalis seeds, which demonstrated higher contents in total phenolics, stronger in vitro antioxidant activities, better α-glucosidase and butyrylcholinesterase inhibitory activities, and stronger anticancer activities, exhibited considerable potential for food and health fields. This work provided insight into the metabolites and bioactivities of C. officinalis pericarps and seeds, contributing to their precise development and utilization.

Quantitative Neuromuscular Monitoring Permits Early Diagnosis of Abnormal Butyrylcholinestrase: Two Case Studies Demonstrating Prevention of Awareness from Premature Awakening.

Administration of succinylcholine to patients with a variant in the butyrylcholinesterase (BChE) gene increases the risk of anesthesia emergence prior to recovery from neuromuscular blockade (NMB). Application of quantitative neuromuscular monitoring (NMM) can identify residual NMB. We present two patients with abnormal BChE gene variants. In the first case, quantitative monitoring was applied too late to prevent awareness, but allowed diagnosis and prevented admission to the intensive care unit. In the second case, monitoring was applied prior to NMB, which enabled early diagnosis and prevented premature awakening from anesthesia. These cases illustrate the importance of quantitative NMM, even in short cases and with short-acting depolarizing agents such as succinylcholine. The clinical implications of this report include a more consistent use of NMM to identify and manage patients with undiagnosed abnormal BChE and to prevent premature anesthesia emergence.

Coumarin-azasugar-benzyl conjugates as non-neurotoxic dual inhibitors of butyrylcholinesterase and cancer cell growth.

We have applied the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction to prepare a library of ten coumarin-azasugar-benzyl conjugates and two phthalimide-azasugar-benzyl conjugates with potential anti-Alzheimer and anti-cancer properties. The compounds were evaluated as cholinesterase inhibitors, demonstrating a general preference, of up to 676-fold, for the inhibition of butyrylcholinesterase (BuChE) over acetylcholinesterase (AChE). Nine of the compounds behaved as stronger BuChE inhibitors than galantamine, one of the few drugs in clinical use against Alzheimer's disease. The most potent BuChE inhibitor (IC50 = 74 nM) was found to exhibit dual activities, as it also showed high activity (GI50 = 5.6 ± 1.1 µM) for inhibiting the growth of WiDr (colon cancer cells). In vitro studies on this dual-activity compound on Cerebellar Granule Neurons (CGNs) demonstrated that it displays no neurotoxicity.

4-Amidophenol Quinone Methide Precursors: Effective and Broad-Scope Nonoxime Reactivators of Organophosphorus-Inhibited Cholinesterases and Resurrectors of Organophosphorus-Aged Acetylcholinesterase.

Acetylcholinesterase (AChE) inhibition by organophosphorus (OP) compounds poses a serious health risk to humans. While many therapeutics have been tested for treatment after OP exposure, there is still a need for efficient reactivation against all kinds of OP compounds, and current oxime therapeutics have poor blood-brain barrier penetration into the central nervous system, while offering no recovery in activity from the OP-aged forms of AChE. Herein, we report a novel library of 4-amidophenol quinone methide precursors (QMP) that provide effective reactivation against multiple OP-inhibited forms of AChE in addition to resurrecting the aged form of AChE after exposure to a pesticide or some phosphoramidates. Furthermore, these QMP compounds also reactivate OP-inhibited butyrylcholinesterase (BChE) which is an in vivo, endogenous scavenger of OP compounds. The in vitro efficacies of these QMP compounds were tested for reactivation and resurrection of soluble forms of human AChE and BChE and for reactivation of cholinesterases within human blood as well as blood and brain samples from a humanized mouse model. We identify compound 10c as a lead candidate due to its broad-scope efficacy against multiple OP compounds as well as both cholinesterases. With methylphosphonates, compound 10c (250 µM, 1 h) shows >60% recovered activity from OEt-inhibited AChE in human blood as well as mouse blood and brain, thus highlighting its potential for future in vivo analysis. For 10c, the effective concentration (EC50) is less than 25 µM for reactivation of three different methylphosphonate-inhibited forms of AChE, with a maximum reactivation yield above 80%. Similarly, for OP-inhibited BChE, 10c has EC50 values that are less than 150 µM for two different methylphosphonate compounds. Furthermore, an in vitro kinetic analysis show that 10c has a 2.2- and 92.1-fold superior reactivation efficiency against OEt-inhibited and OiBu-inhibited AChE, respectively, when compared to an oxime control. In addition to 10c being a potent reactivator of AChE and BChE, we also show that 10c is capable of resurrecting (ethyl paraoxon)-aged AChE, which is another current limitation of oximes.

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.

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.

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.

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.

Butyrylcholinesterase-Activated Near-Infrared Fluorogenic Probe for In Vivo Theranostics of Alzheimer's Disease.

Butyrylcholinesterase (BChE) is a promising biomarker and effective therapeutic target for Alzheimer's disease (AD). Herein, we designed a BChE-activated near-infrared (NIR) probe, DTNP, which could be activated by BChE and inhibit its enzymatic activity. DTNP is composed of a cyclopropane moiety as the recognition unit, a NIR fluorophore hemicyanine as the NIR reporter, and a BChE inhibitor as the therapeutic unit. DTNP specifically binds BChE with high sensitivity and exhibits strong "turn-on" NIR fluorescence as well as nerve cell protection. In vivo imaging shows DTNP has favorable blood-brain barrier permeability and long-term tracking ability with preliminary competence in AD diagnosis. DTNP can significantly inhibit BChE activity, promote the release of ACh, and rescue learning deficits and cognitive impairment. Therefore, DTNP, the first reported and partially validated theranostic probe for the detection of BChE in AD, may provide a foundation and inspiration for imaging and therapy in AD.