Effects of selegiline on neuronal autophagy involving α-synuclein secretion.

Cell-to-cell transmission of α-synuclein (α-syn) pathology underlies the spread of neurodegeneration in Parkinson's disease. α-Syn secretion is an important factor in the transmission of α-syn pathology. However, it is unclear how α-syn secretion is therapeutically modulated. Here, we investigated effects of monoamine oxidase (MAO)-B inhibitor selegiline on α-syn secretion. Treatment with selegiline promoted α-syn secretion in mouse primary cortical neuron cultures, and this increase was kept under glial cell-eliminated condition by Ara-C. Selegiline-induced α-syn secretion was blocked by cytosolic Ca2+ chelator BAPTA-AM in primary neurons. Selegiline-induced α-syn secretion was retained in MAOA siRNA knockdown, whereas it was abrogated by ATG5 knockdown in SH-SY5Y cells. Selegiline increased LC3-II generation with a reduction in intracellular p62/SQSTM1 levels in primary neurons. The increase in LC3-II generation was blocked by co-treatment with BAPTA-AM in primary neurons. Additionally, fractionation experiments showed that selegiline-induced α-syn secretion occurred in non-extracellular vesicle fractions of primary neurons and SH-SY5Y cells. Collectively, these findings show that selegiline promotes neuronal autophagy involving secretion of non-exosomal α-syn via a change of cytosolic Ca2+ levels.

Identification and Evaluation of Olive Phenolics in the Context of Amine Oxidase Enzyme Inhibition and Depression: In Silico Modelling and In Vitro Validation.

The Mediterranean diet well known for its beneficial health effects, including mood enhancement, is characterised by the relatively high consumption of extra virgin olive oil (EVOO), which is rich in bioactive phenolic compounds. Over 200 phenolic compounds have been associated with Olea europaea, and of these, only a relatively small fraction have been characterised. Utilising the OliveNetTM library, phenolic compounds were investigated as potential inhibitors of the epigenetic modifier lysine-specific demethylase 1 (LSD1). Furthermore, the compounds were screened for inhibition of the structurally similar monoamine oxidases (MAOs) which are directly implicated in the pathophysiology of depression. Molecular docking highlighted that olive phenolics interact with the active site of LSD1 and MAOs. Protein-peptide docking was also performed to evaluate the interaction of the histone H3 peptide with LSD1, in the presence of ligands bound to the substrate-binding cavity. To validate the in silico studies, the inhibitory activity of phenolic compounds was compared to the clinically approved inhibitor tranylcypromine. Our findings indicate that olive phenolics inhibit LSD1 and the MAOs in vitro. Using a cell culture model system with corticosteroid-stimulated human BJ fibroblast cells, the results demonstrate the attenuation of dexamethasone- and hydrocortisone-induced MAO activity by phenolic compounds. The findings were further corroborated using human embryonic stem cell (hESC)-derived neurons stimulated with all-trans retinoic acid. Overall, the results indicate the inhibition of flavin adenine dinucleotide (FAD)-dependent amine oxidases by olive phenolics. More generally, our findings further support at least a partial mechanism accounting for the antidepressant effects associated with EVOO and the Mediterranean diet.

Are Terminal Alkynes Necessary for MAO-A/MAO-B Inhibition? A New Scaffold Is Revealed.

A versatile family of quaternary propargylamines was synthesized employing the KA2 multicomponent reaction, through the single-step coupling of a number of amines, ketones, and terminal alkynes. Sustainable synthetic procedures using transition metal catalysts were employed in all cases. The inhibitory activity of these molecules was evaluated against human monoaminoxidase (hMAO)-A and hMAO-B enzymes and was found to be significant. The IC50 values for hMAO-B range from 152.1 to 164.7 nM while the IC50 values for hMAO-A range from 765.6 to 861.6 nM. Furthermore, these compounds comply with Lipinski's rule of five and exhibit no predicted toxicity. To understand their binding properties with the two target enzymes, key interactions were studied using molecular docking, all-atom molecular dynamics (MD) simulations, and MM/GBSA binding free energy calculations. Overall, herein, the reported family of propargylamines exhibits promise as potential treatments for neurodegenerative disorders, such as Parkinson's disease. Interestingly, this is the first time a propargylamine scaffold bearing an internal alkyne has been reported to show activity against monoaminoxidases.

Antidepressant-like Effects of Chinese Quince (Chaenomeles sinensis) Fruit Based on In Vivo and Molecular Docking Studies.

In this study, we examined the potential antidepressant-like effects of Chinese quince fruit extract (Chaenomeles sinensis fruit extract, CSFE) in an in vivo model induced by repeated injection of corticosterone (CORT)-induced depression. HPLC analysis determined that chlorogenic acid (CGA), neo-chlorogenic acid (neo-CGA), and rutin (RT) compounds were major constituents in CSFE. Male ICR mice (5 weeks old) were orally administered various doses (30, 100, and 300 mg/kg) of CSFE and selegiline (10 mg/kg), a monoamine oxidase B (MAO-B) inhibitor, as a positive control following daily intraperitoneal injections of CORT (40 mg/kg) for 21 days. In our results, mice treated with CSFE exhibited significant improvements in depressive-like behaviors induced by CORT. This was evidenced by reduced immobility times in the tail suspension test and forced swim test, as well as increased step-through latency times in the passive avoidance test. Indeed, mice treated with CSFE also exhibited a significant decrease in anxiety-like behaviors as measured by the elevated plus maze test. Moreover, molecular docking analysis indicated that CGA and neo-CGA from CSFE had stronger binding to the active site of MAO-B. Our results indicate that CSFE has potential antidepressant effects in a mouse model of repeated injections of CORT-induced depression.

Discovery of novel benzimidazole derivatives as selective and reversible monoamine oxidase B inhibitors for Parkinson's disease treatment.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. The development of novel scaffolds for human monoamine oxidase B (hMAO-B) inhibitors with reversible properties represents an important strategy to improve the efficacy and safety for PD treatment. In the current work, we have devised and assessed two innovative derivative series serving as hMAO-B inhibitors. These series have utilized benzimidazole as a scaffold and strategically incorporated a primary amide group, which is recognized as a pivotal pharmacophore in subsequent activity screening and reversible mode of action. Among these compounds, 16d has emerged as the most potent hMAO-B inhibitor with an IC50 value of 67.3 nM, comparable to safinamide (IC50 = 42.6 nM) in vitro. Besides, 16d demonstrated good selectivity towards hMAO-B isoenzyme with a selectivity index over 387. Importantly, in line with the design purpose, 16d inhibited hMAO-B in a competitive and reversible manner (Ki = 82.50 nM). Moreover, 16d exhibited a good safety profile in both cellular and acute toxicity assays in mice. It also displayed ideal pharmacokinetic properties and blood-brain barrier permeability in vivo, essential prerequisites for central nervous system medicines. In the MPTP-induced PD mouse model, 16d significantly alleviated the motor impairment, especially muscle relaxation and motor coordination. Therefore, 16d, serving as a lead compound, holds instructive significance for subsequent investigations regarding its application in the treatment of PD.

Synthesis, Molecular Electron Density Theory Study, Molecular Docking, and Pharmacological Evaluation of New Coumarin-Sulfonamide-Nitroindazolyl-Triazole Hybrids as Monoamine Oxidase Inhibitors.

Inhibitors of monoamine oxidases (MAOs) are of interest for the treatment of neurodegenerative disorders and other human pathologies. In this frame, the present work describes different synthetic strategies to obtain MAO inhibitors via the coupling of the aminocoumarin core with arylsulfonyl chlorides followed by copper azide-alkyne cycloaddition, leading to coumarin-sulfonamide-nitroindazolyl-triazole hybrids. The nitration position on the coumarin moiety was confirmed through nuclear magnetic resonance spectroscopy and molecular electron density theory in order to elucidate the molecular mechanism and selectivity of the electrophilic aromatic substitution reaction. The coumarin derivatives were evaluated for their inhibitory potency against monoamine oxidases and cholinesterases. Molecular docking calculations provided a rational binding mode of the best compounds in the series with MAO A and B. The work identified hybrids 14a-c as novel MAO inhibitors, with a selective action against isoform B, of potential interest to combat neurological diseases.

Non-cytotoxic 3-Acetylcoumarin as an Attractive Target for Human Monoamine Oxidase (HMAO) Enzymes.

BACKGROUND/AIM: Coumarins are a broad class of naturally occurring oxygen-heterocyclic compounds found in plants with diverse biological properties, making them attractive for evaluation as novel therapeutic agents. We herein report the in vitro cytotoxic and monoamine oxidase (MAO) inhibitory activities of 3-acetylcoumarins (6a-e). MATERIALS AND METHODS: The cytotoxic activity was evaluated using crystal violet dye binding assay, and those compounds unable to induce cytotoxicity were further tested for the monoamine oxidase (MAO) activity using the MAO-GloTM kit. RESULTS: The 3-acetylcoumarins (6a-e) were non-cytotoxic (inactive) against MDA MB-231 (estrogen receptor-negative, ER-, highly invasive) and MCF-7 (estrogen receptor-positive, ER+, weakly invasive) breast cancer cell lines, but showed interesting MAOs inhibition activities. Among the synthesized compounds, 3-acetylcoumarin bearing dichloro (-diCl) (6d; IC50=0.31±0.04 µM) at Carbon-7, 8 positions showed higher inhibition, MAO B/A non-selectivity (selectivity index, SI=3.10), reversible inhibition against the hMAO-B enzyme, and neuroprotection against H2O2-treated human neuroblastoma (N2a) cells. CONCLUSION: Compound (6d) can be considered a promising scaffold for further investigation in developing hMAO-B inhibitors (MAOIs).

Design and discovery of anthranilamide derivatives as a potential treatment for neurodegenerative disorders via targeting cholinesterases and monoamine oxidases.

Neurodegenerative diseases with progressive cellular loss of the central nervous system and elusive disease etiology provide a continuous impetus to explore drug discovery programmes aiming at identifying robust and effective inhibitors of cholinesterase and monoamine oxidase enzymes. We herein present a concise library of anthranilamide derivatives involving a palladium-catalyzed Suzuki-Miyaura cross-coupling reaction to install the diverse structural diversity required for the desired biological action. Using Ellman's method, cholinesterase inhibitory activity was performed against AChE and BuChE enzymes. In vitro assay results demonstrated that anthranilamides are potent inhibitors with remarkable potency. Compound 6k emerged as the lead candidate and dual inhibitor of both enzymes with IC50 values of 0.12 ± 0.01 and 0.49 ± 0.02 µM against AChE and BuChE, respectively. Several other compounds were found as highly potent and selective inhibitors. Anthranilamide derivatives were also tested against monoamine oxidase (A and B) enzymes using fluorometric method. In vitro data revealed compound 6h as the most potent inhibitor against MAO-A, showing an IC50 value of 0.44 ± 0.02 µM, whereas, compound 6k emerged as the top inhibitor of MAO-B with an IC50 value of 0.06 ± 0.01 µM. All the lead inhibitors were analyzed for the identification of their mechanism of action using Michaelis-Menten kinetics experiments. Compound 6k and 6h depicted a competitive mode of action against AChE and MAO-A, whereas, a non-competitive and mixed-type of inhibition was observed against BuChE and MAO-B by compounds 6k. Molecular docking analysis revealed remarkable binding affinities of the potent inhibitors with specific residues inside the active site of receptors. Furthermore, molecular dynamics simulations were performed to explore the ability of potent compounds to form energetically stable complexes with the target protein. Finally, in silico ADME calculations also demonstrated that the potent compounds exhibit promising pharmacokinetic profile, satisfying the essential criteria for drug-likeness. Altogether, the findings reported in the current work clearly suggest that the identified anthranilamide derivatives have the potential to serve as effective drug candidates for future investigations.

Comprehensive safety evaluation of a novel multitargeting compound XYY-CP1106: A candidate for Alzheimer's disease.

Multitargeting has become a promising strategy for the development of anti-Alzheimer's disease (AD) drugs, considering the complexity of molecular mechanisms in AD pathology. In most pre-clinical studies, the effectiveness of these multi-targeted anti-AD drugs has been demonstrated but comprehensive safety assessments are lacking. Here, the safety evaluation of a novel multi-targeted candidate in AD (XYY-CP1106), characterized by its dual-property of iron chelation and monoamine oxidase B inhibition, was conducted by multifaceted analysis. Acute toxicity in mice was conducted to investigate the safety of oral administration and the maximum tolerated dose of the agent. In vitro Ames analysis, CHL chromosomal aberration analysis, and bone marrow micronucleus analysis were executed to evaluate the genotoxicity. A teratogenesis investigation in pregnant mice were meticulously performed to evaluate the teratogenesis of XYY-CP1106. Furthermore, a 90-day long-term toxicity analysis in rats was investigated to evaluate the cumulative toxicity after long-term administration. Strikingly, no toxic phenomena were found in all investigations, demonstrating relatively high safety profile of the candidate compound. The securing of safety heightened the translational significance of XYY-CP1106 as a novel multi-targeted anti-AD candidate, supporting the rationality of multitargeting strategy in the designs of smart anti-AD drugs.

Identification and optimization of nitrophenolic analogues as dopamine metabolic enzyme inhibitors for the treatment of Parkinson's disease.

Progressive loss of dopaminergic neurons leads to the depletion of the striatal neurotransmitter dopamine, which is the main cause of Parkinson's disease (PD) motor symptoms. Simultaneous inhibition of the two key dopamine metabolic enzymes, catechol-O-methyltransferase (COMT) and monoamine oxidase B (MAO-B), could potentially be a breakthrough in achieving clinical efficacy. Representative compound C12 exhibits good COMT inhibitory activity (IC50 = 0.37 µM), metal chelation ability, and BBB permeability. Furthermore, results from in vivo biological activity evaluations indicate that C12 can improve dopamine levels and ameliorate MPTP-induced PD symptoms in mice. Preliminary in vivo and in vitro study results highlight the potential of compound C12 in PD treatment.

A magnetic beads-based ligand fishing method for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from Hunteria zeylanica.

In this study, a novel magnetic bead-based ligand fishing method was developed for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from natural products. In order to improve the screening efficiency, two different magnetic beads, i.e. amine and carboxyl terminated magnetic beads, were comprehensively compared in terms of their ability to immobilize monoamine oxidase A (MAOA), biocatalytic activity and specific adsorption rates for affinity ligands. Carboxyl terminated magnetic beads performed better for MAOA immobilization and demonstrated superior performance in ligand fishing. The MAOA immobilized magnetic beads were applied to screen novel monoamine oxidase inhibitors in an alkaloid-rich plant, Hunteria zeylanica. Twelve MAOA affinity ligands were screened out, and ten of them were identified as monoterpene indole alkaloids by HPLC-Obitrap-MS/MS. Among them, six ligands, namely geissoschizol, vobasinol, yohimbol, dihydrocorynanthenol, eburnamine and (+)-isoeburnamine which exhibited inhibitory activity against MAOA with low IC50 values. To further explore their inhibitory mechanism, enzyme kinetic analysis and molecular docking studies were conducted.

Catechol-O-methyltransferase and monoamine oxidase B inhibitory activities of Australian bee pollen.

Bee pollen is an apicultural product collected by honeybees from flower stamens and used as a functional food worldwide. In the present study, we aim to elucidate the functions of Australian bee pollen. Australian bee pollen extracts and their main components were tested for catechol-O-methyltransferase (COMT) and monoamine oxidase B (MAOB) inhibitory activities. These enzymes are key neurotransmitters involved in Parkinson's disease and depression. Myricetin (5), tricetin (6), and luteolin (7) exhibited high COMT inhibitory activities (half maximal inhibitory concentration [IC50] = 23.3, 13.8, and 47.4 µM, respectively). In contrast, 5, 7, and annulatin (8) exhibited MAOB inhibitory activities (IC50 = 89.7, 32.8, and 153 µM, respectively). Quantitative analysis via high-performance liquid chromatography revealed that 5 was abundant in Australian bee pollen extracts. Our findings suggest that 5 contributes to the COMT and MAOB inhibitory activities of Australian bee pollen.

Two New Phenylpropanoid Compounds from Lilium Brownii and Their Anti-monoamine Oxidase Activity.

Baihe is a commonly used Chinese medicine for the treatment of neurological disorders. Clinically, the bulbs of Lilium brownii are used to act as Baihe. In the study, two new phenylpropanoid compounds including 3-O-acetyl-1-O-caffeoylglycerol (1) and 3-O-acetyl-1-O-p-coumaroylglycerol (2) were isolated from the bulbs of L. brownii. Their structures were identified by spectroscopic method and the effect on monoamine oxidase activity was determined using an enzyme labeling method. The results show 1 and 2 have anti-monoamine oxidase activity with 20.96 % and 22.31 % inhibition rates at 50 µg/ml, respectively.

Machine learning accelerates pharmacophore-based virtual screening of MAO inhibitors.

Nowadays, an efficient and robust virtual screening procedure is crucial in the drug discovery process, especially when performed on large and chemically diverse databases. Virtual screening methods, like molecular docking and classic QSAR models, are limited in their ability to handle vast numbers of compounds and to learn from scarce data, respectively. In this study, we introduce a universal methodology that uses a machine learning-based approach to predict docking scores without the need for time-consuming molecular docking procedures. The developed protocol yielded 1000 times faster binding energy predictions than classical docking-based screening. The proposed predictive model learns from docking results, allowing users to choose their preferred docking software without relying on insufficient and incoherent experimental activity data. The methodology described employs multiple types of molecular fingerprints and descriptors to construct an ensemble model that further reduces prediction errors and is capable of delivering highly precise docking score values for monoamine oxidase ligands, enabling faster identification of promising compounds. An extensive pharmacophore-constrained screening of the ZINC database resulted in a selection of 24 compounds that were synthesized and evaluated for their biological activity. A preliminary screen discovered weak inhibitors of MAO-A with a percentage efficiency index close to a known drug at the lowest tested concentration. The approach presented here can be successfully applied to other biological targets as target-specific knowledge is not incorporated at the screening phase.

Probing the inhibition of MAO-B by chalcones: an integrated approach combining molecular docking, ADME analysis, MD simulation, and MM-PBSA calculations.

CONTEXT: Monoamine oxidase B (MAO-B), an enzyme of significant relevance in the realm of neurodegenerative disorders, has garnered considerable attention as a potential target for therapeutic intervention. Natural compounds known as chalcones have shown potential as MAO-B inhibitors. In this particular study, we employed a multimodal computational method to evaluate the inhibitory effects of chalcones on MAO-B. METHODS: Molecular docking methods were used to study and assess the complicated binding interactions that occur between chalcones and MAO-B. This extensive analysis provided a valuable and deep understanding of possible binding methods as well as the key residues implicated in the inhibition process. Furthermore, the ADME investigation gave valuable insights into the pharmacokinetic properties of chalcones. This allowed them to be assessed in terms of drug-like attributes. The use of MD simulations has benefited in the research of ligand-protein interactions' dynamic behaviour and temporal stability. MM-PBSA calculations were also done to estimate the binding free energies and acquire a better knowledge and understanding of the binding affinity between chalcones and MAO-B. Our thorough method gives a thorough knowledge of chalcones' potential as MAO-B inhibitors, which will be useful for future experimental validation and drug development efforts in the context of neurodegenerative illnesses.

Novel 6-hydroxybenzothiazol-2-carboxamides as potent and selective monoamine oxidase B inhibitors endowed with neuroprotective activity.

In neurodegenerative diseases, using a single molecule that can exert multiple effects to modify the disease may have superior activity over the classical "one molecule-one target" approach. Herein, we describe the discovery of 6-hydroxybenzothiazol-2-carboxamides as highly potent and selective MAO-B inhibitors. Variation of the amide substituent led to several potent compounds having diverse side chains with cyclohexylamide 40 displaying the highest potency towards MAO-B (IC50 = 11 nM). To discover new compounds with extended efficacy against neurotoxic mechanisms in neurodegenerative diseases, MAO-B inhibitors were screened against PHF6, R3 tau, cellular tau and α-synuclein (α-syn) aggregation. We identified the phenethylamide 30 as a multipotent inhibitor of MAO-B (IC50 = 41 nM) and α-syn and tau aggregation. It showed no cytotoxic effects on SH-SY5Y neuroblastoma cells, while also providing neuroprotection against toxicities induced by α-syn and tau. The evaluation of key physicochemical and in vitro-ADME properties revealed a great potential as drug-like small molecules with multitarget neuroprotective activity.

Enzyme Inhibition Assays for Monoamine Oxidase.

Monoamine oxidase (MAO) catalyzes the oxidative deamination of monoamines with two isoforms, namely, MAO-A and MAO-B, in mitochondrial outer membranes. These two types of MAO-A and MAO-B participate in changes in levels of neurotransmitter such as serotonin (5-hydroxytryptamine) and dopamine. Selective MAO-A inhibitors have been targeted for anti-depression treatment, while selective MAO-B inhibitors are targets of therapeutic agents for Alzheimer's disease and Parkinson's disease. For this reason, study on the development of MAO inhibitors has recently become important. Here, we describe methods of MAO activity assay, especially continuous spectrophotometric methods, which give relatively high accuracy. MAO-A and MAO-B can be assayed using kynuramine and benzylamine as substrates, respectively, at 316 nm and 250 nm, respectively, to measure their respective products, 4-hydroxyquinoline and benzaldehyde. Inhibition degree and pattern can be analyzed by using the Lineweaver-Burk and secondary plots in the presence of inhibitor, and reversibility of inhibitor can be determined by using the dialysis method.

Synergistic utility of NBD-Cl fluorogenic loading activity and salting-out assisted liquid-liquid extraction as sample pretreatment in rasagiline tracking in different matrices.

A typical drug used to treat Parkinson's disease is called rasagiline. It belongs to an assortment of drugs known as monoamine oxidase inhibitors, which function by raising dopamine levels in the brain. This work created a unique spectrofluorimetric method for the analytical assay of rasagiline for the first time. The approach utilized the synergistic utility of the fluorogenic properties of benzofurazan and salting-out assisted liquid-liquid extraction. By combining these techniques an ultrasensitive, and highly selective methodology for the assay of rasagiline was established. Measurements were made of the resultant yellow fluorescent product at 533 nm by applying an excitation wavelength of 475.3 nm. The calibration graph was examined to assess its linearity across a range of 30-600 ng/ml. Through estimation, the limit of detection was discovered to be 8.9 ng/ml, while the quantitation limit was estimated to be 27 ng/ml. All relevant parameters influencing the fulfillment of the developed method were thoroughly examined and tuned. Following the directives set by the (ICH) the suggested approach was confirmed and demonstrated its capability for the accurate determination of rasagiline in tablets, as well as for testing content uniformity. The incorporation of salting-out assisted liquid-liquid extraction technology enables effective tracking of rasagiline in plasma samples, providing a novel and innovative approach for its analysis in biological matrices.

Dual Inhibitors of Brain Carbonic Anhydrases and Monoamine Oxidase-B Efficiently Protect against Amyloid-ß-Induced Neuronal Toxicity, Oxidative Stress, and Mitochondrial Dysfunction.

We report here the first dual inhibitors of brain carbonic anhydrases (CAs) and monoamine oxidase-B (MAO-B) for the management of Alzheimer's disease. Classical CA inhibitors (CAIs) such as methazolamide prevent amyloid-ß-peptide (Aß)-induced overproduction of reactive oxygen species (ROS) and mitochondrial dysfunction. MAO-B is also implicated in ROS production, cholinergic system disruption, and amyloid plaque formation. In this work, we combined a reversible MAO-B inhibitor of the coumarin and chromone type with benzenesulfonamide fragments as highly effective CAIs. A hit-to-lead optimization led to a significant set of derivatives showing potent low nanomolar inhibition of the target brain CAs (KIs in the range of 0.1-90.0 nM) and MAO-B (IC50 in the range of 6.7-32.6 nM). Computational studies were conducted to elucidate the structure-activity relationship and predict ADMET properties. The most effective multitarget compounds totally prevented Aß-related toxicity, reverted ROS formation, and restored the mitochondrial functionality in an SH-SY5Y cell model surpassing the efficacy of single-target drugs.