Static Binding and Dynamic Transporting-Based Design of Specific Ring-Chain-Ring Acetylcholinesterase Inhibitor: From Galantamine to Natural Product.

Acetylcholinesterase (AChE) is a key target for the current symptomatic treatment of Alzheimer's disease, and galantamine is a clinical anticholinesterase drug with transiently acting characteristic and good selectivity for AChE. The present theoretical-experimental work improves the drug's residence time without reducing the inhibition effect, thus providing a crucial breakthrough for modifying the inhibitor of AChE with better kinetic behavior. The static binding and dynamic delivery properties acquired from atomic view reveal that the galantamine simply occupies a catalytic anionic site, and its release from AChE needs only ∼8.6 kcal/mol. Both of these may cause the short residence time of galantamine. The hotspots and most favorable transport mechanism are identified, and the hydrogen bond and aromatic stacking interactions are observed to play crucial roles for galantamine binding and release in AChE. The typical peripheral anionic site arisen at the delivery process would provide another key occupation to enhance the anti-release ability for inhibitors. The compound with "specific-ring-chain-ring" framework with detailed beneficial modification scheme is summarized, which may improve the residence time of the inhibitor in AChE. The thermodynamic and dynamic properties of galantamine derivatives are also studied. Based on dictamnine, a natural alkaloid, two novel eligible derivatives are designed, synthesized and evaluated, which verifies our prediction. Multiple computational approaches and experimental combinations probably provide a train of thought from both static and dynamic views to modify or design appropriate inhibitors on the basis of specific binding and transportation features.

A Comparative Study between Lycorine and Galantamine Abilities to Interact with AMYLOID ß and Reduce In Vitro Neurotoxicity.

Galantamine is a natural alkaloid extracted from the Amaryllidaceae plants and is used as the active ingredient of a drug approved for the treatment of the early stages of Alzheimer's disease. It mainly acts as an acetylcholinesterase (AChE) inhibitor, increasing concentrations of the acetylcholine neurotransmitter. Recent cellular studies have also shown the ability of galantamine to protect SH-SY5Y cell lines against amyloid-ß (Aß)-induced toxicity. Such investigations have supported and validated further in-depth studies for understanding the chemical and molecular features associated with galantamine-protective abilities. In addition to galantamine, other natural alkaloids are known to possess AChE inhibitory activity; among them lycorine has been extensively investigated for its antibacterial, anti-inflammatory and antitumoral activities as well. Despite its interesting biological properties, lycorine's neuroprotective functions against Aß-induced damages have not been explored so far. In this research study, the ability of galantamine and lycorine to suppress Aß-induced in vitro neuronal toxicity was evaluated by investigating the chemical interactions of the two alkaloids with Aß peptide. A multi-technique spectroscopic analysis and cellular cytotoxicity assays were applied to obtain new insights on these molecular associations. The comparison between the behaviors exhibited by the two alkaloids indicates that both compounds possess analogue abilities to interact with the amyloidogenic peptide and protect cells.

Synthesis and biological study of new galanthamine-peptide derivatives designed for prevention and treatment of Alzheimer's disease.

The Alzheimer's disease leads to neurodegenerative processes and affecting negatively million people worldwide. The treatment of the disease is still difficult and incomplete in practice. Galanthamine is one of the most commonly used drugs against the illness. The main aim of this work is design and synthesis of new derivatives of galanthamine comprising peptide moiety as well as study of their ß-secretase inhibitory activity and the anti-aggregating effect. All new derivatives of galanthamine containing analogues of Leu-Val-Phe-Phe (Aß17-Aß20) were synthesized in solution using fragment and consecutive condensation approaches. The new derivatives were characterized by melting points, NMR, and HPLC/MS. They were tested in vitro for ß-secretase inhibition activity by means of fluorescent method and were investigated in vitro for anti-aggregation activity on sheep platelet-rich plasma. Although the new compounds do not contain a structural element responsible for the ß-secretase inhibition, five of them show high or good ß-secretase inhibitory activity between 19.98 and 51.19% with IC50 between 1.95 and 5.26 nM. Four of the new molecules were able to inhibit platelet aggregation between 55.0 and 90.0% with IC50 between 0.69 and 1.36 µM. Four of the compounds were able to inhibit platelet aggregation and two of them have high anti-aggregating effects.

Revealing the cholinergic inhibition mechanism of Alzheimer's by galantamine: a metadynamics simulation study.

Galantamine is one of the approved drugs based on the cholinergic hypothesis for the symptomatic treatment of mild to moderate Alzheimer's disease (AD). The etiology of AD is not fully known; however, the reported cholinergic hypothesis suggests the inadequate synthesis of the neurotransmitter acetylcholine (ACh) is responsible for this disease. The crystal structure of galantamine bound human acetylcholinesterase (hAChE) has been reported; however, the inhibition mechanism of hAChE by galantamine is not well understood. A Well-tempered metadynamics (WTMtD) simulation study has been performed with the crystal structure of galantamine bound hAChE. The reported mechanism for the degradation of ACh is suggested through a proton transfer process from a carboxylic group of Glu334 to the hydroxyl group of Ser203, which attacks ACh for the degradation to acetic acid and choline. Such proton transfer process is lowered in the presence of galantamine due to the separation of catalytic triad inside the gorge of AChE as observed with WTMtD. A docking study has been performed to examine the ACh's binding with the catalytic triad of galantamine bound hAChE. The docking results reveal that the approach of ACh to the catalytic triad is interrupted due to the galantamine's presence in the gorge of the enzyme.

A Galantamine-Curcumin Hybrid Decreases the Cytotoxicity of Amyloid-Beta Peptide on SH-SY5Y Cells.

Misfolded amyloid beta (Aß) peptides aggregate and form neurotoxic oligomers. Membrane and mitochondrial damages, calcium dysregulation, oxidative stress, and fibril deposits are among the possible mechanisms of Aß cytotoxicity. Galantamine (GAL) prevents apoptosis induced by Aß mainly through the ability to stimulate allosterically the α7 nAChRs and to regulate the calcium cytosolic concentration. Here, we examined the cytoprotective effects of two GAL derivatives, namely compounds 4b and 8, against Aß cytotoxicity on the human neuroblastoma cell line SH-SY5Y. The protective effects were tested at simultaneous administration, pre-incubation and post-incubation, with Aß. GAL and curcumin (CU) were used in the study as reference compounds. It was found that 4b protects cells in a similar mode as GAL, while compound 8 and CU potentiate the toxic effects of Aß. Allosteric stimulation of α7 nAChRs is suggested as a possible mechanism of the cytoprotectivity of 4b. These and previous findings characterize 4b as a prospective non-toxic multi-target agent against neurodegenerative disorders with inhibitory activity on acetylcholinesterase, antioxidant, and cytoprotective properties.

LC-MS bioanalysis of targeted nasal galantamine bound chitosan nanoparticles in rats' brain homogenate and plasma.

Validated LC-MS method for the direct quantitative analysis of galantamine (acetylcholinesterase inhibitor) was developed in rat cerebrospinal fluid and brain homogenate besides rat plasma, utilizing structurally close nalbuphine as an internal standard. After a simple protein precipitation step, samples are separated on 2-µm C18 column kept at 40 °C, using isocratic flow of 80% methanol in pH 9.5 ammonium formate buffer, and retention times were about 1.8 and 2.9 min for galantamine and nalbuphine, respectively. Mass detection with electrospray ionization (ESI) and positive polarity was able to detect 0.2 ng mL-1 galantamine using single ion monitoring mode (SIM) at m/z 288 for galantamine and m/z 358 for nalbuphine. The method showed linearity within the range of 0.5 - 300 ng mL-1. The proposed method was validated according to FDA guidelines. Trueness and precision showed acceptable values at all quality control levels, and recoveries were within 85.6 - 114.3% in all matrices at all runs and with relative standard deviations within 0.2 - 12.4%. The method was used to study in vivo brain uptake and pharmacokinetics of galantamine from brain homogenate and plasma samples following the administration of nasal galantamine-bound chitosan nanoparticles compared to oral and nasal galantamine solutions, in scopolamine-induced Alzheimer's disease rat model.

A Novel Galantamine-Curcumin Hybrid as a Potential Multi-Target Agent against Neurodegenerative Disorders.

The acetylcholinesterase (AChE) inhibitors are the main drugs for symptomatic treatment of neurodegenerative disorders like Alzheimer's disease. A recently designed, synthesized and tested hybrid compound between the AChE inhibitor galantamine (GAL) and the antioxidant polyphenol curcumin (CU) showed high AChE inhibition in vitro. Here, we describe tests for acute and short-term toxicity in mice as well as antioxidant tests on brain homogenates measured the levels of malondialdehide (MDA) and glutathione (GSH) and in vitro DPPH, ABTS, FRAP and LPO inhibition assays. Hematological and serum biochemical analyses were also performed. In the acute toxicity tests, the novel AChE inhibitor given orally in mice showed LD50 of 49 mg/kg. The short-term administration of 2.5 and 5 mg/kg did not show toxicity. In the ex vivo tests, the GAL-CU hybrid performed better than GAL and CU themselves; in a dose of 5 mg/kg, it demonstrates 25% reduction in AChE activity, as well as a 28% and 73% increase in the levels of MDA and GSH, respectively. No significant changes in blood biochemical data were observed. The antioxidant activity of 4b measured ex vivo was proven in the in vitro tests. In the ABTS assay, 4b showed radical scavenging activity 10 times higher than the positive control butylhydroxy toluol (BHT). The GAL-CU hybrid is a novel non-toxic AChE inhibitor with high antioxidant activity which makes it a prospective multitarget drug candidate for treatment of neurodegenerative disorders.

Discovery of a Novel Acetylcholinesterase Inhibitor by Fragment-Based Design and Virtual Screening.

Despite extensive and intensive research efforts in recent decades, there is still no effective treatment for neurodegenerative diseases. On this background, the use of drugs inhibiting the enzyme acetylcholinesterase (AChE) remains an eternal evergreen in the symptomatic treatment of mild to moderate cognitive impairments. Even more, the cholinergic hypothesis, somewhat forgotten in recent years due to the shift in focus on amyloid cascade, is back to life, and the search for new, more effective AChE inhibitors continues. We generated a fragment-based library containing aromatic moieties and linkers originating from a set of novel AChE inhibitors. We used this library to design 1220 galantamine (GAL) derivatives following the model GAL (binding core) - linker (L) - aromatic fragment (Ar). The newly designed compounds were screened virtually for blood-brain barrier (BBB) permeability and binding to AChE. Among the top 10 best-scored compounds, a representative lead molecule was selected and tested for anti-AChE activity and neurotoxicity. It was found that the selected compound was a powerful non-toxic AChE inhibitor, 68 times more active than GAL, and could serve as a lead molecule for further optimization and development.

Profiling of acetylcholinesterase inhibitory alkaloids from some Crinum, Habranthus and Zephyranthes species by GC-MS combined with multivariate analyses and in silico studies.

Acetylcholinesterase (AChE) inhibitors remain the class of drugs used for the treatment of Alzheimer disease (AD). For the aim of discovering new sources of potent AChE inhibitors, a combined AChE-inhibitory activity together with alkaloid profiles by GC-MS, combined with multivariate statistical analysis for biomarkers determination and in silico studies were attempted. Strategy was applied on leaves, roots and bulbs of six aquatic and terrestrial Amaryllidaceae species. Thirty alkaloids were identified and the AChE inhibitory activities of the extracts were tested by in-vitro Ellman method. Principal bioactive markers were discovered by correlating AChE inhibitory activity with chemical fingerprints via PLS and OPLS modeling which revealed that galanthamine, lycoramine, caranine, tazettine and N-demethylgalanthamine were the most bio-significant markers. Furthermore, the molecular docking was performed to illustrate binding orientations of the top scoring alkaloids in the active site of human acetylcholinesterase. Suggested strategy revealed that, beside galanthamine, caranine, N-demethylgalanthamine, and lycoramine are promising AChE inhibitors.

Gas-phase basicity and proton affinity measurements of Alzheimer's disease drugs by the extended kinetic method and a theoretical investigation.

This study has been carried out to obtain the thermochemical parameters of drugs used for Alzheimer's disease. The measurement of gas-phase basicity (GB) and proton affinity (PA) values of four important and commercially available drugs for Alzheimer's disease namely, rivastigmine, galantamine, memantine, and tacrine, is attempted for the first time. This study also includes the measurement of GB and PA values for the proposed drug curcumin, a natural product. We calculated the GB and PA values for all these drugs by applying electrospray ionization tandem mass spectrometry (ESI-MS/MS) with the extended kinetic method. Since, all these drugs possessing amino groups (basic nature), the PA values for all these drugs are high i.e., the PA values range from 923.6 to 979.7 kJ/mol and the GB values range from 886.2 to 943.3 kJ/mol. The GB and PA values obtained from the mass spectrometric experiments are well supported with the theoretical calculations. A high-level theoretical B3LYP/6-311 + G(d,p) method is used for the PA and GB calculation and the deviations are in the acceptable range.

Mass Spectrometric Enzyme Histochemistry Method Developed for Visualizing In Situ Cholinesterase Activity in Mus musculus and Drosophila melanogaster.

Enzyme histochemistry facilitates enzyme activity visualization in situ; however, as it is a color-based method, molecular quantification is prohibitive. This study aimed to develop a semiquantitative, mass spectrometry imaging (MSI)-based enzyme histochemistry method to determine endogenous cholinesterase (ChE) activity. Using deuterium-labeled acetylcholine (ACh-d9) as a substrate to distinguish ACh-d9 and choline-d9 from endogenous acetylcholine and choline, respectively, the heterogeneous localization of de novo ChE activity was visualized using MSI, devoid of interferences from in situ factors. Furthermore, a tissue inhibitor assay involving two ChE inhibitors in the mouse brain revealed specific ChE inhibition in the corpus callosum. To the best of our knowledge, this study is the first to report a visualization method for total ChE activity in the ganglia and abdomen in Drosophila melanogaster, indicating its applicability among different animals. The present results provide novel insights into the applicability of enzyme histochemistry via MSI to the metabolism of low-molecular-weight organic compounds (i.e., "small molecules") and semiquantitative capability, suggesting that MSI enzyme histochemistry may become a powerful tool for heterogeneous tissue studies.

Galantamine-Curcumin Hybrids as Dual-Site Binding Acetylcholinesterase Inhibitors.

Galantamine (GAL) and curcumin (CU) are alkaloids used to improve symptomatically neurodegenerative conditions like Alzheimer's disease (AD). GAL acts mainly as an inhibitor of the enzyme acetylcholinesterase (AChE). CU binds to amyloid-beta (Aß) oligomers and inhibits the formation of Aß plaques. Here, we combine GAL core with CU fragments and design a combinatorial library of GAL-CU hybrids as dual-site binding AChE inhibitors. The designed hybrids are screened for optimal ADME properties and BBB permeability and docked on AChE. The 14 best performing compounds are synthesized and tested in vitro for neurotoxicity and anti-AChE activity. Five of them are less toxic than GAL and CU and show activities between 41 and 186 times higher than GAL.

Chemical and Biological Aspects of Montanine-Type Alkaloids Isolated from Plants of the Amaryllidaceae Family.

Plants of the Amaryllidaceae family are promising therapeutic tools for human diseases and have been used as alternative medicines. The specific secondary metabolites of this plant family, called Amaryllidaceae alkaloids (AA), have attracted considerable attention due to their interesting pharmacological activities. One of them, galantamine, is already used in the therapy of Alzheimer's disease as a long acting, selective, reversible inhibitor of acetylcholinesterase. One group of AA is the montanine-type, such as montanine, pancracine and others, which share a 5,11-methanomorphanthridine core. So far, only 14 montanine-type alkaloids have been isolated. Compared with other structural-types of AA, montanine-type alkaloids are predominantly present in plants in low concentrations, but some of them display promising biological properties, especially in vitro cytotoxic activity against different cancerous cell lines. The present review aims to summarize comprehensively the research that has been published on the Amaryllidaceae alkaloids of montanine-type.

Galantamine transdermal patch shows higher tolerability over oral galantamine in rheumatoid arthritis rat model.

Rheumatoid arthritis (RA) is a chronic autoimmune disease of idiopathic etiology that triggers inflammatory cytokines compromising the joint mobility. Epidemiological evidences recommend the utilization of galantamine (GH) to reverse the anti-inflammatory reactions induced RA. Oral administration of GH is non-selectivity due to its association with serious gastrointestinal symptoms which, could hinder its therapeutic success. Therefore, the present study aimed to validate the therapeutic potential of GH transdermal patches as a novel application to constitute an effective and tolerable delivery system for managing RA in adjuvant arthritis model. RA was induced in Sprague-Dawley rats intradermally by Heat-killed M (0.12 ml/day). Oral GH (1.25 mg/kg/day) and GH transdermal patch (2.5 mg/kg/2 days) were administrated for 14 days, during which the hind paw and body weight (BW) were assessed. Effects of C-reactive protein (CRP), inflammatory cytokines (TNF-α, IL-10 and IL-1ß) and Janus kinase (JAK-2) were evaluated. Oral- and transdermal GH significantly improved the hind paw edema in arthritis animal model and offered a protective impact against RA. Oral GH group showed marked decrease in BW than that of transdermal patches group. Transdermal patch group showed a significant decrease in the level of IL-1ß more than the oral group. However, no significant difference was detected in the levels of TNF-α and IL-10 between the two groups. It is concluded that GH transdermal patch can be a promising drug delivery system that copes with side effects better than oral GH consequently represents novel strategy in management of RA.

Comparative evaluation of fish oil and butter oil in modulating delivery of galantamine hydrobromide to brain via intranasal route: pharmacokinetic and oxidative stress studies.

The present study investigates the role of fish oil (FO)- and butter oil (BO)-enriched microemulsion-based system of galantamine hydrobromide (GH), an anti-Alzheimer drug, for its potential role in brain permeation enhancement and neuroprotection against oxidative stress. Microemulsion (ME)-based system of GH was prepared using water phase titration. The prepared ME was characterized by several physicochemical parameters like particle size, polydispersity index, and ex vivo drug permeation. Cell-based oxidative stress assays and pharmacokinetic studies were performed using C6 glial cell lines, and Sprague Dawley rats, respectively. The optimized ME comprised 5.3% v/v of Capmul MCM EP (as oil),15.8% v/v of Tween-80 (as surfactant), 5.3% v/v of Transcutol P (as co-surfactant), and 73.6% v/v of water (as aqueous phase). The addition of FO and BO resulted in a slight increase in the droplet size and decrease in transparency of ME. Cell-based anti-oxidative stress assays (glutathione assay, nitrite assay, and lipid peroxidation assay) showed the efficacy of formulation in the order of ME, BO ME, and FO ME, respectively. A similar trend was also observed in in vivo animal studies, wherein GH FO ME showed a comparatively higher percentage of drug reaching the brain when administered by intranasal route than by IV route. The study concluded the potential benefits of co-administering FO- and BO-enriched microemulsion is not only enhancing the permeation of drugs across BBB but also improving efficacy against lipopolysaccharide-induced oxidative stress. Graphical abstract.

Aporphinoid Alkaloids Derivatives as Selective Cholinesterases Inhibitors: Biological Evaluation and Docking Study.

Alzheimer's dementia is a neurodegenerative disease that affects the elderly population and causes memory impairment and cognitive deficit. Manifestation of this disease is associated to acetylcholine decrease; thus, Cholinesterase inhibition is the main therapeutic strategy for the treatment of Alzheimer's disease. In the present study, a series of aporphinoid alkaloids were tested as potential acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors in vitro. Alkaloids liriodenine (3) and cassythicine (10) were the best inhibitors of both cholinesterases with IC50 values lower than 10 µM. In addition, these alkaloids demonstrated better inhibition of BChE than reference drug galantamine. In addition, some alkaloids showed selective inhibition. Laurotetatine clorhydrate (13) selectively inhibit AChE over BChE. On the contrary, pachyconfine (7) interacted more efficiently with BChE active site. Molecular modelling studies were performed in order to illustrate key interactions between most active compounds and the enzymes and to explain their selectivity. These studies reveal that the benzodioxole moiety exhibits strong interactions due to hydrogen bonds that form with the Glu201 (AChE) and Tyr440 (BChE) residues, which is reflected in the IC50 values.

A fluorescent probe for butyrylcholinesterase activity in human serum based on a fluorophore with specific binding affinity for human serum albumin.

Non-specific binding of a fluorescent probe to human serum albumin is problematic because it induces signal interference when the probe detects the target biomarker in human serum. To eliminate this problem, we used intrinsically problematic non-specific fluorescence in designing a fluorescent probe for butyrylcholinesterase activity in serum. The probe containing a fluorophore with specific binding affinity for albumin could sensitively detect butyrylcholinesterase activity in serum with high selectivity to acetylcholinesterase and screen the efficiency of butyrylcholinesterase inhibitors.

Catalytic Asymmetric Total Syntheses of (-)-Galanthamine and (-)-Lycoramine.

The catalytic asymmetric total syntheses of the biologically important and therapeutically valuable Amaryllidaceae alkaloids (-)-galanthamine and (-)-lycoramine have been divergently achieved from commercially available 3-butyn-1-ol. A newly developed spirocyclic pyrrolidine (SPD)-catalyzed enantioselective Robinson annulation rapidly constructs the key cis-hydrodibenzofuran core, which bears an all-carbon quaternary stereocenter of the target molecules with an excellent stereoselective control. Additionally, the current asymmetric synthetic strategy provides an alternative approach toward the syntheses of (-)-galanthamine and its analogues.

Simultaneous quantification and identification of Amaryllidaceae alkaloids in Narcissus tazetta by ultra performance liquid chromatography-diode array detector-electrospray ionisation tandem mass spectrometry.

Narcissus tazetta is used traditionally for treatment of sores, wounds, skin diseases, cancer in different parts of world. Present study focus on the analysis of amaryllidaceae alkaloids in this plant using an ultra-performance liquid chromatography-diode array detection method. The method was developed for simultaneous quantification of eight Amaryllidaceae alkaloids i.e. pseudolycorine (1), lycorine (2), galanthamine (3), 8-O-demethylhomolycorine (4), N-methylhaemanthidine chloride (5), homolycorine (6), narciclasine (7) and zefbetaine (8) in Narcissus tazetta. The method was validated using a BEH C18 column with linear gradient. Standard calibration curve for the analytes showed good linearity ( r2≥0.999). The method was validated for intra-day (RSDs<0.91%) and inter-day (RSDs<0.65%) precisions and accuracy (recovery 92.2-112.5%). The developed method was successively applied for studying the variation of alkaloids in different parts of Narcissus tazetta, i.e. bulbs, roots, flowers, flower stalks and leaves. The study showed a significant variation of these alkaloids in different parts of the plant. Among the alkaloids under investigation, pseudolycorine had highest content in all the parts. Furthermore, application of the developed method to the identification of phytocomponents allowed the identification of sixteen alkaloids.

A novel on-flow mass spectrometry-based dual enzyme assay.

This work describes a new simultaneous on-flow dual parallel enzyme assay based on immobilized enzyme reactors (ICERs) with mass spectrometry detection. The novelty of this work relies on the fact that two different enzymes can be screened at the same time with only one single sample injection and in less than 6 min. The system consisted of two immobilized capillary enzyme reactors (ICERs). More specifically, the ICERs comprised two different enzymes that were accommodated in parallel and were placed between a liquid chromatography (LC) system and a mass spectrometer (MS). The resulting system could be adapted to other types of enzyme reactors with different supports. All the elements in the system were interfaced by means of two 10-port/two-position switching valves. Different tubing dimensions allowed us to monitor the activity of each enzyme independently during the same analysis. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) bioreactors were chosen as proof of concept. Acetylcholine (ACh) was used as substrate; the area of its protonated enzymatic hydrolysis product ion, choline, [M+H]+m/z 104.0, was monitored in the presence and absence of the standard cholinesterase inhibitor galantamine. This method proved to be an interesting tool for fast, simultaneous, and independent label-free dual enzyme inhibitor assay.