New Galantamine Derivatives with Inhibitory Effect on Acetylcholinesterase Activity

Abstract

Background:

Inhibitors of acetylcholinesterase (AChE) are used to treat many disorders, among which are neurodegenerative upsets, like Alzheimer’s disease (AD). One of the limited licensed AChE inhibitors (AChEIs) used as drugs is the natural compound galantamine (Gal).

Objective:

As Gal is a toxic compound, here we expose data about its four derivatives in hybrid peptide-norgalantamine molecules, which have shown 100 times lower toxicity.

Methods:

Four newly synthesized galantamine derivatives have been involved in docking analysis made by Molegro Virtual Docker. Biological assessments were performed on ICR male mice. The change in short and long-term memory performance was evaluated by passive avoidance test. AChE activity and levels of main oxidative stress parameters: lipid peroxidation, total glutathione (GSH), enzyme activities of catalase (CAT), superoxide dismutase, and glutathione peroxidase were measured in brain homogenates.

Results:

Our experimental data revealed that the new hybrid molecules did not impair memory performance in healthy mice. Two of the compounds demonstrated better than Gal AChE inhibitory activity in the brain. None of them changed the level of lipid peroxidation products, one of the compounds increased GSH levels, and all of them increased CAT enzyme activity.

Conclusion:

The new galantamine-peptide hybrids demonstrated a potential for inhibition of AChE and antioxidant activity and deserve further attention.

Keywords

Acetylcholine esterase galantaminederivatives inhibition peptide

INTRODUCTION

Alzheimer’s disease (AD) is one of the most complex and challenging neurodegenerative disorders all over the world. Its development is related to extracellular amyloid-β (Aβ) deposition, intracellular hyperphosphorylated tau protein aggregation, and acetylcholinergic neuronal deterioration [1 –4]. All these changes in the brain of affected persons lead to increased oxidative stress [5] and diminished acetylcholine (ACh) levels in brain synapses causing loss of cognitive functions, such as memory, communication skills, judgments, and reasoning [6]. Acetylcholinesterase (AChE) is the enzyme responsible for the hydrolysis of acetylcholine in central nervous system (CNS). At this stage, the treatment of AD is extremely symptomatic and not very effective. One of the main goals in this disease management is to increase ACh levels in the brain by inhibition of AChE activity [7]. Up to now only a few AChE inhibitors (AChEIs) for the AD treatment have been approved, among which are donepezil, rivastigmine, and galantamine [8]. The molecular structure of AChE has been studied by many researchers using AChE from different sources [9 –16]. Although some small variations related to the origin of the enzyme have been established, the main data for its structure are similar. The binding site of the enzyme is deep and narrow with several domains: catalytic anionic site (CAS) situated at the bottom of the binding gorge where the positively charged head of ACh is binding, and peripheral ionic site (PAS), whose block prevents AChE-induced Aβ aggregation [9 –16].

Galantamine (Gal) is a drug licensed for AD treatment and acts as AChE inhibitor. It is a natural, tertiary amarylidacea alkaloid originally isolated from the bulbs of snowdrop and Narcissus species. It was firstly registered as a drug under the trade name Nivalin (Sopharma, Bulgaria) by Professor Paskov, who discovered its properties [17]. In general, it is a reversible, competitive AChEI [18] with effects on central and peripheral nervous systems. The molecule of Gal fits perfectly into the AChE binding gorge [19], but it is short enough to block the PAS. Actually, it is considered as a multitarget drug with nicotinic acetylcholine receptors modulatory activity, ability for microglial Aβ phagocytosis enhancing effect, and possibility for improving cognitive abilities in AD patients [19 –21]. Gal’s structure can be modified by substituting the methyl group at the azepine tertiary nitrogen, at the hydroxyl group of the cyclohexene ring, as well as at the methoxy group of the benzofurane [22]. As Gal is a rather toxic compound, which limits its application, our efforts have been directed to find new derivatives comprising its structure but with diminished toxicity.

Here we report investigations of four recently synthesized galantamine–peptide derivatives (GalDs 34, 43, 44, and 46) [23] as AChEIs. Their hybrid molecules include norgalantamine linked to a peptide fragment (norgalantamine is demethylated at the N- atom galantamine, and its structure is presented in the Fig. 1A). The peptide fragment is a shortened analogue of β-secretase inhibitor OM 99-2. In addition, the new molecules contain either nicotinic or isonicotinic acid moiety in accordance to the literature molecules including nicotinic acid residues readily crossing the blood-brain barrier [24 –26].

Fig. 1

Chemical structures of A) norgalantamine and B) the peptide chains of the newly compounds - GalDs.

Our preliminary data revealed that designed and synthesized in this way hybrid compounds possess very low toxicity, about 100 times lower than those of Gal itself [23]. Toxicity studies have been performed both in vivo on mice and in vitro on a panel of transformed cell lines from the following different tissues: human hepatocellular carcinoma HEP-G2 as a model of liver tissue; murine neuroblastoma Neuro-2a as a model of nerve tissue; and lymphoblastic crisis of human chronic myeloleukaemia BV-173 as a model of hematopoietic tissue [23].

The aim of this study is to test the newly designed Gal derivatives both theoretically and experimentally. Theoretically: to dock them into AChE, creating high-resolution models of complexes between the molecule of galantamine with flexible peptide fragments, bounded either to nicotinic or isonicotinic moiety and AChE structure. Experimentally: to use the compounds with good characteristics for in vivo experiments, testing AChE inhibitory activity, antioxidant capacity and possibility to affect short- and long- term memory processes in healthy mice.

Chemical structures of studied compounds are shown in Fig. 1. All four compounds are isomers (mw = 981) and can be considered as built by two main parts: norgalantamine (Fig. 1A) and peptide chain, modified with two aromatic rings of benzyle and pyridile nature (Fig. 1B). All new compounds demonstrated very low solubility (lower than 10^–4 M) in water, ethanol, and octanol, and relatively high solubility in aprotic solvents, like dimethyl sulfoxide (DMSO) and dimethylformamide (DMF). They can be dissolved also in mixtures of DMSO and buffers with basic pH. The four new compounds are stable as solids at normal conditions and also in solutions for time of 2 h, but for a longer period, it is better to keep them in a refrigerator.

MATERIALS AND METHODS

Docking protocol

In order to analyze the interaction of the four new molecules with AChE, the structures published in Protein Data Bank were used. They have been obtained by X-ray diffraction of the crystal structure of the mouse acetylcholinesterase-TZ2PA6 anti-complex 1Q84 [27] and crystal structure of recombinant human acetylcholinesterase in complex with (–)-galantamine 4EY6 [28].

For the purpose of the docking analysis, the Molegro Virtual Docker (2006) [29] was applied.

Synthesis of the newly designed compounds

Compounds were synthesized by applying synthesis in solutions, a procedure recently reported [23]. They consist of two functional parts: norgalantamine and peptide fragments (Fig. 1). The necessary reagents are as follows: Galantamine substance provided by Sopharma Pharmaceuticals AD, Bulgaria; Amino acids, condensing reagents, and solvents purchased from Merck, Alfa Aesar, and Novabiochem and used without further purification.

Laboratory animals

Male Albino IRC mice (18–20 g, n = 8 in each group) were used. They were housed three per cage under conditions of controlled temperature and 12/12 h light/dark cycle, with food and water available ad libitum. The experiments have been performed strictly according to the national regulations and European Communities Council Directive (86/609/EEC) also “Principles of laboratory animal care” (NIH publication No. 85–23) concerning the protection of animals used for scientific and experimental purposes.

Drug treatment and experimental design

Experimental groups: control received saline (0.1 ml/b.w., i.p) and tested groups received one of four GalDs (50 mg/kg, i.p) for 7 days. The referent group was treated with galantamine (1 mg/kg i.p) for 7 days.

Passive avoidance test

This test creates a conditioned reflex of avoidance through negative reinforcement (electricity) according to Jarvik and Kopp (1967) [30]. The apparatus consists of two separate chambers: illuminated and dark chamber. The floor in the dark compartment consists of steel grids for delivering electric shocks.

Acquisition phase: Mice were individually placed on a platform in the illuminated chamber, taking into account the time that they enter in the dark chamber (initial latency, IL). When the animal entered into the dark box with the four paws, an electrical shock (1 mA, 1 s) was given and then the rodent was removed from the apparatus.

Test phase: The interval between the placement in the illuminated chamber and the entry into the dark chamber (step through latency time, STL) was measured in seconds. STL in each group was measured on the 1st hour and 8th day after first treatment.

Analytical methods

For biochemical analyses, animals were sacrificed by decapitation under CO₂ mild inhalation, and the brain tissues were quickly removed on ice, cleaned with ice-cold saline, and stored at –20°C.

Acetylcholine esterase activity in the brain. Prior to assay, the brain tissue was thawed at room temperature and then 10% homogenates in 0.1 M phosphate buffer (pH 8; 1000 rpm) were made. The acetylcholine activity assay in the brain is based on Ellman’s method [31] in which thiocholine produced by the action of acetylcholinesterase forms a yellow color with of 5,5-Dithiobis 2-nitrobenzoic acid (DTNB). Briefly, 10 % tissue homogenate were centrifuged at 4500 rpm 10 min. 100μl of supernatant was incubated with the Ellman reagent 0.01 M DTNB, 0.1 M phosphate buffer, and 0.075 M freshly prepared acetylthiocholine iodide. 500μl of the reaction mixture was injected into the Semi-auto Chemistry Analyzer and kinetic of the reaction was monitored for 3 min at 405 nm.

Dynamic of AChE activity alteration was measured in brain homogenates on the 1st hour and 8th day after first treatment.

Lipid peroxidation was determined by the amount of thiobarbituric acid reactive substances (TBARs) formed in fresh biological preparations using Lipid Peroxidation (MDA Assay Kit, Cat. No. MAK085) from Sigma-Aldrich Co. LLC, USA. The absorbance was read at 532 nm against appropriate blank. The values were expressed in nmoles malondialdehyde (MDA) per mg protein, with a molar extinction coefficient of 1.56×10⁵ M^–1cm^–1.

Total glutathione content was measured by the method of Rahman et al. (2006) [32] and was expressed in ng/mg protein, with glutathione oxidized as a reference standard.

Catalase activity was determined using Catalase Activity Assay Kit (Cat. No. CAT 100) from Sigma-Aldrich Co. LLC, USA the enzyme activity was expressed as Δ E₂₄₀/min/mg protein.

Cu, Zn-superoxide dismutase (SOD) activity, determined according Peskin and Winterbourn (2017) [33] was expressed in U/mg protein (one unit of SOD activity is the amount of the enzyme producing a 50% inhibition of Nitrobluetetrazolium reduction).

Glutathione peroxidase activity was measured by using Glutathione Peroxidase Cellular Activity Assay (Cat. No. CGP 1) from Sigma-Aldrich Co. LLC, USA and was expressed in nmoles NADPH oxidized per minute per mg protein, with a molar extinction coefficient of 6.22×10⁶ M^–1cm^–1.

Protein content was measured applying the method of Lowry et al. [34] and was determined using a calibration curve obtained with bovine serum albumin - Pentex USA.

Statistical analysis

The results were expressed as means±the standard error of the mean (SEM) or as a percentage change over the mean compared to the control. Statistical analyses of the data were performed by one-way analysis of variance (ANOVA) followed by Dunnett post-hoc comparison test. Differences were considered significant at p < 0.05.

RESULTS

Docking of galantamine-peptides hybrids

The catalytic domain of AChE lies on the deep and narrow gorge, about 20 Å long, which penetrates more than halfway into the enzyme, and widens out close to its base. It consists of the catalytic triad: Ser203, Glu334, and His447 and the aromatic amino acids Trp86, Tyr124, Tyr337, and Phe338, which form a catalytic center [35].

The reaction proceeds by nucleophilic addition of oxygen from Ser203 to carbonyl of acetylcholine, accelerated by the simultaneous transfer of a proton from Ser203 to His447 [36].

The other site actively involved in the inhibition of AChE is called peripheral anionic site (PAS). Composed of five residues (Tyr 70, Asp 72, Tyr 124, Trp 279, and Tyr 334, it is located at the top of the gorge [16, 37].

All of newly synthesized molecules enter the active site of AChE, so that the peptide residue reacts with the PAS site (Fig. 2).

Fig. 2

AChE molecular surface is docked inside the gorge with inhibitors.

In order to analyze the interaction of the molecules, galantamine analogues, molecular docking with recombinant human acetylcholinesterase (rhAChE) (4EY6) was performed. Only GalD 43 (43LD) and GalD 34 (34LD) can interact with Ser203 from the catalyst center (Fig. 3). Actually, the difference of alpha aminoacid sequences between the pairs GalDs 34/43 and GalDs 44/46 is the α aminoacid L-Ala (in 34 and 43), substituted by β-Ala (in 44 and 46), which difference reflex on the length and flexibility of the carbon chain. In addition, 34 and 43 contain pyridyl residue, which is bound in m-position (nicotinoyl structure) in 43 and in p-position (isonicotinoyl structure) in 34. The complex formed with 43 is more stable than this with 34 and the possible explanation for this is the presence of a nicotinoyl residue, which is extremely mobile near the galantamine structure. It is involved by hydrogen bonding to Ser125 or Tyr337 from the PAS site. This interaction, often repetitive is observed in all molecules with both rhAChE and mAChE proteins. The peptide residue also acts as a PAS inhibitor and binds to Tyr124, Tyr 337, Tyr341, and Ser125 and Ser293.

Fig. 3

Structures of the Gal Ds 43, 34, 44, and 46 complexes with hrAChE (4EY6).

According to the docking complexes analysis with mAChE (1Q84), the 43LD molecule again showed the largest similarity to galantamine inhibitors, with the nicotinoyl residue involved in the same hydrogen bonds with the PAS center Tyr337 and Tyr72 (Fig. 4).

Fig. 4

Structures of the GalDs 43, 34, 44, and 46 complexes with mAChE (1Q84).

Each peptide residue of the four new GalDs interacts with Tyr124, Tyr341, and Ser293 forming hydrogen bonds.

Assessment of memory formation in healthy mice

Our results have shown that Gal, used as a referent and the four new GalDs (34, 43, 44, and 46) did not change the latent time of the reaction in step-through test as compared to the control (Fig. 5). Based on these results, we can conclude that Gal and newly synthesized GalDs do not cause memory impairment in experimental rodents in both single and repeated administration.

Fig. 5

Effects of Gal and GalDs 34, 43, 44, and 46 on STL in single-trial passive avoidance test in healthy mice. Data are expressed as the mean±SEM (n = 8 animals per group).

Assessment of AChE inhibitory activity

As it is shown on Fig. 6A, 1 h after the first treatment, GalDs 34, 43, 44, and 46 inhibited AChE activity by 17%, 40%, 37%, and 37% respectively, as compared to the control. As far the inhibitory activity of galantamine itself was 16% versus control, it is obvious that the four derivatives exhibited equal or higher inhibitory activity.

Fig. 6

Effect of Gal and four new GalDs 34, 43, 44, and 46 on AChE activity in mice: A) one hour after the first treatment; B) after 7 days of treatment. Data are expressed as percentage changes in AChE as an average value (n = 8 animals per group).

On Fig 6B, data for the activity of Gal and GalDs after 7 consecutive days of treatment are presented. GalDs 43 and 34 revealed better AChEI activity, with 43% and 27% versus control, while GalDs 44 and 46 have shown 11% and 8%. For comparison, Gal exhibited 13% inhibition versus control. Two of the compounds demonstrated better activity as compared to Gal, namely GalDs 43 and 34, of which 43 exerted better effect. The effect of GalDs 44 and 46 observed 1 h after the first treatment decreased with time, so that on the 7th day of constant administration fall significantly, but still remained comparable to that of galantamine under the same conditions.

Assessment of pro/antioxidant capacity

Malone dialdehyde (MDA) is one of the main biomarkers for determining the levels of lipid peroxidation in the body, respectively for assessing the oxidative status. Results of our experiments regarding antioxidant activities are presented on Fig. 7A. Gal used as a referent showed tendency to increase MDA level, injected in healthy animals. All selected GalDs did not significantly alter the levels of MDA in the brains of healthy mice, with the exception of Gal 46, which increased the brain glutathione (GSH) content by 63% (p < 0.05, n = 8). No derivatives, including Gal, affected the non-enzymatic antioxidant defense system of the cells (Fig. 7A).

Fig. 7A

Effects of Gal (referent) and new GalDs 34, 43, 44, and 46 on MDA levels and GSH content in brain homogenates of ICR male mice.

Mice used in the experiments presented on Fig. 7A were treated with Gal or GalDs for 7 days. Controls were treated with 0.9 % NaCl solution. The oxidative stress indicators were measured one day after the last treatment. Data are expressed as the mean±SEM (n = 6 animals per group). Asterisk above bars indicates a significant difference in lipid peroxidation (LPO) or GSH levels between experimental and control groups at ^* p < 0.05. Statistical analysis was made by one-way ANOVA and Dunnett’s as the post hoc comparison test.

All selected GalDs and Gal (used as a referent) affect enzyme defense system of the cells (Fig. 7B). SOD activity was decreased as follows: Gal, GalD 34, 43, 44, and 46 by 46% (p < 0.01), 43% (p < 0.001), 43% (p < 0.01), 33% (p < 0.01), and 49% (p < 0.001), respectively versus control.

Fig. 7B

Effects of Gal and new GalDs 34, 43, 44, and 46 on SOD, CAT, and GPx activities in brain homogenates of ICR male mice.

Glutathione peroxidase (GPx) activity also decreased due to the application of GalD 34, 43, 44, and 46 by 39%, 47% (p < 0.05), 35%, and 57% (p < 0.01), respectively versus control. Gal increased the GPx activity by 60% (p < 0.01) versus control.

All newly synthesized substances increased the CAT activity. This effect was more pronounced in GalD 44 (by 140%, p < 0.001) followed by GalD 34 (by 94, p < 0.001) and GalD 46 (by 78%, p < 0.001) groups versus controls (Fig. 7B). The effect of GalD 43 was not significant and Gal itself did not change the CAT activity.

Mice used in experiments presented on the Fig. 7B were treated with Gal (1 mg/kg, i.p.) or GalD (50 mg/kg, i.p) for 7 days. Controls were treated with 0.9% NaCl solution. The antioxidant enzyme activities were measured one day after the last treatment. Asterisk above bars indicates a significant difference in levels of enzyme activity between experimental and control groups at ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001. Statistical analysis was made by one-way ANOVA and Dunnett’s as the post hoc comparison test.

DISCUSSION

The subject of the present study is a set of four new GalDs (34, 43, 44, and 46), with different peptide substituents, designed to be AChEIs with lower toxicity than Gal. The desired effect was reached by including peptide fragments at position 11 of Gal. The moistures of nicotinoyl and isonicotinoyl were included in order to increase the permeability through the cell membrane.

Docking analysis has shown that all tested molecules enter in the active site of AChE, with the peptide residue reacting with the PAS site. The best results were found for GalDs 43 and less for 34 (Figs. 3 and 4).

Evaluation of the biological activity of the newly synthesized Gal derivatives was performed behaviorally and biochemically on healthy experimental mice. The effects on memory, AChE activity, as well as pro/antioxidant capacity in the brain of healthy mice were examined.

First symptoms of AD in patients are short-term memory loss, progressively developing deficits in speaking, communication, orientation, and judgment [38]. Hence, the effect of the new derivatives on the short and long-term learning and memory performance in healthy experimental mice was evaluated by passive avoidance test. This is a fear-aggravated test where passive learning and memory of animals is measured as the latency of the dark box entrance [39]. We found that under conditions of this experiment, Gal itself and all tested compounds did not significantly change the control level of latency time of reaction in the passive avoidance test after single and repeated treatment. This means that they did not cause a short- or long-term memory impairment in healthy mice.

A major approach to the prevention and treatment of memory impairment in AD have been aimed at improving ACh activity, and the most successful goal for this is the inhibition of enzyme AChE [40]. Cholinesterase inhibitors are the only class of compounds consistently proven efficacious in treating the cognitive and functional symptoms of AD [41]. In this study, we assessed the effects of new GalDs on AChE activity. We found that all derivatives possess AChE inhibitory activity, which was the same or better than that of Gal, used as a referent. The most enhanced and stable was the inhibitory effect of GalD 43, both after single and multiple treatments.

This result was also confirmed by the docking analysis (Figs. 3 and 4). Explanation of this fact can be found in the chemical formulas of the derivatives used. Considering the structures of the four new compounds, it should be noticed that 43 and 34 have the same peptide consistency and differ only in nicotinoyl or isonicotinoyl residue. It seems that the nicotinoyl residue (in GalDs 43) possess stabilizing effect on the forming complex with AChE as compared to the isonicotonoyl (GalD 34), which complex contains weaker bonds. On the other hand, the other two derivatives 44 and 46 contain equal peptide chain, which compared to the peptide chain of 43 and 34 differ by one amino acid. Instead of Ala (in 43 and 34), β-Ala is included in 44 and 46 and namely that difference makes their peptide chain to exert lower inhibitory activity.

High levels of LPO products in the CNS and peripheral tissues have been determined for patients both with dementia of the Alzheimer type and mild cognitive impairment [42, 43]. A reduction in the antioxidant enzymatic defense reflected by a decreasing specific activity of the main antioxidant enzymes, GPx and SOD, has also been demonstrated for such patients [44]. There is also evidence that in AD direct binding between CAT and amyloid-β may decrease enzyme activity, a situation that increases the hydrogen peroxide level in cells and provokes oxidative stress [45 –47]. These findings emphasize the important role of antioxidant agents in the treatment of AD.

Our investigations on the new GalDs to impact antioxidant defense systems have shown that the studied compounds did not increase the level of LPO, assessed by the change of TBARs in mouse brain homogenates. This means that the long-term treatment with them did not cause oxidative stress. In comparison, Gal exhibited a tendency to increase LPO levels in brain homogenates. Total GSH levels were unchanged by any of the used GalDs and Gal itself. Only GalD 46 increased the non-enzymatic antioxidant defense system of cells by as much as 60%. Therefore, at this stage, we could conclude that galantamine derivatives with the design used do not cause oxidative stress in cells.

The new GalDs also affected the enzymes of the antioxidant defense system of the cell. In our experiments, we have chosen to measure the activity of primary enzymes, involved in direct elimination of active oxygen species (hydroxyl radical, superoxide radical, hydrogen peroxide), SOD, CAT, and GPx. It is well known that SOD plays a crucial role in turning free radicals into H₂O₂ and water [48]. The enzymes CAT and GPx transform H₂O₂ into H₂O by assisting of various cofactors [49, 50].

Overall, we can summarize that GalDs, used in this study, decreased the SOD and GPx activity and increased that of CAT. We believe that the increased CAT activity is an indicator for the antioxidant capacity of the newly synthesized derivatives, mostly pronounced in GalD 44, followed by 34 and 46. In comparison, Gal itself did not significantly change CAT activity.

CONCLUSIONS

Docking studies show that the newly synthesized galantamine derivatives form complexes with AChE, and because of pyridyl moiety, the link of Gal residue with CAS is not as strong as this of Gal-molecule. On the other hand, the entry of the Gal residue in the gorge inside the AChE molecule in order to reach the CAS, leads to deployment of the polypeptide chain of the compounds in the PAS site, forming additional links. For these reasons, the compounds used react as dual inhibitors. Indeed, two of the compounds practically demonstrated better AChE inhibitory activity in the brain as compared to Gal. The docking results also reveal that the structure of the chain inhibiting PAS center is of importance, showing that the best results are in case of typical peptide structure, as in beta Ala (two linked methylene groups in the chain) this effect was weaker.

The experiments described here show that the new GalDs do not impair memory performance in healthy mice after single and repeated treatment, which is an effect comparable to those of Gal. GalD 43 and partially GalD 34 confirmed better AChE inhibitory activity in the brain in comparison to Gal, as predicted by docking studies. Derivatives did not cause oxidative stress in brain of healthy animals. All of them, but not Gal, increased CAT enzyme activity in brain homogenates, indication for an antioxidant effect. GalD 46 increased GSH levels, affecting the non-enzyme antioxidant defense system of the cells.

Based on the results obtained and presented here, we consider that new galantamine-peptide hybrids demonstrate potential for AChEIs and antioxidant compounds and deserve further study on animal neurodegenerative models as promising therapeutic agents for treatment of patients with AD.

Footnotes

ACKNOWLEDGMENTS

Authors acknowledge the support of this study by the Grant DH 03/8 “Galantamine’s and 4-aminopyridine’s derivatives containing peptide motif with expected effect on the Alzheimer’s disease and multiple sclerosis” funded by the Scientific Research Fund of Bulgaria.

Special acknowledgments to the financial support of the National Program “European Scientific Networks” project D01-278/05.10.2020 “Drug molecule” MON – Bulgaria are also expressed by the authors.

Authors’ disclosures available online ().

References

Goedert

, Spillantini

(2008) A century of Alzheimer’s disease. Science 314, 777–781.

Kung

(2012) The β-amyloid hypothesis in Alzheimer’s disease: Seeing is believing. ACS Med Chem Lett 3, 265–267.

Ballatore

, Lee

, Trojanowski

(1951) Tau-mediated neurodegeneration in Alzheimer’s disease and related disorders. Nat Rev Neurosci 8, 663–672.

Mashkovsky

, Kruglikova-Lvova

(1951) On the pharmacology of the new alkaloid galantamine. Farmakol Toxicol (Moscow) 14, 27–30.

Gella

, Durany

(2009) Oxidative stress in Alzheimer disease. Cell Adh Migr 3, 88–93.

Lanctôt

, Herrmann

, Yau

, Khan

, Liu

, Loulou

, Einarson

(2003) Efficacy and safety of cholinesterase inhibitors in Alzheimer’s disease: A meta-analysis. CMAJ 169, 557–564.

Martins

, Silva

, Pinto

MMM

, Sousa

(2020) Marine natural products, multitarget therapy and repurposed agents in Alzheimer’s disease. Pharmaceuticals (Basel) 13, 242.

Campos

, Rocha

, Vieira

, Rocha

, Telles-Correia

, Paes

, Yuan

, Nardi

, Arias-Carrión

, Machado

, Caixeta

(2016) Treatment of cognitive deficits in Alzheimer’s disease: A psychopharmacological review. Psychiatr Danub 28, 2–12.

Harel

, Schalk

, Ehret-Sabatier

, Bouet

, Goeldner

(1993) Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase. Proc Natl Acad Sci U S A 90, 9031–9035.

10.

Ordentlich

, Barak

, Kronman

, Flashner

, Leitner

, Segall

, Ariel

, Cohen

, Velan

, Shafferman

(1993) Dissection of the human acetylcholinesterase active center determinants of substrate specificity. Identification of residues constituting the anionic site, the hydrophobic site, and the acyl pocket. J Biol Chem 268, 17083–17095.

11.

Radić

, Pickering

, Vellom

, Camp

, Taylor

(1993) Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors. Biochemistry 32, 12074–12084.

12.

Sussman

, Harel

, Frolow

, Oefner

, Goldman

, Toker

, Silman

(1991) Atomic structure of acetylcholinesterase from Torpedo californica: A prototypic acetylcholine-binding protein. Science 253, 872–879.

13.

Kitz

, Braswell

, Ginsburg

(1970) On the question: Is acetylcholinesterase an allosteric protein? Mol Pharmacol 6, 108–121.

14.

De Ferrari

, Canales

, Shin

, Weiner

, Silman

, Inestrosa

(2001) A structural motif of acetylcholinesterase that promotes amyloid beta-peptide fibril formation. Biochemistry 40, 10447–10457.

15.

Johnson

, Moore

(2004) Identification of a structural site on acetylcholinesterase that promotes neurite outgrowth and binds laminin-1 and collagen IV. Biochem Biophys Res Commun 319, 448–455.

16.

Johnson

, Moore

(2006) The peripheral anionic site of acetylcholinesterase: Structure, functions, and potential role in rational drug design. Curr Pharm Des 12, 217–225.

17.

Paskov

(1959) Nivalin: Pharmacology and Clinical Application, Medicina i Fizkultura, Sofia, Bulgaria.

18.

Marco-Contelles

, do Carmo Carreiras

, Rodríguez

, Villarroya

, García

(2006) Synthesis and pharmacology of galantamine. Chem Rev 106, 116–133.

19.

Takata

, Kitamura

, Saeki

, Terada

, Kagitani

, Kitamura

, Fujikawa

, Maelicke

, Tomimoto

, Taniguchi

, Shimohama

(2010) Galantamine-induced amyloid-beta clearance mediated via stimulation of microglial nicotinic acetylcholine receptors. J Biol Chem 285, 40180–40191.

20.

Dajas-Bailador

, Heimala

, Wonnacott

(2003) The allosteric potentiation of nicotinic acetylcholine receptors by galantamine is transduced into cellular responses in neurons: Ca2+ signals and neurotransmitter release. Mol Pharmacol 64, 1217–1226.

21.

Guillou

, Bernard

, Gras

, Thal

(2001) An efficient total synthesis of (±) galantamine. Angew Chem 40, 4745–4746.

22.

Han

, Sweeney

, Bachman

, Schweiger

, Forloni

, Coyle

, Davis

, Joullié

(1992) Chemical and pharmacological characterization of galantamine, an acetylcholinesterase inhibitor, and its derivatives. A potential application in Alzheimer’s disease? Eur J Med Chem 27, 673–687.

23.

Vezenkov

, Tsekova

, Kostadinova

, Mihaylova

, Vassilev

, Danchev

(2019) Synthesis of new galantamine-peptide derivatives designed for prevention and treatment of Alzheimer’s disease. Curr Alzheimer Res 16, 183–192.

24.

Lemeignan

, Millart

, Lamiable

, Molgo

, Lechat

(1984) Evaluation of 4-aminopyridine and 3,4-diaminopyridine penetrability into cerebrospinal fluid in anesthetized rats. Brain Res 304, 166–169.

25.

Hankes

, Coenen

, Rota

, Langen

, Herzog

, Wutz

, Stoecklin

, Feinendegen

(1991) Effect of Huntington’s and Alzheimer’s diseases on the transport of nicotinic acid or nicotinamide across the human blood-brain barrier. Adv Exp Med Biol. 294, 675–678.

26.

Penberthy

(2009) Nicotinic acid-mediated activation of both membrane and nuclear receptors towards therapeutic glucocorticoid mimetics for treating multiple sclerosis. PPAR Res 2009, 853707.

27.

Bourne

, Kolb

, Radić

, Sharpless

, Taylor

, Marchot

(2014) Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation. Proc Natl Acad Sci U S A 101, 1449–1454.

28.

Cheung

, Rudolph

, Burshteyn

, Cassidy

, Gary

, Love

, Franklin

, Height

(2012) Structures of human acetylcholinesterase in complex with pharmacologically important ligands height. J Med Chem 55, 10282–10286.

29.

Bitencourt-Ferreira

, de Azevedo

Jr (2019) Molegro virtual docker for docking. Methods Mol Biol 2053, 149–167.

30.

Jarvik

, Kopp

(1967) An improved one-trial passive avoidance learning situation. Psychol Rep 21, 221–224.

31.

Ellman

, Courtney

, Andres

Jr , Feather-Stone

(1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7, 88–95.

32.

Rahman

, Kode

, Biswas

(2006) Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc 1, 3159–3165.

33.

Peskin

, Winterbourn

(2017) Assay of superoxide dismutase activity in a plate assay using WST-1. Free Radic Biol Med 103, 188–191.

34.

Lowry

, Rosenbrough

, Farr

, Randall

(1951) Protein measurement with folin phenol reagent. J Biol Chem 193, 256–275.

35.

Dvir

, Silman

, Harel

, Rosenberry

, Sussman

(2010) Acetylcholinesterase: From 3D structure to function. Chem Biol Interact 187, 10–22.

36.

Zhang

, Kua

, McCammon

(2002) Role of the catalytic triad and oxyanion hole in acetylcholinesterase catalysis: An ab initio QM/MM Study. J Am Chem Soc 124, 10572–10577.

37.

Bourne

, Taylor

, Radić

, Marchot

(2003) Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site. EMBO J 22, 1–12.

38.

Sperling

, Aisen

, Beckett

, Bennett

, Craft

, Fagan

, Iwatsubo

, Jack

Jr , Kaye

, Montine

, Park

, Reiman

, Rowe

, Siemers

, Stern

, Yaffe

, Carrillo

, Thies

, Morrison-Bogorad

, Wagster

, Phelps

(2011) Toward defining the preclinical stages of Alzheimer’s disease: Recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement 7, 280–292.

39.

Binder

, Hirokawa

, Windhorst

(2009) Passive avoidance learning. In Encyclopedia of Neuroscience, Binder

, Hirokawa

, Windhorst

, eds. Springer, Berlin, Heidelberg.

40.

Agrawal

, Tyagi

, Saxena

, Nath

(2009) Cholinergic influence on memory stages: A study on scopolamine amnesic mice. Indian J Pharmacol 41, 192–196.

41.

Hake

(2001) Use of cholinesterase inhibitors for treatment of Alzheimer disease. Clev Clin J Med 68, 608–609.

42.

Baldeiras

, Santana

, Proença

, Garrucho

, Pascoal

, Rodrigues

, Duro

, Oliveria

(2008) Peripheral oxidative damage in mild cognitive impairment and mild Alzheimer’s disease. J Alzheimers Dis 15, 117–128.

43.

Greilberger

, Koidl

, Greilberger

, Lamprecht

, Schroecksnadel

, Ledlhuber

, Fuchs

, Oetll

(2008) Malondialdehyde, carbonyl proteins and albumindisulphide as useful oxidative markers in mild cognitive impairment and Alzheimer’s disease. Free Radic Res 42, 633–638.

44.

Padurariu

, Ciobica

, Hritcu

, Stoica

, Bild

, Stefanescu

(2010) Changes of some oxidative stress markers in the serum of patients with mild cognitive impairment and Alzheimer’s disease. Neurosci Lett 469, 6–10.

45.

Mattson

, Lovell

, Furukawa

, Markesbery

(1995) Neurotrophic factors attenuate glutamate induced accumulation of peroxides, elevation of intracellular Ca2+ concentration, and neurotoxicity and increase antioxidant enzyme activities in hippocampal neurons. J Neurochem 65, 1740–1751.

46.

Behl

, Davis

, Lesley

, Schubert

(1994) Hydrogen peroxide mediates amyloid β protein toxicity. Cell 77, 817–827.

47.

Habib

, Lee

MTC

, Yang

(2010) Inhibitors of catalase amyloid interactions protect cells from β-amyloid induced oxidative stress and toxicity. J Biol Chem 285, 38933–38943.

48.

Celino

, Yamaguchi

, Miura

, Ohta

, Tozawa

, Iwai

, Mirua

(2011) Tolerance of spermatogonia to oxidative stress is due to high levels of Zn and Cu/Zn superoxide dismutase. PLoS One 6, e16938.

49.

Bild

, Ciobica

, Padurariu

, Bild

(2012) The interdependence of the reactive species of oxygen, nitrogen, and carbon. J Physiol Biochem 69, 147–154.

50.

Xia

, Zheng

, Shao

, Wang

, Liu

, Wang

(2013) Cloning and functional analysis of glutathione peroxidase gene in red swamp crayfish Procambarus clarkii. Fish Shellfish Immunol 34, 1587–1595.