Sustainable Drug Discovery of Multi-Target-Directed Ligands for Alzheimer's Disease.

The multifactorial nature of Alzheimer's disease (AD) is a reason for the lack of effective drugs as well as a basis for the development of "multi-target-directed ligands" (MTDLs). As cases increase in developing countries, there is a need of new drugs that are not only effective but also accessible. With this motivation, we report the first sustainable MTDLs, derived from cashew nutshell liquid (CNSL), an inexpensive food waste with anti-inflammatory properties. We applied a framework combination of functionalized CNSL components and well-established acetylcholinesterase (AChE)/butyrylcholinesterase (BChE) tacrine templates. MTDLs were selected based on hepatic, neuronal, and microglial cell toxicity. Enzymatic studies disclosed potent and selective AChE/BChE inhibitors (5, 6, and 12), with subnanomolar activities. The X-ray crystal structure of 5 complexed with BChE allowed rationalizing the observed activity (0.0352 nM). Investigation in BV-2 microglial cells revealed antineuroinflammatory and neuroprotective activities for 5 and 6 (already at 0.01 µM), confirming the design rationale.

Structure-activity relationships of dually-acting acetylcholinesterase inhibitors derived from tacrine on N-methyl-d-Aspartate receptors.

Tacrine is a classic drug whose efficacy against neurodegenerative diseases is still shrouded in mystery. It seems that besides its inhibitory effect on cholinesterases, the clinical benefit is co-determined by NMDAR-antagonizing activity. Our previous data showed that the direct inhibitory effect of tacrine, as well as its 7-methoxy derivative (7-MEOTA), is ensured via a "foot-in-the-door" open-channel blockage, and that interestingly both tacrine and 7-MEOTA are slightly more potent at the GluN1/GluN2A receptors when compared with the GluN1/GluN2B receptors. Here, we report that in a series of 30 novel tacrine derivatives, designed for assessment of structure-activity relationship, blocking efficacy differs among different compounds and receptors using electrophysiology with HEK293 cells expressing the defined types of NMDARs. Selected compounds (4 and 5) potently inhibited both GluN1/GluN2A and GluN1/GluN2B receptors; other compounds (7 and 23) more effectively inhibited the GluN1/GluN2B receptors; or the GluN1/GluN2A receptors (21 and 28). QSAR study revealed statistically significant model for the data obtained for inhibition of GluN1/Glu2B at -60 mV expressed as IC50 values, and for relative inhibition of GluN1/Glu2A at +40 mV caused by a concentration of 100 µM. The models can be utilized for a ligand-based virtual screening to detect potential candidates for inhibition of GluN1/Glu2A and/or GluN1/Glu2B subtypes. Using in vivo experiments in rats we observed that unlike MK-801, the tested novel compounds did not induce hyperlocomotion in open field, and also did not impair prepulse inhibition of startle response, suggesting minimal induction of psychotomimetic side effects. We conclude that tacrine derivatives are promising compounds since they are centrally available subtype-specific inhibitors of the NMDARs without detrimental behavioral side-effects.

Tacrine-sugar mimetic conjugates as enhanced cholinesterase inhibitors.

We have used the Cu(i)-catalyzed azide-alkyne Huisgen cycloaddition reaction to obtain two families of bivalent heterodimers where tacrine is connected to an azasugar or iminosugar, respectively, via linkers of variable length. The heterodimers were investigated as cholinesterase inhibitors and it was found that their activity increased with the length of the linker. Two of the heterodimers were significantly stronger acetylcholinesterase inhibitors than the monomeric tacrine. Molecular modelling indicated that the longer heterodimers fitted better into the active gorge of acetylcholinesterase than the shorter counterparts and the former provided more efficient simultaneous interaction with the tryptophan residues in the catalytic anionic binding site (CAS) and the peripheral anionic binding site (PAS).

Tacrines as Therapeutic Agents for Alzheimer's Disease. V. Recent Developments.

Herein we have reviewed our recent developments for the identification of new tacrine analogues for Alzheimer's disease (AD) therapy. Tacrine, the first cholinesterase inhibitor approved for AD treatment, did not stop the progression of AD, producing only some cognitive improvements, but exhibited secondary effects mainly due to its hepatotoxicity. Thus, the drug was withdrawn from the clinics administration. Since then, many publications have described non-hepatotoxic tacrines, and in addition, important efforts have been made to design multitarget tacrines by combining their cholinesterase inhibition profile with the modulation of other biological targets involved in AD.

Sensitive and reversible perylene derivative-based fluorescent probe for acetylcholinesterase activity monitoring and its inhibitor.

A reversible fluorescence probe for acetylcholinesterase activity detection was developed based on water soluble perylene derivative, N,N'-di(2-aspartic acid)-perylene-3,4,9,10-tetracarboxylic diimide (PASP). Based on the photo-induced electron transfer (PET), PASP fluorescence in aqueous is quenched after combining with copper ions (Cu2+). Acetylcholinesterase (AChE) is well known to catalyze the hydrolysis of acetylcholine (ATCh) to produce thiocholine, whose affinity is strong enough to capture Cu2+ by thiol (-SH) group from the complex PASP-Cu, resulting in the fluorescence signal of PASP recovers up to 90%. This optical switch is highly sensitive depended on the coordination and dissociation between PASP and Cu2+. We proposed its application for AChE activity detection, as well as its inhibitor screening. According to the change of fluorescence intensity, quantifying the detection limit of AChE was 1.78 mU·mL-1. Classical inhibitors, tacrine and organophosphate pesticide diazinon, were further evaluated for drug screening. The IC50 value of tacrine was calculated to be 0.43 µM, and the detection limit of diazinon was 0.22 µM. Both of these performances were much better than previous results, revealing our probe is sensitive and reversible for screening applications.

Physicochemical evaluation of new tetrahydroacridine and iodobenzoic acid hybrids as the next step in the design of potential drugs for treating Alzheimer's disease.

Tacrine derivatives containing iodobenzoic acid were developed as a novel multitarget-directed ligand and find potential application in the treatment of Alzheimer's disease. The aim of this study is to perform a physicochemical profile of this series. Experimental log P and pKa values were determined and compared with those already calculated. The results indicated better values of the tested compounds than the values predicted using computer software. The stability report was obtained using the developed HPLC method. The stability assay in different environment conditions provided information about the photosensitivity of these compounds and a proper method for the storage of this series of compounds.

A new fluorescent probe for sensing of biothiols and screening of acetylcholinesterase inhibitors.

A new N2O-type BODIPY probe (LF-Bop) has been proposed for the selective and sensitive detection of biologically relevant small molecular thiols. This detection is based on the Michael addition reaction between the thiol and nitrostyrene groups in the probe, which decreases the quenching effect from the nitro group, thus resulting in the recovery of the deep-red fluorescence from the BODIPY structure. The results show that LF-Bop is able to detect all tested free thiols through a fluorescence turn-on assay. The lowest limit of detection (LOD) for glutathione was found to be down to nanomolar levels (220 nM). Based on this probe, we have developed a new fluorescence assay for the screening of acetylcholinesterase inhibitors. In total, 11 natural and synthetic alkaloids have been evaluated. Both experimental measurements and theoretical molecular docking results reveal that both natural berberine and its synthetic derivative dihydroberberine are potential inhibitors of acetylcholinesterase.

Three Dimensional Quantitative Structure Activity Relationship and Pharmacophore Modeling of Tacrine Derivatives as Acetylcholinesterase Inhibitors in Alzheimer's Treatment.

BACKGROUND: Three dimensional quantitative structure activity relationship and pharmacophore modeling are studied for tacrine derivatives as acetylcholinesterase inhibitors. METHODS: The three dimensional quantitative structure-activity relationship and pharmacophore methods were used to model the 68 derivatives of tacrine as human acetylcholinesterase inhibitors. The effect of the docked conformer of each molecule in the enzyme cavity was investigated on the predictive ability and statistical quality of the produced models. RESULTS: The whole data set was divided into two training and test sets using hierarchical clustering method. 3D-QSAR model, based on the comparative molecular field analysis has good statistical parameters as indicated by q2 =0.613, r2 =0.876, and r2pred =0.75. In the case of comparative molecular similarity index analysis, q2, r2 and r2pred values were 0.807, 0.96, and 0.865 respectively. The statistical parameters of the models proved that the inhibition data are well fitted and they have satisfactory predictive abilities. CONCLUSION: The results from this study illustrate the reliability of using techniques in exploring the likely bonded conformations of the ligands in the active site of the protein target and improve the understanding over the structural and chemical features of AChE.

Colorimetric assay of acetylcholinesterase inhibitor tacrine based on MoO2 nanoparticles as peroxidase mimetics.

Molybdenum dichalcogenides MoX2 (X=S, Se) have been found to possess intrinsic peroxidase-like activity. However, molybdenum oxides (MoO2) as peroxidase mimetics have not been exploited yet. Herein, MoO2 nanoparticles were synthesized by a simple hydrothermal method and found to possess the peroxidase-like activity for the first time. MoO2 nanoparticles could catalyze the oxidation of 3,3',5,5'-tetrametylbenzidine (TMB) by H2O2 to produce a blue-color product (oxTMB). The catalytic property and mechanism were investigated by stead-state kinetics experiment and free radicals scavenging experiment, respectively. Acetylcholinesterase (AChE) could catalyze the hydrolysis of acetylthiocholine chloride (ATCh) into thiocholine (TCh), which could reduce oxTMB to decrease the absorbance in solution. In the presence of AChE inhibitor tacrine, the generation of TCh was inhibited and the absorbance was preserved. Based on these properties, a colorimetric assay method was developed for AChE inhibitor tacrine. This work not only broadens the application of the peroxidase mimetics, but also overcome the disadvantages of traditional methods such as expensive, complex and vulnerable to background interference for colorimetric assay of AChE inhibitor.

Computational Studies on Acetylcholinesterase Inhibitors: From Biochemistry to Chemistry.

Acetylcholinesterase inhibitors are the most promising therapeutics for Alzheimer's disease treatment as these prevent the loss of acetylcholine and slows the progression of the disease. The drugs approved for the management of Alzheimer's disease by the FDA are acetylcholinesterase inhibitors but are associated with side effects. Consistent and stringent efforts by the researchers with the help of computational methods opened new ways of developing novel molecules with good acetylcholinesterase inhibitory activity. In this manuscript, we reviewed the studies that identified the essential structural features of acetylcholinesterase inhibitors at the molecular level as well as the techniques like molecular docking, molecular dynamics, quantitative structure-activity relationship, virtual screening, and pharmacophore modelling that were used in designing these inhibitors.

Synthesis, physicochemical and biological evaluation of tacrine derivative labeled with technetium-99m and gallium-68 as a prospective diagnostic tool for early diagnosis of Alzheimer's disease.

Design, physicochemical and biological studies of novel radioconjugates for the early diagnosis of Alzheimer's disease, based on the newly synthesized tacrine derivatives were performed. Novel tacrine analogues were labeled with technetium-99m and gallium-68. For all obtained radioconjugates ([99mTc]Tc-Hynic-(tricine)2NH(CH2)ntacrine and [68Ga]Ga-DOTA-NH(CH2)9tacrine, where n = 2-9 denotes the number of methylene groups CH2) the studies of physicochemical properties (lipophilicity, stability in the presence of an excess of standard amino acids cysteine or histidine, human serum and in cerebrospinal fluid) were performed. For two selected radioconjugates [99mTc]Tc-Hynic-(tricine)2NH(CH2)9Tac and [68Ga]Ga-DOTA-NH(CH2)9tacrine (characterized with the highest lipophilicity values) the biological tests (inhibition of cholinesterases action, molecular docking and biodistribution studies) have been performed. All novel radioconjugates showed high stability in biological solutions used. Both selected radioconjugates proved to be good inhibitors of cholinesterases and be able to cross the blood-brain barrier. Radioconjugates [99mTc]Tc-Hynic-(tricine)2NH(CH2)9tacrine and [68Ga]Ga-DOTA-NH(CH2)9tacrine fulfil the conditions for application in nuclear medicine. Radiopharmaceutical [68Ga]Ga-DOTA-NH(CH2)9tacrine, due to increased accuracy and improved sensitivity in PET imaging, may be better potential diagnostic tool for early diagnosis of Alzheimer's disease.

Alignment-independent 3D-QSAR and molecular docking studies of tacrine-4-oxo-4H-Chromene hybrids as anti-Alzheimer's agents.

A series of novel tacrine derivatives as multifunctional agents with potential inhibitory effects on both acetylcholinesterase(AChE) and butyrylcholinesterase (BuChE) enzymes for the treatment of Alzheimer's disease(AD), were applied to alignment independent 3D-QSAR methods using Pentacle software. In this studies, GRID-independent molecular descriptors (GRIND) analysis have been applied to characterize important interactions between enzymes and the studied compounds. Two H-bond acceptor groups as well as hydrophobic properties of tacrine rings for AChE and two H-bond acceptor on the carbonyl group of chromene and NH of amid group for BuChE, with positive effects on their inhibitory potency have been identified. The obtained 3D-QSAR models have been analyzed and validated. The statistical quality of the QSAR model for AChE, r2 = 0.87, q2 = 0.56 and for BuChE, r2 = 0.96, q2 = 0.70 was resulted. Using these models, novel structures have been designed and pIC50 of them were predicted. Molecular docking studies were also conducted on AChE (1ACJ) and BuChE (4BDS) and promising results in good agreement with 3D-QSAR studies were obtained.

Molecular modelling studies on the interactions of 7-methoxytacrine-4-pyridinealdoxime with VX-inhibited human acetylcholinesterase. A near attack approach to assess different spacer-lengths.

The novel prophylactic agent 7-methoxytacrine-4-pyridinealdoxime is a hybrid compound formerly designed to keep acetylcholinesterase resistant to organophosphates by reactivating it in case of intoxication by such inhibitors. In rational design, a 5-carbon length-spacer hybrid compound was synthesized to evaluate its inhibitory and reactivation capabilities. In this work, theoretical results were achieved through molecular modelling techniques, taking for granted the enzymatic reactivation reaction through nucleophilic substitution. Based on the near attack conformation approach, docking studies were performed to assess the spacer-length from 1 to 10 carbons long of a series of analogues of 7-methoxytacrine-4-pyridinealdoxime. Consequently, the hybrids with length-spacer of 4 and 5 carbons long were the best assessed and subsequently subjected to further molecular dynamics simulations, complemented by Poisson-Boltzmann surface area calculations. As a result, intermolecular interactions with the different binding sites inside human acetylcholinesterase were elucidated. Besides, thermodynamics and kinetics concepts pointed to the 4-carbon linker as optimum for enzymatic reactivation. Further studies, based on quantum mechanics in conjunction with molecular mechanics, were recommended to the presented near attack conformations to achieve more thermodynamics results between the hybrids with 4- and 5-carbon linkers, like values of activation energy for the reactivation reaction. All of those in silico evaluations could be considered as a set of tools for theoretically investigate novel enzymatic reactivators with different shape of spacers.

The muscarinic agonist pilocarpine modifies cocaine-reinforced and food-reinforced responding in rats: comparison with the cholinesterase inhibitor tacrine.

Activation of muscarinic receptors in the brain antagonizes the actions of cocaine, blocking both its discriminative stimulus and reinforcing properties. Pilocarpine is a nonselective muscarinic agonist that is used clinically, but has not been well characterized for its actions during cocaine-reinforced behavior. This study evaluated its effects on cocaine-reinforced and food-reinforced behaviors in rats, using the cholinesterase inhibitor tacrine as a comparator. Intraperitoneal pilocarpine or tacrine at doses of 1.0 mg/kg or more attenuated self-administration of low-dose cocaine (0.1 mg/kg injection) but also increased oral movements. Pilocarpine was less potent than tacrine in decreasing responding supported by low or intermediate amounts of liquid food. Combined treatment with pilocarpine and tacrine was more effective than either compound alone in attenuating self-administration of intermediate-dose cocaine. At a low (0.66 mg/kg) dose which did not modify reinforced responding, pilocarpine increased nonspecific behavior (sniffing, rearing, and activity) in cocaine-reinforced but not in food-reinforced animals; with greater doses increasing cholinergic or gastrointestinal signs. These effects were most consistently correlated with changes in reinforcement in rats responding for cocaine relative to food-reinforced animals. Overall, pilocarpine exhibited modest selectivity for attenuating self-administration of low-dose cocaine without affecting a nondrug reinforcer.

Novel tacrine-coumarin hybrids linked to 1,2,3-triazole as anti-Alzheimer's compounds: In vitro and in vivo biological evaluation and docking study.

A new series of tacrine-coumarin hybrids linked to 1,2,3-triazole were designed, synthesized, and tested as potent dual binding site cholinesterase inhibitors (ChEIs) for the treatment of Alzheimer's disease (AD). Among them, compound 8e was the most potent anti-AChE derivative (IC50 = 27 nM) and compound 8m displayed the best anti-BChE activity (IC50 = 6 nM) much more active than tacrine and donepezil as the reference drugs. Compound 8e was also evaluated for its BACE1 inhibitory activity and neuroprotectivity against PC12 cells exposed to Aß25-35 which indicated low activity. Finally, in vivo studies by Morris water maze task showed that compound 8e significantly reversed scopolamine-induced memory deficit in rats.

The New Acetylcholinesterase Inhibitors PC-37 and PC-48 (7-Methoxytacrine-Donepezil-Like Compounds): Characterization of Their Metabolites in Human Liver Microsomes, Pharmacokinetics and In Vivo Formation of the Major Metabolites in Rats.

The objective of this study was to elucidate the pharmacokinetics and metabolite formation of newly developed non-selective AChE/BChE 7-MEOTA-donepezil-like inhibitors for potential therapeutic use in Alzheimer's disease (AD) patients. The chemical structures of metabolites were defined during incubation with human liver microsomes, and subsequently, the metabolization was verified in in vivo study. In vitro metabolic profiling revealed the formation of nine major metabolites in the case of PC-37 and eight metabolites of PC-48. Hydroxylation and the enzymatic hydrolysis of bonds close to the piperazine ring appeared to be the principal metabolic pathways in vitro. Of these metabolites, M1-M7 of PC-37 and M1-M6 of PC-48 were confirmed under in vivo conditions. Pilot pharmacokinetic experiments in rats were focused on the absorption, distribution and elimination of these compounds. Absorption after i.m. application was relatively fast; the bioavailability expressed as AUCtotal was 28179 ± 4691 min.ng/mL for PC-37 and 23374 ± 4045 min.ng/mL for PC-48. Both compounds showed ability to target the central nervous system, with brain concentrations exceeding those in plasma. The maximal brain concentrations are approximately two times higher than the plasma concentrations. The relatively high brain concentrations persisted throughout the experiment until 24 hr after application. Elimination via the kidneys (urine) significantly exceeded elimination via the liver (bile). All these characteristics are crucial for new candidates intended for AD treatment. The principle metabolic pathways that were verified in the in vivo study do not show any evidence for formation of extremely toxic metabolites, but this needs to be confirmed by further studies.

Advances toward multifunctional cholinesterase and ß-amyloid aggregation inhibitors.

The emergence of a multitarget design approach in the development of new potential anti-Alzheimer's disease agents has resulted in the discovery of many multifunctional compounds focusing on various targets. Among them the largest group comprises inhibitors of both cholinesterases, with additional anti-ß-amyloid aggregation activity. This review describes recent advances in this research area and presents the most interesting compounds reported over a 2-year span (2015-2016). The majority of hybrids possess heterodimeric structures obtained by linking structurally active fragments interacting with different targets. Multipotent cholinesterase inhibitors with ß-amyloid antiaggregating activity may additionally possess antioxidative, neuroprotective or metal-chelating properties or less common features such as anti-ß-secretase or τ-antiaggregation activity.

Tacrine-resveratrol fused hybrids as multi-target-directed ligands against Alzheimer's disease.

Multi-target drug discovery is one of the most followed approaches in the active central nervous system (CNS) therapeutic area, especially in the search for new drugs against Alzheimer's disease (AD). This is because innovative multi-target-directed ligands (MTDLs) could more adequately address the complexity of this pathological condition. In a continuation of our efforts aimed at a new series of anti-AD MTDLs, we combined the structural features of the cholinesterase inhibitor drug tacrine with that of resveratrol, which is known for its purported antioxidant and anti-neuroinflammatory activities. The most interesting hybrid compounds (5, 8, 9 and 12) inhibited human acetylcholinesterase at micromolar concentrations and effectively modulated Aß self-aggregation in vitro. In addition, 12 showed intriguing anti-inflammatory and immuno-modulatory properties in neuronal and glial AD cell models. Importantly, the MTDL profile is accompanied by high-predicted blood-brain barrier permeability, and low cytotoxicity on primary neurons.

Synthesis and structure-activity relationship study of tacrine-based pyrano[2,3-c]pyrazoles targeting AChE/BuChE and 15-LOX.

A series of tacrine-based pyrazolo[4',3':5,6]pyrano[2,3-b]quinolines and related compounds were designed and synthesized for targeting AChE, BuChE and 15-LOX enzymes in the field of Alzheimer's disease therapy. Most of compounds showed potent activity against cholinesterases and mild potency toward 15-LOX enzyme. In particular, compounds 29, 32 and 40 displayed inhibition at nano-molar level against AChE and BuChE (IC50s = 0.005-0.08 µM), being more potent than reference drug tacrine. Moreover, compound 32 with IC50 value of 31 µM was the most potent compound against 15-LOX. The cytotoxicity assay on HepG2 cells revealed that compounds 29 and 32 showed no significant cytotoxic activity even at concentration of 50 µM. The cytotoxicity of compounds 29 and 32 was significantly less than that of tacrine at higher concentrations.

Design, Synthesis and in vitro Evaluation of Indolotacrine Analogues as Multitarget-Directed Ligands for the Treatment of Alzheimer's Disease.

Novel indolotacrine analogues were designed, synthesized, and evaluated as potential drugs for the treatment of Alzheimer's disease. By using a multitarget-directed ligand approach, compounds were designed to act simultaneously as cholinesterase (ChE) and monoamine oxidase (MAO) inhibitors. The compounds were also evaluated for antioxidant, cytotoxic, hepatotoxic, and blood-brain barrier (BBB) permeability properties. Indolotacrine 9 b (9-methoxy-2,3,4,6-tetrahydro-1H-indolo[2,3-b]quinolin-11-amine) showed the most promising results in the in vitro assessment; it is a potent inhibitor of acetylcholinesterase (AChE IC50 : 1.5 µm), butyrylcholinesterase (BChE IC50 : 2.4 µm) and MAO A (IC50 : 0.49 µm), and it is also a weak inhibitor of MAO B (IC50 : 53.9 µm). Although its cytotoxic (IC50 : 5.5±0.4 µm) and hepatotoxic (IC50 : 1.22±0.11 µm) profiles are not as good as those of the standard 7-methoxytacrine (IC50 : 63±4 and 11.50±0.77 µm, respectively), the overall improvement in the inhibitory activities and potential to cross the BBB make indolotacrine 9 b a promising lead compound for further development and investigation.