Differential tyrosine and serine/threonine phosphorylation/dephosphorylation pathways regulate the expression of α7 versus α3ß4 nicotinic receptor subtypes in mouse hippocampal neurons.

We have recently reported that α7 and α3ß4 nicotinic acetylcholine receptor (nAChR) subtypes are expressed in human chromaffin cells in the plasma membrane where they colocalize and physically interact. The present study was designed to evaluate whether those receptor subtypes also colocalize at the central nervous system to mutually interact, and whether their expression and colocalization are regulated by phosphorylation/dephosphorylation processes, as they are in human chromaffin cells. We have here found that in isolated and maintained in culture mouse hippocampal neurons, nAChR expression and colocalization of α7, but not α3ß4, nAChR subtypes decreased by tyrosine (Tyr)- and serine/threonine (Ser/Thr)-phosphatase inhibition. However, Tyr-kinase inhibition or protein-phosphatase 2A (PP2A) activation increased α3ß4 nAChR expression, diminishing receptor subtypes colocalization. Furthermore, colocalization is not recovered if the inhibitors of Tyr-phosphatase and kinases, or the inhibitor of Ser/Thr-phosphatases and the activator of PP2A are applied together. Therefore, regulation of α7 and α3ß4 nAChR subtypes expression by Tyr- and Ser/Thr kinases and phosphatases exhibit differential mechanisms in mouse hippocampal neurons. Colocalization of nAChR subtypes, however, is altered by any maneuver that affects these kinases or phosphatases, which might have consequences in the functional activity of nAChR subtypes.

C-glycosides analogues of the okadaic acid central fragment exert neuroprotection via restoration of PP2A-phosphatase activity: A rational design of potential drugs for Alzheimer's disease targeting tauopathies.

Protein phosphatase 2A (PP2A) is an important Ser/Thr phosphatase that participates in the regulation of multiple cellular processes. This implies that any deficient activity of PP2A is the responsible of severe pathologies. For instance, one of the main histopathological features of Alzheimer's disease is neurofibrillary tangles, which are mainly comprised by hyperphosphorylated forms of tau protein. This altered rate of tau phosphorylation has been correlated with PP2A depression AD patients. With the goal of preventing PP2A inactivation in neurodegeneration scenarios, we have aimed to design, synthesize and evaluate new ligands of PP2A capable of preventing its inhibition. To achieve this goal, the new PP2A ligands present structural similarities with the central fragment C19-C27 of the well-established PP2A inhibitor okadaic acid (OA). Indeed, this central moiety of OA does not exert inhibitory actions. Hence, these compounds lack PP2A-inhibiting structural motifs but, in contrast, compete with PP2A inhibitors, thus recovering phosphatase activity. Proving this hypothesis, most compounds showed a good neuroprotective profile in neurodegeneration models related to PP2A impairment, highlighting derivative 10, named ITH12711, as the most promising one. This compound (1) restored in vitro and cellular PP2A catalytic activity, measured on a phospho-peptide substrate and by western-blot analyses, (2) proved good brain penetration measured by PAMPA, and (3) prevented LPS-induced memory impairment of mice in the object recognition test. Thus, the promising outcomes of the compound 10 validate our rational approach to design new PP2A-activating drugs based on OA central fragment.

Synthesis and Biological Assessment of 4,1-Benzothiazepines with Neuroprotective Activity on the Ca2+ Overload for the Treatment of Neurodegenerative Diseases and Stroke.

In excitable cells, mitochondria play a key role in the regulation of the cytosolic Ca2+ levels. A dysregulation of the mitochondrial Ca2+ buffering machinery derives in serious pathologies, where neurodegenerative diseases highlight. Since the mitochondrial Na+/Ca2+ exchanger (NCLX) is the principal efflux pathway of Ca2+ to the cytosol, drugs capable of blocking NCLX have been proposed to act as neuroprotectants in neuronal damage scenarios exacerbated by Ca2+ overload. In our search of optimized NCLX blockers with augmented drug-likeness, we herein describe the synthesis and pharmacological characterization of new benzothiazepines analogues to the first-in-class NCLX blocker CGP37157 and its further derivative ITH12575, synthesized by our research group. As a result, we found two new compounds with an increased neuroprotective activity, neuronal Ca2+ regulatory activity and improved drug-likeness and pharmacokinetic properties, such as clog p or brain permeability, measured by PAMPA experiments.

(±)-BIGI-3h: Pentatarget-Directed Ligand combining Cholinesterase, Monoamine Oxidase, and Glycogen Synthase Kinase 3ß Inhibition with Calcium Channel Antagonism and Antiaggregating Properties for Alzheimer's Disease.

Multitarget-directed ligands (MTDLs) are considered a promising therapeutic strategy to address the multifactorial nature of Alzheimer's disease (AD). Novel MTDLs have been designed as inhibitors of human acetylcholinesterases/butyrylcholinesterases, monoamine oxidase A/B, and glycogen synthase kinase 3ß and as calcium channel antagonists via the Biginelli multicomponent reaction. Among these MTDLs, (±)-BIGI-3h was identified as a promising new hit compound showing in vitro balanced activities toward the aforementioned recognized AD targets. Additional in vitro studies demonstrated antioxidant effects and brain penetration, along with the ability to inhibit the aggregation of both τ protein and ß-amyloid peptide. The in vivo studies have shown that (±)-BIGI-3h (10 mg/kg intraperitoneally) significantly reduces scopolamine-induced cognitive deficits.

In Silico Prediction of the Toxic Potential of Neuroprotective Bifunctional Molecules Based on Chiral N-Propargyl-1,2-amino Alcohol Derivatives.

N-Propargylamines are useful synthetic scaffolds for the synthesis of bioactive molecules, and in addition, they possess important pharmacological activities. We obtained several neuroprotective molecules, chiral 1,2-amino alcohols and 1,2-diamines, able to reduce by almost 70% the rotenone and oligomycin A-induced damage in SH-SY5Y cells. Furthermore, some molecules assessed also counteracted the toxicity evoked by the Ser/Thr phosphatase inhibitor okadaic acid. Before extrapolating these data to preclinical studies, we analyze the molecules through an in silico prediction system to detect carcinogenicity risk or other toxic effects. In light of these promising results, these molecules may be considered as a lead family of neuroprotective and relatively safe compounds.

An okadaic acid fragment analogue prevents nicotine-induced resistance to cisplatin by recovering PP2A activity in non-small cell lung cancer cells.

We herein report the design, synthesis, and functional impact of an okadaic acid (OA) small analogue, ITH12680, which restores the activity of phosphoprotein phosphatase 2A (PP2A), whose deficient activity has been implicated in nicotine-mediated tumor progression and chemoresistance in non-small cell lung cancer (NSCLC). For its design, we paid attention to the structure of the PP2A-OA complex, where the C16-C38 OA fragment confers PP2A affinity and selectivity, but it is not involved in the inhibitory effect. Confirming this hypothesis, PP2A activity was not inhibited by ITH12680. By contrast, the compound partially restored OA-exerted PP2A inhibition in vitro. Moreover, flow cytometry and immunoblotting experiments revealed that ITH12680 reversed nicotine-induced cisplatin resistance in NSCLC cells, as it prevented nicotine-induced reduction of Bax expression and inhibited nicotine-mediated activation of cell survival and proliferation kinases, Akt and ERK1/2. Our findings suggest that the rescue of nicotine-inhibited PP2A activity could diminish the resistance to cisplatin treatment observed in NSCLC patients who continue smoking.

New Dual Small Molecules for Alzheimer's Disease Therapy Combining Histamine H3 Receptor (H3R) Antagonism and Calcium Channels Blockade with Additional Cholinesterase Inhibition.

New tritarget small molecules combining Ca2+ channels blockade, cholinesterase, and H3 receptor inhibition were obtained by multicomponent synthesis. Compound 3p has been identified as a very promising lead, showing good Ca2+ channels blockade activity (IC50 = 21 ± 1 µM), potent affinity against hH3R (Ki = 565 ± 62 nM), a moderate but selective hBuChE inhibition (IC50 = 7.83 ± 0.10 µM), strong antioxidant power (3.6 TE), and ability to restore cognitive impairment induced by lipopolysaccharide.

Anti-tumoral activity of the human-specific duplicated form of α7-nicotinic receptor subunit in tobacco-induced lung cancer progression.

OBJECTIVES: Tobacco smoking is strongly correlated with the onset and progression of non-small cell lung cancer (NSCLC). By activating α7 nicotinic acetylcholine receptors (α7-nAChRs) in these tumors nicotine and its tobacco-derived nitrosamine, NNK, contribute to these oncogenic processes. Here, we investigated whether the human-specific duplicated form of the α7-nAChR subunit (dupα7) behaves as an endogenous negative regulator of α7-nAChR-mediated tumorigenic activity induced by tobacco in NSCLC cells, similarly to its influence on other α7-nAChR-controlled functions in non-tumor cells. METHODS: Two human NSCLC cell lines, lung adenocarcinoma (A549) and squamous cell carcinoma of the lung (SK-MES-1), both wild-type or with stable overexpression of dupα7 (A549dupα7 or SK-MES-1dupα7), were used to investigate in vitro anti-tumor activity of dupα7 on nicotine- or NNK-induced tumor progression. For this purpose, migration, proliferation or epithelial-mesenchymal transition (EMT) were examined. The anti-tumor effect of dupα7 on nicotine-promoted tumor growth, proliferation or angiogenesis was also assessed in vivo in an athymic mouse model implanted with A549dupα7 or A549 xenografts. RESULTS: Overexpression of dupα7 in both cell lines almost completely suppresses the in vitro tumor-promoting effects induced by nicotine (1 µM) or NNK (100 nM) in wild-type cells. Furthermore, in mice receiving nicotine, A549dupα7 xenografts show: (i) a significant reduction of tumor growth, and (ii) decreased expression of cell markers for proliferation (Ki67) or angiogenesis (VEGF) compared to A549 xenografts. CONCLUSION: Our study demonstrates, for the first time, the in vitro and in vivo anti-tumor capacity of dupα7 to block the α7-nAChR-mediated tumorigenic effects of tobacco in NSCLC, suggesting that up-regulation of dupα7 expression in these tumors could offer a potential new therapeutic target in smoking-related cancers.

Polycyclic maleimide-based derivatives as first dual modulators of neuronal calcium channels and GSK-3ß for Alzheimer's disease treatment.

Current healthcare has significantly increased the average life expectancy, leading to a consequently greater incidence of age-related diseases, such as Alzheimer's disease. Following a multitarget approach, in this paper a series of polycyclic maleimide-based derivatives were designed and synthesized aimed at simultaneously modulate neuronal calcium channels and glycogen synthase kinase 3-beta (GSK-3ß), validated targets to combat Alzheimer' disease. Different structural modifications were performed on the polycyclic scaffold in order to investigate the structure-activity relationships and compound 10 emerged as a promising non-toxic lead compound, endowed with calcium modulating brain-addressed properties and significant GSK-3ß inhibitory activity. Moreover, the easily affordable polycyclic core appears as a new appealing privileged structure in medicinal chemistry.

Design and synthesis of multipotent 3-aminomethylindoles and 7-azaindoles with enhanced protein phosphatase 2A-activating profile and neuroprotection.

We report the synthesis and pharmacological evaluation of new 3-aminomethylindoles derivatives with neuroprotective properties designed to present multi-target activity centered on reducing the neuronal Ca2+ overload and preventing phosphatase 2A (PP2A) inhibition, which are two important early physiophathological events observed in neurodegenerative scenarios. Chemical syntheses of proposed compounds were achieved in two straightforward reaction steps with high yields. Most of the compounds mitigated the okadaic acid-provoked inhibition of PP2A and protected SH-SY5Y cells against toxic stimuli related to Tau-hyperphosphorylation and oxidative stress, similarly to the observed in Alzheimer's disease (AD). In addition, some of them mitigated the Ca2+ overload induced by depolarization. The derivative 1-(1-benzyl-5-chloro-1H-indol-3-yl)-N,N-dimethylmethanamine (19) outstood by its high recovery of the PP2A activity and blockade of voltage-gated Ca2+ channels, accompanied by good neuroprotective profile. These findings make this compound eligible for further preclinical assays with the goal of positioning new innovative drugs for the treatment of AD.

Modulation of serine/threonine phosphatases by melatonin: therapeutic approaches in neurodegenerative diseases.

Melatonin is an endogenous hormone produced by the pineal gland as well as many other tissues and organs. The natural decline in melatonin levels with ageing contributes significantly to the development of neurodegenerative disorders. Neurodegenerative diseases share common mechanisms of toxicity such as proteinopathy, mitochondrial dysfunction, metal dyshomeostasis, oxidative stress, neuroinflammation and an imbalance in the phosphorylation/dephosphorylation ratio. Several reports have proved the usefulness of melatonin in counteracting the events that lead to a neurodegenerative scenario. In this review, we have focused on the fact that melatonin could rectify the altered phosphorylation/dephosphorylation rate found in some neurodegenerative diseases by influencing the activity of phosphoprotein phosphatases. We analyse whether melatonin offers any protective activity towards these enzymes through a direct interaction. LINKED ARTICLES: This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.

Substituent effect of N-benzylated gramine derivatives that prevent the PP2A inhibition and dissipate the neuronal Ca2+ overload, as a multitarget strategy for the treatment of Alzheimer's disease.

Following the premises of the multitarget-directed ligands approach for the drug R&D against neurodegenerative diseases, where Alzheimer's disease (AD) outstands, we have synthesized and evaluated analogues of the gramine derivative ITH12657 (1-benzyl-5-methyl-3-(piperidin-1-ylmethyl-1H-indole, 2), which had shown important neuroprotective properties, such as blocking effect of voltage-gated Ca2+ channels (VGCC), and prevention of phosphoprotein phosphatase 2A (PP2A) inhibition. The new analogues present different substitutions at the pending phenyl ring, what slightly modified their pharmacological characteristics. The VGCC blockade was enhanced in derivatives possessing nitro groups, while the pro-PP2A feature was ameliorated by the presence of fluorine. Chlorine atoms supplied good activities over the two biological targets aimed; nevertheless that substitution provoked loss of viability at 100-fold higher concentrations (10 µM), what discards them for a deeper pharmacological study. Overall, the para-fluorine derivative of ITH12657 was the most promising candidate for further preclinical assays.

Gramine Derivatives Targeting Ca(2+) Channels and Ser/Thr Phosphatases: A New Dual Strategy for the Treatment of Neurodegenerative Diseases.

We describe the synthesis of gramine derivatives and their pharmacological evaluation as multipotent drugs for the treatment of Alzheimer's disease. An innovative multitarget approach is presented, targeting both voltage-gated Ca(2+) channels, classically studied for neurodegenerative diseases, and Ser/Thr phosphatases, which have been marginally aimed, even despite their key role in protein τ dephosphorylation. Twenty-five compounds were synthesized, and mostly their neuroprotective profile exceeded that offered by the head compound gramine. In general, these compounds reduced the entry of Ca(2+) through VGCC, as measured by Fluo-4/AM and patch clamp techniques, and protected in Ca(2+) overload-induced models of neurotoxicity, like glutamate or veratridine exposures. Furthermore, we hypothesize that these compounds decrease τ hyperphosphorylation based on the maintenance of the Ser/Thr phosphatase activity and their neuroprotection against the damage caused by okadaic acid. Hence, we propose this multitarget approach as a new and promising strategy for the treatment of neurodegenerative diseases.

Ligands for Ser/Thr phosphoprotein phosphatases: a patent review (2005-2015).

INTRODUCTION: The role played by phosphoprotein phosphatases (PPP) enzymes makes them of interest as therapeutic targets to treat pathologies including neurodegenerative diseases, cancer and autoimmune diseases, but also liable to cause severe side effects. This fact has hindered the study of PPP ligands as potential drugs. Fortunately, recent advances in the comprehension of PPP biochemistry have given rise to the development of refined pharmacological strategies to selectively target phosphatases and limit the possible generation of adverse reactions. AREAS COVERED: This review summarizes the most relevant patents claiming the use of PPP ligands to treat human diseases in the last decade (2005-2015). It also includes some pharmacological strategies aiming to indirectly modulate PPP functionality by interacting with PPP-regulating enzymes. EXPERT OPINION: There is still much work to be done to validate PPP enzymes as eligible targets for the development of new drugs. The most significant barrier is likely to be persuading the majority of the scientific community that PPP enzymes are not too unspecific. Few patents disclosed the rational design of direct PPP ligands, while many inventions relied on long chain peptides-based approaches. Overall, the future of ligands for PPP enzymes as therapeutics seems both challenging and exciting.