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La proteína fosfatasa 2A (PP2A) es una importante Ser/Thr fosfatasa que participa en la regulación de múltiples procesos celulares. Esto implica que cualquier actividad deficiente de PP2A es responsable de graves patologías graves. Por ejemplo, una de las principales características histopatológicas de la enfermedad de Alzheimer son los ovillos neurofibrilares, que están compuestos principalmente por formas hiperfosforiladas de la proteína tau. Esta tasa alterada de fosforilación de tau se ha correlacionado con la depresión PP2A en pacientes con EA. Con el objetivo de prevenir la inactivación de PP2A en escenarios de neurodegeneración, nos hemos propuesto diseñar, sintetizar y evaluar nuevos ligandos de PP2A capaces de impidir su inhibición. Para lograr este objetivo, los nuevos ligandos PP2A presentan similitudes estructurales con el fragmento central C19-C27 del conocido inhibidor de PP2A ácido okadaico (AO). De hecho, este fragmento central de OA no ejerce acciones inhibidoras. Así, estos compuestos carecen de motivos estructurales inhibidores de PP2A pero, por contra, compiten con los inhibidores de PP2A, recuperando así la actividad fosfatasa. Demostrando esta hipótesis, la mayoría los compuestos mostraron un buen perfil neuroprotector en modelos de neurodegeneración relacionados con el deterioro de PP2A, destacando el derivado 10, denominado ITH12711, como el más prometedor. Este compuesto (1º) restauró la actividad catalítica de PP2A, medida en un sustrato fosfopéptido por Western blot, (2º) demostró una buena penetración cerebral medida por PAMPA, y (3º) evitó el deterioro de la memoria inducido por LPS en ratones sometidos a la prueba de reconocimiento de objetos. Por lo tanto, los prometedores resultados del compuesto 10 validan nuestra aproximación para diseñar nuevos fármacos activadores de PP2A basados en el fragmento central de OA.

In this review we focused our attention on sulfur mustard [bis(2-chloroethyl) sulphide], the main vesicant chemical warfare agent (CWA), which has been widely used in different military conflicts, including World War I and the Iran-Iraq war. Moreover, the evolution of the recent Iraq and Syria conflicts suggests that terrorist groups are aware of the significant psychological and media effects that would be produced by the mere attempt to use CWAs. Sulfur mustard can produce the alkylation of macromolecules bearing sulfhydryl groups, such as DNA and proteins. This vesicant can also generate free radicals which can develop toxicity in the areas exposed, such as the eyes, skin, respiratory tract (inhalation) and gastrointestinal tract (ingestion). In this respect, we advance and propose three salvage mechanisms through which a broad-spectrum multipotent molecule, melatonin, could facilitate neutralization of the toxic damage induced by sulfur mustard radical scavenging. We also speculate that the long-term effects of varying severity can appear after acute poisoning. Besides, melatonin-based therapy strategies can modulate of epigenetic mechanisms and become very suitable for the clinical treatment of victimized patients. However, the utilization of melatonin as a “therapeutic bullet” addressed to counteract the vesicant CWAs needs much additional in vitro research as well as systematic animal studies and controlled translational trials.

We investigated the role of triterpene barbinervic acid from Eugenia punicifolia dichloromethane extract in vasopressor responses. Renal arteries were cannulated and perfused with Krebs-Hepes solut...

Upon depolarization of chromaffin cells (CCs), a prompt release of catecholamines occurs. This event is triggered by a subplasmalemmal high-Ca2+ microdomain (HCMD) generated by Ca2+ entry through nearby voltage-activated calcium channels. HCMD is efficiently cleared by local mitochondria that avidly take up Ca2+ through their uniporter (MICU), then released back to the cytosol through mitochondrial Na+/Ca2+ exchanger (MNCX). We found that newly synthesized derivative ITH15004 facilitated the release of catecholamines triggered from high K+-depolarized bovine CCs. Such effect seemed to be due to regulation of mitochondrial Ca2+ circulation because: (i) FCCP-potentiated secretory responses decay was prevented by ITH15004; (ii) combination of FCCP and ITH15004 exerted additive secretion potentiation; (iii) such additive potentiation was dissipated by the MICU blocker ruthenium red (RR) or the MNCX blocker CGP37157 (CGP); (iv) combination of FCCP and ITH15004 produced both additive augmentation of cytosolic Ca2+ concentrations ([Ca2+]c) K+-challenged BCCs, and (v) non-inactivated [Ca2+]c transient when exposed to RR or CGP. On pharmacological grounds, data suggest that ITH15004 facilitates exocytosis by acting on mitochondria-controlled Ca2+ handling during K+ depolarization. These observations clearly show that ITH15004 is a novel pharmacological tool to study the role of mitochondria in the regulation of the bioenergetics and exocytosis in excitable cells.

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.

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.

Blister or vesicant chemical warfare agents (CWAs) have been widely used in different military conflicts, including World War I and the Iran-Iraq War. However, their mechanism of action is not fully understood. Sulfur and nitrogen mustard exert toxic effects not only through the alkylation of thiol-bearing macromolecules, such as DNA and proteins, but also produce free radicals that can develop direct toxic effects in target organs such as the eyes, skin, and respiratory system. The lack of effective treatments against vesicant CWAs-induced injury makes us consider, in this complex scenario, the use and development of melatonin-based therapeutic strategies. This multifunctional indoleamine could facilitate neutralization of the oxidative stress, modulate the inflammatory response, and prevent the DNA damage, as well as the long-term health consequences mediated by vesicant CWAs-induced epigenetic mechanisms. In this context, it would be essential to develop new galenic formulations for the use of orally and/or topically applied melatonin for the prophylaxis against vesicant CWAs, as well as the development of post-exposure treatments in the near future. Universidad Camilo José Cela | Ref. QUIMELTER Ministerio de Defensa | Ref. MELVES

The ATP-gated P2X7 purinergic receptor (P2X7) is involved in the pathogenesis of many neurodegenerative diseases (NDDs). Several P2X7 antagonists have been developed, though none of them reached clinical trials for this indication. In this work, we designed and synthesized novel blood-brain barrier (BBB)-permeable derivatives as potential P2X7 antagonists. They comprise purine or xanthine cores linked to an aryl group through different short spacers. Compounds were tested in YO-PRO-1 uptake assays and intracellular calcium dynamics in a human P2X7-expressing HEK293 cell line, two-electrode voltage-clamp recordings in Xenopus laevis oocytes, and in interleukin 1β release assays in mouse peritoneal macrophages. BBB permeability was assessed by parallel artificial membrane permeability assays and P-glycoprotein ATPase activity. Dichloroarylpurinylethanones featured a certain P2X7 blockade, being compound 6 (2-(6-chloro-9H-purin-9-yl)-1-(2,4-dichlorophenyl)ethan-1-one), named ITH15004, the most potent, selective, and BBB-permeable antagonist. Compound 6 can be considered as a first non-nucleotide purine hit for future drug optimizations, This work has been supported by the following grants: EU Horizon 2020 Research and Innovation Program under Marie Skłodowska-Curie, Grant Agreement N. 766124 to AGG and AN, and Ministerio de Economía y Competitividad, Spain, Grant Number SAF2016-78892R to AGG; Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) Project-ID: 335447717, SFB 1328 (TP15) to A.N.; Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain, Grant Numbers PI16/01041 and PI19/01724 (Cofunded by FEDER) to CdlR; Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain, Grant Numbers PI16/00735 and PI19/00082 (Co-funded by FEDER) to J.E.

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.

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 3β 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 β-amyloid peptide. The in vivo studies have shown that (±)-BIGI-3h (10 mg/kg intraperitoneally) significantly reduces scopolamine-induced cognitive deficits., L.I. thanks the Regional Council of Franche-Comté (2016YC- 04540 and 04560) for financial support, Mrs. M.-J. Henriot (PHV Pharma) for her support in the HPLC analyses, and Vincent Luzet for preliminary results in synthesis. O.S., J.J., and J.K. acknowledge the support from the grant by Czech Science Foundation no. 20-29633J. T.K., D.J., and V.H. acknowledge support from the Long-term Development Plan (Faculty of Military Health Sciences). R.A. and K.M. thank the Ministry of Education, Youth and Sports of the Czech Republic (ERDF no. CZ.02.1.01/0.0/0.0/16_025/0007444).

Neurodegenerative diseases (NDDs) represent a huge social burden, particularly in Alzheimer's disease (AD) in which all proposed treatments investigated in murine models have failed during clinical trials (CTs). Thus, novel therapeutic strategies remain crucial. Neuroinflammation is a common pathogenic feature of NDDs. As purinergic P2X7 receptors (P2X7Rs) are gatekeepers of inflammation, they could be developed as drug targets for NDDs. Herein, we review this challenging hypothesis and comment on the numerous studies that have investigated P2X7Rs, emphasizing their molecular structure and functions, as well as their role in inflammation. Then, we elaborate on research undertaken in the field of medicinal chemistry to determine potential P2X7R antagonists. Subsequently, we review the state of neuroinflammation and P2X7R expression in the brain, in animal models and patients suffering from AD, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, and retinal degeneration. Next, we summarize the in vivo studies testing the hypothesis that by mitigating neuroinflammation, P2X7R blockers afford neuroprotection, increasing neuroplasticity and neuronal repair in animal models of NDDs. Finally, we reviewed previous and ongoing CTs investigating compounds directed toward targets associated with NDDs; we propose that CTs with P2X7R antagonists should be initiated. Despite the high expectations for putative P2X7Rs antagonists in various central nervous system diseases, the field is moving forward at a relatively slow pace, presumably due to the complexity of P2X7Rs. A better pharmacological approach to combat NDDs would be a dual strategy, combining P2X7R antagonism with drugs targeting a selective pathway in a given NDD., We would like to acknowledge the support received from the EU Horizon 2020 Research and Innovation Programme under Maria Sklodowska-Curie Grant Agreement N. 766124. We would also like to thank the support received from the Ministerio de Economía y Competitividad (MINECO, Spain; grant SAF2016-78892-R to Luis Gandía and Antonio G. García) and Fundación Teófilo Hernando

N-Methyl-D-aspartate receptors (NMDARs) are crucial for the normal function of the central nervous system (CNS), and fundamental in memory and learning-related processes. The overactivation of these receptors is associated with numerous neurodegenerative and psychiatric disorders. Therefore, NMDAR is considered a relevant therapeutic target for many CNS disorders. Herein, we report the synthesis and pharmacological evaluation of a new scaffold with antagonistic activity for NMDAR. Specifically, a chemical library of eighteen 1-aminoindan-2-ol tetracyclic lactams was synthesized and screened as NMDAR antagonists. The compounds were obtained by chiral pool synthesis using enantiomerically pure 1-aminoindan-2-ols as chiral inductors, and their stereochemistry was proven by X-ray crystallographic analysis of two target compounds. Most compounds reveal NMDAR antagonism, and eleven compounds display IC values in a Ca entry-sensitive fluo-4 assay in the same order of magnitude of memantine, a clinically approved NMDAR antagonist. Docking studies suggest that the novel compounds can act as NMDAR channel blockers since there is a compatible conformation with MK-801 co-crystallized with NMDAR channel. In addition, we show that the tetracyclic 1-aminoindan-2-ol derivatives are brain permeable and non-toxic, and we identify promising hits for further optimization as modulators of the NMDAR function., This work was supported by FCT (Fundaç~ao para a Ci^encia e a Tecnologia, I.P.) through iMed.ULisboa (UID/DTP/04138/2019), Principal Researcher grant CEECIND/01772/2017 (M. M. M. Santos), and PhD fellowships SFRH/BD/117931/2016 (M. Espadinha) and SFRH/BD/121664/2016 (R. Lopes). Financial support from FCT and Portugal 2020 to the Portuguese Mass Spectrometry Network (Rede Nacional de Espectrometria de Massa e RNEM; LISBOA-01-0145- FEDER-402-022125) is also acknowledged. M.I.R.-F. thanks funding from the Spanish Ministry of Science, Innovation and Universities (grant RTI2018-093955-B-C21) and the technical assistance of Ms. Cristina Tortosa (European contract for young professionals). C.d.l.R. thanks funding from Instituto de Salud Carlos III, Madrid, Spain (grant PI16/01041 and PhD fellowship FI17/00079 for L. Viejo).

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.

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.

Following the premises of the multitarget-directed ligands approach for the drug RD 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.

Cardiovascular side effects of varenicline and a case report of a hypertensive crisis in a varenicline-prescribed patient with pheochromocytoma have been reported. The goal of the present study was to determine whether such side effects might derive, in part, from increased exocytosis of secretory vesicles and subsequent catecholamine release triggered by varenicline in human chromaffin cells of the adrenal gland. In this study, we performed electrophysiological plasma membrane capacitance and carbon fiber amperometry experiments to evaluate the effect of varenicline on exocytosis and catecholamine release, respectively, at concentrations reached during varenicline therapy (100 nM). Experiments were conducted in the absence or presence of nicotine, at plasma concentrations achieved right after smoking (250 nM) or steady-state concentrations (110 nM), in chromaffin cells of the adrenal gland obtained from human organ donors. Cells were stimulated with short pulses (10 ms) of acetylcholine (ACh; 300 μM) applied at 0.2 Hz, in order to closer mimic the physiological situation at the splanchnic nerve-chromaffin cell synapse. In addition, rat chromaffin cells were used to compare the effects obtained in cells from a more readily available species. Varenicline increased the exocytosis of secretory vesicles in human and rat chromaffin cells in the presence of nicotine, effects that were not due to an increase of plasma membrane capacitance or currents triggered by the nicotinic agonists alone. These results should be considered in nicotine addiction therapies when varenicline is used, This work was supported by grants from the Spanish Ministry of Science and Innovation [grant number BFU2015-69092 to A.A.] and the U.S. National Institutes of Health [GM136430 and GM103801 to J.M.M]

In this review, we show how chromaffin cells have contributed to evaluate neuroprotective compounds with diverse mechanisms of action. Chromaffin cells are considered paraneurons, as they share many common features with neurons: (i) they synthesize, store, and release neurotransmitters upon stimulation and (ii) they express voltage-dependent calcium, sodium, and potassium channels, in addition to a wide variety of receptors. All these characteristics, together with the fact that primary cultures from bovine adrenal glands or chromaffin cells from the tumor pheochromocytoma cell line PC12 are easy to culture, make them an ideal model to study neurotoxic mechanisms and neuroprotective drugs. In the first part of this review, we will analyze the different cytotoxicity models related to calcium dyshomeostasis and neurodegenerative disorders like Alzheimer's or Parkinson's. Along the second part of the review, we describe how different classes of drugs have been evaluated in chromaffin cells to determine their neuroprotective profile in different neurodegenerative-related models.

Melatonin is an indoleamine produced mainly in the pineal gland. The natural decline of melatonin levels with aging strongly contributes to the development of neurodegenerative disorders. Pleiotropic actions displayed by melatonin prevent several processes involved in neurodegeneration such as neuroinflammation, oxidative stress, excitotoxicity and/or apoptosis. This review focuses on a number of melatonin hybrids resulting from the juxtaposition of tacrine, berberine, tamoxifen, curcumin, N,N-dibenzyl(N-methyl)amine, among others, with potential therapeutic effects for the treatment of neurodegenerative diseases.

Stroke represents one of the most common causes of brain's vulnerability for many millions of people worldwide. The plethora of physiopathological events associated with brain ischemia are regulate through multiple signaling pathways leading to the activation of oxidative stress process, Ca dyshomeostasis, mitochondrial dysfunction, proinflammatory mediators, excitotoxicity and/or programmed neuronal cell death. Understanding this cascade of molecular events is mandatory in order to develop new therapeutic strategies for stroke. In this review article, we have highlighted the pleiotropic effects of melatonin to counteract the multiple processes of the ischemic cascade. Additionally, experimental evidence supports its actions to ameliorate ischemic long-term behavioural and neuronal deficits, preserving the functional integrity of the blood-brain barrier, inducing neurogenesis and cell proliferation through receptor-dependent mechanism, as well as improving synaptic transmission. Consequently, the synthesis of melatonin derivatives designed as new multitarget-directed products has focused a great interest in this area. This latter has been reinforced by the low cost of melatonin and its reduced toxicity. Furthermore, its spectrum of usages seems to be wide and with the potential for improving human health. Nevertheless, the molecular and cellular mechanisms underlying melatonin¿s actions need to be further exploration and accordingly, new clinical studies should be conducted in human patients with ischemic brain pathologies., CdlR thanks IS Carlos III, research contract under Miguel Servet Program (CP10/00531) and FIS project (grant PI13/00789IS) for support. JE thanks IS Carlos III, research contract under Miguel Servet Program (CP14/00008) for support.

KEY POINTS Upon repeated application of short ACh pulses to C57BL6J mouse chromaffin cells, the amperometrically monitored secretory responses promptly decayed to a steady-state level of around 25% of the initial response. A subsequent K+ pulse, however, overcame such decay. These data suggest that mouse chromaffin cells have a ready release-vesicle pool that is selectively recruited by the physiological neurotransmitter ACh. The ACh-sensitive vesicle pool is refilled and maintained by the rate of Ca2+ delivery from mitochondria to the cytosol, through the mitochondrial Na+ /Ca2+ exchanger (mNCX). ITH12662, a novel blocker of the mNCX, prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca2+ ]c clearance. This regulatory pathway may be physiologically relevant in situations of prolonged stressful conflicts where a sustained catecholamine release is regulated by mitochondrial Ca2+ circulation through the mNCX, which couples respiration and ATP synthesis to long-term stimulation of chromaffin cells by endogenously released ACh. ABSTRACT Using caged-Ca2+ photorelease or paired depolarising pulses in voltage-clamped chromaffin cells (CCs), various pools of secretory vesicles with different readiness to undergo exocytosis have been identified. Whether these pools are present in unclamped CCs challenged with ACh, the physiological neurotransmitter at the splanchnic nerve-CC synapse, is unknown. We have explored here whether an ACh-sensitive ready-release vesicle pool (ASP) is present in C57BL6J mouse chromaffin cells (MCCs). Single cells were fast perfused with a Tyrode solution at 37°C, and challenged with 12 sequential ACh pulses (100 μm, 2 s, every 30 s) plus a K+ pulse given at the end (75 mm K+ ). After the first 2-3 ACh pulses the amperometrically monitored secretory responses promptly decayed to a steady-state level of around 25% of the initial response. The last K+ pulse, however, overcame such decay. Repeated ACh pulses to voltage-clamped cells elicited non-desensitising nicotinic currents. Also, the [Ca2+ ]c transients elicited by repeated ACh pulses that were superimposed on a stable baseline elevation did not undergo decay. The novel blocker of the mitochondrial Na+ /Ca2+ exchanger (mNCX) ITH12662 prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca2+ ]c clearance. The experiments are compatible with the idea that C57BL6J MCCs have an ASP vesicle pool that is selectively recruited by the physiological neurotransmitter ACh and is regulated by the rate of Ca2+ delivery from mitochondria to the cytosol, through the mNCX.

New tritarget small molecules combining Ca

Vesicular monoamine transporter-2 is expressed in the presynaptic secretory vesicles membrane in the brain. Its blockade by tetrabenazine (TBZ) causes depletion of dopamine at striatal basal ganglia; this is the mechanism underlying its long-standing use in the treatment of Huntington’s disease. In the frame of a project aimed at investigating the kinetics of exocytosis from vesicles with partial emptying of their neurotransmitter, we unexpectedly found that TBZ facilitates exocytosis; thus, we decided to characterize such effect. We used bovine chromaffin cells (BCCs) challenged with repeated pulses of high K+. Upon repeated K+ pulsing, the exocytotic catecholamine release responses were gradually decaying. However, when cells were exposed to TBZ, responses were mildly augmented and decay rate delayed. Facilitation of exocytosis was not due to Ca2+ entry blockade through voltage-activated calcium channels (VACCs) because, in fact, TBZ mildly blocked the whole-cell Ca2+ current. However, TBZ mimicked the facilitatory effects of exocytosis elicited by BayK8644 (L-subtype VACC agonist), an effect blocked by nifedipine (VACC antagonist). On the basis that TBZ augmented the secretory responses to caffeine (but not those of histamine), we monitored its effects on cytosolic Ca2+ elevations ([Ca2+]c) triggered by caffeine or histamine. While the responses to caffeine were augmented twice by TBZ, those of histamine were unaffected; the same happened in rat cortical neurons. Hence, we hypothesize that TBZ facilitates exocytosis by increasing Ca2+ release through the endoplasmic reticulum ryanodine receptor channel (RyR). Confirming this hypothesis are docking results, showing an interaction of TBZ with RyRs. This is consonant with the existence of a healthy Ca2+-induced-Ca2+-release mechanism in BCCs. SIGNIFICANCE STATEMENT A novel mechanism of action for tetrabenazine (TBZ), a drug used in the therapy of Huntington’s disease (HD), is described here. Such mechanism consists of facilitation by combining TBZ with the ryanodine receptor of the endoplasmic reticulum, thereby increasing Ca2+-induced Ca2+ release. This novel mechanism should be taken into account when considering the efficacy and/or safety of TBZ in the treatment of chorea associated with HD and other disorders. Additionally, it could be of interest in the development of novel medicines to treat these pathological conditions.

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-3β), 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-3β inhibitory activity. Moreover, the easily affordable polycyclic core appears as a new appealing privileged structure in medicinal chemistry.

Tacrine was the first drug approved for the treatment of Alzheimer's disease (AD) in 1993, which was withdrawn in 2013 due to its hepatotoxicity. However, new, non-hepatotoxic tacrine derivatives have been constantly searched for. In this context, since 1997, we have prepared a number of diversely functionalized tacrines by changing the benzene ring present in tacrine to five- or six-membered aromatic ring cores that could present anticholinesterasic activity and additional pharmacological properties. The new compounds were designed as juxtaposed structures between tacrine and the well-known Ca antagonists 1,4-dihydropyridines, with the goal of obtaining multi-target directed ligands for AD. In this account, we present our results on the PyridoTacrine (PyrTac) family of tacrine analogues, resulting from the substitution of the benzene ring by a pyridine. We highlight their pharmacological profile and review similar analogues in the literature. A first set of PyrTac showed inhibitory activity of cholinesterases (ChE) and a blocking profile of voltage-gated Ca channels (VGCC). A second family with improved ChE inhibition lost VGCC blocking activity. However, the lead compound of this family (5f) presented an activating profile of the phosphatase 2A (PP2A) and showed interesting outcomes in experimental in vivo models of AD and stroke. We have identified the PyrTac ethyl 5-amino-2-methyl-6,7,8,9-tetrahydrobenzo[b] [1,8]naphthyridine-3-carboxylate (5f), which presents additional pharmacological properties beyond the mere cholinergic improvement. These new properties warrant attention to 5f and its further development as a new potential therapeutic agent for AD therapy., The authors thank all the co-workers for their effort, commitment and contribution for the synthesis and biological evaluation of the tacrine derivatives shown in this account.

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.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.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.

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. ispartof: Bioorganic Chemistry vol:100 ispartof: location:United States status: published

Hybrid systems based on clay minerals and 7-azaindole derivatives, which show an interesting neuroprotective profile in several in vitro models of neurodegeneration, were developed in order to facilitate the oral administration of these drugs in the treatment of Alzheimer's disease and provide a controlled release. Two 7-azaindole derivatives with different substitution at position 5 (-OCH3, -H) were synthesized and adsorbed on nanocarriers of different morphology, the lamellar clay montmorillonite (MMT) and the halloysite nanotubes (HNT). The drugs intercalation in MMT was confirmed by X-ray diffraction, obtaining basal spacing values around 1.9–2.0 nm in the hybrids, with a drug loading around 31–36 g per 100 g of MMT, while the HNT-based hybrids show an uptake of 15–21 g per 100 g of HNT. Studies carried out in cultures of human neuroblastoma cells confirmed the lack of toxicity of the hybrids and their neuroprotective effect against okadaic acid (OA), the inhibitor of the protein phosphatase 2A (PP2A), similarly to the non-encapsulated drugs. The release assays carried out in systems that simulate the gastrointestinal tract show a rapid release of the drugs from the hybrid materials, making necessary their encapsulation in a protective biopolymer matrix to slow down the release kinetics. A blend of alginate and zein was chosen for this purpose, exploiting the gelation of alginate with calcium ions to process the material as microbeads and the hydrophobic character of zein to reduce the water uptake of the material. Therefore, these bionanocomposite systems integrating the drug/clay hybrids seem promising for the oral administration of drugs with multitarget activity for the treatment of Alzheimer's disease.

Resumen del trabajo presentado al 5th Symposium on Biomedical Research: "Advances and Perspectives In Pharmacology, Drug Toxicity and Pharmacogenetics", celebrado en Madrid del 15 al 16 de marzo de 2018.

The 1,8-naphthyridine CR80 (ethyl 5-amino-2-methyl-6,7,8,9-tetrahydrobenzo[b] [1,8]naphthyridine-3-carboxylate) has shown interesting neuroprotective properties in in vitro and in vivo models of neurodegeneration. In spite of these promising outcomes, the molecular and cellular mechanisms underlying CR80 actions need to be further explored. Materials & methods: We herein report the signal transduction pathways involved in developmental, neuroprotective and stress-activated processes, as well as the gene expression regulation by CR80 in SH-SY5Y neuroblastoma cells. Results: The CR80 exposure upregulated several antioxidant enzymes (HO-1, GSR, SQSTM1, and TRXR1) and anti-apoptotic proteins (Bcl-xL, Bcl-2, P21, and Wnt6). Conclusion: The observed changes in gene expression would afford new insights on the neuroprotective profile of CR80.

The benzothiazepine CGP37157 has shown neuroprotective effects in several in vitro models of excitotoxicity involving dysregulation of intracellular Ca2+ homeostasis. Although its mechanism of neuroprotection is unclear, it is probably related with some of its effects on Ca2+ homeostasis. CGP37157 is a well-known inhibitor of the mitochondrial Na+/Ca2+ exchanger (mNCX). However, it is not very specific and also blocks several other Ca2+ channels and transporters, including voltage-gated Ca2+ channels, plasma membrane Na+/Ca2+ exchanger and the Ca2+ homeostasis modulator 1 channel (CALHM1). In the present work, we have studied if CGP37157 could also induce changes in life expectancy. We now report that CGP37157 extends C. elegans lifespan by 10%–15% with a bell-shaped concentration-response, with high concentrations producing no effect. The effect was even larger (25% increase in life expectancy) in worms fed with heat-inactivated bacteria. The worm CGP37157 concentration producing maximum effect was measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and was close to the IC50 for inhibition of the Na+/Ca2+ exchanger. CGP37157 also extended the lifespan in eat-2 mutants (a model for caloric restriction), suggesting that caloric restriction is not involved in the mechanism of lifespan extension. Actually, CGP37157 produced no effect in mutants of the TOR pathway (daf15/unc24) or the insulin/insulin-like growth factor-1 (IGF-1) pathway (daf-2), indicating that the effect involves these pathways. Moreover, CGP37157 was also ineffective in nuo-6 mutants, which have a defect in the mitochondrial respiratory chain complex I. Since it has been described that neuroprotection by this compound in cell cultures is abolished by mitochondrial inhibitors, this suggests that life extension in C. elegans and neuroprotection in cell cultures may share a similar mechanism involving mitochondria., This work was supported by grants from the Spanish Ministry of Economy, Industry and Competitiveness, Government of Spain (Ministerio de Economía, Industria y Competitividad, Gobierno de España) to MM and JA (BFU2014-55731-R and BFU2017-83509-R), projects co-financed by the European Union through the European Regional Development Fund, a grant from the Ministry of Education, Board of Castilla y León (Consejería de Educación, Junta de Castilla y León; VA011G18) to JA and a grant from Instituto de Salud Carlos III to CR (Proyectos de Investigación en Salud: PI16/01041, co-financed by FEDER). Some C. elegans strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). JA-V has a fellowship from Junta de Castilla y León (JCyL), co-financed by the Fondo Social Europeo (FSE). PG-C has a FPI fellowship from Ministry of Economy, Industry and Competitiveness.

CALHM1 is a Ca(2+) channel discovered in 2008, which plays a key role in the neuronal electrical activity, among other functions. However, there are no known efficient blockers able to modulate its Ca(2+) handling ability. We herein describe that benzothiazepine CGP37157 and its newly synthesized analogue ITH12575 reduced Ca(2+) influx through CALHM1 at low micromolar concentrations. These results could serve as a starting point for the development of more selective CALHM1 ligands using CGP37157 as a hit compound, which would help to study the physiological role of CALHM1 in the control of [Ca(2+)]cyt in excitable cells, as well as its implication in CNS 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.

N-Methyl-d-aspartate (NMDA) receptors are fundamental for the normal function of the central nervous system (CNS), and play an important role in memory and learning. Over-activation of these receptors leads to neuronal loss associated with major neurological disorders such as Parkinson's disease, Alzheimer's disease, schizophrenia, and epilepsy. In this study, 22 novel enantiopure bicyclic lactams were designed, synthesized, and evaluated as NMDA receptor antagonists. Most of the new compounds displayed NMDA receptor antagonism, and the most promising compound showed an IC value on the same order of magnitude as that of memantine, an NMDA receptor antagonist in clinical use for the treatment of Alzheimer's disease. Further biological evaluation indicated that this compound is brain permeable (determined by an in vitro assay) and non-hepatotoxic. All these results indicate that (3S,7aS)-7a-(4-chlorophenyl)-3-(4-hydroxybenzyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (compound 5 b) is a potential candidate for the treatment of pathologies associated with the over-activation of NMDA receptors., This work was supported by the FundaÇao para a Ciência e a Tecnologia (FCT, Portugal) through iMed.ULisboa (UID/DTP/04138/ 2013), and the research project PTDC/QUI-QUI/111664/2009. M.M.M.S. acknowledges FCT “Programa Operacional Potencial Humano” and the European Social Fund for the IF Program (IF/ 00732/2013). The authors also thank FCT fellowship SFRH/BD/ 117931/2016 (M. Espadinha). C.d.l.R. acknowledges financial support from the Instituto de Salud Carlos III, Spanish Ministry of Health (PI13/00789). M.I.R.-F. acknowledges financial support from the Spanish Ministry of Economy and Competitiveness (MINECO, grants SAF2012-31035 and SAF2015-64948-C2-1-R), partially financed by FEDER funds, and Consejo Superior de Investigaciones Cient&ficas (CSIC, grant PIE-201580E109).

ITH12246 (ethyl 5-amino-2-methyl-6,7,8,9-tetrahydrobenzo[b][1,8] naphthyridine-3-carboxylate) is a 1,8-naphthyridine described to feature an interesting neuroprotective profile in in vitro models of Alzheimer's disease. These effects were proposed to be due in part to a regulatory action on protein phosphatase 2A inhibition, as it prevented binding of its inhibitor okadaic acid. We decided to investigate the pharmacological properties of ITH12246, evaluating its ability to counteract the memory impairment evoked by scopolamine, a muscarinic antagonist described to promote memory loss, as well as to reduce the infarct volume in mice suffering phototrombosis. Prior to conducting these experiments, we confirmed its in vitro neuroprotective activity against both oxidative stress and Ca2+ overload-derived excitotoxicity, using SH-SY5Y neuroblastoma cells and rat hippocampal slices. Using a predictive model of blood-brain barrier crossing, it seems that the passage of ITH12246 is not hindered. Its potential hepatotoxicity was observed only at very high concentrations, from 0.1 mM. ITH12246, at the concentration of 10 mg/kg i.p., was able to improve the memory index of mice treated with scopolamine, from 0.22 to 0.35, in a similar fashion to the well-known Alzheimer's disease drug galantamine 2.5 mg/kg. On the other hand, ITH12246, at the concentration of 2.5 mg/kg, reduced the phototrombosis-triggered infarct volume by 67%. In the same experimental conditions, 15 mg/kg melatonin, used as control standard, reduced the infarct volume by 30%. All of these findings allow us to consider ITH12246 as a new potential drug for the treatment of neurodegenerative diseases, which would act as a multifactorial neuroprotectant.

This document is the unedited author's version of a Submitted Work that was subsequently accepted for publication in Journal of Medicinal Chemistry , copyright © American Chemical Society after peer review. To access the final edited and published work, see http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.6b00478, 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 Ca2+ 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 Ca2+ through VGCC, as measured by Fluo-4/AM and patch clamp techniques, and protected in Ca2+ 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, This work was supported by the following grant: Proyectos de Investigación en Salud (PI13/00789, IS Carlos III). R.L.C is granted by Universidad Autónoma de Madrid

Cholinesterase inhibitors participate in the maintenance of the levels of the neurotransmitter acetylcholine by inhibiting the enzymes implicated in its degradation, namely, butyrylcholinesterase and acetylcholinesterase. This pharmacological action has an important role in several diseases, including neurodegenerative diseases such as Alzheimer's.This article reviews recent advances in the development of cholinesterase enzyme inhibitors, covering the development of new chemical entities, new pharmaceutical formulations with known inhibitors or treatments in combination with other drug families.The development of cholinesterase inhibitors has to face several issues, including the fact that the principal indication for these drugs, Alzheimer's disease, is not currently believed to derivate from a cholinergic deficiency, although most of the drugs clinically used for these disease are cholinesterase inhibitors. Moreover, the adverse effects found when administering cholinesterase inhibitors limit their use in other diseases, such as gastrointestinal diseases, glaucoma, or analgesia.