Derivados de gramina con actividad dual sobre canales de calcio y serina/treonina fosfatasas: nueva estrategia multidiana para el tratamiento de la enfermedad de Alzheimer

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Farmacología y Terapéutica. Fecha de lectura: 02-12-2016Esta tesis tiene embargado el acceso al texto completo hasta el 02-06-2018Given the multifactorial nature of Alzheimer's disease, it exists an increasing interest in the development of neuroprotective drugs able to interact with two or more biological targets involved in the neurodegenerative process. The Ca2+ ion plays a central role in many cellular processes, so its intracellular concentration must be tightly regulated to avoid the outbreak of pathological events. Thus, compounds capable of regulating the intracellular Ca2+ signaling have been classically studied for the treatment of neurodegenerative diseases, such as Alzheimer's disease. Furthermore, one of the pathophysiological features of the disease is the neurofibrillary tangles, formed by hyperphosphorylated tau proteins. The imbalance in the phosphorylation of tau is due to, among other causes, a decrease in the activity of the Ser/Thr phosphatase enzymes that catalyze the removal of phosphate groups at tau, among which the phosphoprotein phosphatase 2A outstands. The research of this PhD dissertation aims the discovery of novel neuroprotective compounds, proposing an innovative strategy based on the simultaneous therapeutic approach over Ca2+ dyshomeostasis and Ser/Thr phosphatase activity. Taking the natural alkaloid gramine as hit compound, for its potential activity on both biological targets, the design and synthesis of a total of 44 compounds is presented, as well as the evaluation of their pharmacological activity. In general, gramine and its derivatives decrease the Ca2+ entry into the cell, measured by the Fluo- 4 fluorescent probe, presumably through a blockade of the voltage-gated Ca2+ channels, based on the results obtained by patch-clamp techniques. Moreover, they are able to maintain the Ser/Thr phosphatase activity, compromised by the presence of the selective inhibitor okadaic acid, being their affinity for PP2A enzyme confirmed by molecular docking studies. Finally, the dual activity of the compounds can protect neurons against toxic stimuli related to Alzheimer's disease, such as hyperphosphorylation of tau protein, oxidative stress, Ca2+ overload and excitotoxicity. Based on the obtained results, we propose this multitarget strategy as an innovative and promising approach for the treatment of Alzheimer's disease, being its modulation feasible through this new family of neuroprotective compounds derived from gramine.Dada la naturaleza multifactorial de la enfermedad de Alzheimer, existe un creciente interés en el desarrollo de compuestos neuroprotectores capaces de interaccionar con dos o más dianas biológicas involucradas en el proceso neurodegenerativo. El ión Ca2+ juega un papel central en muchos procesos celulares, por lo que su concentración intracelular debe estar estrechamente regulada, para evitar el desencadenamiento de eventos patológicos. Por esto, compuestos capaces de regular su señalización intracelular han sido clásicamente estudiados para el tratamiento de las enfermedades neurodegenerativas, como la enfermedad de Alzheimer. Por otro lado, una de las características histopatológicas de la enfermedad son los ovillos neurofibrilares, formados por la proteína tau hiperfosforilada. Este desequilibrio en los niveles de fosforilación de tau se debe, entre otras causas, a una disminución de la actividad de las enzimas Ser/Thr fosfatasas, responsables de catalizar la eliminación de los grupos fosfato, entre las que destaca la fosfoproteína fosfatasa 2A. La línea de investigación de esta Memoria de Tesis está dirigida al descubrimiento de nuevos compuestos neuroprotectores, proponiéndose una estrategia innovadora basada en el abordaje simultáneo de la dishomeostasis del Ca2+ y la actividad enzimática de las Ser/Thr fosfatasas. Tomando el alcaloide natural gramina como compuesto de partida, por su potencial actividad dual sobre ambas dianas de interés, se presenta el diseño y síntesis de un total de 44 compuestos, así como la evaluación de su actividad farmacológica. En general, la gramina y sus derivados disminuyen la entrada de Ca2+ al interior celular, medido mediante la sonda fluorescente Fluo-4, presumiblemente a través de un bloqueo de los CCDV, en base a los resultados obtenidos mediante técnicas de patch-clamp. Además, son capaces de mantener la actividad Ser/Thr fosfatasa, comprometida por la presencia del inhibidor AO, confirmándose su afinidad por la enzima PP2A mediante estudios de docking molecular. Por último, la actividad dual de los compuestos es capaz de proteger a las neuronas frente a estímulos tóxicos relacionados con la enfermedad de Alzheimer, como son la hiperfosforilación de la proteína tau, el estrés oxidativo, la sobrecarga de Ca2+ y la excitotoxicidad. En base a los resultados obtenidos, se propone esta estrategia multidiana como una aproximación innovadora y prometedora para el tratamiento de la enfermedad de Alzheimer, siendo su abordaje factible gracias a esta nueva familia de compuestos neuroprotectores derivada de gramina

Gramine derivatives targeting Ca2+ channels and Ser/Thr phosphatases: A new dual strategy for the treatment of neurodegenerative diseases

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.6b00478We 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 diseasesThis 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 Madri

PP2A ligand ITH12246 protects against memory impairment and focal cerebral ischemia in mice

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.Peer Reviewe

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

Synthesis, pharmacological assessment, and molecular modeling of acetylcholinesterase/butyrylcholinesterase inhibitors: Effect against amyloid-β-induced neurotoxicity

The synthesis, molecular modeling, and pharmacological analysis of phenoxyalkylamino-4-phenylnicotinates (2-7), phenoxyalkoxybenzylidenemalononitriles (12, 13), pyridonepezils (14-18), and quinolinodonepezils (19-21) are described. Pyridonepezils 15-18 were found to be selective and moderately potent regarding the inhibition of hAChE, whereas quinolinodonepezils 19-21 were found to be poor inhibitors of hAChE. The most potent and selective hAChE inhibitor was ethyl 6-(4-(1-benzylpiperidin-4-yl) butylamino)-5-cyano-2-methyl-4-phenylnicotinate (18) [IC50 (hAChE) = 0.25 ± 0.02 μM]. Pyridonepezils 15-18 and quinolinodonepezils 20-21 are more potent selective inhibitors of EeAChE than hAChE. The most potent and selective EeAChE inhibitor was ethyl 6-(2-(1-benzylpiperidin-4-yl)ethylamino)-5- cyano-2-methyl-4-phenylnicotinate (16) [IC50 (EeAChE) = 0.0167 ± 0.0002 μM], which exhibits the same inhibitory potency as donepezil against hAChE. Compounds 2, 7, 13, 17, 18, 35, and 36 significantly prevented the decrease in cell viability caused by Aβ1-42. All compounds were effective in preventing the enhancement of AChE activity induced by Aβ1-42. Compounds 2-7 caused a significant reduction whereas pyridonepezils 17 and 18, and compound 16 also showed some activity. The pyrazolo[3,4-b]quinolines 36 and 38 also prevented the upregulation of AChE induced by Aβ1-42. Compounds 2, 7, 12, 13, 17, 18, and 36 may act as antagonists of voltage sensitive calcium channels, since they significantly prevented the Ca2+ influx evoked by KCl depolarization. Docking studies show that compounds 16 and 18 adopted different orientations and conformations inside the active-site gorges of hAChE and hBuChE. The structural and energetic features of the 16-AChE and 18-AChE complexes compared to the 16-BuChE and 18-BuChE complexes account for a higher affinity of the ligand toward AChE. The present data indicate that compounds 2, 7, 17, 18, and 36 may represent attractive multipotent molecules for the potential treatment of Alzheimer's disease.Peer Reviewe