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CK1δ is a serine-threonine kinase involved in several pathological conditions including neuroinflammation and neurodegenerative disorders like Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis. Specifically, it seems that an inhibition of CK1δ could have a neuroprotective effect in these conditions. Here, a series of [1,2,4]triazolo[1,5-a][1,3,5]triazines were developed as ATP-competitive CK1δ inhibitors. Both positions 2 and 5 have been explored leading to a total of ten compounds exhibiting IC50s comprised between 29.1 µM and 2.08 µM. Three of the four most potent compounds (IC50 < 3 µM) bear a thiophene ring at the 2 position. All compounds have been submitted to computational studies that identified the chain composed of at least 2 atoms (e.g., nitrogen and carbon atoms) at the 5 position as crucial to determine a key bidentate hydrogen bond with Leu85 of CK1δ. Most potent compounds have been tested in vitro, resulting passively permeable to the blood-brain barrier and, safe and slight neuroprotective on a neuronal cell model. These results encourage to further structural optimize the series to obtain more potent CK1δ inhibitors as possible neuroprotective agents to be tested on models of the above-mentioned neurodegenerative diseases.
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Alzheimer's disease (AD) involves a neurodegenerative process that has not yet been prevented, reversed, or stopped. Continuing with the search for natural pharmacological treatments, flavonoids are a family of compounds with proven neuroprotective effects and multi-targeting behavior. The American genus Dalea L. (Fabaceae) is an important source of bioactive flavonoids. In this opportunity, we tested the neuroprotective potential of three prenylated flavanones isolated from Dalea species in a new in vitro pre-clinical AD model previously developed by us. Our approach consisted in exposing neural cells to conditioned media (3xTg-AD ACM) from neurotoxic astrocytes derived from hippocampi and cortices of old 3xTg-AD mice, mimicking a local neurodegenerative microenvironment. Flavanone 1 and 3 showed a neuroprotective effect against 3xTg-AD ACM, being 1 more active than 3. The structural requirements to afford neuroprotective activity in this model are a 5'-dimethylallyl and 4'-hydroxy at the B ring. In order to search the mechanistic performance of the most active flavanone, we focus on the flavonoid-mediated regulation of GSK-3ß-mediated tau phosphorylation previously reported. Flavanone 1 treatment decreased the rise of hyperphosphorylated tau protein neuronal levels induced after 3xTg-AD ACM exposure and inhibited the activity of GSK-3ß. Finally, direct exposure of these neurotoxic 3xTg-AD astrocytes to flavanone 1 resulted in toxicity to these cells and reduced the neurotoxicity of 3xTg-AD ACM as well. Our results allow us to present compound 1 as a natural prenylated flavanone that could be used as a precursor to development and design of future drug therapies for AD.
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Enfermedad de Alzheimer , Flavanonas , Fármacos Neuroprotectores , Ratones , Animales , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Ratones Transgénicos , Proteínas tau/metabolismo , Flavanonas/farmacología , Flavanonas/uso terapéutico , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Modelos Animales de Enfermedad , Fosforilación , Péptidos beta-Amiloides/metabolismoRESUMEN
Multitarget ligands (MTLs) have emerged as an interesting alternative for addressing complex multifactorial pathologies such as neurodegenerative diseases. However, a common challenge associated with these compounds is often their high molecular weight and low solubility, which becomes a hurdle when trying to permeate over the blood-brain barrier (BBB). In this study, we have designed two new MTLs that modulate three pharmacological targets simultaneously (tau, beta-amyloid and TAR DNA-binding protein 43). To enhance their brain penetration, we have formulated organic polymeric nanoparticles using poly(lactic-co-glycolic acid). The characterization of the formulations, evaluation of their permeability through an in vitro BBB model, and assessment of their activity on disease-representative cellular models, such as Alzheimer's disease and amyotrophic lateral sclerosis, have been conducted. The results demonstrate the potential of the new MTLs and their nanoparticle encapsulation for the treatment of neurodegenerative diseases.
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Barrera Hematoencefálica , Enfermedades Neurodegenerativas , Permeabilidad , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Ligandos , Humanos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Nanopartículas/química , Diseño de Fármacos , Composición de Medicamentos , Péptidos beta-Amiloides/metabolismo , Animales , Proteínas tau/metabolismoRESUMEN
Amyotrophic lateral sclerosis is a fatal neurodegenerative disease without a cure to reverse its progression. Its main hallmark is the nuclear protein TDP-43, which undergoes different post-translational modifications leading to a loss of function in the nucleus and an increase in toxicity in the cytoplasm. Previous reports have indicated that pathogenic TDP-43 exhibits prion-like propagation in various contexts. With the aim of advancing therapeutics focused on preventing the propagation of TDP-43 pathology, we studied the potential role of pathogenic TDP-43 in lymphoblasts from sporadic ALS patients. We used lymphoblastoid cell lines from sporadic ALS patients as a source of pathogenic forms of TDP-43, and healthy human cells (lymphoblasts, myoblasts, neuroblastoma SH-SY5Y, or osteosarcoma U2OS) as recipient cells to investigate the seeding and spread of TDP-43 proteinopathy. Furthermore, we evaluated the potential of targeting TDP-43 phosphorylation with a CK-1 inhibitor to prevent the propagation of the pathology. The results presented herein indicate that pathogenic forms of TDP-43 are secreted into the extracellular medium of sporadic ALS lymphoblasts and could be transported by extracellular vesicles, spreading TDP-43 pathology to healthy cells. Moreover, tunneling nanotubes have also been discovered in pathological cells and may be involved in the transport of TDP-43. Interestingly, targeting TDP-43 phosphorylation with an in-house designed CK-1 inhibitor (IGS2.7) was sufficient to halt TDP-43 pathology transmission, in addition to its known effects on restoring the homeostasis of TDP-43 protein in patients-derived cells.
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Esclerosis Amiotrófica Lateral , Neuroblastoma , Enfermedades Neurodegenerativas , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Quinasa de la Caseína I , Proteínas de Unión al ADN/metabolismoRESUMEN
Cell division cycle 7 kinase (CDC7) has been found overexpressed in many cancer cell lines being also one of the kinases involved in the nuclear protein TDP-43 phosphorylation in vivo. Thus, inhibitors of CDC7 are emerging drug candidates for the treatment of oncological and neurodegenerative unmet diseases. All the known CDC7 inhibitors are ATP-competitives, lacking of selectivity enough for success in clinical trials. As allosteric sites are less conserved among kinase proteins, discovery of allosteric modulators of CDC7 is a great challenge and opportunity in this field.Using different computational approaches, we have here identified new druggable cavities on the human CDC7 structure and subsequently selective CDC7 inhibitors with allosteric modulation mainly targeting the pockets where the interaction between this kinase and its activator DBF4 takes place.
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Proteínas Nucleares , Proteínas Serina-Treonina Quinasas , Humanos , Fosforilación , Sitio Alostérico , Línea Celular , Ciclo Celular , Proteínas de Ciclo CelularRESUMEN
Introduction: TDP-43 proteinopathy in Alzheimer's disease (AD) patients is recently emerging as a relevant pathomolecular event that may have been overlooked. Recent results in immortalized lymphocytes from AD patients have shown not only an increase of post-translational modifications in TDP-43, such as hyperphosphorylation and fragmentation, but also its prionic behaviour and cell-to-cell disease transmission. With the main goal to advance therapeutic interventions, we present in this work different kinase inhibitors with potential to restore this pathological mechanism. Methodology: We have used immortalized lymphocytes from healthy controls and AD severe patients to evaluate the correction of TDP-43 pathology after the treatment with previously synthetized TTBK1 and CK1 inhibitors. Moreover we used the conditioned mediums of these cells to perform different disease propagation experiments. Results: TDP-43 pathology observed in lymphoblasts from severe AD patients is reduced after the treatment with TTBK1 and CK1 inhibitors (decreasing phosphorylation and increasing nuclear localisation), Furthermore, the significant increase in TDP-43 phosphorylation, cytoplasmic accumulation and aberrant F-actin protrusions (TNT-like structures) observed in control cells growing in CM from AD lymphoblasts were abolished when the CM from AD lymphoblasts treated with previously reported TTBK1 and CK1 inhibitors were used. In addition, the cytosolic transport mediated by molecular motors of the receptor cells was altered with the induced TDP-43 pathology, but it was not produced with the abovementioned pretreated CMs. Conclusion: TTBK1 and CK1 inhibitors, specially VNG1.47 and IGS2.7 compounds, restore TDP-43 pathology and avoid cell-to-cell propagation in immortalized lymphocytes from AD patients, being excellent candidates for the future therapy of this prevalent and devastating disease.
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INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder caused by motoneuron death with a median survival time of 3-5 years since disease onset. There are no effective treatments to date. However, a variety of innovative investigational drugs and biological-based therapies are under clinical development. AREAS COVERED: This review provides an overview of the clinical investigational small molecules as well as a brief summary of the biological-based therapies that are currently undergoing clinical trials for the treatment of ALS. All the data were obtained from ClinicalTrials.gov (registered through November 1). EXPERT OPINION: Drug discovery for ALS is an active and evolving field, where many investigational clinical drugs are in different trials. There are several mechanisms of action supporting all these new therapies, although proteostasis is gaining stage. Probably, small orally bioavailable molecules able to recover functional TDP-43 homeostasis may have solid chances to modify ALS progression.
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Esclerosis Amiotrófica Lateral , Humanos , Drogas en Investigación/farmacología , Drogas en Investigación/uso terapéutico , Descubrimiento de Drogas , Neuronas Motoras , Resultado del TratamientoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease without any effective treatment. Protein TDP-43 is a pathological hallmark of ALS in both sporadic and familiar patients. Post-translational modifications of TDP-43 promote its aggregation in the cytoplasm. Tau-Tubulin kinase (TTBK1) phosphorylates TDP-43 in cellular and animal models; thus, TTBK1 inhibitors emerge as a promising therapeutic strategy for ALS. The design, synthesis, biological evaluation, kinase-ligand complex structure determination, and molecular modeling studies confirmed novel pyrrolopyrimidine derivatives as valuable inhibitors for further development. Moreover, compound 29 revealed good brain penetration in vivo and was able to reduce TDP-43 phosphorylation not only in cell cultures but also in the spinal cord of transgenic TDP-43 mice. A shift to M2 anti-inflammatory microglia was also demonstrated in vivo. Both these activities led to motor neuron preservation in mice, proposing pyrrolopyrimidine 29 as a valuable lead compound for future ALS therapy.
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Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Inflamación/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Encéfalo/metabolismo , Estudios de Casos y Controles , Humanos , Inflamación/metabolismo , Inflamación/patología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Fosforilación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Ratas Wistar , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Distribución TisularRESUMEN
Amyotrophic Lateral Sclerosis (ALS) is the most common degenerative motor neuron disease in adults. About 97% of ALS patients present TDP-43 aggregates with post-translational modifications, such as hyperphosphorylation, in the cytoplasm of affected cells. GSK-3ß is one of the protein kinases involved in TDP-43 phosphorylation. Up-regulation of its expression and activity is reported on spinal cord and cortex tissues of ALS patients. Here, we propose the repurposing of Tideglusib, an in-house non-ATP competitive GSK-3ß inhibitor that is currently in clinical trials for autism and myotonic dystrophy, as a promising therapeutic strategy for ALS. With this aim we have evaluated the efficacy of Tideglusib in different experimental ALS models both in vitro and in vivo. Moreover, we observed that GSK-3ß activity is increased in lymphoblasts from sporadic ALS patients, with a simultaneous increase in TDP-43 phosphorylation and cytosolic TDP-43 accumulation. Treatment with Tideglusib decreased not only phospho-TDP-43 levels but also recovered its nuclear localization in ALS lymphoblasts and in a human TDP-43 neuroblastoma model. Additionally, we found that chronic oral treatment with Tideglusib is able to reduce the increased TDP-43 phosphorylation in the spinal cord of Prp-hTDP-43A315T mouse model. Therefore, we consider Tideglusib as a promising drug candidate for ALS, being proposed to start a clinical trial phase II by the end of the year.
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Proteínas de Unión al ADN/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Tiadiazoles/farmacología , Anciano , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/fisiología , Modelos Animales de Enfermedad , Reposicionamiento de Medicamentos , Glucógeno Sintasa Quinasa 3 beta/fisiología , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Neuronas Motoras/metabolismo , Preparaciones Farmacéuticas/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Médula Espinal/metabolismoRESUMEN
A potent cell division cycle 7 (CDC7) kinase inhibitor, known as PHA-767491, has been described to reduce the transactive response DNA binding protein of 43 KDa (TDP-43) phosphorylation in vitro and in vivo, which is one of the main proteins found to aggregate and accumulate in the cytoplasm of motoneurons in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. However, the main drawback of this compound is its low permeability to the central nervous system (CNS), limiting its use for the treatment of neurological conditions. In this context, the use of drug delivery systems like nanocarriers has become an interesting approach to improve drug release to the CNS. In this study, we prepared and characterized biodegradable nanoparticles in order to encapsulate PHA-767491 and improve its permeability to the CNS. Our results demonstrate that poly (lactic-co-glycolic acid) (PLGA) nanoparticles with an average radius between 145 and 155 nm could be used to entrap PHA-767491 and enhance the permeability of this compound through the blood-brain barrier (BBB), becoming a promising candidate for the treatment of TDP-43 proteinopathies such as ALS.
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TDP-43 has been identified as the major component of protein aggregates found in affected neurons in FTLD-TDP and amyotrophic lateral sclerosis (ALS) patients. TDP-43 is hyperphosphorylated, ubiquitinated, and cleaved in the C-terminus. CDC-7 was reported to phosphorylate TDP-43. There are no effective treatments for either FTLD-TDP or ALS, being a pressing need for the search of new therapies. We hypothesized that modulating CDC-7 activity with small molecules that are able to interfere with TDP-43 phosphorylation could be a good therapeutic strategy for these diseases. Here, we have studied the effects of novel brain penetrant, thiopurine-based, CDC-7 inhibitors in TDP-43 homeostasis in immortalized lymphocytes from FTLD-TDP patients, carriers of a loss-of-function GRN mutation, as well as in cells derived from sporadic ALS patients. We found that selective CDC-7 inhibitors, ERP1.14a and ERP1.28a, are able to decrease the enhanced TDP-43 phosphorylation in cells derived from FTLD-TDP and ALS patients and to prevent cytosolic accumulation of TDP-43. Moreover, treatment of FTLD-TDP lymphoblasts with CDC-7 inhibitors leads to recovering the nuclear function of TDP-43-inducing CDK6 repression. We suggest that CDC-7 inhibitors, mainly the heterocyclic compounds here shown, may be considered as promising drug candidates for the ALS/FTD spectrum.
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Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/metabolismo , Degeneración Lobar Frontotemporal/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Anciano , Células Cultivadas , Proteínas de Unión al ADN/efectos de los fármacos , Femenino , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no known cure. Aggregates of the nuclear protein TDP-43 have been recognized as a hallmark of proteinopathy in both familial and sporadic cases of ALS. Post-translational modifications of this protein, include hyperphosphorylation, cause disruption of TDP-43 homeostasis and as a consequence, promotion of its neurotoxicity. Among the kinases involved in these changes, cell division cycle kinase 7 (CDC7) plays an important role by directly phosphorylating TDP-43. In the present manuscript the discovery, synthesis, and optimization of a new family of selective and ATP-competitive CDC7 inhibitors based on 6-mercaptopurine scaffold are described. Moreover, we demonstrate the ability of these inhibitors to reduce TDP-43 phosphorylation in both cell cultures and transgenic animal models such as C. elegans and Prp-hTDP43 (A315T) mice. Altogether, the compounds described here may be useful as versatile tools to explore the role of CDC7 in TDP-43 phosphorylation and also as new drug candidates for the future development of ALS therapies.
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Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Ratones Transgénicos , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Common copathogenic factors, including oxidative stress and neuroinflammation, are found to play a vital role in the development of neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD). Nowadays, owing to the multifactorial character of the diseases, no effective therapies are available, thus underlying the need for new strategies. Overexpression of the enzyme GSK-3ß and downregulation of the Nrf2/ARE pathway are responsible for a decrease in antioxidant defense effects. These pieces of evidence underline the usefulness of dual GSK-3ß inhibitors/Nrf2 inducers. In this regard, to design a dual modulator, the structures of a curcumin-based analogue, as GSK-3ß inhibitor, and a diethyl fumarate fragment, as Nrf2 inducer, were combined. Among the hybrids, 5 and 6 proved to effectively inhibit GSK-3ß, while 4 and 5 showed a marked ability to activate Nrf2 together to increase the neuronal resistance to oxidative stress. These last pieces of evidence translated into specific neuroprotective effects of 4 and 5 against PD pathological events including neurotoxicity elicited by α-synuclein aggregates and 6-hydroxydopamine. Hybrid 5 also showed neuroprotective effects in a C. elegans model of PD where the activation of GSK-3ß is intimately involved in Nrf2 regulation. In summary, 5 emerged as an interesting multitarget derivative, valuable to be exploited in a multitarget PD perspective.
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Curcumina , Enfermedad de Parkinson , Animales , Caenorhabditis elegans , Curcumina/farmacología , Fumaratos , Glucógeno Sintasa Quinasa 3 beta , Factor 2 Relacionado con NF-E2 , Enfermedad de Parkinson/tratamiento farmacológicoRESUMEN
Pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating disease where no treatment exists, involves the compartmentalization of the nuclear protein TDP-43 (TAR DNA-binding protein 43) in the cytoplasm which is promoted by its aberrant phosphorylation and others posttranslational modifications. Recently, it was reported that CK-1δ (protein casein kinase-1δ) is able to phosphorylate TDP-43. Here, the preclinical efficacy of a benzothiazole-based CK-1δ inhibitor IGS-2.7, both in a TDP-43 (A315T) transgenic mouse and in a human cell-based model of ALS, is shown. Treatment with IGS-2.7 produces a significant preservation of motor neurons in the anterior horn at lumbar level, a decrease in both astroglial and microglial reactivity in this area, and in TDP-43 phosphorylation in spinal cord samples. Furthermore, the recovery of TDP-43 homeostasis (phosphorylation and localization) in a human-based cell model from ALS patients after treatment with IGS-2.7 is also reported. Moreover, we have shown a trend to increase in CK-1δ mRNA in spinal cord and significantly in frontal cortex of sALS cases. All these data show for the first time the in vivo modulation of TDP-43 toxicity by CK-1δ inhibition with IGS-2.7, which may explain the benefits in the preservation of spinal motor neurons and point to the relevance of CK-1δ inhibitors in a future disease-modifying treatment for ALS.
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Esclerosis Amiotrófica Lateral/patología , Quinasa Idelta de la Caseína/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Neuronas Motoras/citología , Inhibidores de Proteínas Quinasas/farmacología , Médula Espinal/citología , Anciano , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Fosforilación , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismoRESUMEN
Dynamic combinatorial chemistry (DCC) has proven its potential in drug discovery speeding the identification of modulators of biological targets. However, the exchange chemistries typically take place under specific reaction conditions, with limited tools capable of operating under physiological parameters. Here we report a catalyzed protein-directed DCC working at low temperatures that allows the calcium sensor NCS-1 to find the best ligands in situ. Ultrafast NMR identifies the reaction intermediates of the acylhydrazone exchange, tracing the molecular assemblies and getting a real-time insight into the essence of DCC processes at physiological pH. Additionally, NMR, X-ray crystallography and computational methods are employed to elucidate structural and mechanistic aspects of the molecular recognition event. The DCC approach leads us to the identification of a compound stabilizing the NCS-1/Ric8a complex and whose therapeutic potential is proven in a Drosophila model of disease with synaptic alterations.
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Calcio/metabolismo , Biblioteca de Genes , Proteínas Sensoras del Calcio Neuronal/metabolismo , Animales , Catálisis , Células Cultivadas , Técnicas Químicas Combinatorias , Drosophila/fisiología , Imagen por Resonancia Magnética , Masculino , Membranas Artificiales , Ratones , Proteínas Sensoras del Calcio Neuronal/genética , Neuronas/metabolismo , Palmitoil-CoA Hidrolasa , Permeabilidad , Conformación Proteica , ProteínasRESUMEN
Twelve derivatives 1a-1m of the ß-crinane-type alkaloid haemanthamine were developed. All the semisynthetic derivatives were studied for their inhibitory potential against both acetylcholinesterase and butyrylcholinesterase. In addition, glycogen synthase kinase 3ß (GSK-3ß) inhibition potency was evaluated in the active derivatives. In order to reveal the availability of the drugs to the CNS, we elucidated the potential of selected derivatives to penetrate through the blood-brain barrier (BBB). Two compounds, namely 11-O-(2-methylbenzoyl)-haemanthamine (1j) and 11-O-(4-nitrobenzoyl)-haemanthamine (1m), revealed the most intriguing profile, both being acetylcholinesterase (hAChE) inhibitors on a micromolar scale, with GSK-3ß inhibition properties, and predicted permeation through the BBB. In vitro data were further corroborated by detailed inspection of the compounds' plausible binding modes in the active sites of hAChE and hBuChE, which led us to provide the structural determinants responsible for the activity towards these enzymes.
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Enfermedad de Alzheimer/metabolismo , Alcaloides de Amaryllidaceae/química , Alcaloides de Amaryllidaceae/metabolismo , Amaryllidaceae/química , Amaryllidaceae/metabolismo , Fenantridinas/química , Fenantridinas/metabolismo , Barrera Hematoencefálica/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Ligandos , Modelos Moleculares , Conformación Molecular , Simulación del Acoplamiento Molecular , Estructura Molecular , Permeabilidad , Relación Estructura-ActividadRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disorder of still unknown etiology that results in loss of motoneurons, paralysis, and death, usually between 2 and 4 years from onset. There are no currently available ALS biomarkers to support early diagnosis and to facilitate the assessment of the efficacy of new treatments. Since ALS is considered a multisystemic disease, here we have investigated the usefulness of immortalized lymphocytes from sporadic ALS patients to study TDP-43 homeostasis as well as to provide a convenient platform to evaluate TDP-43 phosphorylation as a novel therapeutic approach for ALS. We report here that lymphoblasts from ALS patients recapitulate the hallmarks of TDP-43 processing in affected motoneurons, such as increased phosphorylation, truncation, and mislocalization of TDP-43. Moreover, modulation of TDP-43 by an in-house designed protein casein kinase-1δ (CK-1δ) inhibitor, IGS3.27, reduced phosphorylation of TDP-43, and normalized the nucleo-cytosol translocation of TDP-43 in ALS lymphoblasts. Therefore, we conclude that lymphoblasts, easily accessible cells, from ALS patients could be a useful model to study pathological features of ALS disease and a suitable platform to test the effects of potential disease-modifying drugs even in a personalized manner.
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Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Proteínas de Unión al ADN/metabolismo , Linfocitos/metabolismo , Linfocitos/patología , Estudios de Casos y Controles , Línea Celular Transformada , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Numerous studies have highlighted the implications of the glycogen synthase kinase 3 (GSK-3) in several processes associated with Alzheimer's disease (AD). Therefore, GSK-3 has become a crucial therapeutic target for the treatment of this neurodegenerative disorder. Hereby, we report the design and multistep synthesis of ethyl 4-oxo-pyrazolo[4,3-d][1-3]triazine-7-carboxylates and their biological evaluation as GSK-3 inhibitors. Molecular modelling studies allow us to develop this new scaffold optimising the chemical structure. Potential binding mode determination in the enzyme and the analysis of the key features in the catalytic site are also described. Furthermore, the ability of pyrazolotriazinones to cross the blood-brain barrier (BBB) was evaluated by passive diffusion and those who showed great GSK-3 inhibition and permeation to the central nervous system (CNS) showed neuroprotective properties against tau hyperphosphorylation in a cell-based model. These new brain permeable pyrazolotriazinones may be used for key in vivo studies and may be considered as new leads for further optimisation for the treatment of AD.
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Diseño Asistido por Computadora , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Triazinas/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Cinética , Modelos Moleculares , Estructura Molecular , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Ácido Ocadaico/farmacología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirazoles/química , Relación Estructura-Actividad , Triazinas/química , Proteínas tau/antagonistas & inhibidores , Proteínas tau/metabolismoRESUMEN
Overcoming the lack of effective treatments and the continuous clinical trial failures in neurodegenerative drug discovery might require a shift from the prevailing paradigm targeting pathogenesis to the one targeting simultaneously neuroprotection and neuroregeneration. In the studies reported herein, we sought to identify small molecules that might exert neuroprotective and neuroregenerative potential as tools against neurodegenerative diseases. In doing so, we started from the reported neuroprotective/neuroregenerative mechanisms of psychotropic drugs featuring a tricyclic alkylamine scaffold. Thus, we designed a focused-chemical library of 36 entries aimed at exploring the structural requirements for efficient neuroprotective/neuroregenerative cellular activity, without the manifestation of toxicity. To this aim, we developed a synthetic protocol, which overcame the limited applicability of previously reported procedures. Next, we evaluated the synthesized compounds through a phenotypic screening pipeline, based on primary neuronal systems. Phenothiazine 2Bc showed improved neuroregenerative and neuroprotective properties with respect to reference drug desipramine (2Aa). Importantly, we have also shown that 2Bc outperformed currently available drugs in cell models of Alzheimer's and Parkinson's diseases and attenuates microglial activation by reducing iNOS expression.
Asunto(s)
Descubrimiento de Drogas/métodos , Regeneración Nerviosa/efectos de los fármacos , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Psicotrópicos/química , Psicotrópicos/farmacología , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiología , Relación Dosis-Respuesta a Droga , Células Hep G2 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Regeneración Nerviosa/fisiología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , Ratas WistarRESUMEN
Several findings propose the altered tau protein network as an important target for Alzheimer's disease (AD). Particularly, two points of pharmacological intervention can be envisaged: inhibition of phosphorylating tau kinase GSK-3ß and tau aggregation process. On the basis of this consideration and on our interest in multitarget paradigms in AD, we report on the discovery of 2,4-thiazolidinedione derivatives endowed with such a profile. 28 and 30 displayed micromolar IC50 values toward GSK-3ß, together with the capacity of inhibiting AcPHF6 aggregation of 60% and 80% at 10 µM, respectively. In addition, they showed PAMPA-BBB permeability, together with a suitable cellular safety profile. 30 also displayed inhibition of both K18 and full-length tau aggregations. Finally, both compounds were able to improve cell viability in an okadaic acid-induced neurodegeneration cell model. To the best of our knowledge, 28 and 30 are the first balanced, nontoxic, dual-acting compounds hitting tau cascade at two different hubs.