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Introduction: Proteolytic processing of amyloid protein precursor by ß-site secretase enzyme (BACE1) is dependent on the cellular lipid composition and is affected by endomembrane trafficking in dementia and Alzheimer's disease (AD). Stearoyl-CoA desaturase 1 (SCD1) is responsible for the synthesis of fatty acid monounsaturation (MUFAs), whose accumulation is strongly associated with cognitive dysfunction. Methods: In this study, we analyzed the relationship between BACE1 and SCD1 in vivo and in vitro neurodegenerative models and their association in familial AD (FAD), sporadic AD (SAD), and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) using microscopy, biochemical, and mass SPECT approach. Results: Our findings showed that BACE1 and SCD1 immunoreactivities were increased and colocalized in astrocytes of the hippocampus in a rat model of global cerebral ischemia (2-VO). A synergistic effect of double BACE1/SCD1 silencing on the recovery of motor and cognitive functions was obtained. This neuroprotective regulation involved the segregation of phospholipids (PLs) associated with polyunsaturated fatty acids in the hippocampus, cerebrospinal fluid, and serum. The double silencing in the sham and ischemic groups was stronger in the serum, inducing an inverse ratio between total phosphatydilcholine (PC) and lysophosphatidylcholine (LPC), represented mainly by the reduction of PC 38:4 and PC 36:4 and an increase in LPC 16:0 and LPC 18:0. Furthermore, PC 38:4 and PC:36:4 levels augmented in pathological conditions in in vitro AD models. BACE1 and SCD1 increases were confirmed in the hippocampus of FAD, SAD, and CADASIL. Conclusion: Therefore, the findings suggest a novel convergence of BACE-1 and SCD1 in neurodegeneration, related to pro-inflammatory phospholipids.
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Introduction: Alzheimer's disease (AD) is the main type of dementia, caused by the accumulation of amyloid plaques, formed by amyloid peptides after being processed from amyloid precursor protein (APP) by γ- and ß-secretases (BACE-1). Although amyloid peptides have been well established for AD, they have been found in other neurodegenerative diseases, such as Parkinson's disease, Lewy body dementia, and amyotrophic lateral sclerosis. Inhibitors of BACE-1 have been searched and developed, but clinical trials failed due to lack of efficacy or toxicity. Nevertheless, it is still considered a good therapeutic target, as it was proven to remove amyloid peptides and improve memory. Methods: In this work, we designed a peptide based on a sequence obtained from the marine fish Merluccius productus and evaluated it by molecular docking to verify its binding to BACE-1, which was tested experimentally by enzymatic kinetics and cell culture assays. The peptide was injected in healthy mice to study its pharmacokinetics and toxicity. Results: We could obtain a new sequence in which the first N-terminal amino acids and the last one bound to the catalytic site of BACE-1 and showed high stability and hydrophobicity. The synthetic peptide showed a competitive inhibition of BACE-1 and Ki = 94 nM, and when injected in differentiated neurons, it could reduce Aß42o production. In plasma, its half-life is â¼1 h, clearance is 0.0015 µg/L/h, and Vss is 0.0015 µg/L/h. The peptide was found in the spleen and liver 30 min after injection and reduced its level after that, when it was quantified in the kidneys, indicating its fast distribution and urinary excretion. Interestingly, the peptide was found in the brain 2 h after its administration. Histological analysis showed no morphological alteration in any organ, as well as the absence of inflammatory cells, indicating a lack of toxicity. Discussion: We obtained a new BACE-1 inhibitor peptide with fast distribution to the tissues, without accumulation in any organ, but found in the brain, with the possibility to reach its molecular target, BACE-1, contributing to the reduction in the amyloid peptide, which causes amyloid-linked neurodegenerative diseases.
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The accumulation of amyloid-ß (Aß) is the main event related to Alzheimer's disease (AD) progression. Over the years, several disease-modulating approaches have been reported, but without clinical success. The amyloid cascade hypothesis evolved and proposed essential targets such as tau protein aggregation and modulation of ß-secretase (ß-site amyloid precursor protein cleaving enzyme 1 - BACE-1) and γ-secretase proteases. BACE-1 cuts the amyloid precursor protein (APP) to release the C99 fragment, giving rise to several Aß peptide species during the subsequent γ-secretase cleavage. In this way, BACE-1 has emerged as a clinically validated and attractive target in medicinal chemistry, as it plays a crucial role in the rate of Aß generation. In this review, we report the main results of candidates in clinical trials such as E2609, MK8931, and AZD-3293, in addition to highlighting the pharmacokinetic and pharmacodynamic-related effects of the inhibitors already reported. The current status of developing new peptidomimetic, non-peptidomimetic, naturally occurring, and other class inhibitors are demonstrated, considering their main limitations and lessons learned. The goal is to provide a broad and complete approach to the subject, exploring new chemical classes and perspectives.
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Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Humanos , Secretases da Proteína Precursora do Amiloide/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Peptídeos beta-Amiloides/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêuticoRESUMO
Alzheimer's disease (AD) is a neurodegenerative, progressive, and fatal disorder characterized by marked atrophy of the cerebral cortex and loss of basal forebrain cholinergic neurons. The main pathological features of AD are related to neuronal degeneration and include extracellular deposition of amyloid beta plaques (Aß plaques), intracellular formation of neurofibrillary tangles (NFTs), and neuroinflammation. So far, drugs used to treat AD have symptomatic and palliative pharmacological effects, disappearing with continued use due to neuron degeneration and death. Therefore, there are still problems with an effective drug for treating AD. Few approaches evaluate the action of natural products other than alkaloids on the molecular targets of ß-amyloid protein (Aß protein) and/or tau protein, which are important targets for developing neuroprotective drugs that will effectively contribute to finding a prophylactic drug for AD. This review gathers and categorizes classes of natural products, excluding alkaloids, which in silico analysis (molecular docking) and in vitro and/or in vivo assays can inhibit the BACE1 and GSK-3ß enzymes involved in AD.
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Doença de Alzheimer , Produtos Biológicos , Humanos , Peptídeos beta-Amiloides/metabolismo , Glicogênio Sintase Quinase 3 beta , Simulação de Acoplamento Molecular , Secretases da Proteína Precursora do Amiloide/metabolismo , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Ácido Aspártico Endopeptidases/uso terapêutico , Doença de Alzheimer/metabolismo , Proteínas tau/metabolismo , Proteínas tau/uso terapêuticoRESUMO
Alzheimer's disease (AD) is a neurodegenerative pathology responsible for 70% of dementia cases worldwide. Despite its relevance, the few drugs available for the treatment of this disease offer only symptomatic relief, with limited efficacy and serious adverse effects. The most accepted hypothesis about the pathogenesis involves the aggregation and deposition of ß-amyloid peptides, mainly in the cerebral cortex and hippocampus, through the catalytic action of beta-secretase 1 (BACE-1), making this enzyme a promising target for the development of new drugs. In order to prioritize candidates for BACE-1 inhibitors, a hierarchical virtual screening by pharmacophore model and molecular docking was performed against the 216,833 molecules contained in several databases. Our previously built pharmacophore model was used for the first filtering step, which resulted in the selection of 399 molecules. The remaining molecules were filtered through molecular docking with GOLD 5.4.0. In this step, molecules with scoring values ââgreater than the mean plus standard deviation were evaluated for commercial availability and absence of asymmetric centers. Four molecules were selected and evaluated for mutagenic potential by the AMES test with the help of the pkCSM server. Finally, they were tested against the descriptors on Lipinski and Veber rules, and ZINC01589617 (QFIT = 56.52/Score = 44.95) satisfied all requirements, being subjected to molecular dynamics simulations (t = 100 ns) in order to obtain robust data on the mode of bonding and profile of intermolecular interactions. Those in silico strategies demonstrated that ZINC01589617 is a potential candidate for biological tests.Communicated by Ramaswamy H. Sarma.
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Doença de Alzheimer , Simulação de Dinâmica Molecular , Humanos , Simulação de Acoplamento Molecular , Secretases da Proteína Precursora do Amiloide , Doença de Alzheimer/tratamento farmacológicoRESUMO
Introduction: Alzheimer’s disease (AD) is the main type of dementia, caused by the accumulation of amyloid plaques, formed by amyloid peptides after being processed from amyloid precursor protein (APP) by γ- and ß-secretases (BACE-1). Although amyloid peptides have been well established for AD, they have been found in other neurodegenerative diseases, such as Parkinson’s disease, Lewy body dementia, and amyotrophic lateral sclerosis. Inhibitors of BACE-1 have been searched and developed, but clinical trials failed due to lack of efficacy or toxicity. Nevertheless, it is still considered a good therapeutic target, as it was proven to remove amyloid peptides and improve memory. Methods: In this work, we designed a peptide based on a sequence obtained from the marine fish Merluccius productus and evaluated it by molecular docking to verify its binding to BACE-1, which was tested experimentally by enzymatic kinetics and cell culture assays. The peptide was injected in healthy mice to study its pharmacokinetics and toxicity. Results: We could obtain a new sequence in which the first N-terminal amino acids and the last one bound to the catalytic site of BACE-1 and showed high stability and hydrophobicity. The synthetic peptide showed a competitive inhibition of BACE-1 and Ki = 94 nM, and when injected in differentiated neurons, it could reduce Aβ42o production. In plasma, its half-life is ∼1 h, clearance is 0.0015 μg/L/h, and Vss is 0.0015 μg/L/h. The peptide was found in the spleen and liver 30 min after injection and reduced its level after that, when it was quantified in the kidneys, indicating its fast distribution and urinary excretion. Interestingly, the peptide was found in the brain 2 h after its administration. Histological analysis showed no morphological alteration in any organ, as well as the absence of inflammatory cells, indicating a lack of toxicity. Discussion: We obtained a new BACE-1 inhibitor peptide with fast distribution to the tissues, without accumulation in any organ, but found in the brain, with the possibility to reach its molecular target, BACE-1, contributing to the reduction in the amyloid peptide, which causes amyloid-linked neurodegenerative diseases.
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Alzheimer's disease (AD) is characterized by the deposition of aggregated species of amyloid beta (Aß) in the brain, which leads to progressive cognitive deficits and dementia. Aß is generated by the successive cleavage of the amyloid precursor protein (APP), first by ß-site APP cleaving enzyme 1 (BACE1) and subsequently by the γ-secretase complex. Those conditions which enhace or reduce its clearance predispose to Aß aggregation and the development of AD. In vitro studies have demonstrated that Aß assemblies spark a feed-forward loop heightening Aß production. However, the underlying mechanism remains unknown. Here, we show that oligomers and fibrils of Aß enhance colocalization and physical interaction of APP and BACE1 in recycling endosomes of human neurons derived from induced pluripotent stem cells and other cell types, which leads to exacerbated amyloidogenic processing of APP and intracellular accumulation of Aß42. In cells that are overexpressing the mutant forms of APP which are unable to bind Aß or to activate Go protein, we have found that treatment with aggregated Aß fails to increase colocalization of APP with BACE1 indicating that Aß-APP/Go signaling is involved in this process. Moreover, inhibition of Gßγ subunit signaling with ßARKct or gallein prevents Aß-dependent interaction of APP and BACE1 in endosomes, ß-processing of APP, and intracellular accumulation of Aß42. Collectively, our findings uncover a signaling mechanism leading to a feed-forward loop of amyloidogenesis that might contribute to Aß pathology in the early stages of AD and suggest that gallein could have therapeutic potential.
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Alzheimer's disease (AD) is an age-related neurodegenerative disorder. Sever cognitive and memory impairments, huge increase in the prevalence of the disease, and lacking definite cure have absorbed worldwide efforts to develop therapeutic approaches. Since many drugs have failed in the clinical trials due to multifactorial nature of AD, symptomatic treatments are still in the center attention and now, nootropic medicinal plants have been found as versatile ameliorators to reverse memory disorders. In this work, anti-Alzheimer's activity of aqueous extract of areca nuts (Areca catechu L.) was investigated via in vitro and in vivo studies. It depicted good amyloid ß (Aß) aggregation inhibitory activity, 82% at 100 µg/mL. In addition, it inhibited beta-secretase 1 (BACE1) with IC50 value of 19.03 µg/mL. Evaluation of neuroprotectivity of the aqueous extract of the plant against H2O2-induced cell death in PC12 neurons revealed 84.5% protection at 1 µg/mL. It should be noted that according to our results obtained from Morris Water Maze (MWM) test, the extract reversed scopolamine-induced memory deficit in rats at concentrations of 1.5 and 3 mg/kg.
La enfermedad de Alzheimer (EA) es un trastorno neurodegenerativo relacionado con la edad. Los severos deterioros cognitivos y de la memoria, el enorme aumento de la prevalencia de la enfermedad y la falta de una cura definitiva han absorbido los esfuerzos mundiales para desarrollar enfoques terapéuticos. Dado que muchos fármacos han fallado en los ensayos clínicos debido a la naturaleza multifactorial de la EA, los tratamientos sintomáticos siguen siendo el centro de atención y ahora, las plantas medicinales nootrópicas se han encontrado como mejoradores versátiles para revertir los trastornos de la memoria. En este trabajo, se investigó la actividad anti-Alzheimer del extracto acuoso de nueces de areca (Areca catechu L.) mediante estudios in vitro e in vivo. Representaba una buena actividad inhibidora de la agregación de amiloide ß (Aß), 82% a 100 µg/mL. Además, inhibió la beta-secretasa 1 (BACE1) con un valor de CI50 de 19,03 µg/mL. La evaluación de la neuroprotección del extracto acuoso de la planta contra la muerte celular inducida por H2O2 en neuronas PC12 reveló una protección del 84,5% a 1 µg/mL. Cabe señalar que, de acuerdo con nuestros resultados obtenidos de la prueba Morris Water Maze (MWM), el extracto revirtió el déficit de memoria inducido por escopolamina en ratas a concentraciones de 1,5 y 3 mg/kg.
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Animais , Ratos , Areca/química , Extratos Vegetais/administração & dosagem , Doença de Alzheimer/tratamento farmacológico , beta-Amilase/antagonistas & inibidores , Peptídeos beta-Amiloides/efeitos dos fármacos , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/efeitos dos fármacos , Fármacos Neuroprotetores , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/efeitos dos fármacos , Doença de Alzheimer/enzimologia , Doença de Alzheimer/prevenção & controle , Teste do Labirinto Aquático de Morris , Medicina TradicionalRESUMO
Dysfunction in the neurovascular unit (NVU) is a key component in the progressive deterioration of Alzheimer's disease (AD) and is critical in vascular dementia. Recent studies have shown that inflammation plays early and perhaps causal roles in the pathogenesis of AD related to NVU damage, possibly in part by overactivating the aspartic acid protease activity of ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1), which until now has almost solely been studied in the context of the ß-amyloid cascade. In this study, we analyzed the relationship of BACE1 with astrocytes and blood vessels in human brains with sporadic and familial dementia [Autosomal dominant cerebral arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), sporadic Alzheimer's disease (SAD), and familial Alzheimer's disease (FAD)] and how BACE1 inhibition affects astrocytes and endothelial cells under conditions of glutamate toxicity. Our results show increased BACE1, PHF (Paired helical filaments)-tau and GFAP (Glial Fibrillary Acid Protein) immunoreactivity (IR) in the CA1 hippocampal regions of FAD and SAD brains. Furthermore, BACE1 immunoprecipitated with GFAP in tissue samples from all study cases, but their immunofluorescence close to (10 µm3) or overlapping blood vessels was only increased in FAD and SAD brains, and PHF-tau was present around the vessels mainly in FAD brains. Interestingly, the increased BACE1 levels were associated with reactive astrocytes, characterized by morphological changes and upregulation of GFAP under pathological and stressful conditions, and endothelial disruption by glutamate excitotoxicity, and these effects were reversed by BACE1 inhibition; further, BACE1-inhibited astrocytes protected endothelial cell integrity by preserving zonula occludens-1 (ZO-1) distribution and decreasing the expression of inflammatory markers. Taken together, these findings suggest that BACE1 dysregulation in astrocytes may have a role in the alterations in NVU integrity implicated in neurodegeneration.
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BACKGROUND: Alzheimer's disease (AD) involves an irreversible and progressive neurodegeneration, with multifactorial pathophysiology, including the cholinergic deficit, amyloid plaques, neurofibrillary tangles, oxidative stress, and neurodegeneration. Despite the severity of the disease, the therapeutic arsenal is limited, arousing the interest of researchers to search for substances that can act on these markers. OBJECTIVE: In this review, we highlight some relevant points, such as the ability of chalcones to act on different targets related to the pathophysiology of Alzheimer's disease; cholinesterases, amyloid peptide, beta-secretase and other biomarkers. METHOD: This mini-review covered the literature concerning chalcones bioactivity from 2010 until now. In addition to the theoretical review, we included the prediction of physicochemical properties using SwissADME software. RESULTS: We found that the majority of the chalcones have been tested against cholinesterases, with moderate to good potencies, but in recent years, the number of publications related to targets of the amyloid hypothesis has been growing. Regarding the physicochemical properties, chalcones have a good profile, except for the water solubility, which is not favorable. CONCLUSION: The most important characteristic of these molecules is that many of the examples mentioned here act on more than one target, characterizing them as multi-target compounds. Regarding predicted properties, solubility stands out as the most problematic one; however, these structures can incorporate functional groups that circumvent this problem of solubility without interfering in the biological activity.
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Doença de Alzheimer , Chalconas , Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides , Chalconas/farmacologia , Chalconas/uso terapêutico , Colinesterases , HumanosRESUMO
Alzheimer's disease (AD) is a neurodegenerative process that compromises cognitive functions. The physiopathology of AD is multifactorial and is mainly supported by the cholinergic and amyloid hypotheses, which allows the identification the fundamental role of some markers, such as the enzymes acetylcholinesterase (AChE) and ß-secretase (BACE-1), and the ß-amyloid peptide (Aß). In this work, we prepared a series of chalcones and 2'-aminochalcones, which were tested against AChE and BACE-1 enzymes and on the aggregation of Aß. All compounds inhibited AChE activity with different potencies. We have found that the majority of chalcones having the amino group are able to inhibit BACE-1, which was not observed for chalcones without this group. The most active compound is the one derived from 2,3-dichlorobenzaldeyde, having an IC50 value of 2.71 µM. A molecular docking study supported this result, showing a good interaction of the amino group with aspartic acid residues of the catalytic diade of BACE-1. Thioflavin-T fluorescence emission is reduced in 30 - 40%, when Aß42 is incubated in the presence of some chalcones under aggregation conditions. In vitro cytotoxicity and in silico prediction of pharmacokinetic properties were also conducted in this study.
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Chalconas/farmacologia , Inibidores da Colinesterase/farmacologia , Inibidores de Proteases/farmacologia , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Linhagem Celular Tumoral , Chalconas/síntese química , Chalconas/metabolismo , Chalconas/farmacocinética , Chlorocebus aethiops , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacocinética , Electrophorus , Humanos , Camundongos , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/metabolismo , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacocinética , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Células VeroRESUMO
Therapeutic drugs for Alzheimer's disease have been extensively studied due to its recurrence and abundance among neurodegenerative diseases. It is thought that the accumulation of amyloid precursor protein (APP) products, a consequence of an up-regulation of the ß-site APP-cleaving enzyme 1 (BACE1), is the main triggering mechanism during the early stages of the disease. This study aims to explore the ability of a multifunctional conjugate based on magnetite nanoparticles for the cellular delivery of siRNA against the expression of the BACE1 gene. We immobilized the siRNA strand on PEGylated magnetite nanoparticles and investigated the effects on biocompatibility and efficacy of the conjugation. Similarly, we co-immobilized the translocating protein OmpA on PEGylated nanoparticles to enhance cellular uptake and endosomal escape. BACE1 suppression was statistically significant in HFF-1 cells, without any presence of a cytotoxic effect. The delivery of the nanoconjugate was achieved through endocytosis pathways, where endosome formation was likely escaped due to the proton-sponge effect characteristic of PEGylated nanoparticles or mainly by direct translocation in the case of OmpA/PEGylated nanoparticles.
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Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Inativação Gênica , Nanopartículas de Magnetita/uso terapêutico , RNA Interferente Pequeno/uso terapêutico , Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Animais , Encéfalo/metabolismo , Linhagem Celular , Endocitose/fisiologia , Endossomos/metabolismo , Técnicas de Transferência de Genes , Humanos , Teste de MateriaisRESUMO
Increased levels of circulating fatty acids, such as palmitic acid (PA), are associated with the development of obesity, insulin resistance, type-2 diabetes and metabolic syndrome. Furthermore, these diseases are linked to an increased risk of cancer, cardiovascular diseases, mild cognitive impairment and even Alzheimer's disease (AD). However, the precise actions of elevated PA levels on neurons and their association with neuronal metabolic disruption that leads to the expression of pathological markers of AD, such as the overproduction and accumulation of the amyloid-ß peptide, represent an area of intense investigation. A possible molecular mechanism involved in the effects of PA may be through dysfunction of the NAD+ sensor enzyme, SIRT1. Therefore, the aim of the present study was to analyze the relationship between the effects of PA metabolism on the function of SIRT1 and the upregulation of BACE1 in cultured hippocampal neurons. PA reduced the total amount of NAD+ in neurons that caused an increase in p65 K310 acetylation due to inhibition of SIRT1 activity and low protein content. Furthermore, BACE1 protein and its activity were increased, and BACE1 was relocated in neurites after PA exposure.
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Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Hipocampo/metabolismo , NAD/metabolismo , Neurônios/metabolismo , Ácido Palmítico/farmacologia , Sirtuína 1/metabolismo , Acetilação , Animais , Ratos Wistar , Fator de Transcrição RelA/química , Fator de Transcrição RelA/metabolismo , Regulação para CimaRESUMO
In recent years, the design, development, and evaluation of several inhibitors of the BACE1 enzyme, as part of Alzheimer's treatment, have gathered the scientific community's interest. Here, a linear regression model was built using binding free energy calculations through the Bennett acceptance ratio method for 20 known inhibitors of the BACE1 enzyme, with a Pearson coefficient of R = 0.88 and R2 = 0.78. The validation of this model was verified employing eight additional random inhibitors, which also gave a linear correlation with R = 0.97 and R2 = 0.93. Furthermore, this linear regression model was also used for proposing the structure of four potential BACE1 inhibitors, and the most active of them gave a theoretical Kd = 10 nM. However, these molecules have not been synthesized yet. Our team used a total time of more than 800 ns for the Molecular Dynamics to carry out this study, and all the software used were freely available.
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Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Desenho de Fármacos , Modelos Teóricos , Inibidores de Proteases/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/metabolismo , Reprodutibilidade dos TestesRESUMO
The clinical diagnosis of Alzheimer's disease (AD) is a probabilistic formulation that may lack accuracy particularly at early stages of the dementing process. Abnormalities in amyloid-beta precursor protein (APP) metabolism and in the level of APP secretases have been demonstrated in platelets, and to a lesser extent in leukocytes, of AD patients, with conflicting results. The aim of the present study was to compare the protein level of the APP secretases A-disintegrin and metalloprotease 10 (ADAM10), Beta-site APP-cleaving enzyme 1 (BACE1), and presenilin-1 (PSEN1) in platelets and leukocytes from 20 non-medicated older adults with AD and 20 healthy elders, and to determine the potential use of these biomarkers to discriminate cases of AD from controls. The protein levels of all APP secretases were significantly higher in platelets compared to leukocytes. We found statistically a significant decrease in ADAM10 (52.5%, p < 0.0001) and PSEN1 (32%, p = 0.02) in platelets from AD patients compared to controls, but not in leukocytes. Combining all three secretases to generate receiver-operating characteristic (ROC) curves, we found a good discriminatory effect (AD vs. controls) when using platelets (the area under the curve-AUC-0.90, sensitivity 88.9%, specificity 66.7%, p = 0.003), but not in leukocytes (AUC 0.65, sensitivity 77.8%, specificity 50.0%, p = 0.2). Our findings indicate that platelets represent a better biological matrix than leukocytes to address the peripheral level of APP secretases. In addition, combining the protein level of ADAM10, BACE1, and PSEN1 in platelets, yielded a good accuracy to discriminate AD from controls.
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Proteína ADAM10/sangue , Doença de Alzheimer/sangue , Secretases da Proteína Precursora do Amiloide/sangue , Ácido Aspártico Endopeptidases/sangue , Plaquetas/química , Leucócitos/química , Proteínas de Membrana/sangue , Presenilina-1/sangue , Idoso , Doença de Alzheimer/diagnóstico , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Humanos , MasculinoRESUMO
We report in this work new substituted aminopyrimidine derivatives acting as inhibitors of the catalytic site of BACE1. These compounds were obtained from a molecular modeling study. The theoretical and experimental study reported here was carried out in several steps: docking analysis, Molecular Dynamics (MD) simulations, Quantum Theory Atom in Molecules (QTAIM) calculations, synthesis and bioassays and has allowed us to propose some compounds of this series as new inhibitors of the catalytic site of BACE1. The QTAIM study has allowed us to obtain an excellent correlation between the electronic densities and the experimental data of IC50. Also, using combined techniques (MD simulations and QTAIM calculations) enabled us to describe in detail the molecular interactions that stabilize the different L-R complexes. In addition, our results allowed us to determine what portion of these compounds should be changed in order to increase their affinity with the BACE1. Another interesting result is that a sort of synergism was observed when the effects of these new catalytic site inhibitors were combined with Ac-Tyr5-Pro6-Tyr7-Asp8-Ile9-Pro10-Leu11-NH2, which we have recently reported as a modulator of BACE1 acting on its exosite.
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Secretases da Proteína Precursora do Amiloide/química , Ácido Aspártico Endopeptidases/química , Pirimidinas/química , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Sítios de Ligação , Bioensaio , Domínio Catalítico , Desenho de Fármacos , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Pirimidinas/síntese química , Pirimidinas/farmacologia , Relação Quantitativa Estrutura-AtividadeRESUMO
Inflammation has been suggested to play early and perhaps causative roles in Alzheimer's disease (AD) pathogenesis possibly in part by the overactivation of the aspartic acid protease named ß-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1), which is responsible for the ß-amyloid cascade. We have described that BACE1 is involved in the lysophosphatidylethanolamine (LPE) (18:1/20:4/22:6) upregulation associated with tauopathy and inflammation signaling (cPLA2/arachidonic acid/COX2) in a triple transgenic model of Alzheimer's disease, where BACE1 silencing reversed the imbalanced profile and produced cognitive function improvement. In this study, we analyze the role of cPLA2 and desaturases (SCD1, FAD6) in the BACE1 knockdown-induced protective action under a glutamate excitotoxicity model. Glutamate (125⯵M) produced hyperphosphorylation of tau in cortical primary cultures along with increased apoptotic nuclei, LDH release, and cPLA2 expression, which were all reversed by BACE1-KD. This beneficial effect was reinforced by the silencing of cPLA2 but attenuated by the reduction in SCD1 and partially attenuated by the reduction in FAD6. Inversely, overexpression SCD1 and FAD6 recovered the neuroprotective effect produced by BACE1-KD, which was not achieved by the overexpression of each desaturase alone. These findings suggest that the hyperphosphorylation of tau and the creation of a pro-inflammatory cell environment are blocked in a desaturase-dependent manner by targeting BACE1.
Assuntos
Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Fosfolipases A2 do Grupo IV/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/etiologia , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Apoptose/genética , Ácido Aspártico Endopeptidases/genética , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Potenciais Pós-Sinápticos Excitadores , Técnicas de Silenciamento de Genes , Ácido Glutâmico/metabolismo , Fosfolipases A2 do Grupo IV/genética , Neurônios/metabolismo , Fosforilação/genética , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Estearoil-CoA Dessaturase/genética , Proteínas tau/toxicidadeRESUMO
BACE1 is an aspartyl protease with a very relevant role in medicinal chemistry related to Alzheimer Disease since it has demonstrated to be a promising therapeutic target for inhibition and possible control for the progress of the peptide accumulation characteristic of this pathology. The enzymatic activity of this protein is given by the aspartic dyad, Asp93 and Asp289, which can adopt several protonation states depending on the chemical nature of its inhibitors, this is, monoprotonated, diprotonated and di-deprotonated states. In the present study, the analysis of the population density, for a series of protein-inhibitor molecular dynamics simulations, was carried out to identify the most feasible protonation state adopted by the catalytic dyad in the presence of tertiary carbinamine (TC) transition state analog inhibitors. The results revealed that the monoprotonated Asp289i state, in which the Asp93 and Asp289 residue side chains are deprotonated and protonated on the inner oxygen, respectively, is the most preferred in the presence of TC family inhibitors. This result was obtained after evaluating, for all 9 possible protonation state configurations, the individual and combined population densities of a set of parameters sensitive to protonation state of the Aspartic dyad, using an X-ray experimental BACE1/TC crystallographic structure as reference. This case study demonstrates again the usefulness of the concept of population density as a quantitative tool to establish the most stable system settings, among all possible, by measuring the level of occurrence of simultaneous events obtained from a sampling over time. These results will help to clear the phenomena related to the TCs inhibitory pathway, as well as assist in the design of better TC inhibitors against Alzheimer's protease.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Metilaminas/química , Doença de Alzheimer/patologia , Cristalografia por Raios X , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica/fisiologia , PrótonsRESUMO
ß-amyloid (Aß) is produced by the ß-secretase 1 (BACE1)-mediated enzymatic cleavage of the amyloid precursor protein through the amyloidogenic pathway, making BACE1 a therapeutic target against Alzheimer's disease (AD). Alterations in lipid metabolism are a risk factor for AD by an unknown mechanism. The objective of this study was to determine the effect of RNA interference against BACE1 (shBACEmiR) on the phospholipid profile in hippocampal CA1 area in aged 3xTg-AD mice after 6 and 12 months of treatment compared to aged PS1KI mice. The shBACEmiR treatment induced cognitive function recovery and restored mainly the fatty acid composition of lysophosphatidylethanolamine and etherphosphatidylethanolamine, reduced the cPLA2's phosphorylation, down-regulated the levels of arachidonic acid and COX2 in the hippocampi of 3xTg-AD mice. Together, our findings suggest, for the first time, that BACE1 silencing restores phospholipids composition which could favor the recovery of cellular homeostasis and cognitive function in the hippocampus of triple transgenic AD mice.
RESUMO
BACE1 is an aspartyl protease which is a therapeutic target for Alzheimer's disease (AD) because of its participation in the rate-limiting step in the production of Aß-peptide, the accumulation of which produces senile plaques and, in turn, the neurodegenerative effects associated with AD. The active site of this protease is composed in part by two aspartic residues (Asp93 and Asp289). Additionally, the catalytic site has been found to be covered by an antiparallel hairpin loop called the flap. The dynamics of this flap are fundamental to the catalytic function of the enzyme. When BACE1 is inactive (Apo), the flap adopts an open conformation, allowing a substrate or inhibitor to access the active site. Subsequent interaction with the ligand induces flap closure and the stabilization of the macromolecular complex. Further, the protonation state of the aspartic dyad is affected by the chemical nature of the species entering the active site, so that appropriate selection of protonation states for the ligand and the catalytic residues will permit the elucidation of the inhibitory pathway for BACE1. In the present study, comparative analysis of different combinations of protonation states for the BACE1-hydroxyethylamine (HEA) system is reported. HEAs are potent inhibitors of BACE1 with favorable pharmacological and kinetic properties, as well as oral bioavailability. The results of Molecular Dynamics (MD) simulations and population density calculations using 8 different parameters demonstrate that the LnAsp289 configuration (HEA with a neutral amine and the Asp289 residue protonated) is the only one which permits the expected conformational change in BACE1, from apo to closed form, after flap closure. Additionally, differences in their capacities to establish and maintain interactions with residues such as Asp93, Gly95, Thr133, Asp289, Gly291, and Asn294 during this step allow differentiation among the inhibitory activities of the HEAs. The results and methodology here reported will serve to elucidate the inhibitory pathway of other families of compounds that act as BACE1 inhibitors, as well as the design of better leader compounds for the treatment of AD.