RESUMO
Alzheimer's disease (AD) is a significant global healthcare challenge, particularly in the elderly population. This neurodegenerative disorder is characterized by impaired memory and progressive decline in cognitive function. BACE1, a transmembrane protein found in neurons, oligodendrocytes, and astrocytes, exhibits varying levels across different neural subtypes. Abnormal BACE1 activity in the brains of individuals with AD leads to the formation of beta-amyloid proteins. The complex interplay between myelin sheath formation, BACE1 activity, and beta-amyloid accumulation suggests a critical role in understanding the pathological mechanisms of AD. The primary objective of this study was to identify molecular inhibitors that target Aß. Structure-based virtual screening (SBVS) was employed using the MCULE database, which houses over 2 million chemical compounds. A total of 59 molecules were selected after the toxicity profiling. Subsequently, five compounds conforming to the Egan-Egg permeation predictive model of the ADME rules were selected and subjected to molecular docking using AutoDock Vina on the Mcule drug discovery platform. The top two ligands and the positive control, 5HA, were subjected to molecular dynamics simulation for five nanoseconds. Toxicity profiling, physiochemical properties, lipophilicity, solubility, pharmacokinetics, druglikeness, medicinal chemistry attributes, average potential energy, RMSD, RMSF, and Rg analyses were conducted to identify the ligand MCULE-9199128437-0-2 as a promising inhibitor of BACE1.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Humanos , Ligantes , Descoberta de Drogas/métodos , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/químicaRESUMO
ß-site amyloid precursor protein cleaving enzyme (BACE1) represents a key target for Alzheimer's disease (AD) therapy because it is essential for producing the toxic amyloid ß (Aß) peptide that plays a crucial role in the disease's development. BACE1 inhibitors are a promising approach to reducing Aß levels in the brain and preventing AD progression. However, systemic delivery of such inhibitors to the brain demonstrates limited efficacy because of the presence of the blood-brain barrier (BBB). Nose-to-brain (NtB) delivery has the potential to overcome this obstacle. Liposomal drug delivery systems offer several advantages over traditional methods for delivering drugs and nucleic acids from the nose to the brain. The current study aims to prepare, characterize, and evaluate in vitro liposomal forms of donepezil, memantine, BACE-1 siRNA, and their combination for possible treatment of AD via NtB delivery. All the liposomal formulations were prepared using the rotary evaporation method. Their cellular internalization, cytotoxicity, and the suppression of beta-amyloid plaque and other pro-inflammatory cytokine expressions were studied. The Calu-3 Transwell model was used as an in vitro system for mimicking the anatomical and physiological conditions of the nasal epithelium and studying the suitability of the proposed formulations for possible NtB delivery. The investigation results show that liposomes provided the effective intracellular delivery of therapeutics, the potential to overcome tight junctions in BBB, reduced beta-amyloid plaque accumulation and pro-inflammatory cytokine expression, supporting the therapeutic potential of our approach.
Assuntos
Administração Intranasal , Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Donepezila , Lipossomos , RNA Interferente Pequeno , Doença de Alzheimer/tratamento farmacológico , Humanos , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , Donepezila/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Piperidinas/administração & dosagem , Piperidinas/farmacocinética , Piperidinas/farmacologia , Mucosa Nasal/metabolismo , Mucosa Nasal/efeitos dos fármacos , Indanos/administração & dosagem , Indanos/farmacocinética , Peptídeos beta-Amiloides/metabolismoRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects the elderly population globally and there is an urgent demand for developing novel anti-AD agents. In this study, a new series of indole-isoxazole carbohydrazides were designed and synthesized. The structure of all compounds was elucidated using spectroscopic methods including FTIR, 1H NMR, and 13C NMR as well as mass spectrometry and elemental analysis. All derivatives were screened for their acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activity. Out of all synthesized compounds, compound 5d exhibited the highest potency as AChE inhibitor with an IC50 value of 29.46 ± 0.31 µM. It showed significant selectivity towards AChE, with no notable inhibition against BuChE. A kinetic study on AChE for compound 5d indicated a competitive inhibition pattern. Also, 5d exhibited promising BACE1 inhibitory potential with an IC50 value of 2.85 ± 0.09 µM and in vitro metal chelating ability against Fe3+. The molecular dynamic studies of 5d against both AChE and BACE1 were executed to evaluate the behavior of this derivative in the binding site. The results showed that the new compounds deserve further chemical optimization to be considered potential anti-AD agents.
Assuntos
Acetilcolinesterase , Doença de Alzheimer , Butirilcolinesterase , Inibidores da Colinesterase , Indóis , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Acetilcolinesterase/metabolismo , Acetilcolinesterase/química , Butirilcolinesterase/metabolismo , Butirilcolinesterase/química , Indóis/química , Indóis/farmacologia , Indóis/síntese química , Humanos , Simulação de Acoplamento Molecular , Isoxazóis/química , Isoxazóis/farmacologia , Isoxazóis/síntese química , Relação Estrutura-Atividade , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Modelos Moleculares , Sítios de Ligação , Simulação de Dinâmica Molecular , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Cinética , HidrazinasRESUMO
Brain-derived neurotrophic factor (BDNF) content and signaling has been identified as one potential regulator of amyloid precursor protein (APP) processing. Recently published work has demonstrated that BDNF reduces BACE1 activity while also elevating the inhibition of GSK3ß in the prefrontal cortex of male C57BL/6J mice. These results provide evidence that BDNF alters APP processing by reducing BACE1 activity, which may act through GSK3ß inhibition. The purpose of this study was to further explore the role of GSK3ß in BDNF-induced regulation on BACE1 activity. We utilized a cell culture and an in vitro activity assay model to pharmacologically target BDNF and GSK3ß signaling to confirm its involvement in the BDNF response. Treatment of differentiated SH-SY5Y neuronal cells with 75 ng/mL BDNF resulted in elevated pTrkB content, pAkt content, pGSK3ß content, and reduced BACE1 activity. An in vitro BACE1 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF reduced BACE1 activity; however, in the presence of TrkB or Akt inhibition, this effect was abolished. An in vitro ADAM10 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF did not alter ADAM10 activity. However, inhibiting BDNF signaling reduced ADAM10 activity. Collectively these studies suggest that GSK3ß inhibition may be necessary for BDNF-induced reductions in BACE1 activity. These findings will allow for the optimization of future therapeutic strategies by selectively targeting TrkB activation and GSK3ß inhibition.
Assuntos
Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Fator Neurotrófico Derivado do Encéfalo , Glicogênio Sintase Quinase 3 beta , Camundongos Endogâmicos C57BL , Neurônios , Proteínas Proto-Oncogênicas c-akt , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Humanos , Ácido Aspártico Endopeptidases/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Camundongos , Masculino , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Transdução de Sinais , Linhagem Celular Tumoral , Receptor trkB/metabolismo , Receptor trkB/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismoRESUMO
Production, aggregation, and clearance of the amyloid ß peptide (Aß) are important processes governing the initial pathogenesis of Alzheimer's disease (AD). Inhibition of ß-site amyloid precursor protein (APP) cleaving enzyme (BACE1) (one of two key proteases responsible for Aß production) as an AD-therapeutic approach so far has failed to yield a successful drug. BACE1 and its homologue BACE2 are frequently inhibited by the same inhibitors. Several genetic and cerebral organoid modeling studies suggest that BACE2 has dose-dependent AD-suppressing activity, which makes its unwanted inhibition potentially counterproductive for AD treatment. The in vivo effects of an unwanted cross inhibition of BACE2 have so far been impossible to monitor because of the lack of an easily accessible pharmacodynamic marker specific for BACE2 cleavage. In this issue of the JCI, work led by Stefan F. Lichtenthaler identifies soluble VEGFR3 (sVEGFR3) as a pharmacodynamic plasma marker for BACE2 activity not shared with BACE1.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Ácido Aspártico Endopeptidases/genética , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Animais , Peptídeos beta-Amiloides/metabolismo , Biomarcadores/metabolismoRESUMO
Alzheimer's disease is age-related multifactorial neurodegenerative disease manifested by gradual loss of memory, cognitive decline and changes in personality. Due to rapid and continuous growth of its prevalence, the treatment of Alzheimer's disease calls for development of new and efficacies drugs, especially those that could be able to simultaneously act on more than one of possible targets of action. Aminoquinolines have proven to be a highly promising structural scaffold in the design of such a drug as cholinesterases and ß-secretase 1 inhibitors. In this study, we synthesised twenty-two new 4-aminoquinolines with different halogen atom and its position in the terminal N-benzyl group or with a trifluoromethyl or a chlorine as C(7)-substituents on the quinoline moiety. All compounds were evaluated as multi-target-directedligands by determining their inhibition potency towards human acetylcholinesterase, butyrylcholinesterase and ß-secretase 1. All of the tested derivatives were very potent inhibitors of human acetylcholinesterase and butyrylcholinesterase with inhibition constants (Ki) in the nM to low µM range. Most were estimated to be able to cross the blood-brain barrier by passive transport and were nontoxic toward cells that represented the main models of individual organs.
Assuntos
Acetilcolinesterase , Aminoquinolinas , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Butirilcolinesterase , Inibidores da Colinesterase , Humanos , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Aminoquinolinas/farmacologia , Aminoquinolinas/química , Aminoquinolinas/síntese química , Butirilcolinesterase/metabolismo , Relação Estrutura-Atividade , Acetilcolinesterase/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Estrutura Molecular , Halogênios/química , Relação Dose-Resposta a DrogaRESUMO
The fruits of Alpinia oxyphylla (Alpiniae Oxyphyllae Fructus, AOF) are one of the "Four Famous South Medicines" in China. In this study, beta-site amyloid protein precursor cleaving enzyme 1 (BACE1) was applied to explore the active components in AOF responsible for type 2 diabetes mellitus (T2DM)-related cognitive disorder. As a result, 24 compounds including three unreported ones (1, 3, 4) were isolated from AOF. Compound 1 is an unusual carboncarbon linked diarylheptanoid dimer, and compound 4 is the first case of 3,4-seco-eudesmane sesquiterpenoid with a 5/6-bicyclic skeleton. Four diarylheptanoids (3, 5-7), one flavonoid (9) and two sesquiterpenoids (14 and 20) showed BACE1 inhibitory activity, of which the most active 6 was revealed to be a non-competitive and anti-competitive mixed inhibitor. Docking simulation suggested that OH-4' of 6 played important roles in maintaining activity by forming hydrogen bonds with Ser36 and Ile126 residues. Compounds 3, 5, 9 and 20 displayed neuroprotective effects against amyloid ß (Aß)-induced damage in BV2 cells. Mechanism study revealed that compounds 5 and 20 downregulated the expression of BACE1 and upregulated the expression of Lamp2 to exert effects. Thus, the characteristic diarylheptanoids and sesquiterpenoids in AOF had the efficacy to alleviate T2DM-related cognitive disorder by inhibiting BACE1 activity and reversing Aß-induced neuronal damage.
Assuntos
Alpinia , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Diabetes Mellitus Tipo 2 , Frutas , Sesquiterpenos , Alpinia/química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Frutas/química , Estrutura Molecular , Diabetes Mellitus Tipo 2/tratamento farmacológico , Sesquiterpenos/farmacologia , Sesquiterpenos/isolamento & purificação , Simulação de Acoplamento Molecular , Diarileptanoides/farmacologia , Diarileptanoides/isolamento & purificação , Diarileptanoides/química , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Humanos , Animais , China , Flavonoides/farmacologia , Flavonoides/isolamento & purificação , Flavonoides/química , Transtornos Cognitivos/tratamento farmacológico , Camundongos , Extratos VegetaisRESUMO
BACE-1 plays a pivotal role in the production of ß-amyloid (Aß) peptides, implicated in Alzheimer's Disease (AD) pathology. We previously described edaravone N-benzyl pyridinium derivatives (EBPDs) that exhibited multifunctional activity against multiple AD targets. In this study we explored the EBPDs BACE-1 inhibitory activity to potentially enhance the compounds therapeutic profile. The EBPDs exhibited moderate BACE-1 inhibitory activity (IC50 = 44.10 µM - 123.70 µM) and obtained IC50 values between 2.0 and 5.8-fold greater than resveratrol, a known BACE-1 inhibitor (IC50 = 253.20 µM), in this assay. Compound 3 was the most potent inhibitor with an IC50 of 44.10 µM and a Ki of 19.96 µM and a mixed-type mode of inhibition that favored binding in a competitive manner. Molecular docking identified crucial interactions with BACE-1 active site residues, supported by 100 ns MD simulations. The study highlighted the EBPDs therapeutic potential as BACE-1 inhibitors and multifunctional anti-AD therapeutic agents.
Assuntos
Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Edaravone , Simulação de Acoplamento Molecular , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Edaravone/farmacologia , Edaravone/química , Humanos , Cinética , Compostos de Piridínio/farmacologia , Compostos de Piridínio/química , Simulação de Dinâmica Molecular , Ligação ProteicaRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disorder marked by the accumulation of amyloid-beta plaques and hyperphosphorylated tau proteins, leading to cognitive decline and neuronal death. However, despite extensive research, there are still no effective treatments for this condition. In this study, a series of chloride-substituted Ramalin derivatives is synthesized to optimize their antioxidant, anti-inflammatory, and their potential to target key pathological features of Alzheimer's disease. The effect of the chloride position on these properties is investigated, specifically examining the potential of these derivatives to inhibit tau aggregation and beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1) activity. Our findings demonstrate that several derivatives, particularly RA-3Cl, RA-4Cl, RA-26Cl, RA-34Cl, and RA-35Cl, significantly inhibit tau aggregation with inhibition rates of approximately 50%. For BACE-1 inhibition, Ramalin and RA-4Cl also significantly decrease BACE-1 expression in N2a cells by 40% and 38%, respectively, while RA-23Cl and RA-24Cl showed inhibition rates of 30% and 35% in SH-SY5Y cells. These results suggest that chloride-substituted Ramalin derivatives possess promising multifunctional properties for AD treatment, warranting further investigation and optimization for clinical applications.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Proteínas tau , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Humanos , Proteínas tau/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Cloretos/química , Antioxidantes/farmacologia , Antioxidantes/síntese química , Antioxidantes/química , Agregados Proteicos/efeitos dos fármacos , Linhagem Celular Tumoral , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/químicaRESUMO
This study investigates the clustering patterns of human ß-secretase 1 (BACE-1) inhibitors using complex network methodologies based on various distance functions, including Euclidean, Tanimoto, Hamming, and Levenshtein distances. Molecular descriptor vectors such as molecular mass, Merck Molecular Force Field (MMFF) energy, Crippen partition coefficient (ClogP), Crippen molar refractivity (MR), eccentricity, Kappa indices, Synthetic Accessibility Score, Topological Polar Surface Area (TPSA), and 2D/3D autocorrelation entropies are employed to capture the diverse properties of these inhibitors. The Euclidean distance network demonstrates the most reliable clustering results, with strong agreement metrics and minimal information loss, indicating its robustness in capturing essential structural and physicochemical properties. Tanimoto and Hamming distance networks yield valuable clustering outcomes, albeit with moderate performance, while the Levenshtein distance network shows significant discrepancies. The analysis of eigenvector centrality across different networks identifies key inhibitors acting as hubs, which are likely critical in biochemical pathways. Community detection results highlight distinct clustering patterns, with well-defined communities providing insights into the functional and structural groupings of BACE-1 inhibitors. The study also conducts non-parametric tests, revealing significant differences in molecular descriptors, validating the clustering methodology. Despite its limitations, including reliance on specific descriptors and computational complexity, this study offers a comprehensive framework for understanding molecular interactions and guiding therapeutic interventions. Future research could integrate additional descriptors, advanced machine learning techniques, and dynamic network analysis to enhance clustering accuracy and applicability.
Assuntos
Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Humanos , Análise por Conglomerados , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Inibidores de Proteases/metabolismo , Modelos Moleculares , Relação Estrutura-Atividade , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologiaRESUMO
Within drug development, high off-target promiscuity as well as potent cytotoxicity, are associated with a high attrition rate. We investigated the safety profile of novel plasmepsin X (PMX) inhibitors for the treatment of malaria. In our screening cascade, a total of 249 PMX compounds were profiled in a panel of in vitro secondary pharmacology assays containing 44 targets (SafetyScreen44 panel) and in a cytotoxicity assay in HepG2 cells using ATP as an endpoint. Six of the lead compounds were subsequently tested in a 7-d rat toxicology study, and/or in a cardiovascular study in guinea pigs. Overall, compounds with high cytotoxicity in HepG2 cells correlated with high promiscuity (off-target hit rate >20%) in the SafetyScreen44 panel and were associated with poor tolerability in vivo (decedents, morbidity, adverse clinical signs, or severe cardiovascular effects). Some side effects observed in rats or guinea pigs could putatively be linked with hits in the secondary pharmacological profiling, such as the M1 or M2 muscarinic acetylcholine receptor, opioid µ and/or κ receptors or hERG/CaV1.2/Na+ channels, which were common to >50% the compounds tested in vivo. In summary, compounds showing high cytotoxicity and high promiscuity are likely to be poorly tolerated in vivo. However, such associations do not necessarily imply a causal relationship. Identifying the targets that cause these undesirable effects is key for early safety risk assessment. A tiered approach, based on a set of in vitro assays, helps selecting the compounds with highest likelihood of success to proceed to in vivo toxicology studies.
Assuntos
Antimaláricos , Ácido Aspártico Endopeptidases , Animais , Feminino , Cobaias , Humanos , Masculino , Ratos , Antimaláricos/toxicidade , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Hep G2RESUMO
Quinoline-related antimalarial drugs have been associated with cardiotoxicity risk, in particular QT prolongation and QRS complex widening. In collaboration with Medicines for Malaria Venture, we discovered novel plasmepsin X (PMX) inhibitors for malaria treatment. The first lead compounds tested in anesthetized guinea pigs (GPs) induced profound QRS widening, although exhibiting weak inhibition of NaV1.5-mediated currents in standard patch clamp assays. To understand the mechanism(s) underlying QRS widening to identify further compounds devoid of such liability, we established a set of in vitro models including CaV1.2, NaV1.5 rate-dependence, and NaV1.8 patch clamp assays, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), and Langendorff-perfused isolated GP hearts. Six compounds were tested in all models including anesthetized GP, and 8 additional compounds were tested in vitro only. All compounds tested in anesthetized GP and isolated hearts showed a similar cardiovascular profile, consisting of QRS widening, bradycardia, negative inotropy, hypotension, and for some, QT prolongation. However, a left shift of the concentration-response curves was noted from in vitro to in vivo GP data. When comparing in vitro models, there was a good consistency between decrease in sodium spike amplitude in hiPSC-CM and QRS widening in isolated hearts. Patch clamp assay results showed that the QRS widening observed with PMX inhibitors is likely multifactorial, primarily due to NaV1.8 and NaV1.5 rate-dependent sodium blockade and/or calcium channel-mediated mechanisms. In conclusion, early de-risking of QRS widening using a set of different in vitro assays allowed to identify novel PMX inhibitors with improved cardiac safety profile.
Assuntos
Antimaláricos , Ácido Aspártico Endopeptidases , Cardiotoxicidade , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Animais , Cobaias , Antimaláricos/toxicidade , Miócitos Cardíacos/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Potenciais de Ação/efeitos dos fármacos , Masculino , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Técnicas de Patch-Clamp , EletrocardiografiaRESUMO
The ß-secretase ß-site APP cleaving enzyme (BACE1) is a central drug target for Alzheimer's disease. Clinically tested, BACE1-directed inhibitors also block the homologous protease BACE2. Yet little is known about physiological BACE2 substrates and functions in vivo. Here, we identify BACE2 as the protease shedding the lymphangiogenic vascular endothelial growth factor receptor 3 (VEGFR3). Inactivation of BACE2, but not BACE1, inhibited shedding of VEGFR3 from primary human lymphatic endothelial cells (LECs) and reduced release of the shed, soluble VEGFR3 (sVEGFR3) ectodomain into the blood of mice, nonhuman primates, and humans. Functionally, BACE2 inactivation increased full-length VEGFR3 and enhanced VEGFR3 signaling in LECs and also in vivo in zebrafish, where enhanced migration of LECs was observed. Thus, this study identifies BACE2 as a modulator of lymphangiogenic VEGFR3 signaling and demonstrates the utility of sVEGFR3 as a pharmacodynamic plasma marker for BACE2 activity in vivo, a prerequisite for developing BACE1-selective inhibitors for safer prevention of Alzheimer's disease.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Transdução de Sinais , Receptor 3 de Fatores de Crescimento do Endotélio Vascular , Peixe-Zebra , Animais , Humanos , Camundongos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Células Endoteliais/metabolismo , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The prevailing but not undisputed amyloid cascade hypothesis places the ß-site of APP cleaving enzyme 1 (BACE1) center stage in Alzheimer's Disease pathogenesis. Here, we investigated functional properties of BACE1 with novel tag- and antibody-free labeling tools, which are conjugates of the BACE1-inhibitor IV (also referred to as C3) linked to different impermeable Alexa Fluor dyes. We show that these fluorescent small molecules bind specifically to BACE1, with a 1:1 labeling stoichiometry at their orthosteric site. This is a crucial property especially for single-molecule and super-resolution microscopy approaches, allowing characterization of the dyes' labeling capabilities in overexpressing cell systems and in native neuronal tissue. With multiple colors at hand, we evaluated BACE1-multimerization by Förster resonance energy transfer (FRET) acceptor-photobleaching and single-particle imaging of native BACE1. In summary, our novel fluorescent inhibitors, termed Alexa-C3, offer unprecedented insights into protein-protein interactions and diffusion behavior of BACE1 down to the single molecule level.
Assuntos
Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Humanos , Corantes Fluorescentes/química , Animais , Células HEK293 , Imagem Individual de Molécula/métodosRESUMO
In this study, the synthesis of N-(5,6-methylenedioxybenzothiazole-2-yl)-2-[(substituted)thio/piperazine]acetamide/propanamide derivatives (3a-3k) and to investigate their acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and ß-secretase 1 (BACE-1) inhibition activity were aimed. Mass, 1H NMR, and 13C NMR spectra were utilized to determine the structure of the synthesized compounds. Compounds 3b, 3c, 3f, and 3j showed AChE inhibitory activity which compound 3c (IC50 = 0.030 ± 0.001 µM) showed AChE inhibitory activity as high as the reference drug donepezil (IC50 = 0.0201 ± 0.0010 µM). Conversely, none of the compounds showed BChE activity. Compounds 3c and 3j showed the highest BACE-1 inhibitory activity and IC50 value was found as 0.119 ± 0.004 µM for compound 3j whereas IC50 value was 0.110 ± 0.005 µM for donepezil, which is one of the reference substance. Molecular docking studies have been carried out using the data retrieved from the server of the Protein Data Bank (PDBID: 4EY7 and 2ZJM). Using in silico approach behavior active compounds (3c and 3j) and their binding modes clarified.
Assuntos
Acetilcolinesterase , Secretases da Proteína Precursora do Amiloide , Butirilcolinesterase , Inibidores da Colinesterase , Simulação de Acoplamento Molecular , Humanos , Acetamidas/síntese química , Acetamidas/farmacologia , Acetamidas/química , Acetilcolinesterase/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/química , Piperazinas/farmacologia , Piperazinas/química , Piperazinas/síntese química , Relação Estrutura-Atividade , Benzotiazóis/síntese química , Benzotiazóis/química , Benzotiazóis/farmacologiaRESUMO
A previous study reported that the ethanolic extract of the edible fern, Diplazium esculentum (Retz.) Sw. (DE), obtained from a non-optimized extraction condition exhibited anti-Alzheimer's disease (AD) properties through the inhibition of a rate-limiting enzyme in amyloid peptide formation, ß-secretase-1 (BACE-1). Nevertheless, a non-optimized or suboptimal extraction may lead to several issues, such as a reduction in extraction efficiency and increased time and plant materials. In this study, extraction of the DE was optimized to obtain appropriate BACE-1 inhibition using a Box-Behnken design (BBD) and response surface methodology (RSM). Data revealed that the optimal extraction condition was 70% (v/v) aqueous ethanol, 50 min extraction time, 30 °C extraction temperature, and 1:30 g/mL solid/liquid ratio, giving BACE-1 inhibition at 56.33%. In addition, the extract also exhibited significant antioxidant activities compared to the non-optimized extraction. Metabolomic phytochemical profiles and targeted phytochemical analyses showed that kaempferol, quercetin, and their derivatives as well as rosmarinic acid were abundant in the extract. The optimized DE extract also acted synergistically with donepezil, an AD drug suppressing BACE-1 activities. Data received from Drosophila-expressing human amyloid precursor proteins (APPs) and BACE-1, representing the amyloid hypothesis, showed that the optimized DE extract penetrated the fly brains, suppressed BACE-1 activities, and improved locomotor functions. The extract quenched the expression of glutathione S transferase D1 (GSTD1), inositol-requiring enzyme (IRE-1), and molecular chaperone-binding immunoglobulin (Bip), while donepezil suppressed these genes and other genes involved in antioxidant and endoplasmic reticulum (ER) stress response, including superoxide dismutase type 1 (SOD1), activating transcription factor 6 (ATF-6), and protein kinase R-like endoplasmic reticulum kinase (PERK). To sum up, the optimized extraction condition reduced extraction time while resulting in higher phytochemicals, antioxidants, and BACE-1 inhibitors.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Antioxidantes , Compostos Fitoquímicos , Extratos Vegetais , Antioxidantes/farmacologia , Antioxidantes/química , Antioxidantes/isolamento & purificação , Doença de Alzheimer/tratamento farmacológico , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Gleiquênias/química , Humanos , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismoRESUMO
Given continued failure of BACE1 inhibitor programs at symptomatic and prodromal stages of Alzheimer's disease (AD), clinical trials need to target the earlier preclinical stage. However, trial design is complex in this population with negative diagnosis of classical hippocampal amnesia on standard memory tests. Besides recent advances in brain imaging, electroencephalogram, and fluid-based biomarkers, new cognitive markers should be established for earlier diagnosis that can optimize recruitment to BACE1 inhibitor trials in presymptomatic AD. Notably, accelerated long-term forgetting (ALF) is emerging as a sensitive cognitive measure that can discriminate between asymptomatic individuals with high risks for developing AD and healthy controls. ALF is a form of declarative memory impairment characterized by increased forgetting rates over longer delays (days to months) despite normal storage within the standard delays of testing (20-60âmin). Therefore, ALF may represent a harbinger of preclinical dementia and the impairment of systems memory consolidation, during which memory traces temporarily stored in the hippocampus become gradually integrated into cortical networks. This review provides an overview of the utility of ALF in a rational design of next-generation BACE1 inhibitor trials in preclinical AD. I explore potential mechanisms underlying ALF and relevant early-stage biomarkers useful for BACE1 inhibitor evaluation, including synaptic protein alterations, astrocytic dysregulation and neuron hyperactivity in the hippocampal-cortical network. Furthermore, given the physiological role of the isoform BACE2 as an AD-suppressor gene, I also discuss the possible association between the poor selectivity of BACE1 inhibitors and their side effects (e.g., cognitive worsening) in prior clinical trials.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Diagnóstico Precoce , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/diagnóstico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , AnimaisRESUMO
Alzheimer's disease (AD) is a chronic neurological condition that has become a leading cause of cognitive decline in elder individuals. Hardly any effective medication has been developed to halt the progression of AD due to the disease's complexity. Several theories have been put forward to clarify the mechanisms underlying AD etiology. The identification of amyloid plaques as a hallmark of AD has sparked the development of numerous drugs targeting the players involved in the amyloidogenic pathway, such as the ß-site of amyloid precursor protein cleavage enzyme 1 (BACE1) blockers. Over the last ten years, preclinical and early experimental research has led several pharmaceutical companies to prioritize producing BACE1 inhibitors. Despite all these efforts, earlier discovered inhibitors were discontinued in consideration of another second-generation small molecules and recent BACE1 antagonists failed in the final stages of clinical trials because of the complications associated either with toxicity or effectiveness. In addition to discussing the difficulties associated with development of BACE1 inhibitors, this review aims to provide an overview of BACE1 and offer perspectives on the causes behind the failure of five recent BACE1 inhibitors, that would be beneficial for choosing effective treatment approaches in the future.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Humanos , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/tratamento farmacológico , Ácido Aspártico Endopeptidases/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Animais , Descoberta de DrogasRESUMO
In binding free energy calculations, simulations must sample all relevant conformations of the system in order to obtain unbiased results. For instance, different ligands can bind to different metastable states of a protein, and if these protein conformational changes are not sampled in relative binding free energy calculations, the contribution of these states to binding is not accounted for and thus calculated binding free energies are inaccurate. In this work, we investigate the impact of different beta-sectretase 1 (BACE1) protein conformations obtained from x-ray crystallography on the binding of BACE1 inhibitors. We highlight how these conformational changes are not adequately sampled in typical molecular dynamics simulations. Furthermore, we show that insufficient sampling of relevant conformations induces substantial error in relative binding free energy calculations, as judged by a variation in calculated relative binding free energies up to 2 kcal/mol depending on the starting protein conformation. These results emphasize the importance of protein conformational sampling and pose this BACE1 system as a challenge case for further method development in the area of enhanced protein conformational sampling, either in combination with binding calculations or as an endpoint correction.
Assuntos
Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Termodinâmica , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Humanos , Cristalografia por Raios X , LigantesRESUMO
Members of the Signal Peptide-Peptidase (SPP) and Signal Peptide-Peptidase-like (SPPL) family are intramembrane aspartyl-proteases like their well-studied homologs, the presenilins, which comprise the catalytically active subunit within the γ-secretase complex. The lack of in vitro cleavage assays for SPPL proteases limited their biochemical characterization as well as substrate identification and validation. So far, SPPL proteases have been analyzed exclusively in intact cells or membranes, restricting mechanistic analysis to co-expression of enzyme and substrate variants colocalizing in the same subcellular compartments. We describe the details of developing an in vitro cleavage assay for SPPL2b and its model substrate TNFα and analyzed the influence of phospholipids, detergent supplements, and cholesterol on the SPPL2b in vitro activity. SPPL2b in vitro activity resembles mechanistic principles that have been observed in a cellular context, such as cleavage sites and consecutive turnover of the TNFα transmembrane domain. The novel in vitro cleavage assay is functional with separately isolated protease and substrate and amenable to a high throughput plate-based readout overcoming previous limitations and providing the basis for studying enzyme kinetics, catalytic activity, substrate recognition, and the characteristics of small molecule inhibitors. As a proof of concept, we present the first biochemical in vitro characterization of the SPPL2a and SPPL2b specific small molecule inhibitor SPL-707.