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1.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360969

RESUMO

Physical exercise reduces the extent, duration, and frequency of drug use in drug addicts during the drug initiation phase, as well as during prolonged addiction, withdrawal, and recurrence. However, information about exercise-induced neurobiological changes is limited. This study aimed to investigate the effects of forced moderate endurance exercise training on methamphetamine (METH)-induced behavior and the associated neurobiological changes. Male Sprague Dawley rats were subjected to the administration of METH (1 mg/kg/day, i.p.) and/or forced moderate endurance exercise (treadmill running, 21 m/min, 60 min/day) for 2 weeks. Over the two weeks, endurance exercise training significantly reduced METH-induced hyperactivity. METH and/or exercise treatment increased striatal dopamine (DA) levels, decreased p(Thr308)-Akt expression, and increased p(Tyr216)-GSK-3ß expression. However, the phosphorylation levels of Ser9-GSK-3ß were significantly increased in the exercise group. METH administration significantly increased the expression of NMDAr1, CaMKK2, MAPKs, and PP1 in the striatum, and exercise treatment significantly decreased the expression of these molecules. Therefore, it is apparent that endurance exercise inhibited the METH-induced hyperactivity due to the decrease in GSK-3ß activation by the regulation of the striatal glutamate signaling pathway.


Assuntos
Ácido Glutâmico/metabolismo , Metanfetamina/toxicidade , Condicionamento Físico Animal/métodos , Agitação Psicomotora/terapia , Corrida , Animais , Corpo Estriado/metabolismo , Dopamina/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Masculino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Agitação Psicomotora/etiologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais
2.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361045

RESUMO

Phosphoinositides (PIs) play important roles in the structure and function of the brain. Associations between PIs and the pathophysiology of schizophrenia have been studied. However, the significance of the PI metabolic pathway in the pathology of schizophrenia is unknown. We examined the expression of PI signaling-associated proteins in the postmortem brain of schizophrenia patients. Protein expression levels of phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C), phosphatidylinositol 4-kinase alpha (PIK4CA, also known as PIK4A), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), protein kinase B (Akt), and glycogen synthase kinase 3ß (GSK3ß) were measured using enzyme-linked immunosorbent assays and multiplex fluorescent bead-based immunoassays of the prefrontal cortex (PFC) of postmortem samples from 23 schizophrenia patients and 47 normal controls. We also examined the association between PIK4CA expression and its genetic variants in the same brain samples. PIK4CA expression was lower, whereas Akt expression was higher, in the PFC of schizophrenia patients than in that of controls; PIP5K1C, PTEN, and GSK3ß expression was not different. No single-nucleotide polymorphism significantly affected protein expression. We identified molecules involved in the pathology of schizophrenia via this lipid metabolic pathway. These results suggest that PIK4CA is involved in the mechanism underlying the pathogenesis of schizophrenia and is a potential novel therapeutic target.


Assuntos
Fosfatidilinositóis/metabolismo , Córtex Pré-Frontal/metabolismo , Esquizofrenia/metabolismo , 1-Fosfatidilinositol 4-Quinase/metabolismo , Idoso , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
3.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361017

RESUMO

Glycogen synthase kinase-3 beta (GSK-3ß) is an enzyme pertinently linked to neurodegenerative diseases since it is associated with the regulation of key neuropathological features in the central nervous system. Among the different kinds of inhibitors of this kinase, the allosteric ones stand out due to their selective and subtle modulation, lowering the chance of producing side effects. The mechanism of GSK-3ß allosteric modulators may be considered still vague in terms of elucidating a well-defined binding pocket and a bioactive pose for them. In this context, we propose to reinvestigate and reinforce such knowledge by the application of an extensive set of in silico methodologies, such as cavity detection, ligand 3D shape analysis and docking (with robust validation of corresponding protocols), and molecular dynamics. The results here obtained were consensually consistent in furnishing new structural data, in particular by providing a solid bioactive pose of one of the most representative GSK-3ß allosteric modulators. We further applied this to the prospect for new compounds by ligand-based virtual screening and analyzed the potential of the two obtained virtual hits by quantum chemical calculations. All potential hits achieved will be subsequently tested by in vitro assays in order to validate our approaches as well as to unveil novel chemical entities as GSK-3ß allosteric modulators.


Assuntos
Sítio Alostérico , Glicogênio Sintase Quinase 3 beta/química , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/farmacologia , Regulação Alostérica , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Fármacos Neuroprotetores/química , Ligação Proteica
4.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360874

RESUMO

Osteoarthritis (OA) is still a recalcitrant musculoskeletal disease on account of its complex biochemistry and mechanical stimulations. Apart from stimulation by external mechanical forces, the regulation of intracellular mechanics in chondrocytes has also been linked to OA development. Recently, visfatin has received significant attention because of the clinical finding of the positive correlation between its serum/synovial level and OA progression. However, the precise mechanism involved is still unclear. This study determined the effect of visfatin on intracellular mechanics and catabolism in human primary chondrocytes isolated from patients. The intracellular stiffness of chondrocytes was analyzed by the particle-tracking microrheology method. It was shown that visfatin damages the microtubule and microfilament networks to influence intracellular mechanics to decrease the intracellular elasticity and viscosity via glycogen synthase kinase 3ß (GSK3ß) inactivation induced by p38 signaling. Further, microtubule network destruction in human primary chondrocytes is predominantly responsible for the catabolic effect of visfatin on the cyclooxygenase 2 upregulation. The present study shows a more comprehensive interpretation of OA development induced by visfatin through biochemical and biophysical perspectives. Finally, the role of GSK3ß inactivation, and subsequent regulation of intracellular mechanics, might be considered as theranostic targets for future drug development for OA.


Assuntos
Condrócitos , Citocinas/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Nicotinamida Fosforribosiltransferase/fisiologia , Osteoartrite , Citoesqueleto de Actina/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Humanos , Microtúbulos/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Cultura Primária de Células
5.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445680

RESUMO

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.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Tiadiazóis/farmacologia , Idoso , Esclerose Amiotrófica Lateral/tratamento farmacológico , Esclerose Amiotrófica Lateral/metabolismo , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/fisiologia , Modelos Animais de Doenças , Reposicionamento de Medicamentos , Glicogênio Sintase Quinase 3 beta/fisiologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Neurônios Motores/metabolismo , Preparações Farmacêuticas/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Medula Espinal/metabolismo
6.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445773

RESUMO

Inadequate vessel maintenance or growth causes ischemia in diseases such as myocardial infarction, stroke, and neurodegenerative disorders. Therefore, developing an effective strategy to salvage ischemic tissues using a novel compound is urgent. Drug repurposing has become a widely used method that can make drug discovery more efficient and less expensive. Additionally, computational virtual screening tools make drug discovery faster and more accurate. This study found a novel drug candidate for pro-angiogenesis by in silico virtual screening. Using Gene Expression Omnibus (GEO) microarray datasets related to angiogenesis studies, differentially expressed genes were identified and characteristic direction signatures extracted from GEO2EnrichR were used as input data on L1000CDS2 to screen pro-angiogenic molecules. After a thorough review of the candidates, a list of compounds structurally similar to TWS-119 was generated using ChemMine Tools and its clustering toolbox. ChemMine Tools and ChemminR structural similarity search tools for small-molecule analysis and clustering were used for second screening. A molecular docking simulation was conducted using AutoDock v.4 to evaluate the physicochemical effect of secondary-screened chemicals. A cell viability or toxicity test was performed to determine the proper dose of the final candidate, ellipticine. As a result, we found ellipticine, which has pro-angiogenic effects, using virtual computational methods. The noncytotoxic concentration of ellipticine was 156.25 nM. The phosphorylation of glycogen synthase kinase-3ß was decreased, whereas the ß-catenin expression was increased in human endothelial cells treated with ellipticine. We concluded that ellipticine at sublethal dosage could be successfully repositioned as a pro-angiogenic substance by in silico virtual screening.


Assuntos
Elipticinas/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos/métodos , Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Neovascularização Patológica/metabolismo , Ligação Proteica/efeitos dos fármacos , beta Catenina/metabolismo
7.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445804

RESUMO

Protein kinases (PKs) have been recognized as central nervous system (CNS)-disease-relevant targets due to their master regulatory role in different signal transduction cascades in the neuroscience space. Among them, GSK-3ß, FYN, and DYRK1A play a crucial role in the neurodegeneration context, and the deregulation of all three PKs has been linked to different CNS disorders with unmet medical needs, including Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal lobar degeneration (FTLD), and several neuromuscular disorders. The multifactorial nature of these diseases, along with the failure of many advanced CNS clinical trials, and the lengthy approval process of a novel CNS drug have strongly limited the CNS drug discovery. However, in the near-decade from 2010 to 2020, several computer-assisted drug design strategies have been combined with synthetic efforts to develop potent and selective GSK-3ß, FYN, and DYRK1A inhibitors as disease-modifying agents. In this review, we described both structural and functional aspects of GSK-3ß, FYN, and DYRK1A and their involvement and crosstalk in different CNS pathological signaling pathways. Moreover, we outlined attractive medicinal chemistry approaches including multi-target drug design strategies applied to overcome some limitations of known PKs inhibitors and discover improved modulators with suitable blood-brain barrier (BBB) permeability and drug-like properties.


Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Doenças Neurodegenerativas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos
8.
Nutrients ; 13(7)2021 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-34371878

RESUMO

Alzheimer's disease (AD) is characterized by the aberrant processing of amyloid precursor protein (APP) and the accumulation of hyperphosphorylated tau, both of which are accompanied by neuroinflammation. Dietary supplementation with spray-dried porcine plasma (SDP) has anti-inflammatory effects in inflammation models. We investigated whether dietary supplementation with SDP prevents the neuropathological features of AD. The experiments were performed in 2- and 6-month-old SAMP8 mice fed a control diet, or a diet supplemented with 8% SDP, for 4 months. AD brain molecular markers were determined by Western blot and real-time PCR. Senescent mice showed reduced levels of p-GSK3ß (Ser9) and an increase in p-CDK5, p-tau (Ser396), sAPPß, and the concentration of Aß40, (all p < 0.05). SDP prevented these effects of aging and reduced Bace1 levels (all p < 0.05). Senescence increased the expression of Mme1 and Ide1 and pro-inflammatory cytokines (Il-17 and Il-18; all p < 0.05); these changes were prevented by SDP supplementation. Moreover, SDP increased Tgf-ß expression (p < 0.05). Furthermore, in aged mice, the gene expression levels of the microglial activation markers Trem2, Ym1, and Arg1 were increased, and SDP prevented these increases (all p < 0.05). Thus, dietary SDP might delay AD onset by reducing its hallmarks in senescent mice.


Assuntos
Doença de Alzheimer/prevenção & controle , Encéfalo/efeitos dos fármacos , Suplementos Nutricionais , Plasma , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ração Animal , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Quinase 5 Dependente de Ciclina/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Mediadores da Inflamação/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Emaranhados Neurofibrilares/efeitos dos fármacos , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Fragmentos de Peptídeos/metabolismo , Fosforilação , Transdução de Sinais , Secagem por Atomização , Sus scrofa , Proteínas tau/metabolismo
9.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204449

RESUMO

We recently found that, in human osteoblasts, Homer1 complexes to Calcium-sensing receptor (CaSR) and mediates AKT initiation via mechanistic target of rapamycin complex (mTOR) complex 2 (mTORC2) leading to beneficial effects in osteoblasts including ß-catenin stabilization and mTOR complex 1 (mTORC1) activation. Herein we further investigated the relationship between Homer1 and CaSR and demonstrate a link between the protein levels of CaSR and Homer1 in human osteoblasts in primary culture. Thus, when siRNA was used to suppress the CaSR, we observed upregulated Homer1 levels, and when siRNA was used to suppress Homer1 we observed downregulated CaSR protein levels using immunofluorescence staining of cultured osteoblasts as well as Western blot analyses of cell protein extracts. This finding was confirmed in vivo as the bone cells from osteoblast specific CaSR-/- mice showed increased Homer1 expression compared to wild-type (wt). CaSR and Homer1 protein were both expressed in osteocytes embedded in the long bones of wt mice, and immunofluorescent studies of these cells revealed that Homer1 protein sub-cellular localization was markedly altered in the osteocytes of CaSR-/- mice compared to wt. The study identifies additional roles for Homer1 in the control of the protein level and subcellular localization of CaSR in cells of the osteoblast lineage, in addition to its established role of mTORC2 activation downstream of the receptor.


Assuntos
Proteínas de Arcabouço Homer/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Osteoblastos/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Animais , Cálcio/metabolismo , Linhagem da Célula , Sobrevivência Celular , Células Cultivadas , Feminino , Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Detecção de Cálcio/genética
10.
J Chem Theory Comput ; 17(8): 5287-5300, 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34260233

RESUMO

Computational capabilities are rapidly increasing, primarily because of the availability of GPU-based architectures. This creates unprecedented simulative possibilities for the systematic and robust computation of thermodynamic observables, including the free energy of a drug binding to a target. In contrast to calculations of relative binding free energy, which are nowadays widely exploited for drug discovery, we here push the boundary of computing the binding free energy and the potential of mean force. We introduce a novel protocol that leverages enhanced sampling, machine learning, and ad hoc algorithms to limit human intervention, computing time, and free parameters in free energy calculations. We first validate the method on a host-guest system, and then we apply the protocol to glycogen synthase kinase 3 beta, a protein kinase of pharmacological interest. Overall, we obtain a good correlation with experimental values in relative and absolute terms. While we focus on protein-ligand binding, the strategy is of broad applicability to any complex event that can be described with a path collective variable. We systematically discuss key details that influence the final result. The parameters and simulation settings are available at PLUMED-NEST to allow full reproducibility.


Assuntos
Aprendizado de Máquina , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Glicogênio Sintase Quinase 3 beta/química , Glicogênio Sintase Quinase 3 beta/metabolismo , Imidazóis/química , Imidazóis/metabolismo , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Termodinâmica
11.
Cell Death Dis ; 12(7): 701, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262025

RESUMO

The mitochondrial DNA m.3243A > G mutation is well-known to cause a variety of clinical phenotypes, including diabetes, deafness, and osteoporosis. Here, we report isolation and expansion of urine-derived stem cells (USCs) from patients carrying the m.3243A > G mutation, which demonstrate bimodal heteroplasmy. USCs with high levels of m.3243A > G mutation displayed abnormal mitochondrial morphology and function, as well as elevated ATF5-dependent mitochondrial unfolded protein response (UPRmt), together with reduced Wnt/ß-catenin signaling and osteogenic potentials. Knockdown of ATF5 in mutant USCs suppressed UPRmt, improved mitochondrial function, restored expression of GSK3B and WNT7B, and rescued osteogenic potentials. These results suggest that ATF5-dependent UPRmt could be a core disease mechanism underlying mitochondrial dysfunction and osteoporosis related to the m.3243A > G mutation, and therefore could be a novel putative therapeutic target for this genetic disorder.


Assuntos
Fatores Ativadores da Transcrição/genética , DNA Mitocondrial/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , Mutação , Osteoporose/genética , Células-Tronco/metabolismo , Fatores Ativadores da Transcrição/metabolismo , Adulto , Estudos de Casos e Controles , Separação Celular , Células Cultivadas , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Heteroplasmia , Humanos , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/urina , Osteogênese , Osteoporose/diagnóstico , Osteoporose/urina , Fenótipo , Células-Tronco/ultraestrutura , Resposta a Proteínas não Dobradas , Urina/citologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Adulto Jovem
12.
Life Sci ; 281: 119754, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34174323

RESUMO

AIM: The present study was undertaken to elucidate the potential protective mechanism of berberine (BBR) and/or zinc (Zn) against methotrexate (MTX)-induced intestinal injury. METHODS: Five groups of rats were assigned; normal group (received vehicle), MTX group (20 mg/kg; i.p. single dose), and the other three groups received a single daily oral dose of BBR (50 mg/kg), Zn (5 mg/kg), and BBR plus Zn respectively, for 5 days before MTX and 5 days after. RESULTS: Our results emphasized the toxic effect of MTX on rat's intestine as shown by disturbance of oxidant/antioxidant status, down-regulation of NRF2, SIRT1, FOXO-3, Akt, and mTOR expressions, along with up-regulation of GSK-3ß, JAK1, and STAT-3 expressions. Besides, severe intestinal histopathological changes were also observed. On the contrary, BBR and/or Zn produced marked protection against MTX-induced intestinal toxicity via amelioration of oxidative stress, improving NRF2, SIRT1, FOXO-3, GSK-3ß, Akt, mTOR, JAK1, and STAT-3 alterations. Moreover, our treatments significantly restored histopathological abnormalities. Interestingly, combination therapy of BBR plus Zn exhibited higher effectiveness than mono-therapy. SIGNIFICANCE: BBR plus Zn could be used as a novel therapy for the treatment of MTX-induced intestinal damage through modulation of GSK-3ß/NRF2, Akt/mTOR, JAK1/STAT-3, and SIRT1/FOXO-3 signaling pathways.


Assuntos
Berberina/farmacologia , Proteína Forkhead Box O3/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Intestinos/efeitos dos fármacos , Janus Quinase 1/metabolismo , Metotrexato/toxicidade , Fator 2 Relacionado a NF-E2/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Zinco/farmacologia , Animais , Inflamação/prevenção & controle , Intestinos/enzimologia , Intestinos/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Sirtuína 1/metabolismo
13.
Eur J Med Chem ; 222: 113554, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34098466

RESUMO

Alzheimer's disease (AD) is a chronic and progressive neurodegenerative disease, characterized by irreversible cognitive impairment, memory loss and behavioral disturbances, ultimately leading to death. Glycogen synthase kinase 3ß (GSK-3ß) and dual-specificity tyrosine phosphorylation regulated kinase1A (DYRK1A) have gained a lot of attention for its role in tau pathology. To search for potential dual GSK-3ß/DYRK1A inhibitors, we focused on harmine, a natural ß-carboline alkaloid, which has been extensively studied for its various biological effects on the prevention of AD. In this study, a new series of harmine derivatives were designed, synthesized and evaluated as dual GSK-3ß/DYRK1A inhibitors for their multiple biological activities. The in vitro results indicated that most of them displayed promising activity against GSK-3ß and DYRK1A. Among them, compound ZDWX-25 showed potent inhibitory effects on GSK-3ß and DYRK1A with IC50 values of 71 and 103 nM, respectively. Molecular modelling and kinetic studies verified that ZDWX-25 could interact with the ATP binding pocket of GSK-3ß and DYRK1A. Western blot analysis revealed that ZDWX-25 inhibited hyperphosphorylation of tau protein in okadaic acid (OKA)-induced SH-SY5Y cells. In addition, ZDWX-25 showed good blood-brain barrier penetrability in vitro. More importantly, ZDWX-25 could ameliorate the impaired learning and memory in APP/PS1/Tau transgenic mice. These results indicated that the harmine-based compounds could be served as promising dual-targeted candidates for AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Harmina/farmacologia , Fármacos Neuroprotetores/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Doença de Alzheimer/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desenho de Fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Harmina/síntese química , Harmina/química , Humanos , Modelos Moleculares , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Relação Estrutura-Atividade
14.
Genes (Basel) ; 12(5)2021 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066668

RESUMO

The plant glycogen synthase kinase 3 (GSK3)-like kinases are highly conserved protein serine/threonine kinases that are grouped into four subfamilies. Similar to their mammalian homologs, these kinases are constitutively active under normal growth conditions but become inactivated in response to diverse developmental and environmental signals. Since their initial discoveries in the early 1990s, many biochemical and genetic studies were performed to investigate their physiological functions in various plant species. These studies have demonstrated that the plant GSK3-like kinases are multifunctional kinases involved not only in a wide variety of plant growth and developmental processes but also in diverse plant stress responses. Here we summarize our current understanding of the versatile physiological functions of the plant GSK3-like kinases along with their confirmed and potential substrates.


Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas de Plantas/metabolismo , Brassinosteroides/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Desenvolvimento Vegetal , Proteínas de Plantas/genética , Plantas/genética , Plantas/metabolismo , Transdução de Sinais , Estresse Fisiológico
15.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070360

RESUMO

Adenosine is a cellular metabolite with diverse derivatives that possesses a wide range of physiological roles. We investigated the molecular mechanisms of adenosine and cordycepin for their promoting effects in wound-healing process. The mitochondrial energy metabolism and cell proliferation markers, cAMP responsive element binding protein 1 (CREB1) and Ki67, were enhanced by adenosine and cordycepin in cultured dermal fibroblasts. Adenosine and cordycepin stimulated adenosine receptor signaling via elevated cAMP. The phosphorylation of mitogen-activated protein kinase kinase (MEK) 1/2, mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3 beta (Gsk3b) and Wnt target genes such as bone morphogenetic protein (BMP) 2/4 and lymphoid enhancer binding factor (Lef) 1 were activated. The enhanced gene expression by adenosine and cordycepin was abrogated by adenosine A2A and A2B receptor inhibitors, ZM241385 and PSH603, and protein kinase A (PKA) inhibitor H89, indicating the involvement of adenosine receptor A2A, A2B and PKA. As a result of Wnt/ß-catenin pathway activation, the secretion of growth factors such as insulin-like growth factor (IGF)-1 and transforming growth factor beta (TGFß) 3 was increased, previously reported to facilitate the wound healing process. In addition, in vitro fibroblast migration was also increased, demonstrating their possible roles in facilitating the wound healing process. In conclusion, our data strongly demonstrate that adenosine and cordycepin stimulate the Wnt/ß-catenin signaling through the activation of adenosine receptor, possibly promoting the tissue remodeling process and suggest their therapeutic potential for treating skin wounds.


Assuntos
Adenosina/farmacologia , Desoxiadenosinas/farmacologia , Fibroblastos/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Linhagem Celular , Fibroblastos/patologia , Humanos , Pele/lesões , Pele/metabolismo , Pele/patologia , Cicatrização/efeitos dos fármacos , beta Catenina/metabolismo
16.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069111

RESUMO

In colon cancer, wingless (Wnt)/ß-catenin signaling is frequently upregulated; however, the creation of a molecular therapeutic agent targeting this pathway is still under investigation. This research aimed to study how nitazoxanide can affect Wnt/ß-catenin signaling in colon cancer cells (HCT-116) and a mouse colon cancer model. Our study included 2 experiments; the first was to test the cytotoxic activity of nitazoxanide in an in vitro study on a colon cancer cell line (HCT-116) versus normal colon cells (FHC) and to highlight the proapoptotic effect by MTT assay, flow cytometry and real-time polymerase chain reaction (RT-PCR). The second experiment tested the in vivo cytotoxic effect of nitazoxanide against 1,2-dimethylhydrazine (DMH) prompted cancer in mice. Mice were grouped as saline, DMH control and DMH + nitazoxanide [100 or 200 mg per kg]. Colon levels of Wnt and ß-catenin proteins were assessed by Western blotting while proliferation was measured via immunostaining for proliferating cell nuclear antigen (PCNA). Treating HCT-116 cells with nitazoxanide (inhibitory concentration 50 (IC50) = 11.07 µM) revealed that it has a more cytotoxic effect when compared to 5-flurouracil (IC50 = 11.36 µM). Moreover, it showed relatively high IC50 value (non-cytotoxic) against the normal colon cells. Nitazoxanide induced apoptosis by 15.86-fold compared to control and arrested the cell cycle. Furthermore, nitazoxanide upregulated proapoptotic proteins (P53 and BAX) and caspases but downregulated BCL-2. Nitazoxanide downregulated Wnt/ß-catenin/glycogen synthase kinase-3ß (GSK-3ß) signaling and PCNA staining in the current mouse model. Hence, our findings highlighted the cytotoxic effect of nitazoxanide and pointed out the effect on Wnt/ß-catenin/GSK-3ß signaling.


Assuntos
Antiparasitários/farmacologia , Neoplasias do Colo/tratamento farmacológico , Nitrocompostos/farmacologia , Tiazóis/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Antiparasitários/química , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HCT116 , Humanos , Masculino , Camundongos , Simulação de Acoplamento Molecular , Nitrocompostos/química , Antígeno Nuclear de Célula em Proliferação/imunologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Tiazóis/química , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismo
17.
Nat Commun ; 12(1): 3500, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108491

RESUMO

WSX1, a receptor subunit for IL-27, is widely expressed in immune cells and closely involved in immune response, but its function in nonimmune cells remains unknown. Here we report that WSX1 is highly expressed in human hepatocytes but downregulated in hepatocellular carcinoma (HCC) cells. Using NRAS/AKT-derived spontaneous HCC mouse models, we reveal an IL-27-independent tumor-suppressive effect of WSX1 that largely relies on CD8+ T-cell immune surveillance via reducing neoplastic PD-L1 expression and the associated CD8+ T-cell exhaustion. Mechanistically, WSX1 transcriptionally downregulates an isoform of PI3K-PI3Kδ and thereby inactivates AKT, reducing AKT-induced GSK3ß inhibition. Activated GSK3ß then boosts PD-L1 degradation, resulting in PD-L1 reduction. Overall, we demonstrate that WSX1 is a tumor suppressor that reinforces hepatic immune surveillance by blocking the PI3Kδ/AKT/GSK3ß/PD-L1 pathway. Our results may yield insights into the host homeostatic control of immune response and benefit the development of cancer immunotherapies.


Assuntos
Antígeno B7-H1/metabolismo , Carcinoma Hepatocelular/imunologia , Neoplasias Hepáticas/imunologia , Receptores de Interleucina/imunologia , Proteínas Supressoras de Tumor/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Vigilância Imunológica , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Interleucina/metabolismo , Transdução de Sinais/imunologia , Proteínas Supressoras de Tumor/metabolismo
18.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064895

RESUMO

Skeletal muscles, being one of the most abundant tissues in the body, are involved in many vital processes, such as locomotion, posture maintenance, respiration, glucose homeostasis, etc. Hence, the maintenance of skeletal muscle mass is crucial for overall health, prevention of various diseases, and contributes to an individual's quality of life. Prolonged muscle inactivity/disuse (due to limb immobilization, mechanical ventilation, bedrest, spaceflight) represents one of the typical causes, leading to the loss of muscle mass and function. This disuse-induced muscle loss primarily results from repressed protein synthesis and increased proteolysis. Further, prolonged disuse results in slow-to-fast fiber-type transition, mitochondrial dysfunction and reduced oxidative capacity. Glycogen synthase kinase 3ß (GSK-3ß) is a key enzyme standing at the crossroads of various signaling pathways regulating a wide range of cellular processes. This review discusses various important roles of GSK-3ß in the regulation of protein turnover, myosin phenotype, and oxidative capacity in skeletal muscles under disuse/unloading conditions and subsequent recovery. According to its vital functions, GSK-3ß may represent a perspective therapeutic target in the treatment of muscle wasting induced by chronic disuse, aging, and a number of diseases.


Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Elevação dos Membros Posteriores , Músculo Esquelético/fisiopatologia , Atrofia Muscular/patologia , Miosinas/metabolismo , Estresse Oxidativo , Proteólise , Animais , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Fenótipo
19.
J Biochem Mol Toxicol ; 35(8): e22815, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34047419

RESUMO

Oxidative stress of retinal ganglion cells (RGCs) has been established as a main contributor to retinal degeneration in the pathogenesis of glaucoma. Polo-like kinase 2 (PLK2) has recently been reported to be a potent antioxidant protein that enhances cell survival in response to oxidative stress. To date, the involvement of PLK2 in RGC-associated oxidative stress is undermined. In the present work, we evaluated whether PLK2 regulates oxidative stress evoked by hydrogen peroxide (H2 O2 ) in RGCs. PLK2 expression was induced by H2 O2 stimulation in RGCs. Upregulation of PLK2 had a profoundly cytoprotective effect on H2 O2 -stimulated RGCs by attenuating cellular apoptosis and reactive oxygen species (ROS) level. Further data revealed that upregulation of PLK2 strikingly enhanced the activation of Nrf2 signaling. Moreover, PLK2 overexpression promoted glycogen synthase kinase (GSK)-3ß phosphorylation, whereas PLK2 knockdown reduced the levels of GSK-3ß phosphorylation. Notably, GSK-3ß inhibition using a chemical inhibitor markedly abrogated the suppressive effects of PLK2 knockdown on Nrf2 activation. Repression of Nrf2 blocked the PLK2 overexpression-induced protective effects in H2 O2 -stimulated RGCs. Overall, this study elucidates that upregulation of PLK2 protects RGCs against H2 O2 -induced oxidative stress injury by upregulating Nrf2 activation via modulation of GSK-3ß phosphorylation. These findings underline the pivotal role of PLK2 in mediating oxidative stress-evoked retinal degeneration in the pathogenesis of glaucoma.


Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/metabolismo , Células Ganglionares da Retina/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Peróxido de Hidrogênio , Ratos
20.
Life Sci ; 278: 119581, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33961854

RESUMO

AIMS: For most human cancers, the expression pattern and biological function of ADORA1 (Adenosine A1 Receptor) are largely unknown. This study has been designed to explore the clinical significance and the mechanism of ADORA1 in nasopharyngeal carcinoma (NPC) cells. MATERIALS AND METHODS: The level of ADORA1 in NPC and its adjacent tissues was analyzed by IHC, real-time PCR and western blotting. MTT and colony formation assays were used to determine the cell viability post ADORA1 overexpression or knockdown. Wound-healing assay and Transwell assay were used to analyze the effect of ADORA1 on migration and invasion. Moreover, the effect of ADORA1 on tumor growth was also studied in vivo by using xenograft mouse model. The regulation of ADORA1 on PI3K/AKT/GSK-3ß/ß-catenin pathway was determined by western blotting and TOP-Flash luciferase assay. KEY FINDINGS: Primary NPC exhibits overexpression of ADORA1, which is related to the overexpression of its mRNA. Ectopic expression of ADORA1 promotes the proliferation, invasion and migration in NPC cells. The apoptosis, however, is suppressed. ADORA1 silencing was found to exert opposite effects in in vitro studies and produced a significant inhibitory effect on murine xenograft tumor growth in vivo experiments. Besides, ADORA1 also triggers the PI3K/AKT/GSK-3ß/ß-catenin intracellular oncogenic pathway for signal transduction. Inhibition of this pathway by PI3K inhibitor LY294002 obstructed the impact of ADORA1 on tumor development in cells with ADORA1-overexpression. SIGNIFICANCE: ADORA1 has been identified as an important oncoprotein, promoting tumor cell proliferation via PI3K/AKT/GSK-3ß/ß-catenin signaling pathway in NPC.


Assuntos
Perfilação da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Receptor A1 de Adenosina/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Colágeno/química , Combinação de Medicamentos , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Laminina/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Proteoglicanas/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Cicatrização , beta Catenina/metabolismo
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