Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.863
Filtrar
1.
Am J Physiol Heart Circ Physiol ; 318(3): H715-H722, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32083971

RESUMO

Thin filament hypertrophic cardiomyopathy (HCM) mutations increase myofilament Ca2+ sensitivity and alter Ca2+ handling and buffering. The myosin inhibitor mavacamten reverses the increased contractility caused by HCM thick filament mutations, and we here test its effect on HCM thin filament mutations where the mode of action is not known. Mavacamten (250 nM) partially reversed the increased Ca2+ sensitivity caused by HCM mutations Cardiac troponin T (cTnT) R92Q, and cardiac troponin I (cTnI) R145G in in vitro ATPase assays. The effect of mavacamten was also analyzed in cardiomyocyte models of cTnT R92Q and cTnI R145G containing cytoplasmic and myofilament specific Ca2+ sensors. While mavacamten rescued the hypercontracted basal sarcomere length, the reduced fractional shortening did not improve with mavacamten. Both mutations caused an increase in peak systolic Ca2+ detected at the myofilament, and this was completely rescued by 250 nM mavacamten. Systolic Ca2+ detected by the cytoplasmic sensor was also reduced by mavacamten treatment, although only R145G increased cytoplasmic Ca2+. There was also a reversal of Ca2+ decay time prolongation caused by both mutations at the myofilament but not in the cytoplasm. We thus show that mavacamten reverses some of the Ca2+-sensitive molecular and cellular changes caused by the HCM mutations, particularly altered Ca2+ flux at the myofilament. The reduction of peak systolic Ca2+ as a consequence of mavacamten treatment represents a novel mechanism by which the compound is able to reduce contractility, working synergistically with its direct effect on the myosin motor.NEW & NOTEWORTHY Mavacamten, a myosin inhibitor, is currently in phase-3 clinical trials as a pharmacotherapy for hypertrophic cardiomyopathy (HCM). Its efficacy in HCM caused by mutations in thin filament proteins is not known. We show in reductionist and cellular models that mavacamten can rescue the effects of thin filament mutations on calcium sensitivity and calcium handling although it only partially rescues the contractile cellular phenotype and, in some cases, exacerbates the effect of the mutation.


Assuntos
Benzilaminas/farmacologia , Cálcio/metabolismo , Cardiomiopatia Hipertrófica/metabolismo , Coração/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Sarcômeros/efeitos dos fármacos , Uracila/análogos & derivados , Animais , Cardiomiopatia Hipertrófica/genética , Camundongos , Mutação , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Sarcômeros/metabolismo , Troponina I/genética , Troponina I/metabolismo , Troponina T/genética , Troponina T/metabolismo , Uracila/farmacologia
2.
Org Biomol Chem ; 18(3): 557-568, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31894828

RESUMO

The isoquinolinequinone (IQQ) pharmacophore is a privileged framework in known cytotoxic natural product families, caulibugulones and mansouramycins. Exploiting both families as a chemical starting point, we report on the structured development of an IQQ N-oxide anticancer framework which exhibits growth inhibition in the nM range across melanoma, ovarian and leukaemia cancer cell lines. A new lead compound (16, R6 = benzyl, R7 = H) exhibits nM GI50 values against 31/57 human tumour cell lines screened as part of the NCI60 panel and shows activity against doxorubicin resistant tumour cell lines. An electrochemical study highlights a correlation between electropositivity of the IQQ N-oxide framework and cytotoxicity. Adduct binding to sulfur based biological nucleophiles glutathione and cysteine was observed in vitro. This new framework possesses significant anticancer potential.


Assuntos
Antineoplásicos/farmacologia , Óxidos N-Cíclicos/farmacologia , Isoquinolinas/farmacologia , Quinonas/farmacologia , Antineoplásicos/síntese química , Benzilaminas/síntese química , Benzilaminas/farmacologia , Linhagem Celular Tumoral , Óxidos N-Cíclicos/síntese química , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Isoquinolinas/síntese química , Quinonas/síntese química
3.
Nat Neurosci ; 22(11): 1782-1792, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31636451

RESUMO

Microglia are the brain's resident innate immune cells and also have a role in synaptic plasticity. Microglial processes continuously survey the brain parenchyma, interact with synaptic elements and maintain tissue homeostasis. However, the mechanisms that control surveillance and its role in synaptic plasticity are poorly understood. Microglial dynamics in vivo have been primarily studied in anesthetized animals. Here we report that microglial surveillance and injury response are reduced in awake mice as compared to anesthetized mice, suggesting that arousal state modulates microglial function. Pharmacologic stimulation of ß2-adrenergic receptors recapitulated these observations and disrupted experience-dependent plasticity, and these effects required the presence of ß2-adrenergic receptors in microglia. These results indicate that microglial roles in surveillance and synaptic plasticity in the mouse brain are modulated by noradrenergic tone fluctuations between arousal states and emphasize the need to understand the effect of disruptions of adrenergic signaling in neurodevelopment and neuropathology.


Assuntos
Microglia/fisiologia , Plasticidade Neuronal/fisiologia , Norepinefrina/fisiologia , Córtex Visual/fisiologia , Animais , Benzilaminas/farmacologia , Receptor 1 de Quimiocina CX3C/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Ritmo Circadiano/fisiologia , Clembuterol/farmacologia , Dexmedetomidina/farmacologia , Dominância Ocular , Feminino , Fentanila/farmacologia , Locus Cerúleo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Microglia/citologia , Microglia/efeitos dos fármacos , Nadolol/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Norepinefrina/metabolismo , Propanolaminas/farmacologia , Restrição Física/fisiologia , Terbutalina/farmacologia , Vigília , Ferimentos e Lesões/fisiopatologia
4.
Eur J Med Chem ; 182: 111588, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31421630

RESUMO

Intrinsically disordered proteins are an emerging class of proteins without a folded structure and currently disorder-based drug targeting remains a challenge. p53 is the principal regulator of cell division and growth whereas MDM2 consists its main negative regulator. The MDM2-p53 recognition is a dynamic and multistage process that amongst other, employs the dissociation of a transient α-helical N-terminal ''lid'' segment of MDM2 from the proximity of the p53-complementary interface. Several small molecule inhibitors have been reported to inhibit the formation of the p53-MDM2 complex with the vast majority mimicking the p53 residues Phe19, Trp23 and Leu26. Recently, we have described the transit from the 3-point to 4-point pharmacophore model stabilizing this intrinsically disordered N-terminus by increasing the binding affinity by a factor of 3. Therefore, we performed a thorough SAR analysis, including chiral separation of key compound which was evaluated by FP and 2D NMR. Finally, p53-specific anti-cancer activity towards p53-wild-type cancer cells was observed for several representative compounds.


Assuntos
Antineoplásicos/farmacologia , Proteínas Intrinsicamente Desordenadas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Benzilaminas/síntese química , Benzilaminas/química , Benzilaminas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cianetos/síntese química , Cianetos/química , Cianetos/farmacologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Formiatos/síntese química , Formiatos/química , Formiatos/farmacologia , Humanos , Indóis/síntese química , Indóis/química , Indóis/farmacologia , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Estrutura Molecular , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo
5.
Med Sci Monit ; 25: 4225-4232, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31171764

RESUMO

BACKGROUND The aim of this study was to assess the efficacy and safety of "on-demand" dapoxetine in the treatment of premature ejaculation (PE). MATERIAL AND METHODS We performed a meta-analysis of intravaginal ejaculatory latency time (IELT), patient-reported global impression of change (PGIC), perceived control over ejaculation (PCOE), and drug-related adverse effects (AEs). We searched Medline, PubMed, Embase, CNKI, Wanfang, and VIP databases up to May 30, 2018 with the following search terms: "dapoxetine" or "SSRIs" and "premature ejaculation" or "sexual dysfunction". RESULTS Our analysis included 11 RCTs (8521 cases and 4338 controls). We found that IELT, PGIC, and PCOE in PE patients with "on-demand" dapoxetine were significantly higher than in the control group, and we observed higher proportions in 60 mg vs. 30 mg dapoxetine. The AEs were mild and tolerable. CONCLUSIONS "On-demand" dapoxetine is effective and safe for patients with PE, and a dose of 60 mg may be more effective than 30 mg.


Assuntos
Benzilaminas/administração & dosagem , Naftalenos/administração & dosagem , Ejaculação Precoce/tratamento farmacológico , Adulto , Benzilaminas/farmacologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Ejaculação/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Naftalenos/farmacologia , Inibidores de Captação de Serotonina/uso terapêutico , Resultado do Tratamento
6.
Int J Mol Sci ; 20(12)2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31226744

RESUMO

The relevance of nitric oxide synthase 2 (NOS2) as a prognostic factor in Glioblastoma Multiforme (GBM) malignancy is emerging. We analyzed the effect of NOS2 inhibitor 1400W on the autophagic flux and extracellular vesicle (EV) secretion in U87MG glioma cells. The effects of glioma stem cells (GSC)-derived EVs on adherent U87MG were evaluated. Cell proliferation and migration were examined while using Cell Counting Kit-8 assay (CCK-8) and scratch wound healing assay. Cell cycle profile and apoptosis were analyzed by flow cytometry. Autophagy-associated acidic vesicular organelles were detected and quantified by acridine orange staining. The number and size of EVs were assessed by nanoparticle tracking analysis. EV ultrastructure was verified by transmission electron microscopy (TEM). WB was used to analyze protein expression and acid sphingomyelinase was determined through ceramide levels. 1400W induced autophagy and EV secretion in both adherent U87MG and GSCs. EVs secreted by 1400W-treated GSC, but not those from untreated cells, were able to inhibit adherent U87MG cell growth and migration while also inducing a relevant level of autophagy. The hypothesis of NOS2 expression as GBM profile marker or interesting therapeutic target is supported by our findings. Autophagy and EV release following treatment with the NOS2 inhibitor could represent useful elements to better understand the complex biomolecular frame of GBM.


Assuntos
Amidinas/farmacologia , Autofagia/efeitos dos fármacos , Benzilaminas/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Glioblastoma/tratamento farmacológico , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Glioblastoma/metabolismo , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo
7.
Eur J Med Chem ; 178: 214-231, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31185412

RESUMO

Discovery of antimicrobial agents with a novel model of action is in urgent need for the clinical management of multidrug-resistant bacterial infections. Recently, we reported the identification of a first-in-class bacterial ribosomal RNA synthesis inhibitor, which interrupted the interaction between the bacterial transcription factor NusB and NusE. In this study, a series of diaryl derivatives were rationally designed and synthesized based on the previously established pharmacophore model. Inhibitory activity against the NusB-NusE binding, circular dichroism of compound treated NusB, antimicrobial activity, cytotoxicity, hemolytic property and cell permeability using Caco-2 cells were measured. Structure-activity relationship and quantitative structure-activity relationship were also concluded and discussed. Some of the derivatives demonstrated improved antimicrobial activity than the hit compound against a panel of clinically important pathogens, lowering the minimum inhibition concentration to 1-2 µg/mL against Staphylococcus aureus, including clinical strains of methicillin-resistant Staphylococcus aureus at a level comparable to some of the marketed antibiotics. Given the improved antimicrobial activity, specific inhibition of target protein-protein interaction and promising pharmacokinetic properties without significant cytotoxicity, this series of diaryl compounds have high potentials and deserve for further studies towards a new class of antimicrobial agents in the future.


Assuntos
Compostos de Anilina/farmacologia , Antibacterianos/farmacologia , Benzilaminas/farmacologia , Ligação Proteica/efeitos dos fármacos , Bases de Schiff/farmacologia , Compostos de Anilina/síntese química , Compostos de Anilina/química , Compostos de Anilina/toxicidade , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/toxicidade , Proteínas de Bactérias/metabolismo , Benzilaminas/síntese química , Benzilaminas/química , Benzilaminas/toxicidade , Células CACO-2 , Desenho de Fármacos , Eritrócitos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Células HeLa , Hemólise/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Bases de Schiff/síntese química , Bases de Schiff/química , Bases de Schiff/toxicidade , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo
8.
Brain Stimul ; 12(6): 1517-1525, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31217080

RESUMO

BACKGROUND: Animal models of Parkinson's Disease (PD) demonstrated increased facilitatory cortico-striatal activity, reflecting overactive glutamatergic neurotransmission and contributing to the pathophysiology of l-dopa induced dyskinesias (LIDs). OBJECTIVE: To assess different facilitatory intracortical circuits in the primary motor cortex (M1) in patients with PD and LIDs by means of a combination of transcranial magnetic stimulation (TMS) protocols. METHODS: We tested the Input/Output (I/O) curve, intracortical facilitation (ICF) and short-interval intracortical facilitation (SICF) at baseline (T0), 'OFF' and 'ON' state, in 20 PD patients with LIDs. The same parameters were examined after 2 weeks of chronic intake of 50 mg (T1) and 100 mg/day (T2) of safinamide. Finally, we tested SICF in a further group of patients without LIDs. RESULTS: At T0, patients with LIDs showed increased I/O curve steepness, which was partly ameliorated by l-dopa. These patients also had normal ICF, and abnormally increased SICF, which did not change with l-dopa. Safinamide improved the I/O curve both at T1 and T2, it reduced SICF at T1 and normalized this measure at T2. In patients with PD and LIDs, SICF correlated with the severity of dyskinesia. In patients without LIDs, SICF was less prominently abnormal and responsive to l-dopa. CONCLUSIONS: Patients with PD and LIDs have abnormal cortical facilitation, possibly suggesting overactive glutamatergic neurotransmission in specific circuits within M1. Although not responsive to l-dopa, this dysfunction is restored by the anti-glutamatergic properties of safinamide 100 mg. The results suggest that the abnormal cortical facilitation in M1 contributes to the pathophysiology of LIDs.


Assuntos
Discinesia Induzida por Medicamentos/diagnóstico , Discinesia Induzida por Medicamentos/fisiopatologia , Levodopa/efeitos adversos , Doença de Parkinson/diagnóstico , Doença de Parkinson/fisiopatologia , Estimulação Magnética Transcraniana/métodos , Idoso , Idoso de 80 Anos ou mais , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Animais , Benzilaminas/farmacologia , Benzilaminas/uso terapêutico , Discinesia Induzida por Medicamentos/tratamento farmacológico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Córtex Motor/efeitos dos fármacos , Córtex Motor/fisiologia , Doença de Parkinson/tratamento farmacológico
9.
Am J Physiol Endocrinol Metab ; 316(6): E1136-E1145, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30964702

RESUMO

Liver X receptors (LXRs) are ligand-dependent transcription factors activated by cholesterol metabolites. These receptors induce a suite of target genes required for de novo synthesis of triglycerides and cholesterol transport in many tissues. Two different isoforms, LXRα and LXRß, have been well characterized in liver, adipocytes, macrophages, and intestinal epithelium among others, but their contribution to cholesterol and fatty acid efflux in the lactating mammary epithelium is poorly understood. We hypothesize that LXR regulates lipogenesis during milk fat production in lactation. Global mRNA analysis of mouse mammary epithelial cells (MECs) revealed multiple LXR/RXR targets upregulated sharply early in lactation compared with midpregnancy. LXRα is the primary isoform, and its protein levels increase throughout lactation in MECs. The LXR agonist GW3965 markedly induced several genes involved in cholesterol transport and lipogenesis and enhanced cytoplasmic lipid droplet accumulation in the HC11 MEC cell line. Importantly, in vivo pharmacological activation of LXR increased the milk cholesterol percentage and induced sterol regulatory element-binding protein 1c (Srebp1c) and ATP-binding cassette transporter a7 (Abca7) expression in MECs. Cumulatively, our findings identify LXRα as an important regulator of cholesterol incorporation into the milk through key nodes of de novo lipogenesis, suggesting a potential therapeutic target in women with difficulty initiating lactation.


Assuntos
Colesterol/metabolismo , Epitélio/metabolismo , Lactação/genética , Receptores X do Fígado/genética , Glândulas Mamárias Animais/metabolismo , Leite/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Benzoatos/farmacologia , Benzilaminas/farmacologia , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Lactação/metabolismo , Lipogênese/genética , Receptores X do Fígado/metabolismo , Camundongos , RNA Mensageiro/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
10.
J Exp Clin Cancer Res ; 38(1): 157, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975171

RESUMO

BACKGROUND: Prostate cancer (PCa) remains a challenge worldwide. Due to the development of castration-resistance, traditional first-line androgen deprivation therapy (ADT) became powerlessness. Epidermal growth factor receptor (EGFR) is a well characterized therapeutic target to treat colorectal carcinoma and non-small cell lung cancer. Increasing studies have unraveled the significance of EGFR and its downstream signaling in the progression of castration-resistant PCa. METHOD: MTS, colony formation and Edu staining assays were used to analyze the cell proliferation of PCa cells. Flow cytometry was used to analyze PCa cell cycle distribution and cell apoptosis. Western blot was used to measure the expression of key proteins associated with cell cycle progression, apoptosis and EGFR signaling pathways. Transfection of exogenous small interfering RNA (siRNA) or plasmid was used to intervene specific gene expression. Nude mouse model was employed to test the in vivo effect of Spautin-1. RESULTS: The current study reveals that Spautin-1, a known inhibitor of ubiquitin-specific peptidase 10 (USP10) and USP13, inhibits EGFR phosphorylation and the activation of its downstream signaling. Inhibition of EGFR signaling induced by Spautin-1 leads to cell cycle arrest and apoptosis of PCa in a USP10/USP13 independent manner. The application of Spautin-1 reduces the expression of glucose transporter 1 (Glut1) and dramatically induces cell death under glucose deprivation condition. In vivo experiments show a potent anti-tumor effect of Spautin-1 alone and in combination with Enzalutamide. CONCLUSION: This study demonstrates the therapeutic potential of EGFR signaling inhibition by the use of Spautin-1 for PCa treatment.


Assuntos
Benzilaminas/farmacologia , Neoplasias da Próstata/metabolismo , Quinazolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Biomarcadores Tumorais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Modelos Animais de Doenças , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Modelos Biológicos , Terapia de Alvo Molecular , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Mediators Inflamm ; 2019: 6217548, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30944547

RESUMO

Liver X receptors (LXRs) have emerged as important regulators of inflammatory gene expression. Previously, we had reported that an LXRα gene promoter polymorphism (-1830 T > C) is associated with systemic lupus erythematosus (SLE). Therefore, we assessed cytokine expression in relation to LXRα polymorphism in monocyte-derived macrophages from patients with SLE. Macrophages were obtained after 72 hours of culture of human monocytes supplemented with phorbol 12-myristate 13-acetate. Cells were transfected with LXRα promoter constructs. Additionally, peripheral blood mononuclear cell- (PBMC-) derived macrophages from the patients were evaluated for proinflammatory cytokines in relation to the genotypes of LXRα -1830 T > C. The expression of LXRα was increased in macrophages; levels of proinflammatory cytokines were decreased with LXRα expression. Production of proinflammatory cytokines varied depending on LXRα -1830 T > C genotype. In particular, expression of LXRα was decreased and that of proinflammatory cytokines was increased for LXRα -1830 TC genotype compared to that for TT genotype. The data were consistent in PBMC-derived macrophages from patients with SLE. Increased proinflammatory cytokines is related to TLR7 and TLR9 expression. These data suggest that the expression levels of LXRα, according to LXRα -1830 T > C genotype, may contribute to the inflammatory response by induction of inflammatory cytokines in SLE.


Assuntos
Leucócitos Mononucleares/metabolismo , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Macrófagos/metabolismo , Benzoatos/farmacologia , Benzilaminas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Citocinas/metabolismo , Genótipo , Humanos , Hidrocarbonetos Fluorados/farmacologia , Immunoblotting , Leucócitos Mononucleares/efeitos dos fármacos , Receptores X do Fígado/agonistas , Macrófagos/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Sulfonamidas/farmacologia , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo
13.
Int J Mol Med ; 43(5): 1927-1938, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896855

RESUMO

The farnesoid X receptor (FXR) is known to regulate the gene expression of SR­BI, which mediates plasma high­density lipoprotein (HDL)­cholesterol uptake. Our previous study demonstrated that the activation of FXR by obeticholic acid (OCA) lowered plasma HDL­cholesterol levels and increased the hepatic mRNA and protein expression levels of SR­BI in hypercholesterolemic hamsters, but not in normolipidemic hamsters, suggesting that dietary cholesterol may be involved in the OCA­induced transcription of SR­BI. In the present study, a functional 90­base­pair regulatory region was identified in the first intron of the SR­BI gene of hamster and mouse that contains a FXR response element (IR­1) and an adjacent liver X receptor (LXR) response element (LXRE). By in vitro DNA binding and luciferase reporter gene assays, it was demonstrated that FXR and LXR bind to their recognition sequences within this intronic region and transactivate the SR­BI reporter gene in a synergistic manner. It was also shown that mutations at either the IR­1 site or the LXRE site eliminated OCA­mediated gene transcription. Utilizing chow­fed hamsters as an in vivo model, it was demonstrated that treating normolipidemic hamsters with OCA or GW3965 alone did not effectively induce levels of SR­BI, whereas their combined treatment significantly increased the mRNA and protein levels of SR­BI in the liver. The study further investigated effects of FXR and LXR coactivation on the gene expression of SR­BI in human liver cells. The intronic FXRE and LXRE regulatory region was not conserved in the human SR­BI genomic sequence, however, higher mRNA expression levels of SR­BI were observed in human primary hepatocytes and HepG2 cells exposed to combined treatments of FXR and LXR agonists, compared with those in cells exposed to individual ligand treatment. Therefore, these results suggest that human SR­BI gene transcription may also be subject to concerted activation by FXR and LXR, mediated via currently unidentified regulatory sequences.


Assuntos
Antígenos CD36/genética , Ácido Quenodesoxicólico/análogos & derivados , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores X do Fígado/metabolismo , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição Genética/efeitos dos fármacos , Animais , Sequência de Bases , Benzoatos/farmacologia , Benzilaminas/farmacologia , Antígenos CD36/metabolismo , Ácido Quenodesoxicólico/farmacologia , Genes Reporter , Células Hep G2 , Humanos , Íntrons/genética , Fígado/efeitos dos fármacos , Masculino , Mesocricetus , Motivos de Nucleotídeos/genética , Elementos de Resposta/genética
14.
Eur J Med Chem ; 168: 221-231, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30822711

RESUMO

Growing evidence confirms the potential of PDE4 inhibitors for the treatment of Parkinson's disease. Our reported PDE4 inhibitors FCPR16 and FCPR03 have displayed neuroprotective effects in SH-SY5Y cells, but have very low oral bioavailability. To access analogues with improved bioavailability, a new series of arylbenzylamine derivatives were designed and synthesized. Preliminary screening results of the series showed that arylbenzylamine derivatives bearing a pyridin-3-amine side chain displayed good inhibitory activities against human PDE4B1 and PDE4D7 isoforms. Moreover, kinetic studies revealed that the most potent compounds 11r and 11s with mid-nanomolar IC50 values partially bind to PDE4B1 (Imax = 93% and 90% respectively). Molecular docking results revealed the possible interactions of compounds 11r and 11s with upstream conserved region 2 (UCR2) of PDE4B1, which illuminate possible reasons for their partial inhibition against PDE4. Using a cell-based model of PD, compounds 11r and 11s were found to alleviate cellular apoptosis in SH-SY5Y cells induced by MPP+ (1-methyl-4-phenylpyridinium), with this neuroprotective effect being greater than PDE4 inhibitor rolipram. Furthermore, compound 11r displayed nearly sevenfold oral bioavailability (8.20%) than FCPR03 (1.23%).


Assuntos
Benzilaminas/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Descoberta de Drogas , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/tratamento farmacológico , Inibidores da Fosfodiesterase 4/farmacologia , Animais , Benzilaminas/síntese química , Benzilaminas/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Inibidores da Fosfodiesterase 4/síntese química , Inibidores da Fosfodiesterase 4/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Células Tumorais Cultivadas
15.
Biochim Biophys Acta Gene Regul Mech ; 1862(4): 447-456, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30831268

RESUMO

PDZK1 (NHERF3) interacts with membrane proteins whereby modulating their spatial arrangement, membrane stability, and function. One of the membrane proteins shown to be stabilized by interaction with PDZK1 is the HDL-receptor SR-BI (SCARB1). Testing the influence of TO 901317, a known activator of liver X receptor alpha (LXRα, NR1H3) which is a central regulator of the lipid homeostasis, Grefhorst et al. reported in 2012 that administration of TO 901317 did not affect PDZK1 expression and reduced the amount of SR-BI protein in mouse liver. Considering that TO 901317 also activates the xenosensor pregnane X receptor (PXR, NR1I2), it was aim of this study to further investigate the influence of LXRα and PXR activation on transcription of PDZK1. First, we tested the transactivation of PDZK1 by LXRα or PXR in cell-based reporter gene assays comparing the effect of prototypical ligands to that of TO 901317. Ligand mediated activation of LXRα increased, while that of PXR lowered luciferase activity. Further, we located the most likely binding site for LXRα and PXR on the PDZK1 promoter between -85 bp and -54 bp. The transcriptional regulation by LXRα was further supported showing enhanced mRNA expression of PDZK1 in HepG2 cells treated with the selective LXRα-agonist GW3965, while treatment with TO 901317 reduced the protein amount of PDZK1. Taken together, we provide evidence that both LXRα and PXR are transcriptional regulators of PDZK1 supporting the previous notion that the scaffold protein is part of cholesterol homeostasis and drug metabolism.


Assuntos
Proteínas de Transporte/genética , Receptores X do Fígado/metabolismo , Receptor de Pregnano X/metabolismo , Ativação Transcricional , Benzoatos/farmacologia , Benzilaminas/farmacologia , Sítios de Ligação , Proteínas de Transporte/biossíntese , Simulação por Computador , Células Hep G2 , Humanos , Hidrocarbonetos Fluorados/farmacologia , Receptores X do Fígado/agonistas , Proteínas de Membrana , Motivos de Nucleotídeos , Receptor de Pregnano X/agonistas , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Deleção de Sequência , Sulfonamidas/farmacologia
16.
Circ Res ; 124(8): 1228-1239, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30732532

RESUMO

RATIONALE: Subcellular Ca2+ indicators have yet to be developed for the myofilament where disease mutation or small molecules may alter contractility through myofilament Ca2+ sensitivity. Here, we develop and characterize genetically encoded Ca2+ indicators restricted to the myofilament to directly visualize Ca2+ changes in the sarcomere. OBJECTIVE: To produce and validate myofilament-restricted Ca2+ imaging probes in an adenoviral transduction adult cardiomyocyte model using drugs that alter myofilament function (MYK-461, omecamtiv mecarbil, and levosimendan) or following cotransduction of 2 established hypertrophic cardiomyopathy disease-causing mutants (cTnT [Troponin T] R92Q and cTnI [Troponin I] R145G) that alter myofilament Ca2+ handling. METHODS AND RESULTS: When expressed in adult ventricular cardiomyocytes RGECO-TnT (Troponin T)/TnI (Troponin I) sensors localize correctly to the sarcomere without contractile impairment. Both sensors report cyclical changes in fluorescence in paced cardiomyocytes with reduced Ca2+ on and increased Ca2+ off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localized Ca2+ handling conferred by MYK-461 and levosimendan, including an increase in Ca2+ binding rates with both levosimendan and MYK-461 not detected by an unrestricted protein sensor. Coadenoviral transduction of RGECO-TnT/TnI with hypertrophic cardiomyopathy causing thin filament mutants showed that the mutations increase myofilament [Ca2+] in systole, lengthen time to peak systolic [Ca2+], and delay [Ca2+] release. This contrasts with the effect of the same mutations on cytoplasmic Ca2+, when measured using unrestricted RGECO where changes to peak systolic Ca2+ are inconsistent between the 2 mutations. These data contrast with previous findings using chemical dyes that show no alteration of [Ca2+] transient amplitude or time to peak Ca2+. CONCLUSIONS: RGECO-TnT/TnI are functionally equivalent. They visualize Ca2+ within the myofilament and reveal unrecognized aspects of small molecule and disease-associated mutations in living cells.


Assuntos
Cálcio/metabolismo , Cardiomiopatia Hipertrófica/genética , Mutação , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Sarcômeros/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Adenoviridae , Animais , Benzilaminas/farmacologia , Cardiomiopatia Hipertrófica/metabolismo , Cobaias , Técnicas In Vitro , Masculino , Miofibrilas/efeitos dos fármacos , Miosinas/efeitos dos fármacos , Miosinas/metabolismo , Simendana/farmacologia , Transdução Genética/métodos , Troponina I/genética , Troponina I/metabolismo , Troponina T/genética , Troponina T/metabolismo , Uracila/análogos & derivados , Uracila/farmacologia , Ureia/análogos & derivados , Ureia/farmacologia
17.
Circ Res ; 124(8): 1172-1183, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30700234

RESUMO

RATIONALE: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in combination with CRISPR/Cas9 genome editing provide unparalleled opportunities to study cardiac biology and disease. However, sarcomeres, the fundamental units of myocyte contraction, are immature and nonlinear in hiPSC-CMs, which technically challenge accurate functional interrogation of contractile parameters in beating cells. Furthermore, existing analysis methods are relatively low-throughput, indirectly assess contractility, or only assess well-aligned sarcomeres found in mature cardiac tissues. OBJECTIVE: We aimed to develop an analysis platform that directly, rapidly, and automatically tracks sarcomeres in beating cardiomyocytes. The platform should assess sarcomere content, contraction and relaxation parameters, and beat rate. METHODS AND RESULTS: We developed SarcTrack, a MatLab software that monitors fluorescently tagged sarcomeres in hiPSC-CMs. The algorithm determines sarcomere content, sarcomere length, and returns rates of sarcomere contraction and relaxation. By rapid measurement of hundreds of sarcomeres in each hiPSC-CM, SarcTrack provides large data sets for robust statistical analyses of multiple contractile parameters. We validated SarcTrack by analyzing drug-treated hiPSC-CMs, confirming the contractility effects of compounds that directly activate (CK-1827452) or inhibit (MYK-461) myosin molecules or indirectly alter contractility (verapamil and propranolol). SarcTrack analysis of hiPSC-CMs carrying a heterozygous truncation variant in the myosin-binding protein C ( MYBPC3) gene, which causes hypertrophic cardiomyopathy, recapitulated seminal disease phenotypes including cardiac hypercontractility and diminished relaxation, abnormalities that normalized with MYK-461 treatment. CONCLUSIONS: SarcTrack provides a direct and efficient method to quantitatively assess sarcomere function. By improving existing contractility analysis methods and overcoming technical challenges associated with functional evaluation of hiPSC-CMs, SarcTrack enhances translational prospects for sarcomere-regulating therapeutics and accelerates interrogation of human cardiac genetic variants.


Assuntos
Algoritmos , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Sarcômeros/fisiologia , Software , Benzilaminas/antagonistas & inibidores , Benzilaminas/farmacologia , Fármacos Cardiovasculares/farmacologia , Proteínas de Transporte/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desenho Assistido por Computador , Fluorescência , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Microscopia de Força Atômica/métodos , Contração Miocárdica , Miócitos Cardíacos/efeitos dos fármacos , Miosinas/efeitos dos fármacos , Miosinas/metabolismo , Propranolol/farmacologia , Uracila/análogos & derivados , Uracila/antagonistas & inibidores , Uracila/farmacologia , Ureia/análogos & derivados , Ureia/farmacologia , Verapamil/farmacologia , Gravação em Vídeo
18.
Toxicol Lett ; 304: 50-57, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30658151

RESUMO

Two emerging psychoactive substances, 2-(2,5-dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine (25D-NBOMe) and N-(2-methoxybenzyl)-2,5-dimethoxy-4-chlorophenethylamine (25C-NBOMe), are being abused, leading to fatal and non-fatal intoxications. However, most of their adverse effects have been reported anecdotally. In the present study, cardiotoxicity was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, rat electrocardiography (ECG), and human ether-a-go-go-related gene (hERG) assay. Expression levels of p21 (CDC42/RAC)-activated kinase 1 (PAK1), one of known biomarkers for cardiotoxicity, were also analyzed. Both 25D-NBOMe and 25C-NBOMe at 100 µM reduced cell viability in MTT assay. At 2.0 mg/kg and 0.75 mg/kg, they prolonged QT intervals in rat ECG. PAK1 was down-regulated by treatment with these two test compounds. Furthermore, potassium channels were inhibited by 25D-NBOMe treatment in hERG assay. Taken together, these results suggest that both 25D-NBOMe and 25C-NBOMe have potential cardiotoxicity, especially regarding cardiac rhythm. Further studies are needed to confirm the relationship between PAK1 down-regulation and cardiotoxicity.


Assuntos
Benzilaminas/efeitos adversos , Etilaminas/toxicidade , Cardiopatias/induzido quimicamente , Frequência Cardíaca/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Fenetilaminas/farmacologia , Psicotrópicos/efeitos adversos , Potenciais de Ação , Animais , Benzilaminas/farmacologia , Células CHO , Cardiotoxicidade , Sobrevivência Celular , Cricetulus , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/metabolismo , Cardiopatias/metabolismo , Cardiopatias/fisiopatologia , Masculino , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenetilaminas/efeitos adversos , Psicotrópicos/farmacologia , Ratos Sprague-Dawley , Quinases Ativadas por p21/metabolismo
19.
PLoS One ; 14(1): e0211281, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30689673

RESUMO

Diabetes is a very strong predictor of chronic systemic vascular diseases and acute cardiovascular events. Recently, associations between metabolic disorders and pulmonary hypertension have also been reported in both humans and animal models. In order to get some further insight into the relationship of pulmonary hypertension with obesity, insulin resistance and hyperglycemia, herein we have used the Zucker diabetic fatty rats (ZDF/clr-lepr fa) at 20 weeks fed a standard diet and compared to their lean Zucker littermates (ZL). ZDF rats were obese, had elevated plasma glucose levels and insulin resistance, i.e. a clinically relevant model of type 2 diabetes. They presented elevated systolic, diastolic and mean pulmonary arterial pressures and a parallel increase in the Fulton index. Systemic arterial pressures were also increased but the left ventricle plus septum weight was similar in both groups and the heart rate was reduced. Wall media thickening was observed in the small pulmonary arteries from the ZDF rats. Isolated pulmonary arteries mounted in a wire myograph showed similar vasoconstrictor responses to phenylephrine and 5-HT and similar responses to the endothelium-dependent vasodilator acetylcholine. However, the iNOS inhibitor 1400W enhanced the vasoconstrictor responses in ZDF but not in ZL rats. The protein expression of eNOS and iNOS was not significantly different in the lungs of the two groups. The lung expression of Bmpr2 mRNA was downregulated. However, the mRNA expression of Kcna5, Kcnk3, Kcnq1, Kcnq4 or Kcnq5, which encode for the potassium channels Kv1.5, TASK-1, Kv7.1, Kv7.4 and Kv7.5, respectively, was similar in ZL and ZDF rats. In conclusion, ZDF rats show increased pulmonary arterial pressure, right ventricular hypertrophy, pulmonary arterial medial thickening and downregulated lung Bmpr2 despite leptin resistance. These changes were mild but are consistent with the view that diabetes is a risk factor for pulmonary hypertension.


Assuntos
Pressão Arterial/fisiologia , Artéria Pulmonar/fisiologia , Amidinas/farmacologia , Animais , Benzilaminas/farmacologia , Glicemia/análise , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Citocinas/sangue , Regulação da Expressão Gênica , Hemodinâmica , Pulmão/metabolismo , Pulmão/patologia , Obesidade/patologia , Obesidade/veterinária , Canais de Potássio/genética , Canais de Potássio/metabolismo , Ratos , Ratos Zucker , Vasoconstrição/efeitos dos fármacos
20.
Bioorg Med Chem ; 27(1): 161-166, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30503782

RESUMO

Inspite of progress made for the discovery of novel antiepileptic drugs, epilepsy remains an unmet medical need. We synthesized nine trifluoromethylated enaminone derivatives and tested them for their anticonvulsant activity using maximal electroshock seizure (MES) test, subcutaneous pentylenetetrazole (scPTZ) test, and rotorod test for neurotoxicity. Among the compounds tested 3-(4-fluoro-3-(trifluomethyl)benzylamino)-5-(trifluoromethyl)cyclohex-2-enone (4f) showed ED50 of 23.47 mg/kg, when given orally to rats, 3-(4-chlorophenylamino)-5-(trifluoromethyl)cyclohex-2-enone (5a), which was previously reported by us but for which no quantitative data was available at the time, exhibited an ED50 of 62.39 mg/kg. Under the same conditions commercially available carbamazepine showed an ED50 of 28.20 mg/kg. There were no neurotoxicity observed upto a dose of 300 mg/kg for all the tested compounds. Compounds 4f and 5a represent good lead compounds for further development.


Assuntos
Anticonvulsivantes/farmacologia , Benzilaminas/farmacologia , Cicloexanonas/farmacologia , Cicloexilaminas/farmacologia , Hidrocarbonetos Fluorados/farmacologia , Convulsões/prevenção & controle , Animais , Anticonvulsivantes/síntese química , Anticonvulsivantes/farmacocinética , Anticonvulsivantes/toxicidade , Benzilaminas/síntese química , Benzilaminas/farmacocinética , Benzilaminas/toxicidade , Simulação por Computador , Cicloexanonas/síntese química , Cicloexanonas/farmacocinética , Cicloexanonas/toxicidade , Cicloexilaminas/síntese química , Cicloexilaminas/farmacocinética , Cicloexilaminas/toxicidade , Desenho de Fármacos , Hidrocarbonetos Fluorados/síntese química , Hidrocarbonetos Fluorados/farmacocinética , Hidrocarbonetos Fluorados/toxicidade , Masculino , Camundongos , Estrutura Molecular , Ratos , Relação Estrutura-Atividade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA