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1.
Nat Commun ; 11(1): 5108, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037189

RESUMO

The spectrin-based membrane skeleton is a major component of the cell cortex. While expressed by all metazoans, its dynamic interactions with the other cortex components, including the plasma membrane or the acto-myosin cytoskeleton, are poorly understood. Here, we investigate how spectrin re-organizes spatially and dynamically under the membrane during changes in cell mechanics. We find spectrin and acto-myosin to be spatially distinct but cooperating during mechanical challenges, such as cell adhesion and contraction, or compression, stretch and osmolarity fluctuations, creating a cohesive cortex supporting the plasma membrane. Actin territories control protrusions and contractile structures while spectrin territories concentrate in retractile zones and low-actin density/inter-contractile regions, acting as a fence that organize membrane trafficking events. We unveil here the existence of a dynamic interplay between acto-myosin and spectrin necessary to support a mesoscale organization of the lipid bilayer into spatially-confined cortical territories during cell mechanoresponse.


Assuntos
Actomiosina/metabolismo , Membrana Celular/metabolismo , Espectrina/metabolismo , Actinas/metabolismo , Animais , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose/fisiologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Camundongos , Microscopia Confocal , Células NIH 3T3 , Espectrina/genética , Estresse Mecânico
2.
Pharmacol Res Perspect ; 8(6): e00674, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33124786

RESUMO

SARS-CoV-2, a member of the coronavirus family, has caused a global public health emergency. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously reasoned that the FDA-approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) should inhibit coronaviruses, including SARS-CoV-2. Here, using model polymerase extension experiments, we demonstrate that the active triphosphate form of Sofosbuvir is incorporated by low-fidelity polymerases and SARS-CoV RNA-dependent RNA polymerase (RdRp), and blocks further incorporation by these polymerases; the active triphosphate form of Sofosbuvir is not incorporated by a host-like high-fidelity DNA polymerase. Using the same molecular insight, we selected 3'-fluoro-3'-deoxythymidine triphosphate and 3'-azido-3'-deoxythymidine triphosphate, which are the active forms of two other anti-viral agents, Alovudine and AZT (an FDA-approved HIV/AIDS drug) for evaluation as inhibitors of SARS-CoV RdRp. We demonstrate the ability of two of these HIV reverse transcriptase inhibitors to be incorporated by SARS-CoV RdRp where they also terminate further polymerase extension. Given the 98% amino acid similarity of the SARS-CoV and SARS-CoV-2 RdRps, we expect these nucleotide analogues would also inhibit the SARS-CoV-2 polymerase. These results offer guidance to further modify these nucleotide analogues to generate more potent broad-spectrum anti-coronavirus agents.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , RNA Replicase/antagonistas & inibidores , Betacoronavirus/enzimologia , Carbamatos/farmacologia , Infecções por Coronavirus/virologia , Didesoxinucleotídeos/farmacologia , Combinação de Medicamentos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Pandemias , Pneumonia Viral/virologia , Sofosbuvir/farmacologia , Nucleotídeos de Timina/farmacologia , Zidovudina/análogos & derivados , Zidovudina/farmacologia
3.
Pharm Res ; 37(10): 194, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32918191

RESUMO

PURPOSE: We characterized three canine P-gp (cP-gp) deficient MDCKII cell lines. Their relevance for identifying efflux transporter substrates and predicting limitation of brain penetration were evaluated. In addition, we discuss how compound selection can be done in drug discovery by using these cell systems. METHOD: hMDR1, hBCRP-transfected, and non-transfected MDCKII ZFN cells (all with knock-down of endogenous cP-gp) were used for measuring permeability and efflux ratios for substrates. The compounds were also tested in MDR1_Caco-2 and BCRP_Caco-2, each with a double knock-out of BCRP/MRP2 or MDR1/MRP2 transporters respectively. Efflux results were compared between the MDCK and Caco-2 models. Furthermore, in vitro MDR1_ZFN efflux data were correlated with in vivo unbound drug brain-to-plasma partition coefficient (Kp,uu). RESULTS: MDR1 and BCRP substrates are correctly classified and robust transporter affinities with control substrates are shown. Cell passage mildly influenced mRNA levels of transfected transporters, but the transporter activity was proven stable for several years. The MDCK and Caco-2 models were in high consensus classifying same efflux substrates. Approx. 80% of enlisted substances were correctly predicted with the MDR1_ZFN model for brain penetration. CONCLUSION: cP-gp deficient MDCKII ZFN models are reliable tools to identify MDR1 and BCRP substrates and useful for predicting efflux liability for brain penetration.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/deficiência , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Neoplasias/metabolismo , Farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular , Dibenzocicloeptenos/farmacologia , Dicetopiperazinas/farmacologia , Cães , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Células Madin Darby de Rim Canino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Prazosina/farmacocinética , Quinidina/farmacocinética , Quinolinas/farmacologia , Especificidade por Substrato , Transfecção
4.
Nat Commun ; 11(1): 4902, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994402

RESUMO

Living cells and tissues experience various complex modes of forces that are important in physiology and disease. However, how different force modes impact gene expression is elusive. Here we apply local forces of different modes via a magnetic bead bound to the integrins on a cell and quantified cell stiffness, chromatin deformation, and DHFR (dihydrofolate reductase) gene transcription. In-plane stresses result in lower cell stiffness than out-of-plane stresses that lead to bead rolling along the cell long axis (i.e., alignment of actin stress fibers) or at different angles (90° or 45°). However, chromatin stretching and ensuing DHFR gene upregulation by the in-plane mode are similar to those induced by the 45° stress mode. Disrupting stress fibers abolishes differences in cell stiffness, chromatin stretching, and DHFR gene upregulation under different force modes and inhibiting myosin II decreases cell stiffness, chromatin deformation, and gene upregulation. Theoretical modeling using discrete anisotropic stress fibers recapitulates experimental results and reveals underlying mechanisms of force-mode dependence. Our findings suggest that forces impact biological responses of living cells such as gene transcription via previously underappreciated means.


Assuntos
Cromatina/química , Fibras de Estresse/química , Tetra-Hidrofolato Desidrogenase/genética , Transcrição Genética/fisiologia , Regulação para Cima/fisiologia , Animais , Anisotropia , Fenômenos Biomecânicos/genética , Células CHO , Cromatina/metabolismo , Cricetulus , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Microscopia Intravital , Microscopia de Fluorescência , Miosina Tipo II/antagonistas & inibidores , Miosina Tipo II/metabolismo , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Estresse Mecânico , Transcrição Genética/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
5.
J Vis Exp ; (162)2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32831305

RESUMO

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors. This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment. The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting. The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.


Assuntos
Bioensaio/métodos , Histona Acetiltransferases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Acetilação/efeitos dos fármacos , Ácidos Anacárdicos/farmacologia , Ciclina D1/genética , Ciclina D1/metabolismo , Proteína p300 Associada a E1A/antagonistas & inibidores , Proteína p300 Associada a E1A/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Histonas/metabolismo , Humanos , Lisina/metabolismo , Células MCF-7 , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes , Sonicação
6.
Science ; 369(6505): 858-862, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32792401

RESUMO

The conversion of neural stem cells into neurons is associated with the remodeling of organelles, but whether and how this is causally linked to fate change is poorly understood. We examined and manipulated mitochondrial dynamics during mouse and human cortical neurogenesis. We reveal that shortly after cortical stem cells have divided, daughter cells destined to self-renew undergo mitochondrial fusion, whereas those that retain high levels of mitochondria fission become neurons. Increased mitochondria fission promotes neuronal fate, whereas induction of mitochondria fusion after mitosis redirects daughter cells toward self-renewal. This occurs during a restricted time window that is doubled in human cells, in line with their increased self-renewal capacity. Our data reveal a postmitotic period of fate plasticity in which mitochondrial dynamics are linked with cell fate.


Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Mitocôndrias/fisiologia , Dinâmica Mitocondrial , Mitose , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Neurônios/citologia , Animais , Córtex Cerebral/citologia , Feminino , Células HEK293 , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Masculino , Camundongos , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Sirtuínas/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(34): 20803-20813, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32764148

RESUMO

Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric vesicular stomatitis virus (VSV) containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small-molecule inhibitors of the main endosomal phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.


Assuntos
Betacoronavirus/efeitos dos fármacos , Ebolavirus/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases , Triazinas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Betacoronavirus/fisiologia , Células Cultivadas , Infecções por Coronavirus , Ebolavirus/fisiologia , Edição de Genes , Humanos , Pandemias , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Pneumonia Viral , Proteínas do Envelope Viral/genética
8.
Anticancer Res ; 40(7): 3781-3792, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32620617

RESUMO

BACKGROUND/AIM: Canine B-cell lymphoma represents a useful in vivo model for human diffuse large B-cell lymphoma (DLBCL). Pan-Bromodomain and extra-terminal (BET) inhibition targeting BRD2/3/4 and selective inhibition of BRD4, as well as spleen tyrosine kinase (SYK) inhibition, are currently evaluated as haematologic cancer therapy. Herein, we characterized the differences in the biologic response of isoform-specific or pan-BET inhibition alone or in combination with SYK inhibition. MATERIALS AND METHODS: I-BET151 (pan-inhibitor) and AZD5153 (BRD4 inhibitor) were combined with Entospletinib (SYK inhibitor) and comparatively analysed in the canine DLBCL cell line CLBL-1. Dose- and time-dependent cellular responses were analysed by cell number, metabolic activity, apoptosis/necrosis, and cell morphology. The synergistic potential was evaluated through the Bliss independence model. RESULTS: I-BET151 and AZD5153 showed significant dose- and time-dependent inhibitory effects. Adding Entospletinib to I-BET151 or AZD5153 had no additional synergistic effects. CONCLUSION: Entospletinib did not enhance the inhibitory effects of the pan- or isoform-specific BET.


Assuntos
Indazóis/farmacologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Isoformas de Proteínas/antagonistas & inibidores , Pirazinas/farmacologia , Animais , Linhagem Celular Tumoral , Cães , Compostos Heterocíclicos com 2 Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Linfoma Difuso de Grandes Células B/metabolismo , Piperazinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Quinase Syk/metabolismo
9.
Front Immunol ; 11: 1518, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32655582

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of the genus Betacoronavirus within the family Coronaviridae. It is an enveloped single-stranded positive-sense RNA virus. Since December of 2019, a global expansion of the infection has occurred with widespread dissemination of coronavirus disease 2019 (COVID-19). COVID-19 often manifests as only mild cold-like symptomatology, but severe disease with complications occurs in 15% of cases. Respiratory failure occurs in severe disease that can be accompanied by a systemic inflammatory reaction characterized by inflammatory cytokine release. In severe cases, fatality is caused by the rapid development of severe lung injury characteristic of acute respiratory distress syndrome (ARDS). Although ARDS is a complication of SARS-CoV-2 infection, it is not viral replication or infection that causes tissue injury; rather, it is the result of dysregulated hyperinflammation in response to viral infection. This pathology is characterized by intense, rapid stimulation of the innate immune response that triggers activation of the Nod-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome pathway and release of its products including the proinflammatory cytokines IL-6 and IL-1ß. Here we review the literature that describes the pathogenesis of severe COVID-19 and NLRP3 activation and describe an important role in targeting this pathway for the treatment of severe COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Inflamassomos/antagonistas & inibidores , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pneumonia Viral/metabolismo , Animais , Infecções por Coronavirus/complicações , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Síndrome da Liberação de Citocina/tratamento farmacológico , Síndrome da Liberação de Citocina/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Humanos , Imunidade Inata , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/metabolismo , Camundongos , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Piroptose/efeitos dos fármacos , Síndrome do Desconforto Respiratório do Adulto/tratamento farmacológico , Síndrome do Desconforto Respiratório do Adulto/etiologia , Síndrome do Desconforto Respiratório do Adulto/metabolismo , Sesquiterpenos de Guaiano/farmacologia , Sesquiterpenos de Guaiano/uso terapêutico , Sulfonas/farmacologia , Sulfonas/uso terapêutico
10.
Am J Respir Cell Mol Biol ; 63(4): 519-530, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32628869

RESUMO

KD025 is a ROCK2 inhibitor currently being tested in clinical trials for the treatment of fibrotic lung diseases. The therapeutic effects of KD025 are partly due to its inhibition of profibrotic pathways and fat metabolism. However, whether KD025 affects pulmonary microvascular endothelial cell (PMVEC) function is unknown, despite evidence that alveolar-capillary membrane disruption constitutes major causes of death in fibrotic lung diseases. We hypothesized that KD025 regulates PMVEC metabolism, pH, migration, and survival, a series of interrelated functional characteristics that determine pulmonary barrier integrity. We used PMVECs isolated from Sprague Dawley rats. KD025 dose-dependently decreased lactate production and glucose consumption. The inhibitory effect of KD025 was more potent compared with other metabolic modifiers, including 2-deoxy-glucose, extracellular acidosis, dichloroacetate, and remogliflozin. Interestingly, KD025 increased oxidative phosphorylation, whereas 2-deoxy-glucose did not. KD025 also decreased intracellular pH and induced a compensatory increase in anion exchanger 2. KD025 inhibited PMVEC migration, but fasudil (nonspecific ROCK inhibitor) did not. We tested endothelial permeability in vivo using Evans Blue dye in the bleomycin pulmonary fibrosis model. Baseline permeability was decreased in KD025-treated animals independent of bleomycin treatment. Under hypoxia, KD025 increased PMVEC necrosis as indicated by increased lactate dehydrogenase release and propidium iodide uptake and decreased ATP; it did not affect Annexin V binding. ROCK2 knockdown had no effect on PMVEC metabolism, pH, and migration, but it increased nonapoptotic caspase-3 activity. Together, we report that KD025 promotes oxidative phosphorylation; decreases glycolysis, intracellular pH, and migration; and strengthens pulmonary barrier integrity in a ROCK2-independent manner.


Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Pulmão/efeitos dos fármacos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Anexina A5/metabolismo , Movimento Celular/efeitos dos fármacos , Desoxiglucose/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Glicólise/efeitos dos fármacos , Concentração de Íons de Hidrogênio , L-Lactato Desidrogenase/metabolismo , Pulmão/metabolismo , Masculino , Fosforilação Oxidativa/efeitos dos fármacos , Propídio/farmacologia , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/metabolismo
11.
Nat Commun ; 11(1): 2814, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499508

RESUMO

Food consumption is fundamental for life, and eating disorders often result in devastating or life-threatening conditions. Anorexia nervosa (AN) is characterized by a persistent restriction of energy intake, leading to lowered body weight, constant fear of gaining weight, and psychological disturbances of body perception. Herein, we demonstrate that SIRT1 inhibition, both genetically and pharmacologically, delays the onset and progression of AN behaviors in activity-based anorexia (ABA) models, while SIRT1 activation accelerates ABA phenotypes. Mechanistically, we suggest that SIRT1 promotes progression of ABA, in part through its interaction with NRF1, leading to suppression of a NMDA receptor subunit Grin2A. Our results suggest that AN may arise from pathological positive feedback loops: voluntary food restriction activates SIRT1, promoting anxiety, hyperactivity, and addiction to starvation, exacerbating the dieting and exercising, thus further activating SIRT1. We propose SIRT1 inhibition can break this cycle and provide a potential therapy for individuals suffering from AN.


Assuntos
Anorexia Nervosa/metabolismo , Regulação da Expressão Gênica , Fator 1 Nuclear Respiratório/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sirtuína 1/metabolismo , Animais , Peso Corporal , Carbazóis/farmacologia , Modelos Animais de Doenças , Feminino , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Resveratrol/farmacologia , Estresse Mecânico , Regulação para Cima
12.
Virus Res ; 286: 198068, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32565126

RESUMO

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a renewed interest in studying the role of the spike S glycoprotein in regulating coronavirus infections in the natural host. Taking advantage of the cryo-electron microscopy structure of SARS-CoV-2 S trimer in the prefusion conformation, we performed a virtual screening simulation with the aim to identify novel molecules that could be used as fusion inhibitors. The spike glycoprotein structure has been completed using modeling techniques and its inner cavity, needful for the postfusion transition of the trimer, has been scanned for the identification of strongly interacting available drugs. Finally, the stability of the protein-drug top complexes has been tested using classical molecular dynamics simulations. The free energy of interaction of the molecules to the spike protein has been evaluated through the MM/GBSA method and per-residue decomposition analysis. Results have been critically discussed considering previous scientific knowledge concerning the selected compounds and sequence alignments have been carried out to evaluate the spike glycoprotein similarity among the betacoronavirus family members. Finally, a cocktail of drugs that may be used as SARS-CoV-2 fusion inhibitors has been suggested.


Assuntos
Antivirais/química , Betacoronavirus/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Indóis/química , Perileno/análogos & derivados , Glicoproteína da Espícula de Coronavírus/química , Sulfonamidas/química , Antivirais/farmacologia , Betacoronavirus/patogenicidade , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Expressão Gênica , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Indóis/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Perileno/química , Perileno/farmacologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Sulfonamidas/farmacologia , Termodinâmica , Interface Usuário-Computador , Internalização do Vírus/efeitos dos fármacos
13.
Am J Physiol Endocrinol Metab ; 319(2): E330-E337, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32543942

RESUMO

Ghrelin is a predominantly stomach-derived peptide hormone with many actions including regulation of food intake, body weight, and blood glucose. Plasma ghrelin levels are robustly regulated by feeding status, with its levels increasing upon caloric restriction and decreasing after food intake. At least some of this regulation might be due to direct responsiveness of ghrelin cells to changes in circulating nutrients, including glucose. Indeed, oral and parental glucose administration to humans and mice lower plasma ghrelin. Also, dissociated mouse gastric mucosal cell preparations, which contain ghrelin cells, decrease ghrelin secretion when cultured in high ambient glucose. Here, we used primary cultures of mouse gastric mucosal cells in combination with an array of pharmacological tools to examine the potential role of changed intracellular oxidative stress in glucose-restricted ghrelin secretion. The antioxidants resveratrol, SRT1720, and curcumin all markedly increased ghrelin secretion. Furthermore, three different selective activators of Nuclear factor erythroid-derived-2-like 2 (Nrf2), a master regulator of the antioxidative cellular response to oxidative stress, increased ghrelin secretion. These antioxidant compounds blocked the inhibitory effects of glucose on ghrelin secretion. Therefore, we conclude that lowering oxidative stress within ghrelin cells stimulates ghrelin secretion and blocks the direct effects of glucose on ghrelin cells to inhibit ghrelin secretion.


Assuntos
Mucosa Gástrica/metabolismo , Grelina/metabolismo , Estresse Oxidativo/fisiologia , Animais , Antioxidantes/farmacologia , Células Cultivadas , Curcumina/farmacologia , Mucosa Gástrica/efeitos dos fármacos , Glucose/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/fisiologia , Resveratrol/farmacologia
14.
Clin Sci (Lond) ; 134(12): 1357-1376, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32490513

RESUMO

Non-specific inhibition of Rho-associated kinases (ROCKs) alleviated renal fibrosis in the unilateral ureteral obstruction (UUO) model, while genetic deletion of ROCK1 did not affect renal pathology in mice. Thus, whether ROCK2 plays a role in renal tubulointerstitial fibrosis needs to be clarified. In the present study, a selective inhibitor against ROCK2 or genetic approach was used to investigate the role of ROCK2 in renal tubulointerstitial fibrosis. In the fibrotic kidneys of chronic kidney diseases (CKDs) patients, we observed an enhanced expression of ROCK2 with a positive correlation with interstitial fibrosis. In mice, the ROCK2 protein level was time-dependently increased in the UUO model. By treating CKD animals with KD025 at the dosage of 50 mg/kg/day via intraperitoneal injection, the renal fibrosis shown by Masson's trichrome staining was significantly alleviated along with the reduced expression of fibrotic genes. In vitro, inhibiting ROCK2 by KD025 or ROCK2 knockdown/knockout significantly blunted the pro-fibrotic response in transforming growth factor-ß1 (TGF-ß1)-stimulated mouse renal proximal tubular epithelial cells (mPTCs). Moreover, impaired cellular metabolism was reported as a crucial pathogenic factor in CKD. By metabolomics analysis, we found that KD025 restored the metabolic disturbance, including the impaired glutathione metabolism in TGF-ß1-stimulated tubular epithelial cells. Consistently, KD025 increased antioxidative stress enzymes and nuclear erythroid 2-related factor 2 (Nrf2) in fibrotic models. In addition, KD025 decreased the infiltration of macrophages and inflammatory response in fibrotic kidneys and blunted the activation of macrophages in vitro. In conclusion, inhibition of ROCK2 may serve as a potential novel therapy for renal tubulointerstitial fibrosis in CKD.


Assuntos
Células Epiteliais/enzimologia , Túbulos Renais Proximais/patologia , Doenças Metabólicas/enzimologia , Quinases Associadas a rho/antagonistas & inibidores , Adolescente , Animais , Anti-Inflamatórios/farmacologia , Criança , Pré-Escolar , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Feminino , Fibrose , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Lactente , Inflamação/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Doenças Metabólicas/patologia , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Células RAW 264.7 , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Regulação para Cima/efeitos dos fármacos , Obstrução Ureteral/enzimologia , Obstrução Ureteral/patologia , Quinases Associadas a rho/metabolismo
15.
J Cancer Res Clin Oncol ; 146(9): 2219-2229, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32507974

RESUMO

PURPOSE: Pancreatic cancer is a lethal form of cancer that can be triggered by prolonged or acute inflammation of the pancreas. Inflammation have been shown to be regulated by a group of key protein molecules known as the inflammasomes. The NLRP3 inflammasome is the most studied inflammasome and have been strongly implicated to regulate cancer cell proliferation. Therefore, this study aimed to examine the regulation of NLRP3 inflammasome under LPS-induced inflammation and its role in modulating cell proliferation in a panel of pancreatic cancer cells. METHODS: The effects of LPS-induced NLRP3 activation in the presence or absence of MCC950, NLRP3-specific inhibitor, was tested on a panel of three pancreatic cancer cell lines (SW1990, PANC1 and Panc10.05). Western blotting, cell viability kits and ELISA kits were used to examine the effects of LPS-induced NLRP3 activation and inhibition by MCC950 on NLRP3 expression, cell viability, caspase-1 activity and cytokine IL-1ß, respectively. RESULTS: LPS-induced inflammation in the presence of ATP activates NLRP3 that subsequently increases pancreatic cancer cell proliferation by increasing caspase-1 activity leading to overall production of IL-1ß. The inhibition of the NLRP3 inflammasome activation via the specific NLRP3 antagonist MCC950 was able to reduce the cell viability of pancreatic cancer cells. However, the efficacy of MCC950 varies between cell types which is most probably due to the difference in ASC expressions which have a different role in inflammasome activation. CONCLUSION: There is a dynamic interaction between inflammasome that regulates inflammasome-mediated inflammation in pancreatic adenocarcinoma cells.


Assuntos
Adenocarcinoma/tratamento farmacológico , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Inflamassomos/efeitos dos fármacos , Inflamação/tratamento farmacológico , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Sulfonas/farmacologia , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Inflamassomos/metabolismo , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Neoplasias Pancreáticas/induzido quimicamente , Neoplasias Pancreáticas/metabolismo
16.
Toxicol Appl Pharmacol ; 400: 115075, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32470352

RESUMO

NLRP3, one of the HSP-90 clients, has been defined as a critical component of IBD. In a rat model of DSS-induced colitis, we investigated the anti-inflammatory potential of the combined therapy with CP-456773 (CP), an NLRP3 inhibitor, and celastrol (CSR), an NF-κB inhibitor. Our results revealed that the CSR/CP combined therapy (CCCT) attenuated colon shortening, DAI and MDI in addition to improvement of the colonic histological picture. Moreover, the CCCT increased the antioxidant defense machinery of the colonic tissue and decreased MPO activity. Furthermore, the inflammation markers such as TNF-α and IL-6 were downregulated. These effects might be attributed to the inhibitory effect of CSR on the priming step of the NLRP3 inflammasome activation by interrupting NF-κB signalling and inhibition of HSP-90 (at the protein and mRNA levels) along with inhibitory effect of CP on the expression of the NLRP3. These latter effects resulted in decreased tissue expression and activity of the caspase-1 and repressing the subsequent release of the active forms of IL-1ß and IL-18, hence, the pyroptosis process is restrained. Additionally, the CCCT resulted in inducing autophagy by AMPK/mTOR-dependent mechanisms leading to the accumulation of BECN1 protein and a significant decrease in the levels of p62 SQSTM1. The inhibitory effect on HSP-90 in conjunction with induction of autophagy suggest increased autophagic degradation of NLRP3. This novel approach provides a basis for the clinical application of this combination in IBD treatment and might also be promising for the pharmacological intervention of other NLRP3 inflammasome-dependent inflammatory conditions.


Assuntos
Anti-Inflamatórios/farmacologia , Autofagia/efeitos dos fármacos , Colite/tratamento farmacológico , Proteínas de Choque Térmico HSP90/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Sulfonas/farmacologia , Triterpenos/farmacologia , Animais , Anti-Inflamatórios/administração & dosagem , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Citocinas/metabolismo , Sulfato de Dextrana/farmacologia , Modelos Animais de Doenças , Quimioterapia Combinada , Proteínas de Choque Térmico HSP90/sangue , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Inflamassomos/efeitos dos fármacos , Inflamassomos/imunologia , Masculino , Ratos Sprague-Dawley , Sulfonas/administração & dosagem , Sulfonas/uso terapêutico , Triterpenos/administração & dosagem , Triterpenos/uso terapêutico
17.
Inflamm Res ; 69(7): 697-710, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32350570

RESUMO

OBJECTIVE: Sepsis-associated encephalopathy (SAE) is a major cause of mortality worldwide. Oxidative stress, inflammatory response and apoptosis participate in the pathogenesis of SAE. Nuclear factor erythroid 2-related factor 2 (Nrf2) and nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) pathway is involved in oxidative stress and inflammatory response. We reported that hydrogen gas protected against sepsis in wild-type (WT) but not Nrf2 knockout (KO) mice. Therefore, it is vital to identify the underlying cause of hydrogen gas treatment of sepsis-associated encephalopathy. METHODS: SAE was induced in WT and Nrf2 KO mice by cecal ligation and puncture (CLP). As a NLRP3 inflammasome inhibitor, MCC950 (50 mg/kg) was administered by intraperitoneal (i.p.) injection before operation. Hydrogen gas (H2)-rich saline solution (5 mL/kg) was administered by i.p. injection at 1 h and 6 h after sham and CLP operations. Brain tissue was collected to assess the NLRP3 and Nrf2 pathways by western blotting, reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. RESULTS: SAE increased NLRP3 and Nrf2 expression in microglia. MCC950 inhibited SAE-induced NLRP3 expression, interleukin (IL)-1ß and IL-18 cytokine release, neuronal apoptosis and mitochondrial dysfunction. SAE increased NLRP3 and caspase-1 expression in WT mice compared to Nrf2 KO mice. Hydrogen increased Nrf2 expression and inhibited the SAE-induced expression of NLRP3, caspase-1, cytokines IL-1ß and IL-18, neuronal apoptosis, and mitochondrial dysfunction in WT mice but not Nrf2 KO mice. CONCLUSION: SAE increased NLRP3 and Nrf2 expression in microglia. Hydrogen alleviated inflammation, neuronal apoptosis and mitochondrial dysfunction via inhibiting Nrf2-mediated NLRP3 pathway.


Assuntos
Hidrogênio/administração & dosagem , Fator 2 Relacionado a NF-E2/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Encefalopatia Associada a Sepse/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Química Encefálica , Ceco , Córtex Cerebral/ultraestrutura , Citocinas/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Masculino , Camundongos , Camundongos Knockout , Microglia/fisiologia , Mitocôndrias/fisiologia , Fator 2 Relacionado a NF-E2/deficiência , Proteína 3 que Contém Domínio de Pirina da Família NLR/análise , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Punções , Encefalopatia Associada a Sepse/patologia , Sulfonas/farmacologia
18.
J Smooth Muscle Res ; 56(0): 19-28, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32350168

RESUMO

Blebbistatin, a potent inhibitor of myosin II, is known to suppress smooth muscle contraction without affecting myosin light chain phosphorylation level. In order to clarify the regulatory mechanisms of blebbistatin on phasic and tonic smooth muscles in detail, we examined the effects of blebbistatin on relaxation process by Ca2+ removal after Ca2+-induced contraction of ß-escin skinned (cell membrane permeabilized) trachea and taenia cecum preparations from guinea pigs. Blebbistatin significantly suppressed the force during relaxation both in skinned trachea and taenia cecum. The data fitting analysis of the relaxation processes indicates that blebbistatin accelerates slow (latch-like) bridge dissociation.


Assuntos
Ceco/citologia , Ceco/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Traqueia/citologia , Traqueia/efeitos dos fármacos , Animais , Cálcio/fisiologia , Membrana Celular , Células Cultivadas , Escina , Cobaias , Masculino
19.
Oncogene ; 39(24): 4770-4779, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32366905

RESUMO

Nuclear protein of the testis (NUT) midline carcinoma (NMC), is a rare and highly aggressive form of undifferentiated squamous cell carcinoma. NMC is molecularly characterized by chromosomal rearrangement of the NUT gene to another gene, most commonly the bromodomain and extraterminal domain (BET) gene BRD4, forming the BRD4-NUT fusion oncogene. Therefore, inhibiting BRD4-NUT oncogenic function directly by BET inhibitors represents an attractive therapeutic approach but toxicity may limit the use of pan-BET inhibitors treating this cancer. We thus performed a drug screening approach using a library consisting of epigenetic compounds and 'Donated Chemical Probes' collated by the Structural Genomics Consortium (SGC) and identified the p300/CBP HAT inhibitor A-485, in addition to the well-known BET inhibitor JQ1, to be the most active candidate for NMC treatment. In contrast to JQ1, A-485 was selectively potent in NMC compared to other cell lines tested. Mechanistically, A-485 inhibited p300-mediated histone acetylation, leading to disruption of BRD4-NUT binding to hyperacetylated megadomains. Consistently, BRD4-NUT megadomain-associated genes MYC, CCAT1 and TP63 were downregulated by A-485. A-485 strongly induced squamous differentiation, cell cycle arrest and apoptosis. Combined inhibition of p300/CBP and BET showed synergistic effects. In summary, we identified the p300/CBP HAT domain as a putative therapeutic target in highly therapy-resistant NMC.


Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas , Sistemas de Liberação de Medicamentos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Fragmentos de Peptídeos , Sialoglicoproteínas , Antineoplásicos/química , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios Proteicos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sialoglicoproteínas/antagonistas & inibidores , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
20.
PLoS One ; 15(4): e0232055, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324796

RESUMO

Chronic kidney diseases affect more than 800 million people globally and remain a high unmet need. Various therapeutic targets are currently under evaluation in pre-clinical and clinical studies. Because the growth arrest specific gene 6 (Gas6)/AXL pathway has been implicated in the pathogenesis of kidney diseases, we generated a novel selective and potent AXL inhibitor, CH5451098, and we evaluated its efficacy and elucidated its mechanism in an NEP25 mouse model that follows the clinical course of glomerular nephritis. In this model, CH5451098 significantly ameliorated the excretion of urinary albumin and elevation of serum creatinine. Additionally, it also inhibited tubulointerstitial fibrosis and tubular damage. To elucidate the mechanism behind these changes, we analyzed the effect of CH5451098 against transforming growth factor ß1 (TGFß1) and Gas6, which is a ligand of AXL receptor, in NRK-52E renal tubular epithelial cells. CH5451098 inhibited epithelial-to-mesenchymal transition (EMT) caused by the synergistic effects of TGFß1 and Gas6 in NRK-52E cells. This inhibition was also observed in NEP25 mice. Taken together, these results suggest that CH5451098 could ameliorate kidney dysfunction in glomerular nephritis by inhibiting EMT in tubular cells. These results reveal that AXL strongly contributes to the disease progression of glomerular nephritis.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glomerulonefrite/tratamento farmacológico , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Túbulos Renais/citologia , Rim/fisiopatologia , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Albuminas/análise , Animais , Linhagem Celular , Creatinina/sangue , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Glomerulonefrite/genética , Glomerulonefrite/metabolismo , Glomerulonefrite/fisiopatologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Rim/efeitos dos fármacos , Testes de Função Renal , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Camundongos , Camundongos Transgênicos , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Ratos , Fator de Crescimento Transformador beta1/genética
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