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1.
Nat Commun ; 12(1): 76, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397953

RESUMO

Full development of IL-17 producing CD4+ T helper cells (TH17 cells) requires the transcriptional activity of both orphan nuclear receptors RORα and RORγt. However, RORα is considered functionally redundant to RORγt; therefore, the function and therapeutic value of RORα in TH17 cells is unclear. Here, using mouse models of autoimmune and chronic inflammation, we show that expression of RORα is required for TH17 cell pathogenicity. T-cell-specific deletion of RORα reduces the development of experimental autoimmune encephalomyelitis (EAE) and colitis. Reduced inflammation is associated with decreased TH17 cell development, lower expression of tissue-homing chemokine receptors and integrins, and increased frequencies of Foxp3+ T regulatory cells. Importantly, inhibition of RORα with a selective small molecule antagonist mostly phenocopies our genetic data, showing potent suppression of the in vivo development of both chronic/progressive and relapsing/remitting EAE, but with no effect on overall thymic cellularity. Furthermore, use of the RORα antagonist effectively inhibits human TH17 cell differentiation and memory cytokine secretion. Together, these data suggest that RORα functions independent of RORγt in programming TH17 pathogenicity and identifies RORα as a safer and more selective therapeutic target for the treatment of TH17-mediated autoimmunity.


Assuntos
Inflamação/imunologia , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Células Th17/imunologia , Animais , Autoimunidade/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Doença Crônica , Colo/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental , Células HEK293 , Humanos , Inflamação/genética , Camundongos Endogâmicos C57BL , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Tamanho do Órgão/efeitos dos fármacos , Índice de Gravidade de Doença , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Tiofenos/química , Tiofenos/farmacologia
2.
Nat Commun ; 12(1): 51, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397955

RESUMO

Identifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin-Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Inibidores de Proteínas Quinases/farmacologia , Ubiquitina Tiolesterase/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Fusão bcr-abl , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiofenos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína 1 de Ligação a Y-Box/metabolismo , Proteínas ras/metabolismo
3.
Life Sci ; 269: 119026, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33444617

RESUMO

Morphine is a commonly used opioid drug to treat acute pain by binding to the mu-opioid receptor (MOR), but its effective analgesic efficacy via triggering of the heterotrimeric Gi protein pathway is accompanied by a series of adverse side effects via triggering of the ß-arrestin pathway. Recently, PZM21, a recently developed MOR biased agonist, shows preferentially activating the G protein pathway over ß-arrestin pathway. However, there is no high-resolution receptor structure in complex with PZM21 and its action mechanism remains elusive. In this study, PZM21 and Morphine were docked to the active human MOR-1 homology structure and then subjected to the molecular dynamics (MD) simulations in two different situations (i.e., one situation includes the crystal waters but another does not). Detailed comparisons between the two systems were made to characterize the differences in protein-ligand interactions, protein secondary and tertiary structures and dynamics networks. PZM21 could strongly interact with Y3287.43 of TM7, besides the residues (Asp1493.32 and Tyr1503.33) of TM3. The two systems' network paths to the intracellular end of TM6 were roughly similar but the paths to the end of TM7 were different. The PZM21-bound MOR's intracellular ends of TM5-7 bent outward more along with the distance changes of the three key molecular switches (ionic lock, transmission and Tyr toggle) and the distance increase of some conserved inter-helical residue pairs. The larger intracellular opening of the receptor could potentially facilitate G protein binding.


Assuntos
Simulação de Dinâmica Molecular , Receptores Opioides mu/agonistas , Tiofenos/farmacologia , Ureia/análogos & derivados , Regulação Alostérica , Animais , Ácido Aspártico/química , Análise por Conglomerados , Sequência Conservada , Cristalização , Humanos , Ligantes , Camundongos , Morfina/farmacologia , Análise de Componente Principal , Conformação Proteica , Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Transdução de Sinais , Homologia Estrutural de Proteína , Tiofenos/química , Tirosina/química , Ureia/química , Ureia/farmacologia , Água/química
4.
Life Sci ; 269: 119028, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33444618

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) is the second most common cancer-related death in the world. No effective curative option exists for the treatment of HCC. The available drugs exhibit severe toxic effects and low therapeutic index. AIM: This work aimed to examine different monocationic arylthiophene derivatives for possible use as chemotherapeutic agents against HCC. METHODS: The IC50 values for the compounds were determined. The mechanism of cytotoxicity was further investigated using different methods. RESULTS: Compound 2j proved to retain the highest cytotoxicity in comparison to as a positive control. The selectivity index of compound 2j revealed the safety to normal cells. Moreover, compound 2j was able to inhibit HepG2 cells´ migration and division. The anticancer effect of compound 2j was found to be partially via cell cycle arrest, activation of the tumour suppressor p53 protein, and induction of apoptosis via both intrinsic and extrinsic pathways. Compound 2j has a potential sensitization activity and significantly reduced the IC50 values for the anticancer drugs doxorubicin, cisplatin, and taxol. CONCLUSION: The tested arylthiophenes showed a potent cytotoxicity against HepG2 cells and were safe to normal cells. The most active compound 2j was found to be able to inhibit cell division and migration and also to induce apoptosis. Compound 2j also proved to have a sensitization effect on standard anticancer drugs.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Tiofenos/uso terapêutico , Antineoplásicos/química , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/patologia , Caspases/metabolismo , Cátions , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Mitose/efeitos dos fármacos , Paclitaxel/farmacologia , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacologia , Ensaio Tumoral de Célula-Tronco , Cicatrização/efeitos dos fármacos
5.
Metabolism ; 114: 154409, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33096076

RESUMO

BACKGROUND AND OBJECTIVES: The gut-liver axis plays an important role in the pathogenesis of nonalcoholic steatohepatitis (NASH), and increased intestinal permeability causes transfer of endotoxin to the liver, which activates the immune response, ultimately leading to hepatic inflammation. Nuclear receptor Rev-erbα is a critical regulator of circadian rhythm, cellular metabolism, and inflammatory responses. However, the role and mechanism of Rev-erbα in gut barrier function and NASH remain unclear. In the present study, we investigated the involvement of Rev-erbα in the regulation of intestinal permeability and the treatment of NASH. METHODS AND RESULTS: The expression of tight junction-related genes and Rev-erbs decreased in the jejunum, ileum and colon of mice with high cholesterol, high fat diet (CL)-induced NASH. Chromatin immunoprecipitation analysis indicated that REV-ERBα directly bound to the promoters of tight junction genes to regulate intestinal permeability. Pharmacological activation of REV-ERBα by SR9009 protected against lipopolysaccharide-induced increased intestinal permeability both in vitro and in vivo, and these effects were associated with the activation of autophagy and decreased apoptotic signaling of epithelial cells. In addition, the chronopharmacological effects of SR9009 were more potent at Zeitgeber time 0 (ZT0) than at ZT12, which was contrary to the rhythm of Rev-erbs in the gastrointestinal tract. The administration of SR9009 attenuated hepatic lipid accumulation, insulin resistance, inflammation, and fibrosis in mice with CL diet-induced NASH, which might be partly attributed to the enhancement of intestinal barrier function. CONCLUSION: Chronopharmacological activation of REV-ERBα might be a potential strategy to treat intestinal barrier dysfunction-related disorders and NASH.


Assuntos
Mucosa Intestinal/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Pirrolidinas/uso terapêutico , Receptores Citoplasmáticos e Nucleares/agonistas , Proteínas Repressoras/agonistas , Tiofenos/uso terapêutico , Junções Íntimas/efeitos dos fármacos , Animais , Autofagia/efeitos dos fármacos , Glicemia , Células CACO-2 , Colesterol/sangue , Humanos , Insulina/sangue , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Permeabilidade/efeitos dos fármacos , Pirrolidinas/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiofenos/farmacologia , Junções Íntimas/metabolismo , Triglicerídeos/sangue
6.
Zhongguo Dang Dai Er Ke Za Zhi ; 22(12): 1331-1337, 2020 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-33328006

RESUMO

OBJECTIVE: To study the expression and significance of ubiquitin-specific protease 7 (USP7) and the key factors of the Wnt signaling pathway in the lung tissue of preterm rats after hyperoxia exposure. METHODS: A total of 180 preterm neonatal Wistar rats were randomly divided into an air control group, an air intervention group, a hyperoxia control group, and a hyperoxia intervention group, with 45 rats in each group. Lung injury was induced by hyperoxia exposure in the hyperoxia groups. The preterm rats in the intervention groups were given intraperitoneal injection of the USP7 specific inhibitor P5091 (5 mg/kg) every day. The animals were sacrificed on days 3, 5, and 9 of the experiment to collect lung tissue specimens. Hematoxylin-eosin staining was used to observe the pathological changes of lung tissue. RT-PCR and Western blot were used to measure the mRNA and protein expression levels of USP7 and the key factors of the Wnt signaling pathway ß-catenin and α-smooth muscle actin (α-SMA) in lung tissue. RESULTS: The air groups had normal morphology and structure of lung tissue; on days 3 and 5, the hyperoxia control group showed obvious alveolar compression and disordered structure, with obvious inflammatory cells, erythrocyte diapedesis, and interstitial edema. On day 9, the hyperoxia control group showed alveolar structural disorder and obvious thickening of the alveolar septa. Compared with the hyperoxia control group at the corresponding time points, the hyperoxia intervention group had significantly alleviated disordered structure, inflammatory cell infiltration, and bleeding in lung tissue. At each time point, the hyperoxia groups had a significantly lower radial alveolar count (RAC) than the corresponding air groups (P < 0.05), and the hyperoxia intervention group had a significantly higher RAC than the hyperoxia control group (P < 0.05). On days 3, 5, and 9 of the experiment, the hyperoxia groups had significantly higher mRNA expression of USP7 and ß-catenin and protein expression of USP7, ß-catenin, and α-SMA than the corresponding air groups (P < 0.05). Compared with the hyperoxia control group, the hyperoxia intervention group had significant reductions in the mRNA expression of ß-catenin and the protein expression of ß-catenin and α-SMA (P < 0.05), while there were no significant differences in the mRNA and protein expression of USP7 between the hyperoxia intervention and hyperoxia control groups (P > 0.05). There were no significant differences in the mRNA expression of USP7 and ß-catenin and the protein expression of USP7, ß-catenin, and α-SMA between the air intervention and air control groups (P > 0.05). CONCLUSIONS: Hyperoxia exposure can activate the Wnt/ß-catenin signaling pathway, and USP7 may participate in hyperoxic lung injury through the Wnt/ß-catenin signaling pathway. The USP7 specific inhibitor P5091 may accelerate the degradation of ß-catenin by enhancing its ubiquitination, reduce lung epithelial-mesenchymal transition, and thus exert a certain protective effect against hyperoxic lung injury.


Assuntos
Hiperóxia/fisiopatologia , Pulmão/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Animais , Animais Recém-Nascidos , Pulmão/fisiopatologia , Distribuição Aleatória , Ratos , Ratos Wistar , Tiofenos/farmacologia , Proteases Específicas de Ubiquitina , Via de Sinalização Wnt
7.
PLoS One ; 15(10): e0240044, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33007022

RESUMO

Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for the treatment of diabetes and obesity. Ertiprotafib is a PTP1B inhibitor that reached the clinical trial stage for the treatment of diabetes. Interestingly, Ertiprotafib reduces the melting temperature of PTP1B in differential scanning fluorimetry (DSF) assays, different from most drugs that increase the stability of their target upon binding. No molecular data on how Ertiprotafib functions has been published. Thus, to gain molecular insights into the mode of action of Ertiprotafib, we used biomolecular NMR spectroscopy to characterize the molecular details of the PTP1B:Ertiprotafib interaction. Our results show that Ertiprotafib induces aggregation of PTP1B in a concentration dependent manner. This shows that the insufficient clinical efficacy and adverse effects caused by Ertiprotafib is due to its tendency to cause aggregation of PTP1B.


Assuntos
Inibidores Enzimáticos/farmacologia , Fenilpropionatos/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Tiofenos/farmacologia , Domínio Catalítico , Humanos , Proteína Tirosina Fosfatase não Receptora Tipo 1/química
8.
PLoS One ; 15(10): e0236000, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33002003

RESUMO

Non-alcoholic fatty liver disease (NAFLD) affects a significant number of people worldwide and currently there are no pharmacological treatments. NAFLD often presents with obesity, insulin resistance, and in some cases cardiovascular diseases. There is a clear need for treatment options to alleviate this disease since it often progresses to much more the much more severe non-alcoholic steatohepatitis (NASH). The REV-ERB nuclear receptor is a transcriptional repressor that regulates physiological processes involved in the development of NAFLD including lipogenesis and inflammation. We hypothesized that pharmacologically activating REV-ERB would suppress the progression of fatty liver in a mouse model of NASH. Using REV-ERB agonist SR9009 in a mouse NASH model, we demonstrate the beneficial effects of REV-ERB activation that led to an overall improvement of hepatic health by suppressing hepatic fibrosis and inflammatory response.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/efeitos dos fármacos , Pirrolidinas/farmacologia , Tiofenos/farmacologia , Animais , Modelos Animais de Doenças , Humanos , Inflamação/metabolismo , Lipogênese/fisiologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/patologia , Camundongos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/metabolismo
9.
Anticancer Res ; 40(9): 5141-5149, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878802

RESUMO

BACKGROUND/AIM: This study investigated the effects of temozolomide (TMZ) and/or checkpoint kinase inhibitor AZD7762 in human glioma cells. MATERIALS AND METHODS: Glioma cells were treated with TMZ and/or AZD7762 for 24 or 48 h, then the cellular survival was studied and the expression of various proteins was investigated. RESULTS: Both TMZ and AZD7762 induced concentration- and time-dependent cytotoxic effects, and combined TMZ and AZD7762 (TMZ+AZD) caused synergistic cytotoxic effects in glioma cells (p<0.05). AZD7762 suppressed the O6-methylguanine-DNA-methyltransferase (MGMT) expression. TMZ+AZD increased the expression of phospho-p53 (p-p53), p-p38 mitogen-activated protein kinase, and phosphatase and tensin homolog; and decreased the expression of p-extracellular signal-regulated kinase 1/2 and p-signal transducer and activator of transcription 3 in glioma cells. CONCLUSION: TMZ and AZD7762 combined induced synergistic cytotoxic effects on human glioma cells and such effects may be related to the AZD7762-induced suppression of MGMT expression and the modulation of multiple signaling pathways.


Assuntos
Antineoplásicos/farmacologia , Temozolomida/farmacologia , Tiofenos/farmacologia , Ureia/análogos & derivados , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/genética , Glioma/metabolismo , Humanos , Ureia/farmacologia
10.
PLoS One ; 15(9): e0238156, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946510

RESUMO

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) represent a technical revolution in integrative neuroscience. However, the first used ligands exhibited dose-dependent selectivity for their molecular target, leading to potential unspecific effects. Compound 21 (C21) was recently proposed as an alternative, but in vivo characterization of its properties is not sufficient yet. Here, we evaluated its potency to selectively modulate the activity of nigral dopaminergic (DA) neurons through the canonical DREADD receptor hM4Di using TH-Cre rats. In males, 1 mg.kg-1 of C21 strongly increased nigral neurons activity in control animals, indicative of a significant off-target effect. Reducing the dose to 0.5 mg.kg-1 circumvented this unspecific effect, while activated the inhibitory DREADDs and selectively reduced nigral neurons firing. In females, 0.5 mg.kg-1 of C21 induced a transient and residual off-target effect that may mitigated the inhibitory DREADDs-mediated effect. This study raises up the necessity to test selectivity and efficacy of chosen ligands for each new experimental condition.


Assuntos
Drogas Desenhadas/farmacologia , Terapia de Alvo Molecular , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos , Ratos
11.
Nat Commun ; 11(1): 4678, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938916

RESUMO

Diabetic foot ulcers (DFUs) are a life-threatening disease that often result in lower limb amputations and a shortened lifespan. However, molecular mechanisms contributing to the pathogenesis of DFUs remain poorly understood. We use next-generation sequencing to generate a human dataset of pathogenic DFUs to compare to transcriptional profiles of human skin and oral acute wounds, oral as a model of "ideal" adult tissue repair due to accelerated closure without scarring. Here we identify major transcriptional networks deregulated in DFUs that result in decreased neutrophils and macrophages recruitment and overall poorly controlled inflammatory response. Transcription factors FOXM1 and STAT3, which function to activate and promote survival of immune cells, are inhibited in DFUs. Moreover, inhibition of FOXM1 in diabetic mouse models (STZ-induced and db/db) results in delayed wound healing and decreased neutrophil and macrophage recruitment in diabetic wounds in vivo. Our data underscore the role of a perturbed, ineffective inflammatory response as a major contributor to the pathogenesis of DFUs, which is facilitated by FOXM1-mediated deregulation of recruitment of neutrophils and macrophages, revealing a potential therapeutic strategy.


Assuntos
Pé Diabético/genética , Pé Diabético/imunologia , Proteína Forkhead Box M1/imunologia , Cicatrização/imunologia , Adulto , Idoso , Animais , Proliferação de Células , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/imunologia , Pé Diabético/patologia , Modelos Animais de Doenças , Feminino , Proteína Forkhead Box M1/antagonistas & inibidores , Proteína Forkhead Box M1/metabolismo , Humanos , Inflamação/genética , Inflamação/imunologia , Masculino , Camundongos Endogâmicos , Pessoa de Meia-Idade , Mucosa Bucal/fisiologia , Piridinas/farmacologia , Tiofenos/farmacologia , Transcriptoma/fisiologia , Cicatrização/genética
12.
Mol Carcinog ; 59(11): 1256-1268, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32885857

RESUMO

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis selectively in cancer cells. For melanoma, the targeting of TRAIL signaling appears highly attractive, due to pronounced TRAIL receptor expression in tumor tissue. However, mechanisms of TRAIL resistance observed in melanoma cells may limit its clinical use. The Bcl-2 family members are critical regulators of cell-intrinsic apoptotic pathways. Thus, the antiapoptotic Bcl-2 protein myeloid cell leukemia 1 (Mcl-1) is overexpressed in many tumor types and was linked to chemotherapy resistance in melanoma. In this study, we evaluated the involvement of antiapoptotic Bcl-2 proteins (Bcl-2, Bcl-xL , Bcl-w, Mcl-1, Bcl-A1, and Bcl-B) in TRAIL resistance. They were targeted by small interfering RNA-mediated silencing in TRAIL-sensitive (A-375, Mel-HO) and in TRAIL-resistant melanoma cell lines (Mel-2a, MeWo). This highlighted Mcl-1 as the most efficient target to overcome TRAIL resistance. In this context, we investigated the effects of Mcl-1-targeting microRNAs as well as the Mcl-1-selective inhibitor S63845. Both miR-193b and S63845 resulted in significant enhancement of TRAIL-induced apoptosis, associated with decreased cell viability. Apoptosis induction was mediated by caspase-3 processing as well as by Bax and Bak activation, indicating the critical involvement of intrinsic apoptosis pathways. These data may indicate a high relevance of Mcl-1 targeting also in melanoma therapy. Furthermore, the data may suggest to consider the use of the tumor suppressor miR-193b as a strategy for countering TRAIL resistance in melanoma.


Assuntos
Apoptose , Resistencia a Medicamentos Antineoplásicos , Melanoma/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , RNA Interferente Pequeno/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , MicroRNAs/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirimidinas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Tiofenos/farmacologia , Células Tumorais Cultivadas
13.
Nat Commun ; 11(1): 4527, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913197

RESUMO

Evasion of programmed cell death represents a critical form of oncogene addiction in cancer cells. Understanding the molecular mechanisms underpinning cancer cell survival despite the oncogenic stress could provide a molecular basis for potential therapeutic interventions. Here we explore the role of pro-survival genes in cancer cell integrity during clonal evolution in non-small cell lung cancer (NSCLC). We identify gains of MCL-1 at high frequency in multiple independent NSCLC cohorts, occurring both clonally and subclonally. Clonal loss of functional TP53 is significantly associated with subclonal gains of MCL-1. In mice, tumour progression is delayed upon pharmacologic or genetic inhibition of MCL-1. These findings reveal that MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Evolução Clonal , Variações do Número de Cópias de DNA , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Progressão da Doença , Humanos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Cultura Primária de Células , Estudos Prospectivos , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , RNA-Seq , Estudos Retrospectivos , Esferoides Celulares , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Proteína Supressora de Tumor p53/genética , Microtomografia por Raio-X
14.
Cell Prolif ; 53(10): e12901, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32960500

RESUMO

OBJECTIVES: To assess the expression, prognostic value, and functionality of T-lymphokine-activated killer (T-LAK) cell-originated protein kinase (TOPK) in chordoma pathogenesis. MATERIALS AND METHODS: TOPK expression in chordoma was assessed via immunohistochemical staining of a tissue microarray (TMA) and correlated with patient clinicopathology. TOPK expression in chordoma cell lines and fresh patient tissues was then evaluated by Western blot. TOPK small interfering RNA (siRNA) and the specific inhibitor OTS514 were applied to determine the roles of TOPK in chordoma pathogenicity. The effect of TOPK expression on chordoma cell clonogenicity was also investigated using clonogenic assays. A 3D cell culture model was utilized to mimic in vivo environment to validate the effect of TOPK inhibition on chordoma cells. RESULTS: TOPK was highly expressed in 78.2% of the chordoma specimens in the TMA and all chordoma cell lines. High TOPK expression significantly correlated with metastasis, recurrence, disease status and shorter overall survival. Knockdown of TOPK with specific siRNA resulted in significantly decrease chordoma cell viability. Inhibition of TOPK with OTS514 significantly inhibited chordoma cell growth and proliferation, colony-forming capacity and ex vivo spheroid growth. CONCLUSIONS: High expression of TOPK is an important predictor of poor prognosis in chordoma. Inhibition of TOPK resulted in significantly decrease chordoma cell proliferation and increase apoptosis. Our results indicate TOPK as a novel prognostic biomarker and therapeutic target for chordoma.


Assuntos
Neoplasias Ósseas/patologia , Cordoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cordoma/metabolismo , Cordoma/mortalidade , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Metástase Neoplásica , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Quinolonas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Tiofenos/farmacologia
15.
Br J Anaesth ; 125(4): 596-604, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32819621

RESUMO

BACKGROUND: A novel G-protein signalling-biased mu opioid peptide (MOP) receptor agonist, PZM21, was recently developed with a distinct chemical structure. It is a potent Gi/o activator with minimal ß-arrestin-2 recruitment. Despite intriguing activity in rodent models, PZM21 function in non-human primates is unknown. The aim of this study was to investigate PZM21 actions after systemic or intrathecal administration in primates. METHODS: Antinociceptive, reinforcing, and pruritic effects of PZM21 were compared with those of the clinically used MOP receptor agonists oxycodone and morphine in assays of acute thermal nociception, capsaicin-induced thermal allodynia, itch scratching responses, and drug self-administration in gonadally intact, adult rhesus macaques (10 males, six females). RESULTS: After subcutaneous administration, PZM21 (1.0-6.0 mg kg-1) and oxycodone (0.1-0.6 mg kg-1) induced dose-dependent thermal antinociceptive effects (P<0.05); PZM21 was 10 times less potent than oxycodone. PZM21 exerted oxycodone-like reinforcing effects and strength as determined by two operant schedules of reinforcement in the intravenous drug self-administration assay. After intrathecal administration, PZM21 (0.03-0.3 mg) dose-dependently attenuated capsaicin-induced thermal allodynia (P<0.05). Although intrathecal PZM21 and morphine induced MOP receptor-mediated antiallodynic effects, both compounds induced robust, long-lasting itch scratching. CONCLUSIONS: PZM21 induced antinociceptive, reinforcing, and pruritic effects similar to clinically used MOP receptor agonists in primates. Although structure-based discovery of PZM21 identified a novel avenue for studying G-protein signalling-biased ligands, biasing an agonist towards G-protein signalling pathways did not determine or alter reinforcing (i.e. abuse potential) or pruritic effects of MOP receptor agonists in a translationally relevant non-human primate model.


Assuntos
Analgésicos/farmacologia , Prurido/induzido quimicamente , Receptores Opioides mu/agonistas , Reforço Psicológico , Tiofenos/farmacologia , Ureia/análogos & derivados , Animais , Relação Dose-Resposta a Droga , Feminino , Macaca mulatta , Masculino , Ureia/farmacologia
16.
Anesthesiology ; 133(3): 559-568, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32788558

RESUMO

BACKGROUND: To improve understanding of the respiratory behavior of oliceridine, a µ-opioid receptor agonist that selectively engages the G-protein-coupled signaling pathway with reduced activation of the ß-arrestin pathway, the authors compared its utility function with that of morphine. It was hypothesized that at equianalgesia, oliceridine will produce less respiratory depression than morphine and that this is reflected in a superior utility. METHODS: Data from a previous trial that compared the respiratory and analgesic effects of oliceridine and morphine in healthy male volunteers (n = 30) were reanalyzed. A population pharmacokinetic-pharmacodynamic analysis was performed and served as basis for construction of utility functions, which are objective functions of probability of analgesia, P(analgesia), and probability of respiratory depression, P(respiratory depression). The utility function = P(analgesia ≥ 0.5) - P(respiratory depression ≥ 0.25), where analgesia ≥ 0.5 is the increase in hand withdrawal latency in the cold pressor test by at least 50%, and respiratory depression ≥ 0.25 is the decrease of the hypercapnic ventilatory response by at least 25%. Values are median ± standard error of the estimate. RESULTS: The two drugs were equianalgesic with similar potency values (oliceridine: 27.9 ± 4.9 ng/ml; morphine 34.3 ± 9.7 ng/ml; potency ratio, 0.81; 95% CI, 0.39 to 1.56). A 50% reduction of the hypercapnic ventilatory response by morphine occurred at an effect-site concentration of 33.7 ± 4.8 ng/ml, while a 25% reduction by oliceridine occurred at 27.4 ± 3.5 ng/ml (potency ratio, 2.48; 95% CI, 1.65 to 3.72; P < 0.01). Over the clinically relevant concentration range of 0 to 35 ng/ml, the oliceridine utility function was positive, indicating that the probability of analgesia exceeds the probability of respiratory depression. In contrast, the morphine function was negative, indicative of a greater probability of respiratory depression than analgesia. CONCLUSIONS: These data indicate a favorable oliceridine safety profile over morphine when considering analgesia and respiratory depression over the clinical concentration range.


Assuntos
Analgésicos Opioides/farmacologia , Morfina/farmacologia , Insuficiência Respiratória/induzido quimicamente , Compostos de Espiro/farmacologia , Tiofenos/farmacologia , Adulto , Analgésicos Opioides/efeitos adversos , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Morfina/efeitos adversos , Valores de Referência , Medição de Risco , Compostos de Espiro/efeitos adversos , Tiofenos/efeitos adversos , Adulto Jovem
17.
Life Sci ; 257: 118053, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32634424

RESUMO

AIMS: Vascular smooth muscle cells (VSMCs) play a crucial role in the progression of atherosclerosis. Paired box 9 (Pax9) is a member of the Pax gene family which participates in the development of various tissues and organs. However, the effect of Pax9 on atherosclerosis and VSMCs and the underlying mechanisms remain unclear. MAIN METHODS: Western blotting was performed to assess Pax9 expression in atherosclerosis and VSMCs. Pax9 siRNA and overexpression plasmid were constructed to explore the biological function. Cell proliferation assay, phalloidin staining, and Transwell assay, accompanied by the sonic hedgehog (Shh) signaling pathway antagonist, cyclopamine (5 µM) and agonist, SAG (100 nM), were used to evaluate the VSMC phenotype, proliferation, and migration, as well as explore the associated mechanisms. KEY FINDINGS: We first discovered Pax9 to be significantly increased in atherosclerotic mice and platelet-derived growth factor-BB (PDGF-BB)-induced VSMCs. Pax9 knockdown inhibited the phenotypic transformation, proliferation, and migration of VSMCs, whereas the opposite effect was observed when Pax9 was overexpressed. Next, we established that Shh was activated in PDGF-BB-induced VSMCs. Moreover, Pax9 overexpression further activated Shh and exacerbated the phenotypic transformation, proliferation, and migration of PDGF-BB-induced VSMCs. These changes were effectively inhibited by treatment with the Shh signaling pathway antagonist. Consistently, Pax9 knockdown down-regulated Shh expression and inhibited the phenotypic transformation, proliferation, and migration of PDGF-BB-induced VSMCs. Treatment with the Shh signaling pathway agonist prevented these changes. SIGNIFICANCE: Pax9 regulated VSMC phenotypic transformation, proliferation, and migration via Shh, which may represent a novel target for the treatment of atherosclerosis.


Assuntos
Aterosclerose/genética , Proteínas Hedgehog/genética , Miócitos de Músculo Liso/citologia , Fator de Transcrição PAX9/genética , Animais , Aterosclerose/patologia , Becaplermina/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Cicloexilaminas/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Transdução de Sinais/genética , Tiofenos/farmacologia , Alcaloides de Veratrum/farmacologia
18.
Nucleic Acids Res ; 48(14): 7914-7923, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32652039

RESUMO

Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter -10 element, while not affecting interactions with -35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of -10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with -10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials.


Assuntos
Antibacterianos/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Regiões Promotoras Genéticas , Tiofenos/farmacologia , Iniciação da Transcrição Genética/efeitos dos fármacos , Antibacterianos/química , Bactérias/enzimologia , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fator sigma/antagonistas & inibidores , Fator sigma/química , Tiofenos/química
19.
Int J Nanomedicine ; 15: 4079-4090, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606665

RESUMO

Purpose: The aim of this study is to develop efficient localized therapy of sertaconazole nitrate for the treatment of vaginal candidiasis. Methods: Sertaconazole nitrate-loaded cationic liposomes were prepared by thin-film hydration method and coated with different concentrations of pectin (0.05%, 0.1% and 0.2%) to develop mucoadhesive liposomes. The formulated mucoadhesive vesicles were characterized in terms of morphology, entrapment efficiency, particle size, zeta value, mucoadhesive properties and drug release. The selected formula was incorporated into a gel base and further characterized by an ex vivo permeation study in comparison with conventional sertaconazole gel. Also, the in vivo study was performed to assess the efficacy of sertaconazole mucoadhesive liposomal gel in treating rats with vaginal candidiasis. Results: The mucoadhesive liposomes were spherical. Coating liposomes with pectin results in increased entrapment efficiency and particle size compared with uncoated vesicles. On the contrary, zeta values were reduced upon coating liposomes with pectin indicating efficient coating of liposomes with pectin. Mucoadhesive liposomes showed a more prolonged and sustained drug release compared with uncoated liposomes. Ex vivo study results showed that mucoadhesive liposomal gel increased sertaconazole tissue retention and reduced drug tissue penetration. In the invivo study, the mucoadhesive liposomal gel showed a significant reduction in the microbial count with a subsequent reduction in inflammatory responses with the lowest histopathological change compared with conventional gel. Conclusion: The study confirmed the potentiality of employing mucoadhesive liposomes as a successful carrier for the vaginal delivery of antifungal drugs.


Assuntos
Antifúngicos/uso terapêutico , Candidíase Vulvovaginal/tratamento farmacológico , Imidazóis/uso terapêutico , Muco/química , Tiofenos/uso terapêutico , Adesividade , Animais , Anti-Infecciosos/farmacologia , Biomarcadores/metabolismo , Preparações de Ação Retardada , Liberação Controlada de Fármacos , Feminino , Géis , Humanos , Imidazóis/farmacologia , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Mediadores da Inflamação/metabolismo , Lipossomos/ultraestrutura , Mucinas/metabolismo , Tamanho da Partícula , Ratos Sprague-Dawley , Ovinos , Eletricidade Estática , Tiofenos/farmacologia , Vagina/patologia , beta-Glucanas/metabolismo
20.
Mol Pharmacol ; 98(4): 475-486, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32680919

RESUMO

Methadone is a synthetic opioid agonist with notoriously unique properties, such as lower abuse liability and induced relief of withdrawal symptoms and drug cravings, despite acting on the same opioid receptors triggered by classic opioids-in particular the µ-opioid receptor (MOR). Its distinct pharmacologic properties, which have recently been attributed to the preferential activation of ß-arrestin over G proteins, make methadone a standard-of-care maintenance medication for opioid addiction. Although a recent biophysical study suggests that methadone stabilizes different MOR active conformations from those stabilized by classic opioid drugs or G protein-biased agonists, how this drug modulates the conformational equilibrium of MOR and what specific active conformation of the receptor it stabilizes are unknown. Here, we report the results of submillisecond adaptive sampling molecular dynamics simulations of a predicted methadone-bound MOR complex and compare them with analogous data obtained for the classic opioid morphine and the G protein-biased ligand TRV130. The model, which is supported by existing experimental data, is analyzed using Markov state models and transfer entropy analysis to provide testable hypotheses of methadone-specific conformational dynamics and activation kinetics of MOR. SIGNIFICANCE STATEMENT: Opioid addiction has reached epidemic proportions in both industrialized and developing countries. Although methadone maintenance treatment represents an effective therapeutic approach for opioid addiction, it is not as widely used as needed. In this study, we contribute an atomic-level understanding of how methadone exerts its unique function in pursuit of more accessible treatments for opioid addiction. In particular, we present details of a methadone-specific active conformation of the µ-opioid receptor that has thus far eluded experimental structural characterization.


Assuntos
Analgésicos Opioides/farmacologia , Metadona/farmacologia , Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Compostos de Espiro/farmacologia , Tiofenos/farmacologia , Analgésicos Opioides/química , Animais , Sítios de Ligação , Entropia , Humanos , Cadeias de Markov , Metadona/química , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Compostos de Espiro/química , Tiofenos/química
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