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1.
FASEB J ; 33(4): 4780-4789, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30601680

RESUMO

Prostaglandin E2 (PGE2) is produced in the airway during allergic lung inflammation and both promotes and inhibits features of asthma pathology. These mixed effects relate to 4 E-prostanoid (EP) receptor subtypes (EP1, 2, 3 and 4) expressed at different levels on different resident and infiltrating airway cells. Although studies have asserted both EP2 and EP4 expression in human airway smooth muscle (HASM), a recent study asserted EP4 to be the functionally dominant EP subtype in HASM. Herein, we employ recently-developed subtype-selective ligands to investigate singular or combined EP2 and EP4 receptor activation in regulating HASM signaling and proliferation. The subtype specificity of ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) was first demonstrated in human embryonic kidney 293 cells stably expressing different EP receptor subtypes. EP receptor knockdown and subtype-selective antagonists demonstrated EP2 and EP4 receptor responsiveness in HASM cells to the specific ONO compounds, whereas PGE2 appeared to preferentially signal via the EP4 receptor. Both singular EP2 and EP4 receptor agonists inhibited HASM proliferation, and combined EP2 and EP4 receptor agonism exhibited positive cooperativity in both chronic Gs-mediated signaling and inhibiting HASM proliferation. These findings suggest both EP2 and EP4 are functionally important in HASM, and their combined targeting optimally inhibits airway smooth muscle proliferation.-Michael, J. V. Gavrila, A., Nayak, A. P., Pera, T., Liberato, J. R., Polischak, S. R., Shah, S. D., Deshpande, D. A., Penn, R. B. Cooperativity of E-prostanoid receptor subtypes in regulating signaling and growth inhibition in human airway smooth muscle.


Assuntos
Músculo Liso/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP3/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Cálcio/metabolismo , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Immunoblotting , Microscopia Confocal , RNA Interferente Pequeno/genética , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP3/genética , Receptores de Prostaglandina E Subtipo EP4/genética , Transdução de Sinais/fisiologia
2.
FASEB J ; 32(2): 862-874, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29042451

RESUMO

GPCRs have diverse signaling capabilities, based on their ability to assume various conformations. Moreover, it is now appreciated that certain ligands can promote distinct receptor conformations and thereby bias signaling toward a specific pathway to differentially affect cell function. The recently deorphanized G protein-coupled receptor OGR1 [ovarian cancer G protein-coupled receptor 1 ( GPR68)] exhibits diverse signaling events when stimulated by reductions in extracellular pH. We recently demonstrated airway smooth muscle cells transduce multiple signaling events, reflecting a diverse capacity to couple to multiple G proteins. Moreover, we recently discovered that the benzodiazepine lorazepam, more commonly recognized as an agonist of the γ-aminobutyric acid A (GABAA) receptor, can function as an allosteric modulator of OGR1 and, similarly, can promote multiple signaling events. In this study, we demonstrated that different benzodiazepines exhibit a range of biases for OGR1, with sulazepam selectively activating the canonical Gs of the G protein signaling pathway, in heterologous expression systems, as well as in several primary cell types. These findings highlight the potential power of biased ligand pharmacology for manipulating receptor signaling qualitatively, to preferentially activate pathways that are therapeutically beneficial.-Pera, T., Deshpande, D. A., Ippolito, M., Wang, B., Gavrila, A., Michael, J. V., Nayak, A. P., Tompkins, E., Farrell, E., Kroeze, W. K., Roth, B. L., Panettieri, R. A. Jr Benovic, J. L., An, S. S., Dulin, N. O., Penn, R. B. Biased signaling of the proton-sensing receptor OGR1 by benzodiazepines.


Assuntos
Benzodiazepinas/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Células HEK293 , Humanos , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Receptores Acoplados a Proteínas G/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genética
3.
Am J Respir Cell Mol Biol ; 53(5): 728-37, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25897650

RESUMO

Preclinical models of human conditions including asthma showed the therapeutic potential of Compound A (CpdA), a dissociated glucocorticoid (GC) receptor (GRα) ligand. Whether CpdA inhibits GC resistance, a central feature of severe asthma, has not been addressed. We investigated whether CpdA modulates cytokine-induced GC resistance in human airway smooth muscle (ASM) cells. Healthy and asthmatic ASM cells were treated with TNF-α/IFN-γ for 24 hours in the presence or absence of CpdA. ELISA and quantitative PCR assays were used to assess the effect of CpdA on chemokine expression. Activation of GRα by CpdA was assessed by quantitative PCR, immunostaining, and receptor antagonism using RU486. An effect of CpdA on the transcription factor interferon regulatory factor 1 (IRF-1) was investigated using immunoblot, immunostaining, and small interfering RNA (siRNA) knockdown. CpdA inhibited production of fluticasone-resistant chemokines CCL5, CX3CL1, and CXCL10 at protein and mRNA levels in both asthmatic and healthy cells. CpdA failed to induce expression of GC-induced Leucine Zipper while transiently inducing mitogen-activated protein kinase phosphatase 1 (MKP-1) at both mRNA and protein levels. CpdA inhibitory action was not associated with GRα nuclear translocation, nor was it prevented by RU486 antagonism. Activation of IRF-1 by TNF-α/IFN-γ was inhibited by CpdA. IRF-1 siRNA knockdown reduced cytokine-induced CCL5 and CX3CL1 production. siRNA MKP-1 prevented the inhibitory effect of CpdA on cytokine-induced CXCL10 production. For the first time, we show that CpdA inhibits the production of GC-resistant chemokines via GRα-independent mechanisms involving the inhibition of IRF-1 and up-regulation of MKP-1. Thus, targeting CpdA-sensitive pathways in ASM cells represents an alternative therapeutic approach to treat GC resistance in asthma.


Assuntos
Acetatos/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Tiramina/análogos & derivados , Adulto , Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Asma/genética , Asma/imunologia , Asma/patologia , Estudos de Casos e Controles , Quimiocina CCL5/genética , Quimiocina CCL5/imunologia , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Fosfatase 1 de Especificidade Dupla/genética , Fosfatase 1 de Especificidade Dupla/imunologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Fluticasona/farmacologia , Expressão Gênica/imunologia , Humanos , Fator Regulador 1 de Interferon/antagonistas & inibidores , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/imunologia , Interferon gama/farmacologia , Masculino , Pessoa de Meia-Idade , Mifepristona/farmacologia , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/imunologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Fator de Necrose Tumoral alfa/farmacologia , Tiramina/farmacologia
4.
ChemMedChem ; 8(5): 709-18, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23495190

RESUMO

Greater than the sum of its parts: Artemisinins are currently in phase I-II clinical trials against breast, colorectal and non-small-cell lung cancers. In an attempt to offer increased specificity, a series of hybrid artemisinin-polypyrrole minor groove binder conjugates are described. DNA binding/modelling studies and preliminary biological evaluation give insights into their mechanism of action and the potential of this strategy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/síntese química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Artemisininas/farmacologia , DNA/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Polímeros/farmacologia , Pirróis/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/química , Artemisininas/química , Sítios de Ligação/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HL-60 , Células HT29 , Humanos , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Testes de Sensibilidade Parasitária , Polímeros/química , Pirróis/química , Relação Estrutura-Atividade , Termodinâmica
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