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1.
Mol Pharmacol ; 83(1): 283-93, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23115324

RESUMO

Epigenetic alterations, such as histone acetylation, regulate the signaling outcomes and phenotypic responses of fibroblasts after growth factor stimulation. The bromodomain and extra-terminal domain-containing proteins (Brd) bind to acetylated histone residues, resulting in recruitment of components of the transcriptional machinery and subsequent gene transcription. Given the central importance of fibroblasts in tissue fibrosis, this study sought to determine the role of Brd proteins in human lung fibroblasts (LFs) after growth factor stimulation and in the murine bleomycin model of lung fibrosis. Using small interfering RNA against human Brd2 and Brd4 and pharmacologic Brd inhibitors, this study found that Brd2 and Brd4 are essential in mediating the phenotypic responses of LFs downstream of multiple growth factor pathways. Growth factor stimulation of LFs causes increased histone acetylation, association of Brd4 with growth factor-responsive genes, and enhanced transcription of these genes that could be attenuated with pharmacologic Brd inhibitors. Of note, lung fibrosis induced after intratracheal bleomycin challenge in mice could be prevented by pretreatment of animals with pharmacologic inhibitors of Brd proteins. This study is the first demonstration of a role for Brd2 and Brd4 proteins in mediating the responses of LFs after growth factor stimulation and in driving the induction of lung fibrosis in mice in response to bleomycin challenge.


Assuntos
Fibroblastos/fisiologia , Pulmão/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-sis/farmacologia , Fibrose Pulmonar/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Actinas/biossíntese , Administração Oral , Animais , Becaplermina , Bleomicina , Proteínas de Ciclo Celular , Movimento Celular , Proliferação de Células , Citocinas/metabolismo , Epigênese Genética , Proteínas da Matriz Extracelular/biossíntese , Fibroblastos/efeitos dos fármacos , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-sis/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/prevenção & controle , RNA Interferente Pequeno/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcrição Gênica , Fator de Crescimento Transformador beta1/metabolismo
2.
Bioinformatics ; 28(3): 301-5, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22155864

RESUMO

MOTIVATION: Cell-based phenotypic screens using small molecule inhibitors is an important technology for early drug discovery if the relationship between the disease-related cellular phenotype and inhibitors' biological targets can be determined. However, chemical inhibitors are rightfully believed to be less specific than perturbation by biological agents, such as antibody and small inference RNA. Therefore, it is often a challenge in small molecule phenotypic screening to infer the causality between a particular cellular phenotype and the inactivation of the responsible protein due to the off-target effect of the inhibitors. RESULTS: In this article, we present a Roche in-house effort of screening 746 structurally diverse compounds for their cytotoxicity in HeLa cells measured by high content imaging technology. These compounds were also systematically profiled for the targeted and off-target binding affinity to a panel of 25 pre-selected protein kinases in a cell-free system. In an effort to search for the kinases whose activities are crucial for cell survival, we found that the simple association method such as the chi-square test yields a large number of false positives because the observed cytotoxic phenotype is likely to be the result of promiscuous action of less specific inhibitors instead of true consequence of inactivation of single relevant target. We demonstrated that a stratified categorical data analysis technique such as the Cochran-Mantel-Haenszel test is an effective approach to extract the meaningful biological connection from the spurious correlation resulted from confounding covariates. This study indicates that, empowered by appropriate statistical adjustment, small molecule inhibitor perturbation remains a powerful tool to pin down the relevant biomarker for drug safety and efficacy research. CONTACT: xin.wei@roche.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Inibidores de Proteínas Quinases/farmacologia , Sobrevivência Celular , Sistema Livre de Células , Células HeLa , Humanos
3.
Prostaglandins Other Lipid Mediat ; 92(1-4): 33-43, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20214997

RESUMO

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.


Assuntos
Benzodioxóis/farmacologia , Fenilpropionatos/farmacologia , Pneumonia/tratamento farmacológico , Primatas , Receptores do Leucotrieno B4/antagonistas & inibidores , Animais , Benzodioxóis/uso terapêutico , Benzodioxóis/toxicidade , Cães , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Feminino , Cobaias , Células HL-60 , Humanos , Hipersensibilidade/complicações , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Ozônio/farmacologia , Fenilpropionatos/uso terapêutico , Fenilpropionatos/toxicidade , Pneumonia/induzido quimicamente , Pneumonia/complicações , Pneumonia/metabolismo , Ratos , Receptores do Leucotrieno B4/metabolismo , Fumar/efeitos adversos , Testes de Toxicidade
4.
Sci Adv ; 6(20): eaay1057, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32440537

RESUMO

The transcription factor interferon regulatory factor 5 (IRF5) plays essential roles in pathogen-induced immunity downstream of Toll-, nucleotide-binding oligomerization domain-, and retinoic acid-inducible gene I-like receptors and is an autoimmune susceptibility gene. Normally, inactive in the cytoplasm, upon stimulation, IRF5 undergoes posttranslational modification(s), homodimerization, and nuclear translocation, where dimers mediate proinflammatory gene transcription. Here, we report the rational design of cell-penetrating peptides (CPPs) that disrupt IRF5 homodimerization. Biochemical and imaging analysis shows that IRF5-CPPs are cell permeable, noncytotoxic, and directly bind to endogenous IRF5. IRF5-CPPs were selective and afforded cell type- and species-specific inhibition. In plasmacytoid dendritic cells, inhibition of IRF5-mediated interferon-α production corresponded to a dose-dependent reduction in nuclear phosphorylated IRF5 [p(Ser462)IRF5], with no effect on pIRF5 levels. These data support that IRF5-CPPs function downstream of phosphorylation. Together, data support the utility of IRF5-CPPs as novel tools to probe IRF5 activation and function in disease.


Assuntos
Peptídeos Penetradores de Células , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/farmacologia , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Fosforilação
5.
Methods Mol Biol ; 356: 19-31, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-16988392

RESUMO

It is early to fully reflect on the state of the art in high content screening (HCS), because it is still a relatively new approach in drug discovery. Although the development of the first microscopes are a century old and the first confocal microscope is only 20 yr old, the fluorescent probes used within HCS along with the combination of robotic automation and integrated software technologies are quite new. HCS will require a few more years to fully demonstrate its potential power in drug discovery. Within the last year, however, one has seen this ever-expanding field lure participants in from all areas of science, introducing newer versions of instruments and reagents such that the combined efforts result in platforms and tools that meet many organizational goals in multiple ways. The potential of HCS today lies in its versatility. HCS can be used for primary screening, basic research, target identification, biomarkers, cytotoxicity, and helping to predict clinical outcomes. HCS is being applied to stem cells, patient cells, primary hepatocytes, and immortalized cultured cells. We have noted for individual specialized assays, there are multiple solutions just as there are for those standardized universally accepted assays. Whether we have needed to query cellular processes under live conditions or wanted to follow kinetically the course of a compound's effects on particular cellular reactions, we have been hampered by only a few limitations. This chapter offers a glimpse inside the use of HCS in our drug discovery environment.


Assuntos
Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Indústria Farmacêutica/métodos , Análise Serial de Tecidos/métodos , Animais , Humanos , Testes para Micronúcleos , Transporte Proteico , RNA Interferente Pequeno , Receptores Acoplados a Proteínas G/análise , Software , Fatores de Transcrição/metabolismo
6.
Methods Enzymol ; 414: 99-120, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17110189

RESUMO

Ligand-activated G protein-coupled receptors (GPCRs) are known to regulate a myriad of homeostatic functions. Inappropriate signaling is associated with several pathophysiological states. GPCRs belong to a approximately 800 member superfamily of seven transmembrane-spanning receptor proteins that respond to a diversity of ligands. As such, they present themselves as potential points of therapeutic intervention. Furthermore, orphan GPCRs, which are GPCRs without a known cognate ligand, offer new opportunities as drug development targets. This chapter describes a systems-based biological approach, one that combines in silico bioinformatics, genomics, high-throughput screening, and high-content cell-based confocal microscopy strategies to (1) identify a relevant subset of protein family targets, (2) within the therapeutic area of energy metabolism/obesity, (3) and to identify small molecule leads as tractable combinatorial and medicinal chemistry starting points. Our choice of screening platform was the Transfluor beta-arrestin-green fluorescent protein translocation assay in which full-length human orphan GPCRs were stably expressed in a U-2 OS cell background. These cells lend themselves to high-speed confocal imaging techniques using the Evotec Technologies Opera automated microscope system. The basic assay system can be implemented in any laboratory using a fluorescent probe, a stably expressed GPCR of interest, automation-assisted plate and liquid-handling techniques, an optimized image analysis algorithm, and a high-speed confocal microscope with sophisticated data analysis tools.


Assuntos
Arrestinas/química , Proteínas de Fluorescência Verde/química , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Receptores Acoplados a Proteínas G/química , Algoritmos , Animais , Automação , Biologia Computacional/métodos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico , Software , beta-Arrestinas
11.
Mol Cancer Ther ; 10(11): 2104-14, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21878655

RESUMO

A major problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or recurrence when they reenter the cell cycle. Earlier studies showed that levels of the serine/theronine kinase Mirk/dyrk1B are elevated up to 10-fold in quiescent G(0) tumor cells. Mirk uses several mechanisms to block cell cycling, and Mirk increases expression of antioxidant genes that decrease reactive oxygen species (ROS) levels and increase quiescent cell viability. We now show that a novel small molecule Mirk kinase inhibitor blocked tumor cells from undergoing reversible arrest in a quiescent G(0) state and enabled some cells to exit quiescence. The inhibitor increased cycling in Panc1, AsPc1, and SW620 cells that expressed Mirk, but not in HCT116 cells that did not. Mirk kinase inhibition elevated ROS levels and DNA damage detected by increased phosphorylation of the histone protein H2AX and by S-phase checkpoints. The Mirk kinase inhibitor increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold by gemcitabine and cisplatin. A phenocopy of these effects occurred following Mirk depletion, showing drug specificity. In previous studies Mirk knockout or depletion had no detectable effect on normal tissue, suggesting that the Mirk kinase inhibitor could have a selective effect on cancer cells expressing elevated levels of Mirk kinase.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Pancreáticas/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Apoptose , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Senescência Celular , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Éxons , Humanos , Neoplasias Pancreáticas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Gencitabina , Quinases Dyrk
12.
J Med Chem ; 53(9): 3502-16, 2010 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-20380377

RESUMO

The inhibition of LTB(4) binding to and activation of G-protein-coupled receptors BLT1 and BLT2 is the premise of a treatment for several inflammatory diseases. In a lead optimization effort starting with the leukotriene B(4) (LTB(4)) receptor antagonist (2), members of a series of 3,5-diarylphenyl ethers were found to be highly potent inhibitors of LTB(4) binding to BLT1 and BLT2 receptors, with varying levels of selectivity depending on the substitution. In addition, compounds 33 and 38 from this series have good in vitro ADME properties, good oral bioavailability, and efficacy after oral delivery in guinea pig LTB(4) and nonhuman primate allergen challenge models. Further profiling in a rat non-GLP toxicity experiment provided the rationale for differentiation and selection of one compound (33) for clinical development.


Assuntos
Descoberta de Drogas , Antagonistas de Leucotrienos/química , Éteres Fenílicos/farmacologia , Receptores do Leucotrieno B4/antagonistas & inibidores , Animais , Avaliação Pré-Clínica de Medicamentos , Cobaias , Células HL-60 , Humanos , Antagonistas de Leucotrienos/farmacologia , Éteres Fenílicos/química , Primatas , Ligação Proteica , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Receptores do Leucotrieno B4/metabolismo , Relação Estrutura-Atividade
13.
Comb Chem High Throughput Screen ; 12(9): 888-98, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19531000

RESUMO

Widely diverse biological queries are now routinely analyzed on the various optical platforms: laser line scanners, nonconfocal imagers and confocal imagers. These analyses may be performed to query a limited number of samples or range to include the evaluation of a million samples as is the goal of many screening departments in the pharmaceutical drug discovery area. First we review the key elements that distinguish the optical pathway and the hardware features used amongst the three classifications of automated imaging platforms. Recognizing the need for both resolution and throughput and maximizing the use of optics we discuss some of the influences that address how to best match some of the more common biological assays with the current hardware imaging platforms. We describe here some considerations with respect to the biology, the cell types, and the goals of the screening efforts.


Assuntos
Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala/instrumentação , Preparações Farmacêuticas/química , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Descoberta de Drogas/instrumentação , Descoberta de Drogas/métodos , Corantes Fluorescentes/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Humanos
14.
Expert Opin Investig Drugs ; 16(12): 1909-20, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18042000

RESUMO

Leukotriene B(4) (LTB(4)) is a lipid inflammatory mediator derived from membrane phospholipids by the sequential actions of cytosolic phospholipase A2 (PLA2), 5-lipoxygenase (5-LO) and leukotriene A(4) (LTA(4)) hydrolase. Several inflammatory diseases, including asthma, chronic obstructive pulmonary disease, arthritis and inflammatory bowel disease, have been associated with elevated levels of LTB(4). As a result, pharmacological strategies to modulate the synthesis of LTB(4) (inhibition of PLA2, 5-LO or LTA(4) hydrolase) or the effects of LTB(4) itself (antagonism of LTB(4) receptors) are being developed by several companies. Two G-protein-coupled receptors mediate the effects of LTB(4), namely BLT1 and BLT2. The pharmacology, expression and function of these two receptors were last reviewed by Tager and Luster in 2004. Since then, there has been an increased understanding of the function of these receptors, in particular for the lesser understood of the two receptors, BLT2. Furthermore, since last reviewed in 1996, there have been several clinical developments in the use of BLT receptor antagonists for inflammatory diseases. This review summarizes the latest preclinical and clinical developments in BLT antagonism for inflammatory diseases and discusses potential future developments.


Assuntos
Drogas em Investigação/uso terapêutico , Inflamação/tratamento farmacológico , Receptores do Leucotrieno B4/antagonistas & inibidores , Animais , Ensaios Clínicos como Assunto/métodos , Ensaios Clínicos como Assunto/tendências , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/tendências , Drogas em Investigação/química , Drogas em Investigação/farmacologia , Humanos , Inflamação/metabolismo , Receptores do Leucotrieno B4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
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