Anti-inflammatory Assay for Cystic Fibrosis

囊性纤维化的抗炎测定

基本信息

批准号：
8134500
负责人：
RANGAN MAITRA
金额：
$ 5.22万
依托单位：
RESEARCH TRIANGLE INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-30 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8134500
关键词：
Adverse effects Affect Anions Anti-Inflammatory Agents Anti-inflammatory Antibiotic Resistance Apical Apoptosis Apoptotic Bacterial Infections Biological Assay Biotechnology Cell Line Cell Nucleus Cells Cessation of life Chronic Code Complex Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Deoxyribonuclease I Deterioration Disease Disease Management Doctor of Philosophy Engineering Epithelial Cells Flagellin Foundations Funding Genes Genetic Transcription Green Fluorescent Proteins Haemophilus influenzae Hereditary Disease Human Ibuprofen Image Immunity Individual Infection Inflammation Inflammatory Inflammatory Response Institutes Ion Transport Laboratories Lead Letters Libraries Lipopolysaccharides Lung Lung Inflammation Mediating Methotrexate Microbe Molecular Mechanisms of Action Monitor Mutation NF-kappa B Natural Immunity New Agents Normal Cell Opportunistic Infections Participant Pathway interactions Patients Pharmaceutical Preparations Physiological Process Proteins Proteomics Pseudomonas Pseudomonas aeruginosa Qualifying Regulation Regulator Genes Reporting Research Research Personnel Resistance Respiratory physiology Role Scientist Screening procedure Staphylococcus aureus Surface Techniques Testing Toll-Like Receptor Pathway Toxic effect Tracheobronchial United States National Institutes of Health Validation Work antimicrobial assay development base cellular engineering cellular imaging clinical efficacy combat cystic fibrosis airway cystic fibrosis patients drug discovery innovation medical schools methotrexate analog microbial p65 programs recombinant human DNase research clinical testing response tool transcription factor

项目摘要

DESCRIPTION (provided by applicant): The overall aim of this project is to develop a disease-relevant HTS assay for the discovery of anti-inflammatory drugs to manage chronic lung inflammation in Cystic Fibrosis (CF). The phenotypic hallmark of CF is chronic lung infection from opportunistic microbes such as Pseudomonas aeruginosa. Resultant inflammation eventually leads to deterioration of lung function and death in afflicted individuals. Accordingly, anti-inflammatory agents and antibiotics are used to manage chronic inflammation and infection in CF patients. However, most inflammatory agents currently used in patients have severe limitations due to adverse side effects. Several studies indicate that the toll-like receptor pathway (TLR) mediates bacterial infection-induced inflammation in CF through hyper-activation of the transcription factor nuclear factor-kappa B (NF?B). Thus, we will develop and optimize a Pseudomonas aeruginosa bacterial filtrate (PAF)-induced image-based translocation assay for the NF?B subunit p65 in CF airway epithelial cells. We have already created and partially characterized a CF airway cell-line stably expressing the p65 subunit of NF?B fused to green fluorescent protein (GFP-p65). Our preliminary data indicate that GFP-p65 is functional in the engineered cells and translocates to the nucleus upon stimulation with PAF. We will develop a comprehensive strategy for imaging these cells in a format suitable for HTS. The assay will be further validated and optimized for HTS through the screening of a pilot library of pharmacologically active molecules. We will also develop and test a strategy to prioritize active compounds from HTS efforts based upon activity and pathway selectivity. Finally, we will define and test our approach to determine the molecular mechanism of action of a test agent and reveal its intracellular targets.

描述（由申请人提供）：该项目的总体目标是开发一种与疾病相关的 HTS 检测方法，以发现抗炎药物来控制囊性纤维化 (CF) 的慢性肺部炎症。 CF 的表型特征是机会性微生物（如铜绿假单胞菌）引起的慢性肺部感染。由此产生的炎症最终导致患者肺功能恶化和死亡。因此，抗炎剂和抗生素用于控制 CF 患者的慢性炎症和感染。然而，目前用于患者的大多数炎症药物由于不良副作用而具有严重的局限性。多项研究表明，Toll 样受体通路 (TLR) 通过转录因子核因子 kappa B (NF?B) 的过度激活介导细菌感染诱导的 CF 炎症。因此，我们将开发和优化铜绿假单胞菌细菌滤液 (PAF) 诱导的基于图像的 CF 气道上皮细胞中 NFκB 亚基 p65 易位测定。我们已经创建并部分表征了稳定表达与绿色荧光蛋白融合的 NFκB p65 亚基 (GFP-p65) 的 CF 气道细胞系。我们的初步数据表明 GFP-p65 在工程细胞中具有功能，并在 PAF 刺激后易位至细胞核。我们将制定一个全面的策略，以适合 HTS 的格式对这些细胞进行成像。该检测将通过筛选药理活性分子的试点库进一步针对 HTS 进行验证和优化。我们还将开发和测试一项策略，根据活性和途径选择性优先考虑 HTS 工作中的活性化合物。最后，我们将定义和测试我们的方法，以确定测试药物的分子作用机制并揭示其细胞内靶点。