Anti-inflammatory Assay for Cystic Fibrosis

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，以发现治疗囊性纤维化（CF）慢性肺部炎症的抗炎药物。CF的表型标志是慢性肺部感染的机会性微生物，如铜绿假单胞菌。由此产生的炎症最终导致肺功能恶化和患者死亡。因此，使用抗炎药和抗生素来控制CF患者的慢性炎症和感染。然而，目前在患者中使用的大多数炎症剂由于不良副作用有严重的局限性。一些研究表明，toll样受体途径（TLR）通过转录因子核因子κ B （NF?B）的过度激活介导CF中细菌感染诱导的炎症。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NF？CF气道上皮细胞中的B亚基p65。我们已经创建并部分表征了一种CF气道细胞系，该细胞系稳定表达NF的p65亚基。B与绿色荧光蛋白（GFP-p65）融合。我们的初步数据表明GFP-p65在工程细胞中起作用，并在PAF刺激下易位到细胞核。我们将制定一项综合策略，以适合HTS的格式对这些细胞进行成像。通过筛选药理学活性分子的先导文库，将进一步验证和优化该检测方法。我们还将开发和测试一种策略，根据活性和途径选择性从HTS工作中优先考虑活性化合物。最后，我们将定义并测试我们的方法来确定测试剂的分子作用机制并揭示其细胞内靶标。

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.