A Cell Line for Anti-Asthma Drug Discovery

描述(由申请人提供)：哮喘患者的特征是气道高反应性、组织炎症、呼吸道重塑以及随之而来的粘液纤毛运输受损。这一建议是基于一种假设，即IL-4和IL-13激活的STAT6通路在特应性哮喘患者中上调，从而导致致病的上皮功能变化，从而损害粘液纤毛清除。这一途径对呼吸道上皮细胞具有较高的特异性，这表明具有STAT6结合基序功能过度表达的永生化上皮细胞株与STAT6激活发光报告一起将提供一个使能平台来寻找该途径的修饰物，从而寻找可用药的靶点。此外，在新的化学物质(NCE‘s)的初步筛选过程中，激活或抑制IL-4或IL-13激活的STAT6通路的新化学实体(NCE’s)的初步筛选，如果将其用于基于荧光的机械生理学分析，则该细胞系的实用性将显著增强。在该项目的第一阶段，我们开发并验证了第一个稳定的IL-4通过将人C/EBP-STAT6结合基序的5个串联拷贝整合到具有可报告的荧光素酶活性的染色体上来验证第一个稳定的IL-4诱导的人支气管上皮细胞系的基因组序列，命名为亚D1Beas-2。根据荧光素酶的测量，所得到的亚D1BEAS-2细胞系的Z分数为0.65；完全在基于细胞的高通量筛选药物发现平台的适用范围内。在第二阶段，我们建议a)进一步验证这种IL-4、IL-13激活的STAT6通路在亚D1BEAS-2细胞系中的分子信号转导，b)随后证明该细胞系适合于高通量筛选(HTS)药物的发现，使用荧光素酶试验以及细胞内离子和细胞膜电位的荧光功能分析作为呼吸道上皮细胞功能变化的预测因子；以及(C)建立亚D1BEAS-2细胞的原代和次级细胞库。该项目将广泛影响哮喘、过敏和炎症方面的药物发现研究。它的申请解决了国家卫生研究院几个研究所的SBIR征集的需求和任务。这些应用包括：1)NHLBI对COPD和哮喘的理解和治疗；2)国家过敏和传染病研究所(NIAID)的哮喘、过敏和炎症计划；以及3)基于与信号转导(NCI)相互作用的分子靶标的新分析方法的开发和使用。

DESCRIPTION (provided by applicant): Persons with asthma are characterized by hyper-responsive airways, tissue inflammation, airway remodeling and the consequent impaired mucociliary transport. This proposal is based on the hypothesis that the IL-4 and IL-13 activated STAT6 pathway is up-regulated in persons with atopic asthma and subsequently responsible for pathogenic epithelial functional changes with the consequent impairment of mucociliary clearance. The relatively high specificity of this pathway to airway respiratory epithelial cells indicates that an immortalized epithelial cell line with functional over-expression of STAT6 binding motif together with a STAT6 activation luminescent reporter will provide an enabling platform to search for modifiers of this pathway and thus "druggable" target sites. Additionally, the utility of this cell line will be markedly enhanced by using it in fluorescence-based mechanistic physiological assays coincidentally with, or immediately following the primary screening of new chemical entity's (NCE's) which either activate or suppress the IL-4 or IL-13 activated STAT6 pathway. In Phase I of this Project we developed and validated the genomic sequence the first stable IL-4 elicited "hyper-responsive" human bronchial epithelial cell line by incorporating 5 tandem copies of human C/EBP-STAT6 binding motif into the chromosome with reportable luciferase activities, designated, sub-D1 BEAS-2¿. The Z-score of the resulting sub-D1BEAS-2¿ cell line based on luciferase measurements was 0.65; well within the applicable range for use in cell-based high throughput screening drug discovery platform. In Phase II, we propose to a) further validate the molecular signal transduction of this IL-4, IL-13, activated STAT6 pathway in the sub-D1 BEAS-2¿ cell line, b) subsequently demonstrate that this cell line is suitable for high throughput screening (HTS) drug discovery using both the luciferase assay as well as the fluorescent functional assays of intracellular ions and cell membrane potential as predictors of pathogenic changes in respiratory airway epithelial cellular function; and (c) establish the primary and secondary cell banks of sub-D1 BEAS-2¿. This project will broadly impact drug discovery research in asthma, allergy and inflammation. Its applications address the needs and missions of the SBIR solicitations by several institutes of the NIH. These applications include: 1) the understanding and treatment of COPD and asthma at NHLBI; 2) the Asthma, Allergy and Inflammation Program at the National Institutes of Allergy and Infectious Diseases (NIAID), and 3) the development and use of new assays based on molecular targets that interact with signal transduction (NCI).