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反应过度、组织炎症、气道重塑以及随之而来的粘膜纤毛运输受损。这一建议是基于这样的假设：IL-4和IL-13激活的STAT6通路在特应性哮喘患者中上调，并随后导致致病性上皮功能改变，从而导致粘膜纤毛清除受损。该通路对气道呼吸上皮细胞具有相对较高的特异性，这表明具有STAT6结合基模功能性过表达的永生化上皮细胞系以及STAT6激活发光报告基因将为寻找该通路的修饰因子以及“可药物化”靶点提供有利平台。此外，将该细胞系用于基于荧光的机械生理试验，同时或紧随新化学实体（NCE）的初步筛选，激活或抑制IL-4或IL-13激活的STAT6途径，将显着增强该细胞系的效用。在该项目的第一阶段，我们开发并验证了基因组序列，通过将人类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中，第一个稳定的IL-4诱导的“超反应”人支气管上皮细胞系，命名为sub-D1 BEAS-2¿。基于荧光素酶测量得到的sub-D1BEAS-2¿细胞系的z分数为0.65；完全在适用范围内，用于基于细胞的高通量筛选药物发现平台。在II期，我们建议a)进一步验证这种IL-4、IL-13激活的STAT6通路在sub-D1 BEAS-2¿细胞系中的分子信号转导，b)随后证明该细胞系适用于高通量筛选（HTS）药物发现，使用荧光素酶测定以及细胞内离子和细胞膜电位的荧光功能测定作为呼吸道上皮细胞功能致病性变化的预测因子；(c)建立sub-D1 BEAS-2¿的初级和次级细胞库。该项目将广泛影响哮喘、过敏和炎症的药物发现研究。它的应用程序解决了NIH的几个研究所的SBIR请求的需求和任务。这些应用包括：1)NHLBI对COPD和哮喘的认识和治疗；2)美国国家过敏和传染病研究所（NIAID）的哮喘、过敏和炎症项目，以及3)基于与信号转导（NCI）相互作用的分子靶点的新检测方法的开发和使用。

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).