A Cell Line for Anti-Asthma Drug Discovery

用于发现抗哮喘药物的细胞系

• 批准号: 7052458
• 负责人: Hua Mao
• 金额: $ 32.95万
• 依托单位: BIOTECHPLEX CORPORATION
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7052458
• 关键词: asthma; bioassay; biological signal transduction; cell bank /registry; cell line; chloride ion; confocal scanning microscopy; cryopreservation; drug discovery /isolation; enzyme activity; fluorescence; high throughput technology; interleukin 13; interleukin 4; luciferin monooxygenase; membrane potentials; reporter genes; respiratory epithelium; sodium ion; tissue /cell preparation; transcription factor

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).

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、气道重塑和随后的粘膜纤毛转运受损。这一建议是基于这样的假设，即IL-4和IL-13激活的STAT 6通路在特应性哮喘患者中上调，随后导致致病性上皮功能变化，从而损害粘膜纤毛清除。该途径对气道呼吸道上皮细胞的相对高的特异性表明，具有功能性过表达的STAT 6结合基序以及STAT 6活化发光报道基因的永生化上皮细胞系将提供一个能够搜索该途径的修饰剂的平台，从而提供“可药物化”靶位点。此外，通过在激活或抑制IL-4或IL-13激活的STAT 6途径的新化学实体（NCE）的初步筛选同时或紧接在其之后将其用于基于荧光的机制生理学测定中，该细胞系的效用将显著增强。在该项目的I期，我们开发并验证了基因组序列，通过将5个串联拷贝的人C/EBP-STAT 6结合基序整合到染色体中，并具有可报告的荧光素酶活性，命名为sub-D1 BEAS-2？，第一个稳定的IL-4引起“高反应性”人支气管上皮细胞系。基于荧光素酶测量，所得亚D1 BEAS-2“细胞系的Z分数为0.65;完全在用于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STAT 6通路在sub-D1 BEAS-2中的分子信号转导。细胞系，B）随后证明该细胞系适合于高通量筛选（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的致病性变化;和（c）建立sub-D1 BEAS-2 <$的原代和次级细胞库。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美国国立卫生研究院的几个研究所的SBIR招标的需求和任务。这些应用包括：1）在NHLBI了解和治疗COPD和哮喘; 2）在过敏和传染病（NIAID）的国家研究所哮喘，过敏和炎症计划，和3）开发和使用基于分子靶点与信号转导（NCI）相互作用的新测定。