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). ? ?
