Trans-NIH Research Support

描述（由申请人提供）：囊性纤维化（CF）是高加索人中最常见的致死性遗传疾病。它是由CF跨膜传导调节因子（CFTR）基因突变引起的，该基因编码调节上皮分泌物的体积和组成所需的顶膜阴离子通道。当CFTR不存在时，气道表面液体耗尽，粘液和细菌从肺部的清除受损，炎症和支气管扩张导致呼吸衰竭。最常见的CFTR突变，存在于>90%的CF患者中的至少一个等位基因上，在位置508处缺失苯丙氨酸（F508 del），这导致蛋白质错误折叠。内质网（ER）质量控制eliminates突变CFTR的降解，消除其运输到上皮细胞顶端膜。 尽管高通量筛选已经鉴定出可以恢复F508 del CFTR的阴离子转运功能的小分子，但它们校正了不到15%的WT CFTR活性，产生的临床益处不足。迄今为止，主要的CF药物发现测定已经采用CFTR的阴离子转运功能的测量，这是一种依赖于功能性CFTR向细胞表面的募集的方法，涉及多个洗涤步骤，并且依赖于快速饱和的信号。到目前为止，还没有简单的方法来直接确定CF中的主要缺陷的校正：突变蛋白向细胞表面的运输。我们最近开发了报告通过当前可用的小分子校正突变CFTR运输的工具和细胞系，并将该测定扩展到96孔格式。 使用这种方法，我们将：1）与MLSCN合作进行HTS以鉴定F508 del CFTR运输的新校正剂，2）采用二次筛选以验证来自HTS的命中并评估其选择性，以及3）使用来自F508 del患者的人气道细胞原代培养物确定命中功效，这是目前可用于将CF药物推进到临床的最相关测定。这种在细胞表面的F508 del CFTR的新的和简单的测定应该允许发现更有效的药物，从而防止这种疾病的灾难性影响。此外，该平台的模块化设计将使其适用于其他疾病，其中功能丧失是由膜蛋白的折叠和/或运输缺陷引起的。

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians. It is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, which encodes an apical membrane anion channel that is required for regulating the volume and composition of epithelial secretions. When CFTR is absent, airway surface liquid is depleted, the clearance of mucus and bacteria from the lungs is impaired, and inflammation and bronchiectasis lead to respiratory failure. The most common CFTR mutation, present on at least one allele in >90% of CF patients, deletes phenylalanine at position 508 (F508del), which causes the protein to misfold. Endoplasmic reticulum (ER) quality control elicits the degradation of mutant CFTR, eliminating its trafficking to the epithelial cell apical membrane. Although High Throughput Screening has identified small molecules that can restore the anion transport function of F508del CFTR, they correct less than 15% of WT CFTR activity, yielding insufficient clinical benefit. To date, primary CF drug discovery assays have employed measurements of CFTR's anion transport function, a method that depends on the recruitment of a functional CFTR to the cell surface, involves multiple wash steps, and relies on a signal that saturates rapidly. Until now, there has been no simple method to directly determine correction of the primary defect in CF: trafficking of the mutant protein to the cell surface. We have recently developed tools and cell lines that report the correction of mutant CFTR trafficking by currently available small molecules and have extended this assay to the 96-well format. Using this approach, we will: 1) Perform HTS in collaboration with the MLSCN to identify new correctors of F508del CFTR trafficking, 2) Employ secondary screens to verify hits from HTS and evaluate their selectivity, and 3) Determine hit efficacy using human airway cell primary cultures from F508del patients, the most relevant assay available currently for advancing CF drugs to the clinic. This new and simple assay of F508del CFTR at the cell surface should permit the discovery more efficacious drugs and thereby prevent the catastrophic effects of this disease. In addition, the modular design of this platform will make it useful for other diseases where loss-of-function results from folding and/or trafficking defects in membrane proteins.