Trans-NIH Research Support

描述(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病。它是由CF跨膜电导调节器(CFTR)基因突变引起的，CFTR基因编码顶膜阴离子通道，调节上皮分泌物的数量和组成。当CFTR缺失时，呼吸道表面液体耗尽，肺部粘液和细菌的清除受到损害，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患者的至少一个等位基因上，它删除了508位(F508del)的苯丙氨酸，导致蛋白质错误折叠。内质网(ER)质量控制导致突变的CFTR的降解，消除了它向上皮细胞顶膜的转运。尽管高通量筛选已经发现了可以恢复F508del CFTR阴离子转运功能的小分子，但它们纠正了不到15%的WT CFTR活性，产生的临床益处不足。到目前为止，主要的CF药物发现分析采用了CFTR阴离子传输功能的测量，这种方法依赖于功能性CFTR在细胞表面的招募，涉及多个洗涤步骤，并依赖于快速饱和的信号。到目前为止，还没有一种简单的方法来直接确定CF中主要缺陷的纠正：突变蛋白向细胞表面的运输。我们最近开发了工具和细胞系，报告了目前可用的小分子对突变CFTR贩运的纠正，并将这一分析扩展到96孔格式。使用这种方法，我们将：1)与MLSCN合作进行HTS，以确定F508del CFTR贩运的新校正者；2)使用二次筛查来验证HTS的命中结果并评估其选择性；3)使用来自F508del患者的人呼吸道细胞原代培养来确定命中效果，这是目前可用于将CF药物推向临床的最相关的检测方法。这种新的、简单的细胞表面F508del CFTR分析应该可以发现更有效的药物，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因膜蛋白折叠和/或运输缺陷而导致功能丧失的疾病。

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.