Molecular Pathophysiology of Cystic Fibrosis

描述（由申请人提供）：CFTR（囊性纤维化跨膜传导调节器）是一种氯离子通道，在介导上皮氯离子分泌和吸收中起关键作用。CFTR属于ABC（ATP Binding Cassette）转运蛋白超家族，具有两个核苷酸结合域（NBD 1和NBD 2）和一个功能尚不清楚的特征序列，其中NBD 1和NBD 2分别具有典型的步行者A和B基序。标签序列的功能重要性通过以下事实得到证明：在NBD 1的标签序列中发现许多疾病相关突变（例如，G551 D）或NBD 2（例如，G1349D）。有趣的是，虽然G551 D突变与严重形式CF相关，但G1349 D突变导致轻度形式疾病，表明这两个特征序列在控制CFTR功能中发挥不同的作用。由于ABC转运蛋白超家族包括发挥各种生理作用的成员，如胆固醇转运，癌症耐药性，心脏膜兴奋性和胰岛素分泌，因此了解CFTR在分子水平上的工作方式将对基础科学和临床医学产生广泛影响。CFTR N-末端核苷酸结合结构域（NBD 1）的X射线晶体结构的最新解决方案为详细研究签名序列在控制CFTR功能中的作用打开了大门。目前的提案将采用电生理学、分子生物学和结构生物学技术的组合来解决特征序列中的突变如何导致CFTR功能障碍（目标1）。由于这些突变的缺陷很可能被小分子药物试剂修正，我们将研究一些已知化合物对CFTR起作用的机制（目的2）。一旦成功，我们将启动基于结构的药物设计，以发现具有高效力和功效的新化合物。对突变引起的CFTR功能障碍的分子机制和药物对CFTR作用的物理/化学机制的清楚理解将有助于设计用于治疗CF和其他CFTR相关疾病的治疗试剂。 公共卫生相关性 囊性纤维化是美国最常见的致命遗传性疾病，由CFTR蛋白突变引起。该应用程序的目标是了解疾病相关突变如何导致CFTR功能障碍以及小分子化合物如何恢复突变CFTR的功能。

DESCRIPTION (provided by applicant): CFTR (Cystic Fibrosis Transmembrane conductance Regulator) is a chloride channel that plays a critical role in mediating epithelial chloride secretion and absorption. Being a member of the ABC (ATP Binding Cassette) transporter superfamily, CFTR possesses two nucleotide binding domains (NBD1 and NBD2) characterized by the canonical Walker A and B motifs for ATP binding/hydrolysis, and the signature sequence whose function remains unknown. The functional importance of the signature sequence is attested by the fact that many disease-associated mutations are found in the signature sequence of either NBD1 (e.g., G551D) or NBD2 (e.g., G1349D). Interestingly, while the G551D mutation is associated with severe form CF, the G1349D mutation causes mild form disease, indicating that these two signature sequences play distinct roles in controlling CFTR function. Since the ABC transporter superfamily encompasses members that play a variety of physiological roles such as transport of cholesterol, drug resistance in cancers, cardiac membrane excitability and insulin secretion, understanding how CFTR works at a molecular level will have a broad impact on both basic sciences and clinical medicine. Recent solution of X-ray crystal structure of CFTR's N-terminal nucleotide binding domain (NBD1) has opened the door for detailed studies of the role of signature sequences in controlling CFTR function. The current proposal will employ a combination of electrophysiolgical, molecular biological and structural biological techniques to address how mutations in the signature sequences cause CFTR dysfunction (Aim 1). Since defects of these mutations are likely to be amended by small-molecule, pharmacological reagents, we will investigate the mechanism by which some of the known compounds work on CFTR (Aim 2). Once succeeded, we will launch structure- based drug design to discover new compounds with high potency and efficacy. A clear understanding of the molecular mechanisms of CFTR dysfunction caused by mutations and the physical/chemical mechanism of drug actions on CFTR will aid in design of therapeutical reagents for the treatment of CF and other CFTR-associated diseases. PUBLIC HEALTH RELEVANCE Cystic fibrosis, the most common fatal genetic disease in the US, is caused by mutations of the CFTR protein. The goal of the application is to understand how disease-associated mutations cause dysfunction of CFTR and how small-molecule compounds restore the function of mutant CFTR.