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剂）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。作为ABC （ATP Binding Cassette）转运体超家族的成员，CFTR具有两个核苷酸结合结构域（NBD1和NBD2），其特征为ATP结合/水解的典型Walker a和B基序，以及功能未知的特征序列。在NBD1（如G551D）或NBD2（如G1349D）的特征序列中发现了许多疾病相关突变，这一事实证明了特征序列的功能重要性。有趣的是，G551D突变与严重形式CF相关，而G1349D突变导致轻度形式CF，这表明这两个特征序列在控制CFTR功能方面发挥着不同的作用。由于ABC转运蛋白超家族包括发挥多种生理作用的成员，如胆固醇转运、癌症耐药、心膜兴奋性和胰岛素分泌，因此了解CFTR在分子水平上的工作原理将对基础科学和临床医学产生广泛影响。最近对CFTR n端核苷酸结合域（NBD1） x射线晶体结构的研究为深入研究特征序列在控制CFTR功能中的作用打开了大门。目前的提案将结合电生理学、分子生物学和结构生物学技术来解决特征序列中的突变如何导致CFTR功能障碍（目的1）。由于这些突变的缺陷可能被小分子药理学试剂修正，我们将研究一些已知化合物作用于CFTR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疗效的新化合物。明确突变导致CFTR功能障碍的分子机制以及药物作用于CFTR的物理/化学机制，将有助于设计治疗CF及其他CFTR相关疾病的治疗试剂。

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.