Potentiation of Mutant CFTR Activity

描述(申请人提供)：囊性纤维化(CF)是影响高加索人群的最常见的致命性遗传病。这种疾病是由囊性纤维化跨膜传导调节因子(CFTR)的异常引起的，CFTR是一种氯离子通道，在维持肺健康和动态平衡方面发挥着关键作用。正常的CFTR门控或“激活”需要调节结构域(RD)的磷酸化和ATP驱动的两个核苷酸结合结构域(NBD)的二聚化。Cftr突变可能导致减少氯离子转运的一系列缺陷中的一种，包括表达减少、膜上存在减少以及门控不足。高通量药物筛选程序已经发现了能够改善突变CFTR门控的“电位剂”化合物，其中第一种化合物VX-770最近在CF受试者的第一阶段测试中显示出相当大的前景。然而，cftr增强子打开突变氯离子通道的机制(S)尚不清楚。这项研究的第一个目标是详细分析几种CFTR型增效剂化合物的作用机理。分离的RD的生化研究、全长CFTR的电生理分析和酶研究将确定对基于cAMP的RD磷酸化级联的影响。量热(热熔化)实验将评估增效剂与单个NBD的结合情况。在第二个目标中，我们将调查这些发现与小鼠体内CFTR增强子激活的相关性。这既包括最常见的致病突变AF508-CFTR，也包括不太常见的突变，如G551D和R177H。Cftr的增强子提供了一个更好地了解这一重要离子通道激活的基本方式，以及cftr和其他ATP结合盒基因家族成员所利用的结构域相互作用的机会。对增效剂作用机理的了解也是更合理和有效地优化化合物的方法所必需的，例如人类使用的这些化合物。公共卫生相关性：囊性纤维化在美国是一种常见的遗传性疾病。在这个项目中，我们的目标是更好地定义通过纠正上皮离子通道功能缺陷来帮助缓解疾病的药物类型。

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is the most common lethal genetic disease affecting Caucasian populations. The illness is caused by abnormalities in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that serves a critical role maintaining lung health and homeostasis. Normal CFTR gating or "activation" requires phosphorylation of the regulatory domain (RD) and ATP-driven dimerization of the two nucleotide binding domains (NBDs). CFTR mutations may engender one of a number of defects that reduce chloride transport, including reduced expression, decreased presence at the membrane, and insufficient gating. 'Potentiator' compounds capable of improving the gating of mutant CFTR have been discovered by high-throughput drug screening programs, and the first of these compounds, VX-770, has recently shown considerable promise as part of Phase I testing in CF subjects. However, the mechanism(s) by which CFTR potentiators open the mutant chloride channel are not known. The first Aim of this study encompasses a detailed analysis of the mechanisms underlying several CFTR potentiator compounds. Biochemical study of isolated RD, electrophysiologic analysis of full-length CFTR, and enzymatic studies will determine effects on the cAMP-based RD phosphorylation cascade. Calorimetric (thermal melting) experiments will evaluate potentiator binding to individual NBDs. In the second Aim, we will investigate the relevance of these findings to in vivo potentiator activation of CFTR in mice. This will include both AF508-CFTR, the most common disease-causing CFTR mutation, and less frequent mutations, e.g. G551D and R177H. Potentiators of CFTR present an opportunity to better understand the basic means underlying activation of this important ion channel, and the domain interactions utilized by CFTR and other ATP binding cassette gene family members. An understanding of potentiator mechanism of action is also required for a more rational and efficient approach to optimizing compounds such as these for human use. PUBLIC HEALTH RELEVANCE: Cystic Fibrosis is a common genetic disease in the U.S. In this project we aim to better define the types of drugs that will help alleviate the disease by correcting defective function of the epithelial ion channel.