Correction of defective functional expression of CFTR in cystic fibrosis

 描述(申请人提供)：囊性纤维化(CF)是高加索人群中最常见的致死性遗传病。它是由编码囊性纤维化跨膜电导调节因子(CFTR)的CF基因突变引起的，CFTR是cAMP调节的氯离子通道。最常见的CF突变，苯丙氨酸508(∆F508)的缺失会损害CFTRs的翻译后折叠、门控特性以及生物合成和内吞加工。CFTR在细胞膜上的功能表达缺陷导致分泌上皮细胞的氯离子、碳酸氢盐和液体转运受损，表现为反复肺部感染，这是导致CF死亡的主要原因。CF研究的一个主要焦点是识别针对∆F508和其他CFTR突变的有缺陷的cftr加工的小分子校正子和有缺陷的门控增强剂。现有的最好的校正剂(VX-809)的效果很低，以至于处理后的细胞与非CF细胞相比，只显示出∼15%的氯离子电导。我们和其他人已经证明，长期接触门控增强剂(例如VX-770)会破坏∆F508-cftr的稳定，并降低矫正效果。实际上，第三阶段的临床数据显示，VX-809和VX-770联合治疗的临床疗效不大，但意义重大。这一竞争性更新建立在我们最近发现的以下几个方面：a)通过高通量筛选检测校正子和增强子分子；b)纠正∆F508-cftr的两个初级折叠缺陷，包括在协同结构域折叠的背景下nbd1构象和nbd1-MSD2界面的不稳定；c)初步鉴定不会破坏∆F508-cftr稳定的新型增强子分子；以及d)通过校正子和增强子的组合巧妙地纠正W128X-cftr无义协同突变的功能表达缺陷。为了确定能够将∆F508-cftr折叠和氯离子通道功能恢复到野生型的50%的校正子-增强剂组合，我们建议在目标1中鉴定和验证缺乏∆F508-cftr不稳定作用的有效增强剂。在目标2中，我们将利用新的局部结构缺陷靶向筛选方法来鉴定不同的结构特异性校正子作为药理伴侣，稳定NBD1并与界面稳定药物(例如VX-809)协同作用。设计的原代呼吸道上皮细胞将提高这些筛查的成功率。新型校正剂的作用机理将通过生物物理、生化和细胞生物学检测来确定。在目标1-2的基础上，在目标3对W1282X-CFTR功能表达缺陷的新机制研究中，将实施突变特异性生化和功能HTS检测，以识别小分子校正子和增强子，并建立它们的作用机制。

 DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is the most common lethal genetic disease in the Caucasian population. It is caused by mutations in the CF gene, encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The most prevalent CF mutation, deletion of phenylalanine 508 (∆F508) impairs the posttranslational folding, gating characteristics, and the biosynthetic and endocytic processing of CFTR. The functional expression defect of CFTR at the plasma membrane leads to impaired chloride, bicarbonate and fluid transport in secretory epithelia, manifesting in recurrent lung infection, the primary cause of mortality in CF. A major focus of CF research is the identification of small-molecule correctors of defective CFTR processing and potentiators of defective gating that target that the ∆F508 and other CFTR mutations. The efficacy of the best correctors available (VX-809) is low, such that treated cells show only ∼15% of chloride conductance compared to non-CF cells. We and others have demonstrated that chronic exposure to gating potentiators (e.g. VX-770) destabilizes ∆F508-CFTR and reduces correction efficacy. Phase III clinical data, indeed, showed only modest but significant clinical efficacy of combination VX-809 and VX-770 therapy. This competitive renewal builds on our recent discoveries of: a) corrector and potentiator molecules by high throughput screening (HTS) assays; b) the correction requirement of both primary folding defects of ∆F508-CFTR, including destabilization of the NBD1 conformation and the NBD1-MSD2 interface in the context of cooperative domain folding; c) preliminary identification of novel potentiator molecules that do not destabilize ∆F508-CFTR; and d) correction of the functional expression defect of the W128X-CFTR nonsense mutation synergistically by corrector and potentiator combination. To identify corrector-potentiator combinations that restore ∆F508-CFTR folding and chloride channel function to >50% of its wild-type counterpart, we propose to identify and validate efficacious potentiators that lack destabilizing effect of the ∆F508-CFTR in Aim 1. In Aim 2 we will utilize novel localized structure defect-targeted (LSDT) screening approaches to identify distinct, structure-specific correctors as pharmacological chaperones that stabilize NBD1 and act synergistically with interface stabilizing drugs (e.g. VX-809). Engineered primary airways epithelial cells will increase the success rate of these screens. The mechanism of action of novel correctors will be established by biophysical, biochemical and cell biological assays. Based on the results of Aim 1-2, in Aim 3 novel mechanistic studies on the W1282X-CFTR functional expression defect, mutation-specific biochemical and functional HTS assays will be implemented to identify small-molecule correctors and potentiator and establish their mechanism of action.