Correction of defective functional expression of CFTR in cystic fibrosis

 描述（由申请人提供）：囊性纤维化（CF）是高加索人群中最常见的致死性遗传疾病。它是由CF基因突变引起的，CF基因编码囊性纤维化跨膜传导调节因子（CFTR），一种cAMP调节的氯离子通道。最普遍的CF突变，苯丙氨酸508（CFNF 508）的缺失损害CFTR的翻译后折叠，门控特性，以及生物合成和内吞加工。CFTR在质膜上的功能表达缺陷导致分泌上皮中氯化物、碳酸氢盐和液体转运受损，表现为复发性肺部感染，这是CF死亡的主要原因。CF研究的一个主要焦点是鉴定缺陷CFTR加工的小分子校正剂和靶向CFNF 508和其他CFTR突变的缺陷门控的增效剂。可用的最佳校正剂（VX-809）的功效较低，使得经处理的细胞与非CF细胞相比仅显示约15%的氯离子电导。我们和其他人已经证明，长期暴露于门控增强剂（例如VX-770）使CF 3F 508-CFTR不稳定，并降低校正功效。事实上，III期临床数据显示，VX-809和VX-770联合治疗的临床疗效虽然不高，但意义重大。这种竞争性更新建立在我们最近的以下发现上：a）通过高通量筛选（HTS）测定的校正剂和增效剂分子; B）在协同结构域折叠的背景下，校正NF 508-CFTR的两种主要折叠缺陷的需要，包括NBD 1构象和NBD 1-MSD 2界面的不稳定; d）通过校正剂和增效剂组合协同地校正W128 X-CFTR无义突变的功能表达缺陷。为了鉴定将IFF 508-CFTR折叠和氯离子通道功能恢复至其野生型对应物的>50%的校正剂-增效剂组合，我们提出鉴定和验证在目标1中缺乏IFF 508-CFTR的去稳定作用的有效增效剂。在目标2中，我们将利用新的局部结构缺陷靶向（LSDT）筛选方法来鉴定不同的结构特异性校正剂作为药理学伴侣，其稳定NBD 1并与界面稳定药物（例如VX-809）协同作用。工程原代气道上皮细胞将增加这些筛选的成功率。新型校正剂的作用机制将通过生物物理、生物化学和细胞生物学测定来确定。基于目标1-2的结果，在目标3中，将对W1282 X-CFTR功能表达缺陷进行新的机制研究，实施突变特异性生化和功能HTS试验，以鉴定小分子校正剂和增效剂，并建立其作用机制。

 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.