Correction of defective deltaF508-CFTR processing 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。最常见的CF突变，苯丙氨酸508（？F508）破坏CFTR的翻译后折叠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离子、碳酸氢盐和水的转运受损，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小分子校正剂的鉴定。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<15%的氯离子电导。与此雅阁的是，II期临床试验的初始数据表明VX-809的临床疗效有限。识别高效校正分子是阻碍我们的不完整的理解？F508 CFTR错误折叠。虽然最近的研究结果表明，？F508突变大力不稳定的孤立的核苷酸结合域1（NBD 1），这意味着？F508-NBD 1构象稳定代表了理想的药物靶标，仍有待在全长CFTR的背景下验证。事实上，我们的初步数据表明，额外的机制（S）发挥同样重要的作用？F508 CFTR错误折叠。这种竞争性的更新建立在我们的发现a）校正剂和增效剂分子的高通量筛选（HTS）测定，B）CFTR合作域折叠机制，以及c）初步数据表明，热力学校正？F508-NBD 1是必要的，但不足以恢复？F508 CFTR生物合成折叠、加工和质膜稳定性。以隔离恢复的校正器？由于F508 CFTR折叠和质膜氯离子通道功能超过其野生型对应物的50%，我们提出鉴定不同的结构特异性校正剂作为药物分子伴侣，其通过使用新的局部结构缺陷靶向筛选（LSDS）方法协同作用。目标1.会不会说明造成这种情况的主要结构缺陷？F508-CFTR错误折叠通过量化结构域-结构域相互作用和NBD 1能量学对通道折叠和功能的贡献。分离的NBD 1和全长CFTR中不同结构缺陷的第二位点抑制突变的结果将通过生物物理、生物化学和细胞生物学测定来确定。目标2.将通过多个局部结构缺陷靶向的HTS来鉴定小分子矫正剂，并建立它们在体外和体内的作用机制。目标3.将决定基于LSDS的组合的转化潜力？F508-CFTR校正剂治疗使用创新的替代人类细胞和动物模型的CF。 公共卫生相关性：目前可用于囊性纤维化（CF）的疗法不能纠正潜在的CFTR缺陷，并且没有将中位预期寿命提高到40岁以上。该建议将建立一个概念新颖的，基于结构的高通量筛选技术，利用我们的知识，由CF中最常见的突变，F508残基缺失引起的生化和结构缺陷。这项研究的结果将包括提高对CF蛋白的理解，并分离出潜在的新的、更有效的囊性纤维化治疗方法。

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) disrupts the posttranslational folding, as well as the biosynthetic and endocytic processing of CFTR. The functional expression defect of CFTR at the plasma membrane leads to impaired chloride, bicarbonate and water 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 corrector. 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. In accord, initial data of phase II clinical trials indicate marginal clinical efficiency of VX-809. Identification of highly efficient corrector molecules is impeded by our incomplete understanding of the ?F508 CFTR misfolding. While recent results suggest that the ?F508 mutation energetically destabilizes the isolated nucleotide binding domain 1 (NBD1), the implication that ?F508-NBD1 conformational stabilization represents the ideal drug target remains to be validated in the context of full-length CFTR. Indeed, our preliminary data indicate that additional mechanism(s) play equally important role in the ?F508 CFTR misfolding. This competitive renewal builds on our discoveries of a) corrector and potentiator molecules by high throughput screening (HTS) assays, b) the CFTR cooperative domain folding mechanism, as well as c) preliminary data indicating that thermodynamic correction of the ?F508-NBD1 is necessary, but not sufficient to restore the ?F508 CFTR biosynthetic folding, processing and plasma membrane stability. To isolate correctors that restore the ?F508 CFTR folding and plasma membrane chloride channel function to >50% of its wild-type counterpart, we propose to identify distinct, structure-specific correctors as pharmaco- chaperones that act synergistically by using a novel, localized structure defect-targeted screening (LSDS) approach. Aim 1. Will elucidate the major structural defects responsible for ?F508-CFTR misfolding by quantifying the contribution of domain-domain interactions and the NBD1 energetics to the channel folding and function. The consequence of second site suppressor mutations of distinct structural defects in isolated NBD1 and full-length CFTR will be established by biophysical, biochemical and cell biological assays. Aim 2. will identify small-molecule correctors by multiple localized structure defect-targeted HTS and establish their mechanism of action in vitro and in vivo. Aim 3. Will determine the translational potential of LSDS-based combination ?F508-CFTR corrector therapy by using innovate surrogate human cellular and animal models of CF. PUBLIC HEALTH RELEVANCE: The presently available therapies for cystic fibrosis (CF) do not correct the underlying CFTR defect, and have not improved median life expectancy beyond 40 years. This proposal will establish a conceptually novel, structure-based high throughput screening techniques utilizing our knowledge about the biochemical and structural defects caused by the most common mutation in CF, deletion of F508 residue. The outcome of this research will include improved understanding of the CF protein and the isolation of potentially new, more efficient therapies for cystic fibrosis.