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命性遗传疾病。它是由CF基因突变引起的，该基因编码囊性纤维化跨膜传导调节因子（CFTR），这是camp调节的氯离子通道。最常见的CF突变，苯丙氨酸508 (？F508)破坏翻译后折叠，以及CFTR的生物合成和内吞加工。CFTR在质膜处的功能表达缺陷导致分泌上皮中氯化物、碳酸氢盐和水分运输受损，表现为肺部复发性感染，是CF死亡的主要原因。CF研究的一个重点是小分子纠正剂的鉴定。目前可用的最佳校正剂（VX-809）的有效性很低，因此与非cf细胞相比，处理过的细胞仅显示出<15%的氯化物电导。与此一致的是，VX-809的II期临床试验初始数据显示其边际临床效率。我们对dna的不完全理解阻碍了高效校正分子的鉴定。F508 CFTR折叠错误。虽然最近的研究结果表明？F508突变在能量上破坏了分离的核苷酸结合结构域1 （NBD1）的稳定性，这意味着？F508-NBD1构象稳定性代表了理想的药物靶点，在全长CFTR的背景下仍有待验证。事实上，我们的初步数据表明，其他机制在？F508 CFTR折叠错误。这种竞争性更新建立在我们通过高通量筛选（HTS）分析发现的a)校正剂和增强剂分子，b) CFTR合作结构域折叠机制，以及c)表明热力学校正的初步数据之上？F508-NBD1是必要的，但不足以恢复？F508 CFTR生物合成折叠、加工及质膜稳定性。隔离恢复？F508 CFTR折叠和质膜氯离子通道的功能比其野生型对应物低50%，我们建议使用一种新颖的局部结构缺陷靶向筛选（LSDS）方法来识别独特的、结构特异性的校正物作为协同作用的药物伴侣。目的1。将阐明主要的结构性缺陷的责任？通过量化结构域相互作用和NBD1能量学对通道折叠和功能的贡献来研究F508-CFTR错误折叠。分离的NBD1和全长CFTR中不同结构缺陷的第二位点抑制基因突变的后果将通过生物物理、生化和细胞生物学检测来确定。目标2。将通过多个局部结构缺陷靶向HTS识别小分子纠正剂，并建立其体外和体内作用机制。目标3。将决定基于lsds的组合的转化潜力？F508-CFTR校正治疗，利用创新的替代人细胞和动物CF模型。

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.