Correction of defective deltaF508-CFTR processing 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)的缺失会扰乱CFTR的翻译后折叠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、碳酸氢盐和水的转运受损，表现为反复肺部感染，这是导致CF死亡的主要原因。小分子校正子的鉴定是目前化学校正剂研究的一个重要方向。现有最好的校正剂(VX-809)的效率很低，以至于处理过的细胞与非CF细胞相比，只显示出&lt；15%的氯离子电导。与之一致的是，II期临床试验的初步数据表明，VX-809的临床疗效微乎其微。由于我们对？F508CFTR错误折叠的不完全理解，高效校正子分子的鉴定受到阻碍。虽然最近的结果表明？F508突变使分离的核苷酸结合域1(NBD1)失去了稳定性，但？F508-NBD1构象稳定代表着理想的药物靶点的暗示仍有待在全长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)在？F508CFTR错误折叠中起着同样重要的作用。这一竞争性更新建立在我们通过高通量筛选(HTS)试验发现的a)校正子和增强子分子，b)CFTR合作结构域折叠机制，以及c)初步数据表明？F508-NBD1的热力学校正是必要的，但不足以恢复？F508 CFTR生物合成折叠、加工和质膜稳定性。为了分离将？F508CFTR折叠和质膜氯通道功能恢复到野生型的50%的校正子，我们建议通过使用一种新的、局部结构缺陷靶向筛选(LSD)的方法来鉴定不同的、结构特异的校正子作为协同作用的药物伴侣。目的1.通过量化结构域-结构域相互作用和NBD1能量学对通道折叠和功能的贡献，阐明导致F508-CFTR错误折叠的主要结构缺陷。分离的NBD1和全长CFTR中不同结构缺陷的第二位点抑制物突变的结果将通过生物物理、生化和细胞生物学分析来确定。目的2.通过多个局部结构缺陷靶向HTS识别小分子校正子，并建立其在体内外的作用机制。目的3.用创新的替代人细胞和动物CF模型来确定基于LSD的联合F508-CFTR矫正器疗法的翻译潜力。

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.