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儿科遗传病。囊性纤维化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是一种cAMP调节的氯通道，属于ATP结合盒家族。大量的突变，包括最常见的苯丙氨酸508(DeltaF508)缺失，导致CFTR的折叠和加工缺陷。使用功能性高通量筛选(HTS)试验，我们已经鉴定出四类小分子(“校正子”)，它们可以部分逆转转基因的非极化和极化细胞以及原代培养的人呼吸道上皮细胞的加工缺陷。我们的长期目标是为CF治疗寻找临床上有用的小分子。为了实现这一目标，我们有三个具体目标。1)全面的细胞生物学和生化分析将确定deltaF508-CFTR校正器作用的分子机制。需要分析的潜在作用位点包括：翻译、翻译后折叠、伴侣相互作用、内质网稳定性、细胞表面稳定性、内化和再循环效率以及内质网和高尔基体后间隔的泛素化。了解校正机制对于确定小分子类的优先顺序以用于进一步的临床开发和选择协同组合是重要的。2)将使用具有最大化学多样性的100,000个类药物小分子的集合进行额外的HTS检测，以确定高效的校正子。HITS将通过药物化学进行优化，并通过生化和电生理测试进行验证。随后将进行一系列二次和三次筛选，以确定进一步开发的潜在先导化合物的优先顺序：对人类呼吸道上皮细胞原代和永生化培养的功效研究，对啮齿动物的药理和功效分析。3)为了寻找发现小分子的新靶点，将进行纠正deltaF508-CFTR加工缺陷的功能遗传学。通过siRNA敲除筛选全基因组功能丧失(LOF)和通过cDNA转导筛选功能增强(GOF)将使用生化/功能性HTS分析。所提出的研究将有助于了解小分子校正剂的作用机理，并为开发新的先导化合物和发现小分子化合物提供新的靶点。

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is the most common lethal pediatric genetic disease in the Caucasian population. CF is caused by mutations in the CF gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel that belongs to the ATP-binding cassette family. A large number of mutations, including the most common deletion of phenylalanine 508 (deltaF508), cause folding and processing defect of CFTR. Using a functional high-throughput screening (HTS) assay, we have identified four classes of small molecules ('correctors') that partially revert the processing defect in transfected non-polarized and polarized cells, as well as in primary cultures of human airway epithelia. Our long-term goal is to identify clinically useful small molecules for CF therapy. Toward this goal we have three specific aims. 1) A comprehensive cell biological and biochemical analysis will establish the molecular mechanisms of deltaF508-CFTR corrector action. Potential sites of action to be analyzed include: translation, posttranslational folding, chaperone interactions, ER stability, cell surface stability, internalization and recycling efficiency and ubiquitination at the ER and post-Golgi compartments. Understanding the corrector mechanism is important to prioritize small molecule classes for further clinical development and in selecting synergistic combinations. 2) Additional HTS assays will be performed using a collection of >100,000 drug-like small molecules with maximum chemical diversity to identify highly efficient correctors. Hits will be optimized by medicinal chemistry and validated by biochemical and electrophysiological assays. A series of secondary and tertiary screens will be subsequently carried out to prioritize potential lead compounds for further development: efficacy studies on primary and immortalized cultures of human airway epithelia, pharmacological and efficacy analysis in rodents. 3) To identify novel targets for small molecule discovery, functional genetics for correction of the deltaF508-CFTR processing defect will be perfromed. Genome-wide loss-of-function (LOF) screens by siRNA knock-down and gain-of function (GOF) screens by cDNA transduction will be carried out using the biochemical/functional HTS assays. The proposed studies will provide an understanding of the small molecule correctors mechanism, as well as provide new classes of lead compounds for development and novel targets for small-molecule discovery in CF.