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儿科遗传性疾病。CF是由编码囊性纤维化跨膜传导调节因子（CFTR）的CF基因突变引起的，CFTR是一种cAMP调节的氯离子通道，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）的缺失，导致CFTR的折叠和加工缺陷。使用功能性高通量筛选（HTS）测定，我们已经鉴定了四类小分子（"校正物"），其部分地逆转转染的非极化和极化细胞以及人气道上皮的原代培养物中的加工缺陷。我们的长期目标是确定用于CF治疗的临床有用的小分子。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物化学分析将建立deltaF508-CFTR校正剂作用的分子机制。待分析的潜在作用位点包括：翻译，翻译后折叠，伴侣相互作用，ER稳定性，细胞表面稳定性，内化和再循环效率以及ER和高尔基体后区室的泛素化。了解校正剂机制对于优先考虑小分子类别用于进一步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种药物样小分子的集合进行额外的HTS测定，以鉴定高效的校正剂。命中将通过药物化学进行优化，并通过生物化学和电生理测定进行验证。随后将进行一系列的二级和三级筛选，以优先考虑潜在的先导化合物进行进一步开发：对人气道上皮细胞的原代和永生化培养物的疗效研究，啮齿动物的药理学和疗效分析。3)为了鉴定小分子发现的新靶点，将进行用于校正deltaF508-CFTR加工缺陷的功能遗传学。将使用生物化学/功能HTS试验，通过siRNA敲低进行全基因组功能丧失（LOF）筛选，并通过cDNA转导进行功能获得（GOF）筛选。拟议的研究将提供对小分子校正剂机制的理解，并为CF中的小分子发现提供新的先导化合物和新的靶点。

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.