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儿科遗传疾病。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camp调节的氯离子通道，属于atp结合盒家族。大量突变，包括最常见的苯丙氨酸508缺失（deltaF508），导致CFTR折叠和加工缺陷。使用功能性高通量筛选（HTS）试验，我们已经确定了四类小分子（“校正者”），这些小分子部分地恢复了转染的非极化和极化细胞以及人气道上皮原代培养中的加工缺陷。我们的长期目标是确定临床上有用的CF治疗小分子。为了实现这一目标，我们有三个具体目标。1)通过全面的细胞生物学和生化分析，建立deltaF508-CFTR纠偏作用的分子机制。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、内质网稳定性、细胞表面稳定性、内化和循环效率以及内质网和后高尔基区室的泛素化。了解校正机制对于进一步临床开发和选择协同组合优先考虑小分子类别非常重要。2)额外的HTS分析将使用10万个具有最大化学多样性的药物样小分子进行，以确定高效的校正剂。将通过药物化学优化hit，并通过生化和电生理试验进行验证。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，以进一步开发：对人气道上皮原代和永生化培养物的功效研究，啮齿动物的药理和功效分析。3)为了确定小分子发现的新靶点，将进行功能遗传学以纠正deltaF508-CFTR加工缺陷。通过siRNA敲除的全基因组功能缺失（LOF）筛选和cDNA转导的功能获得（GOF）筛选将使用生化/功能HTS分析进行。这些研究将有助于了解小分子校正机制，并为CF的小分子发现提供新的先导化合物类别和新的靶点。

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.