Rescue of CFTR-deltaF508 misfolding by pharmacological and genetic suppressors

描述(申请人提供)：囊性纤维化是一种危及生命的遗传性疾病，其特征是体内分泌上皮细胞的电解质分泌改变。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是一种细胞表面定位的完整膜蛋白，起氯离子通道的作用。在北美近90%的囊性纤维化患者中发现的CF病等位基因包含一个三核苷酸缺失，该缺失消除了CFTR肽序列508位的苯丙氨酸。这种蛋白质cftr-deltaF508是一种构象突变，被细胞蛋白质质量控制系统识别，并在内质网相关的降解过程中通过泛素-蛋白酶体系统被消除。然而，CFTR-deltaF508在允许CFTR-deltaF508正确折叠并运输到质膜的条件下发挥氯通道作用的能力，例如在降低孵育温度(30℃)下，表明挽救CFTRdeltaF508折叠可能成为治疗囊性纤维化的可行靶点。由于缺乏适用于高通量筛选(HTS)平台的技术来直接测量细胞表面存在的CFTR-delta508，因此通过基因敲除研究识别与CFTR-deltaF508(药理伴侣)或参与CFTR-deltaF508加工的蛋白质相结合并稳定的小分子一直受到阻碍。为了克服这些限制，我们开发了一种快速和强大的基于细胞的系统，以直接检测错误折叠的CFTR-deltaF508对细胞表面的挽救，该系统结合了高亲和力配体结合、酶互补/信号放大和灵敏的基于FRET的荧光检测系统。该系统的能力在初步的HTS研究中得到了例证，在该研究中，我们发现了一种从植物提取物中提取的新的小分子，它可以挽救CFTRdeltaF508的错误折叠。因此，我们建议使用这一工具：1)评估先前发现的CFTR通道活性调节剂拯救CFTR-deltaF508错误折叠的能力；2)筛选一组天然产品提取物，寻找纠正CFTR-deltaF508折叠的潜在化合物；3)在人类细胞系中建立一个符合遗传学研究的系统，以确定CFTR-deltaF508 ER保留和降解的抑制子。这项提案的短期目标是更好地了解CFTR-deltaF508生物学，确定CFTR-deltaF508药理伴侣，并发现可以作为药物靶点的蛋白质，以拯救CFTR-deltaF508贩运。我们研究的广泛目标是发现可以转化为囊性纤维化新疗法的线索。

DESCRIPTION (provided by applicant): Cystic fibrosis is a life-threatening genetic disease characterized by the altered secretion of electrolytes by the secretory epithelial cells of the body. It is caused by mutations in the CF gene, which encodes the cystic fibrosis transmembrane conductance regulator (CFTR), a cell surface localized integral membrane protein that functions as chloride ion channel. The CF disease allele found in nearly 90% of cystic fibrosis patients in North America contains a trinucleotide deletion that removes the phenylalanine at position 508 of the CFTR peptide sequence. This protein, CFTR-deltaF508, is a conformational mutant that is recognized by the cellular protein quality control system and is eliminated via the ubiquitin-proteasome system in a process known as endoplasmic reticulum-associated degradation. However, the ability of CFTR-deltaF508 to function as a chloride channel under conditions that permit CFTR-deltaF508 to properly fold and traffic to the plasma membrane, such as at reduced incubation temperatures (<30¿C), indicate that the rescue of CFTRdeltaF508 folding may serve as a feasible target for the treatment of cystic fibrosis. The identification of small molecules that bind to and stabilize the native fold of CFTR-deltaF508 (pharmacological chaperones) or of proteins involved in CFTR-deltaF508 processing by gene-knockdown studies have been hindered by the lack of technology to directly measure CFTR-delta508 present on the cell surface that is suited to high throughput screening (HTS) platforms. To overcome these limitations we have developed a rapid and robust cell-based system to directly detect the rescue of misfolded CFTR-deltaF508 to the cell surface that combines high affinity ligand binding, enzymatic complementation/signal amplification and a sensitive FRET based fluorescence detection system. The capability of this system is exemplified in a preliminary HTS study, in which we identified a novel small molecule derived from plant extracts that rescues CFTRdeltaF508 misfolding. Therefore, we propose to use this tool to 1) assess the ability of previously identified modulators of CFTR channel activity to rescue CFTR-deltaF508 misfolding , 2) perform screen of a set of natural products extracts for potential compounds that correct the folding of CFTR-deltaF508 and 3) set up a system in human cell line amenable to genetic studies to identify suppressors of CFTR-deltaF508 ER retention and degradation. The short-term goals of this proposal are to provide a greater understanding of the CFTR-deltaF508 biology, to identify CFTR-deltaF508 pharmacological chaperones and to discover proteins that could serve as drug targets for the rescue of CFTR-deltaF508 trafficking. The broad objective of our studies is to uncover leads that could translate into new treatments for cystic fibrosis.