HTS for Detection of deltaF508 CFTR at the Cell Surface

用于检测细胞表面 deltaF508 CFTR 的 HTS

• 批准号: 6912479
• 负责人: JOHN R RIORDAN
• 金额: $ 25.55万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6912479
• 关键词: biotechnology; cell line; cell membrane; chloride channels; cystic fibrosis; drug discovery /isolation; endoplasmic reticulum; gene mutation; high throughput technology; protein transport; small molecule; technology /technique development

DESCRIPTION (provided by applicant): The past two decades have provided major advances in understanding of the genetics, physiology and biochemistry of cystic fibrosis. However, as yet this information has not been fully exploited to provide new molecular therapies that benefit the patients. Although the identification of the CFTR gene and disease associated mutations has enabled DNA-based screening which is now the standard of care, progress towards gene replacement therapy has not proceeded as rapidly as anticipated. Therefore alternative approaches to the development of new treatments are required. Despite the many complexities and challenging aspects of the disease there is at least one feature that provides an opportunity for manipulation at the molecular level. Approximately 90% of patients have a mutation, deltaF508 that allows a potentially functional CFTR protein to be synthesized. The protein is detected as abnormal by endoplasmic reticulum quality control and prevented from trafficking to the apical plasma membrane of epithelial cells where its chloride channel activity is required. However, this mutation is temperature sensitive and its effects can be circumvented in cells grown at reduced temperature or by protein stabilizing agents such as some ampholytes. Hence the search for small molecule drugs that could have these effects becomes an attractive strategy which already has been taken by two other groups that have initiated high throughput screening (HTS) efforts using assays that indirectly measure restoration of deltaF508 CFTR chloride channel activity with fluorescent probes that sense changes in membrane potential or chloride concentration. These assays are useful and one has already identified new modulators of CFTR channel activity but not yet agents that overcome AF508 misprocessing. However many changes other than mutant CFTR maturation can give signals in these indirect readouts (false positives). Therefore we have initiated development of an HTS that directly measures the appearance and stability of the deltaF508 protein at the cell surface by insertion of an exogenous epitome into a modified extracytoplasmic loop of CFTR without perturbing its synthesis, glycosylation or function. Our specific objectives in this proposal are to further develop and optimize this assay as a highly sensitive luminescence cell-based HTS to provide stringent validation assays and to initiate screening of a diverse library of compounds in collaboration with a Molecular Libraries Screening Center.

描述(由申请人提供)： 在过去的二十年里，对囊性纤维化的遗传学、生理学和生物化学的理解取得了重大进展。然而，到目前为止，这些信息还没有被充分利用来提供使患者受益的新的分子疗法。尽管cftr基因和疾病相关突变的识别使基于DNA的筛查成为现在护理的标准，但基因替代治疗的进展并没有像预期的那样迅速。因此，开发新疗法的替代方法是必要的。尽管这种疾病有许多复杂和具有挑战性的方面，但至少有一个特征为在分子水平上进行操作提供了机会。大约90%的患者有一个突变，deltaF508，它允许合成一个潜在的功能性CFTR蛋白。内质网质量控制检测到该蛋白异常，并阻止其转运到需要其氯通道活性的上皮细胞的顶端质膜。然而，这种突变是温度敏感的，它的影响可以在低温下生长的细胞中规避，或者通过一些蛋白质稳定剂，如一些两性离子来规避。因此，寻找可能具有这些作用的小分子药物成为一种有吸引力的策略，这一策略已经被另外两个小组采用，这两个小组已经启动了高通量筛选(HTS)工作，使用检测膜电位或氯浓度变化的荧光探针间接测量deltaF508 CFTR氯通道活性的恢复。这些分析是有用的，并且已经发现了CFTR通道活动的新的调节剂，但还没有克服AF508误处理的药物。然而，除了突变的CFTR成熟以外的许多变化都可以在这些间接读数中给出信号(假阳性)。因此，我们启动了HTS的开发，通过将外源表位插入到修改的CFTR胞外环路中，直接测量DeltaF508蛋白在细胞表面的外观和稳定性，而不干扰其合成、糖基化或功能。我们在这项提案中的具体目标是进一步开发和优化这一基于高灵敏发光单元的HTS，以提供严格的验证分析，并与分子文库筛选中心合作启动对多样化化合物文库的筛选。