HTS for Detection of deltaF508 CFTR at the Cell Surface

描述（由申请人提供）： 过去二十年来，在囊性纤维化的遗传学、生理学和生物化学方面取得了重大进展。然而，到目前为止，这些信息还没有被充分利用，以提供新的分子疗法，使患者受益。虽然CFTR基因和疾病相关突变的鉴定已经使基于DNA的筛查成为可能，这是现在的护理标准，但基因替代疗法的进展并没有像预期的那样迅速。因此，需要开发新治疗的替代方法。尽管该疾病有许多复杂性和挑战性方面，但至少有一个特征提供了在分子水平上操纵的机会。大约90%的患者有一个突变，deltaF508，允许合成一个潜在的功能CFTR蛋白。通过内质网质量控制将蛋白质检测为异常，并阻止其运输到上皮细胞的顶端质膜，在那里需要其氯离子通道活性。然而，这种突变是温度敏感的，其影响可以在降低的温度下生长的细胞中或通过蛋白质稳定剂如一些两性电解质来规避。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，这已经被另外两个小组采用，这两个小组已经开始了高通量筛选（HTS）的努力，使用检测膜电位或氯离子浓度变化的荧光探针间接测量deltaF508 CFTR氯离子通道活性的恢复。这些测定是有用的，并且已经鉴定了CFTR通道活性的新调节剂，但还没有克服AF 508错误加工的试剂。然而，除了突变体CFTR成熟之外的许多变化可以在这些间接读数中给出信号（假阳性）。因此，我们已经开始开发HTS，其通过将外源表位插入CFTR的修饰的胞质外环中而不干扰其合成、糖基化或功能来直接测量deltaF508蛋白在细胞表面的外观和稳定性。我们在该提案中的具体目标是进一步开发和优化该检测方法，作为一种高灵敏度的基于发光细胞的HTS，以提供严格的验证检测方法，并与分子库筛选中心合作，开始筛选不同的化合物库。

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.