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: 7251883
• 负责人: JOHN R RIORDAN
• 金额: $ 24.23万
• 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 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7251883
• 关键词: Ampholytes; Apical; Appearance; Biochemistry; Biological Assay; Caring; Cell membrane; Cell surface; Cells; Chloride Channels; Chloride Ion; Chlorides; Collaborations; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Detection; Development; Disease; Endoplasmic Reticulum; Epithelial Cells; Fluorescent Probes; Genes; Genetic; Lead; Libraries; Measures; Membrane Potentials; Molecular; Molecular Bank; Mutation; Patients; Pharmaceutical Preparations; Physiology; Proteins; Quality Control; Replacement Therapy; Screening procedure; Signal Transduction; Stabilizing Agents; Standards of Weights and Measures; Temperature; Validation; base; cystic fibrosis patients; deltaF508-CFTR protein; gene replacement; glycosylation; high throughput screening; improved; luminescence; mutant; prevent; restoration; scaffold; small molecule; trafficking

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）： 过去的二十年来，在理解囊性纤维化的遗传学，生理和生物化学方面已提供了重大进步。但是，目前尚未完全利用此信息来提供受益于患者的新分子疗法。尽管CFTR基因和疾病相关的突变的鉴定已成为基于DNA的筛查，这是现在的护理标准，但基因置换疗法的进展尚未像预期的那样快速进行。因此，需要采用新疗法的替代方法。尽管该疾病有许多复杂性和挑战性的方面，但至少有一个特征为分子水平的操纵提供了机会。大约90％的患者具有突变的Deltaf508，该突变允许合成潜在的功能性CFTR蛋白。通过内质网质量控制检测到该蛋白质异常，并阻止了需要其氯化物通道活性的上皮细胞的顶端质膜。但是，该突变是温度敏感的，并且可以通过在降低温度或蛋白质稳定剂（例如某些两性溶解剂）生长的细胞中绕过其作用。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，这些策略已经被其他两个组采取了高吞吐量筛选（HTS）的努力，该测定方法是通过间接测量Deltaf508 CFTR氯化物通道活性的荧光探针的荧光探针的恢复，从而感觉到膜潜力或氯化物浓度的变化。这些测定很有用，并且已经确定了CFTR通道活动的新调节剂，但尚未克服AF508错误处理的代理。但是，除突变体CFTR成熟以外的许多变化都可以在这些间接读数（假阳性）中产生信号。因此，我们通过将外源性表现因子插入CFTR的修饰外质质环中，而无需扰动其合成，糖基化或功能，从而直接开发了HTS的HTS，该HTS直接测量了Deltaf508蛋白在细胞表面的外观和稳定性。我们在本提案中的具体目标是进一步开发并优化该测定法作为高度敏感的发光基于细胞的HTS，以提供严格的验证测定法，并与分子库筛选中心合作筛选各种化合物库。

项目成果

Sedentary Patterns Are Associated with Bone Mineral Density and Physical Function in Older Adults: Cross-Sectional and Prospective Data.

• DOI: 10.3390/ijerph17218198
• 发表时间: 2020-11-06
• 期刊: International journal of environmental research and public health
• 作者: Gobbo LA;Júdice PB;Hetherington-Rauth M;Sardinha LB;Dos Santos VR
• 通讯作者: Dos Santos VR

COVID-19 pandemic impacts physical activity levels and sedentary time but not sleep quality in young badminton athletes.

• DOI: 10.1007/s11332-021-00763-6
• 发表时间: 2021
• 期刊: Sport sciences for health
• 影响因子: 1.5
• 作者: da Silva Santos AM;Rossi FE;Dos Santos Nunes de Moura HP;de Sousa Junior AVM;Machado DCD;Neves LM;Brito AS;Moura P;Monteiro PA;Freitas Junior IF;Dos Santos MAP;Ribeiro SLG
• 通讯作者: Ribeiro SLG