Molecular fingerprinting of GPCR ligands in Cancer

癌症中 GPCR 配体的分子指纹图谱

• 批准号: 6861075
• 负责人: LAWRENCE S. BARAK
• 金额: $ 7.7万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6861075
• 关键词: arrestins; biomarker; biosensor device; biotechnology; cell line; cell surface receptors; diagnosis design /evaluation; early diagnosis; genetically modified animals; high throughput technology; laboratory mouse; ligands; microcapsule; neoplasm /cancer diagnosis; polystyrenes; proteomics; transfection

DESCRIPTION (provided by applicant): Cancer remains an endemic problem in this country extracting a toll on both the quality of life and the economy. Short of outright prevention, the early detection of malignancies through the development of novel biological and analytical techniques potentially provides an optimal approach for saving life and healthcare costs. Different cancers possess, respond to, or secrete distinct molecular markers and may presumably be identified by these molecules which provide molecular signatures for their identification. G protein-coupled receptors are the most common targets of pharmacological therapy and regulate many critical physiological processes including cell growth and differentiation. Endogenous GPCR ligands have been proposed as tumor biomarkers but generalized high throughput assays to screen biological samples for GPCR ligands do not exist. The ability to analyze a small biological sample simultaneously for all known GPCR ligands could provide a novel high-throughput proteomic-based detection technique for screening known biomarkers and identifying or discovering new ones. In particular if the detection system utilizes the receptors themselves as sensors, this would provide a validated screening platform of great sensitivity and specificity. The goal of this proposal is to provide the biological arm for such a high-throughput system that will utilize fluorescence imaging as readout of sensor bioactivity. The specific aims are: (1) Validate the use of polystyrene microspheres as both a growth and analysis platform for the biosensors. (2) Validate the use of U2OS cells containing fluorescent arrestins, distinct GPCRs, and quantum microdots as barcoded biosensors. (3) Demonstrate that biosensors can be mutiplexed such that potentially upwards of 100 distinct GPCR biosensors can be used to analyze drop-sized volumes of 100 microliters or less. This type of platform for biological analysis of GPCR ligands would have utility for either basic research and clinical screening. Moreover, automated imaging systems for the analysis of the biosensors are currently available and their throughput rate for identifying GPCR ligands useful in cancer therapy could be increased upwards of 100 fold.

描述（由申请人提供）： 在这个国家，癌症仍然是一个流行的问题，对生活质量和经济的质量都造成了损失。除了完全预防的情况下，通过开发新型的生物学和分析技术对恶性肿瘤进行了早期检测，这可能为节省生命和医疗保健成本提供了最佳的方法。不同的癌症具有，响应或分泌不同的分子标记物，并且可能会通过这些分子为其鉴定提供分子特征来识别。 G蛋白偶联受体是药理治疗的最常见靶标，并调节许多关键的生理过程，包括细胞生长和分化。已经提出内源性GPCR配体为肿瘤生物标志物，但不存在筛选GPCR配体的生物样品的广义高通量测定法。同时分析所有已知GPCR配体的小型生物样品的能力可以提供一种新型的高通量基于蛋白质组学的检测技术，用于筛选已知的生物标志物并识别或发现新的生物标志物。特别是，如果检测系统利用受体本身作为传感器，则将提供一个良好灵敏度和特异性的验证筛选平台。该提案的目的是为这种高通量系统提供生物臂，该系统将利用荧光成像作为传感器生物活性的读数。具体目的是：（1）验证使用聚苯乙烯微球作为生物传感器的生长和分析平台。 （2）验证使用含有荧光抑制蛋白，独特的GPCR和量子微量点作为条形码生物传感器的U2OS细胞。 （3）证明生物传感器可以被静止，以便可以使用100个不同的GPCR生物传感器来分析100微升或更少的滴量体积。这种用于GPCR配体生物学分析的平台将具有基础研究和临床筛查的实用性。此外，目前可以使用用于分析生物传感器的自动成像系统，并且它们用于识别有用的GPCR配体的吞吐量可以增加100倍。