Tissue Lysate Arrays for Molecular Screening (RMI)

用于分子筛选 (RMI) 的组织裂解物阵列

• 批准号: 6879791
• 负责人: YILING LU
• 金额: $ 7.55万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879791
• 关键词: antibody; antineoplastics; biological signal transduction; biotechnology; breast neoplasms; cell line; chemical genetics; drug discovery /isolation; enzyme inhibitors; fluorescent dye /probe; high throughput technology; immunofluorescence technique; neoplasm /cancer chemotherapy; ovary neoplasms; phosphatidylinositol 3 kinase; phosphorylation; proteomics; small molecule

DESCRIPTION (provided by applicant): Molecular therapeutics targeting the underlying defects in signaling pathways that contribute to cancer initiation and progression is a rapidly emerging avenue for patient therapy. The success of Imatinib Mesylate (Gleevec) has galvanized the field to identify and implement additional targeted therapeutics. A large number of inhibitors against specific kinases are currently in development. Most screening approaches analyze one or at most two criteria usually with purified enzymes or with a phenotypic assay. Many of these inhibitors demonstrated unexpected effects in intact cells, which reflect off target responses and the robust characteristics of signaling networks. There is an urgent need for function-based assays to prioritize and validate candidate targeting reagents. We propose the study of "Tissue lysate array as a high throughput assay for molecular screening". The assay can rapidly assess over 100 different characteristics of functional proteomics, pathways and networks in intact cells, which greatly assists in selection of molecules for chemical genomics or lead compounds for pharmaceutical development. The assay is based on lysis of drug treated cells under stringent conditions followed by arraying on a solid matrix. The matrix can then be probed with pairs of antibodies identifying activation state and total amount of the protein. Based on this technology, we are able to gather multiple information from each compound and integrated into a "fingerprint" database to allow rapid assessment of on and off target activity. We propose: Specific Aim 1) To develop feasible and robust approaches to adapt tissue lysate arrays to a 96-well format, which will be applied to screen cellular responses to pharmacological standard. Aim 2) To develop multiplex technology to improve the cost, efficacy and robustness of the tissue lysate array. Aim 3) To develop and validate cell lines appropriate for high throughput screening by tissue lysate array. Aim 4) To validate the technology in a synthetic lethality analysis by combinations of screening molecule with drug of known function.

描述（由申请人提供）：靶向导致癌症发生和进展的信号通路中的潜在缺陷的分子治疗是一种快速出现的患者治疗途径。 甲磺酸伊马替尼（格列卫）的成功激发了该领域的识别和实施其他靶向治疗。 目前正在开发大量针对特定激酶的抑制剂。 大多数筛选方法通常使用纯化的酶或表型测定来分析一个或至多两个标准。 这些抑制剂中的许多在完整细胞中表现出意想不到的作用，这反映了脱靶反应和信号网络的稳健特征。 迫切需要基于功能的测定来优先考虑和验证候选靶向试剂。 我们提出了“组织裂解物芯片作为高通量分子筛选方法”的研究。 该分析可以快速评估完整细胞中功能蛋白质组学、途径和网络的100多种不同特征，这极大地有助于选择化学基因组学分子或药物开发的先导化合物。 该测定基于在严格条件下裂解药物处理的细胞，然后在固体基质上排列。 然后可以用识别蛋白质的活化状态和总量的抗体对探测基质。 基于这项技术，我们能够从每种化合物中收集多种信息，并将其整合到“指纹”数据库中，以快速评估靶向和非靶向活性。 我们建议：具体目的1）开发可行且稳健的方法以使组织裂解物阵列适应96孔格式，其将被应用于筛选对药理学标准的细胞反应。 目的2）发展多重检测技术，以提高组织裂解物芯片的成本、效率和稳定性。 目的3）建立和验证适合组织裂解物芯片高通量筛选的细胞系。 目的4）将筛选分子与已知功能的药物结合，验证该技术在综合致死率分析中的应用。