Tissue Lysate Arrays for Molecular Screening (RMI)

描述（由申请人提供）：分子疗法针对导致癌症发生和进展的信号通路中的潜在缺陷，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用纯化酶或表型分析分析一个或最多两个标准。许多这些抑制剂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强大特性。迫切需要基于功能的分析来优先考虑和验证候选靶向试剂。我们提出研究“组织裂解物阵列作为一种高通量的分子筛选方法”。该方法可以快速评估完整细胞中100多种功能蛋白质组学、通路和网络的不同特征，极大地有助于化学基因组学分子的选择或药物开发先导化合物的选择。该分析是基于在严格条件下裂解药物处理细胞，然后在固体基质上排列。然后，可以用抗体对检测基质的激活状态和蛋白质的总量。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，以便快速评估目标活动。我们提出：具体目标1)开发可行且稳健的方法，使组织裂解物阵列适应96孔格式，这将用于筛选细胞对药理学标准的反应。目的2)开发多种技术以提高组织裂解物阵列的成本、效率和稳健性。目的3)开发和验证适合组织裂解物阵列高通量筛选的细胞系。目的4)通过筛选分子与已知功能药物的组合，验证该技术在合成致死性分析中的应用。

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.