High Throughput Assay for Detecting Protein Modifications in Cell Lysates

描述（由申请人提供）：翻译后修饰（PTM），如磷酸化、糖基化和甲基化，在细胞信号转导中发挥着核心和普遍的作用，催化这些反应的酶，如激酶和甲基转移酶，是几乎所有治疗领域的密集药物发现努力的目标，其中最集中的是癌症。特异性检测细胞提取物中的翻译后修饰（PTM）是药物发现和蛋白质组学研究人员面临的一个基本技术挑战。虽然许多位点特异性抗体是可用的，但它们通常没有足够的选择性来区分密切相关的PTM位点，导致不可靠的结果。此外，用于检测PTM的免疫细胞化学或蛋白质印迹方法难以以自动化、高通量方式进行，这阻碍了它们用于使用高通量筛选（HTS）鉴定潜在药物分子。为了克服这些技术障碍，我们建议开发一种通用平台，用于快速、体外开发用于高度特异性、同质检测PTM的测定法，该测定法利用了大量可用的PTM抗体。I期可行性将包括：a）使用来自噬菌体展示文库的肽增加PTM免疫检测方法的特异性，所述噬菌体展示文库特异性识别抗体-磷蛋白复合物，和B）使用称为淬灭共振能量转移的新的单标记方法证明三价免疫复合物的均相检测。在第二阶段，贝尔布鲁克将为许多最具治疗相关性的激酶和甲基转移酶途径开发“高还原-PTM”检测试剂盒，并将其作为具有简单裂解和检测格式的HTS细胞检测试剂盒进行商业化。这些产品可能对癌症和其他疾病的药物发现产生重大影响，通过使用靶酶活性的最直接终点（特定PTM）在完整细胞的生理环境中大规模筛选激酶和甲基转移酶抑制剂。此外，我们还将研究如何将高重复率-PTM平台的特异性和检测增强应用于改进蛋白质组学方法，例如PTM的组织化学检测，从而加快在健康和患病组织中绘制PTM的努力。 公共卫生相关性：细胞中大多数蛋白质的功能是由共价修饰调节的，并且异常修饰是许多疾病病理学的基础，特别是癌症。我们建议开发检测方法，以加速药物分子的识别，防止特定的异常蛋白质修饰，而不影响正常的。

DESCRIPTION (provided by applicant): Post translational modifications (PTMs) such as phosphorylation, glycosylation and methylation play a central and ubiquitous role in cellular signal transduction, and the enzymes that catalyze these reactions such as kinases and methyltransferases are the targets of intense drug discovery efforts for virtually every therapeutic area, with the most intense focus on cancer. Specific detection of posttranslational modifications (PTMs) in cell extracts is a fundamental technical challenge facing drug discovery and proteomics researchers working in this area. Though many site-specific antibodies are available, they often are not selective enough to differentiate between closely related PTM sites, leading to unreliable results. Moreover, the immunocytochemical or Western blot methods used to detect PTMs are difficult to perform in an automated, high throughput fashion, which discourages their use for identifying potential drug molecules using high throughput screening (HTS). To overcome these technical barriers, we propose to develop a generic platform for rapid, in vitro development of assays for highly specific, homogenous detection of PTMs that leverages the extensive pool of available PTM antibodies. Phase I feasibility will include: a) increasing the specificity of PTM immunodetection methods using peptides from phage display libraries that specifically recognize antibody-phosphoprotein complexes and b) demonstrating homogenous detection of the trivalent immune complex using a new single label method called Quenched Resonance Energy Transfer. In Phase II, BellBrook will develop panels of "High Throughput-PTM" assays for many of the most therapeutically relevant kinase and methyltransferase pathways and commercialize them as HTS cellular assay kits with a simple lyse-and-detect format. These products could have a significant impact on drug discovery for cancer and other diseases by enabling large scale screening for kinase and methyltransferase inhibitors in the physiological context of intact cells using the most immediate endpoint of target enzyme activity: a specific PTM. Additionally, we will investigate how the specificity and detection enhancements of the High Throughput-PTM platform can be applied to improve proteomic methods, such as histochemical detection of PTMs, and thus accelerate efforts to map PTMs in healthy and diseased tissues. PUBLIC HEALTH RELEVANCE: The function of most proteins in the cell is regulated by covalent modifications, and aberrant modifications underlie many disease pathologies, especially cancer. We propose to develop detection methods to accelerate the identification of drug molecules that prevent specific aberrant protein modifications without affecting normal ones.