High Throughput Assay for Detecting Protein Modifications in Cell Lysates

描述(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，而催化这些反应的酶，如激酶和甲基转移酶，几乎是每个治疗领域密集药物发现工作的目标，最关注的是癌症。细胞提取物中翻译后修饰(PTM)的特异性检测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。虽然有许多部位特异性抗体可用，但它们往往没有足够的选择性来区分密切相关的PTM部位，导致结果不可靠。此外，用于检测PTM的免疫细胞化学或Western印迹方法很难以自动化、高通量的方式执行，这阻碍了它们用于使用高通量筛选(HTS)来识别潜在的药物分子。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、体外开发高特异性、同质性的PTM检测方法，利用广泛的PTM抗体库。第一阶段的可行性将包括：a)使用噬菌体展示文库中识别抗体-磷蛋白复合体的多肽来提高PTM免疫检测方法的特异性，以及b)使用一种名为猝灭共振能量转移的新的单标记法展示对三价免疫复合体的均一检测。在第二阶段，BellBrook将为许多与治疗最相关的激酶和甲基转移酶途径开发“高通量-PTM”分析小组，并将其商业化为HTS细胞分析试剂盒，具有简单的裂解和检测格式。这些产品可能会对癌症和其他疾病的药物开发产生重大影响，因为它们能够使用靶酶活性的最直接终点：特定的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，我们将研究如何应用高通量-PTM平台的特异性和检测增强来改进蛋白质组学方法，例如PTM的组织化学检测，从而加快在健康和疾病组织中定位PTM的努力。 与公共健康相关：细胞中大多数蛋白质的功能受共价修饰的调节，而异常修饰是许多疾病病理的基础，尤其是癌症。我们建议开发检测方法来加速药物分子的鉴定，以防止特定的异常蛋白修饰而不影响正常的修饰。

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.