High Throughput Assay for Detecting Protein Modifications in Cell Lysates

用于检测细胞裂解物中蛋白质修饰的高通量测定

基本信息

批准号：
8124311
负责人：
Robert G Lowery
金额：
$ 27.24万
依托单位：
BELLBROOK LABS, LLC
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-26 至 2013-09-25
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8124311
关键词：
Affect Amino Acids Antibodies Antigen-Antibody Complex Antigens Area Binding Biochemical Biological Assay Cell Extracts Cell physiology Cells Cellular Assay Complex Cytolysis Detection Disease Energy Transfer Environment Enzymes Epidermal Growth Factor Receptor Epitope Mapping Event Fluorescence Generations Generic Drugs Immunoblotting In Vitro Internet Label Lanthanoid Series Elements Liquid substance Malignant Neoplasms Maps Measures Methods Methylation Methyltransferase Modeling Modification Noise Pathology Pathway interactions Peptide Phage Display Library Peptides Pharmaceutical Preparations Phase Phospho-Specific Antibodies Phosphopeptides Phosphoproteins Phosphorylation Phosphorylation Site Phosphotransferases Physiological Play Post-Translational Modification Site Post-Translational Protein Processing Proteins Proteomics Protocols documentation Reaction Research Personnel Role Screening procedure Signal Transduction Site Solid Specificity Therapeutic Time Tissue Sample Tissues Western Blotting Work assay development base drug discovery enzyme activity gel electrophoresis glycosylation high throughput screening immunocytochemistry improved inhibitor/antagonist mutant prevent tool

项目摘要

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.

描述（由申请人提供）：磷酸化、糖基化和甲基化等翻译后修饰（PTM）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（例如激酶和甲基转移酶）是密集药物发现的目标几乎每个治疗领域都在努力，其中最关注的是癌症。细胞提取物中翻译后修饰 (PTM) 的特异性检测是该领域药物发现和蛋白质组学研究人员面临的基本技术挑战。尽管有许多位点特异性抗体可用，但它们通常没有足够的选择性来区分密切相关的 PTM 位点，从而导致结果不可靠。此外，用于检测 PTM 的免疫细胞化学或蛋白质印迹方法很难以自动化、高通量的方式进行，这阻碍了它们通过高通量筛选 (HTS) 来识别潜在药物分子。为了克服这些技术障碍，我们建议开发一个通用平台，利用大量可用的 PTM 抗体，快速、体外开发高度特异性、同质的 PTM 检测检测方法。第一阶段的可行性将包括：a) 使用噬菌体展示文库中专门识别抗体-磷蛋白复合物的肽来提高 PTM 免疫检测方法的特异性，b) 使用称为淬灭共振能量转移的新单标记方法展示三价免疫复合物的同质检测。在第二阶段，BellBrook 将为许多治疗上最相关的激酶和甲基转移酶途径开发“高通量-PTM”检测组合，并将其商业化为具有简单裂解和检测格式的 HTS 细胞检测试剂盒。这些产品可以使用目标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，从而对癌症和其他疾病的药物发现产生重大影响。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功能来改进蛋白质组学方法，例如 PTM 的组织化学检测，从而加快在健康和患病组织中绘制 PTM 的工作。公共健康相关性：细胞中大多数蛋白质的功能是通过共价修饰来调节的，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识别，以防止特定的异常蛋白质修饰而不影响正常蛋白质修饰。