Kinome-wide cell-based assays

全激酶组细胞分析

• 批准号: 8780886
• 负责人: REENA ZUTSHI
• 金额: $ 23.99万
• 依托单位: LUCEOME BIOTECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8780886
• 关键词: Active Sites; Adaptor Signaling Protein; Apoptosis; Architecture; Binding; Biochemical; Biological; Biological Assay; Cardiovascular Diseases; Catalytic Domain; Cell Cycle; Cell Proliferation; Cell division; Cell membrane; Cell physiology; Cells; Chemicals; Clinic; Communities; Complex; DHFR gene; Dasatinib; Data; Development; Diabetes Mellitus; Dihydrofolate Reductase; Dimerization; Disease; Drug Design; Early identification; Economics; Escherichia coli; Evaluation; Failure; Fingerprint; Genome; Goals; Human; Human Genome; Hybrids; In Vitro; Inflammation; Lead; Length; Libraries; Luciferases; Luminescent Measurements; Measures; Mediating; Mediator of activation protein; Methodology; Methods; Monitor; Neoplasm Metastasis; Normal Cell; Pathway interactions; Permeability; Pharmaceutical Preparations; Phase; Phosphorylation; Phosphotransferases; Physiology; Play; Property; Protein Kinase; Protein Tyrosine Kinase; Protein-Serine-Threonine Kinases; Reporting; Role; Scaffolding Protein; Serine; Services; Signal Transduction; Testing; Threonine; Toxic effect; Trees; Trimethoprim; Tyrosine; Work; base; cost; cross reactivity; design; drug candidate; drug development; drug discovery; effective therapy; genetic regulatory protein; high throughput screening; human disease; inhibitor/antagonist; kinase inhibitor; luminescence; meetings; neoplastic cell; public health relevance; screening; stem; success; therapeutic target; trafficking

DESCRIPTION (provided by applicant): Protein kinases serve as critical mediators of signal transduction in human cells and impact virtually all aspects of cellular physiology, from the coordination of the cell cycle and cell division to apoptosis. The deregulation of kinase mediated signaling is now known to be involved in a variety of human diseases that include, diabetes, inflammation, cardiovascular diseases, tumor cell proliferation and metastasis making them a target for drug development. Structurally, kinases share a very similar architecture at the active-site (ATP-binding domain), making selectivity an issue in drug discovery and development. Thus, the development of rapid and economic methods for profiling drug candidates against a large panel of kinases in whole cells will not only aid in anticipating potential long term toxicit, but also help in understanding kinase targeted polypharmacology and aid in identifying new targets for old compounds. In this Phase I application, we will develop a split-luciferase based luminescent assay for kinase profiling in a cellular setting. These cell-based assays will meet a critical need for directly monitoring the effect of compounds on a target kinase not only to ascertain cell-permeability and cellular toxicity, but also to establish efficacy in the cellular mlieu in the presence of adaptor and regulatory proteins. We will test the generality of the approach for kinases across various groups and evaluate the feasibility of our assay for high throughput screening using a panel of 80 known kinase inhibitors. Our goal is to make cellular kinase profiling assays both easily available and affordable, so that compound profiling in a cellular or native context can be done earlier and thereby lead to early identification of failures, resulting n many more opportunities for success.

描述（由申请人提供）：蛋白激酶作为人类细胞中信号转导的关键介质，影响细胞生理学的几乎所有方面，从细胞周期和细胞分裂的协调到细胞凋亡。现在已知激酶介导的信号传导的失调涉及多种人类疾病，包括糖尿病、炎症、心血管疾病、肿瘤细胞增殖和转移，使其成为药物开发的靶标。在结构上，激酶在活性位点（ATP结合结构域）具有非常相似的结构，使得选择性成为药物发现和开发中的一个问题。因此，开发快速和经济的方法来分析候选药物对整个细胞中的一大组激酶的作用，不仅有助于预测潜在的长期毒性，而且有助于理解激酶靶向的多药理学，并有助于识别旧化合物的新靶标。在第一阶段的申请中，我们将开发一种基于分裂荧光素酶的发光检测方法，用于细胞环境中的激酶分析。这些基于细胞的测定将满足直接监测化合物对靶激酶的作用的关键需求，不仅确定细胞渗透性和细胞毒性，而且确定在衔接蛋白和调节蛋白存在下在细胞内的功效。我们将测试跨不同群体的激酶的方法的一般性，并使用一组80种已知激酶抑制剂评估我们的高通量筛选测定的可行性。我们的目标是使细胞激酶谱分析既容易获得又负担得起，以便可以更早地在细胞或天然环境中进行化合物谱分析，从而导致早期识别失败，从而获得更多的成功机会。