Biosensor Assay to Screen for Signaling Pathway Inhibition in Cancer

用于筛选癌症信号通路抑制的生物传感器测定

• 批准号: 8956420
• 负责人: Laurie L. Parker
• 金额: $ 17.39万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8956420
• 关键词: Acute Myelocytic Leukemia; Address; Antineoplastic Agents; Area; Award; Binding; Bioinformatics; Biological; Biological Assay; Biosensor; Cell Line; Cells; Chronic; Chronic Lymphocytic Leukemia; Clinic; Clinical; Color; Companions; Complex; Data; Detection; Development; Diagnostic; Disease; Disease model; Drug Combinations; Drug resistance; Dyes; Energy Transfer; Environment; Evaluation; Exhibits; Face; Failure; Family; Future; Goals; Hematologic Neoplasms; Imatinib; In Vitro; JAK2 gene; Lanthanoid Series Elements; Lead; Libraries; Life; Luminescent Measurements; Malignant Neoplasms; Measures; Methods; Monitor; Mutation; Myelogenous; Oncology; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Pharmacology; Phenotype; Phosphorylation; Phosphotransferases; Point Mutation; Preclinical Drug Evaluation; Process; Protein Kinase; Protein Kinase Inhibitors; Proteins; Proteomics; Protocols documentation; Relapse; Resistance; Sampling; Signal Pathway; Signal Transduction; Staging; System; Techniques; Technology; Testing; Time; Treatment Effectiveness; Treatment outcome; Up-Regulation; Validation; Work; base; chemotherapy; clinically relevant; design; drug action; drug development; drug discovery; drug sensitivity; drug-sensitive; fluorophore; human SYK protein; improved; in vivo; inhibitor/antagonist; insight; kinase inhibitor; knowledge base; leukemia; luminescence; next generation; novel; pre-clinical; preclinical study; prevent; protein kinase inhibitor; public health relevance; reconstitution; resistance mechanism; response; screening; sensor; small molecule; success; therapy development; time use; tool; tool development; ward

Kinase inhibitors created a new paradigm in chemotherapy and are a major focus of new oncology drug development, but developmental success rates are low (5-10%). Resistance to kinase inhibitors occurs through target-dependent mechanisms (e.g. point mutations that abrogate drug binding) and target-independent mechanisms (e.g. upregulation of alternative signaling pathways, termed "kinome reprogramming"). Therefore, combinations of inhibitors that target several kinases at once are desirable to have a better chance of avoiding the resistance/relapse cycle. Detecting protein kinase activity inside living cells (rather than in lysates or reconstituted systems) is important for understanding kinase inhibitor drug sensitivity and resistance mechanisms, and would lead to better screening for inhibitors likely to make it through the development process. We will develop multiplexed, cell-based assays for specific kinase activities that are important to inhibitor response and kinome reprogramming, and use them to detect kinase activation profiles in patient cells and for inhibitor screens. Phosphorylation of the biosensors is detected using time-resolved lanthanide luminescence measurements, in which Tb3+ emission energy is measured directly via small molecule fluorophores to give different emission colors depending on the fluorophore. In Aim 1, we will establish quantitative assays for activity (and therefore inhibition) of key kinases in drug sensitive and drug resistant CML cells, profiling the pathway activation phenotypes in therapeutically relevant cellular states. We will use the set of biosensors we have already established in preliminary work, and add biosensors for other kinases as they are developed. The assays will be established with cell lines and samples from CML patients (comparing to healthy controls), and validated with RT-qPCR and SWATH" proteomics. In Aim 2, we will screen for synergies between existing kinase inhibitor drugs and new compounds (via libraries). We will also develop homogenous multiplexed analysis of biosensor phosphorylation using energy transfer from lanthanides to organic dyes. In Aim 3, we expand the biosensor design pipeline to develop new, non-natural peptide substrates to use as biosensors for other kinases. The work described in this aim will add to the set of biosensors we already have available. Completion of the work described in this proposal will give us a novel assay for multiple kinases, a suite of new biosensors as well as new and refined detection strategies to use in screening. Drug discovery will benefit from this technology's ability to address kinome reprogramming mechanisms by targeting several kinases at a time. Drug development will benefit from companion assays for multiple targets that could follow a drug or drug combination through the hit to lead transition, target validation, pre-clinical studies, clinical trials, and beyond into treatment management. This assay and its associated tools will contribute to the next generation of targeted therapy development in cancer by breaking new ground in our ability to model the disease environment during drug screening and development.

激酶抑制剂在化疗中创造了一个新的范例，并且是新的肿瘤药物的主要焦点 发展，但发展的成功率很低（5-10%）。对激酶抑制剂的耐药性是通过 靶点依赖性机制（例如，消除药物结合的点突变）和靶点非依赖性机制 这些机制（例如上调替代信号传导途径，称为“激酶组重编程”）。 因此，期望同时靶向几种激酶的抑制剂的组合具有更好的机会。 避免耐药/复发循环。检测活细胞内的蛋白激酶活性（而不是裂解物中 或重组系统）对于理解激酶抑制剂药物敏感性和耐药性是重要的 机制，并将导致更好地筛选抑制剂可能使其通过发展 过程我们将开发针对特定激酶活性的多路复用的、基于细胞的检测方法， 抑制剂反应和激酶组重编程，并使用它们检测患者中激酶活化谱 细胞和抑制剂筛选。磷酸化的生物传感器检测使用时间分辨镧系元素 发光测量，其中Tb 3+发射能量通过小分子直接测量 荧光团，以根据荧光团给出不同的发射颜色。在目标1中，我们将 用于药物敏感和耐药细胞中关键激酶活性（以及因此抑制）的定量测定 CML细胞，分析治疗相关细胞状态下的通路激活表型。我们将使用 我们已经在初步工作中建立了一套生物传感器，并添加了其他激酶的生物传感器， 它们是发达的。将用来自CML患者的细胞系和样品建立测定法（与对照组相比）。 健康对照），并用RT-qPCR和SWATH蛋白质组学验证。在目标2中，我们将筛选 现有激酶抑制剂药物和新化合物之间的协同作用（通过文库）。我们还将开发 使用从镧系元素到 有机染料在目标3中，我们扩展了生物传感器设计管道，以开发新的非天然肽 底物用作其他激酶的生物传感器。这一目标中所述的工作将增加一套 我们已经有了生物传感器完成本建议书中所描述的工作将给我们带来一部小说。 多种激酶的测定，一套新的生物传感器以及新的和完善的检测策略，用于 筛选药物发现将受益于这项技术解决激酶组重编程的能力 一次靶向几种激酶的机制。药物开发将受益于伴随测定， 多个目标，可以遵循药物或药物组合，通过命中导致过渡，目标 验证、临床前研究、临床试验以及治疗管理。该测定及其 相关工具将有助于下一代癌症靶向治疗的发展， 我们在药物筛选和开发过程中模拟疾病环境的能力的新基础。