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.

激酶抑制剂开创了化疗的新范例，是新肿瘤药物的主要焦点 发育，但发育成功率较低（5-10%）。对激酶抑制剂的耐药性通过以下方式发生： 靶点依赖性机制（例如消除药物结合的点突变）和靶点无关机制 机制（例如替代信号通路的上调，称为“激酶组重编程”）。 因此，需要同时靶向多种激酶的抑制剂组合才能有更好的机会 避免耐药/复发循环。检测活细胞内的蛋白激酶活性（而不是裂解液中的活性） 或重构系统）对于了解激酶抑制剂药物敏感性和耐药性很重要 机制，并将导致更好地筛选可能通过开发的抑制剂 过程。我们将开发针对特定激酶活性的多重、基于细胞的检测方法，这对 抑制剂反应和激酶组重编程，并用它们来检测患者的激酶激活谱 细胞和抑制剂筛选。使用时间分辨镧系元素检测生物传感器的磷酸化 发光测量，其中 Tb3+ 发射能量通过小分子直接测量 荧光团根据荧光团给出不同的发射颜色。在目标 1 中，我们将建立 对药物敏感和耐药的关键激酶的活性（以及抑制作用）进行定量分析 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 我们已经在前期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 他们是发达的。该检测将使用 CML 患者的细胞系和样本进行（与 健康对照），并通过 RT-qPCR 和 SWATH”蛋白质组学进行验证。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（通过库）。我们还将开发 利用从稀土元素到稀土元素的能量转移对生物传感器磷酸化进行同质多重分析 有机染料。在目标 3 中，我们扩展了生物传感器设计流程，以开发新的非天然肽 用作其他激酶生物传感器的底物。该目标中描述的工作将添加到 我们已经有了生物传感器。完成本提案中描述的工作将为我们提供一个新颖的 多种激酶的测定、一套新的生物传感器以及新的和改进的检测策略用于 筛选。药物发现将受益于该技术解决激酶组重编程的能力 通过一次靶向多个激酶的机制。药物开发将受益于伴随检测 可以跟随药物或药物组合通过命中到先导转变的多个目标，目标 验证、临床前研究、临床试验等进入治疗管理。该测定及其 相关工具将通过打破 我们在药物筛选和开发过程中模拟疾病环境的能力有了新的突破。