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.

激酶抑制剂在化学疗法方面产生了新的范式，是新肿瘤药物的主要重点 发展，但发展成功率较低（5-10％）。对激酶抑制剂的抗性通过 靶依赖机制（例如废除药物结合的点突变）和靶非依赖性 机理（例如，替代信号通路的上调，称为“ Kinome重新编程”）。 因此，希望立即靶向几种激酶的抑制剂的组合是有更好的机会 避免阻力/复发周期。检测活细胞内蛋白激酶活性（而不是裂解物 或重构系统）对于理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抑制剂 过程。我们将开发针对特定激酶活性的多路复用，基于细胞的测定 抑制剂反应和Kinome重新编程，并使用它们检测患者的激酶激活谱 细胞和抑制剂筛选。使用时间分辨的灯笼检测到生物传感器的磷酸化 发光测量值，其中TB3+发射能直接通过小分子测量 根据荧光团的不同，荧光团具有不同的发射颜色。在AIM 1中，我们将建立 对药物敏感和抗药性的关键激酶的活性（以及抑制）的定量测​​定 CML细胞，分析治疗相关的细胞状态中的途径激活表型。我们将使用 我们已经在初步工作中已经建立的一组生物传感器，并为其他激酶添加生物传感器 它们是开发的。该测定将使用CML患者的细胞系和样品建立（相比 健康控制），并用RT-QPCR和Swath“蛋白质组学。在AIM 2中，我们将筛选 现有激酶抑制剂药物与新化合物（通过库）之间的协同作用。我们还将发展 使用从灯笼到的能量转移对生物传感器磷酸化的同质多路复用分析 有机染料。在AIM 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 用作其他激酶的生物传感器的底物。此目标中描述的工作将增加 生物传感器我们已经有可用。本提案中描述的工作的完成将为我们提供一本小说 多种激酶，一套新的生物传感器以及新的和精制的检测策略的测定 筛选。药物发现将受益于这项技术解决Kinome重新编程的能力 一次靶向几个激酶来实现机制。药物开发将受益于同伴测定 可以通过命中率进行过渡的多个目标，可以跟随药物或药物组合，目标 验证，临床前研究，临床试验以及治疗管理。这个测定及其 相关工具将通过破坏癌症的下一代有针对性的治疗发展 我们在药物筛查和发育过程中对疾病环境建模的能力的新基础。