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式，是肿瘤新药的主要焦点 发展方面，但发展成功率很低(5-10%)。对激酶抑制剂的抗药性是通过 靶标依赖机制(例如，取消药物结合的点突变)和靶标非依赖机制 机制(例如，替代信号通路的上调，称为“动态组重新编程”)。 因此，一次靶向几种酶的抑制剂的组合是可取的，以有更好的机会 避免耐药/复发循环。检测活细胞内的蛋白激酶活性(而不是在裂解物中 或重组系统)对于了解激酶抑制剂的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 进程。我们将开发多重的、基于细胞的分析方法，以检测特定的激酶活性，这些活性对 抑制反应和动态组重编程，并用它们来检测患者的激酶激活谱 细胞和抑制物筛选。使用时间分辨的稀土元素检测生物传感器的磷酸化 发光测量，其中Tb3+的发射能量是通过小分子直接测量的 荧光团根据荧光团的不同而产生不同的发射颜色。在目标1中，我们将确立 对药物敏感和耐药的关键激酶活性(从而抑制)的定量测定 慢性粒细胞白血病细胞，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，并添加了用于其他酶的生物传感器，如 它们是被开发出来的。检测将以CML患者的细胞系和样本为基础(与 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 现有的激酶抑制剂药物和新化合物之间的协同作用(通过文库)。我们还将发展 基于稀土元素能量转移的生物传感器磷酸化均相多重分析 有机染料。在目标3中，我们扩展了生物传感器设计流水线，以开发新的、非天然的多肽。 用作其他酶的生物传感器的底物。本目标中描述的工作将增加一套 我们已经有可用的生物传感器。完成这项提案中描述的工作将给我们带来一部小说 多种酶的分析，一套新的生物传感器以及新的和改进的检测策略用于 放映。药物发现将受益于这项技术解决动态组重新编程的能力 通过一次靶向几个激酶来实现这一机制。药物开发将受益于与之配套的检测 多个目标可以跟随药物或药物组合通过命中到主导过渡，目标 验证、临床前研究、临床试验，甚至更深入的治疗管理。这一检测方法及其应用 相关工具将为下一代癌症靶向治疗的开发做出贡献 我们在药物筛选和开发过程中对疾病环境进行建模的能力取得了新的进展。