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

基本信息

批准号：
9445128
负责人：
Laurie L. Parker
金额：
$ 15万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2019-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9445128
关键词：
Acute Myelocytic Leukemia Address Antineoplastic Agents Area Award Binding Bioinformatics Biological Biological Assay Biosensor Cell Line Cells Chronic Lymphocytic Leukemia Chronic Myeloid Leukemia Clinic Clinical Color Companions Complex Data Detection Development Disease Disease model Drug Combinations Drug effect disorder Drug resistance Drug-sensitive Dyes Energy Transfer Environment Evaluation Exhibits Face Failure Family Future Goals Hematologic Neoplasms Imatinib In Vitro JAK2 gene Lanthanoid Series Elements Lead Libraries Luminescent Measurements Malignant Neoplasms Measures Methods Monitor Mutation Pathway interactions Patients Peptides Pharmaceutical Preparations Pharmacology Phenotype Phosphorylation Phosphotransferases Point Mutation Preclinical Drug Evaluation Process Protein Kinase Protein Kinase Inhibitors Proteomics Protocols documentation Relapse Resistance Sampling Signal Pathway Signal Transduction System Techniques Technology Testing Therapeutic Time Treatment Effectiveness Treatment outcome Up-Regulation Validation Work base chemotherapy clinical development clinically relevant companion diagnostics design drug development drug discovery drug sensitivity fluorophore improved in vivo inhibitor/antagonist insight kinase inhibitor knowledge base leukemia treatment luminescence multiplex detection next generation novel novel drug combination novel therapeutics oncology preclinical study prevent profiles in patients protein biomarkers protein kinase inhibitor public health relevance reconstitution resistance mechanism response screening sensor small molecule success synergism targeted treatment therapy development time use tool tool development

项目摘要

DESCRIPTION (provided by applicant): 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 or 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, clinial 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.

描述（由申请人提供）：激酶抑制剂开创了化疗的新范例，是新肿瘤药物开发的主要焦点，但开发成功率较低（5-10%）。对激酶抑制剂的耐药性通过靶点依赖性机制（例如消除药物结合的点突变）和靶点独立机制（例如替代信号通路的上调，称为“激酶组重编程”）发生。因此，需要同时靶向多种激酶的抑制剂组合，以更好地避免耐药/复发循环。检测活细胞内（而不是裂解物或重组系统中）的蛋白激酶活性对于了解激酶抑制剂药物敏感性和耐药机制非常重要，并且将有助于更好地筛选可能通过开发过程的抑制剂。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，这些激酶活性对于抑制剂反应和激酶组重编程很重要，并使用它们来检测患者细胞中的激酶激活谱和抑制剂筛选。使用时间分辨镧系元素发光测量来检测生物传感器的磷酸化，其中通过小分子荧光团直接测量 Tb3+ 发射能量，根据荧光团给出不同的发射颜色。在目标 1 中，我们将建立药物敏感和耐药 CML 细胞中关键激酶活性（以及抑制作用）的定量分析，分析治疗相关细胞状态下的通路激活表型。我们将使用我们在前期工作中已经建立的一组生物传感器，并在开发时添加针对其他激酶的生物传感器。这些测定将使用 CML 患者的细胞系和样本（与健康对照相比）建立，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（通过我们还将利用从镧系元素到有机染料的能量转移来开发生物传感器磷酸化的均质多重分析。在目标 3 中，我们扩展了生物传感器设计流程，以开发新的非天然材料。用作其他激酶生物传感器的肽底物。该目标中描述的工作将添加到我们已有的生物传感器组中。完成本提案中描述的工作将为我们提供一种针对多种激酶的新型检测方法，这是一套新的方法。生物传感器以及用于筛选的新的和完善的检测策略将受益于该技术通过一次靶向多个激酶来解决激酶组重编程机制的能力，药物开发将受益于可遵循的多个靶点的伴随检测。药物或药物组合通过从命中到先导的转变、目标验证、临床前研究、临床试验以及后续的治疗管理。该测定及其相关工具将为我们在药物筛选和开发过程中模拟疾病环境的能力开辟新天地，从而为下一代癌症靶向治疗的开发做出贡献。