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：
9076374
负责人：
Laurie L. Parker
金额：
$ 5.4万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
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/9076374
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 Health Hematologic Neoplasms Imatinib In Vitro JAK2 gene Lanthanoid Series Elements Lead Libraries Life 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 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 companion diagnostics design drug development drug discovery drug sensitivity fluorophore human SYK protein improved in vivo inhibitor/antagonist insight kinase inhibitor knowledge base leukemia treatment luminescence resonance energy transfer next generation novel oncology pre-clinical preclinical study prevent protein kinase inhibitor reconstitution resistance mechanism response screening sensor small molecule success 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）：激酶抑制剂在化学疗法方面产生了新的范式，是新的肿瘤药物开发的主要重点，但发育成功率较低（5-10％）。对激酶抑制剂的抗性是通过靶依赖性机制（例如消除药物结合的点突变）和目标无关机制（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抗药性/复发周期的机会。检测活细胞内蛋白激酶活性（而不是在裂解物或重构系统中）很重要或了解激酶抑制剂药物敏感性和耐药机制，并会导致更好地筛选在开发过程中可能使其抑制的抑制剂。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，这些激酶活性对抑制剂反应和活化组重编程很重要，并使用它们来检测患者细胞和抑制剂筛查中的激酶激活谱。使用时间分辨的灯笼发光测量值检测到生物传感器的磷酸化，其中直接通过小分子荧光团直接测量TB3+发射能，以根据荧光团的形式获得不同的发射颜色。在AIM 1中，我们将建立对药物敏感和耐药性CML细胞中关键激酶活性（并因此抑制）的定量测定，从而分析了治疗性相关的细胞状态中途径激活表型。我们将使用我们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。该测定法将使用来自CML患者的细胞系和样品（与健康对照组相比），并用RT-QPCR和Swath“蛋白质组学进行验证。在AIM 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（通过图书馆）（通过图书馆）（通过图书馆），我们还将使用类似的分析分析型元素分析。 3，我们将生物传感器的设计管线扩展到新的非天然肽底物来作为其他激酶的生物传感器，这将增加我们已经在此提案中所描述的工作的一组生物传感器。一次针对多个激酶来解决活性重编程机制的能力将受益于多个靶标，这些靶标可以通过铅或药物组合来通过铅过渡，目标验证，临床前研究，临床试验，以及以外的治疗管理。该测定及其相关工具将通过在药物筛查和发育过程中为疾病环境建模的能力来为癌症的下一代靶向治疗发展做出贡献。