权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

A new multi-pathway kinase activity assay applied to compound library screening in cancer biology

一种新的多途径激酶活性测定应用于癌症生物学化合物库筛选

基本信息

批准号：
10505710
负责人：
Tian Zhang
金额：
$ 11.74万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10505710
关键词：
3-Dimensional Advisory Committees Affect Antineoplastic Agents Binding Biological Assay Cancer Biology Cancer Model Cancer cell line Cell Culture Techniques Cells Chemicals Communication Communities Complex Cyclic AMP-Dependent Protein Kinases Cysteine Data Drug Compounding Drug Screening Drug resistance Elements Ensure Environment Foundations Future Goals Human In Vitro Incubated Label Lead Leadership Libraries Ligands MAP Kinase Gene Malignant Neoplasms Malignant neoplasm of ovary Mass Spectrum Analysis Measurement Measures Mentors Methods Modeling Morphology Pathway interactions Patients Peptides Phase Phosphorylated Peptide Phosphorylation Phosphotransferases Process Protein Kinase Proteome Proto-Oncogene Proteins c-akt Reagent Reproducibility Research Research Personnel Sampling Screening for cancer Serous Signal Pathway Signal Transduction Solid System Technology Training Work Writing anticancer research base cancer cell cancer therapy cancer type career development chemotherapy data acquisition drug development drug discovery druggable target experience genetic regulatory protein high throughput screening high-throughput drug screening in vivo innovation instrumentation kinase inhibitor knowledge base mass spectrometer medical schools method development neoplasm resource novel research and development response screening skills small molecule libraries targeted cancer therapy targeted treatment therapeutically effective three dimensional cell culture three-dimensional modeling tool training opportunity triple-negative invasive breast carcinoma tumor tumor heterogeneity two-dimensional

项目摘要

A new multi-pathway kinase activity assay applied to compound library screening in cancer biology Project Abstract Dysregulation of protein kinase signaling pathways is a hallmark of most human cancers. Although targeting kinases has been an effective therapeutic strategy in diverse cancer types, limited druggable targets, tumor heterogeneity and drug resistance remain problems in kinase-targeted cancer therapies. Finding new modulators of kinase activity in complex cancer models is therefore an urgent need, but current kinase activity measurement methods suffer from being low throughput and only provide surrogates for kinase activity. This proposal aims to apply the latest sample multiplexing technologies using Tandem Mass Tag (TMT) reagents and mass spectrometry to create a novel, multi-pathway, high-throughput, direct kinase activity readout assay and apply it to profile kinase activity for drug screening. An in vitro peptide phosphorylation assay provides a way to directly measure kinase activities for many pathways, while TMT increases throughput by sample multiplexing. First, I will select peptide substrates to represent specific kinase pathways (e.g., AKT, SRC, MAPK, CDK, etc.) and create a high-throughput in vitro kinase assay based on the TMT reagents. This assay will then be adapted to a 96-well plate format (Aim1). In Aim2, I will expand the assay to a three-dimensional (3D) cell culture model, profile and compare the kinase activities in response to 137 known kinase inhibitors in both standard two-dimensional (2D) and 3D cell culture models. In Aim3, I will perform high-throughput profiling of kinase activities across a library of 3200 compounds in both 2D and 3D ovarian cancer models using the 96-well plate platform for drug screening. Upon completion of these Aims, I will have developed and applied a highly optimized method for direct kinase activity measurement. This novel assay will result in the ability to quantitatively measure the activation state of dozens of signaling pathways directly from cellular lysates and, therefore, identify kinase modulators for future drug development. This proposal draws on my training in mass spectrometry-based method development and kinase and cancer biology. However, to ensure completion of the goals and successful transition to independence, I have a detailed training plan, under the guidance of my mentors Dr. Steven Gygi and Dr. Joan Brugge, as well as my advisory team, Dr. Peter Sicinski, Dr. Jennifer Smith and Dr. Joao Paulo. During the mentored phase, I plan to (1) deepen my understanding of mass spectrometry instrumentation, (2) develop new expertise in drug screening, (3) strengthen my knowledge base in cancer biology, especially in 3D cell culture and cellular signaling, and (4) enhance my scientific writing and communication skills while gaining experience in lab management and leadership. The environment at Harvard Medical School presents an excellent opportunity for training in drug screening and cancer biology. The proposed research strategy combined with the career development training outlined herein will provide the necessary elements for my successful transition to an independent investigator in the fields of mass spectrometry and cancer biology.

一种新的多途径激酶活性测定方法用于肿瘤生物学化合物库筛选项目摘要蛋白激酶信号通路的失调是大多数人类癌症的标志。虽然针对激酶已经成为多种癌症类型、有限的可药物靶点、肿瘤异质性和耐药性仍然是激酶靶向癌症治疗中问题。寻找新的调节剂因此，迫切需要在复杂的癌症模型中检测激酶活性，但目前的激酶活性测量这些方法的缺点是通量低，并且仅提供激酶活性的替代物。该提案旨在应用使用串联质量标签（TMT）试剂的最新样品多重技术和质谱法来创建一种新的、多途径、高通量、直接的激酶活性读出测定并将其应用于药物筛选的激酶活性分析。体外肽磷酸化测定提供了一种新的方法。直接测量许多途径的激酶活性的方法，而TMT通过样品增加通量多路复用首先，我将选择肽底物来代表特定的激酶途径（例如，AKT，SRC，MAPK， CDK等）并基于TMT试剂创建高通量体外激酶测定。然后，该测定将适用于96孔板格式（Aim 1）。在Aim 2中，我将试验扩展到三维（3D）细胞培养模型，分析并比较两种细胞中对137种已知激酶抑制剂的激酶活性标准二维（2D）和3D细胞培养模型。在Aim3中，我将执行高通量分析，使用96孔板在2D和3D卵巢癌模型中跨3200种化合物库的激酶活性用于药物筛选的平板平台。完成这些目标后，我将开发和应用一个高度用于直接激酶活性测量的优化方法。这种新的测定方法将导致定量的能力，直接从细胞裂解物中测量数十种信号通路的激活状态，用于未来药物开发的激酶调节剂。这个建议借鉴了我在基于质谱的方法开发和激酶与癌症方面的培训生物学然而，为了确保完成各项目标和成功地向独立过渡，在我的导师Steven Gygi博士和Joan Brugge博士的指导下，团队，彼得·西辛斯基博士，詹妮弗·史密斯博士和若昂·保罗博士。在指导阶段，我计划（1）加深我对质谱仪器的理解，（2）开发药物筛选的新专业知识，（3）加强我在癌症生物学方面的知识基础，特别是在3D细胞培养和细胞信号传导方面，以及（4）提高我的科学写作和沟通技巧，同时获得实验室管理经验，领导哈佛医学院的环境为药物培训提供了极好的机会筛查和癌症生物学。提出了与职业发展培训相结合的研究策略本文概述的内容将为我成功过渡到独立调查员提供必要的要素在质谱和癌症生物学领域。