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

一种新的用于肿瘤生物学化合物文库筛选的多途径激酶活性测定法 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。虽然目标是 对于不同的癌症类型、有限的药物靶点、肿瘤，激酶一直是一种有效的治疗策略 异质性和耐药性仍然是以激酶为靶点的癌症治疗中的问题。寻找新的调制器 因此，迫切需要对复杂癌症模型中的激酶活性进行研究，但目前的激酶活性测量 方法的缺点是产量低，并且只提供激酶活性的替代方法。 该提案旨在应用使用串联质量标签(TMT)试剂的最新样品多路传输技术 和质谱学来创建一种新的、多途径、高通量、直接的激酶活性读出分析 并将其应用于药物筛选中的激酶活性分析。体外多肽磷酸化试验提供了一种 一种直接测量许多途径的激酶活性的方法，而TMT通过样本增加吞吐量 多路传输。首先，我将选择代表特定激酶途径的多肽底物(例如，AKT、SRC、MAPK、 CDK等)并建立了基于TMT试剂的高通量体外激酶检测方法。这个化验结果将会 适用于96孔板格式(Aim1)。在AIM2中，我将把分析扩展到三维(3D)细胞 建立培养模型，分析和比较137种已知的激酶抑制剂对两者的激酶活性的影响 标准二维(2D)和3D细胞培养模型。在Aim3中，我将执行以下内容的高通量分析 使用96孔在2D和3D卵巢癌模型中的3200个化合物库中的激酶活性 用于药物筛选的平板平台。在完成这些目标后，我将开发和应用一个高度的 直接测定激酶活性的优化方法。这一新的分析方法将使我们能够定量地 直接从细胞裂解物中测量数十条信号通路的激活状态，从而识别 用于未来药物开发的激酶调节剂。 这项建议借鉴了我在基于质谱学的方法开发和激酶与癌症方面的培训。 生物学。然而，为了确保目标的完成和向独立的成功过渡，我有一个详细的 在我的导师Steven Gygi博士和Joan Brugge博士的指导下以及我的建议下，制定了培训计划 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)深化 我对质谱仪的了解，(2)在药物筛选方面发展新的专业知识，(3) 加强我在癌症生物学方面的知识基础，特别是3D细胞培养和细胞信号传递方面的知识，以及(4) 提高我的科技写作和沟通能力，同时积累实验室管理和 领导力。哈佛医学院的环境为药物培训提供了绝佳的机会 筛查和癌症生物学。提出了结合职业发展培训的研究策略 这里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 在质谱学和癌症生物学领域。

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.