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Probing RAS-mediated signaling mechanisms with monobody inhibitors

使用单体抑制剂探索 RAS 介导的信号传导机制

基本信息

批准号：
10220892
负责人：
SHOHEI KOIDE
金额：
$ 55.1万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-02-01 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10220892
关键词：
Address Advisory Committees Affinity Amino Acids Antibodies BRAF gene Binding Binding Proteins Biochemical Biochemistry Biological Biology Biophysics Cause of Death Cells Charge Chimera organism Clinical Communities Coupled Development Dimerization Environment Event Extracellular Domain FDA approved Family GTP Binding Genetically Engineered Mouse Goals Grant Growth Growth Factor Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Human In Vitro Individual International KRAS2 gene Laboratories Malignant Neoplasms Malignant neoplasm of pancreas Mediating Mission Modality Modeling Molecular Mutate Mutation NRAS gene National Cancer Institute Oncogenes Oncogenic Oncoproteins Oxidation-Reduction Pathway interactions Patients Pharmaceutical Preparations Phosphotransferases Play Prevalence Process Protein Engineering Protein Isoforms Proteins Public Health RAS genes RAS inhibition Ras Inhibitor Reagent Research Risk Role Series Signal Transduction Site Specificity Structure Surface System Talents Technology Tertiary Protein Structure Testing Therapeutic United States Work anticancer research base cancer therapy cell growth chemical genetics empowered experimental study genetic approach in vivo inhibitor/antagonist innovation insight mutant novel novel strategies pre-clinical prevent protein function raf Kinases ras Proteins receptor recruit steroid hormone receptor success targeted treatment therapeutically effective tool tumor tumorigenesis tumorigenic

项目摘要

Project Summary: Cancer is a leading cause of death in the United States and worldwide. As such the President of the United States has recently established the White House Cancer Moonshot Task Force, the mission of which is to eliminate cancer as we know it. Part of this mission is to encourage development of novel cancer treatments. This innovative multi-PI proposal represents a novel approach to this challenge. Oncogenic activation of the RAS family of GTPases occurs in ~30% of cancers making it the most frequently mutated oncogene in human cancers. Despite a great deal of progress in our understanding of the biochemistry of RAS and it's role in tumorigenesis, development of effective therapeutic inhibitors of RAS to date has been disappointing. Thus, there remains a critical need to develop targeted inhibitors of this oncoprotein for treatment of patients with Ras-positive tumors. Using an unbiased, protein engineering approach, we have developed a highly specific and potent inhibitor of H-RAS and K-RAS based on the monobody platform. Monobodies are single-domain proteins of ~95 amino acids that achieve levels of affinity and selectivity similar to antibodies yet are insensitive to the redox potential of their environment. High affinity monobodies have been isolated to a diverse array of targets including the extracellular domain of receptors, kinases, steroid hormone receptors, and modular protein domains. Using this protein-based monobody inhibitor as a powerful experimental tool, we will probe the function of RAS in the tumorigenic process in ways that have not previously been possible. We propose three major aims to accomplish our goal of interrogating RAS function in oncogenesis. In Aim 1, we will use genetically encoded versions of our monobody inhibitor, termed NS1, to address unanswered questions regarding RAS function. In particular, NS1 blocks RAS through binding an allosteric interface important for dimerization of RAS and stimulation of signaling and transformation. Using NS1, we will address the importance of RAS dimerization in activation of multiple RAS effector pathways as well as probe the isoform specific difference in effector engagement. Aim 2 will employ a unique chemical-genetic approach to regulate NS1 expression in vivo to address whether targeting this novel allosteric interface interferes with RAS- dependent tumorigenesis. Building on our recent success with NS1, Aim 3 will develop isoform specific inhibitory monobodies to each RAS isoform and determine their mechanism of action. These studies represent a unique and powerful approach toward studying RAS and defining potential novel approaches to blocking RAS action. Thus, our work has the potential to make a major impact on cancer therapy. In addition, this project is highly relevant to the mission of National Cancer Institute's RAS Initiative at the Frederick National Laboratory for Cancer Research which is charged with targeting RAS-dependent cancers as well as President Obama's Cancer Moonshot initiative. We anticipate that our studies will answer important questions regarding RAS function in cancer while also providing powerful new experimental tools for the wider scientific community to utilize in interrogating RAS function.

项目摘要：癌症是美国和全世界的主要死亡原因。作为美国总统，美国最近成立了白宫癌症登月特别工作组，其使命是消除我们所知道的癌症。这个使命的一部分是鼓励开发新的癌症治疗方法。这种创新的多PI提案代表了应对这一挑战的新方法。致癌激活的约30%的癌症中存在RAS家族的GTP酶，使其成为人类中最常见的突变癌基因癌的尽管我们对RAS的生物化学及其作用的了解取得了很大进展，然而，尽管RAS抑制剂与肿瘤发生密切相关，但迄今为止RAS的有效治疗性抑制剂的开发一直令人失望。因此，在本发明中，仍然迫切需要开发这种癌蛋白的靶向抑制剂， Ras阳性肿瘤。使用无偏见的蛋白质工程方法，我们开发了一种高度特异性的和K-RAS的有效抑制剂。单体是单结构域约95个氨基酸的蛋白质，达到与抗体相似的亲和力和选择性水平，但不敏感与环境的氧化还原电位有关。高亲和力的单体已经被分离到各种各样的抗体中。靶点包括受体、激酶、类固醇激素受体和模块的细胞外结构域，蛋白质结构域。使用这种蛋白质为基础的单体抑制剂作为一个强大的实验工具，我们将探索 RAS在肿瘤发生过程中的作用是以前不可能的。我们提出三个主要目标来完成我们的目标，询问RAS在肿瘤发生中的功能。在目标1中，我们将使用我们的单体抑制剂的基因编码版本，称为NS 1，以解决悬而未决的问题，关于RAS功能特别是，NS 1通过结合一个重要的变构界面来阻断RAS， RAS的二聚化以及信号传导和转化的刺激。使用NS 1，我们将解决 RAS二聚化在多种RAS效应子通路激活中的重要性以及探测亚型效应器接合的特定差异。目标2将采用一种独特的化学遗传方法来调节 NS 1在体内的表达，以解决靶向这种新型变构界面是否干扰RAS-1。依赖性肿瘤发生基于我们最近在NS 1上的成功，Aim 3将开发针对NS 1的亚型特异性抗体。本发明的目的在于检测针对每种RAS同种型的抑制性单体并确定其作用机制。这些研究代表了一种独特而强大的方法，用于研究RAS并定义潜在的阻断新方法 RAS行动。因此，我们的工作有可能对癌症治疗产生重大影响。另外这款该项目与弗雷德里克国家癌症研究所RAS倡议的使命高度相关癌症研究实验室负责靶向RAS依赖性癌症，奥巴马的癌症登月计划。我们希望我们的研究能够回答一些重要问题， RAS在癌症中的功能，同时也为更广泛的科学界提供了强大的新实验工具用于询问RAS功能。