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.

项目概要：癌症是美国和全世界的主要死亡原因。因此，联合国主席各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的癌症。该使命的一部分是鼓励开发新型癌症治疗方法。这项创新的多 PI 提案代表了应对这一挑战的新颖方法。致癌激活 GTP 酶的 RAS 家族存在于约 30% 的癌症中，使其成为人类中最常见的突变癌基因癌症。尽管我们对 RAS 的生物化学及其作用的理解取得了很大进展迄今为止，有效的 RAS 治疗抑制剂的开发一直令人失望。因此，仍然迫切需要开发这种癌蛋白的靶向抑制剂来治疗患有癌症的患者 Ras 阳性肿瘤。使用公正的蛋白质工程方法，我们开发了一种高度特异性的以及基于单体平台的 H-RAS 和 K-RAS 的有效抑制剂。单体是单域的约 95 个氨基酸的蛋白质可达到与抗体相似的亲和力和选择性水平，但不敏感其环境的氧化还原电位。高亲和力单体已被分离到多种靶标包括受体、激酶、类固醇激素受体和模块的胞外域蛋白质结构域。使用这种基于蛋白质的单体抑制剂作为强大的实验工具，我们将探究 RAS 在致瘤过程中以以前不可能的方式发挥作用。我们建议实现我们探究 RAS 在肿瘤发生中的功能的目标的三个主要目标。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 NS1，用于解决尚未解答的问题关于 RAS 功能。特别是，NS1 通过结合一个对 RAS 至关重要的变构界面来阻断 RAS。 RAS 二聚化以及信号传导和转化的刺激。使用 NS1，我们将解决 RAS 二聚化在多个 RAS 效应通路激活以及探测异构体中的重要性效应器参与度的具体差异。目标 2 将采用独特的化学遗传方法来调节 NS1 体内表达，以解决靶向这种新型变构界面是否会干扰 RAS- 依赖性肿瘤发生。基于我们最近在 NS1 方面取得的成功，Aim 3 将开发特定的异构体每种 RAS 亚型的抑制性单体并确定其作用机制。这些研究代表一种独特而强大的方法，用于研究 RAS 并定义潜在的新型阻断方法 RAS 行动。因此，我们的工作有可能对癌症治疗产生重大影响。此外，这该项目与弗雷德里克国家癌症研究所 RAS 计划的使命高度相关癌症研究实验室，负责靶向 RAS 依赖性癌症，并担任主席奥巴马的癌症登月计划。我们预计我们的研究将回答以下重要问题 RAS 在癌症中发挥作用，同时还为更广泛的科学界提供强大的新实验工具用于询问 RAS 功能。