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：癌症是美国和全球的主要死亡原因。因此，联合总统各州最近建立了白宫癌月份特遣队，其任务是消除我们所知道的癌症。该任务的一部分是鼓励发展新型癌症治疗。这项创新的多PI提案代表了解决这一挑战的一种新方法。致癌激活 RAS家族GTPases发生在约30％的癌症中，使其成为人类最常见的癌基因癌症。尽管在我们对RA的生物化学的理解中取得了很大进步，并且它在肿瘤发生，迄今为止RAS有效的治疗抑制剂的发展令人失望。因此，仍需要开发这种癌蛋白的靶向抑制剂来治疗患者 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。单子是单域〜95个氨基酸的蛋白质达到了类似于抗体的亲和力和选择性水平但不敏感的蛋白质具有其环境的氧化还原潜力。高亲和力单体化已被隔离到各种各样的阵列包括受体外细胞域，激酶，类固醇激素受体和模块化的靶标蛋白质结构域。将此基于蛋白质的单体抑制剂作为强大的实验工具，我们将探测 RA在肿瘤过程中的功能以前是不可能的。我们建议三个主要目的是实现我们在肿瘤发生中询问RAS功能的目标。在AIM 1中，我们将使用我们的单体抑制剂的遗传编码版本称为NS1，以解决未解决的问题关于RAS功能。特别是，NS1通过结合变构界面来阻止RAS RAS的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效应途径的激活中的重要性以及探测同工型效应子参与方面的特定差异。 AIM 2将采用独特的化学遗传学方法来调节 NS1在体内表达以解决针对这种新型变构界面是否会干扰Ras- 依赖性肿瘤发生。在我们最近在NS1成功的基础上，AIM 3将开发同工型对每个RAS同工型的抑制性单体化并确定其作用机理。这些研究代表一种独特而有力的方法，用于研究RAS并定义潜在的新方法来阻止 RAS动作。因此，我们的工作有可能对癌症治疗产生重大影响。此外，这项目与弗雷德里克国家（Frederick National）国家癌症研究所的RAS计划的任务高度相关负责针对RAS依赖性癌症的癌症研究实验室以及总统奥巴马的癌症月亮倡议。我们预计我们的研究将回答有关的重要问题 RAS在癌症中的功能，同时还为更广泛的科学界提供强大的新实验工具利用询问RAS功能。