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

• 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
• 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

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.

项目总结： 癌症是美国和世界范围内的主要死亡原因。因此，美国总统 各州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。这一使命的一部分是鼓励开发新的癌症治疗方法。 这一创新的多PI提案代表了应对这一挑战的新方法。致癌活性的研究 RAS家族的GTP酶存在于约30%的癌症中，使其成为人类最常见的突变癌基因 癌症。尽管在我们对RAS的生物化学及其在 到目前为止，RAS的有效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 RAS阳性肿瘤。使用不偏不倚的蛋白质工程方法，我们已经开发出高度特异的 以及基于单体平台的H-RAS和K-RAS的有效抑制剂。单体体是单域的 ~95个氨基酸组成的蛋白质具有与抗体相似的亲和力和选择性，但不敏感 其环境的氧化还原潜力。高亲和力的单体已经被分离成不同的阵列 靶点包括受体、激酶、类固醇激素受体和模块化的胞外结构域 蛋白质结构域。利用这种基于蛋白质的单体抑制剂作为强大的实验工具，我们将探索 RAS在肿瘤形成过程中的作用是以前不可能实现的。我们建议 三个主要目的是为了实现我们询问RAS在肿瘤发生中的作用的目标。在目标1中，我们将使用 我们的单体抑制物的基因编码版本，称为NS1，以解决未回答的问题 关于RAS的功能。特别是，NS1通过结合变构界面来阻断RAS，该界面对 RAS的二聚化以及信号和转化的刺激。使用NS1，我们将解决 RAS二聚化在激活多个RAS效应通路中的重要性及其异构体的探测 效应器接合的具体差异。Aim 2将使用一种独特的化学遗传方法来调节 NS1在体内的表达，以解决靶向这一新的变构界面是否干扰RAS- 依存性肿瘤发生。在我们最近在NS1上取得成功的基础上，Aim 3将开发特定的异构体 抑制单体对每个RAS异构体的作用，并确定其作用机制。这些研究代表了 研究RAS并定义潜在的阻止新方法的独特而强大的方法 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，这一点 该项目与国家癌症研究所在弗雷德里克国家医院的RAS倡议的使命高度相关 负责靶向RAS依赖癌症的癌症研究实验室以及总裁 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在癌症中的作用，同时也为更广泛的科学界提供了强大的新实验工具 在审问RAS中发挥作用。