Probing RAS-mediated Signaling with Monobody Inhibitors

使用单体抑制剂探测 RAS 介导的信号转导

• 批准号: 10530818
• 负责人: SHOHEI KOIDE
• 金额: $ 60.15万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10530818
• 关键词: Address; Affect; Affinity; Amino Acids; Antibodies; Architecture; Award; Binding; Binding Proteins; Biochemical; Biochemistry; Biological Products; Biological Testing; Biology; Cancer Control; Cancer Patient; Cause of Death; Cells; Colorectal Cancer; Communities; Complex; Data; Development; Dimerization; Disulfides; Engineering; FDA approved; Family; Family member; Funding; GTP Binding; Genetic; Guanine; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Human; KRAS2 gene; Laboratories; Lobe; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of pancreas; Mediating; Molecular; Mutate; Mutation; Nucleotides; Oncogenes; Oncogenic; Pharmaceutical Preparations; Pharmacology; Phase; Process; Productivity; Protein Engineering; Protein Isoforms; Proteins; RAS genes; RAS inhibition; Ras Inhibitor; Reagent; Research Personnel; Role; Series; Signal Transduction; Site; Specificity; Synthetic Genes; System; Technology; Therapeutic; United States; Work; anticancer research; base; cancer therapy; established cell line; in vivo; inhibitor; innovation; insight; mouse model; mutant; nano; novel; novel strategies; pharmacophore; prevent; ras Guanine Nucleotide Exchange Factors; recruit; small molecule inhibitor; success; therapeutic development; therapeutic target; therapeutically effective; tool; tumor addiction; tumorigenesis; ubiquitin ligase

Project Summary Cancer is a leading cause of death in the United States and worldwide. This innovative multi-PI project in its 6th year has established a novel approach to the challenge of discovering strategies to control cancer. Oncogenic activation of the RAS family of GTPases occurs in ~30% of cancers making it the most frequently mutated oncogene in human cancers. Despite impressive progress in our understanding of the biochemistry of RAS and its role in tumorigenesis over the past 3 decades and the excitement of the first approved drug that directly targets a particular oncogenic RAS mutant, development of effective therapeutics targeting RAS remains a grand challenge. We have pioneered the use of monobody technology to define previously unrecognized vulnerabilities in RAS. Monobodies are small synthetic binding proteins that achieve levels of affinity and selectivity for their target similar to antibodies and can be used as tool biologics in biochemical, structural, cellular and in vivo studies. In the current project period, we have developed and used two monobodies, NS1 and R15, to gain new insights into RAS function and vulnerabilities. NS1 revealed the importance of the α4-α5 interface implicated in RAS dimerization. R15 revealed the feasibility of targeting the nucleotide-free (apo) state of a subset of oncogenic RAS mutants, despite the conventional wisdom that one cannot effectively compete against tightly bound nucleotides in RAS. Furthermore, we have established the feasibility of developing monobodies that noncovalently and selectively inhibit oncogenic RAS mutants and of selectively degrading RAS mutants using monobody-VHL fusions. Building on these successes and strong preliminary data, the next phase of this project aims to accomplish the following: 1, We will utilize NS1, R15 and additional monobodies as highly selective perturbants to address important mechanistic questions in RAS biology, including roles of dimerization/self- association in RAS effector activation, roles of wild-type KRAS in heterozygous KRAS mutant cells, and roles of KRAS4A in tumorigenesis. 2, We will establish cell lines and mouse models using genetically encoded monobodies to determine how specific modes of RAS inhibition affect tumorigenesis in vivo driven by oncogenic RAS mutants. 3, We will develop monobodies with new specificity profiles to expand the scope of our project, specifically those selective to NRAS, KRAS4A, and common RAS mutations. Results from this project will advance our mechanistic understanding of RAS function at the biochemical, cellular and in vivo levels and inform the development of therapeutics directly targeting RAS. Furthermore, uniquely powerful tools developed in this project will empower the entire RAS community.

项目摘要 癌症是美国和全球的主要死亡原因。这个创新的多PI项目在其第六个项目中 一年已经建立了一种新的方法来发现控制癌症策略的挑战。致癌 RAS家族GTPases的激活发生在约30％的癌症中，使其成为最常见的突变 人类癌中的癌基因。尽管我们对Ras的生物化学的理解令人印象深刻 它在过去三十年中在肿瘤发生中的作用，以及第一个批准的药物的兴奋，直接 靶向特定的致癌性RAS突变体，开发有效的治疗靶向RAS仍然是一个宏伟的 挑战。我们已经开创了使用单体技术来定义以前未知的漏洞 在拉斯。单体化是小的合成结合蛋白，可实现其亲和力和选择性的水平 与抗体相似的靶标，可以用作生化，结构，细胞和体内研究中的工具生物制剂。 在当前的项目时期，我们已经开发并使用了两个单一版本NS1和R15，以获得新的见解 进入RAS功能和漏洞。 NS1揭示了在RAS中实现的α4-α5界面的重要性 二聚化。 R15揭示了靶向无核苷酸（APO）的可行性 拉斯突变体，传统观念，即无法有效地与紧密束缚竞争 RAS中的核苷酸。此外，我们已经建立了开发单体形成的可行性 非折线和选择性地抑制致癌性RAS突变体，并选择性地降解RAS突变体 单体-VHL融合。以这些成功和强大的初步数据为基础，该项目的下一阶段 旨在完成以下操作：1，我们将使用NS1，R15和其他单一作用作为高度选择性 扰动者在RAS生物学中解决重要的机理问题，包括二聚/自我的作用 RAS效应子激活的关联，野生型KRAS在杂合KRAS突变细胞中的作用以及 KRAS4A在肿瘤发生中。 2，我们将使用基因编码的细胞系和鼠标模型建立细胞系和鼠标模型 单个模板以确定RAS抑制的特定模式如何通过致癌性影响体内驱动的肿瘤发生 RAS突变体。 3，我们将开发具有新的特异性配置文件的单一体形，以扩大我们项目的范围， 该项目的结果将特别是那些对NRA，KRAS4A和常见RAS突变的选择性的结果。 提高我们对生化，细胞和体内水平的RAS功能的机械理解，并告知 治疗剂的发展直接针对RA。此外，在此开发了独特的强大工具 项目将增强整个RAS社区的能力。