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.

项目概要 癌症是美国和全世界的主要死亡原因。这个创新的多 PI 项目已进入第六个阶段 今年制定了一种新方法来应对发现控制癌症策略的挑战。致癌性 GTP 酶 RAS 家族的激活发生在约 30% 的癌症中，使其成为最常发生突变的癌症 人类癌症中的癌基因。尽管我们对 RAS 生物化学的理解取得了令人瞩目的进展， 过去 3 年来它在肿瘤发生中的作用，以及第一个批准的直接作用于肿瘤发生的药物的兴奋 针对特定的致癌 RAS 突变体，开发针对 RAS 的有效疗法仍然是一个重大任务 挑战。我们率先使用单体技术来定义以前未识别的漏洞 在RAS中。单体是小型合成结合蛋白，其亲和力和选择性达到一定水平 目标与抗体相似，可用作生化、结构、细胞和体内研究中的工具生物制剂。 在当前的项目期间，我们开发并使用了两个单体NS1和R15，以获得新的见解 深入了解 RAS 功能和漏洞。 NS1 揭示了 RAS 中涉及的 α4-α5 界面的重要性 二聚化。 R15揭示了靶向致癌基因子集的无核苷酸（apo）状态的可行性 RAS 突变体，尽管传统观点认为 RAS 突变体无法有效地与紧密结合的竞争 RAS 中的核苷酸。此外，我们已经确定了开发单体的可行性 非共价选择性抑制致癌 RAS 突变体和选择性降解 RAS 突变体 单体-VHL 融合。基于这些成功和强有力的初步数据，该项目的下一阶段 旨在实现以下目标： 1、我们将利用 NS1、R15 和其他单体作为高选择性 扰动来解决 RAS 生物学中的重要机制问题，包括二聚化/自- RAS 效应子激活中的关联、野生型 KRAS 在杂合 KRAS 突变细胞中的作用以及 KRAS4A 在肿瘤发生中的作用。 2、我们将利用基因编码建立细胞系和小鼠模型 确定 RAS 抑制的特定模式如何影响致癌驱动的体内肿瘤发生 RAS突变体。 3，我们将开发具有新特异性的单体，以扩大我们的项目范围， 特别是那些对 NRAS、KRAS4A 和常见 RAS 突变具有选择性的突变。该项目的结果将 促进我们在生化、细胞和体内水平上对 RAS 功能的机制理解，并提供信息 直接针对 RAS 的疗法的开发。此外，在此开发了独特的强大工具 项目将为整个 RAS 社区提供支持。