Probing RAS-mediated Signaling with Monobody Inhibitors

项目摘要 癌症是美国和全世界的主要死亡原因。这个创新的多PI项目在其第六届 这一年建立了一种新的方法来应对发现控制癌症的策略的挑战。致癌 约30%的癌症中存在RAS家族GTP酶的激活，使其成为最常见的突变 癌基因在人类癌症中的作用。尽管我们对RAS生物化学的理解取得了令人印象深刻的进展， 在过去的30年里，它在肿瘤发生中的作用以及第一种被批准的药物的兴奋， 靶向特定的致癌RAS突变体，开发有效的靶向RAS的治疗方法仍然是一个重大的挑战。 挑战.我们率先使用单体技术来定义以前未被识别的漏洞 在RAS中。单体是小的合成结合蛋白，其实现对其结合的亲和力和选择性水平。 靶向类似于抗体，并可用作生物化学、结构、细胞和体内研究中工具生物制剂。 在当前的项目期间，我们开发并使用了两个单体，NS 1和R15，以获得新的见解 RAS功能和漏洞。NS 1揭示了RAS中α4-α5界面的重要性 二聚化。R15揭示了靶向一个致癌基因亚型的无核苷酸（apo）状态的可行性。 RAS突变体，尽管传统的智慧，一个人不能有效地竞争， RAS中的核苷酸。此外，我们已经确定了开发单抗体的可行性， 非共价和选择性地抑制致癌RAS突变体，以及使用 单体-VHL融合物。在这些成功和强有力的初步数据的基础上，该项目的下一阶段 目的是实现以下几点：1、我们将利用NS 1、R15和其他单体作为高选择性的 干扰剂，以解决RAS生物学中的重要机制问题，包括二聚化/自我- RAS效应子激活的相关性，野生型KRAS在杂合KRAS突变细胞中的作用，以及 KRAS 4A在肿瘤发生中的作用2、我们将利用基因编码技术建立细胞系和小鼠模型， 单抗体，以确定RAS抑制的特定模式如何影响由致癌基因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谱的单抗体，以扩大我们项目的范围， 特别是对NRAS、KRAS 4A和常见RAS突变具有选择性的那些。该项目的成果将 推进我们在生化，细胞和体内水平上对RAS功能的机制理解， 开发直接靶向RAS的治疗方法。此外，在这方面开发的独特的强大工具 该项目将增强整个RAS社区的能力。

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.