Conformational Regulation and Therapeutic Targeting of Oncogenic KRAS

KRAS是最致命的、但未被下药的癌症蛋白之一，存在于超过30%的人类体内 肿瘤，在胰腺癌、肺癌、甲状腺癌、结肠癌和肝癌中出现的频率更高。因此，实现 对KRAS放松管制的新机制见解和推进中和致癌因素的创新方法 KRAS仍然是癌症领域的最优先事项之一，并代表着这一跨学科的重点 求婚。KRAS是一种GTP酶，它是一系列细胞功能的关键控制点，从 细胞的存活和增殖到内吞和运动。KRAS的功能活性由核苷酸决定 交换，GTP绑定和GDP绑定的表单分别表示打开和关闭状态。癌 细胞通过功能获得性突变或基因扩增劫持并加强KRAS的激活状态。 到目前为止，直接阻断GTP结合位点的小分子方法还没有成功，因为 KRAS对GTP和GDP的亚纳摩尔接触。鸟嘌呤SOS_1与KRAS络合物的结构 核苷酸交换因子通过促进GDP释放来增强KRAS活性，显示出螺旋在槽中 潜在的α-螺旋拟态靶向相互作用。我们应用了全烃多肽装订来产生 稳定的SOS1α螺旋(SAH-SOS1)并鉴定了一个参与致癌的原型化合物 KRAS，包括广泛的临床突变体，抑制ERK-MAP激酶的磷酸化信号级联 在KRAS下游，并损害KRAS驱动的癌细胞的活力。我们发现，不仅是因为 如预期的那样，原型SAH-SOS1构建解离了催化的SOS1/KRAS相互作用，但也直接 并通过一种未知的机制独立阻断核苷酸与KRAS的联系。在这里，我们的目标是申请 用化学、结构、细胞和体内方法研究SAH-SOS1多肽是如何直接阻断 KRAS的酶活性，与SOS1的天然激动剂活性进行比较和对比 蛋白质，从而提供了我们对SOS1/KRAS调控的结构和功能的理解以及一种新的策略 用于治疗抑制人类癌症中的KRAS。为了实现这些目标，我们提出了三个实验目标： (1)模拟KRAS相互作用结构域合成结构增强螺旋的扩展文库 确定与KRAS及其致癌突变体的结合决定因素和功能相互作用； (2)用氢-重离子交换质谱仪研究了SOS1的构象效应 蛋白质和SAH-SOS1多肽对KRAS蛋白的作用，从而确定酶的调节机制； (3)将优化的SAH-SOS1抑制剂用于KRAS驱动的癌症的细胞和体内试验以验证 作用机制和治疗窗口，为临床翻译提供概念验证。通过组合 Engen实验室的生物化学和质谱学专业知识与癌症化学生物学和 瓦伦斯基实验室的翻译方法，我们的目标是提供新的机械洞察 致癌的KRAS途径，并提供了一种新的解除其武装的方式，从而在癌症中发挥治疗作用。

KRAS is one of the most deadly, yet undrugged, cancer proteins and is present in over 30% of all human tumors, with even higher frequencies found in pancreatic, lung, thyroid, colon, and liver cancers. Thus, achieving new mechanistic insights into KRAS deregulation and advancing innovative approaches to neutralize oncogenic KRAS remain among the highest priorities of the cancer field and represent the focus of this interdisciplinary proposal. KRAS is a GTPase that serves as a critical control point for a host of cellular functions ranging from cell survival and proliferation to endocytosis and motility. The functional activity of KRAS is dictated by nucleotide exchange, with the GTP-bound and GDP-bound forms representing the on and off states, respectively. Cancer cells hijack and enforce the activated state of KRAS through gain-of-function mutagenesis or gene amplification. To date, small molecule approaches to directly block the GTP-binding site have been unsuccessful due to subnanomolar engagement of GTP and GDP by KRAS. The structure of KRAS in complex with SOS1, a guanine nucleotide exchange factor that enhances KRAS activity by facilitating GDP release, revealed a helix-in-groove interaction potentially targetable by α-helical mimicry. We applied all-hydrocarbon peptide stapling to generate stabilized alpha-helices of SOS1 (SAH-SOS1) and identified a prototype compound that engaged oncogenic KRAS, including the broad diversity of clinical mutants, inhibited the ERK-MAP kinase phosphosignaling cascade downstream of KRAS, and impaired the viability of KRAS-driven cancer cells. We found that not only did the prototype SAH-SOS1 construct dissociate the catalytic SOS1/KRAS interaction as anticipated, but also directly and independently blocked nucleotide association with KRAS by an unknown mechanism. Here, we aim to apply chemical, structural, cellular, and in vivo approaches to interrogate just how a SAH-SOS1 peptide can directly block the enzymatic activity of KRAS, compare and contrast this mechanism to the natural agonist activity of the SOS1 protein, and thereby inform both our structure-function understanding of SOS1/KRAS regulation and a new strategy for therapeutic inhibition of KRAS in human cancer. To achieve these goals, we propose three experimental aims: (1) Synthesize an expansive library of structurally-reinforced helices modeled after the KRAS-interaction domain of SOS1 to identify the binding determinants and functional interactions with KRAS and its oncogenic mutants; (2) Apply hydrogen-deuterium exchange mass spectrometry to elucidate the conformational effects of the SOS1 protein and SAH-SOS1 peptides on KRAS proteins and thereby define the mechanisms of enzymatic regulation; (3) Advance optimized SAH-SOS1 inhibitors to cellular and in vivo testing in KRAS-driven cancers to validate mechanism of action and therapeutic window, and provide proof-of-concept for clinical translation. By combining the biochemical and mass spectrometry expertise of the Engen laboratory with the cancer chemical biology and translational approaches of the Walensky laboratory, our goal is to provide new mechanistic insight into the oncogenic KRAS pathway and inform a new modality to disarm it for therapeutic benefit in cancer.