Conformational Regulation and Therapeutic Targeting of Oncogenic KRAS

KRAS是最致命的，但undrugged，癌症蛋白质之一，并存在于超过30%的所有人类 肿瘤，在胰腺癌、肺癌、甲状腺癌、结肠癌和肝癌中发现的频率甚至更高。因此，实现 对KRAS失调的新机制见解和推进创新方法来中和致癌性 KRAS仍然是癌症领域的最高优先事项之一，并代表了这一跨学科的重点 提议KRAS是一种GT3，作为一系列细胞功能的关键控制点， 细胞存活和增殖到内吞作用和运动性。KRAS的功能活性由核苷酸决定， 交换，其中GTP绑定和GDP绑定形式分别表示开和关状态。癌 细胞通过功能获得性诱变或基因扩增劫持并加强KRAS的激活状态。 迄今为止，直接阻断GTP结合位点的小分子方法由于以下原因而不成功： 通过KRAS的GTP和GDP的亚纳摩尔接合。KRAS与鸟嘌呤SOS 1复合物的结构 核苷酸交换因子，通过促进GDP的释放增强KRAS活性，揭示了一个螺旋槽 通过α-螺旋模拟潜在靶向相互作用。我们应用全烃肽钉合来产生 稳定的SOS 1 α-螺旋（SAH-SOS 1），并确定了一个原型化合物， KRAS，包括广泛多样的临床突变体，抑制ERK-MAP激酶磷酸化信号级联反应 在KRAS下游，并损害KRAS驱动的癌细胞的活力。我们发现， 原型SAH-SOS 1构建体如预期的那样解离催化SOS 1/KRAS相互作用，但也直接 并通过未知机制独立阻断核苷酸与KRAS的结合。在这里，我们的目标是应用 化学，结构，细胞和体内方法来询问SAH-SOS 1肽如何直接阻断 KRAS的酶活性，将该机制与SOS 1的天然激动剂活性进行比较和对比 蛋白质，从而告知我们对SOS 1/KRAS调控的结构-功能理解和新的策略 用于治疗性抑制人类癌症中的KRAS。为了实现这些目标，我们提出了三个实验目标： (1)合成一个以KRAS相互作用域为模型的结构增强螺旋的扩展库 以确定与KRAS及其致癌突变体的结合决定簇和功能相互作用; (2)应用氢-氘交换质谱法阐明SOS 1的构象效应 蛋白和SAH-SOS 1肽对KRAS蛋白的作用，从而确定酶调节的机制; (3)将优化的SAH-SOS 1抑制剂用于KRAS驱动的癌症的细胞和体内测试，以验证 作用机制和治疗窗，并为临床翻译提供概念验证。通过组合 Engen实验室在癌症化学生物学方面的生物化学和质谱专业知识， 翻译方法的Walensky实验室，我们的目标是提供新的机械洞察力， 致癌KRAS通路，并告知解除其在癌症中的治疗益处的新模式。

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.