Conformational Regulation and Therapeutic Targeting of Oncogenic KRAS

致癌 KRAS 的构象调控和治疗靶向

• 批准号: 10549717
• 负责人: Loren David Walensky
• 金额: $ 47.13万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10549717
• 关键词: Agonist; Alanine; Benchmarking; Binding; Binding Sites; Biochemical; Biology; Catalytic Domain; Cell Proliferation; Cell Survival; Cell physiology; Cells; Chemicals; Clinical; Colon Carcinoma; Complex; Deuterium; Dissociation; Endocytosis; Epitopes; Extracellular Signal Regulated Kinases; Family; Frequencies; GTP Binding; Gene Amplification; Generations; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Human; Hydrocarbons; Hydrogen; Impairment; KRAS oncogenesis; KRAS2 gene; Laboratories; Lead; Libraries; Link; Malignant Neoplasms; Malignant neoplasm of liver; Malignant neoplasm of lung; Malignant neoplasm of pancreas; Malignant neoplasm of thyroid; Mass Spectrum Analysis; Measures; Mediating; Modality; Modeling; Molecular Conformation; Monitor; Mutagenesis; Mutation; Nucleotides; Oncogenic; Pathologic; Pathway interactions; Peptides; Pharmaceutical Chemistry; Pharmaceutical Preparations; Protein Conformation; Protein Family; Proteins; Proto-Oncogenes; RAS inhibition; Regulation; Reporting; Research; Scanning; Signal Transduction; Specificity; Structure; Testing; Therapeutic; Variant; Work; alpha helix; analytical method; cancer cell; cell motility; clinical translation; efficacy testing; enhancing factor; gain of function; human model; in vivo; in vivo evaluation; inhibitor; innovation; insight; mimicry; mouse model; mutant; nanomolar; next generation; novel; novel therapeutic intervention; pharmacologic; prototype; ras Proteins; small molecule; stapled peptide; synergism; targeted treatment; therapeutic target; translational approach; tumor; uptake

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.

KRAS 是最致命但未经药物治疗的癌症蛋白之一，存在于超过 30% 的人类体内 肿瘤，在胰腺癌、肺癌、甲状腺癌、结肠癌和肝癌中发现的频率更高。从而，实现 对 KRAS 放松管制和推进中和致癌性的创新方法的新机制见解 KRAS 仍然是癌症领域的最高优先事项之一，代表了这一跨学科的焦点 提议。 KRAS 是一种 GTP 酶，可作为一系列细胞功能的关键控制点，包括 细胞的存活和增殖依赖于内吞作用和运动。 KRAS 的功能活性由核苷酸决定 交换，GTP 绑定和 GDP 绑定形式分别代表开启和关闭状态。癌症 细胞通过功能获得性突变或基因扩增劫持并强制 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尚未成功，原因如下： KRAS 对 GTP 和 GDP 进行亚纳摩尔参与。 KRAS 与鸟嘌呤 SOS1 复合物的结构 核苷酸交换因子通过促进 GDP 释放来增强 KRAS 活性，揭示了凹槽中的螺旋 相互作用可能是α-螺旋拟态的目标。我们应用全烃肽装订来生成 稳定 SOS1 (SAH-SOS1) 的 α 螺旋并鉴定出一种参与致癌作用的原型化合物 KRAS（包括广泛多样性的临床突变体）抑制 ERK-MAP 激酶磷酸信号级联 KRAS 下游，并损害 KRAS 驱动的癌细胞的活力。我们发现，不仅 原型 SAH-SOS1 构建体按预期解离催化 SOS1/KRAS 相互作用，但也直接解离 并通过未知机制独立阻断核苷酸与 KRAS 的关联。在这里，我们的目标是申请 化学、结构、细胞和体内方法来探究 SAH-SOS1 肽如何直接阻断 KRAS 的酶活性，将此机制与 SOS1 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 SOS1/KRAS 调节的结构功能理解和新策略 用于人类癌症中 KRAS 的治疗性抑制。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 SOS1 以确定与 KRAS 及其致癌突变体的结合决定因素和功能相互作用； (2)应用氢氘交换质谱阐明SOS1的构象效应 KRAS 蛋白上的蛋白质和 SAH-SOS1 肽，从而确定酶促调节机制； (3) 将优化的 SAH-SOS1 抑制剂推进到 KRAS 驱动的癌症的细胞和体内测试中以进行验证 作用机制和治疗窗，并为临床转化提供概念验证。通过结合 Engen 实验室的生化和质谱专业知识以及癌症化学生物学和 瓦伦斯基实验室的转化方法，我们的目标是提供新的机制见解 致癌的 KRAS 通路并提供了一种新的方式来解除它以达到癌症治疗的效果。