Conformational Regulation and Therapeutic Targeting of Oncogenic KRAS

致癌 KRAS 的构象调控和治疗靶向

基本信息

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
项目类别：
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

项目摘要

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，但未腐败的癌症之一，在所有人类中的30％以上。肿瘤，在胰腺，肺，甲状腺，结肠和肝癌中发现甚至更高的频率。那是实现的对KRAS放松管制的新机械洞察力，并推进中和致癌的方法 KRAS仍然是癌症领域的最高优先事项，代表了该跨学科的重点提议。 KRAS是GTPase，它是一系列蜂窝函数的关键控制点细胞存活和增殖到内吞和运动。 KRAS的功能活性由核苷酸决定与GTP结合和GDP结合的形式分别代表ON和OFF状态的交换。癌症细胞通过功能诱变或基因扩增劫持KRAS活化状态。迄今为止，由于直接阻断GTP结合位点的小分子方法由于 KRAS的GTP和GDP的亚植物粒度参与。 KRAS与SOS1复合物的结构，一种鸟嘌呤核苷酸交换因子通过释放GDP的释放来增强KRAS活性相互作用可能通过α-螺旋模拟物来靶向。我们应用了全水合碳胡椒丁香来生成 SOS1（SAH-SOS1）的稳定α-螺旋，并鉴定 KRAS，包括临床突变体的广泛多样性，抑制了ERK-MAP激酶磷酸化级联 KRAS的下游，并损害了KRAS驱动的癌细胞的生存能力。我们发现不仅原型SAH-SOS1构建原型将催化SOS1/KRAS相互作用如预期的，但也直接并通过未知机制独立阻断了与KRAS的核苷酸关联。在这里，我们旨在申请化学，结构，细胞和体内方法，以询问SAH-SOS1胡椒如何直接阻塞 KRAS的酶活性，将这种机制与SOS1的自然激动剂进行比较和对比蛋白质，从而了解我们对SOS1/KRAS调节的结构 - 功能的理解和新策略用于对人类癌症中KRA的治疗抑制作用。为了实现这些目标，我们提出了三个实验目标：（1）合成以KRAS相互作用结构域建模的结构增强螺旋的广泛库 SOS1的识别结合决定剂和与KRAS及其致癌突变体的功能相互作用；（2）应用氢 - 居民交换质谱法以阐明SOS1的构象作用蛋白质和SAH-SOS1在KRAS蛋白上的辣椒，从而定义了酶调节的机制；（3）在KRAS驱动的癌症中对细胞和体内测试的优化优化的SAH-SOS1抑制剂以验证作用机理和治疗窗口，并为临床翻译提供了概念验证。通过组合癌症化学生物学和 Walensky实验室的翻译方法，我们的目标是提供新的机械洞察力致癌的KRAS途径并为新的方式提供了新的方式，以使其在癌症中进行治疗益处。