KRAS G12C: Kinetic and Redox Characterization of Covalent Inhibition

KRAS G12C：共价抑制的动力学和氧化还原表征

• 批准号: 10682167
• 负责人: Sharon L Campbell
• 金额: $ 58.29万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10682167
• 关键词: 3-Dimensional; Acceleration; Acrylamides; Address; Affect; Affinity; Baptist Church; Binding; Biochemical; Biological; Biological Assay; Cancer cell line; Cell Culture Techniques; Cell Line; Cell membrane; Cells; Chemicals; Chronic; Clinical; Clinical Trials; Complex; Comprehensive Cancer Center; Computer Analysis; Computer Models; Crystallography; Cysteine; Data; Development; Drug Design; Environment; Exhibits; FDA approved; Fluorescence; Foundations; Frequencies; Future; Gene Frequency; Genetic; Goals; Guanine Nucleotides; Human; Hydrogen Peroxide; In Situ; In Vitro; KRAS oncogenesis; KRAS2 gene; KRASG12D; Kinetics; Knowledge; Ligands; Ligation; Location; Lung Neoplasms; Malignant Neoplasms; Malignant neoplasm of lung; Measures; Medicine; Methods; Modeling; Modification; Molecular Conformation; Mutate; Mutation; Non-Small-Cell Lung Carcinoma; Nucleotides; Oncogenic; Operative Surgical Procedures; Organoids; Output; Oxidants; Oxidation-Reduction; Pathologic; Patient Care; Patients; Pharmaceutical Preparations; Physiological; Positioning Attribute; Predisposition; Production; Property; Protein Isoforms; Proteins; Publications; RAS genes; Reaction; Recombinant Proteins; Sampling; Series; Signal Transduction; Site; Smoking; Specimen; Spectrum Analysis; Sulfinic Acids; System; Technology; Testing; Therapeutic; Time; Tissues; Treatment Efficacy; Tumor Tissue; Work; adduct; bench to bedside; cell growth; clinical development; clinical efficacy; cold temperature; covalent bond; dosage; drug discovery; experimental study; forest; improved; inhibitor; lung cancer cell; molecular dynamics; mutant; next generation sequencing; novel strategies; oxidation; preclinical efficacy; prevent; ras Proteins; resistance mechanism; response; scaffold; spatiotemporal; targeted treatment; therapy development; therapy resistant; thioether; treatment response; tumor; tumorigenesis

ABSTRACT Inhibition of oncogenic KRAS is a highly pursued goal in drug discovery efforts, as RAS mutations are found in ~25% of human cancers. One key oncogenic KRAS mutation (KRASG12C) contains a reactive cysteine at a hotspot location, is the fourth most prevalent mutation in KRAS-driven tumors and is found at particularly high frequency (40% of RAS mutations, 13% overall) in non-small cell lung cancer (NSCLC). Excitement has accelerated rapidly around the discovery and application of covalent, Cys12-specific inhibitors of KRASG12C in recent years as these compounds have shown efficacy in advanced clinical trials, with Sotorasib (AMG510, LUMAKRAS™) recently receiving FDA approval for treatment of locally advanced or metastatic NSCLC. However, the kinetic mechanisms underlying the activity of this class of inhibitors remain poorly understood impeding rational drug design efforts. To address this gap in knowledge, we developed a new fluorescence- based approach to kinetically characterize the reactions of the proteins with these acrylamide-based inhibitors. Intriguingly, we find that the two clinical compounds, AMG510 (Amgen) and MRTX849/Adagrasib (Mirati Therapeutics) possess distinctly different kinetic properties, which we propose to investigate in further detail here. Recognizing the oxidative environment promoted by oncogenic KRAS signaling and tumorigenesis, we also evaluated the redox sensitivity and oxidative status in cells and found that Cys12 of KRASG12C is prone to oxidation. Moreover, oxidation at this site prevents inhibitor attachment, suggesting that redox modification of KRASG12C may constitute a mechanism of resistance to AMG510 and other covalent inhibitors. Other unanswered questions remain in the field, including the propensity of the protein-drug adducts to undergo chemical reversibility of the Michael addition reactions through which they bind, and how this could be affected by altered acrylamide “warheads”. Aim 1 proposes structural and kinetic studies combined with computational modeling and molecular dynamics simulations to elucidate the differential mechanisms of KRASG12C inhibitor engagement, inactivation and reversal. As development of treatment resistance remains a significant hurdle for targeted inhibition strategies, Aims 2 and 3 propose to investigate the linkage between the redox sensitivity of KRASG12C and inhibitor efficacy by measuring functional, signaling-relevant outputs for recombinant proteins and biological samples (lung cancer cell lines and patient-derived organoids). For Aim 2, lung cancer cells under variably oxidizing conditions, with and without inhibitor present, will be assessed for KRASG12C modifications and downstream signaling outputs; use of HyPer-DAAO genetic constructs with targeting to the plasma membrane will allow spatiotemporal and dosage control over hydrogen peroxide production within cells, near KRASG12C. In Aim 3, the relationship between tumor redox properties and inhibitor efficacy will be investigated using fresh NSCLC tumor specimens carrying KRASG12C mutations. Cumulatively, the results will provide improved under- standing of the drugs and will serve as a platform for characterizing and developing future direct KRAS inhibitors.

抽象的 抑制致癌 KRAS 是药物发现工作中高度追求的目标，因为 RAS 突变存在于 约 25% 的人类癌症中，一个关键的致癌 KRAS 突变 (KRASG12C) 含有一个反应性半胱氨酸。 热点位置，是 KRAS 驱动的肿瘤中第四个最常见的突变，并且发现率特别高 非小细胞肺癌 (NSCLC) 中的频率（RAS 突变为 40%，总体为 13%）令人兴奋。 围绕 KRASG12C 共价 Cys12 特异性抑制剂的发现和应用迅速加速 近年来，这些化合物在高级临床试验中显示出功效，其中 Sotorasib（AMG510、 LUMAKRAS™）最近获得 FDA 批准用于治疗局部晚期或转移性 NSCLC。 然而，此类抑制剂活性的动力学机制仍知之甚少。 为了解决这一知识差距，我们开发了一种新的荧光- 基于动力学的方法来表征蛋白质与这些基于丙烯酰胺的抑制剂的反应。 有趣的是，我们发现两种临床化合物 AMG510 (Amgen) 和 MRTX849/Adagrasib (Mirati 疗法）具有明显不同的动力学特性，我们建议对其进行更详细的研究 认识到致癌 KRAS 信号传导和肿瘤发生促进的氧化环境，我们。 还评估了细胞内的氧化还原敏感性和氧化状态，发现KRASG12C的Cys12容易发生 此外，该位点的氧化阻止了抑制剂的附着，表明氧化还原修饰。 KRASG12C可能构成对AMG510和其他共价抑制剂的耐药机制。 该领域仍然存在未解答的问题，包括蛋白质-药物加合物发生反应的倾向 它们结合的迈克尔加成反应的化学可逆性，以及如何影响它 通过改变丙烯酰胺“弹头”，目标 1 提出了与计算相结合的结构和动力学研究。 建模和分子动力学模拟阐明 KRASG12C 抑制剂的差异机制 参与、失活和逆转治疗耐药性的发展仍然是一个重大障碍。 靶向抑制策略，目标 2 和建议 3 研究氧化还原敏感性之间的联系 通过测量重组蛋白的功能性、信号相关输出和 KRASG12C 和抑制剂功效 生物样品（肺癌细胞系和患者来源的类器官）对于目标 2，肺癌细胞。 将针对 KRASG12C 修饰评估不同的氧化条件（无论是否存在抑制剂） 下游信号输出；使用 HyPer-DAAO 基因构建体靶向质膜 将允许对 KRASG12C 附近细胞内过氧化氢的产生进行时空和剂量控制。 目标 3，将使用新鲜的材料来研究肿瘤氧化还原特性与抑制剂功效之间的关系 携带 KRASG12C 突变的 NSCLC 肿瘤样本累积起来，结果将提供改善的不足。 药物的地位，并将作为表征和开发未来直接 KRAS 抑制剂的平台。