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.

摘要 抑制致癌的KRAS是药物发现工作中高度追求的目标，因为在 约25%的人类癌症。一个关键的致癌突变KRAS(KRASG12C)在 热点位置，是KRAS驱动的肿瘤中第四大最常见的突变，被发现的频率特别高 在非小细胞肺癌(NSCLC)中的频率(40%的RAS突变，总共13%)。令人兴奋的是 随着KRASG12C共价、Cys12特异性抑制剂的发现和应用， 近年来，随着这些化合物在高级临床试验中显示出有效性，Sotorasib(AMG510， LUMAKRAS™)最近获得美国食品和药物管理局批准，用于治疗局部晚期或转移性非小细胞肺癌。 然而，这类抑制剂活性背后的动力学机制仍然知之甚少。 阻碍合理的药物设计努力。为了解决这一知识缺口，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(安进)和MRTX849/Adagrasib(Mirati 治疗)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 这里。认识到致癌KRAS信号促进的氧化环境与肿瘤的发生 还评估了细胞对氧化还原的敏感性和氧化状态，发现KRASG12C的Cys12容易 氧化。此外，在这个位置的氧化阻止了抑制剂的附着，这表明氧化还原修饰 KRASG12C可能构成了对AMG510和其他共价抑制剂的抗性机制。其他 该领域仍有一些未解答的问题，包括蛋白质-药物加合物的倾向。 通过它们结合的迈克尔加成反应的化学可逆性，以及这可能如何受到影响 被改变的丙烯酰胺“弹头”。目标1建议将结构和动力学研究与计算相结合 KRASG12C缓蚀剂差异化机理的建模和分子动力学模拟 接合、失活和反转。由于治疗耐药性的发展仍然是 靶向抑制策略，目标2和3建议调查氧化还原敏感性之间的联系 KRASG12C和抑制物功效通过测量功能，信号相关的重组蛋白和 生物样本(肺癌细胞系和病人来源的有机物)。对于AIM 2，肺癌细胞在 在存在和不存在缓蚀剂的情况下，将评估可变氧化条件，以进行KRASG12C修改和 下游信号输出；以质膜为靶点的Hyper-DAAO基因构建物的使用 将允许在KRASG12C附近的细胞内对过氧化氢的产生进行时空和剂量控制。在……里面 目的3、研究肿瘤氧化还原特性与抑制物疗效的关系。 携带KRASG12C突变的非小细胞肺癌肿瘤标本。总体而言，这些结果将在以下方面有所改善- 药物的地位，并将作为一个平台，以表征和开发未来的直接KRAS抑制剂。