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 在药物发现工作中，抑制致癌性KRAS是一个高度追求的目标，因为在 〜25％的人类癌症。一个关键的致癌KRAS突变（KRASG12C）在A处含有反应性半胱氨酸 热点位置，是KRAS驱动肿瘤中第四大最普遍的突变，发现尤其高 非小细胞肺癌（NSCLC）中的频率（占RAS突变的40％，总体13％）。兴奋有 在发现和应用共价，Cys12特异性抑制剂的Krasg12c中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有效性，sotorasib（amg510，， Lumakras™）最近获得了FDA批准用于治疗局部晚期或转移性NSCLC。 但是，该类别抑制剂活性的基础机制仍然很少了解 阻碍理性的药物设计工作。为了解决这一知识的差距，我们开发了一种新的荧光 - 基于动力学的方法可以表征蛋白质与这些丙烯酸基抑制剂的反应。 有趣的是，我们发现两种临床化合物AMG510（AMGEN）和MRTX849/ADAGRASIB（Mirati 治疗剂具有明显不同的动力学特性，我们提出要进行详细研究 这里。认识到由致癌的KRAS信号传导和肿瘤发生促进的氧化环境，我们 还评估了细胞中的氧化还原敏感性和氧化状态，发现Krasg12c的Cys12容易容易 氧化。此外，该部位的氧化可防止抑制剂附着，表明氧化还原的修饰 KRASG12C可能构成对AMG510和其他共价抑制剂的抗性机制。其他 未解决的问题仍然存在于现场，包括蛋白质 - 药物加合物的承诺 迈克尔添加反应通过其绑定的化学可逆性，以及如何影响它 通过改变Accrylamide的“弹头”。 AIM 1提案结构和动力学研究与计算 建模和分子动力学模拟，以阐明KRASG12C抑制剂的差异机制 参与，失活和逆转。由于治疗的发展仍然是一个重大障碍 有针对性的抑制策略，目标2和3提案，以调查氧化还原灵敏度之间的联系 KRASG12C和抑制剂效率通过测量重组蛋白的功能，信号相关的输出 生物样品（肺癌细胞系和患者衍生的器官）。对于AIM 2，肺癌细胞 将评估有或不存在抑制剂的可变氧化条件，以进行KRASG12C修改和 下游信号输出；使用具有靶向质膜的高daao遗传构建体 将允许在KRASG12C附近的细胞内的过氧化氢生产中进行时空和剂量控制。 AIM 3，将使用Fresh研究肿瘤氧化还原特性与抑制剂效率之间的关系 携带KRASG12C突变的NSCLC肿瘤标本。累积地，结果将提供改善的不足 毒品的地位，将作为表征和发展未来直接KRAS抑制剂的平台。