KRAS G12C: Kinetic and Redox Characterization of Covalent Inhibition

摘要 致癌KRAS的抑制是药物发现努力中高度追求的目标，因为RAS突变发现于 约25%的人类癌症。一个关键的致癌KRAS突变（KRASG 12 C）在2010年12月21日至2011年12月31日期间包含一个反应性半胱氨酸。 热点位置，是KRAS驱动的肿瘤中第四大最普遍的突变，并且在特别高的 非小细胞肺癌（NSCLC）中RAS突变的发生率（40%，总体13%）。兴奋一直 KRASG 12 C的共价Cys 12特异性抑制剂的发现和应用迅速加速， 近年来，由于这些化合物在高级临床试验中显示出功效，使用Sotorasib（AMG 510， LUMAKRAS™）最近获得FDA批准用于治疗局部晚期或转移性NSCLC。 然而，这类抑制剂活性的动力学机制仍然知之甚少 阻碍了合理的药物设计。为了填补这一知识空白，我们开发了一种新的荧光- 的方法来动力学表征的蛋白质与这些丙烯酰胺基抑制剂的反应。 有趣的是，我们发现两种临床化合物，AMG 510（Amgen）和MRTX 849/Adagrasib（Mirati 治疗学）具有明显不同的动力学性质，我们建议进一步详细研究 这里.认识到致癌KRAS信号和肿瘤发生促进的氧化环境，我们 还评估了细胞的氧化还原敏感性和氧化状态，发现KRASG 12 C的Cys 12倾向于 氧化此外，该位点的氧化阻止了抑制剂的附着，这表明， KRASG 12 C可能构成对AMG 510和其他共价抑制剂的耐药机制。其他 在该领域中仍然存在未回答的问题，包括蛋白质-药物加合物经历 它们结合的迈克尔加成反应的化学可逆性，以及如何影响这种可逆性 改变丙烯酰胺的“弹头”目标1提出结构和动力学研究结合计算 模型和分子动力学模拟，以阐明KRASG 12 C抑制剂的差异机制 参与、失活和逆转。由于治疗耐药性的发展仍然是一个重大障碍， 靶向抑制策略，目的2和3提出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， 生物样品（肺癌细胞系和患者来源的类器官）。对于目标2，肺癌细胞在 将评估在存在和不存在抑制剂的情况下的氧化条件下的KRASG 12 C修饰， 下游信号传导输出;使用靶向质膜的Hyper-DAAO遗传构建体 将允许时空和剂量控制过氧化氢生产细胞内，附近KRASG 12 C。在 目的3，肿瘤氧化还原性质和抑制剂功效之间的关系将使用新鲜的 携带KRASG 12 C突变的NSCLC肿瘤标本。累积起来，这些结果将改善- 这些药物的地位，并将作为表征和开发未来直接KRAS抑制剂的平台。

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.