Elucidating the Mechanism of B-Raf Dimerization Inhibition Using (+)-Griffipavixanthone Derivatives

项目摘要 （+）-grifipavixanthone衍生物抑制B-Raf二聚化的机理研究 Ras、Raf、MEK和ERK是致癌表达的常见热点，因为它们是参与肿瘤发生的蛋白质。 控制细胞增殖的高度调节途径。一个突变就能导致细胞被劫持成指数级 无规则复制。开发工具来探测、理解和预防这些突变的影响 由于目前还没有FDA批准的抑制突变型Ras（一种致癌基因）的药物， 约占人类所有癌症的1/3。FDA已经批准了一些突变B-Raf的抑制剂; 然而，随着抗药性的获得，预期寿命仅延长3-8个月。（+）-Griffipavixanthone（GPX）， 一种二聚氧杂蒽酮天然产物，我们可以很容易地通过不对称合成，已经证明， 在各种细胞系中的抗癌活性。特别是，我们已经发现在癌细胞中K- RasG 12和B-RafV 600 E突变。当与FDA批准的药物索拉非尼相比时，GPX显示出 GI 50较低，LC 50相似或较高。我们已经发现，B-Raf二聚化，一个重要的事件介导 Ras在细胞增殖中的作用在用（+）-GPX处理后被抑制。有趣的是， 在（+）-GPX中，其非天然（-）对映体抑制B-Raf二聚化的响应时间较长（18 h）。 目的：本研究将（1）拓展关键的不对称反应， 功能化以产生化合物文库以了解B-Raf二聚化的延迟响应时间 抑制，因为我们认为GPX可能是一种前药，其经历细胞内氧化成反应性β- 醌甲基化物这些进展将对药物开发和抑制剂产生重大影响。 设计，并在化学界;（2）确定的作用机制，因为它减弱了放松管制， 被劫持的道路这将对社区和研究这种致癌基因的人产生更广泛的影响。 （3）使GPX更有效、更像药物、更有靶点特异性。提出了三个目标， 实现上述目标。目的1：具有不同功能的GPX衍生类似物库 将合成具有增加的亲脂性的前药和氧化变体，并以时间依赖性方法进行评价。 B-Raf二聚化抑制的方式。目的2：细胞内效应的完整表征， 将在WT和Raf/Ras突变细胞中进行GPX及其衍生物的下游信号传导。我们还将 体外和体内研究GPX-Ras/Raf表面直接结合。此外，无偏下拉实验 将用于确认细胞内靶点。目标3：我们将开发GPX衍生物的结构模型， 候选受体，包括K-Ras突变体，并使用这些信息来设计更高亲和力的类似物。 总的来说，这项研究将影响化学合成，前药开发和代谢组学，并将提供 Ras/Raf/MEK/ERK是最不受调节的致癌通路，是研究Ras/Raf/MEK/ERK的重要信息和工具。

Project Summary Elucidating the Mechanism of B-Raf Dimerization Inhibition Using (+)-Griffipavixanthone Derivatives Ras, Raf, MEK, and ERK are common hotspots for oncogenic expression as they are proteins involved in a highly-regulated pathway controlling cell proliferation. A single mutation can result in a hijacked cell exponentially replicating without regulation. Developing tools to probe, understand, and prevent the effects of these mutations are of great interest as there are currently no FDA-approved drugs that inhibit mutant Ras, an oncogene accounting for approximately 1/3 of all human cancers. The FDA has approved few inhibitors of mutant B-Raf; however, life expectancy is extended for only 3-8 months as resistance is acquired. (+)-Griffipavixanthone (GPX), a dimeric xanthone natural product that we can readily access by asymmetric synthesis, has demonstrated anticancer activity in various cell lines. In particular, we have found maximal potency in cancer cells with K- RasG12 and B-RafV600E mutations. When compared to the FDA-approved drug, sorafenib, GPX demonstrated a lower GI50 and similar or higher LC50. We have found that B-Raf dimerization, an important event mediated by Ras in cell proliferation, is inhibited upon treatment with (+)-GPX. Interestingly, the response is greater in (+)-GPX than its unnatural (-) enantiomer with a long response time for inhibition of B-Raf dimerization (18 h). Objectives: The proposed study will (1) expand on the key asymmetric reaction and employ late-stage functionalization to generate a compound library to understand the delayed response time of B-Raf dimerization inhibition, as we believe that GPX is likely a prodrug that undergoes intracellular oxidation to a reactive p- quinone methide. These advancements would have considerable impact on drug development and inhibitor design, and in the chemistry community; (2) ascertain the mechanism of action as it attenuates a deregulated, hijacked pathway. This will have high broader impact on the community and those studying this oncogenic pathway; (3) elaborate GPX to be more potent, drug-like, and target specific. Three aims are proposed to address the aforementioned objectives. Aim 1: A library of analogues with varying functionality, GPX-derived prodrugs with increased lipophilicity, and oxidized variants will be synthesized and evaluated in a time-dependent manner for B-Raf dimerization inhibition. Aim 2: A complete characterization of intracellular effects and downstream signaling by GPX and its derivatives will be performed in WT and Raf/Ras mutant cells. We will also study direct GPX-Ras/Raf surface binding in vitro and in vivo. Additionally, an unbiased pull-down experiment will be used to confirm intracellular targets. Aim 3: We will develop structural models of GPX derivatives and candidate receptors, including K-Ras mutants, and use this information to design higher affinity analogues. Overall, this research will impact chemical synthesis, prodrug development and metabolomics, and will provide important information on and tools for study of Ras/Raf/MEK/ERK, the most deregulated oncogenic pathway.