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

使用 ( )-Griffipavixanthone 衍生物阐明 B-Raf 二聚化抑制机制

• 批准号: 9910705
• 负责人: Michael J Smith
• 金额: $ 4.55万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9910705
• 关键词: Accounting; Address; Affinity; American Cancer Society; Attenuated; BAY 54-9085; Binding; Biological; Cell Line; Cell Membrane Permeability; Cell Proliferation; Cells; Cessation of life; Chemistry; Colon Carcinoma; Colorectal Cancer; Communities; Computer software; Cyclization; Data; Development; Dimerization; Docking; Dose; Ethers; Evaluation; Event; FDA approved; Face; Guanosine Triphosphate Phosphohydrolases; HCT-15; Human; In Vitro; Investigation; KRAS2 gene; Libraries; Life; Life Expectancy; MEKs; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Malignant neoplasm of thyroid; Mediating; Melanoma Cell; Methodology; Multiple Myeloma; Mutate; Mutation; National Cancer Institute; Natural Products; Oncogenic; Oxides; Pathway interactions; Pharmaceutical Preparations; Prodrugs; Proteins; Publishing; Ras Inhibitor; Ras/Raf; Reaction; Reaction Time; Regulation; Reporting; Research; Research Proposals; Resistance; Signal Transduction; Structural Models; Structure; Surface; Surface Plasmon Resonance; Testing; Time; Validation; Variant; Xanthones; analog; anticancer activity; anticancer research; cancer cell; cancer diagnosis; chemical synthesis; colon cancer cell line; design; dimer; drug development; enantiomer; experimental study; in vivo; inhibitor/antagonist; insight; interest; lipophilicity; melanoma; metabolomics; mutant; novel; oxidation; prevent; quinone methide; raf Kinases; ras Oncogene; receptor; response; statistics; tool

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.

项目摘要 使用（+） - griffipavixanthone衍生物阐明B-RAF二聚化抑制的机制 RAS，RAF，MEK和ERK是致癌表达的常见热点，因为它们是参与A的蛋白质 高度调节的途径控制细胞增殖。单个突变会导致被劫持的单元格子指数 复制而无需调节。开发工具来探测，理解和防止这些突变的影响 引起了极大的兴趣，因为目前没有FDA批准的药物抑制突变体RAS，一种癌基因 所有人类癌症的大约1/3。 FDA批准了突变B-RAF的抑制剂很少。 但是，由于获得阻力，预期寿命仅延长3-8个月。 （+） - griffipavixanthone（GPX）， 我们可以通过非对称合成很容易访问的二聚体黄体天然产品已证明 在各种细胞系中的抗癌活性。特别是，我们发现具有K-的癌细胞中最大效力 RASG12和B-RAFV600E突变。与FDA批准的药物索拉非尼相比，GPX证明了 较低的GI50和类似或更高的LC50。我们发现B-RAF二聚体是一个重要的事件介导的 通过RAS在细胞增殖中，（+）-GPX处理后，会抑制细胞增殖。有趣的是，回应更大 在（+） - GPX中，比其非自然（ - ）对映异构体具有抑制B-RAF二聚体的响应时间很长（18 h）。 目的：拟议的研究将（1）扩展在关键的不对称反应上并采用晚期 功能化以生成复合库，以了解B-RAF二聚体的延迟响应时间 抑制作用，正如我们认为GPX可能是一种前药，可将细胞内氧化至反应性P- 喹酮甲基。这些进步将对药物开发和抑制剂产生重大影响 设计和化学界； （2）确定动作机理，因为它会衰减受管制， 被劫持的路径。这将对社区和研究这种致癌的人产生更大的影响 途径； （3）精心制作的GPX更有效，像药物一样和特异性。提出三个目标 解决上述目标。 AIM 1：具有不同功能，GPX衍生的类似物库 具有增加亲脂性和氧化变体的前药将在时间依赖性中合成和评估 B-RAF二聚化抑制的方式。目标2：完整的细胞内效应和 GPX及其衍生物的下游信号将在WT和RAF/RAS突变细胞中进行。我们也会 研究在体外和体内直接直接GPX-RAS/RAF表面结合。此外，一个公正的下拉实验 将用于确认细胞内靶标。目标3：我们将开发GPX衍生物的结构模型和 包括K-RAS突变体在内的候选受体，并使用此信息来设计更高的亲和力类似物。 总体而言，这项研究将影响化学合成，前药开发和代谢组学，并将提供 有关RAS/RAF/MEK/ERK的研究和工具的重要信息，这是最失调的致癌途径。