Dissecting and Targeting Deregulated Mitochondrial Apoptosis in Human Cancer

项目总结/摘要 BCL-2蛋白参与了一个动态的相互作用网络，决定了细胞的生存或死亡。 这一重要信号通路的失调是人类癌症发病机制和对肿瘤耐药性的基础。 治疗这个R35研究计划的目标是阐明基本的蛋白质相互作用机制 驱动凋亡程序，并利用这些见解来开发下一代癌症治疗方法。超过 在过去五年的R35支持中，我们应用了新的化学工具和一系列分析技术， 实现机制的发现，揭示了新的药物结合位点和化合物，以他们为目标。我们 发现在促凋亡BAX和抗凋亡MCL-1和BFL-1中不同半胱氨酸的共价修饰 差异调节它们的凋亡功能。我们对模拟这些翻译后的共价配体的追求 这些修饰产生了用于癌症治疗的原型BAX激活剂和MCL-1和BFL-1抑制剂。 解读BAX和巴克如何被直接激活，以及它们的构象机制 从潜在单体到有毒线粒体寡聚体的转化也是我们工作的主要焦点。 事实上，BAX和巴克死亡通道的难以捉摸的结构代表了细胞凋亡的“圣杯 research.我们最近产生了第一个全长同源BAX寡聚体（BAXO），其结构符合- 功能表征，提供了一个功能BAXO的大分子组织一瞥 物种BAXO及其突变体使我们能够精确定位BAX每一步的结构决定因素， 激活途径，从而为细胞凋亡的药理学激活提供新的控制点。除了 解剖这些高优先级，典型的BCL-2蛋白质相互作用，我们已经开发了蛋白质组学工具， 鉴定非典型靶标，最近发现MCL-1直接与脂肪酸氧化相互作用 酶VLCAD，揭示了MCL-1在细胞凋亡和代谢调节交叉点的双重作用。我们 假设MCL-1驱动癌症依赖于凋亡抑制和脂肪酸代谢， 最大限度地提高病理存活率，这可能解释了为什么MCL-1是最广泛表达的抗凋亡蛋白。 人类癌症中的蛋白质。在这里，我们建立在我们最新的机械见解，以询问一系列的 BCL-2家族相互作用驱动人类癌症，并挖掘每一个机会来颠覆它们。 具体来说，我们的下一组R35目标是：（1）确定介导的结构和功能决定因素 线粒体凋亡的“执行阶段”;（2）解决BAX寡聚体的结构;（3）表征 MCL-1在细胞凋亡和癌症代谢的交叉点中的非典型作用;以及（4）促进 BCL-2家族分子调节剂作为人类肿瘤的下一代疗法的开发和体内测试 癌我们使用跨化学，结构生物学， 蛋白质组学、生物化学、细胞生物学和体内测试。作为一名化学生物学家和儿科肿瘤学家，我 致力于将我们的新机制见解转化为复发性和难治性癌症的新疗法。

PROJECT SUMMARY/ABSTRACT BCL-2 proteins participate in a dynamic interaction network that determines whether a cell will live or die. Deregulation of this essential signaling pathway underlies the pathogenesis of human cancer and resistance to treatment. The goal of this R35 research program is to elucidate the fundamental protein interaction mechanisms that drive the apoptotic program and harness these insights to develop next-generation cancer treatments. Over the last five years of R35 support, we applied novel chemical tools and a host of analytical technologies to achieve mechanistic discoveries that revealed new druggable binding sites and compounds to target them. We found that covalent modification of distinct cysteines in pro-apoptotic BAX and anti-apoptotic MCL-1 and BFL-1 differentially regulate their apoptotic functions. Our pursuit of covalent ligands that mimic these post-translational modifications are yielding prototype BAX activators and MCL-1 and BFL-1 inhibitors for cancer therapy. Deciphering how BAX and BAK are directly activated, and the conformational mechanisms that underlie their conversion from latent monomers into toxic mitochondrial oligomers, has also been a major focus of our work. Indeed, the elusive structures of the BAX and BAK death channels represent the “holy grail” of apoptosis research. We recently generated the first full-length homogeneous BAX oligomer (BAXO) amenable to structure- function characterizations, providing a glimpse into the macromolecular organization of a functional BAXO species. BAXO and its mutants are enabling us to pinpoint the structural determinants for each step of the BAX- activation pathway and thus inform new control points for pharmacologic activation of apoptosis. In addition to dissecting these high-priority, canonical BCL-2 protein interactions, we have developed proteomic tools to identify non-canonical targets and recently found that MCL-1 directly interacts with the fatty acid oxidation enzyme VLCAD, revealing a dual role for MCL-1 at the intersection of apoptosis and metabolic regulation. We hypothesize that MCL-1-driven cancers rely on both apoptotic suppression and fatty acid metabolism to maximize pathologic survival, potentially explaining why MCL-1 is the most widely expressed anti-apoptotic protein across human cancers. Here, we build on our newest mechanistic insights to interrogate a spectrum of BCL-2 family interactions that drive human cancer and mine each opportunity to pharmacologically subvert them. Specifically, our next set of R35 goals are: (1) identify the structural and functional determinants that mediate the “execution phase” of mitochondrial apoptosis; (2) solve the structure of a BAX oligomer; (3) characterize the non-canonical role of MCL-1 at the intersection of apoptosis and cancer metabolism; and (4) advance the development and in vivo testing of BCL-2 family molecular modulators as next-generation therapies for human cancer. We tackle these goals using multidisciplinary approaches that span chemistry, structural biology, proteomics, biochemistry, cell biology, and in vivo testing. As a chemical biologist and pediatric oncologist, I am committed to transforming our fresh mechanistic insights into new therapies for relapsed and refractory cancers.