Dissecting and Targeting Deregulated Mitochondrial Apoptosis in Human Cancer

项目摘要/摘要 BCL-2蛋白参与了一个动态的相互作用网络，该网络决定了一个细胞的生存或死亡。 这一重要信号通路的失控是人类癌症发生和抗药性的基础 治疗。R35研究计划的目标是阐明蛋白质相互作用的基本机制 这推动了细胞凋亡计划，并利用这些见解开发下一代癌症治疗方法。完毕 在R35支持的最后五年中，我们应用了新的化学工具和大量分析技术来 实现机械性发现，揭示新的可药物结合部位和化合物以靶向它们。我们 发现不同半胱氨酸在促凋亡的Bax和抗凋亡的MCL-1和BFL-1中的共价修饰 不同地调节它们的凋亡功能。我们对模仿这些翻译后反应的共价配体的追求 改良正在产生用于癌症治疗的Bax激活剂和MCL-1和BFL-1抑制剂的原型。 破译BAX和BAK是如何被直接激活的，以及它们背后的构象机制 从潜伏的单体到有毒的线粒体低聚物的转化也是我们工作的一个主要重点。 事实上，BAX和BAK死亡通道的难以捉摸的结构代表了细胞凋亡的“圣杯” 研究。我们最近合成了第一个全长均相Bax齐聚物(BAXO)，它具有结构- 功能表征，提供了对功能性BAXO的大分子组织的一瞥 物种。BAXO及其突变体使我们能够精确定位BAX- 激活途径，从而为药物激活细胞凋亡提供新的控制点。除了……之外 剖析这些高度优先的、规范的bcl2蛋白相互作用，我们开发了蛋白质组学工具来 识别非典型靶点，最近发现MCL-1与脂肪酸氧化直接相互作用 VLCAD酶，揭示了MCL-1在细胞凋亡和代谢调节的交叉点上的双重作用。我们 假设MCL-1驱动的癌症依赖于凋亡抑制和脂肪酸代谢来 最大限度地提高病理存活率，潜在地解释了为什么MCL-1是表达最广泛的抗细胞凋亡 人类癌症中的蛋白质。在这里，我们基于我们最新的机械论见解来审问一系列 BCL-2家族的相互作用推动了人类癌症，并挖掘了每一个从药物上颠覆它们的机会。 具体地说，我们的下一组R35目标是：(1)确定中介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在细胞凋亡和肿瘤代谢的交叉点上的非典型作用；以及(4)促进 新一代人类治疗药物bcl2家族分子调节剂的研制及体内试验 癌症。我们使用跨越化学、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 致力于将我们新的机械洞察力转化为复发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.