Direct Small Molecule Activation of Pro-apoptotic BAK

摘要： 细胞凋亡是一种高度调节的、能量依赖的细胞死亡形式，对发育和 多细胞生物的动态平衡。当调控失调时，细胞凋亡会导致疾病状态，如 癌症、自身免疫和神经退化。遗传学和生物化学研究已经确定了bcl2 作为线粒体凋亡关键调节因子的蛋白质家族。促凋亡的BAK是一种效应因子 线粒体凋亡，其激活导致线粒体外膜通透性(MOMP)。 当BAK与BH3激活蛋白短暂相互作用时，BAK经历了几次构象变化 包括暴露其自身的BH3-结构域，这允许形成BAK低聚物，负责 MOMP。尽管它在导致细胞死亡方面起着关键作用，但它的激活机制还不完全。 已澄清。小分子细胞凋亡调节剂的发展有助于阐明bcl2的作用 蛋白质的生物化学作用以及疾病中的作用。此外，抗凋亡抑制剂和直接的和选择性的 BAX激活剂在临床和体内研究中显示出了良好的前景。小分子的发展 BAK活性的调节剂将有助于阐明其激活机制，并证明 BAK药物控制的治疗潜力。在这里，我们建议开发小分子激活剂 从基于核磁共振的片段筛选中鉴定的片段7H8中获得BAK。我们的具体目标是1)设计和 合成靶向BAK的7H8小分子类似物并确定其活化机理 评估小分子的生化和细胞活性。为了实现我们的第一个目标，我们将使用 HSQC-核磁共振和计算对接数据以虚拟设计和化学合成精细的7H8 类比。然后，我们将使用微量热电泳法和荧光法确定它们的亲和力和特异性。 偏振结合分析。我们将使用氢-重离子交换质谱仪来确定 与BAK激活相关的构象变化。我们将通过评估来实现我们的第二个目标 体外小分子活性脂质体荧光释放法分离线粒体细胞色素c 释放、齐聚和构象变化分析。然后我们将评估在基因上的活动 改良小鼠胚胎成纤维细胞以评估细胞活性和特异性。通过实现这些目标，这 该提案将促进我们对促凋亡BAK激活机制的理解，并展示一种 通过激活BAK以药物方式诱导细胞凋亡的新范式。在我的指导下 导师们，我将能够实现这些目标，并进一步扩大我们对BAK在细胞凋亡中的作用的了解 同时获得结构生物学、生物物理学、细胞生物学、 生物化学和药物化学。我将通过参加科学会议和 额外的课程和工作坊，同时通过演讲、助学金和 撰写科学手稿，并通过教学来实现我作为内科科学家的目标。

Abstract: Apoptosis is a highly regulated, energy dependent form of cell death critical for the development and homeostasis of multicellular organisms. When dysregulated, apoptosis contributes to disease states such as cancer, autoimmunity, and neurodegeneration. Genetic and biochemical studies have established the BCL-2 family of proteins as critical regulators of mitochondrial apoptosis. Pro-apoptotic BAK is an effector of mitochondrial apoptosis whose activation leads to mitochondrial outer membrane permeabilization (MOMP). Upon transient interaction with a BH3-only activator protein, BAK undergoes several conformational changes including exposure of its own BH3-domain, which allows for the formation of BAK oligomers, responsible for MOMP. Despite its critical role in committing a cell to death, its mechanism of activation has not been fully elucidated. The development of small molecule modulators of apoptosis has helped elucidate the role of BCL-2 proteins biochemically as well as in disease. Furthermore, anti-apoptotic inhibitors and direct and selective BAX activators have shown promise in clinical and in vivo studies. The development of small molecule modulators of BAK activity would aid in the elucidation of its activation mechanism as well as demonstrate the therapeutic potential of pharmacologic control of BAK. Here, we propose to develop small molecule activators of BAK from fragment 7H8 identified in an NMR-based fragment screen. Our specific aims are 1) to design and synthesize small molecule analogs of 7H8 targeting BAK and determine their activation mechanism and 2) to evaluate the biochemical and cellular activity of small molecules. To accomplish our first aim, we will use HSQC-NMR and computational docking data to virtually design and chemically synthesize elaborated 7H8 analogs. We will then determine their affinity and specificity using microscale thermophoresis and fluorescence polarization binding assays. We will use hydrogen-deuterium exchange mass spectrometry to determine the conformational changes associated with BAK activation. We will accomplish our second aim by assessing small molecule activity in vitro using liposomal fluorescence release, isolated mitochondrial cytochrome c release, and oligomerization and conformational change assays. We will then assess activity in genetically modified mouse embryonic fibroblasts to assess for cellular activity and specificity. By realizing these aims, this proposal will advance our understanding of the activation mechanism of pro-apoptotic BAK and demonstrate a new paradigm for pharmacologic induction of apoptosis through BAK activation. Under the guidance of my mentors, I will be able to accomplish these goals and further expand our knowledge of BAK's role in apoptosis and human disease while gaining skills and knowledge in structural biology, biophysics, cellular biology, biochemistry, and medicinal chemistry. I will expand my training through attending scientific meetings and additional coursework and workshops while honing my communications skills through presentations, grant and scientific manuscript writing, and teaching to further my goals as a physician scientist.