Direct Small Molecule Activation of Pro-apoptotic BAK

摘要： 细胞凋亡是一种高度调节的、能量依赖性的细胞死亡形式，对于肿瘤的发生和发展至关重要。 多细胞生物体内平衡。当失调时，细胞凋亡有助于疾病状态，如 癌症、自身免疫和神经变性。遗传和生物化学研究已经确定了BCL-2 作为线粒体凋亡的关键调节因子的蛋白质家族。促凋亡巴克是一种 线粒体凋亡，其活化导致线粒体外膜透化（MOMP）。 在与仅BH 3激活蛋白的瞬时相互作用后，巴克经历几种构象变化 包括暴露其自身的BH 3结构域，这允许形成巴克寡聚体，负责 MOMP。尽管它在细胞死亡中起着关键作用，但其激活机制尚未完全阐明。 阐明。细胞凋亡小分子调节剂的发展有助于阐明BCL-2在细胞凋亡中的作用。 蛋白质生物化学以及疾病。此外，抗凋亡抑制剂和直接的和选择性的 BAX激活剂已在临床和体内研究中显示出前景。小分子的发展 巴克活性的调节剂将有助于阐明其激活机制，并证明 巴克的药理学控制的治疗潜力。在这里，我们建议开发小分子活化剂， 在基于NMR的片段筛选中鉴定的来自片段7 H8的巴克的序列。我们的具体目标是：1）设计和 合成靶向巴克的7 H8的小分子类似物并确定它们的活化机制，以及2） 评估小分子的生物化学和细胞活性。为了实现我们的第一个目标，我们将使用 HSQC-NMR和计算对接数据，用于虚拟设计和化学合成精细的7 H8 类似物然后，我们将确定他们的亲和力和特异性，使用微量热泳和荧光 极化结合测定。我们将使用氢-氘交换质谱法来确定 与巴克激活相关的构象变化。我们将通过评估来实现第二个目标 使用脂质体荧光释放体外小分子活性，分离的线粒体细胞色素c 释放以及寡聚化和构象变化测定。然后我们将评估基因组中 修饰的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， 该提案将促进我们对促凋亡巴克激活机制的理解，并证明 通过巴克激活药理学诱导细胞凋亡的新范例。在我的指导下， 我相信，通过这些努力，我将能够实现这些目标，并进一步扩大我们对巴克在细胞凋亡中的作用的认识 和人类疾病，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来扩大我的训练， 额外的课程和研讨会，同时通过演讲，赠款和 科学手稿的写作和教学，以促进我作为一个医生科学家的目标。

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.