Biophysical and Mechanistic Determinants for Cancer Cell Import of Hydrocarbon-Stapled Peptides

项目总结 解除调控的细胞内蛋白相互作用介导了一个复杂的病理信号事件网络， 会导致人类癌症。用于调节致癌的大、平和复杂界面的药物的发展 信号蛋白仍然是一个巨大的挑战，并激发了传统的小分子 分子发现。尽管定义分子握手的正是多肽结构 蛋白质非常适合于调节这样的信号事件，即蛋白质上下文中的结构化多肽 当与整体分离时，通常会失去生物活性的形状。我们之前开发了碳氢化合物装订 通过插入化学交联键来概括-螺旋相互作用基序的自然形状的多肽。 装订的多肽结构稳定，耐酶，并保留了参与其生物作用的能力 具有天然效力和特异性的靶标。出乎意料的是，部分装订的-螺旋肽也是细胞- 渗透性，为解剖和潜在的麻醉病理打开了一扇全新的模式之门 癌症中的蛋白质相互作用。然而，尽管十年来在创造新的研究工具方面取得了进展，以 研究和靶向致病蛋白，结构肽作为一种新的治疗方法的真正希望 由于我们对两个基本问题的理解非常有限，癌症的平台一直受到阻碍：(1) 什么生物物理特性决定了装订的多肽是否会被癌细胞摄取？(2)什么是 细胞通透性装订多肽的细胞输入和细胞内释放的明确分子机制？ 这些问题的答案不仅具有改变这些研究工具和原型的潜力 治疗药物进入真正的抗癌药物的武器库，但也将提供关键的新洞察力 囊泡运输的细胞过程。例如，某些癌细胞通过以下方式强制代谢永生 通过胞饮作用输入途径上调蛋白质营养吸收--这是一种可以利用的机制 实现以结构多肽为基础的癌症药物的治疗窗口。在这里，我建议双管齐下 利用我们在装订多肽设计和应用方面的丰富经验的研究计划，但存在分歧 从我们传统的蛋白质靶向研究转向关注为什么和如何不同的装订多肽 进入细胞内环境。为了实现我们的目标，我们将(1)使用生物统计和计算 收集哪些生物物理参数在我们的装订文库中赋予细胞外透性的方法 多肽，以及(2)执行基于全基因组CRISPR的筛查以识别并随后检查那些细胞 另一种是削弱和增强装订多肽的进口途径的成分。通过将我们的 实验室在致癌蛋白与拟议的生物统计学相互作用的化学生物学方面的基础， CRISPR筛查和癌细胞进口验证研究，我们希望在理解上取得新的突破 关于如何利用装订的多肽及其摄取机制来治疗癌症的研究。

PROJECT SUMMARY Deregulated intracellular protein interactions mediate a complex network of pathologic signaling events that drive human cancer. The development of drugs to modulate the large, flat, and complex interfaces of oncogenic signaling proteins remains a formidable challenge and has inspired alternative approaches to traditional small molecule discovery. Although the very peptide structures that define the molecular handshakes between proteins are ideally suited to modulate such signaling events, structured peptides in the context of a protein typically lose their bioactive shape when isolated from the whole. We previously developed hydrocarbon-stapled peptides that recapitulate the natural shape of -helical interaction motifs by insertion of chemical crosslinks. Stapled peptides are structurally-stable, protease-resistant, and retain the capacity to engage their biological targets with natural potency and specificity. Unexpectedly, select stapled -helical peptides are also cell- permeable, opening the door to an entirely new modality for dissecting and potentially drugging pathologic protein interactions in cancer. However, despite a decade of progress in the creation of new research tools to investigate and target disease-causing proteins, the true promise of structured peptides as a novel therapeutic platform for cancer has been hampered by our very limited understanding of two fundamental questions: (1) What biophysical properties dictate whether a stapled peptide will be taken up by a cancer cell? (2) What is the explicit molecular mechanism of cellular import and intracellular release for cell-permeable stapled peptides? The answers to these questions not only carry the potential to transform these research tools and prototype therapeutics into an arsenal of bona fide cancer drugs, but will also provide critical new insight into the essential cellular process of vesicle transport. For example, certain cancer cells enforce metabolic immortality by upregulating protein nutrient uptake via the pinocytosis import pathway – a mechanism that could be leveraged to achieve a therapeutic window for structured peptide-based cancer drugs. Here, I propose a two-pronged research plan that harnesses our deep experience with stapled peptide design and application, but diverges from our traditional protein targeting research to instead focus on the why and how distinct stapled peptides access the intracellular environment. To achieve our goals, we will (1) employ biostatistical and computational methods to glean what biophysical parameters confer cellular penetrance among our libraries of stapled peptides, and (2) perform a genome-wide CRISPR-based screen to identify and then vet those cellular components that alternatively impair and enhance the import pathway for stapled peptides. By integrating our laboratory’s foundation in the chemical biology of oncogenic protein interactions with the proposed biostatistical, CRISPR screening, and cancer cell import validation studies, we hope to break new ground in our understanding of just how stapled peptides and their uptake mechanisms can be harnessed for therapeutic benefit in cancer.