Chemical Biology Approaches for Studying Circadian Rhythms

项目概要/摘要 生物钟是一种时间跟踪系统，它允许生物体适应一天中的时间，并驱动许多生物体。 细胞功能。它们的改变会导致各种疾病，包括癌症、心脏病和代谢性疾病。 紊乱在这项工作中，我们建议开发一个化学生物学工具箱，包括成像和 蛋白质靶向平台，以促进在分子水平上研究昼夜节律， 是不可能的。核心时钟由转录-翻译反馈回路组成， 蛋白质组分，包括旁系同源物。已经观察到启动子活性和蛋白质之间的差异， 翻译，核心时钟组件对刺激的反应之间，以及补偿机制， 从击倒和淘汰的策略。为了填补这些空白，并促进对 分子钟，我们将使用化学生物学为基础的策略：1。产生正交荧光 支架跟踪启动子活性和蛋白质翻译的多个昼夜节律基因在一个平行的，高含量 方式; 2.）开发基于小分子和蛋白质的工具，以直接靶向昼夜节律蛋白及其 交互. 对于分子钟的研究，跟踪昼夜节律和靶向核心时钟蛋白是至关重要的。 动态和选择性的方式。基于萤火虫荧光素酶的报告基因已被用于评估启动子活性。 单个昼夜节律基因，大多数基于蛋白质的研究涉及来自单个发光基因的细胞。 小鼠模型我们将使用正交荧光探针（纳米灯）来开发多信号 同时跟踪和关联启动子活性和蛋白质翻译关系的平台， 多重基因昼夜节律蛋白的扰动对于理解机制也是必不可少的，包括 角色扮演目前，除了遗传方法之外，几乎没有其他选择，这可能导致单方面的 改变和激活网络补偿机制。分子工具提供了直接靶向 时钟蛋白的功能成分和/或它们的相互作用。虽然小分子呈现出 尽管存在有吸引力的方法，但直接靶向核心时钟蛋白的相对较少。因此，我们建议生成 询问生物钟系统的新试剂：我们将重新使用经过验证的时钟蛋白结合小分子 分子通过合成将其转化为蛋白质降解剂（PROTAC），并使用酵母表面展示， 识别结合昼夜节律蛋白并阻止特定相互作用的纳米抗体。这些方法加在一起， 提出了一个强大的手段来了解生理时钟的机制，并可用于各种 模型和疾病研究，包括发现新的治疗靶点。

Project Summary/Abstract Circadian clocks are time-tracking systems that allow organisms to adapt to the time of day, and drive many cellular functions. Their alteration can lead to various diseases, including cancer, heart disease, and metabolic disorders. In this work, we propose development of a chemical biology toolbox, consisting of both imaging and protein-targeting platforms to facilitate studies of circadian rhythms at the molecular level that have otherwise not been possible. The core clock is comprised of a transcriptional-translational feedback loop with multiple protein components, including paralogs. Disparities have been observed between promoter activity and protein translation, among responses of core clock components to stimuli, and compensatory mechanisms resulting from knock-down and knock-out strategies. To address these gaps and facilitate additional studies of the molecular clock, we will use chemical biology-based strategies to: 1.) generate orthogonal chemiluminescent scaffolds to track promoter activity and protein translation of multiple circadian genes in a parallel, high-content manner; and 2.) develop small molecule and protein-based tools to directly target circadian proteins and their interactions. For studies of the molecular clock, it is essential to track circadian rhythms and target core clock proteins in a dynamic and selective manner. Firefly luciferase-based reporters have been used to assess promoter activity of individual circadian genes, and most protein-based studies involve cells derived from a single luminescent mouse model. We will use orthogonal chemiluminescent probes (Nano-lanterns) to develop a multi-signal platform to simultaneously track and associate promoter activity and protein translation relationships among multiple genes. Perturbation of circadian proteins is also essential for understanding mechanisms, including paralog roles. Currently, there are few options outside of genetic approaches, which can result in unilateral changes and activation of network compensation mechanisms. Molecular tools offer the ability to directly target the functional components of the clock – proteins, and/or their interactions. While small molecules present an attractive approach, relatively few exist that directly target core clock proteins. Hence, we propose to generate new agents for interrogating the circadian clock system: we will repurpose validated clock protein-binding small molecules by synthetically converting them into protein degraders (PROTACs), and use yeast surface-display to identify nanobodies that bind circadian proteins and prevent specific interactions. Together, these approaches present a powerful means to understand the mechanisms of the circadian clock, and can be used in a variety of models and in studies of diseases, including to uncover new therapeutic targets.