Chemical Biology Approaches for Studying Circadian Rhythms

研究昼夜节律的化学生物学方法

• 批准号: 10623212
• 负责人: Michelle Elizabeth Farkas
• 金额: $ 38.36万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623212
• 关键词: Address; Behavior; Binding; Binding Proteins; Biology; Cell physiology; Cells; Chemicals; Circadian Rhythms; Clock protein; Compensation; Development; Disease; Disparity; Feedback; Firefly Luciferases; Genes; Genetic Transcription; Heart Diseases; Image; Individual; Knock-out; Malignant Neoplasms; Metabolic Diseases; Modeling; Molecular; New Agents; Organism; Physiological; Proteins; Reporter; Role; Signal Transduction; Stimulus; Surface; System; Time; Translations; Work; Yeasts; circadian; circadian pacemaker; genetic approach; knock-down; molecular clock; mouse model; nano; nanobodies; new therapeutic target; paralogous gene; prevent; promoter; response; scaffold; small molecule; tool

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.

项目摘要/摘要 昼夜节律是时间跟踪系统，可让生物适应一天中的时间，并驾驶许多 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 疾病。在这项工作中，我们建议开发化学生物学工具箱，包括成像和 蛋白质靶向平台，以促进分子水平的昼夜节律研究 不可能。核心时钟由带有多个的转录翻译反馈循环组成 蛋白质成分，包括旁系同源物。启动子活性和蛋白质之间已经观察到了差异 翻译，核心时钟组件对刺激的响应以及导致的补偿机制 从淘汰赛和淘汰策略中。解决这些差距，并促进有关 分子时钟，我们将使用基于化学生物学的策略来：1。）产生正交化学发光 脚手架以跟踪启动子活性和蛋白质翻译的蛋白质翻译 方式;和2.）开发小分子和基于蛋白质的工具，以直接靶向昼夜节律蛋白及其 互动。 对于分子时钟的研究，必须跟踪昼夜节律和靶向核心时钟蛋白 动态和选择性的方式。基于萤火虫荧光素酶的记者已被用来评估 单个昼夜节律基因，大多数基于蛋白质的研究涉及衍生自单发光的细胞 鼠标模型。我们将使用正交化学发光问题（Nano-lanterns）来发展多信号 轻松跟踪和关联启动子活动和蛋白质翻译关系的平台 多个基因。昼夜节律蛋白质的扰动对于理解机制，包括 旁学角色。当前，遗传方法之外的选择很少，这可能会导致单方面 网络补偿机制的变化和激活。分子工具提供了直接靶向的能力 时钟的功能成分 - 蛋白质和/或其相互作用。而小分子则是 有吸引力的方法，很少有直接靶向核心时钟蛋白的方法。因此，我们建议生成 询问昼夜节时钟系统的新代理：我们将复制经过验证的时钟蛋白质结合小 通过合成将它们转化为蛋白质降解器（PROTAC），然后使用酵母表面播放到 识别结合昼夜节律蛋白并防止特定相互作用的纳米化。在一起，这些方法 提出一种强大的手段来了解昼夜节律的机制，并且可以用于多种 模型和疾病研究，包括发现新的治疗靶点。