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 生物钟是一种时间跟踪系统，使生物体能够适应一天中的时间，并驱动许多 细胞功能。它们的改变会导致各种疾病，包括癌症、心脏病和新陈代谢。 精神错乱。在这项工作中，我们提出了一个化学生物学工具箱的开发，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 不可能的。核心时钟由转录-翻译反馈环组成，具有多个 蛋白质成分，包括Paralog。已经观察到启动子活性和蛋白质之间的差异。 核心时钟分量对刺激的响应之间的平移以及由此产生的补偿机制 不受击倒和击倒策略的影响。为了弥补这些差距，并促进对 分子时钟，我们将使用以化学生物学为基础的策略来：1)产生正交化学发光 用于跟踪多个昼夜节律基因的启动子活性和蛋白质翻译的支架 举止；及开发小分子和基于蛋白质的工具来直接靶向昼夜节律蛋白质及其 互动。 对于分子时钟的研究，跟踪昼夜节律和靶向核心时钟蛋白是必不可少的。 动态的、有选择的方式。基于萤火虫荧光素酶的记者已被用来评估启动子活性 单个昼夜节律基因，大多数基于蛋白质的研究涉及来自单一发光的细胞 老鼠模型。我们将使用正交化学发光探针(纳米灯笼)来开发一种多信号 同时跟踪和关联启动子活性和蛋白质翻译关系的平台 多个基因。昼夜节律蛋白质的扰动对于理解机制也是必不可少的，包括 Paralog角色。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 改变和激活网络补偿机制。分子工具提供了直接打靶的能力 时钟-蛋白质的功能成分，和/或它们的相互作用。虽然小分子呈现出一种 有吸引力的方法，相对较少的存在，直接针对核心时钟蛋白。因此，我们建议生成 用于询问生物钟系统的新试剂：我们将重新使用有效的时钟蛋白结合小分子 分子通过合成转化为蛋白质降解物(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体。总而言之，这些方法 提供了一种了解生物钟机制的强大手段，并可用于各种 在模型和疾病研究方面，包括发现新的治疗靶点。