Chemical Biology Approaches for Studying Circadian Rhythms

研究昼夜节律的化学生物学方法

• 批准号: 10437920
• 负责人: Michelle Elizabeth Farkas
• 金额: $ 34.95万
• 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/10437920
• 关键词: Address; Behavior; Binding; Binding Proteins; Biology; Cell physiology; Cells; Chemicals; Circadian Rhythms; Clock protein; Development; Disease; Feedback; Financial compensation; Firefly Luciferases; Genes; Genetic Transcription; Heart Diseases; Image; Individual; Knock-out; Lead; Malignant Neoplasms; Metabolic Diseases; Modeling; Molecular; New Agents; Organism; Physiological; Proteins; Reporter; Role; Signal Transduction; Stimulus; Surface; System; Time; Translations; Work; Yeasts; base; 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.

项目概要/摘要 昼夜节律钟是时间跟踪系统，使生物体能够适应一天中的时间，并驱动许多 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，我们建议开发一个化学生物学工具箱，包括成像和诊断工具。 蛋白质靶向平台，以促进分子水平上的昼夜节律研究，否则 核心时钟由具有多个的转录翻译反馈环组成。 已观察到启动子活性和蛋白质之间存在差异，包括旁系同源物。 核心时钟组件对刺激的响应之间的转换以及由此产生的补偿机制 为了解决这些差距并促进进一步的研究。 分子钟，我们将使用基于化学生物学的策略来：1.）生成正交化学发光 支架以并行、高含量的方式跟踪多个昼夜节律基因的启动子活性和蛋白质翻译 方式；和 2.) 开发基于小分子和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。 基于萤火虫荧光素酶的动态和选择性方式已用于评估启动子活性。 个体昼夜节律基因，大多数基于蛋白质的研究涉及源自单一发光的细胞 我们将使用正交化学发光探针（纳米灯笼）来开发多信号。 平台同时跟踪和关联启动子活性和蛋白质翻译关系 昼夜节律蛋白的扰动对于理解机制也至关重要，包括 目前，除了遗传方法之外，几乎没有其他选择，这可能会导致单方面的结果。 分子工具的改变和激活提供了直接靶向的能力。 时钟的功能成分——蛋白质，和/或它们的相互作用。 有吸引力的方法，直接靶向核心时钟蛋白的方法相对较少，因此，我们建议生成。 用于询问生物钟系统的新试剂：我们将重新利用经过验证的时钟蛋白结合小分子 通过将分子合成转化为蛋白质降解剂（PROTAC），并使用酵母表面展示来 这些方法共同鉴定了结合昼夜节律蛋白并防止特定相互作用的纳米抗体。 提供了理解生物钟机制的强大手段，并可用于多种领域 模型和疾病研究，包括发现新的治疗靶点。