Chemical Biology Approaches for Studying Circadian Rhythms

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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.