A Dual Transcriptional and High Content Assay for Cryptochrome

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DESCRIPTION (provided by applicant): In mammals, the circadian clock (or core oscillator) regulates biological rhythms such as temperature, blood pressure, hormone secretion, metabolism, and sleep wake behavior to a precise twenty four hour cycle. Circadian sleep disorders, such as advanced sleep phase syndrome (ASPS) severely disrupt the sleep wake cycle and dependent physiology. Genetic, biochemical, and cellular evidence have demonstrated that these biological rhythms are governed by the mammalian circadian clock, which is composed of a cellular transcriptional/translational feedback loop. In the core loop, a heterodimeric complex of Clock and Bmal transactivators regulate expression of a battery of output genes including the Cryptochromes, Cry1 and Cry2. The Cryptochrome molecules subsequently translocate to the nucleus and potently repress the activity of the Clock/Bmal complex, thereby inhibiting their own mRNA expression. This process takes approximately twenty-four hours and comprises the primary feedback loop of the clock. Cryptochrome deficient mice display arrhythmic locomotor activity behavior, establishing their critical role in clock function. In plants and bacteria, Cryptochromes have also been shown to bind chromophores and act as photoreceptors that communicate light information to the circadian clock. However, conclusive experiments to establish a photoreceptor role for Cryptochrome in mammalian clock function have been complicated by the arrhythmic behavior of the knockout mice. What is needed is a method to transiently inactivate Cryptochrome's repression function. Small molecule tool compounds could address this question, but as yet, specific inhibitors of Cryptochrome have not yet been developed. To address this limitation, we propose to identify small molecule modulators of Cryptochrome that transiently and reversibly inactivate its transcriptional function. The approach we propose takes advantage of its potent repression of Clock and Bmal in cell based assays, and furthermore has the ability to inform the mechanism of this perturbation by following Cryptochrome protein levels and intracellular localization. In addition, these compounds would provide proof of concept in modulation of important circadian clock parameters by small molecules. Thus, successful completion of the proposed research would provide a tool to address an important scientific question, as well as lay the groundwork for mechanism-based perturbation of the clock.