In vitro characterization of a small molecule targeting mammalian CRYs' secondary pocket

Circadian rhythms are endogenous ~24-hour cycles that regulate various physiological and behavioural processes. They are found in organisms ranging from bacteria to mammals. The generation and maintenance of circadian rhythms are made by an internal timekeeping system referred to as the circadian clock. In mammals, the master clock is the suprachiasmatic nucleus (SCN) located in the hypothalamus. The SCN receives environmental light cues from the retina and regulates circadian timing. Peripheral clocks, found in nearly every tissue and cell, are synchronized by the SCN in turn. While peripheral clocks can oscillate independently, they rely on external cues, such as feeding times and temperature, to remain aligned with the environment. At the molecular level, the circadian clock is driven by transcription-translation feedback loops (TTFLs) involving four main core-clock proteins: CLOCK, BMAL1, PERs, and CRYs. CLOCK:BMAL1 complex initiates the transcription of its repressors, PERs and CRYs, which in turn repress the CLOCK:BMAL1-mediated E-box transcription, and thus their transcription. Disruption of circadian rhythms by genetic mutations, irregular light exposure, or lifestyle is associated with various disorders, including metabolic, cardiovascular, neurodegenerative, sleep disorders, and cancer. Recent studies have discovered smallmolecule modulators that target different aspects of the circadian clock, including clock proteins and their post-translational modifications. These molecules hold therapeutic potential for restoring circadian timing and enhancing health outcomes. In this study, we identified and characterized a novel small molecule, designated A7, which targets the secondary pocket of CRY1. A7 enhances the amplitude, reduces the repression activity of CRY1 on CLOCK:BMAL1-mediated transcription by targeting the secondary pocket. Furthermore, transcriptional levels of DBP, PER2, CRY1, and BMAL1 increase upon A7 treatment. A7 does not have any effect on the subcellular localization of core-clock proteins. Importantly, it disrupts the interaction between CRY1 and CLOCK. Altogether, our results demonstrate that A7 regulates the circadian clock by interfering with the binding of CRY1 to CLOCK. These findings offer a new therapeutic intervention for health problems associated with decreased amplitude and facilitate a deeper understanding of the core-clock protein interactions and their impact on circadian rhythms.