M54 selectively stabilizes the circadian clock component of CRY1 and enhances the period of circadian rhythm at cellular level.

Circadian rhythms are daily oscillations in biochemical, physiological, and behavioral processes in living organisms, aligned with the 24-h day-night cycle and governed by an internal molecular clock. This molecular clock functions through transcriptional-translational feedback loops driven by core clock proteins including BMAL1, CLOCK, PERs, and CRYs. CRY1 and CRY2, along with PERs, repress BMAL1:CLOCK-mediated transcriptional activity. Several studies have also suggested that CRY1 and CRY2 play distinct roles within the molecular clock. In our previous work, we identified M54 as a modulator of circadian rhythm at the cellular level via CRY1. Here, we demonstrate that M54 specifically binds to CRY1, but not CRY2, reducing its ubiquitination and thereby enhancing its stability. Consequently, M54 lengthens the period of the U2-OS circadian rhythm and decreases the transcription of clock-controlled genes in a concentration-dependent manner. These findings highlight the potential of M54 as a therapeutic candidate for circadian disorders associated with reduced CRY1 levels.