Targeted screening and identification of chlorhexidine as a pro-myogenic circadian clock activator.

Background: The circadian clock is an evolutionarily conserved mechanism that exerts pervasive temporal control in stem cell behavior. This time-keeping machinery is required for orchestrating myogenic progenitor properties in regenerative myogenesis that ameliorates muscular dystrophy. Here we report a screening platform to discover circadian clock modulators that promote myogenesis and identify chlorhexidine (CHX) as a clock-activating molecule with pro-myogenic activities.

Methods: A high-throughput molecular docking pipeline was applied to identify compounds with a structural fit for a hydrophobic pocket within the key circadian transcription factor protein, Circadian Locomotor Output Cycles Kaput (CLOCK). These identified molecules were further screened for clock-modulatory activities and functional validations for pro-myogenic properties.

Results: CHX was identified as a clock activator that promotes distinct aspects of myogenesis. CHX activated circadian clock that reduced cycling period length and augmented amplitude. This action was mediated by the targeted CLOCK structure via augmented interaction with heterodimer partner Bmal1, leading to enhanced CLOCK/Bmal1-controlled transcription with upregulation of core clock genes. Consistent with its clock-activating function, CHX displayed robust effects on stimulating myogenic differentiation in a clock-dependent manner. In addition, CHX augmented the proliferative and migratory activities of myoblasts.

Conclusion: Our findings demonstrate the feasibility of a screening platform to discover clock modulators with myogenic regulatory activities. Discovery of CHX as a pro-myogenic molecule could be applicable to promote regenerative capacities in ameliorating dystrophic or degenerative muscle diseases.