Chemical regulation of circadian rhythms through inhibition of casein kinase Iδ and α.

Circadian rhythms are generated by transcriptional-translational feedback loops of clock genes and proteins and are further regulated by post-translational modifications. Among these mechanisms, casein kinase I (CKI)-mediated phosphorylation of Period (PER) proteins plays a critical role in circadian period determination by regulating PER stability, degradation, and subcellular localization. Small-molecule CKI inhibitors have been shown to lengthen circadian period; however, the relationship between CKI isoform inhibition profiles and circadian period regulation remains incompletely understood. Here, we identified THBZA as a novel circadian period-lengthening compound through a high-throughput phenotypic screening. THBZA lengthened circadian periods in human cells as well as in mouse lung and spleen explants, and its effect was reversible after compound removal. In vitro kinase assays showed that THBZA preferentially inhibited CKIδ over CKIα. Comparative docking analysis results were consistent with the experimentally observed CKIδ preference and suggested that preference was potentially associated with differences in multiple interactions within the conserved ATP-binding pocket. These findings identify THBZA as a novel CKI-targeting circadian modulator and provide a useful chemical tool for further investigating how different CKI isoform inhibition profiles influence mammalian circadian period regulation.