Received for publication March 16, 2007.
Revised May 10, 2007.
Accepted for publication May 10, 2007.

An inhibitor of casein kinase I induces phase delays in circadian rhythms under free-running and entrained conditions

Abstract

Casein kinase I (CKI) is an essential component of the biological clock, phosphorylating PER proteins and in doing so regulating their turnover and nuclear entry in oscillator cells of the suprachiasmatic nucleus (SCN). Although hereditary decreases in PER phosphorylation have been well characterized, little is known about the consequences of acute enzyme inhibition by pharmacological means. A novel reagent, PF-670462, proved to be both a potent (IC₅₀ = 7.7 ± 2.2 nM) and selective (>30-fold with respect to 42 additional kinases) inhibitor of CKI in isolated enzyme preparations; in transfected whole cell assays, it caused a concentration-related redistribution of nuclear vs. cytosolic PER. When tested in free-running animals, PF-670462 (50 mg/kg sc) produced robust phase delays when dosed at CT9 (-1.97 ± 0.17 h). Entrained rats dosed in normal light-dark (LD) and then released to constant darkness also experienced phase delays that were dose- and time of dosing-dependent. PF-670462 yielded only phase delays across the circadian period with the most sensitive time at CT12 when PER levels are near their peak in the SCN. Most importantly, these drug-induced phase delays persisted in animals entrained and maintained in LD throughout the entire experiment; re-entrainment to the prevailing LD required days in contrast to the drug's rapid elimination (t = 0.46 ± 0.04 h). Together, these results suggest that inhibition of CKI yields a perturbation of oscillator function that forestalls light as a zeitgeber and demonstrates that pharmacologic tools such as PF-670462 may yield valuable insights into clock function.

Key words: PER, biological clock, clock genes, locomotor activity, protein phosphorylation, suprachiasmatic nucleus

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