Selective Inhibition of Casein Kinase 1ϵ Minimally Alters Circadian Clock Period

Abstract

The circadian clock links our daily cycles of sleep and activity to the external environment. Deregulation of the clock is implicated in a number of human disorders, including depression, seasonal affective disorder, and metabolic disorders. Casein kinase 1 epsilon (CK1) and casein kinase 1 delta (CK1␦) are closely related Ser-Thr protein kinases that serve as key clock regulators as demonstrated by mammalian mutations in each that dramatically alter the circadian period. Therefore, inhibitors of CK1␦/ may have utility in treating circadian disorders. Although we previously demonstrated that a pan-CK1␦/ inhibitor, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine (PF-670462), causes a significant phase delay in animal models of circadian rhythm, it remains unclear whether one of the kinases has a predominant role in regulating the circadian clock. To test this, we have characterized 3-(3-, a novel and potent inhibitor of CK1 (IC 50 ϭ 32 nM) with greater than 20-fold selectivity over CK1␦. PF-4800567 completely blocks CK1-mediated PER3 nuclear localization and PER2 degradation. In cycling Rat1 fibroblasts and a mouse model of circadian rhythm, however, PF-4800567 has only a minimal effect on the circadian clock at concentrations substantially over its CK1 IC 50 . This is in contrast to the pan-CK1␦/ inhibitor PF-670462 that robustly alters the circadian clock under similar conditions. These data indicate that CK1 is not the predominant mediator of circadian timing relative to CK1␦. PF-4800567 should prove useful in probing unique roles between these two kinases in multiple signaling pathways.All living things, from fungi to humans, have regular cycles aligning them with the daily events in their environment. These cycles, known as circadian rhythms, are controlled in mammals by the master clock located in the suprachiasmatic nucleus of the hypothalamus (Antle and Silver, 2005;Gallego and Virshup, 2007). At the cellular level, the molecular events behind clock cycling are described by the regular increase and decrease in mRNAs and proteins that define feedback loops, resulting in approximately 24-h cycles. The suprachiasmatic nucleus is primarily regulated, or entrained, directly by light via the retinohypothalamic tract. The cycling outputs of the suprachiasmatic nucleus, not fully identified, regulate multiple downstream rhythms, such as those in sleep and awakening, body temperature, and hormone secretion (Schibler et al., 2003;Ko and Takahashi, 2006). As anyone who has experienced jet lag knows, misalignment of the internal clock with the external environment profoundly affects well being. Furthermore, diseases, such as depression, seasonal affective disorder, and metaArticle, publication date, and citation information can be found at

Csnk1e Is a Genetic Regulator of Sensitivity to Psychostimulants and Opioids

“…Together, these results suggest that both Csnk1e and Csnk1d are important for sensitivity to drugs of abuse. However, because PF-670462 only exhibits six-fold selectivity for Csnk1d over Csnk1e (Walton et al, 2009;Meng et al, 2010), it is unclear if our previous results were mediated by Csnk1d or Csnk1e (Bryant et al, 2009b;Li et al, 2011).…”

“…PF-670462 was previously thought to be slightly more selective for Csnk1e over Csnk1d (Badura et al, 2007), but a recent study showed that PF-670462 was six-fold more selective for Csnk1d over Csnk1e and that PF-4800567 was 20-fold more selective for Csnk1e over Csnk1d (Walton et al, 2009). Because PF-4800567 increased MA and fentanyl sensitivity whereas PF-670462 decreased MA sensitivity, the combined results provide further evidence that Csnk1e and Csnk1d exert opposing regulatory roles on the locomotor stimulant response to drugs of abuse.…”

“…PF-4800567 shows 420-fold selectivity for Csnk1e over Csnk1d and exhibits markedly reduced efficacy at inhibiting off-target proteins relative to PF-670462. Nevertheless, both PF-4800567 and PF-670462 can significantly inhibit epidermal growth factor receptor (EGFR) at micromolar concentrations (Walton et al, 2009). We are unaware of any study examining the acute effect of selective inhibition of EGFR on psychostimulant-or opioidinduced locomotor activity.…”

“…The polypropylene glycol/PF solution was briefly sonicated using a Qsonica Misonix S-4000 (Farmingdale, NY) before adding 30% Captisol solution. The dose of PF was chosen based on previously published studies in mice (Walton et al, 2009;Meng et al, 2010), a limit in solubility at higher concentrations, and pilot studies indicating its effectiveness at modulating drug-induced locomotor activity. PF has been shown to exhibit 20-fold greater selectivity for Csnk1e vs Csnk1d and has been useful in clarifying the importance of the two isoforms in regulating circadian rhythms (Walton et al, 2009;Meng et al, 2010).…”

“…Based on the location of our QTL and prior studies implicating Csnk1e in the response to drugs of abuse, we further hypothesized that Csnk1e regulates sensitivity to psychostimulants and opioids. Thus, we examined the effect of a Csnk1e-null mutation or pharmacological inhibition with the new selective Csnk1e inhibitor PF-4800567 (Walton et al, 2009;Meng et al, 2010) on the locomotor response to MA and the m-opioid receptor agonist fentanyl.…”