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

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-(3chloro-phenoxymethyl)-1-(tetrahydro-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine (PF-4800567), a novel and potent inhibitor of CK1 (IC50 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 IC50. 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 http://jpet.aspetjournals.org. doi:10.1124/jpet.109.151415. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: CK1, casein kinase 1; PF-4800567, 3-(3-chloro-phenoxymethyl)-1-(tetrahydro-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4ylamine; PF-670462, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine; GFP, green fluorescent protein; PBS, phosphatebuffered saline; D4476, 4-[4-(2,3-dihydro-benzo[1,4]dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl] benzamide; TCEP, tris(2-carboxyethyl)phosphine; DMSO, dimethyl sulfoxide. 0022-3565/09/3302-430–439$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 330, No. 2 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 151415/3496115 JPET 330:430–439, 2009 Printed in U.S.A. 430 http://jpet.aspetjournals.org/content/suppl/2009/05/19/jpet.109.151415.DC1 Supplemental material to this article can be found at: at A PE T Jornals on D ecem er 1, 2017 jpet.asjournals.org D ow nladed from bolic disorders, may have a circadian origin (Barnard and Nolan, 2008; Green et al., 2008). The Drosophila kinase double time (Dbt) was among the earliest proteins identified as having a molecular role in the clock, with point mutants producing either short or long daily cycles (Kloss et al., 1998; Price et al., 1998). Soon after the identification of Dbt, its mammalian homolog, CK1ε, was identified as mediating the phenotype of the hamster with the mutation (Ralph and Menaker, 1988; Lowrey et al., 2000). Although a normal hamster has a 24-h period, i.e., the time from the beginning of one active cycle to the next, the hamster has a markedly short period of 20 h. This phenotype caused by a point mutation in the gene for CK1ε indicates that the role of this kinase in the circadian clock is conserved across evolution. Subsequently, a mutation in CK1 , a kinase highly related to CK1ε, was found to mediate a human genetic trait surprisingly similar to the hamster phenotype (Xu et al., 2005). This trait, familial advanced sleep phase syndrome, is characterized by the “morning lark” phenotype, wherein individuals exhibit early sleep times, early-morning awakening, and a short period (Jones et al., 1999). This suggests that CK1 has as prominent a role in the mammalian circadian clock as CK1ε has. CK1ε and CK1 control the timing of the clock via phosphorylation of several proteins, including PER1–3 (Vielhaber et al., 2000; Camacho et al., 2001). PER phosphorylation by CK1ε and/or CK1 in the cytoplasm induces their translocation to the nucleus. Although the action of PER3 in the circadian clock is unclear, PER1 and PER2 bind to the coregulators CRY1 and CRY2. In the nucleus, these multimers repress the activity of the CLOCK/BMAL1 transcriptional complex. This results in decreased expression of the circadian output genes, including repression of the Per and Cry genes themselves. As the PER proteins collect in the nucleus, CK1ε and/or CK1 phosphorylate them further, leading to their ubiquitin-dependent degradation. Ultimately, the transcriptional repression is fully released, and the genes induced by CLOCK/BMAL1, including Per and Cry, start to express at high levels again. In this way, the circadian cycle begins anew. Until recently, unique roles for CK1ε and CK1 in the circadian clock were unclear. Although the CK1ε mutation dramatically shortens period, recent investigations with a CK1ε knockout mouse indicate the clock runs well without the kinase (Meng et al., 2008a). Although this finding suggests that the kinases may be redundant, the potential exists for compensation by CK1 in an animal that never expressed CK1ε. An alternative to address this issue is acute, pharmacologic inhibition. Several previously identified CK1 /ε inhibitors, however, have only modest potency and cell penetration, and none exhibits sufficient selectivity to be useful in identifying unique functions (Badura et al., 2007). This is not surprising because the kinases are 85% similar overall, with 98% similarity in the kinase domain (Knippschild et al., 2005). However, we now report the development of a selective CK1ε inhibitor, PF-4800567. Our studies demonstrate that acute CK1ε inhibition has little affect on circadian period, whereas a pan-CK1 /ε inhibitor dramatically delays the clock. Thus, in the presence of a fully functioning CK1 , CK1ε activity is superfluous for establishing the period of the oscillator. Materials and Methods CK1 / Cloning and Purification. Full-length human CK1 isoform 1 gene (accession number NP_001884A), mammalian codon optimized, was synthesized by DNA2.0 (Menlo Park, CA) and cloned in-frame into pcDNA4/HisA (Invitrogen, Carlsbad, CA) at the PstI and ApaI sites. This expressed residues 2 to 415 of the full-length