PF-670462

PF-670462 is a potent and selective inhibitor of CK1ε and CK1δ with IC50 value of 80 nM and 13 nM, respectively [1].
CK1ε and CK1δ belong to the casein kinase 1 family which is serine/threonine-selective enzyme and plays an important role in regulating circadian rhythm [1].
In COS-7 cells cotransfected with GFP-tagged PER3 and the human CK1ε  enzyme, PF-670462 inhibited PER3 nuclear translocation in a concentration-dependent way [2]. In primary lung fibroblast cells obtained from WT mice, PF-670462 significantly lengthened the circadian period in a dose-dependent way. And with a dose of 1 μM, the period in WT fibroblast cells extended to 33 h [3].
In WT, Ck1ε−/− and Ck1εtau mice, PF-670462 (10 mg/kg/d) significantly lengthened the period of activity rhythms in all three genotypes (WT: 0.44 h, Ck1εtau: 0.39 h, Ck1ε−/−: 0.42 h). In WT mice, the activity onset occurred 10.4 h after 24 d of treatment. Also, PF-670462 caused identical period lengthening in WT and Ck1ε−/− mice, which suggested that the effect of CK1δ on the circadian period [3].   
References:
[1]. Walton KM, Fisher K, Rubitski D, et al. Selective inhibition of casein kinase 1 epsilon minimally alters circadian clock period. J Pharmacol Exp Ther, 2009, 330(2): 430-439.
[2]. Badura L, Swanson T, Adamowicz W, et al. An inhibitor of casein kinase I epsilon induces phase delays in circadian rhythms under free-running and entrained conditions. J Pharmacol Exp Ther, 2007, 322(2): 730-738.
[3]. Meng QJ, Maywood ES, Bechtold DA, et al. Entrainment of disrupted circadian behavior through inhibition of casein kinase 1 (CK1) enzymes. Proc Natl Acad Sci U S A, 2010, 107(34): 15240-15245.

Identification and Profiling of a Selective and Brain Penetrant Radioligand for in Vivo Target Occupancy Measurement of Casein Kinase 1 (CK1) Inhibitors.[Pubmed:28609096]

ACS Chem Neurosci. 2017 Sep 20;8(9):1995-2004.

To enable the clinical development of our CNS casein kinase 1 delta/epsilon (CK1delta/epsilon) inhibitor project, we investigated the possibility of developing a CNS positron emission tomography (PET) radioligand. For this effort, we focused our design and synthesis efforts on the initial CK1delta/epsilon inhibitor HTS hits with the goal of identifying a compound that would fulfill a set of recommended PET ligand criteria. We identified [(3)H]PF-5236216 (9) as a tool ligand that meets most of the key CNS PET attributes including high CNS MPO PET desirability score and kinase selectivity, CNS penetration, and low nonspecific binding. We further used [(3)H]-9 to determine the binding affinity for PF-670462, a literature CK1delta/epsilon inhibitor tool compound. Lastly, [(3)H]-9 was used to measure in vivo target occupancy (TO) of PF-670462 in mouse and correlated TO with CK1delta/epsilon in vivo pharmacology (circadian rhythm modulation).

Transcriptional regulation of NHE3 and SGLT1 by the circadian clock protein Per1 in proximal tubule cells.[Pubmed:26377793]

Am J Physiol Renal Physiol. 2015 Dec 1;309(11):F933-42.

We have previously demonstrated that the circadian clock protein period (Per)1 coordinately regulates multiple genes involved in Na(+) reabsorption in renal collecting duct cells. Consistent with these results, Per1 knockout mice exhibit dramatically lower blood pressure than wild-type mice. The proximal tubule is responsible for a majority of Na(+) reabsorption. Previous work has demonstrated that expression of Na(+)/H(+) exchanger 3 (NHE3) oscillates with a circadian pattern and Na(+)-glucose cotransporter (SGLT)1 has been demonstrated to be a circadian target in the colon, but whether these target genes are regulated by Per1 has not been investigated in the kidney. The goal of the present study was to determine if Per1 regulates the expression of NHE3, SGLT1, and SGLT2 in the kidney. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of SGLT1 and NHE3 but not SGLT2 in the renal cortex of mice. Per1 small interfering RNA and pharmacological blockade of Per1 nuclear entry in human proximal tubule HK-2 cells yielded the same results. Examination of heterogeneous nuclear RNA suggested that the effects of Per1 on NHE3 and SGLT1 expression occurred at the level of transcription. Per1 and the circadian protein CLOCK were detected at promoters of NHE3 and SGLT1. Importantly, both membrane and intracellular protein levels of NHE3 and SGLT1 were decreased after blockade of nuclear Per1 entry. This effect was associated with reduced activity of Na(+)-K(+)-ATPase. These data demonstrate a role for Per1 in the transcriptional regulation of NHE3 and SGLT1 in the kidney.

Systems genetic and pharmacological analysis identifies candidate genes underlying mechanosensation in the von Frey test.[Pubmed:27231153]

Genes Brain Behav. 2016 Jul;15(6):604-15.

Mechanical sensitivity is commonly affected in chronic pain and other neurological disorders. To discover mechanisms of individual differences in punctate mechanosensation, we performed quantitative trait locus (QTL) mapping of the response to von Frey monofilament stimulation in BXD recombinant inbred (BXD) mice. Significant loci were detected on mouse chromosome (Chr) 5 and 15, indicating the location of underlying polymorphisms that cause heritable variation in von Frey response. Convergent evidence from public gene expression data implicates candidate genes within the loci: von Frey thresholds were strongly correlated with baseline expression of Cacna2d1, Ift27 and Csnk1e in multiple brain regions of BXD strains. Systemic gabapentin and PF-670462, which target the protein products of Cacna2d1 and Csnk1e, respectively, significantly increased von Frey thresholds in a genotype-dependent manner in progenitors and BXD strains. Real-time polymerase chain reaction confirmed differential expression of Cacna2d1 and Csnk1e in multiple brain regions in progenitors and showed differential expression of Cacna2d1 and Csnk1e in the dorsal root ganglia of the progenitors and BXD strains grouped by QTL genotype. Thus, linkage mapping, transcript covariance and pharmacological testing suggest that genetic variation affecting Cacna2d1 and Csnk1e may contribute to individual differences in von Frey filament response. This study implicates Cacna2d1 and Ift27 in basal mechanosensation in line with their previously suspected role in mechanical hypersensitivity. Csnk1e is implicated for von Frey response for the first time. Further investigation is warranted to identify the specific polymorphisms involved and assess the relevance of these findings to clinical conditions of disturbed mechanosensation.

Combined Pharmacological and Genetic Manipulations Unlock Unprecedented Temporal Elasticity and Reveal Phase-Specific Modulation of the Molecular Circadian Clock of the Mouse Suprachiasmatic Nucleus.[Pubmed:27605609]

J Neurosci. 2016 Sep 7;36(36):9326-41.

UNLABELLED: The suprachiasmatic nucleus (SCN) is the master circadian oscillator encoding time-of-day information. SCN timekeeping is sustained by a cell-autonomous transcriptional-translational feedback loop, whereby expression of the Period and Cryptochrome genes is negatively regulated by their protein products. This loop in turn drives circadian oscillations in gene expression that direct SCN electrical activity and thence behavior. The robustness of SCN timekeeping is further enhanced by interneuronal, circuit-level coupling. The aim of this study was to combine pharmacological and genetic manipulations to push the SCN clockwork toward its limits and, by doing so, probe cell-autonomous and emergent, circuit-level properties. Circadian oscillation of mouse SCN organotypic slice cultures was monitored as PER2::LUC bioluminescence. SCN of three genetic backgrounds-wild-type, short-period CK1epsilon(Tau/Tau) mutant, and long-period Fbxl3(Afh/Afh) mutant-all responded reversibly to pharmacological manipulation with period-altering compounds: picrotoxin, PF-670462 (4-[1-Cyclohexyl-4-(4-fluorophenyl)-1H-imidazol-5-yl]-2-pyrimidinamine dihydrochloride), and KNK437 (N-Formyl-3,4-methylenedioxy-benzylidine-gamma-butyrolactam). This revealed a remarkably wide operating range of sustained periods extending across 25 h, from 42 h. Moreover, this range was maintained at network and single-cell levels. Development of a new technique for formal analysis of circadian waveform, first derivative analysis (FDA), revealed internal phase patterning to the circadian oscillation at these extreme periods and differential phase sensitivity of the SCN to genetic and pharmacological manipulations. For example, FDA of the CK1epsilon(Tau/Tau) mutant SCN treated with the CK1epsilon-specific inhibitor PF-4800567 (3-[(3-Chlorophenoxy)methyl]-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrim idin-4-amine hydrochloride) revealed that period acceleration in the mutant is due to inappropriately phased activity of the CK1epsilon isoform. In conclusion, extreme period manipulation reveals unprecedented elasticity and temporal structure of the SCN circadian oscillation. SIGNIFICANCE STATEMENT: The master circadian clock of the suprachiasmatic nucleus (SCN) encodes time-of-day information that allows mammals to predict and thereby adapt to daily environmental cycles. Using combined genetic and pharmacological interventions, we assessed the temporal elasticity of the SCN network. Despite having evolved to generate a 24 h circadian period, we show that the molecular clock is surprisingly elastic, able to reversibly sustain coherent periods between 42 h at the levels of individual cells and the overall circuit. Using quantitative techniques to analyze these extreme periodicities, we reveal that the oscillator progresses as a sequence of distinct stages. These findings reveal new properties of how the SCN functions as a network and should inform biological and mathematical analyses of circadian timekeeping.

Acute inhibition of casein kinase 1delta/epsilon rapidly delays peripheral clock gene rhythms.[Pubmed:25245819]

Mol Cell Biochem. 2015 Jan;398(1-2):195-206.

Circadian rhythms are generated through a transcription-translation feedback loop involving clock genes and the casein kinases CSNK1D and CSNK1E. In this study, we investigated the effects of the casein kinase inhibitor PF-670462 (50 mg/kg) on rhythmic expression of clock genes in the liver, pancreas and suprachiasmatic nucleus (SCN) as well as plasma corticosterone, melatonin and running behaviour in rats and compared them to the responses to a 4 h extension of the light phase. PF-670462 acutely phase delayed the rhythmic transcription of Bmal1, Per1, Per2 and Nr1d1 in both liver and pancreas by 4.5 +/- 1.3 and 4.5 +/- 1.2 h, respectively, 1 day after administration. In the SCN, the rhythm of Nr1d1 and Dbp mRNA expression was delayed by 4.2 and 4 h, respectively. Despite these changes, the time of peak plasma melatonin secretion was not delayed, although the plasma corticosterone rhythm and onset of wheel-running activity were delayed by 2.1 and 1.1 h, respectively. These changes are in contrast to the effects of the 4 h light extension, which resulted in delays in peak expression of the clock genes of less than 1 h and no change in the melatonin or corticosterone rhythms. The ability of the casein kinase inhibitor to bring about large phase shifts in the rhythms of major metabolic target tissues may lead to new drugs being developed to rapidly phase adjust circadian rhythms to alleviate the metabolic impact of shift work.

A role for casein kinase 1 epsilon in the locomotor stimulant response to methamphetamine.[Pubmed:19050854]

Psychopharmacology (Berl). 2009 May;203(4):703-11.

RATIONALE: We previously colocalized a quantitative trait locus (QTL) for sensitivity to the locomotor stimulant effects of methamphetamine (MA) with a QTL for expression of casein kinase 1 epsilon (Csnk1-epsilon) in the nucleus accumbens (NAc). Subsequently, we identified a single nucleotide polymorphism in CSNK1E (rs135745) that was associated with increased sensitivity to the subjective effects of d-amphetamine in healthy human subjects. Based on these results, we hypothesized that differential expression of Csnk1-epsilon causes differential sensitivity to MA-induced locomotor activity in mice. OBJECTIVE: In the present study, we used PF-670462 (PF), which is a selective inhibitor of Csnk1-epsilon, to directly evaluate the role of Csnk1-epsilon in the locomotor stimulant response to MA in male C57BL/6J mice. METHODS: We administered vehicle, PF, MA, or MA + PF, either via intraperitoneal injections or bilateral intra-NAc microinjections. We also examined Darpp-32 phosphorylation in mice receiving intraperitoneal injections. RESULTS: Intraperitoneal PF (20-40 mg/kg) attenuated the locomotor stimulant response to MA (2 mg/kg) without affecting baseline activity. The high dose of PF also significantly inhibited MA-induced phosphorylation of Darpp-32, providing a potential mechanism by which Csnk1-epsilon contributes to MA-induced locomotor activity. Furthermore, microinjection of PF (5 microg/side) into the NAc completely blocked the locomotor stimulant response to MA (2.5 microg/side) without affecting baseline activity. CONCLUSIONS: These results provide direct evidence that Csnk1-epsilon is crucial for the locomotor stimulant response to a moderate dose of MA and suggest that genetic polymorphisms affecting Csnk1-epsilon expression or function could influence sensitivity to amphetamines in both mice and humans.

Inhibition of casein kinase I epsilon/delta produces phase shifts in the circadian rhythms of Cynomolgus monkeys.[Pubmed:19277609]

Psychopharmacology (Berl). 2009 Jul;204(4):735-42.

INTRODUCTION: Circadian rhythms in mammals depend upon the cyclic oscillations of transcriptional/translational feedback loops in pacemaker cells of the suprachiasmatic nucleus. The rise and fall of clock-related proteins is a function of synthesis and degradation, the latter involving phosphorylation by casein kinase Iepsilon and delta. OBJECTIVE: Earlier studies by our lab described the actions of a selective CKIepsilon/delta inhibitor, PF-670462, on circadian behavior in rats; the present work extended these studies to a diurnal species, Cynomolgus monkeys. MATERIALS AND METHODS: General cage activity was used to estimate the circadian rhythms of eight telemeterized monkeys under baseline conditions and following s.c. doses of PF-670462. RESULTS AND DISCUSSION: Consolidated bouts of activity were noted during periods of light with a repeating period length of roughly 24 h based on their onset. Reassessment in constant dim light (42 vs. 450 lx) again yielded period lengths of 24 h, in this instance revealing the animals' endogenous rhythm. PF-670462 (10-100 mg/kg s.c.) produced a dose-dependent phase delay in much the same manner as that observed previously in rats. Dosing occurred 1.5 h prior to lights-off, roughly coincident with peaking levels of PER protein, a primary substrate of CKIepsilon/delta. CONCLUSIONS: These findings suggest that the time of dosing, when held constant in both the monkey and rat studies, produced nearly identical effects despite the subjects' diurnal or nocturnal preference. Importantly, these changes in rhythm occurred in the presence of light, revealing the drug as a powerful zeitgeber in a non-human primate and, by extension, in man.

An inhibitor of casein kinase I epsilon induces phase delays in circadian rhythms under free-running and entrained conditions.[Pubmed:17502429]

J Pharmacol Exp Ther. 2007 Aug;322(2):730-8.

Casein kinase Iepsilon (CKIepsilon) 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, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine (PF-670462), proved to be both a potent (IC(50) = 7.7 +/- 2.2 nM) and selective (>30-fold with respect to 42 additional kinases) inhibitor of CKIepsilon in isolated enzyme preparations; in transfected whole cell assays, it caused a concentration-related redistribution of nuclear versus cytosolic PER. When tested in free-running animals, 50 mg/kg s.c. PF-670462 produced robust phase delays when dosed at circadian time (CT)9 (-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 cycle 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 rapid elimination of the drug (t(1/2) = 0.46 +/- 0.04 h). Together, these results suggest that inhibition of CKIepsilon yields a perturbation of oscillator function that forestalls light as a zeitgeber, and they demonstrate that pharmacological tools such as PF-670462 may yield valuable insight into clock function.