Selective Inhibition of Casein Kinase 1 Epsilon Minimally...

JPET #151415

1

Selective Inhibition of Casein Kinase 1 Epsilon Minimally Alters Circadian Clock

Period

Kevin M. Walton, Katherine Fisher, David Rubitski, Michael Marconi, Qing-Jun Meng,

Martin Sládek, Jessica Adams, Michael Bass, Rama Chandrasekaran, Todd Butler, Matt

Griffor, Francis Rajamohan, Megan Serpa, Yuhpyng Chen, Michelle Claffey, Michael

Hastings, Andrew Loudon, Elizabeth Maywood, Jeffrey Ohren, Angela Doran and Travis

T. Wager

Neuroscience Discovery (J.A., K.F., M.M., D.R., K.M.W. ), Medicinal Chemistry (T.B.,

R.C., M.C., Y.C., T.T.W.), Department of Pharmacokinetics, Dynamics, and Metabolism

(M.B., A.D., M.S.), Exploratory Medicinal Sciences (M.G., J.O., F.R.), Pfizer Global

Research and Development, Groton, CT; Faculty of Life Sciences, University of

Manchester, U.K. (Q-J.M, A.L.); Institute of Physiology, Academy of Sciences of the

Czech Republic, Prague, CZ (M.S.); Medical Research Council Laboratory of Molecular

Biology, Cambridge, U.K. (M.H., E.M)

JPET Fast Forward. Published on May 19, 2009 as DOI:10.1124/jpet.109.151415

Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 19, 2009 as DOI: 10.1124/jpet.109.151415

at ASPE

T Journals on January 9, 2020

jpet.aspetjournals.orgD

ownloaded from

http://jpet.aspetjournals.org/

JPET #151415

2

Running Title: Selective inhibition of CK1ε

Corresponding Author:

Dr. Travis T. Wager

Medicinal Chemistry

MS 8220-4045

Pfizer Global Research and Development

Groton, CT 06340

Email: [email protected]

Tel: 860-715-4059

Fax: 860-686-5552

Number of text pages: 25

Number of tables: 5

Number of figures: 8

Number of references: 34

Number of words in the Abstract: 229

Number of words in the Introduction: 696

Number of words in the Discussion: 1182


at ASPE



ownloaded from


JPET #151415

3

ABBREVIATIONS: SCN, suprachiasmatic nucleus; CK1, casein kinase 1; FASPS,

familial advanced sleep phase syndrome; DSPS, delayed sleep phase syndrome; PF-

4800567, 3-(3-chloro-phenoxymethyl)-1-(tetrahydro-pyran-4-yl)-1H-pyrazolo[3,4-

d]pyrimidin-4-ylamine; PF-670462, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-

yl]-pyrimidin-2-ylamine; GFP, green fluorescent protein; EGFR, epidermal growth factor

receptor; PK, protein kinase; 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; EDTA,

ethylenediaminetetraacetic acid; PMSF, phenylmethanesulphonylfluoride.

Recommended section assignment: Cellular and Molecular


at ASPE



ownloaded from


JPET #151415

4

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 (CK1ε) and casein kinase 1 delta (CK1δ) 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 CK1δ/ε may

have utility in treating circadian disorders. Though we previously demonstrated that a

pan-CK1δ/ε inhibitor, 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 PF-4800567, a novel

and potent inhibitor of CK1ε (IC50 = 32 nM) with greater than 20-fold selectivity over

CK1δ. PF-4800567 completely blocks CK1ε-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 CK1ε IC50. This is in contrast to the pan-CK1δ/ε inhibitor PF-

670462 which robustly alters the circadian clock under similar conditions. These data

indicate that CK1ε is not the predominant mediator of circadian timing relative to CK1δ.

PF-4800567 should prove useful in probing unique roles between these two kinases in

multiple signaling pathways.


at ASPE



ownloaded from


JPET #151415

5

Introduction

All living things, from fungi to humans, have regular cycles aligning them to 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 (SCN)

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 roughly 24-hour

cycles. The SCN is primarily regulated, or entrained, directly by light via the

retinohypothalamic tract. The cycling outputs of the SCN, not fully identified, regulates

multiple downstream rhythms, such as those in sleep and awakening, body temperature

and hormone secretion (Ko and Takahashi, 2006; Schibler et al, 2003). 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 metabolic disorders may have a circadian origin (Barnard

and Nolan, 2008; Green et al, 2008).

The Drosophila kinase doubletime (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 homologue, CK1ε, was identified as mediating the

phenotype of the hamster with the tau mutation (Ralph and Menaker, 1988; Lowrey et al.,

2000). While a normal hamster has a 24 h period, i.e., the time from the beginning of one

active cycle to the next, the tau 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


at ASPE



ownloaded from


JPET #151415

6

kinase in the circadian clock is conserved across evolution. Subsequently, a mutation in

CK1δ, a kinase highly related to CK1ε, was found to mediate a human genetic trait

surprisingly similar to the hamster tau phenotype (Xu et al., 2005). Called familial

advanced sleep phase syndrome (FASPS), this trait 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 CK1δ has as prominent a role

in the mammalian circadian clock as CK1ε.

CK1ε and CK1δ 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 CK1δ in the cytoplasm induce their translocation to the

nucleus. Although the action of PER3 in the circadian clock is unclear, PER1 and PER2

bind to the co-regulators 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 CK1δ

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 CK1δ in the circadian clock were

unclear. Although the CK1ε tau mutation dramatically shortens period, recent

investigations with a CK1ε knockout mouse indicate the clock runs well without the

kinase (Meng et al., 2008a). While this suggests the kinases may be redundant, the


at ASPE



ownloaded from


JPET #151415

7

potential exists for compensation by CK1δ in an animal that never expressed CK1ε. An

alternative to address this issue is acute, pharmacologic inhibition. Several previously

identified CK1δ/ε inhibitors, however, have only modest potency and cell penetration

and none exhibit sufficient selectivity to be useful in identifying unique functions

(Badura et al, 2007). This is not surprising as 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 while a pan-CK1δ/ε

inhibitor dramatically delays the clock. Thus, in the presence of a fully functioning

CK1δ, CK1ε activity is superfluous for establishing the period of the oscillator.

Methods

CK1δ/ε cloning and purification. Full length human CK1δ isoform 1 gene

(accession 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 CK1δ protein.

A COOH-terminally truncated kinase domain construct of human CK1δ isoform

including residues 1-317 was sub-cloned into the PET15 vector (Novagen, Gibbstown,

NJ) containing an NH3-terminal 6X-His tag and thrombin cleavage site and was

recombinantly expressed in E. coli at 20 °C. The resulting cell pellet was solubilized at 4

mL/g cells in a lysis buffer consisting of 100 mM Tris, pH 7.5, 0.5 M NaCl, 2 mM TCEP,

EDTA-free protease inhibitors (Roche Biochemical, Indianapolis, IN), 1 mM PMSF, and

10 mM imidazole and lysed with a single pass through a pre-chilled microfluidizer at 18


at ASPE



ownloaded from


JPET #151415

8

Kpsi. The lysate was clarified by centrifugation and loaded at 2 mL/min onto a 5 mL Hi

Trap Ni2+-Sepharose Fast-Flow column (GE Healthcare, Chalfont St. Giles, UK) that was

pre-equilibrated with a wash buffer consisting of 50 mM Tris-HCl, 0.5 M NaCl, 1 mM

TCEP, and 100 mM imidazole, pH 7.8. The column was washed with ~50 column

volumes (CV) of wash buffer and then eluted with a 15 CV gradient of wash buffer

containing 500 mM imidazole. The fractions containing CK1δ based on SDS-PAGE

analysis were pooled and then dialyzed into a size exclusion chromatography (SEC)

buffer consisting of 30 mM Tris-HCl, 0.3 M NaCl, 5 mM β-octylglucoside, 1 mM TCEP,

and 1 mM EDTA, pH 7.8. The dialyzed fractions were then treated with 1 μL of bovine

pancreatic high activity thrombin (cat# BCT-1020, Haematologic Technologies, Inc.,

Essex Junction, VT) for 2 h at room temperature or overnight at 4 oC. The cleavage

reaction was halted by slow addition of a 200 mM stock solution of PMSF to a final

concentration of 1 mM. The sample was concentrated, loaded onto a S200 2660 column

equilibrated in SEC buffer and purified at a flow rate of 1 mL/min. Finally, the SEC

fractions containing CK1δ based on SDS-PAGE analysis were pooled, concentrated to ~1

mg/mL, aliquoted and flash-cooled in liquid nitrogen for storage at -80 °C. All

purification steps were conducted at 4 °C unless otherwise noted.

CK1ε cloning and purification was described previously (Badura et al, 2007).

Kinase Assays. The CK1ε and δ kinase assays were performed in a 20 μL

volume in buffer containing 50 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM DTT, 10 μM

ATP and 42 μM of a peptide substrate, PLSRTLpSVASLPGL (Flotow et. al., 1990). The

final enzyme concentrations were 2.5 nM for CK1ε and 2 nM for CK1δ. Assays were

run in a panel format in the presence 1 μL of CK1 inhibitor or 5% DMSO. The reactions


at ASPE



ownloaded from


JPET #151415

9

were incubated for 2 h at room temperature, followed by detection using 20 μL of the

Kinase-Glo Plus Assay reagent (Promega, Madison, WI) according to the manufacturer’s

instructions. Luminescence was measured using Enhanced Lum detection on an Envision

plate reader (Perkin Elmer, Waltham, MA).

Nuclear Translocation. CK1ε and CK1δ dependent PER3 nuclear translocation

was run as described previously for CK1ε (Badura et. al. 2007) with alterations as

indicated below. CK1δ dependent PER3 nuclear translocation was performed with

human full-length CKIδ-pcDNA4/HisA co-transfected into COS-7 cells with the GFP

tagged murine PER3. The sensitivity of the assay was increased by incorporating an anti-

GFP antibody staining step. After the plates were fixed with 4% paraformaldehyde and

washed in PBS, cells were blocked and permeabilized in PBS with 4% goat serum and

0.1% Triton X-100 for 1 h at room temperature. Blocking buffer was then replaced with

a polyclonal anti-GFP antibody (cat. # A11122, Invitrogen) diluted 1:1000 in blocking

buffer and incubated for 2 h. Following 3 washes in PBS, goat anti-rabbit alexa fluor 488

secondary antibody (cat. # A11008, Invitrogen), diluted 1:1000 in Hoechst staining

solution, was added and incubated for 1 h before 3 final washes in PBS.

Re-optimization of the mPer3::GFP cDNA to CK1δ/ε cDNA ratio used in the

transient transfections resulted in a 1:9 ratio for both kinases. Utilizing the Molecular

Translocation Algorithm, High Content analysis was performed on the ArrayScan VTI

(Thermo-Fisher, Thermo Scientific, Pittsburgh, PA). The new scanning parameters

included a 0.035 second exposure for the detection of the antibody stained mPER3-GFP,

an isodata threshold of 0.8, a cytoplasmic ring width of 5 pixels and zero Nuc-Cyto ring


at ASPE



ownloaded from


JPET #151415

10

separation. Output features and data analysis remained as described previously (Badura

et. al. 2007).

Protein Degradation. Real-time monitoring of PER2::YFP degradation in COS-

7 cells was performed essentially as described previously (Meng et al, 2008a). Cells co-

transfected with PER2::YFP and CK1ε expression plasmids were treated first with

compound and subsequently (1 h later) 20 μg/mL cycloheximide (Sigma). Fluorescence

recording was initiated 30 min later, at 10 min intervals, for 8 h by time-lapse microscopy

using Openlab software (Improvision, Warwick, UK). Single cell fluorescent data were

analyzed by IPlab software (Scanalytics, B.D. Bioscience, Rockville, MD) and

background corrected. Overall, 25-30 separate cells in three independent experiments

were measured for each experimental condition. Normalization, one phase exponential

decay curve fitting and statistics were performed in Prism (GraphPad Software, La Jolla,

CA). Data were plotted as best-fit decay curves or as calculated degradation rate

constants K ± SEM.

In Vitro Circadian Bioluminescence Real-Time Recording. To report in real-

time the transcriptional rhythms of the mouse Per2 gene, Rat1 cells were stably

transfected with an mPer2::luc expression plasmid as described previously (Meng,

2008b). Confluent Rat1 stable cells in 35 mm dishes were synchronized by treatment

with 200 nM dexamethasone (DEX) for 1 h. The medium was then changed to non-

phenol red DMEM supplemented with 0.1 mM Luciferin substrate (Yamazaki and

Takahashi, 2005; Meng et al, 2008a). Each 35 mm dish was sealed with vacuum grease

and placed in a light-tight and temperature controlled environment at 37 °C. Light

emission (bioluminescence) was measured continuously using Photomultiplier tubes


at ASPE



ownloaded from


JPET #151415

11

(PMT, H6240 MOD1, Hamamatsu Photonics, Hertfordshire, UK). Data are presented as

photon counts per minute.

For drug treatment, 24 h after synchronization, individual dishes of cells under

PMT recording were treated with CK1 inhibitors (PF-4800567 or PF-670462 at a range

of doses) or DMSO (vehicle control). The compounds were left continuously with the

samples thereafter while the luminescence patterns were recorded for at least 6 days.

Periods were analyzed using RAP software (Okamoto, 2005). Data are expressed as

means ± SEM.

In Vivo Circadian Timing. All procedures were approved by Pfizer’s

Institutional Animal Care and Use Committee before implementation and they were in

accordance with federal policies on animal care and handling procedures. Adult male

C57BL/6J mice (initial weight 25-30 g; Jackson Laboratories, Inc., Jackson, ME) were

used for all experiments. After acclimating for 1 week, the mice were anesthetized with

2.5% isoflurane and prepped for surgical implantation of an indwelling telemeter unit. A

midline incision through skin and muscle was made in the ventral abdominal region, and

a sterile telemeter (TA10TA-F40; Data Sciences International, St. Paul, MN) was

inserted into the peritoneal cavity. The muscle was closed with absorbable suture and the

skin was closed with wound clips. The animals were given 5 mg/kg s.c. carprofen, and

were allowed to recover in a clean cage beneath a heating lamp. Once ambulatory, the

animals were placed in their home cages, the cages were placed on matrixed receivers,

and the telemeters were activated and tested on the data collection system (Dataquest

Acquisition Software; Data Sciences International). Telemeters were programmed to

record temperature for 10 s every 5 min, and the collected data were uploaded into


at ASPE



ownloaded from


JPET #151415

12

Matlab-based (Mathworks Inc., Natick, MA) analysis software (ClockLab; Actimetrics,

Evanston, IL) containing algorithms to determine and predict activity onsets and

associated circadian parameters. Each cage contained a running wheel to encourage

activity and was housed within a light-tight isolation chamber (four cages/box; Plastic

Design, Inc., Burlington, MA) with constant ventilation. Lighting for each box was set at

250 to 300 lux and the light cycle was controlled with a timer (Chrontrol, San Diego,

CA). The timing of lights on and off in each box was also recorded and checked daily.

The mice were kept in 12:12 LD (light:dark cycle of 12 h each) for at least 2 weeks after

recovery from transmitter implantation surgery prior to initiation of the study to ensure

their sleep/wake cycles were entrained to the LD cycle.

Prior to dosing, baseline data were reviewed to ensure animals met exclusion

criteria. Animals were excluded if their baseline tau (daily period) was <23.90 or >24.10

h. Animals were also excluded if the characteristics of their data were insufficient to

allow for reliable determination of activity onsets. Treatment groups were randomized

across isolation chambers such that each chamber contained animals from different

treatment groups. On the day before dosing began, the lights were disconnected once

they had turned off for that day. Dosing was done at circadian time 11 (CT11), which

was 1 h before the lights would normally have turned off. The mice were dosed for 3

days at CT11 and after the completion of dosing the mice remained in DD (24 h dark,

i.e., with the lights off) conditions for 7 days post-dosing. Data were then downloaded

and phase shifts, i.e., the time difference between activity onset pre-dosing vs post-

dosing, were analyzed by ANOVA.


at ASPE



ownloaded from


JPET #151415

13

Pharmacokinetic Characterization. The concentration-time profiles of PF-

4800567 and PF-670462 in plasma and whole brain were determined in male C57Bl/6J

mice following administration of a single subcutaneous dose. PF-4800567 was

administered at a dose of 100 mg/kg using 20% hydroxypropyl β-cyclodextrin as dosing

vehicle. PF-670462 was administered at a dose of 32 mg/kg using 5/5/90

DMSO:cremophor:20% hydroxypropyl β-cyclodextrin as dosing vehicle. The dosing

volume for both compounds was 10 mL/kg.

Plasma and whole brains were collected from mice while under isoflurane

treatment at 0.5, 1, 2, 4, 8, and 24 h post dose (N=4 per time point). Plasma was obtained

following centrifugation of whole blood. Whole brains were diluted in a 4x volume

(w/v) 60% isopropyl alcohol and homogenized using a Mini-Beadbeater 96 (Biospec

Products, Bartlesville, OK). Both plasma and brain homogenate samples were quantified

using an LC/MS method following protein precipitation. Concentration and

pharmacokinetic results are presented as mean ± SEM. AUC0-Tlast values were calculated

using linear trapezoidal rule.

Protein binding experiments were conducted with mouse plasma and brain

homogenate using high throughput 96-well equilibrium dialysis (Banker et al, 2003).

Aliquots (N=6 per matrix) of plasma and brain homogenate spiked with 1 μM compound

were dialyzed against an equal volume of buffer for 6 h at 37 °C. The dialysis

membranes had a molecular cutoff of 12 to 14 kDa (Spectrum Laboratories Inc., Rancho

Dominguez, CA). Following incubation, donor and receiver samples were transferred to

a 96-well block containing an equal volume of the opposite matrix and internal standard.

The diluted samples were then analyzed by LC/MS following protein precipitation. Free


at ASPE



ownloaded from


JPET #151415

14

fraction in plasma was calculated as the ratio of instrument response between

donor/receiver samples. Determination of undiluted brain free fraction was calculated as

described previously (Kalvass and Maurer, 2002).

Drugs

Synthesis of PF-4800567. The synthesis of PF-4800567 begins with

commercially available starting materials, as illustrated (Supplemental Materials;

Supplementary Fig. 1). Condensation of 2-(benzyloxy)acetyl chloride with malononitrile

using NaH as the base gave 2 in a 75% isolated yield (1.8 kg). Methylation of 2 with

trimethylorthoformate in the presence of PTSA gave 3 in a 44% isolated yield (500g).

Reaction of 3 with known hydrazine, (tetrahydro-pyran-4-yl)-hydrazine (4) (Ranatunge et

al, 2004) as the dihydrochloride in the presence of triethyl amine in methanol gave 5-

amino-3-benzyloxymethyl-1-(tetrahydro-pyran-4-yl)-1H-pyrazole-4-carbonitrile (5) in a

80% crude yield (190 g). Reaction of 5 with formamide at elevated temperature effected

a second ring closure to give the pyrazolpyrimidine (6) in a 73 % (crude) yield (110 g).

Reaction of 6 under standard hydrogenation conditions removed the benzyl group to yield

[4-amino-1-(tetrahydro-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl]-methanol (7) in a

73 % crude yield (73 g). Completion of the synthesis was accomplished by reaction of 7

with 3-chlorophenol under Mitsunobu conditions yielding the title compound, 3-(3-

chloro-phenoxymethyl)-1-(tetrahydro-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-

ylamine (PF-4800567) in a 77% isolated yield. See Supplemental Information for the

detailed synthetic scheme.


at ASPE



ownloaded from


JPET #151415

15

PF-670462 was synthesized by the Department of Medicinal Chemistry (Pfizer

Global Research and Development). Through Pfizer’s Gift program, both PF-670462

and PF-4800567 may be available for those interested in using them as tools to better

understand the role of CK1ε and CK1δ for in vitro and in vivo systems. If interested,

please contact the authors for details on the program.


at ASPE



ownloaded from


JPET #151415

16

Results

Selectivity of PF-4800567. Recently published data suggest that the kinases

CK1ε and CK1δ may have roles distinct from each other (Meng, 2008a). This prompted

the initiation of efforts to identify compounds that preferentially inhibited one of these

kinases. Via directed chemistry efforts, PF-4800567 (Fig. 1) was identified. When tested

in an in vitro assay against purified CK1ε and CK1δ, this compound had an IC50 of 32

nM for CK1ε, while its CK1δ IC50 was 711 nM (Fig. 2; Table 1). This revealed a 22-fold

greater potency towards CK1ε. In contrast, PF-670462 (Fig. 1), a compound we

described previously as a potent inhibitor of both CK1ε and CK1δ, showed a small

preference in the opposite direction and was approximately 6-fold more potent towards

CK1δ. Both compounds were determined to be ATP competitive (data not shown). PF-

4800567 was tested against 50 additional kinases at 1 μM and/or 10 μM (Fig. 3; Table 2).

These kinases represent a highly diverse subset of the major branches of the kinome tree.

At the lower concentration, PF-4800567 showed significant activity, though less potent,

towards only one additional kinase in this panel, EGFR. The broad nature of this kinase

panel suggests that a similar degree of selectivity for CK1ε exists in the rest of the

kinome. PF-4800567 has greater selectivity against several kinases compared to PF-

670462, including PKA, PKC, p38 and GSK3β.

Inhibition of PER3 nuclear translocation. CK1ε and CK1δ have multiple roles

in the circadian clock. One of these is to phosphorylate the PER proteins in the

cytoplasm, resulting in their translocation into the nucleus. We wanted to test whether

the in vitro preference of PF-4800567 for CK1ε would translate to selective inhibition of

this kinase in intact cells. Each kinase effectively promotes the nuclear localization of


at ASPE



ownloaded from


JPET #151415

17

PER3 (Fig. 4A, 4B, DMSO control). At the highest concentration of each compound, 10

μM, the nuclear localization of the GFP-tagged PER3 by each kinase is effectively

blocked, resulting in the absence of nuclear fluorescence (Fig. 4A, 4B). For PF-670462,

the nuclei are clearly visible down to 0.1 μM, and even down to 0.01 μM the nuclei are

more visible than in the DMSO control (Fig. 4B). Similarly, PF-4800567 effectively

blocks nuclear translocation mediated by CK1ε down to 0.01 μM (Fig4A, top).

However, it is less effective against CK1δ at this concentration, and even at 0.1 μM does

not block CK1δ mediated translocation of PER3 (Fig. 4A, bottom). Quantitation of the

relative PER3 nuclear intensity shows that PF-670462 has comparable IC50’s for both

kinases, while PF-4800567 is over 20-fold more potent for CK1ε (Fig. 4C, 4D; Table 1).

For both inhibitors, the ratio of the kinase IC50’s against the purified enzymes was

essentially the same as the ratio of the kinase IC50’s determined in the whole cell assay.

These data support the translatability of the selectivity data measured with the enzymes to

the more complex whole cell environment.

Inhibition of PER2 degradation. Another key role for CK1ε in the circadian

clock is the phosphorylation-dependent degradation of the PER proteins. Subsequent to

the phosphorylation event that occurs in the cytoplasm to induce PER translocation to the

nucleus and repression of CLOCK/BMAL1, this second phosphorylation takes place in

the nucleus to promote ubiquitin-mediated degradation of PER and relief of the

CLOCK/BMAL1 transcriptional repression (Eide et al, 2005). Coexpression of CK1ε

with PER2-YFP significantly increases the rate of PER2 degradation by about 4-fold (Fig

5). Both PF-4800567 and PF-670462 can completely block the enhanced PER2

degradation. These data, along with the nuclear localization studies, show that each of


at ASPE



ownloaded from


JPET #151415

18

the modulatory roles that CK1ε has on the PER proteins can be potently inhibited by

these compounds.

CK1ε inhibition by PF-4800567 does not alter circadian period in vitro. With

confirmation of the selective inhibition of CK1ε by PF-4800567 and its ability to block

the roles of CK1ε in the clock, we then wanted to investigate the effect of CK1ε

inhibition on the cycling clock itself. Extensive studies of the circadian clock have been

done in Rat1 cells (Ripperger and Schibler, 2006; Meng et al, 2008b). Following the

quiescence of these cells by serum starvation, addition of dexamethasone initiates a

synchronized circadian rhythm. In the cells used here, a stable line expressing PER2-

luciferase shows the rhythmic cycling of this signal over several days (Fig. 6A, 6B).

Addition of the pan-CK1δ/ε inhibitor PF-670462 causes a robust lengthening of period,

as shown by the shifting of the peak of activity over several days (Fig. 6B). Under the

same conditions, PF-4800567 causes a minimal shift in the cycling of PER2-luciferase,

even at the highest doses (Fig. 6A). The period of the cycle in the cells can be calculated

be measuring the time peak to peak. The dose responsive effect on the period shows a

dramatic increase by PF-670462 at concentrations as low as 1 μM, but PF-4800567

shows only a minor effect at concentrations up to 30 μM (Fig. 6C). When these data are

analyzed as a function of the compound concentration relative to the whole-cell IC50 for

each kinase, the ability of the compound to dissect the role of each kinase on the cycling

of the circadian clock is highlighted (Fig. 6D, 6E). PF-670462 begins to increase the

period of the clock at about 3- to10-fold over the whole-cell CK1δ IC50 and about 3-fold

lower for CK1ε. PF-4800567 parallels this effect on period up to about 3-fold the CK1δ

IC50 though it falls off at the highest concentration. However, at the doses where PF-


at ASPE



ownloaded from


JPET #151415

19

4800567 shows a small increase in period, its concentration is nearly 100-fold over its

whole-cell CK1ε IC50 (Fig. 6D). These data indicate that in this circadian model, CK1ε

inhibition alone does not drive changes in the period of the circadian clock.

Characterization of PF-4800567 as an in vivo tool. In order to use PF-4800567

to test the role of CK1e in the clock, its in vivo exposure must be sufficient to inhibit the

kinase in the brain. To assess this, we determined in C57BL/6J mice the

pharmacokinetics of this compound after a single 100 mg/kg subcutaneous dose and

compared it to the pan-CK1δ/ε inhibitor PF-670462 dosed at 32 mg/kg (Fig. 7; Table 3).

The Tmax for both compounds in plasma and brain were observed at the first time point,

0.5 h, indicating rapid absorption and distribution. The brain to plasma ratios were

generally constant throughout the 24 h time course, with an average value of 2.1 and 1.3

for PF-4800567 and PF-670462, respectively.

Conversion of the time course data to reflect free tissue concentrations showed

that free brain Cmax of PF-4800567 was nearly 3-fold over the whole-cell CK1ε IC50

(Fig. 7, Table 4). For CK1δ, however, the free Cmax in the brain is below its whole-cell

CK1δ IC50, reaching only 0.2-fold. PF-670462, in contrast, was 3-fold above its CK1δ

whole cell IC50, the same level as PF-4800567 was for CK1ε, along with a 0.7-fold CK1ε

whole cell IC50. This indicates that in vivo, at the doses tested, PF-670462 should inhibit

most of the CK1δ and CK1ε activity, while PF-4800567 should only reach levels to

selectively inhibit CK1ε. Similar studies at both lower and higher doses indicate the

central and peripheral exposure is dose related for both compounds, and that for PF-

4800567, the higher dose would significantly inhibit CK1δ and thereby lose the selective

nature of the compound (data not shown). At the 24 h time point, PF-4800567 is


at ASPE



ownloaded from


JPET #151415

20

undetectable in the brain and free compound in the plasma is below 1 nM. Therefore, no

significant accumulation of the compound would be expected with daily dosing of 100

mg/kg. Likewise, no significant accumulation of PF-670462 dosed at 32 mg/kg is

expected with daily dosing (Badura et al, 2007; data not shown).

Selective inhibition of CK1ε has little effect on circadian timing in vivo.

Confirmation that PF-4800567 and PF-670462 can be dosed in mice to similar multiples

of their whole-cell CK1ε and CK1δ IC50‘s, respectively, allowed us to test the effects of

this inhibition on in vivo circadian rhythm. Mice were kept in a controlled 12:12 LD

(light and dark cycles of 12 h each) environment for several weeks to ensure their

circadian rhythms were synchronized. They were subsequently shifted into DD (24 h,

dark) to isolate them from the entraining effects light has on circadian rhythm, thereby

providing the greatest sensitivity for the effects of the compounds on the endogenous

circadian rhythm. Following the first 24 h shift into DD, the mice were dosed daily for

three days with either 100 mg/kg PF-4800567 or 32 mg/kg PF-670462 and their

respective vehicles at CT11, i.e., one hour before the time that the lights had previously

been turned off, which is also the beginning of their active period. Following the third

day of dosing, the mice were maintained in DD to determine the effects of the

compounds on their endogenous circadian clock, as indicated by the shift in the start of

their active period post-dosing versus pre-dosing. PF-4800567 delayed the active period

by only 0.5 h when dosed at 3- and 0.2-fold its whole-cell CK1ε and CK1δ IC50’s,

respectively (Fig. 8, Table 5). In contrast, PF-670462 delayed the active period by 6.5 h

when dosed to 0.7- and 3-fold its whole-cell CK1ε and CK1δ IC50‘s, respectively. The

compound-induced delay of the circadian clock did not have any post-dose effects on the


at ASPE



ownloaded from


JPET #151415

21

animals’ tau (period), i.e., the time from the start of one active period to the start of the

next active period (Table 5). This suggests that the changes are confined to an acute

effect on the clock only when the compounds are present.


at ASPE



ownloaded from


JPET #151415

22

Discussion

Phosphorylation of circadian clock proteins is an essential element in controlling

the clock’s cyclical rhythm. Genetic analyses of the hamster tau and the human FASPS

mutations confirmed CK1ε and CK1δ, respectively, as primary drivers of the mammalian

clock (Lowrey et al., 2000; Xu et al., 2005). Consistent with the central role of these

kinases in circadian rhythm, chronic administration of a potent, dual inhibitor of CK1ε

and CK1δ, PF-670462, dramatically slows down the clock in cycling fibroblasts in vitro

(Fig. 6). More significantly, acute treatment in vivo induces phase delays in a rodent

model of circadian rhythm (Fig. 8; Badura et al., 2007). These data, however, do not

clarify whether CK1ε and CK1δ are redundant or whether one is the primary driver of the

mammalian circadian clock. The study presented here demonstrates that selective

inhibition of CK1ε by PF-4800567 does not affect the circadian clock in vitro and yields

only a small phase shift in vivo. At 3-fold its whole-cell CK1ε IC50, PF-4800567 caused

less than one-tenth the phase shift that was induced by PF-670462 at 3- and 0.7-fold its

whole-cell CK1δ and CK1ε IC50’s, respectively. It is interesting to note that the large in

vivo effect by PF-670462 is at a concentration that has only a very small effect on Rat1

cells in vitro. This may indicate that the in vivo clock is much more sensitive to changes

in CK1δ/ε inhibition. If so, then the in vivo shift by PF-4800567 may be due to a small

but direct effect on CK1δ added on to its CK1ε inhibition. At the dose tested, PF-

4800567 is at 0.2-fold its whole-cell CK1δ IC50. A 0.5 h phase shift via this low level

CK1δ inhibition is consistent with our observations that phase shift responds in a linear

fashion to compound concentration (data not shown). Additional support for the minor in

vivo activity being due to CK1δ inhibition is that the kinase panel used to characterize


at ASPE



ownloaded from


JPET #151415

23

the selectivity of PF-4800567 indicates it has little activity against most other kinases

tested, including other kinases implicated in circadian function, such as GSK3β and

ERK2 (Gallego and Virshup, 2007).

While it may be that both CK1ε and CK1δ need to be inhibited in order to delay

the clock, the effects of PF-670462 and its preferential inhibition of CK1δ over CK1ε

suggest that CK1δ is the predominant mediator of clock period. The limited separation

between its potency for these kinases, however, makes this claim uncertain. Evaluating

the role of CK1δ in the clock has been difficult, as the knockout of this kinase is

embryonic lethal (Xu et al., 2005). Although it is now clear through selective inhibition

of CK1ε that CK1δ can set clock period by itself, the question remains whether it is also

true for CK1ε in the absence of CK1δ, or is CK1δ the primary controller? Answering

this question requires the development of additional pharmacologic or genetic tools.

Both CK1ε and CK1δ phosphorylate the PER and CRY proteins and CK1ε also

phosphorylates BMAL1, though CK1δ has yet to be tested against BMAL1. Among

these clock pathway substrates, PER phosphorylation is the most extensively studied and

has several specific effects: 1) phosphorylation is required for PER translocation to the

nucleus; 2) in the nucleus, the phosphorylated PERs in combination with the CRYs

inhibit transcription of CLOCK/BMAL1 induced genes, and; 3) further phosphorylation

leads to PER degradation and the restart of transcriptional induction by the

CLOCK/BMAL1 complex (Vielhaber et al., 2000; Akashi et al., 2002; Eide et al., 2005).

In vitro, the lengthening of period by inhibiting both CK1ε and CK1δ with PF-670462

(Fig. 6) is consistent with the slowing of the cycle via chronic inhibition of PER

phosphorylation. This would slow down the rate of movement through each step, but still


at ASPE



ownloaded from


JPET #151415

24

allow the cycle to continue. In vivo, the half-lives of the compounds indicate they would

only slow down the cycle for the short time the compound is present in sufficient

concentration. This would yield an acute delay in the clock each day, with a cumulative

shift over the treatment days. This aligns with the observed effect of PF-670462 (Fig. 8).

The effect of these compounds on the clock is consistent with the characterization

of the hamster tau mutation as having a gain of function CK1ε phenotype (Gallego et al,

2006; Meng et al., 2008a). If the tau mutation induced shorter periods via a kinase with

decreased activity, then it would be expected that a CK1ε kinase inhibitor would have the

same effect. On the contrary, CK1ε inhibition has little or no effect on period. The

lengthening of period by CK1δ/ε inhibition, via decreased PER phosphorylation, aligns

with the shortened period of the tau mutation being mediated by increased PER

phosphorylation. With regards to the lack of an effect with CK1ε inhibition alone, loss of

CK1ε phosphorylation of PER could be compensated simply by the increased

presentation of unphosphorylated PER to CK1δ. In the tau mutation, however,

compensation of increased PER phoshorylation by CK1ε would require the concomitant

decrease of CK1δ activity and the kinase may not be able to respond in this manner.

Despite the observation that selective inhibition of CK1ε has little effect on

setting period, it should not be construed to mean that CK1ε is dispensable in circadian

biology. With the central role that phosphorylation plays in the clock (Gallego and

Virshup, 2007), there are other aspects of circadian function not tested here that CK1ε

may yet reveal a primary role. A recently reported genetic association between CK1ε

and stimulant sensitivity suggests the kinase may have a significant role in the ability of

stimulants to entrain behavior (Falcon and McClung, 2008; Bryant et al, 2009).


at ASPE



ownloaded from


JPET #151415

25

The identification of a selective CK1ε inhibitor opens up other key regulatory

pathways for exploration into the function of this kinase. For example, CK1ε has a key

role in Wnt signaling. The Wnt pathway stimulates changes in gene expression through

the induction of β-catenin translocation to the nucleus, leading to effects on proliferation,

survival and cell fate (Price, 2006). Addition of the peptide Wnt to cells activates CK1ε

(Swiatek et al., 2004). This results in phosphorylation of Dishevelled and translocation of

β-catenin to the nucleus (McKay et al., 2001; Price, 2006). It is unclear whether both

CK1δ and CK1ε have a role in the Wnt pathway. It was claimed that the CK1ε tau

mutation has a loss of function in this pathway, but no related effects were reported in the

tau hamster or tau transgenic mice. It is possible, however, that the loss of activity is

compensated by CK1δ. Although the CK1 inhibitor D4476 can block Wnt signaling, it

does not have selectivity between CK1ε and CK1δ (Bryja et al., 2007). PF-4800567

provides a tool to probe for separate roles of these kinases in this pathway.

CK1ε and CK1δ have generally been treated as interchangeable due to their high

degree of sequence identity and the lack of tools to tease apart their unique functions.

The development of PF-4800567 as a pharmacological tool that can distinguish between

these two kinases now creates an opportunity to explore the differences between these

kinases in greater detail.


at ASPE



ownloaded from


JPET #151415

26

References

Akashi M, Tsuchiya Y, Yoshino T, and Nishida E (2002) Control of intracellular

dynamics of mammalian period proteins by casein kinase I ε (CK1ε) and CK1δ in

cultured cells. Mol Cell Biol 22:1693–1703.

Antle MC and Silver R (2005) Orchestrating time: arrangements of the brain circadian

clock. Trends Neurosci 28:145–151.

Badura L, Swanson T, Adamowicz W, Adams J, Cianfrogna J, Fisher K, Holland J,

Kleiman R, Nelson F, Reynolds L, et al. (2007) An inhibitor of casein kinase Iε

induces phase delays in circadian rhythms under free-running and entrained

conditions. J Pharm Exp Ther 322:730-738.

Banker MJ, Clark TH, and Williams JA (2003) Development and validation of a 96-well

equilibrium dialysis apparatus for measuring plasma protein binding. J Pharm Sci 92:

967-974.

Barnard AR and Nolan PM (2008) When Clocks Go Bad: Neurobehavioural

Consequences of Disrupted Circadian Timing. PLOS Genet 4:e1000040.

Bryant CD, Graham ME, Distler MG, Munoz MB, Li D, Vezina P, Sokoloff G and

Palmer AA (2009) A role for casein kinase 1 epsilon in the locomotor stimulant

response to methamphetamine. Psychopharm doi 10.1007/s00213-008-1417-z.

Bryja V, Schulte G, Rawal N, Grahn A, and Arenas E. (2007) Wnt -5a induces

Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent

mechanism. J Cell Sci 120:586-595.


at ASPE



ownloaded from


JPET #151415

27

Camacho F, Cilio M, Guo Y, Virshup DM, Patel K, Khorkova O, Styren S, Morse B, Yao

Z, and Keesler GA (2001) Human casein kinase Iδ phosphorylation of human

circadian clock proteins period 1 and 2. FEBS Lett 489:159-165.

Green CB, Takahashi JS and Bass J (2008) The meter of metabolism. Cell 134:728-742.

Eide EJ, Woolf MF, Kang H, Woolf P, Hurst W, Camacho F, Vielhaber EL, Giovanni A,

and Virshup DM (2005) Control of mammalian circadian rhythm by CK1ε-Regulated

Proteasome-Mediated PER2 Degradation. Mol Cell Biol 25:2795-2807.

Falcon E and McClung CA (2008) A role for the circadian genes in drug addiction.

Neuropharm 56:91-96

Flotow H, Graves PR, Wang AQ, Fiol CJ, Roeske RW, and Roach PJ (1990) Phosphate

groups as substrate determinants for casein kinase I action. J Biol Chem 265:14264–

14269.

Gallego M, Eide EJ, Woolf MF, Virshup DM, and Forger DB (2006) An opposite role for

tau in circadian rhythms revealed by mathematical modeling. Proc Natl Acad Sci USA

103:10618–10623.

Gallego M and Virshup DM (2007) Post-translational modifications regulate the tiCK1ng

of the circadian clock. Nat Rev Mol Cell Biol 8:139-148.

Jones CR, Campbell SS, Zone SE, Cooper F, DeSano A, Murphy PJ, Jones B,

Czajkowski L, and Ptacek LJ (1999) Familial advanced sleep-phase syndrome: A

short-period circadian rhythm variant in humans. Nat Med 5:1062-1065.

Kalvass JC and Maurer TS (2002) Influence of nonspecific brain and plasma binding on

CNS exposure: implications for rational drug discovery. Biopharm Drug Dispos 23:

327-338.


at ASPE



ownloaded from


JPET #151415

28

Kloss B, Price JL, Saez L, Blau J, Rothenfluh A, Wesley CS, and Young MW (1998) The

Drosophila clock gene double-time encodes a protein closely related to human casein

kinase Iε. Cell 94:97-107.

Knippschild U, Gocht A, Wolff S, Huber N, Lohler J, and Stoter M (2005) The casein

kinase 1 family: participation in multiple cellular processes in eukaryotes. Cell Signal

17:675–689.

Ko CH and Takahashi JS (2006) Molecular components of the mammalian circadian

clock. Hum Mol Gen 15:R271-R277.

Lowrey PL, Swhimomura K, Antoch MP, Yamazaki S, Zemenides PD, Ralph MR,

Menaker M, and Takahashi JS (2000) Positional syntenic cloning and functional

characterization of the mammalian circadian mutation tau. Science 288: 483–490.

McKay RM, Peters JM, and Graff JM. (2001) The Casein Kinase I Family in Wnt

Signaling. Dev Biol 235:388-396.

Meng QJ, Logunova L, Maywood ES, Gallego M, Lebiecki J, Brown TM, Sladek M,

Semikhodskii AS, Glossop NRJ, Piggins HD, et al. (2008a) Setting clock speed in

mammals: The CK1e tau mutation in mice accelerates circadian pacemakers by

selectively destabilizing PERIOD proteins. Neuron 58:78-88.

Meng QJ, McMaster A, Beesley S, Lu WQ, Gibbs J, Parks D, Collins J, Farrow S, Donn

R, Ray D, and Loudon A (2008b) Ligand modulation of REV-ERBα function resets

the peripheral circadian clock in a phasic manner. J Cell Sci 121:3629-3635.

Okamoto K, Onai K, and Ishiura M (2005) RAP, an integrated program for monitoring

bioluminescence and analyzing circadian rhythms in real time. Anal Biochem 340:193-

200.


at ASPE



ownloaded from


JPET #151415

29

Price JL, Blau J, Rothenfluh A, Abodeely M, Kloss B, and Young MW (1998) Double-

time is a novel Drosophila clock gene that regulates PERIOD protein accumulation.

Cell 94:83-95.

Price MA (2006) CK1, there's more than one: casein kinase I family members in Wnt and

Hedgehog signaling. Genes & Dev 20:399-410.

Ralph MR and Menaker M (1988) A mutation of the circadian system in golden

hamsters. Science 241:1225-1227.

Ranatunge RR, Augustyniak M, Bandarage UK, Earl RA, Ellis JL, Garvey DS, Janero

DR, L. Letts G, Martino AM, Murty MG, et al. (2004) Synthesis and Selective

Cyclooxygenase-2 Inhibitory Activity of a Series of Novel, Nitric Oxide Donor-

Containing Pyrazoles. J Med Chem 47:2180-2193.

Ripperger J and Schibler U (2006) Rhythmic CLOCK-BMAL1 binding to multiple E-box

motifs drives circadian Dbp transcription and chromatin transitions. Nat Gen 38:369-

374

Schibler U, Ripperger J and Brown SA (2003) Peripheral Circadian Oscillators in

Mammals: Time and Food. J Biol Rhythms 18:250-260

Swiatek W, Tsai I-C, Klimowski L, Pepler A, Barnette J, Yost HJ, and Virshup DM

(2004) Regulation of Casein Kinase Iε Activity by Wnt Signaling. J Biol Chem 279:

13011-13017.

Vielhaber E, Eide E, Gao Z-H, and Virshup DM (2000) Nuclear entry of the circadian

regulator mPER1 is controlled by mammalian casein kinase I ε. Mol Cell Biol

20:4888–4899.


at ASPE



ownloaded from


JPET #151415

30

Xu Y, Padiath QS, Shapiro RE, Jones CR, Wu SC, Saigoh N, Saigoh K, Ptacek LJ, and

Fu Y-H (2005) Functional consequences of a CK1δ mutation causing familial

advanced sleep phase syndrome. Nature 434:640–644.

Yamazaki S and Takahashi JS (2005) Real-time luminescence reporting of circadian gene

expression in mammals. Methods Enzymol 393:288-301.


at ASPE



ownloaded from


JPET #151415

31

Footnotes

Send reprint requests to Dr. Travis T. Wager, Medicinal Chemistry, MS 8220-4045,

Pfizer Global Research and Development, Groton, CT 06340


at ASPE



ownloaded from


JPET #151415

32

Legends for Figures

Figure 1. Structures of PF-4800567 and PF-670462.

Figure 2. PF-4800567 selectively inhibits CK1ε versus PF-670462 which is more potent

at CK1δ. Purified enzymes were assayed in vitro in the presence of increasing

concentrations of inhibitor. Activity was measured as a function of remaining ATP at the

termination of the kinase reaction and plotted as percent activity inhibited relative to

DMSO control. Data shown are from two separate experiments each performed in

triplicate, mean +/- SEM. IC50 values were determined by analysis in Graphpad PRISM

and are shown in Table 1.

Figure 3. Heat map of PF-4800567 selectivity. Compound was tested at 1 μM at the

indicated kinases using Invitrogen SelectScreen Kinase Profiling. All kinases were tested

at their Km for ATP.

Figure 4. PF-4800567 selectively inhibits CK1ε mediated PER3 nuclear translocation.

Representative examples of COS-7 cells cotransfected with cDNA expression vectors for

GFP-PER3 and CK1ε or CK1δ were incubated with increasing concentrations of either

PF-4800567 (A) or PF-670462 (B); a single DMSO control is used for each kinase for

both compounds. Percent inhibition of GFP signal localized to the nucleus relative to

DMSO control was quantitated and compiled from two separate experiments performed

in triplicate, mean +/- SEM (C, D). IC50 values were determined by analysis in Graphpad

PRISM and are shown in Table 1.

Figure 5. Increased PER2 degradation is inhibited by both PF-4800567 and PF-670462.

A) Decay of PER2::YFP fluorescence in COS-7 cells cotransfected with CK1ε kinase


at ASPE



ownloaded from


JPET #151415

33

dead or CK1ε wild-type incubated either with vehicle or 0.5 μM compound. Cells were

treated with cyclohexamide (20 mg/mL) 0.5 h before recording. B) Degradation rate

constant K for PER2::YFP in presence of CK1ε kinase-dead or wild-type treated with

vehicle or the indicated compounds (mean ± SEM, *p < 0.001 relative to all other

conditions).

Figure 6. Selective CK1ε inhibition by PF-4800567 does not alter the circadian period

while preferential CK1δ inhibition by PF-670462 does alter the period. Representative

PER2::LUC bioluminescence oscillations in Rat1 cells in the presence of increasing

concentrations of either PF-4800567 (A) or PF-670462 (B); arrow indicates time of

compound addition. (C) Quantitation of period as measured peak to peak, data are mean

+/- SEM from 5 independent experiments of single cultures. Data are replotted as fold

over whole cell CK1ε IC50 (D) or as fold over whole cell CK1δ IC50 (E).

Figure 7. CNS exposure of PF-4800567 is sufficient to selectively inhibit CK1ε in vivo.

Time course of PF-4800567 exposure in C57BL/6J mice following a single 100mg/kg

s.c. dose. Brain concentrations were below the limit of quantitation in all animals at the

24 hour time point.

Figure 8. Selective CK1ε inhibition by PF-4800567 has only minor effects on shifting in

vivo phase while preferential CK1δ inhibition by PF-670462 induces a large phase shift.

Mice (N=5) were maintained in 12:12 LD for several weeks to get a baseline of activity.

The lights were subsequently turned off and the mice kept in DD conditions for testing.

They were dosed with 100 mg/kg of PF-4800567 or 32 mg/kg PF-670462, or the

appropriate vehicle, for 3 days, q.d. They were maintained another week in DD and


at ASPE



ownloaded from


JPET #151415

34

shifts in the start of their active periods post-dose were determined relative to the starts

pre-dose; this is defined as the phase shift. A) quantitation of the phase shifts. B)

representative temperature analyses shown as double-plotted actograms from each of the

treatment groups. Asterisks indicate time of dosing. By convention, an advance in the

active period is positive and a delay is negative.


at ASPE



ownloaded from


JPET #151415

35

Table 1: Enzyme and whole cell IC50’s for PF-4800567 and PF-670462. Values with

95% confidence intervals (CI) determined from data shown in Figs. 3 and 4.

ENZYME WHOLE CELL

IC50 (μM) Ratio IC50 (μM) Ratio

CK1ε (95% CI)

CK1δ (95% CI)

.CK1δ CK1ε

CK1ε (95% CI)

CK1δ (95% CI)

CK1δ CK1ε

PF-4800567 0.032 (0.025 to 0.042)

0.711 (0.566 to 0.893)

22.22 0.13 (0.10 to 0.17)

2.65 (1.35 to 5.19)

20.38

PF-670462 0.080 (0.069 to 0.092)

0.013 (0.011 to 0.017)

0.16 0.55 (0.41 to 0.74)

0.14 (0.10 to 0.20)

0.25


at ASPE



ownloaded from


JPET #151415

36

Table 2: Selectivity comparison between PF-4800567 and PF-670462

PF-4800567 PF-670462

1 μM 10 μM 1 μM 10 μM

CK1γ2 1 nd 2 nd

GSK3β nd 0 nd 57

p38 cascade 2 3 100 100

MLCK nd 4 nd 53

PRKC B2 2 7 29 75

PKACα 1 23 78 98

EphA2 16 39 20 68

SRC 9.5 47 nd 58

VEGFR2 11 51 20 70

LCK 32 57 55 88

HGK 20 75 97 99

EGFR 69 83 76 99

Data shown are per cent inhibition at the indicated concentration of each inhibitor.

CK1γ2, casein kinase 1γ2; GSK3β, glycogen synthase kinase 3β; p38 cascade, p38α

coupled to MAPKAPK2; MLCK, myosin light chain kinase; PRKC B2, protein kinase C

B2; PKAC α, protein kinase A catalytic subunit α; VEGFR2, vascular endothelial growth

factor receptor 2 tyrosine kinase; EGFR, epidermal growth factor receptor tyrosine

kinase.


at ASPE



ownloaded from


JPET #151415

37

Table 3: Pharmacokinetic parameters of PF-4800567 and PF-670462 following s.c.

administration to mice.

PF-4800567 PF-670462

Plasma Brain Plasma Brain

Dose (mg/kg) 100 100 32 32

AUC (ng*h/mL) 6030 11400 2880 3410

Total Cmax (ng/mL) 5250 9250 2760 2880

Tmax (h) 0.5 0.5 0.5 0.5

fu 0.069 0.016 0.12 0.046

Free Cmax (nM) 1007 411 982 393


at ASPE



ownloaded from


JPET #151415

38

Table 4: Comparison of the observed free Cmax concentrations in mice relative to

whole-cell CK1δ/ε IC50 values. PF-4800567 and PF-670462 were dosed at 100mpk and

32mpk, respectively.

PF-4800567 PF-670462

Fold over Whole Cell IC50 Free Plasma Free Brain Free Plasma Free Brain

CK1ε 8 3 2 0.7

CK1δ 0.4 0.2 7 3


at ASPE



ownloaded from


JPET #151415

39

Table 5: Selective CK1ε inhibition by PF-4800567 has only a small effect on the

circadian clock in vivo while preferential CK1δ inhibition by PF-670462 dramatically

delays the clock. Phase shift indicates the difference in the time of day of the start of the

active period prior to treatment to the start of the active period following treatment. By

convention, an advance in the active period is positive and a delay is negative. Tau refers

to the time from the start of one active period to the start of the next active period.

Significance testing performed by one-way ANOVA.

Treatment Phase Shift (h) Pre-dose tau Post-dose tau

mean SEM p value mean SEM mean SEM

Veh1 0.17 0.15 24.00 0.01 23.81 0.03

PF-4800567 -0.51 0.08 <0.01 24.01 0.01 23.84 0.03

Veh2 0.19 0.13 24.00 0.01 23.81 0.08

PF-670462 -6.55 0.23 <0.001 23.99 0.01 23.80 0.11


at ASPE



ownloaded from



at ASPE



ownloaded from


Selective Inhibition of Casein Kinase 1 Epsilon Minimally...

Documents

Transcript of Selective Inhibition of Casein Kinase 1 Epsilon Minimally...