A small molecule modulates circadian rhythms through phosphorylation of the period protein.
- SourceAvailable from: Steve A Kay[show abstract] [hide abstract]
ABSTRACT: Impairment of the circadian clock has been associated with numerous disorders, including metabolic disease. Although small molecules that modulate clock function might offer therapeutic approaches to such diseases, only a few compounds have been identified that selectively target core clock proteins. From an unbiased cell-based circadian phenotypic screen, we identified KL001, a small molecule that specifically interacts with cryptochrome (CRY). KL001 prevented ubiquitin-dependent degradation of CRY, resulting in lengthening of the circadian period. In combination with mathematical modeling, our studies using KL001 revealed that CRY1 and CRY2 share a similar functional role in the period regulation. Furthermore, KL001-mediated CRY stabilization inhibited glucagon-induced gluconeogenesis in primary hepatocytes. KL001 thus provides a tool to study the regulation of CRY-dependent physiology and aid development of clock-based therapeutics of diabetes.Science 07/2012; 337(6098):1094-7. · 31.20 Impact Factor
A Small Molecule Modulates Circadian Rhythms through
Phosphorylation of the Period Protein**
Jae Wook Lee, Tsuyoshi Hirota, Eric C. Peters, Michael Garcia, Rodolfo Gonzalez,
Charles Y. Cho, Xu Wu, Peter G. Schultz,* and Steve A. Kay*
Many living organisms including bacteria, fungi, plants, and
animals have observable, genetically determined biological
rhythms. The rhythm affects many aspects of mammalian
behavior and physiology including sleep, physical activity,
alertness, hormone levels, body temperature, immune func-
tion, and digestive activity.These circadian rhythms are
controlled by cell-autonomous circadian clocks.In mam-
mals, the central circadian pacemaker controlling behavioral
rhythms is located within the suprachiasmatic nucleus (SCN)
of the anterior hypothalamus.Peripheral clocks are located
in most major organs and control key physiological processes
such as blood pressure, hepatic glucose levels, and heart
rate.Clock genes and their gene products are involved in
negative and positive regulatory feedback loops.For
example CLOCK (circadian locomotor output cycles kaput)
and BMAL1 (brain and muscle ARNT-like protein 1) hetero-
dimerize and activate the expression of the clock genes period
(Per) 1, 2, and 3, and cryptochrome (Cry) 1 and 2. PER and
CRY proteins enter the nucleus and repress CLOCK-
Perturbation of clock function is implicated in numerous
pathologies including circadian sleep disorders, cardiovascu-
lar disease, cancer, and metabolic disease.Thus, the
identification of small molecules that modulate clock function
may not only provide additional insights into the role of
circadian rhythms in human physiology, but also lead to new
treatments for diseases that have an underlying circadian
disorder.To date, a number of small molecules have been
demonstrated to be modulators of circadian rhythms includ-
ing the CDK inhibitor roscovitine, a JNK inhibitor SP600125,
and longdaysin, which lengthens circadian period by inhibit-
ing CKIa, CKId, and ERK2.[9–11]Furthermore, several intra-
cellular small molecules such as heme,[12a,b]cAMP,[12c]and
NAD[12d,e]are known to be circadian modulators.
To identify novel small molecules that induce circadian
period changes, we exploited a previously developed circa-
dian cell-based assay with a luminescent readout(see the
Supporting Information). Human U2OS cells stably express-
ing a Bmal1-dLuc reporter were treated with approximately
500000 druglike compounds,and luminescence intensity
was measured every two hours for three days. Primary hit
compounds were further tested in assays in an eight point
serial dilution format to determine compound potency and
We identified a number of different scaffolds that
lengthened the circadian period in a dose-dependent
manner in U2OS cells. The benzothiazole derivative LH846
(8 mm) lengthened the period (10 h) with minimal effect on
the amplitude of both Per2-dLuc and Bmal1-dLuc rhythms in
U2OS cells (Figure 1). A preliminary structure activity
relationship (SAR) study was performed to identify sites
which could be derivatized without significant loss in activity
to generate affinity probes for target identification (see
[*] Dr. J. W. Lee,[+]Dr. R. Gonzalez, Prof. P. G. Schultz
Department of Chemistry and
The Skaggs Institute for Chemical Biology
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
Dr. T. Hirota,[+]Prof. S. A. Kay
Cell and Developmental Biology, Division of Biological Sciences
University of California at San Diego
9500 Gilman Drive, La Jolla, CA 92093 (USA)
Dr. E. C. Peters, Dr. M. Garcia, Dr. C. Y. Cho, Dr. X. Wu
Genomics Institute of the Novartis Research Foundation
10675 John Jay Hopkins Drive, San Diego, CA 92121 (USA)
[+ +] These authors contributed equally to this work.
[**] We thank Dr. Warren G. Lewis, Paul Anderson, Jason Matzen, Jeff
Janes, and Dr. Achim Brinker for compound screening and Francis
Peters for critical reading of the manuscript. This work was funded
in part by grants from the NIH (R01 GM074868, R01 MH051573,
and P50 GM085764 to S.A.K.) and the Skaggs Institute for Chemical
Biology (to P.G.S).
Supporting information for this article is available on the WWW
Figure 1. Period effect and dose response of LH846 on Per2-dLuc and
Bmal1-dLuc U2OS cells. LH846 lengthened circadian period of Per2-dLuc
and Bmal1-dLuc rhythms in a dose-dependent manner.
? 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2011, 50, 10608–10611
Table 1S in the Supporting Information). Twenty six benzo-
thiazole analogues were synthesized and tested in a dose
response format in the U2OS cell-based assay. We defined the
potency of the compound by calculating the concentration
which causes 1 h period lengthening. Methyl and methoxy
substitution on the benzothiazole led to a decrease in activity.
In contrast, chloro and bromo substitution at the 5- or 6-
positions of the benzothiazole ring appear to be tolerated or
slightly increase activity, whereas, chloro substitution at the 4-
position decreased activity. Substitution of the phenylaceta-
mide group with benzamide or phenylsulfonamide moieties
resulted in a complete loss in activity. Also, substitution with
benzothiazole and benzamide moieties (LH25 and LH26) led
to a complete loss in activity. Some derivatives of the
phenylacetamide group with methoxy at the meta or para
position had only slightly decreased activity (see Table 1S in
the Supporting Information). Based on the SAR, the para
position of the phenylacetamide moiety was used for attach-
ment of LH846 to solid support through a diethylene glycol
linker. Although the diethylene glycol derivative (LH846-
linker) was around 10-fold less potent than LH846, it retained
a significant period lengthening effect and was therefore used
in pull-down assays.
An agarose conjugate of LH846-linker (Figure 2a) was
prepared and incubated for 3 h with U2OS cell lysate in the
absence or the presence of 300 mm LH846 as competitor.
Proteins that bound to the affinity resin were separated by
sodium dodecylsulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and visualized by silver staining. One band at
about 45 kDa was observed, and binding of the protein(s) to
the LH846-linker-agarose resin was blocked by free LH846
(Figure 2b), indicating specific binding to LH846. Analysis of
the band by linear ion trap mass spectrometry (LTQ)
suggested casein kinase 1 delta (CKId) as the target. An
independent affinity chromatography pull-down experiment
followed by Western blotting with anti-CKId antibody con-
firmed binding of CKId to the affinity resin which was
blocked in the presence of LH846 (Figure 2c). In vitro kinase
profiling against a panel of around 50 kinases revealed that
LH846 strongly inhibited CKId and, with less potency CKIa
and ROCK2 (see Table 2S in the Supporting Information).
Determination of the half maximal inhibitory concentration
(IC50) based on an in vitro assay revealed that LH846
inhibited CKId with an IC50of 290 nm, CKIa with an IC50of
2.5 mm, CKIe with an IC50of 1.3 mm and had no effect on CK2
(see Figure 1S in the Supporting Information). Compounds
with similar structures to LH846 have been reported as
inhibitors of ubiquitination.However, we found that an
active ubiquitination inhibitor (PC4)had no effect on
circadian period and did not inhibit CKId activity (see
Figure 2S in the Supporting Information). In contrast, an
inactive ubiquitination inhibitor (PC43) that is also similar to
LH846 inhibited CKId activity and lengthened circadian
period (see Figure 2S in the Supporting Information). Fur-
thermore, a commercially available cell-permeable ubiqui-
tin E1 inhibitor PYR-41 showed no effect on period length
(see Figure 2S in the Supporting Information). Taken
together, these results suggest that CKId is the biological
target of LH846.
CKIdis awell-characterizedkinase inperiod regulation of
the circadian clock; modulation of its activity by genetic
mutation or CKI inhibitor is known to cause period
change.[17,18]We therefore knocked down expression of the
CKId gene (CSNK1D) by RNA interference and determined
the effect on period length in the Per2-dLuc U2OS cell-based
assay. siRNA-mediated knockdown of CK1d showed a
significant period lengthening effect while reduction of
CK1e did not alter period length (Figure 3a,b). We confirmed
specific knockdown of CK1d (60%) and CK1e (80%) by
measuring cellular mRNA levels (Figure 4c). The observed
difference in period change caused by LH846 (around 10 h)
and CKId knockdown (<1 h) may be due to partial inhibition
of CKId by siRNA-mediated knockdown (Figure 3c) or a
difference because of inhibition of kinase activity (LH846)
versus reduction of mRNA level (siRNA).
Because CKId is known to cause PER1 proteosomal
degradation upon phosphorylation of PER1 protein,[18,20]we
next investigated the effect of LH846 on CKId-mediated
PER1 phosphorylation using a phosphorylation-dependent
mobility shift assay. Coexpression of PER1 with CKId
Figure 2. Identification of a cellular protein that interacts with LH846.
A) Structure of the LH846-linker. B) The LH846-linker matrix was
incubated with cell lysate of U2OS cells in the presence or absence of
LH846 (300 mm). Bound proteins were separated by SDS-PAGE fol-
lowed by silver staining. C) The LH846 matrix was incubated with cell
lysate of U2OS cells in the absence or presence of LH846 (300 mm).
Proteins bound to the LH846 matrix were immunostained with anti-
Angew. Chem. Int. Ed. 2011, 50, 10608–10611? 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
resulted in a mobility shift and lower abundance of PER1
protein (Figure 4). Treatment with LH846 reduced the PER1
mobility shift and PER1 clearance in a dose-dependent
manner, indicating that CKId-dependent phosphorylation
and subsequent degradation of PER1 protein is inhibited by
LH846 treatment. Previous genetic studies demonstrated an
important role of CKI-dependent phosphorylation of PER
protein in period regulation,[17,18]supporting the period
lengthening effect of LH846 through PER1 protein phos-
The CKI family of serine and threonine kinases is
ubiquitously expressed and genetically conserved in eukar-
yotes.CKIa, CKId, and CKIe are all known to play roles in
modulating circadian rhythms. CKId and CKIe phosphorylate
PER and trigger its degradation;mutations in each affect
the clock in vivo.[17b,23]The relatively nonselective casein
kinase inhibitors IC261 (IC50of 6.0, 4.7, and 31 mm for CKIa,
CKId, and CKIe, respectively) and D4476 (IC50of 2.5, 1.1, and
11 mm for CKIa, CKId, and CKIe, respectively) cause period
lengthening in cell culture, but to a significantly lesser degree
than LH846. The CKIe selective inhibitor PF-4800567 showed
that CKIe does not contribute significantly to period regu-
lation,while longdaysinwhich inhibits CKIa, CKId, and
ERK2 showed a combinational effect of these kinases on
period length comparable to that of LH846.
LH846 is therefore a relatively potent and selective
inhibitor of CKId that has a significant effect on period
length (10 h) in cell culture. Because behavioral studies
mediated by CKId activity are restricted because of the
lethality of known CKId-null mutations,LH846 and its
analogues may prove useful tools for studies of clock function
in more complex organismal systems and may ultimately lead
to chronotherapeutic agents.
Received: June 9, 2011
Revised: August 29, 2011
Published online: September 26, 2011
high-throughput screening · period proteins
Keywords: casein kinase · chemical biology · circadian rhythm ·
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