C-H activation generates period-shortening molecules that target cryptochrome in the mammalian circadian clock

Article abstract of DOI:10.1002/anie.201502942

The synthesis and functional analysis of KL001 derivatives, which are modulators of the mammalian circadian clock, are described. By using cutting-edge C￡H activation chemistry, a focused library of KL001 derivatives was rapidly constructed, which enabled

Full text of DOI:10.1002/anie.201502942

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201502942
German Edition: DOI: 10.1002/ange.201502942
Structure–Activity Relationships
À
C H Activation Generates Period-Shortening Molecules That Target
Cryptochrome in the Mammalian Circadian Clock**
Tsuyoshi Oshima, Iori Yamanaka, Anupriya Kumar, Junichiro Yamaguchi, Taeko Nishiwaki-
Ohkawa, Kei Muto, Rika Kawamura, Tsuyoshi Hirota, Kazuhiro Yagita, Stephan Irle,*
Steve A. Kay, Takashi Yoshimura,* and Kenichiro Itami*
Dedicated to Professor Takao Kondo
Abstract: The synthesis and functional analysis of KL001
derivatives, which are modulators of the mammalian circadian
vates the transcription of the period (Per) and cryptochrome
(Cry) genes. PER and CRY form a complex and suppress
their own gene expression by inhibiting CLOCK–BMAL1-
mediated transcription. FBXL3, an F-box-type ubiquitin
ligase subunit, recognizes CRY at the flavin adenine dinucle-
À
clock, are described. By using cutting-edge C H activation
chemistry, a focused library of KL001 derivatives was rapidly
constructed, which enabled the identification of the critical sites
on KL001 derivatives that induce a rhythm-changing activity
along with the components that trigger opposite modes of
action. The first period-shortening molecules that target the
cryptochrome (CRY) were thus discovered. Detailed studies on
the effects of these compounds on CRY stability implicate the
existence of an as yet undiscovered regulatory mechanism.
T
he circadian rhythm is an approximately 24-hour cell-
autonomous biological oscillation observed in almost all
living organisms.[1] This rhythm regulates various physiolog-
ical behaviors, such as sleep–wake cycles, hormone secretion,
metabolism, and seasonal reproduction.[2,3] The circadian
rhythm is driven by a circadian oscillator, which consists of
multiple transcriptional–translational feedback loops.[3,4] The
main feedback loop of the mammalian circadian clock is
shown in Figure 1. The CLOCK–BMAL1 heterodimer acti-
Figure 1. The core feedback loop of the mammalian circadian clock
and the effect of KL001 on the cryptochrome (CRY).
[*] T. Oshima,[+] Dr. I. Yamanaka,[+] Dr. A. Kumar, Dr. J. Yamaguchi,
Dr. T. Nishiwaki-Ohkawa, K. Muto, R. Kawamura, Dr. T. Hirota,
Prof. Dr. S. Irle, Prof. Dr. S. A. Kay, Prof. Dr. T. Yoshimura,
Prof. Dr. K. Itami
Prof. Dr. K. Yagita
Department of Physiology and Systems Bioscience
Kyoto Prefectural University of Medicine
Kyoto 602-8566 (Japan)
Institute of Transformative Bio-Molecules (WPI-ITbM)
Nagoya University
Chikusa, Nagoya, 464-8601 (Japan)
Prof. Dr. S. A. Kay
Dornsife College of Letters, Arts and Sciences
University of Southern California
E-mail: sirle@chem.nagoya-u.ac.jp
3430 S. Vermont Avenue, Los Angeles, CA 90089-3301 (USA)
takashiy@agr.nagoya-u.ac.jp
itami@chem.nagoya-u.ac.jp
T. Oshima,[+] Dr. J. Yamaguchi, K. Muto, Prof. Dr. S. Irle,
Prof. Dr. K. Itami
Department of Chemistry, Graduate School of Science
Nagoya University
Chikusa, Nagoya, 464-8602 (Japan)
Prof. Dr. T. Yoshimura
National Institute for Basic Biology
Myodaiji-cho, Okazaki 444-8585 (Japan)
Prof. Dr. K. Itami
JST, ERATO, Itami Molecular Nanocarbon Project
Chikusa, Nagoya, 464-8602 (Japan)
[+] These authors contributed equally to this work.
Dr. T. Nishiwaki-Ohkawa, R. Kawamura, Prof. Dr. T. Yoshimura
Graduate School of Bioagricultural Sciences
Nagoya University
[**] This work was supported by the Funding Program for Next
Generation World-Leading Researchers of the JSPS (K.I. and T.Y.),
the ERATO program of the JST (K.I.), and a JSPS KAKENHI grant
(26000013 for T.Y.). We thank Dr. Ayako Miyazaki for critical
comments and Dr. Keiko Kuwata for assistance with mass
spectrometry. The ITbM is supported by the World Premier
International Research Center (WPI) Initiative (Japan).
Chikusa, Nagoya, 464-8601 (Japan)
Dr. T. Hirota
JST, PRESTO
Chikusa, Nagoya, 464-8602 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201502942.
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otide (FAD) binding pocket,[5] promoting proteasomal deg-
radation of CRY.[6–8] Recently, a period-lengthening small
molecule, KL001, that directly targets CRY was discovered.[9]
KL001 stabilizes CRY by binding to the FAD-binding pocket
in competition with FBXL3,[10] thereby extending the circa-
dian period in mammals.[9]
respectively. Although not present in the parent KL001
molecule, the introduction of substituents at the furan or
thiophene moieties might enable the tuning of the period-
changing properties of the molecule. An ideal method to
obtain various substituted derivatives would be a late-stage
[11]
À
C H activation reaction on the heterocyclic groups. We
Provided that KL001 is the first-in-class small-molecule
modulator targeting CRY with a unique molecular structure,
there are huge opportunities for KL001 and its derivatives not
only as tools to understand the mechanism of the mammalian
circadian clock, but also in a number of other applications.
However, very limited information regarding the structure–
activity relationships (SARs) of KL001 has been available up
to now. Herein, we report the first functional analysis of
successfully introduced a range of aryl groups at the C5
position of the heterocycles to afford arylated compounds 4
by treating 2 with aryl iodides in the presence of [PdCl2-
(PPh3)2], AgNO3, and KF in DMSO (Moriꢀs conditions).[14]
The introduction of the aryl group at the least reactive
C4 position of the five-membered heterocycle is known to be
extremely challenging. Nevertheless, we achieved this by
employing our recently developed catalytic system.[15] Thus,
the treatment of 2 with aryl boronic acids in the presence of
[Pd(OAc)2], 2,2’-bipyridyl, and 2,2,6,6-tetramethylpiperidine
1-oxyl (TEMPO) in C6H5CF3 afforded the target C4 arylation
products 5 (see the Supporting Information for details).
By following the above-mentioned synthetic scheme, we
synthesized over 50 KL001 derivatives. The effects of these
new compounds on the circadian-clock activity were inves-
tigated by a cell-based luminescence assay using Bmal1–dLuc
reporter U2OS cells.[9,16–18] The results of the SAR study and
the period-changing activities of representative molecules at
a substrate concentration of 10 mm are summarized in Fig-
ure 2b and 2c, respectively (see the Supporting Information
for the results of other compounds). The carbazole moiety of
KL001 was found to be critical for the period-changing
activity: Replacing the carbazole group with diphenylamine
(GO203) or 2-phenylindole led to a complete loss of activity.
À
a range of KL001 derivatives enabled by cutting-edge C H
activation chemistry.[11–13] This campaign led us to uncover the
sites on KL001 derivatives that are critical for its rhythm-
changing activity and the rhythm-lengthening/shortening
selectivity, along with the discovery of the first period-
shortening molecules targeting CRY.
The established general synthetic scheme for KL001
derivatives is shown in Figure 2a. Arylmethylamines 1 were
first mesylated with CH3SO2Cl and then subjected to
N alkylation with epibromohydrin and a subsequent epoxide
ring-opening reaction with amines to afford KL001-like core
structures 2. The furan and carbazole moieties can be easily
modified in this reaction sequence by changing 1 and the
amines in the epoxide ring-opening step. SAR studies on the
hydroxy group of 2 can be carried out by subsequent
oxidation or substitution reactions to provide 3 and 3’,
Figure 2. a) General synthetic scheme for KL001 derivatives. b) Summary of the SAR study. c) Period-changing activity of representative molecules
in the initial screening.
2
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The hydroxy substructure turned out to be a significant, but
non-critical site as ketone (GO152) and alkoxy analogues
sustained the period-lengthening activity, albeit with less
potency. The furan part was found not to be a critical site as
thiophene analogue GO061 and a phenyl-substituted ana-
logue also exhibited period-changing effects. Fortunately, as
expected, it was found that additional substituents on the
C5 position led to an increase in period-changing activities.
For example, the C5-bromo-substituted derivative GO214
induced a higher period-lengthening effect at the same
concentration. C5-aryl-substituted derivative GO216 had an
activity similar to that of KL001. Interestingly, we discovered
that some aryl substituents at the C4 position could induce the
opposite effect (period shortening), albeit with low efficiency.
For example, when a 4-n-butylphenyl group was attached at
the C4 position (GO044), the period was shortened by
0.4 hours. Given the opposite activity of C5 isomer GO216,
the rotation around the thiophene ring may be rather
restricted at the binding site. We also found that the
substituent effect at the C4 position was rather elusive.
Whereas the introduction of some para-substituted aryl
groups (GO200 and GO211) also induced a period-shortening
effect, a small para substituent, such as the methoxy group
(GO058), led to a period-lengthening effect.
With these exciting preliminary results in hand, we
examined the effects of the following representative com-
pounds in further detail: one period-lengthening compound,
GO214, and three period-shortening compounds, GO044,
GO200, and GO211. We first studied the effects of these four
compounds at three different concentrations (Figure 3a,b).
At 10 mm, the period-lengthening effect of GO214 was
significantly greater than that of KL001 (Supporting Infor-
mation, Figure S1). On the other hand, GO044, GO200, and
GO211 shortened the period by 0.3 to 1 hour at 10 mm
(Figure 3a,b). These effects were also confirmed in the
mPer2 promoter-driven luciferase assay (Figure S2).
We next added period-lengthening and -shortening com-
pounds simultaneously to a cell culture to examine whether
these compounds compete with each other. When we added
a 1 mm solution of KL001 together with a 1 mm solution of
either GO044, GO200, or GO211, the period-lengthening
effect of KL001 was attenuated, and the resulting period was
comparable to that observed in vehicle-treated cells (Fig-
ure 3c and Figure S3a). The period-lengthening effect of
GO214 was also cancelled out by the addition of GO200 or
GO211 (Figure S3b). These results suggest that the period-
shortening compounds may compete with KL001 to bind to
the same FAD-binding pocket.
Therefore, to elucidate the binding mode of the deriva-
tives to CRY in more detail, we performed docking simu-
lations for the representative compounds (period-lengthening
GO214 and period-shortening GO044, GO200, and G0211)
with the CRY protein structure (PDB X-ray structure
4MLP).[10] Technical details of these simulations are described
in the Supporting Information. The optimal docking pose of
the ligands was obtained by performing a conformational
search allowing for torsional flexibility. Using this method,
the binding pose of the crystal structure for KL001 was
successfully reproduced. The best docking pose and the most
Figure 3. Discovery of period-lengthening and -shortening molecules.
a) Representative luminescence traces and b) the changes in the
circadian period in Bmal1-dLuc U2OS cell lines in the presence of each
compound. Measurements were done in triplicate or quadruplicate.
Data are presented as meanÆSD (* P<0.05, ** P<0.01; one-way
ANOVA vs. vehicle). c) Competition assay between KL001 and period-
shortening compounds. Bmal1-dLuc U2OS cells were simultaneously
treated with KL001 (1 mm) and either GO044 or GO200 (1 mm).
Measurements were done in triplicate or quadruplicate. Data are
presented as meanÆSD. Different characters (a–d) indicate significant
differences (P<0.05; one-way ANOVA). For each compound, we
performed three independent experiments, and representative results
are shown.
important amino acid residue interactions of GO214, GO044,
GO200, and G0211 are shown as snapshots of the binding
pocket in Figures 4a, 4b, S4a, and S4b, respectively.
The binding free energies of KL001, GO214, GO044,
GO200, and GO211 were determined to be À10.41, À11.39,
À13.58, À8.44, and À12.27 kcalmolÀ1, respectively, indicating
that all four compounds bind to the FAD-binding pocket of
CRY. In a similar manner to KL001, the hydroxy moiety and
the sulfonyl moiety in GO214 and GO044 form a hydrogen
bond with S414 and H377, respectively. Furthermore, the
mesyl moiety forms a CH–p interaction[19] with W417 and
W310 (within < 4 ꢁ), which is similar to the interaction of
P426 in the FBXL3–CRY complex (PDB: 4I6J).[20] In the case
of GO200, two hydrogen bonds were observed, one between
the mesyl group and H377 and the second one between the
hydroxy moiety and H373 instead of S414, owing to a torsional
change to fit into the active site. The superimposition of the
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GO200, there was no flip of the thiophene moiety, but
a hydrogen bond with S414 was lost, which results in a lower
binding free energy. Therefore, novel compounds that probe
new conformational space for additional interactions in the
active site are potential CRY modulators.
Although we cannot disregard other possibilities, both the
competition assay (Figures 3c and S3) and the docking study
(Figure 4) suggest that our representative compounds
(period-lengthening GO214 and period-shortening GO044,
GO200, and G0211) target the FAD-binding pocket of CRY.
KL001 inhibits the FBXL3- and ubiquitin-dependent degra-
dation of the CRY protein.[9,10] Therefore, we further
examined whether KL001 derivatives also affect the stability
of CRY. When we examined the effect of period-lengthening
compound GO214 on the CRY1 degradation rate using
a HEK293 cell line stably expressing the CRY1–luciferase
fusion protein (CRY1–LUC),[9] the half-life of CRY1–LUC
increased in a dose-dependent manner (Figures 5 and S6)
Figure 4. The overlaid docking poses of a) GO214 (light green) and
b) GO044 (dark green) with KL001 (magenta) bound to the CRY
protein (PDB: 4MLP).
Figure 5. Effects of KL001 derivatives on the half-live of CRY1 (t1/2 (h))
in the HEK293 stable cell line expressing the CRY1–LUC fusion
protein. Measurements were done in triplicate or quadruplicate, and
data are presented as meanÆSD. Different characters (a–d) indicate
significant differences (P<0.05; one-way ANOVA vs. vehicle). Data are
representative of four to eight independent experiments.
best binding pose of GO200 to CRY (PDB: 4MLP) with that
of FAD bound to CRY (PDB: 4I6G)[20] shows that the tert-
butyl moiety of GO200 occupies the position of the sugar
moiety of FAD in the FAD-binding site (Figure S5a). Hence,
more hydrogen-bond acceptors at this position could enhance
the binding of GO200 and mimic the interactions of FAD. In
the case of GO211, which showed similar behavior to KL001,
two hydrogen bonds are formed, with one observed between
the mesyl group and H377 and the second one between the
hydroxy moiety and S414. The superposition of the best
binding mode of GO211 to CRY (PDB: 4MLP) with that of
FAD bound to CRY (PDB: 4I6G), shows that the methox-
ymethoxy moiety of GO211 bends towards the flavin ring in
FAD (Figure S5b). The silyloxyphenyl moiety in GO200 and
the methoxymethoxyphenyl moiety in GO211 occupy the
position of a water molecule and form a hydrogen bond with
another water molecule. Surprisingly, the longer side chains of
the period-shortening compounds, that is, GO044, GO200,
and GO211, fit reasonably well into the binding pocket of
FAD. To fit into the binding pocket, the conformation of the
thiophene moiety in GO044 and GO211 was flipped without
a reduction in the binding strength. On the other hand, in
without affecting LUC stability (Figure S7). This suggests that
GO214 lengthens the circadian period in a similar manner to
KL001. Unexpectedly, however, period-shortening molecules
did not exhibit CRY1-destabilizing activity; GO044 had little
effect on the stability of CRY1, and both GO200 and GO211
increased the half-life of CRY1 in a dose-dependent manner
(Figures 5 and S6). It is possible that these molecules regulate
the circadian period not only by the degradation of CRY but
also through some other unknown processes mediated by
CRY. Although further extensive studies are necessary to
elucidate the as yet unknown CRY-mediated period-short-
À
ening mechanism, it is clear that C H activation chemistry
unveiled an untapped chemical space of biological impor-
tance.
In summary, we have succeeded in uncovering the sites of
KL001 derivatives that are critical for their rhythm-changing
4
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Lee, Z. Wang, S. Chang, J. Am. Chem. Soc. 2014, 136, 4141; h) J.
activities and period-lengthening/shortening selectivities
towards the CRY-mediated circadian clock regulation. We
envisage that these compounds provide a tool to study the
regulatory mechanism of CRY in the circadian timekeeping
mechanism. Furthermore, the modulation of the circadian
period is expected to improve animal production and to form
the basis of therapeutic applications. Last but not least, we
wish to emphasize that the present study represents the
fruitful merging of synthetic chemistry, circadian-clock sci-
ence, and theoretical chemistry, and also showcases the power
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À
of new C H activation processes in discovering new biofunc-
tional molecules (the first period-shortening molecules tar-
geting the CRY were discovered in this study). With the
À
recent advent of a number of game-changing C H activation
reactions,[12,13] there are significant opportunities to use these
methods to accelerate circadian-clock science.
À
Keywords: C H activation · circadian clock · cryptochrome ·
small-molecule modulators · structure–activity relationships
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Received: March 30, 2015
Published online: && &&, &&&&
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

.
Angewandte
Communications
Communications
Structure–Activity Relationships
T. Oshima, I. Yamanaka, A. Kumar,
J. Yamaguchi, T. Nishiwaki-Ohkawa,
K. Muto, R. Kawamura, T. Hirota,
K. Yagita, S. Irle,* S. A. Kay,
T. Yoshimura,* K. Itami*
&&&& — &&&&
À
C H Activation Generates Period-
A change in rhythm: The first functional
analysis of KL001 derivatives, which are
mammalian circadian-clock modulators,
that are critical for their rhythm-changing
activity were elucidated, which led to the
discovery of the first period-shortening
molecules that target the cryptochrome.
Shortening Molecules That Target
Cryptochrome in the Mammalian
Circadian Clock
À
was enabled by cutting-edge C H activa-
tion. The sites of the KL001 derivatives
6
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!