Photopharmacological Manipulation of Mammalian CRY1 for Regulation of the Circadian Clock

Article abstract of DOI:10.1021/jacs.0c12280

CRY1 and CRY2 proteins are highly conserved components of the circadian clock that controls daily physiological rhythms. Disruption of CRY functions are related to many diseases, including circadian sleep phase disorder. Development of isoform-selective a

Full text of DOI:10.1021/jacs.0c12280

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Photopharmacological Manipulation of Mammalian CRY1 for
Regulation of the Circadian Clock
†
†
†
̌
Dusan Kolarski, Simon Miller, Tsuyoshi Oshima, Yoshiko Nagai, Yugo Aoki, Piermichele Kobauri,
Ashutosh Srivastava, Akiko Sugiyama, Kazuma Amaike, Ayato Sato, Florence Tama, Wiktor Szymanski,
Ben L. Feringa,* Kenichiro Itami,* and Tsuyoshi Hirota*
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ABSTRACT: CRY1 and CRY2 proteins are highly conserved
components of the circadian clock that controls daily physiological
rhythms. Disruption of CRY functions are related to many diseases,
including circadian sleep phase disorder. Development of isoform-
selective and spatiotemporally controllable tools will facilitate the
understanding of shared and distinct functions of CRY1 and CRY2.
Here, we developed CRY1-selective compounds that enable light-
dependent manipulation of the circadian clock. From phenotypic
chemical screening in human cells, we identified benzophenone
derivatives that lengthened the circadian period. These compounds
selectively interacted with the CRY1 photolyase homology region,
resulting in activation of CRY1 but not CRY2. The benzophenone
moiety rearranged a CRY1 region called the “lid loop” located outside of
the compound-binding pocket and formed a unique interaction with Phe409 in the lid loop. Manipulation of this key interaction was
achieved by rationally designed replacement of the benzophenone with a switchable azobenzene moiety whose cis−trans
isomerization can be controlled by light. The metastable cis form exhibited sufficiently high half-life in aqueous solutions and
structurally mimicked the benzophenone unit, enabling reversible period regulation over days by cellular irradiation with visible light.
This study revealed an unprecedented role of the lid loop in CRY-compound interaction and paves the way for spatiotemporal
regulation of CRY1 activity by photopharmacology for molecular understanding of CRY1-dependent functions in health and disease.
ing, ever since the discovery of a compound targeting both
CRY1 and CRY2.11 Recently identified CRY1- or CRY2-
selective compounds also interact with the FAD-binding
pocket but require the CCT, which is diversified between
CRY1 and CRY2, for their selective effects.12,13 To date, no
compound has been reported to target the conserved PHR in
an isoform-selective manner.
In mammals, Per, Cry, Clock, and Bmal1 genes form a
transcription-translation feedback loop to generate circadian
rhythms at a cellular level. CLOCK-BMAL1 heterodimer
activates transcription of Per and Cry genes, and their protein
products, PER and CRY, inhibit CLOCK-BMAL1 function.
Degradation of PER and CRY results in reactivation of
CLOCK-BMAL1 to start the cycle over again.14 Because these
clock components reside ubiquitously in almost all cells of the
INTRODUCTION
■
CRY proteins belong to the photolyase/cryptochrome family
and consist of a highly conserved photolyase homology region
(PHR) that binds to flavin adenine dinucleotide (FAD) and a
diversified CRY C-terminal domain (CCT).1 In plants and
insects, CRY acts as a blue light photoreceptor by using FAD
as a cofactor. In contrast, mammalian CRYs, CRY1 and CRY2,
are light-independent transcriptional repressors2 that regulate
circadian rhythms and blood glucose levels in partially
overlapping ways.3−5 Mutation of CRY1 and CRY2 genes
results in familial delayed and advanced sleep phase in humans,
respectively, due to the altered period length of the circadian
clock.6,7 Furthermore, increasing evidence indicates distinct
roles of CRY1 and CRY2, as shown, for example, by CRY2-
dependent degradation of a proto-oncogene product, c-MYC.8
Development of small molecule modulators, especially iso-
form-selective tools, will accelerate the understanding of shared
and distinct functions of CRY1 and CRY2 as attractive
therapeutic targets of circadian clock-related diseases. How-
ever, high similarity of the FAD-binding pocket (16 out of 17
residues are identical in CRY1 and CRY2) in the PHR9,10 has
rendered the design of isoform-selective compounds challeng-
Received: November 24, 2020
Published: January 19, 2021
https://dx.doi.org/10.1021/jacs.0c12280
J. Am. Chem. Soc. 2021, 143, 2078−2087
© 2021 American Chemical Society
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Figure 1. Benzophenone derivatives lengthen the circadian period. (A) Chemical structures of TH303, TH129, and KL101. (B−D) Effects on
circadian rhythms in Bmal1-dLuc and Per2-dLuc U2OS cells. Luminescence rhythms in the presence of various concentrations of compounds (B,
mean of n = 3) and changes in period (C) and luminescence intensity (D) compared to a DMSO control are shown (n = 3 biologically
independent samples). (E) Period-lengthening activity of TH129 derivatives in Bmal1-dLuc cells. The concentrations for 2-h period-lengthening of
the derivatives are shown (n ≥ 2 biologically independent samples).
body, spatiotemporal control over compound activity, in
addition to isoform selectivity, is required to precisely regulate
the target protein for a deeper understanding of its functions
and local control of the circadian clock system. Photo-
pharmacology is an emerging approach that utilizes photo-
responsive molecular moieties to control drug activity with
high spatiotemporal resolution using light stimulus.15,16
Azobenzenes are the most commonly used photoswitches
that enable light-dependent reversible isomerization of the
compound structure between the thermally stable trans-form
and thermally unstable cis-form and have been applied to
noninvasively control biological processes ranging from
milliseconds to hours.17−19 Because of the short half-life of
the cis-isomer in most cases, its application to long-term
biological processes, such as circadian rhythms, has been
particularly challenging.
Here, we discovered isoform-selective compounds against
CRY1 PHR, and by meticulous design of their azobenzene
derivatives, achieved light-dependent reversible manipulation
of mammalian CRY1 function to control cellular circadian
rhythms. This study provides benzophenone as a useful
platform to develop azobenzene-based photoswitchable
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Figure 2. TH303 and TH129 are selective against CRY1. (A) Effects of TH303, TH129, and KL101 on CRY degradation in HEK293 cells. The
half-lives of CRY1-luciferase fusion protein CRY1-LUC and CRY2-LUC relative to LUC are plotted by setting a DMSO control to 1 (n = 3
biologically independent samples). Effects of KL001, which stabilizes both CRY1 and CRY2, are also shown (gray). (B) Effects on Per2::Luc knock-
in reporter activity in wild type, Cry1/Cry2 double knockout, Cry1 knockout, and Cry2 knockout fibroblasts. Changes in luminescence intensity
compared to a DMSO control are shown (n = 4 biologically independent samples from two experiments). (C) Effects on circadian period in
Per2::Luc knock-in fibroblasts. Changes in period compared to a DMSO control are shown (n = 4 biologically independent samples from two
experiments). (D) Interaction with CRY1(PHR) and CRY2(PHR) in vitro. Changes in denaturing temperatures of recombinant CRY proteins in
the presence of various concentrations of compounds compared to a DMSO control are shown (n = 4 biologically independent samples from two
experiments). Compound interaction induced thermal stabilization.
molecules, and forms the basis of precise control of the
circadian system.
CRY2 in HEK293 cell degradation assays (Figure 2A), without
affecting the activities of CKIδ, CKIα, or CK2 in in vitro kinase
assays (Figure S1B). Consistent with the repressive effect of
CRY1 on Per2 transcription,14 stabilization of CRY1 by
TH303 and TH129 resulted in the reduction of Per2-dLuc
reporter activity in human U2OS cells (Figure 1D) and
Per2::Luc knock-in reporter activity in mouse fibroblasts
(Figure 2B, wild type). Per2::Luc repression by TH303 and
TH129 was abolished in Cry1 knockout and Cry1/Cry2 double
knockout cells, while it was enhanced in Cry2 knockout cells
(Figure 2B). Consistently, the period lengthening effects of
TH303 and TH129 were blunted by Cry1 knockout but not
Cry2 knockout (Figures 2C and S1C). These results together
indicated CRY1-selectivity of TH303 and TH129.
CRY1 and CRY2 proteins consist of a highly conserved PHR
and a diversified CCT. KL101 requires the CCT for its CRY1-
selective effect and, therefore, interacts with CRY1(PHR) and
CRY2(PHR) similarly in in vitro thermal shift assays12 (Figure
2D). To our surprise, TH303 and TH129 showed strongly
reduced interaction with CRY2(PHR) compared with CRY1-
(PHR) (Figure 2D). Therefore, TH303 and TH129 are the
first-in-class compounds selectively targeting CRY1(PHR).
Compound-Induced Rearrangement of CRY1. To
reveal the molecular basis of CRY1-compound interaction,
we determined the X-ray crystal structures of CRY1(PHR) in
RESULTS
■
Identification of CRY1-Selective Compounds. We
conducted cell-based phenotypic screening of circadian clock
modulators in human U2OS reporter cells20 and identified hit
compounds that affected the circadian period. Here, we focus
on a benzophenone derivative TH303 (Figure 1A, left).
TH303 caused dose-dependent period lengthening in two
clock gene reporter cells Bmal1-dLuc and Per2-dLuc (Figures
1B,C) and repressed Per2-dLuc activity stronger than Bmal1-
dLuc (Figures 1B,D). These biological effects and the chemical
structure of TH303 were similar to a phenylpyrazole derivative
KL101 that we recently identified as a CRY1-selective
compound12 (Figure 1A, right, and B−D). We therefore
tested a hybrid molecule TH129 (a benzophenone derivative
of KL101; Figure 1A, middle) and observed similar circadian
effects with higher potency than TH303 (Figures 1B−D).
Neither TH303 nor TH129 affected cell viability (Figure S1A).
Substitution of the benzoyl group of TH129 severely reduced
the period-lengthening activity (TH124−TH128; Figure 1E),
indicating its essential role in circadian clock regulation.
Among the target protein candidates for period length-
ening,11,21,22 TH303 and TH129 stabilized CRY1 but not
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Figure 3. TH303 and TH129 interact with the FAD-binding pocket and rearrange the lid loop of CRY1. (A) Overall X-ray crystal structures of
CRY1 in apo form and in complex with TH303, TH129, and KL101. The FAD-binding pocket (box) is shown in B. (B) Interactions of TH303 and
TH129 with CRY1. Residues in hydrophobic region 1, hydrophobic region 2, and the affinity region are shown in blue, green, and magenta
characters, respectively. Red nonbounded spheres and yellow dashed lines represent water molecules and hydrogen bonds, respectively. F409
(gray) in the lid loop that interacted with the benzophenone moiety of the compounds is also shown. (C) Effects of TH303 and TH129 on W399
and the lid loop. Conformational changes in W399, F405, and F409 are visualized by dashed arrows. The auxiliary pocket of CRY1-TH303 is
shown by the surfaces of residues F295, F296, A299, F306, I314, M398 and S404. Negative, positive, and sulfur-containing regions are colored red,
blue, and yellow, respectively. (D) Gibbs free energy landscape corresponding to W399, F405, and F409 Chi1 dihedral angle and distance from
center of mass of the FAD-binding pocket, calculated using combined trajectory of three independent MD simulation runs of CRY1-apo, CRY1-
TH303, and CRY1-TH303 (TH303 removed). Red dots represent poses in the corresponding crystal structure. (E, F) Effects of KL101, TH303,
and TH129 on CRY degradation in HEK293T cells. The half-lives of CRY1-LUC, CRY1 F409A-LUC mutant, and CRY2-LUC relative to LUC are
plotted by setting a DMSO control to 1 (E, n = 4 biologically independent samples from two experiments; ****P < 0.0001, ***P < 0.001, **P <
0.01, and *P < 0.05 for CRY1 F409A relative to CRY1; ns, not significant). Basal stability is shown in F (n = 12; ****P < 0.0001 and ***P < 0.001,
relative to CRY1).
complex with TH303 and TH129 (Table S1). The overall
structures of CRY1-TH303 (PDB ID: 7D1C) and CRY1-
TH129 (7D19) were similar to CRY1-KL101 (6KX6) and
CRY1-apo (6KX4) (Figure 3A). The methoxyphenyl sub-
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Figure 4. Rational design and characterization of azobenzene derivatives of TH129. (A) Rational azologization of benzophenone. (B) Distributions
of ring angles and distances of benzophenone and cis- and trans-azobenzene structures in the CSD. (C) Synthesis and photoisomerization scheme
of GO1323, DK551, YG037, and GO1423. (D) Docking poses of cis- (red) and trans- (gray) isomers of the compounds superposed with CRY1-
TH129 (white-cyan). (E) Photophysical properties of the compounds. (F, G) Photoisomerization spectra and reversible photochromism of
GO1323 (F) and GO1423 (G) in DMSO solution (∼30 μM, 25 °C). The photoisomerization UV−vis spectra (left) show thermally adapted
photoswitches (dark), and PSSs reached at 365 nm (180 s), 420 nm (60 s), 530 nm (240 s), and 390 nm (90 s). The reversible photochromism
graphs (right) show cycling between two PSSs of each compound, starting from the thermally adapted state. For GO1323, 365 nm light is used for
trans-to-cis and 420 nm light for cis-to-trans isomerization, while for GO1423, cycles were performed using 530 and 390 nm light.
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Figure 5. Azobenzene derivatives of TH129 regulate the circadian period in a light-dependent manner. (A) Effects of GO1323, DK551, and YG037
on circadian rhythms in Bmal1-dLuc U2OS cells. Changes in period compared to a DMSO control are shown (n = 3−6 biologically independent
samples). Diluted compounds were irradiated in vitro with UV light (60 min; purple) followed by white light (10 min; orange) and then applied to
the cells. (B) Effect of GO1323 and TH129 on circadian rhythms. Changes in period compared to a DMSO control are shown (n = 4 biologically
independent samples). UV light-irradiated compounds were applied to the cells (purple), then irradiated in cellulo with white light (10 min;
orange). (C) Interaction with CRY1(PHR) and CRY2(PHR) in vitro. Changes in denaturing temperatures of recombinant CRY proteins in the
presence of various concentrations of compounds compared to a DMSO control are shown (n = 2 biologically independent samples). (D) Effect of
GO1423 and TH129 on circadian rhythms. Changes in period compared to a DMSO control are shown (n = 5 biologically independent samples).
Compounds were applied to the cells and then irradiated in cellulo with green light (30 min; green) and with violet light for back-isomerization (10
min; violet). Inset of the left panel represents the period changes at 7 μM GO1423 (****P < 0.0001, relative to dark control).
stituent of TH303 and the m-xylene substituent of TH129
located chiefly at hydrophobic region 2 of the FAD-binding
pocket by interacting with R358, A362, F381, and W397. They
also interacted with L400 in hydrophobic region 1 and H355
and H359 in the affinity region (Figure 3B). The
dihydrothienopyrazole moiety in TH303 and TH129 formed
a stacking interaction with H355 in the affinity region, a
hydrogen bond to R358, and hydrophobic interactions with
A388 and I392 in hydrophobic region 2. The amide group of
both compounds formed a canonical H-bond with S396 and an
H-bond with an induced “in” conformation of Q289 in the
affinity region. The benzophenone moiety formed hydro-
phobic and stacking interactions with W292, F296, and L400
in hydrophobic region 1 and F409 in the lid loop outside the
FAD-binding pocket (gray in Figure 3B).
flexible after removal of the compound (TH303 removed), and
W399 exited the auxiliary pocket to re-enter the FAD-binding
pocket. These static and dynamic structures revealed that
TH303 induced unique conformations of W399 and F409.
Because the replacement of the benzoyl group in TH129
severely reduced period-lengthening activity (Figure 1E), we
further analyzed the role of F409 in the effects of the
compounds. A CRY1 F409A mutant showed reduced
responses to TH303 and TH129 compared with wild type
CRY1 in cell-based degradation assays while retaining a KL101
response (Figure 3E). Together, the results suggest that the
interaction of F409 in the lid loop with the benzophenone
moiety plays a key role in the effect of TH303 and TH129 on
CRY1.
Rational Design for Replacement of Benzophenone
with Azobenzene. Incorporation of a photoswitchable
azobenzene moiety into a compound allows reversible
photoisomerization between the two isomers, cis and trans.
Typically, UV light is used for the trans-to-cis isomerization,
and visible light drives the reverse process. Furthermore,
usually the cis isomer is thermally unstable, and will isomerize
back to the trans isomer in time. The structural difference
between the isomers of the photoswitch-modified compound is
expected to cause distinct affinity toward the target protein
and, therefore, enable reversible modulation of its activity.15,16
The essential role of the benzophenone moiety for CRY1
regulation (Figure 3) prompted us to photopharmacologically
manipulate its interaction with F409 for controlling the
circadian clock, under the hypothesis that benzophenone
moiety is structurally and electronically similar to the
azobenzene photoswitch (Figure 4A). Screening of the
Cambridge Structural Database (CSD) showed clear similarity
Compared to CRY1-KL101,12 the significantly increased
steric bulk of the benzophenone caused a large conformational
change in the side chain of W399, which was ejected from the
FAD-binding pocket and inserted into an “auxiliary pocket”
composed of F295, F296, A299, F306, I314, M398, and S404,
leading to a positional shift of ∼6 Å at the ζ3-carbon (Figures
3C and S2). The auxiliary pocket is typically occupied by F405
in the lid loop (in CRY1-apo and CRY1-KL101). Insertion of
W399 into this pocket in CRY1-TH303 and CRY1-TH129
resulted in the ejection of F405 and conformational rearrange-
ment of the lid loop inducing an interaction of F409 with the
benzoyl group (Figure 3C). The flexibility of these residues
was evaluated by molecular dynamics (MD) simulations
(Figures 3D and S3). The movement of W399 and F405,
but not F409, was very limited in CRY1-apo. Interaction with
the compound in CRY1-TH303 restricted the motion of F409
and increased F405 mobility. All three residues became more
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of benzophenone and cis-azobenzene substructures in both
ring angles and distances between the two aromatic rings
(Figure 4B). Through a comparative analysis of protein−ligand
complexes from the Protein Data Bank (PDB), the geometry
distributions in the CSD and the PDB were found to be in very
good agreement (Figure S4A). Moreover, the experimental
dipole moments of benzophenone (3.0 D23) and cis-
azobenzene (3.0 D) are identical, while trans-azobenzene has
a dipole moment of 0 D.24 This striking analogy was confirmed
by density functional theory (DFT) calculations (Table S2).
We therefore designed photoswitchable TH129 analogues
by replacing the benzophenone with the azobenzene moiety.
With the aim of determining the most favorable azobenzene
regioisomer for light modulation, we considered all three
possible structures (GO1323, DK551, and YG037 for para,
meta, and ortho positions, respectively) (Figure 4C). Docking
simulations of the azobenzene derivatives into the CRY1-
TH129 crystal structure showed that the cis-isomers of
GO1323 and DK551 mimicked the bent geometry of the
benzophenone moiety to engage in a π−π interaction with
F409, which was not formed by the largely different
conformations of the other binding poses (Figures 4D and
S4B), suggesting a possibility of light-dependent regulation of
CRY1. Furthermore, the calculated dipole moment of the
entire molecule supported the bioisosteric replacement of
benzophenone with cis-azobenzene, especially attached at the
para position (Table S3).
Based on rational design, we synthesized azobenzene
derivatives of TH129 by acylation of amine S1 with acyl
chloride derivatives of the corresponding azobenzenes (Figure
4C). In addition to the structural change, photophysical
properties of the photoswitchable compounds play an
important role in enabling reversible regulation of the target
protein function. The photostationary state (PSS) determines
the trans/cis ratio under light irradiation, and a high PSS is
necessary to obtain large light-induced changes of the effect of
the compound. Furthermore, in order to control the circadian
period in cellular assays, which requires several days to evaluate
the biological effect, the cis-isomer needs to be highly stable
(i.e., display slow thermal cis-to-trans isomerization) in
aqueous solutions. Achieving both high PSS and high thermal
stability of the metastable cis-isomer presents a major challenge
in photopharmacology.19,25 Interestingly, GO1323 showed
high trans-to-cis PSS after UV light irradiation (82% cis isomer)
and subsequent cis-to-trans PSS after irradiation with white
light (77% trans isomer). Moreover, we observed a long half-
life (>1 d) of the cis-isomer in both DMSO and cell culture
medium (Figures 4E and S4C). In contrast, DK551 showed
low PSS after UV light irradiation (32% cis isomer), while the
half-life of cis-YG037 was short, preventing reliable determi-
nation of the PSS. Performed reversible photochromism of
GO1323 indicated high photostability of this photoswitch
(Figure 4F).
with lower period effects compared with GO1323, and the
effects of neither YG037 nor TH129 were changed by light.
These results were consistent with CRY1 docking simulations
(Figure 4D) and photophysical properties (Figure 4E).
Furthermore, irradiation with white light to cell culture also
resulted in the deactivation of UV-irradiated GO1323 (Figure
5B). In vitro thermal shift assays revealed UV light-dependent
and CRY1-selective interaction of GO1323 with CRY(PHR)
(Figure 5C), supporting the modulation of CRY1 activity by
light for the circadian period control.
GO1323 provided the basis of light-dependent reversible
control of the circadian period. However, trans-to-cis photo-
isomerization in cell culture was hampered because of the
cytotoxicity of UV light as well as a high concentration of
luciferin in cell culture medium for circadian luciferase reporter
assays, resulting in strong absorption of UV light. We therefore
synthesized GO1423 (Figure 4C) by introducing the tetra-o-
fluoro azobenzene moiety whose trans-to-cis and cis-to-trans
isomerization can be achieved by visible light: green and violet,
respectively.26 Similar to GO1323, the docking pose of cis-
GO1423 resembled TH129 (Figure 4D), and GO1423 showed
high PSS as well as a very long half-life of the cis-isomer
(Figures 4E and S4C). Moreover, repeated photoisomerization
cycles indicate high photostability (Figure 4G). In cellular
circadian assays, thermally adapted and dark-kept GO1423
(>98% trans) was almost inactive, while green light-irradiated
(cis-enriched) GO1423 caused period lengthening (Figure S5).
Furthermore, cellular irradiation with green light activated
GO1423 and subsequent irradiation with violet light resulted
in its deactivation, without affecting TH129 (Figure 5D).
Altogether, we developed isoform-selective photoswitchable
modulators of CRY1 and enabled reversible control of the
circadian period by visible light.
DISCUSSION
■
We discovered previously unknown benzophenone derivatives
that selectively target CRY1 for circadian clock regulation. This
finding brought about two breakthroughs: (1) isoform
selectivity against a highly conserved region, and (2) reversible
and noninvasive long-term regulation of biological processes,
specifically the circadian clock, utilizing photopharmacology.
Development of isoform-selective compounds against highly
homologous proteins has been the major challenge in drug
discovery. In contrast to the difficulty of molecular design due
to high (91%) sequence similarity of the entire PHR (∼500
residues) between CRY1 and CRY2, unbiased phenotypic
screening of circadian clock modulators resulted in the
identification of unique tools, TH303 and TH129. CRY(PHR)
contains two functional pockets: the FAD-binding pocket,
which is recognized by the C-terminal region of a ubiquitin
ligase FBXL3 for degradation,10 and the secondary pocket,
which interacts with CLOCK-BMAL1 for transcriptional
repression.27,28 TH303 and TH129 occupied the FAD-binding
pocket, resulting in stabilization of CRY1 and consequently
causing the period lengthening. These compounds also
rearranged the lid loop structure through a unique interaction
of the benzophenone moiety with CRY1 F409. The lid loop is
located at an interaction interface with FBXL3 and PER210,29
and has functional importance: CRY1 F409A mutation
resulted in increased basal stability of CRY1 (Figure 3F) and
hyper repression of Per2 reporter activity.30 Therefore, the lid
loop provides a new target to control CRY function by
compounds.
Photopharmacological Regulation of the Clock. We
then analyzed the effects of GO1323, DK551, and YG037 on
the circadian period using Bmal1-dLuc reporter U2OS cells. All
the compounds were first thermally adapted to obtain their
trans isomers (>98%). Dark-kept GO1323 showed only a
minor effect on the period, while UV-irradiated (cis-enriched)
GO1323 caused significant period lengthening (Figure 5A).
Subsequent white light irradiation in vitro (trans-enriched
GO1323) reduced the period-lengthening activity equivalent
to the dark sample. DK551 exhibited light-dependent changes
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Application of a cutting-edge photopharmacological ap-
proach opens the possibility of spatiotemporal manipulation of
circadian rhythms. We previously introduced photoremovable
protecting groups (PPGs) to the CKI inhibitor longdaysin and
demonstrated inducible regulation of the circadian period by
light.31 This method, however, is limited by the irreversible
nature of PPG activation. In contrast, an azobenzene
photoswitch enables light-induced reversible regulation, but
its successful introduction into the compound of interest is not
always straightforward. One of the greatest challenges in
photopharmacology is to obtain and maximize the difference of
binding affinity between the cis- and trans-isomer of the
photoswitchable drug.32,33 Here, we have shown that
benzophenone is a suitable moiety for rational azologization
of biologically active compounds. The bent geometry and
dipole moment of benzophenone were critical in successfully
obtaining “turn-on” type photoswitches as a result of higher
similarity with the metastable cis-isomer. Detailed structure−
activity relationship and photochemical analyses of all possible
azobenzene regioisomers led to discovery of the ideal
substitution for light-dependent regulation of CRY1. Long
half-life of the cis-isomer, high PSS and stability, and
photoreversibility in cell culture medium were crucial photo-
chemical parameters for successful demonstration of reversible
manipulation of circadian rhythms in long-term cellular assays.
Although the majority of biologically applied photoswitching
molecules to date utilize UV light for trans-to-cis isomer-
ization,15,16,32,33 UV is poorly biocompatible because of
cytotoxicity and negligible tissue penetration. Due to the
deeper penetration and lower toxicity of visible light, GO1423
comprising a tetra-o-fluoroazobenzene moiety has an advant-
age for future application to circadian clock regulation at tissue
levels using green and violet light for isomerization. Given that
CRY1 is related to many diseases such as sleep phase disorder,6
diabetes,4,5 and cancer,34 isoform-specific spatiotemporal
regulation of CRY1 by photopharmacology represents a
promising basis of targeted chronotherapy in the future.
9747 AG, The Netherlands; orcid.org/0000-0003-0588-
8435; Email: b.l.feringa@rug.nl
Authors
̌
Dusan Kolarski − Centre for Systems Chemistry, Stratingh
Institute for Chemistry, University of Groningen, Groningen
9747 AG, The Netherlands
Simon Miller − Institute of Transformative Bio-Molecules,
Nagoya University, Nagoya 464-8601, Japan
Tsuyoshi Oshima − Institute of Transformative Bio-Molecules
and Department of Chemistry, Graduate School of Science,
Nagoya University, Nagoya 464-8601, Japan
Yoshiko Nagai − Institute of Transformative Bio-Molecules,
Nagoya University, Nagoya 464-8601, Japan
Yugo Aoki − Institute of Transformative Bio-Molecules and
Department of Chemistry, Graduate School of Science,
Nagoya University, Nagoya 464-8601, Japan
Piermichele Kobauri − Centre for Systems Chemistry,
Stratingh Institute for Chemistry, University of Groningen,
Groningen 9747 AG, The Netherlands
Ashutosh Srivastava − Institute of Transformative Bio-
Molecules, Nagoya University, Nagoya 464-8601, Japan;
orcid.org/0000-0001-9820-720X
Akiko Sugiyama − Institute of Transformative Bio-Molecules,
Nagoya University, Nagoya 464-8601, Japan
Kazuma Amaike − Institute of Transformative Bio-Molecules
and Department of Chemistry, Graduate School of Science,
Nagoya University, Nagoya 464-8601, Japan
Ayato Sato − Institute of Transformative Bio-Molecules,
Nagoya University, Nagoya 464-8601, Japan
Florence Tama − Institute of Transformative Bio-Molecules
and Department of Physics, Graduate School of Science,
Nagoya University, Nagoya 464-8601, Japan;
Computational Structural Biology Unit, RIKEN-Center for
Computational Science, Hyogo 650-0047, Japan;
orcid.org/0000-0003-2021-5618
Wiktor Szymanski − Centre for Systems Chemistry, Stratingh
Institute for Chemistry and Department of Radiology,
Medical Imaging Center, University of Groningen, Groningen
9747 AG, The Netherlands
ASSOCIATED CONTENT
* Supporting Information
■
sı
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c12280
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c12280.
Figures, tables, methods, compound synthesis, geometry
measurements, dipole moment calculation, PSS distri-
bution determination, photoisomerization, half-life
determination, and references (PDF)
Author Contributions
†D.K., S.M., and T.O. contributed equally.
Notes
The authors declare no competing financial interest.
X-ray data for CRY1-TH129 (CIF)
X-ray data for CRY1-TH303 (CIF)
ACKNOWLEDGMENTS
■
We thank Natsuko Ono, Daniel Bader, Dr. Yoshiki Aikawa,
Ayano Shiba, Naoya Kadofusa, Dr. Shinya Oishi, Dr. Kazuhiro
Abe, Dr. Kunio Hirata, and Dr. Toshiya Senda for technical
assistance. This work was supported in part by JST PRESTO
Grant JPMJPR14LA (T.H.); JSPS Grants 18H02402 and
20K21269 (T.H.), and JP1905463 (K.I.); Takeda Science
Foundation (T.H.); Uehara Memorial Foundation (T.H.);
The Netherlands Organization for Scientific Research NWO−
CW Top grant (B.L.F.) and VIDI Grant 723.014.001 (W.S.);
the Royal Netherlands Academy of Arts and Sciences KNAW
(B.L.F.); the Ministry of Education, Culture and Science
Gravitation program 024.001.035 (B.L.F.); and the European
Research Council Advanced Investigator Grant 227897
AUTHOR INFORMATION
Corresponding Authors
■
Tsuyoshi Hirota − Institute of Transformative Bio-Molecules,
Nagoya University, Nagoya 464-8601, Japan; orcid.org/
0000-0003-4876-3608; Email: thirota@itbm.nagoya-
u.ac.jp
Kenichiro Itami − Institute of Transformative Bio-Molecules
and Department of Chemistry, Graduate School of Science,
Nagoya University, Nagoya 464-8601, Japan; orcid.org/
0000-0001-5227-7894; Email: itami@chem.nagoya-u.ac.jp
Ben L. Feringa − Centre for Systems Chemistry, Stratingh
Institute for Chemistry, University of Groningen, Groningen
2085
https://dx.doi.org/10.1021/jacs.0c12280
J. Am. Chem. Soc. 2021, 143, 2078−2087

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
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Feringa, B. L. Emerging targets in photopharmacology. Angew. Chem.,
Int. Ed. 2016, 55, 10978−10999.
(B.L.F.). X-ray diffraction data collection and preliminary
experiments were carried out at beamlines BL44XU of SPring-
8 (Proposal Nos. 2017A6743, 2017B6743, 2018B6843, and
2019A6942), BL41XU of SPring-8 (Proposal No.
2018B1011), and BL-17A of Photon Factory (Proposal Nos.
2016R-63, 2017G563, and 2019G024). Recombinant CRY
expression and beamline experiments were supported in part
by BINDS from AMED (Support Nos. JP19am0101074-0055
and JP19am0101071-0529).
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