Photolytic Release of a Caged Inhibitor of an Endogenous Transcription Factor Enables Optochemical Control of CREB-Mediated Gene Expression

Article abstract of DOI:10.1021/acs.orglett.9b03568

A direct optochemical method for regulating gene function has been developed based on uncaging of an inactive caged precursor that fragments to produce a CREB (cAMP-response element binding protein) inhibitor that binds to an endogenous transcription factor responsible for regulating CREB-mediated gene expression levels.

Full text of DOI:10.1021/acs.orglett.9b03568

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Photolytic Release of a Caged Inhibitor of an Endogenous
Transcription Factor Enables Optochemical Control of CREB-
Mediated Gene Expression
Takuma Imoto,† Akihiro Kawase,† Masafumi Minoshima,† Tatsushi Yokoyama,‡ Haruhiko Bito,‡
and Kazuya Kikuchi*,†,§
†Division of Advanced Science and Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka
565-0871, Japan
‡Department of Neurochemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
113-0033, Japan
§Immunology Frontier Research Center, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
S
* Supporting Information
ABSTRACT: A direct optochemical method for regulating
gene function has been developed based on uncaging of an
inactive caged precursor that fragments to produce a CREB
(cAMP-response element binding protein) inhibitor that
binds to an endogenous transcription factor responsible for
regulating CREB-mediated gene expression levels.
This study reports the development of a photochemically
ccess to chemical biology tools that can be used to
Amanipulate transcriptional activity is extremely useful for addressable endogenous transcription system, based on
photochemical release of an inhibitor of the cAMP-response
element binding protein (CREB), which is a transcription
factor that is endogenously expressed in various cell lines.12
CREB is known to interact with a coactivator paralog CREB
binding protein (CBP) to promote transcriptional initiation
from cAMP-mediated response elements (CRE) downstream
of cAMP/PKA and Ca2+ signaling pathways that lead to
increased gene expression. CREB-mediated transcription
activity is known to be important for promoting cell
proliferation and cell differentiation, as well as the develop-
ment of long-term memory networks in the nervous system.
Therefore, the availability of a simple optochemical tool that
could be used for the spatiotemporal control of CREB activity
would be very useful for probing the role of CREB in cellular
development. CREB activity has previously been regulated by
gene knockdown using small interfering RNA (siRNA)
technology;13 however, this approach only offers limited
spatiotemporal control of CREB activity.
understanding gene function in cells. Optical tools for genetic
manipulation have been used to generate spatiotemporal
perturbations of targeted gene expression in cellular networks,
which enables the role of the proteins they encode to be
explored.1 Recently, a variety of optogenetic systems have been
developed that employ light-sensitive transcription factors and
proteins to control gene function.2,3 Alternative optochemical
approaches that employ photoactivatable compounds have also
been used as a method to achieve spatiotemporal modulation
of transcription, without the need for genetically modified cells
or animals.4 Caged compounds are inactive precursors of
biologically active molecules that are masked with a photo-
responsive protecting group (PPG) that can be irreversibly
activated by light.5−8 A number of caged ligands/inhibitors
targeting various transcription factors have been developed,
whose ability to modulate transcriptional activity is based on
photolytic generation of an activating or inhibitory ligand. For
example, caged ligand systems based on photoaddressable
doxycycline9 and tamoxifen10,11 precursors have previously
been used for light-stimulated control of gene expression.
However, these ligand systems are generally used in
combination with exogenous gene expression systems (such
as Tet-ON/OFF and CreER), which limits their general
applicability for the direct regulation of endogenous gene
expression. Therefore, the development of simple photo-
activatable caged ligand systems that can be used to target
endogenous gene expression, without the need for exogenous
gene expression systems, is potentially of great interest.
The aim of this study was to develop inactive caged CREB
inhibitors (CCIs) that could be photolytically cleaved to
release an active inhibitor of CREB-mediated transcriptional
activity in cellular systems. Light activation of an inactive CCI
within the cell would trigger intracellular release of the
inhibitor cargo, thus enabling spatiotemporal suppression of
CREB-dependent transcription to be achieved in real time
Received: October 9, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b03568
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
(Figure 1). This would provide a simple chemical method to
achieve controlled knockdown of CREB-mediated gene
expression based on simple light stimulation of living cells.
Scheme 1. Synthesis of CREB Inhibitors CCI-1 (a), CCI-2,
and CCI-3 (b)
Figure 1. Caged inhibitor strategy for optochemical control of CREB-
mediated gene expression.
The target caged CREB inhibitors CCI-1, CCI-2, and CCI-3
were designed based on a naphthol AS-E derivative (1) (Figure
2), which is known to possess high CREB inhibitory activity
(Figure S2).16 However, photolytic cleavage reactions of
amino-protected CCI-2 and CCI-3 at 405 nm proceeded
cleanly, affording inhibitor 1 in high yield, with no evidence of
any photo-Claisen rearrangement products having been
formed (see HPLC traces shown in Figure 3). Analysis of
the time course of the photolytic reactions of CCI-2 and CCI-
3 enabled their cleavage rates to be determined (Figure 4) with
their relatively rapid photocleavage rate constants (Table 1),
indicating that they would be suitable as photolytically labile
caged CREB inhibitors for cellular studies.
The CREB inhibitory activities of uncaged CCI-2 and CCI-
3 were then evaluated in living human embryo kidney
(HEK293T) cells that had been cotransfected to express a
dual luciferase reporter system. These cells contained a firefly
luciferase reporter gene that was under the control of a CREB-
responsive promoter containing a cAMP response element
(CRE), and a renilla luciferase gene whose luminescence
activity served as an internal control. Use of this HEK293T
cell-line enabled CREB-dependent gene expression activity to
be determined by normalizing the luminescent activity of the
firefly luciferase reporter against the known luminescent
activity of the renilla luciferase reporter, thus minimizing any
experimental variability caused by differences in cell viability
and/or transfection efficiency. Prior to photoactivation, CCI-2
(5 μM) and CCI-3 (10 μM) were shown to have no effect on
CREB-mediated firefly luciferase expression levels in
HEK293T cells (Figure S3). In contrast, treatment of
HEK293T cells with CREB inhibitor 1 (IC50 = 614 nM)
(Figure S4), or uncaged CCI-2 (IC50 = 872 nM) or CCI-3
Figure 2. Structures of CREB inhibitor 1 and caged CREB inhibitors
(CCIs) whose PPG fragments are highlighted in red.
(IC50 = 81 nM).14 The 7-(diethylamino)coumarin-4-yl methyl
(DEACM) group was selected as PPG owing to its high
decomposition efficiency at long wavelengths above 400 nm.15
The phenolic hydroxyl group of the CREB inhibitor 2 was then
functionalized with bromo-DEACM 3, followed by TFA-
mediated deprotection of the N-Boc group to afford CCI-1 in
21% yield over two steps (Scheme 1a). The carbamate linkers
of CCI-2 (containing a diethylamino group) and CCI-3
(containing a bis(carboxymethyl)amino group) were synthe-
sized by direct conjugation of the free amino group of 1 with
the corresponding p-nitrophenyl carbonate esters of DEACM
in 71% and 2% yields over two steps, respectively (Scheme
1b).
The photochemical properties of CCI-1, CCI-2, and CCI-3
were then explored through uncaging of their DEACM groups
at 405 nm in HEPES buffer, which resulted in products with
absorption maxima below 380 nm (Figure S1). HPLC analysis
of the photolytic cleavage products of CCI-1 revealed that
inhibitor 1 had been formed in the presence of a number of
other byproducts (Figure S2). Mass spectrometric analysis
revealed that the major byproduct gave an identical molecular
ion to CCI-1 ([M + H]+ = 813.29), indicating that a
competing intramolecular photo-Claisen rearrangement of
CCI-1 had occurred to afford a rearranged phenolic isomer
B
DOI: 10.1021/acs.orglett.9b03568
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in Figure 5 reveal that the background inhibitory activities of
CCI-2 and CCI-3 at a concentration of 5 μM in HEK293T
Figure 5. Inhibition of cellular CREB activity by uncaged CCI-2 and
CCI-3. Reaction conditions: 5 μM CCI-2 or CCI-3 in cells were
illuminated by light. The dual-luciferase reporter assay was performed
at ∼6 h after light illumination with either blank or 5 μM CCI-2 or
CCI-3 in DMEM (1% DMSO) (N = 3). Error bars denote SD.
Figure 3. HPLC traces of the photoreaction products produced from
uncaging of compounds 1, CCI-2, and CCI-3. Reaction conditions:
50 μM compound 1 in 20 mM HEPES (10% DMSO), 50 μM CCI-2
in 20 mM HEPES buffer/CH3CN = 3/7 (10% DMSO), or 50 μM
CCI-3 in 20 mM HEPES buffer/CH3CN = 7/3 (10% DMSO). Light
intensity: 20 mW/cm2 for 15 min, λex = 405 nm.
cells were negligible. However, photoactivation of HEK293T
cells treated with CCI-2 and CCI-3 at 405 nm resulted in a
large decrease in CREB-dependent luciferase expression levels
to 47% and 3% of their original values, respectively. The reason
for the enhanced photoinhibitory activity of CCI-3 is currently
unclear; however, it may be due to its significantly better
aqueous solubility resulting in a more efficient photolysis
cleavage reaction. These results clearly indicated that
illumination of the inactive CCI-2 and CCI-3 precursors had
resulted in photolytic cleavage to afford an active inhibitor 1
that then transiently inhibits CREB-dependent gene expression
in cells. However, since CREB and CBP are both expressed
within cells, we considered the possibility that the low
inhibitory activity of CCI-2 and CCI-3 might be caused by
their low cellular permeabilities, with photolytic cleavage to
afford the cell permeable inhibitor 1 actually occurring outside
the cell. Consequently, we performed a fluorescence cell
imaging experiment on HEK293T cells that had been
incubated with CCI-2 and CCI-3 for 30 min. The resultant
images revealed the presence of fluorescent signals derived
from the DEACM chromophore in the cytosolic fraction of the
cells (Figure S6), thus demonstrating that both CCI-2 and
CCI-3 can effectively penetrate the cell membrane and that
their photolytic cleavage occurs intracellularly. Although CCI-
3 has two anionic carboxyl groups that might cause
electrostatic repulsion with cell membranes, the compound
still maintains its cell permeability. This result might be
attributed to the increased hydrophobicity of CCI-3 upon
conjugation with CREB inhibitor 1.
Figure 4. Photolysis rates of CCI-2 and CCI-3. Reaction conditions:
(a) 50 μM CCI-2 in 20 mM HEPES buffer (pH = 7.2)/CH3CN = 3/
7 (10% DMSO) or (b) 50 μM CCI-3 in 20 mM HEPES buffer (pH =
7.2)/CH3CN = 7/3 (10% DMSO). Light intensity: 10 mW/cm2, λ =
405 nm. Error bars denote SD.
a
Table 1. Photochemical Properties of CCI-2 and CCI-3
CCI-2
CCI-3
ε (405 nm) [M−1 cm−1
k [s−1
k’ [s−1
]
1.0 × 104
0.24
1.0 × 104
0.20
]
]
0.19
0.19
a
k and k′ are the rate constants for consumption of the CCIs and
In conclusion, we have developed new caged derivatives of a
CREB inhibitor (CCIs) that can be used as direct
optochemical tools for light-triggered knockdown of CREB
transcriptional activity in living cells. Use of the more water-
soluble CCI-3 potentially allows for spatiotemporal knock-
down of endogenous CREB-mediated transcription activity in
different cell types. However, light-triggered activation of the
gene expression is highly desired, which could be achieved with
a caged CREB activator. Caged CREB modulators will provide
a useful tool for investigating the role of CREB-controlled
production of inhibitor 1, respectively.
(IC50 = 702 nM) resulted in the gene expression levels being
decreased in a concentration-dependent manner (Figure S3).
We confirmed that CREB inhibitor 1 does not directly affect
the activity of firefly luciferase (Figure S5).
Then, light-induced inhibition of CREB-dependent gene
expression was examined in living cells by using a dual
luciferase reporter system. Calibrated luminescent data shown
C
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b03568.
■
S
Full experimental procedures, characterization data, and
NMR spectra (PDF)
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family transcription factors in the nervous system. Neuron 2002, 35,
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: kkikuchi@mls.eng.osaka-u.ac.jp.
ORCID
Kazuya Kikuchi: 0000-0001-7103-1275
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the following funding sources:
Grant-in-Aid for Scientific Research (Grant 16K01933 to
M.M.; 17H06312 to H.B.; 25220207 and 18H03935 to K.K.);
Innovative Areas “Frontier Research on Chemical Communi-
cations” (Grant 17H06409) from the Ministry of Education,
Culture, Sports, Science, and Technology (MEXT) of Japan,
AMED (19dm0207079h0001 to H.B.; 18he0902005h0004,
17ae0101041h9902, 18fm0208018h0002 to K.K.); Takeda
Science Foundation; JSPS A3 Foresight Program; and JSPS
CORE-to-CORE Program “Asian Chemical Biology Initiative”.
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DOI: 10.1021/acs.orglett.9b03568
Org. Lett. XXXX, XXX, XXX−XXX