A Photo-responsive Small-Molecule Approach for the Opto-epigenetic Modulation of DNA Methylation

Article abstract of DOI:10.1002/anie.201901139

Controlling the functional dynamics of DNA within living cells is essential in biomedical research. Epigenetic modifications such as DNA methylation play a key role in this endeavour. DNA methylation can be controlled by genetic means. Yet there are few c

Full text of DOI:10.1002/anie.201901139

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Accepted Article
Title: Opto-epigenetic modulation of DNA methylation with a photo-
responsive small-molecule approach
Authors: Ha Phuong Nguyen, Sabrina Stewart, Mikiembo N Kukwikila,
Sioned Fôn Jones, Daniel Offenbartl-Stiegert, Shiqing Mao,
Shankar Balasubramanian, Stephan Beck, and Stefan
Howorka
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201901139
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Opto-epigenetic modulation of DNA methylation with a photo-
responsive small-molecule approach**
Ha Phuong Nguyen1, Sabrina Stewart2, Mikiembo N. Kukwikila1,3, Sioned Fôn Jones1, Daniel
Offenbartl-Stiegert1, Shiqing Mao4,5, Shankar Balasubramanian 4,5, Stephan Beck2, Stefan Howorka1**
molecules of tuneable bioactivity[26-31]. These can be used without
Controlling the functional dynamics of DNA within living cells is
essential in biomedical research. Epigenetic modifications such as
DNA methylation play a key role in this process. Controlled DNA
methylation editing can be attained via genetic means. Yet there are
few chemical tools available for the spatial and temporal modulation
of this modification. Here we present a small-molecule approach to
modulate DNA methylation with light. The strategy uses a photo-
tuneable version of a clinically used drug (5-aza-2¢-deoxycytidine) to
alter the catalytic activity of DNA methyltransferases, the enzymes
that methylate DNA. After uptake by cells, the photo-regulated
molecule can be light-controlled to reduce genome-wide DNA
methylation levels in proliferating cells. The chemical tool
complements genetic, biochemical and pharmacological approaches
to study the role of DNA methylation in biology and medicine.
the need for genetic engineering of cells leading to powerful
applications within cell biology[32]. Yet, despite the importance of
DNA methylation in biology, no light-tuneable small-molecule tool
has been developed to manipulate methylation levels in cells.
Here we present a photo-mediated small-molecule strategy that
modulates methylation in light-exposed cells. At the approach’s
centre is an inhibitor that interferes with DNA methyltransferases
(DNMTs), the enzymes responsible for DNA methylation[33]
including the maintenance DNA methyltransferase 1 (DNMT1)[34]
.
The inhibitor’s bioactivity becomes tuneable with light by chemical
derivatization with a photocage. As schematically illustrated in
Figure1a, the attached photocage renders the inhibitor biologically
inactive. However, light exposure cleaves off the photocage to
restore the original inhibitory effect (Figure1a). The photocaged
molecule is hence expected to maintain methylation levels in the
dark, while light should decrease methylation levels following
replication of cells[35] (Figure 1a).
The methylation of DNA at position 5 of cytosines is chemically a
very simple but biologically one of the most important modifications
of DNA. It influences many biological processes in humans such as
the regulation of cell function, cellular reprogramming, and
organismal development[1-7]
.
Biological effects of higher
methylation levels at promoters are mediated by lowering the
transcription of genes either via blocking binding of transcription
factors, or by recruiting unique methyl-recognizing proteins that
lower gene expression. Altered levels of methylation are also
associated with several diseases[8-11] including cancer[8,12-16]
.
Driven by the growing importance of DNA methylation in
biomedical research, there is a strong interest to experimentally
lower or increase methylation levels[17-23] to study, for example, the
role of epigenetic reprogramming in tissue development or
regenerative medicine[24-25]
. Optical control is of particular
relevance given the high spatial and temporal resolution of light.
Often, the approach is implemented with photosensitive small
[*]
Dr. H. P. Nguyen, Dr. M. N. Kukwikila, Sioned Fôn Jones, Daniel
Offenbartl-Stiegert,, Dr. S. Howorka
Department of Chemistry, Institute for Structural and Molecular
Biology, University College London, 20 Gordon Street, London
WC1H 0AJ (UK)
Email: s.howorka@ucl.ac.uk
Dr. S. Stewart, Dr. S. Beck
UCL Cancer Institute, London (UK)
Dr. S. Mao, Dr. S. Balasubramanian
Department of Chemistry, University of Cambridge, Lensfield Road,
Cambridge (UK), Cancer Research UK Cambridge Institute,
University of Cambridge, Robinson Way, Cambridge (UK)
Supporting information for this article is given via a link at the end of
the document.
Funding: H.P.N. was recipient of a BBSRC Case studentship and S.
S. was supported by a MRC CASE studentship (G1000411). S.H. is
supported by EPSRC (EP/N009282/1), the BBSRC (BB/M025373/1,
BB/N017331/1) and the Leverhulme Trust (RPG-2017-015). St.B. is
supported by the Wellcome Trust (84071) and a Royal Society
Wolfson Research Merit Award (WM100023). Sh.B. is supported by
a Wellcome Trust Investigator Award (grant no. 099232/z/12/z) and
an Institute Core Grant from Cancer Research UK
Figure 1. Photocaged derivatives of DNMT inhibitor 5-aza-2¢-deoxycytidine
(dAC) designed to optically modulate the methylation of DNA. (a) Scheme
illustrating the principle of the photo-caging approach. Photocaged inhibitor
dAC is biologically inert and allows DNMT to maintain high methylation levels.
Exposure to light removes the phototag to restore the inhibitory effect on
DNMT to cause lowered DNA methylation with each round of DNA replication.
(b) Caged DNMT inhibitors N-DEACMOC-dAC (1a), N-NPEOC-dAC (1b), N-
DMNPEOC-dAC (1c), bis-NPEOC-AC (1d), 5¢-DEACMOC-dAC (2), 3¢-
DEACMOC-dAC (3).
[**]
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Our approach was implemented with DNMT inhibitor 5-aza-2¢-
deoxycytidine (dAC, decitabine)[35-36] (Figure 1b). The cytidine
analogue is a clinically used drug for myelodysplastic syndromes[37]
and is being tested against leukemia and solid tumors[18,38] and as
sensitizer for immunotherapies[39-40]. dAC is the best choice for the
photocaging approach given its high inhibitory effect on DNMTs[41]
even though it is also known to undergo slow hydrolysis at the 5-
aza-base ring[42]. To exert its inhibitory effect after cellular uptake,
dAC is phosphorylated by deoxycytidine kinase in a rate-limiting
step[43]. Subsequent phosphorylations to triphosphate lead to the
polymerase-mediated incorporation into DNA[43] where the 5-aza-
base ring forms an covalent adduct with DNMT. This adduct
prevents methylation of DNA in replicating cells but also targets
DNMT for proteosomal degradation[44]. Given the tight fit inside the
active site of deoxycytidine kinase (Figure S1), we surmised that
photocaging dAC would block the rate-limiting step of
phosphorylation and hence abolish inhibition of DNMT.
To optically control the activity of dAC, we attached a photocage
to each of all possible coupling sites within the nucleoside: the
exocyclic NH2 group of the base, and the 3¢ and 5¢ OH groups of the
deoxyribose (Figure 1b)[27,31]. All three positions were modified as
the resulting steric blockade was expected to hinder binding of dAC
into the active site of deoxycytidine kinase (Figure S1). For the
chemical derivatization, photocage diethylaminocoumarinyl-4-
methyl (DEACM) (Figure 1b) was used given its favorable high
extinction coefficient (ε = 16,000 M-1 cm-1) and long absorption
wavelength (λ = 385 nm) that ensure biocompatibility by avoiding
mutagenic irradiation at high intensity in the UV spectral region.
Three DEACM derivatives of dAC 1a, 2, and 3 (Figure 1b) were
synthesized. In 1a, the photocage is attached via a carbamate bond
to NH2, while the linkage in 2 and 3 is mediated via a carbonate to
5¢ and 3¢ OH, respectively (Figure 1b). The synthetic routes to 1a, 2,
and 3 are described in the Supporting Methods.
Additional photocaged compounds were made to demonstrate
that the synthetic route is generic. For example, synthesis of 1b and
1c carrying a nitrophenyl group on the amino group (Figure 1b)
showed that a chromophore other than DEACM can be attached to
dAC. 1b and 1c also served as reference compounds for the
spectroscopy analysis (see below). Similarly, preparation of
nitrophenyl-modified azacytidine 1d (Figure 1b) proved that the
clinically used ribonucleotide version of dAC can be equipped with
a photocage (see Supporting Methods for synthetic routes of 1b-d).
DEACM-dAC derivatives 1a, 2, and 3 were examined to probe
whether the spectroscopic properties are influenced by the
chromophore’s attachment site. As shown in Figure 2a, all
compounds exhibited strong absorption at
a biocompatible
wavelength of λ = 365 nm (Table 1) with ε close to unconjugated
DEACM (ε = 7000 M-1 cm-1, Figure S2)[45] implying minimal
influence by coupling to dAC. The data for compounds 1b-d showed
similar findings (Table 1, Figure S2).
Uncaging efficiency, by contrast, was influenced at which site of
dAC the chromophore was attached. The analysis (Figure 2b)
revealed for compound 1a a fast uncaging rate of k = 1.03 x 10-3 s-1
equivalent to a 50% recovery of dAC within a half-life of t1/2 = 11
min (Figure 2c) while 2 was slower (Figure 2c, Figure S3), possibly
due to a quenching interaction between the photocage and proximal
triazine nucleobase. In support, 3 with DEACM at more distant 3¢
OH to triazine had a fast photolysis with t1/2 = 8 min (Figure 2b and
2c, Figure S3). The likely mechanism for uncaging is shown in
Figure S4.
Figure 2. Spectroscopic and photochemical analysis of photocaged dAC versions 1a, 2 and 3. (a) UV-vis absorption spectra of photocaged dAC compounds 1a, 2,
and 3 at 50 μM in DMSO/Water (5/95). (b) HPLC traces for the photodeprotection of 1a. The initial peak corresponding to caged 1a disappears upon irradiation at
365 nm to yield uncaged dAC and free DEACM-OH. The rates for photo-induced uncaging were determined by exposing the DEACM-dAC conjugates to light at λ
= 365 nm of moderate intensity at 145 µW cm-2 and at ambient temperature of 25 ºC. (c) Time course for photo-induced uncaging of 1a, 2 and 3 at λ = 365 nm.
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Table 1. Spectroscopic and photolytic properties of photocaged DNMT inhibitors.
a
a
b
b
c
d
e
λmax
391
233
λmax
ε254
ε365
k / s-1
t1/2 / min
11
Φ365
ε×Φ365
1a
1b
10000
16400
11000
9000
7000
200
1.03×10-3
8.33×10-5
3.33×10-4*
6.67×10-5
1.00×10-4*
n/a
6.11×10-2
4.93×10-3
427
139
0.99
1c
348
5000
10400
4000
173
3.94×10-3
16
1d
2
260
395
392
14800
11000
12000
12100
11400
11700
460
n/a
24
8
n/a
n/a
7300
4.83×10-4
1.50×10-3
2.88×10-2
8.84×10-2
210
716
3
8100
a Wavelength of maximum absorption (nm). b Molar absorptivities (M-1 cm-1) at λmax, 254 nm, or 365 nm. c Deprotection-rate constant for
irradiation at 365 nm, or at 254 nm as indicated by *. d Quantum yield of uncaging at λ = 365 nm. e Product of molar absorption coefficient and
quantum yield of uncaging at λ = 365 nm in M-1 cm-1.
3d and 3d, dAC), while light exposure in the absence of 3 did not
Successful uncoupling of the photocage from the nucleobase was
also found for control nucleotides 1b–d whose spectroscopic and
photolytic properties were in line with literature value for
nitrophenyl (Table 1 and Figure S3). Nevertheless, the uncaging
rates of 1b–d are too low for subsequent cell work. By comparison,
compound 3 has a high absorption wavelength and the fastest
photolysis.
Analysis of 3 determined its stability in the absence of light.
Unmodified dAC is known to have a slightly reduced stability due
to hydrolysis at the 5-aza-base ring leading to a half-life of 2200 min
at 25 °C[42]. By comparison, 3 had a half-life of 690 min 25 °C which
reflects partial hydrolysis of the ring and the carbonate linkage to the
photocage, as determined by MS (Figure S5). This half-life is almost
70-times longer than the half-life for photo-induced uncaging of 3
and 7-times longer than the subsequent incubation duration to cells.
This means that after 1 h of light-induced deprotection, only 3% or
less of compound 3 are still in the caged form. Dark instability is
hence not compromising photouncaging. Reflecting its adequate
stability and fast deprotection rate under illumination, compound 3
was used for subsequent biological investigations.
To test whether methylation levels in cells can be controlled with
light, 3 was added to hypermethylated human cancer cell lines
SaOS2 and T24[46]. Additional exposing cells to light was expected
to induce passive demethylation due to photo-uncaging of 3 and the
resulting non-methylation during DNA replication in dividing cells
(Figure 3b). Lack of illumination was anticipated to maintain
methylation (Figure 3a). Consequently, cells were incubated with
0.1 µM 3 and either illuminated for 1 h at 365 nm and 25 °C, or kept
in the dark at 25 °C. Treatment of cells with unmodified dAC served
as positive control for demethylation (Figure 3c). After incubation
with the small molecules, the medium was changed, cells were
grown at 37 °C for 24 h, genomic DNA was isolated and
enzymatically digested, and the nucleotide content analysed with
Liquid Chromatography coupled with tandem Mass Spectrometry
(LC-MS).
affect methylation (Figure 3d and 3e, 0). The data demonstrate that
our strategy of light-induced demethylation is successful; by
photolysis of 3, dAC’s biological inhibition was reactivated to block
DNA methyl transferases within cells. Our approach was also
confirmed by demethylation at a concentration of 0.5 µM 3 (Figure
S6). At 1.5 µM or higher, the compound leads do demethylation
without light exposure, possibly because 3 is hydrolytically
inactivated by enzymes. Control experiments where cells were
solely exposed to light did not lead to altered methylation levels
(Figure S6; 0 µM 3).
Molecular analysis confirmed the proposed mechanism for 3's
attainment of lower methylation levels in light-exposed cells. First,
an enzymatic assay established that the photocage in 3 interferes
with deoxycytidine kinase activity. The kinase usually
phosphorylates 5¢ OH of uncaged dAC[43] after the compound is
taken up by cells. However, the photocage attached to 3¢ OH of 3
prevents the compound’s phosphorylation (Figure S7) most likely
due sterically hindering access of 3 to the enzyme’s active site
(Figure S1). In addition, Western blot analysis confirmed that
uncaged 3 lowers methylation by decreasing levels of the DNA
methyltransferase 1 (Figure S8). The amount of DNMT1 was
reduced when cells were exposed to 0.1 µM 3 and light to liberate
dAC. The inhibitor’s mode of action is thought to involve its
incorporation into DNA to form a covalent adduct with DNMT1[43]
which prevents methylation of DNA in replicating cells but also
targets DNMT for proteosomal degradation[44]
.
Figure 3d and 3e summarize the cellular levels of methylated C
as percentage of the total cytosine pool for SaSO2 and T24 cells,
respectively. Exposure to 3 without illumination maintained a high
level of methylated DNA (Figure 3d and 3e, 3), thereby confirming
that photocaged dAC was biologically inactive at the tested
conditions. However, incubation with 3 and simultaneous exposure
to light caused a drastic reduction in methylated DNA (Figure 3d
and 3e, 3-light) to a level almost identical to uncaged dAC (Figure
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to be the best in terms of high wavelength absorption and photolytic
efficiency, while carbonate or carbamate-tethered nitrobenzyls 1b-
1d were not suitable, similar to previously tested ether-based
linkages. In practical terms, this insight could improve the future
synthesis of photocaged versions of the clinically tested dAC-related
drugs such as SGI-110[54]. Finally, dAC and related drugs could be
modified with photoswitches that regulate bioactivity via
photoisomerable conformation changes rather than photolysis[26-29]
.
The optically addressable DNMT inhibitor may be developed into a
potentially valuable research tool for studying epigenetic
mechanisms in health and disease. Areas of interest include
regenerative medicine[55]
,
developmental biology[4], development
and progression of cancer[56], and the development of therapeutic
routes[18,38,57-59] to treat surface-accessible tissues[60]
Before
.
realizing the potential, the photocaged nucleoside’s bioavailability
has to be successfully tested and its stability may have to improved,
such as replacing the carbonate tether with self-immolating
linkages[61-63]. In the case of thicker tissues or organs, high-
wavelength photocages active in the optical window need to be
devised. In conclusion, our photocaged DNMT inhibitor opens up
exciting new avenues in basic and clinical research for epigenetics
but also the synthesis of photo-controlled molecules.
Figure 3. DEACMOC-dAC 3 can be photo-deprotected to re-activate its
inhibitory effect on DNMT and lower DNA methylation levels in cells. (a-c)
Schematic representation of cell treatment conditions and expected qualitative
changes in DNA methylation levels. Treatment with 3 in the absence of light
maintains high methylation levels (a), while illumination restores dAC activity to
lower DNA methylation (b) to levels close to unmodified dAC (c). The
concentration of 3 and dAC was 0.1 µM. Cells take up photocaged dAC at up to
4.5 µM within 1 has shown using cell viability read-out. (d, e) Treatment-
dependent changes in methylation levels in SaOS2 (d) and T24 cell lines (e) for
condition in (a-c) and 0 μM dAC, as quantified via LC-MS. DNA methylation
levels (%5mC) are expressed as a percentage of total cytosines and analysed
in biological triplicates.
Acknowledgments
The authors would like to thank Gurpreet Virdi for the synthesis and
characterisation of 3'-DEACMOC-deoxycytidine (3'CdC) and Dr.
Jonathan R. Burns for generating all images.
Keywords: DNA, cytosine, methylation, photo-caging, epigenetics
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This article is protected by copyright. All rights reserved.

10.1002/anie.201901139
Angewandte Chemie International Edition
COMMUNICATION
Keyworks: DNA, methylation, epigenetics, photocage, 5-aza-2¢-
deoxycytidine, decitabine
This article is protected by copyright. All rights reserved.