Light-Controlled Histone Deacetylase (HDAC) Inhibitors: Towards Photopharmacological Chemotherapy

Article abstract of DOI:10.1002/chem.201502809

Cancer treatment suffers from limitations that have a major impact on the patient's quality of life and survival. In the case of chemotherapy, the systemic distribution of cytotoxic drugs reduces their efficacy and causes severe side effects due to nonselective toxicity. Photopharmacology allows a novel approach to address these problems because it employs external, local activation of chemotherapeutic agents by using light. The development of photoswitchable histone deacetylase (HDAC) inhibitors as potential antitumor agents is reported herein. Analogues of the clinically used chemotherapeutic agents vorinostat, panobinostat, and belinostat were designed with a photoswitchable azobenzene moiety incorporated into their structure. The most promising compound exhibits high inhibitory potency in the thermodynamically less stable cis form and a significantly lower activity for the trans form, both in terms of HDAC activity and proliferation of HeLa cells. This approach offers a clear prospect towards local photoactivation of HDAC inhibition to avoid severe side effects in chemotherapy.

Full text of DOI:10.1002/chem.201502809

DOI: 10.1002/chem.201502809
Full Paper
&
Photopharmacology
Light-Controlled Histone Deacetylase (HDAC) Inhibitors: Towards
Photopharmacological Chemotherapy
Wiktor Szymanski,*[a, b] Maria E. Ourailidou,[c] Willem A. Velema,[a] Frank J. Dekker,*[c] and
Ben L. Feringa*[a]
Abstract: Cancer treatment suffers from limitations that
have a major impact on the patient’s quality of life and sur-
vival. In the case of chemotherapy, the systemic distribution
of cytotoxic drugs reduces their efficacy and causes severe
side effects due to nonselective toxicity. Photopharmacology
allows a novel approach to address these problems because
it employs external, local activation of chemotherapeutic
agents by using light. The development of photoswitchable
histone deacetylase (HDAC) inhibitors as potential antitumor
agents is reported herein. Analogues of the clinically used
chemotherapeutic agents vorinostat, panobinostat, and beli-
nostat were designed with a photoswitchable azobenzene
moiety incorporated into their structure. The most promising
compound exhibits high inhibitory potency in the thermo-
dynamically less stable cis form and a significantly lower ac-
tivity for the trans form, both in terms of HDAC activity and
proliferation of HeLa cells. This approach offers a clear pros-
pect towards local photoactivation of HDAC inhibition to
avoid severe side effects in chemotherapy.
Introduction
effects to be avoided by minimizing the concentration of
active compound outside the area of treatment, thereby tre-
mendously increasing the quality of the patient’s life.[5]
Chemotherapy, besides radiotherapy and surgery, is one of the
crucial elements of cancer treatment.[1] It relies on the systemic
administration of cytotoxic antineoplastic agents, which are in-
famous for their severe side effects.[2] Efforts towards targeted
delivery of chemotherapeutics are often insufficient, and there-
fore, the lack of drug selectivity remains a major problem in
care for cancer patients.[3] Ineffective cancer treatment causes
unrelenting emotional and societal burdens to the patients
and their environment and also has a massive economic
impact.[4] In the case of cancer types that warrant localized
treatment, high spatiotemporal control over the activity of the
drug, that is, the possibility to externally modulate the potency
of the chemotherapeutic agent, would allow the systemic side
Photopharmacology[6,7] aims at using light as an external,
noninvasive control element to modulate drug activity
(Figure 1). Light can be delivered with very high spatiotempo-
ral precision, with a wide range of intensities and wave-
lengths[8,9] and very limited effects on the patient’s body. From
a medicinal chemistry perspective, photopharmaceuticals are
developed by the incorporation of photoresponsive molecular
switches into existing drugs to enable alteration of their bio-
logical properties upon light irradiation.[8–10] Photoswitching is
reversible by irradiation with light of different wavelengths or
in a thermal process.[11] Recent examples of bioactive com-
pounds with photomodulated potency include photocontrol-
led ion-channel blockers,[12] antibiotics,[13] and enzyme inhibi-
tors.[14] However, successful examples for switch-on chemother-
apeutics are currently lacking.
[a] Dr. W. Szymanski,+ Dr. W. A. Velema, Prof. Dr. B. L. Feringa
Centre for Systems Chemistry, Stratingh Institute for Chemistry
University of Groningen, Nijenborgh 4
9747 AG, Groningen (The Netherlands)
E-mail: b.l.feringa@rug.nl
[b] Dr. W. Szymanski+
For a possible therapeutic application, the potential photo-
pharmaceuticals must fulfill a number of criteria. First, they
must show high potency that is at least comparable with the
parent, clinically useful drug that inspired their design. Second,
the difference in activity between the inactive and active
states should be sufficiently large to allow for “off–on” switch-
ing of their activity under physiological conditions. Finally, the
thermodynamically less stable form should show higher bio-
logical activity, preferably with a known mechanism of toxicity,
to enable precise local activation, as presented in Figure 1.
Photopharmaceuticals have been described that meet some of
these requirements, that is, high potency,[15] >20 times differ-
ence in activity,[16] and enhanced potency for the unstable
state.[13] However, fulfilling all of these criteria in one molecule
Department of Radiology, University of Groningen
University Medical Center Groningen Hanzeplein 1
9713 GZ, Groningen (The Netherlands)
E-mail: w.szymanski@umcg.nl
[c] M. E. Ourailidou,+ Prof. Dr. F. J. Dekker
Department of Pharmaceutical Gene Modulation
University of Groningen, Antonius Deusinglaan 1
9713 AV Groningen (The Netherlands)
E-mail: f.j.dekker@rug.nl
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201502809.
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large variety of natural and synthetic compounds have been
reported as potential agents for cancer prevention or treat-
ment of different stages of several tumor types. Depending on
their structure, they can be categorized to hydroxamic acids,
cyclic peptides, benzamides, and short-chain fatty acids.[20,22] To
date, there are 11 different HDAC inhibitors (HDACis) undergo-
ing clinical trials as monotherapy or in combination with other
antitumor approaches in cancer patients.[29] The most success-
ful inhibitors, so far, proved to be the hydroxamic acid type
pan-HDACis, from which three have obtained US Food and
Drug Administration (FDA) approval for clinical use: vorinostat
(SAHA), panobinostat, and belinostat.[30] These compounds
nonselectively inhibit class I, II, and IV HDACs with a nanomo-
lar-scale potency[31] and are successfully applied as chemother-
apeutic agents for the treatment of hematologic malignan-
cies.[30,32–34] However, despite significant efforts, the specific
HDACs responsible for the clinical effects of these successful
inhibitors have not been elucidated as yet.[31,34]
The crystal structure of human deacetylase-like protein
(HDLP) with SAHA shows that SAHA binds inside the catalytic
pocket by inserting the chain into the enzymatic channel
(Figure 2).[35] The hydroxamic acid interacts with the zinc cation
at the polar bottom part and also forms hydrogen bonds with
catalytic residues. Moreover, the aliphatic chain makes van der
Waals interactions with residues at the hydrophobic part of the
pocket, whereas the cap group serves in packing the inhibitor
at the rim of the active site.
Figure 1. Comparison of the principles behind classic chemotherapy (a) and
high-precision photopharmacological chemotherapy (b,c). The reversible
photoswitching between the inactive (blue) and active (red) chemothera-
peutic agent (b) allows for local activation of the drug and permits its use at
elevated concentrations, without systemic side effects (c).
The insertion of SAHA into the channel of the deacetylase
and the flexibility of the aliphatic chain suggest that the inhibi-
tory activity may be controlled by changes in length, shape,
and substituents of the molecule. During the studies reported
herein, a patent was published that demonstrated an increased
activity for the cis isomers of azobenzene–benzamide-type
HDAC inhibitors.[36] Despite their potential clinical relevance,
compounds of this class lack the high toxicity for cancer cells
generally reported for hydroxamic-acid-type HDAC inhibitors.
Therefore, we have chosen to employ the clinically approved
inhibitors SAHA, panobinostat, and belinostat as starting
points for the design of photoswitchable HDACis, as potential
photocontrolled chemotherapeutic agents for improved, safer
cancer therapy with less severe side effects. We aimed to
design a potent compound that would show high activity in
the thermodynamically unstable cis state and very little cyto-
toxicity in the stable trans state.
remains a major challenge and is crucial for the development
of a clinically useful, photoactivated drug.
To demonstrate the viability of this novel approach with the
ultimate goal of photoresponsive chemotherapy, we have
chosen histone deacetylases (HDACs) as a pharmacological
target. The function of HDACs is the deacetylation of e-acety-
lated lysine residues on histone tails to restore the positive
charge of the histones and their electrostatic interactions with
DNA, leading to condensed and transcriptionally silent chroma-
tin structures.[17]
The HDAC family members are categorized into four classes
(I–IV), based on their primary structure, size, and sequence ho-
mology to the respective yeast enzymes.[18,19] The mechanism
of deacetylase activity is zinc dependent for classes I (HDAC1–
3 and 8); II, which is subdivided into classes IIa (HDAC4, 5, 7,
and 9) and IIb (HDAC6 and 10); and IV, and nicotinamide ade-
nine dinucleotide (NAD+) dependent for class III. As epigenetic
regulators of both histone and non-histone proteins,[20] HDACs
play a pivotal role in a vast array of biological processes, in-
cluding DNA repair, cell differentiation, proliferation, and apop-
tosis. As a result, alterations in expression or mutations of
genes encoding for HDACs can lead to aberrant gene tran-
scription, disruption of cell homeostasis, and subsequently to
tumorigenesis.[21–23] Recent evidence demonstrates that individ-
ual HDACs are strongly associated with neurodegenerative[24–26]
and inflammatory diseases,[27,28] tissue fibrosis, and metabolic
disorders.[21]
We have chosen the azobenzene photoswitch as a photores-
ponsive element that, when incorporated into the structure of
chemotherapeutic agents, should provide control over their ac-
tivity with light. Azobenzene molecules can be switched, usual-
ly by using UV irradiation, from a flat, trans isomer to the bent
cis isomer (Figure 3a).[11] The latter, which is thermodynamically
less stable than the former, will switch back to the initial state
over time (Figure 3a). This reverse process can also be ach-
ieved by using visible-light irradiation. Importantly, the two
forms show major differences in their shape and polarity, and
therefore, light-induced isomerization will result in switching of
the properties of an azobenzene-modified drug, which may
consequently change the drug’s biological activity.
The link between abnormal HDAC activity and cancer initia-
tion and progression is best shown in classes I, II, and IV.[21]
A
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study the influence of the substitution pattern on the photo-
chemical and pharmacological properties of the obtained pho-
tocontrolled chemotherapeutic agents, all possible attach-
ments of azobenzene to SAHA (para (1), meta (2), and ortho
(3)) were evaluated. Additionally, a para-MeO substituent was
introduced into the terminal aromatic ring in compounds 4
and 5, in an attempt to increase the cis content in the irradiat-
ed samples, as described for other azobenzenes.[39] The influ-
ence of the length of the aliphatic chain in the SAHA core was
tested with compound 6, which was a shortened homologue
of compound 2. Finally, we studied visible-light-switchable
compound 7 to evaluate the possibility of using higher and
more biocompatible wavelengths of light for the photocontrol
of bioactivity.
In the second approach (compounds 8–12), the linker part
of the inhibitor (Figure 2b) was modified because it was more
tightly embedded within the protein. We hypothesized that in-
troducing a photoswitch into this part of the SAHA molecule
would lead to increased photocontrol over binding because
one of the photoisomeric forms should fit the tunnel better
than another. Compounds 8, 9, 10, and 11 were designed to
test this hypothesis. In compound 9, an additional benzyl
moiety was introduced to mimic the cap in the SAHA molecule
(Figure 2b). Homologous compounds 10 and 11 were pre-
pared to test the influence of the inhibitor’s flexibility and
linker length in comparison to 8 and 9. Finally, inspired by the
structure of two known clinically approved inhibitors, belino-
stat and panobinostat,[40] we introduced a double bond into
the linker part of compound 12.
The biological activity assay (see below) we performed was
based on the addition of the respective compound as a stock
solution in DMSO to a buffered solution of enzyme and, after
1 h incubation at RT, measurement of the conversion of a pro-
fluorogenic substrate.[41] Therefore, two photochemical proper-
ties of the inhibitors are of importance: the content of cis
isomer obtained upon irradiation in DMSO and the half-life of
the thermodynamically unstable cis isomer under the assay
conditions (Figure 3c and d, respectively). The latter value is
also crucial for photopharmacological applications (Figure 1),
in which one would envision the use of cis–trans isomerization
for slow auto-inactivation of compounds that have been locally
activated.
Figure 2. Structural design of photoswitchable HDAC inhibitors. a) Crystal
structure of HDLP from Aquifex aeolicus in complex with SAHA. The aliphatic
chain is inserted into the enzymatic channel, in which the hydroxamic group
interacts with the zinc cation, leaving the cap at the rim of the catalytic
pocket. The picture was adapted from PDB file 1C3S.[35] b) Molecular design
of photoswitchable SAHA analogues, with the azobenzene moiety intro-
duced into the cap (first design) or the linker region (second design).
Azobenzene photochromes with different properties have
been used in photopharmacological projects due to their easy
preparation, efficient photoswitching, and low rate of photo-
bleaching.[8,9] Their electronic properties determine the absorp-
tion maxima and the half-lives of the cis isomers. Accordingly,
azobenzenes with various substitution patterns were included
in our newly designed inhibitors to optimize their photophysi-
cal properties and HDAC inhibition with respect to potency
and selectivity. We also used a new class of tetra-ortho-substi-
tuted azobenzenes that could be photoisomerized by using
visible light (up to l=650 nm), which allowed for lower toxici-
ty and deeper tissue penetration.[37,38]
All of the compounds show a satisfactory photostationary
state (PSS, >76%, defined as the content of cis isomer, at equi-
librium, under the l=365 nm light irradiation at ꢀ2 mgmLÀ1
)
in DMSO (Figure 3c). In the isomeric series 1–3, the lowest PSS
was obtained for the meta-substituted compound and the
highest (95%) for the para isomer. The introduction of me-
thoxy substituents in compounds 4 and 5 indeed allowed the
PSS to be increased with respect to parent structures 1 and 2
(from 95 (1) and 83% (3) to 97 (4) and 95% (5)). With com-
pound 7, we were able to achieve 78% PSS by using blue light
(l=400 nm) and 61% PSS by using green light (l=530 nm),
which is in line with similar tetra-ortho-substituted sys-
tems,[37,38] and permits the switching of these compounds with
visible light. For all para-alkoxy-substituted compounds, 8–12,
a high PSS was reached (>92%).
Results and Discussion
Two distinct approaches were taken in the molecular design of
photoswitchable HDACi. In the first one (compounds 1–7; Fig-
ure 3b), the photoswitches were introduced into the cap
moiety of the SAHA molecule (Figure 2b). To systematically
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Figure 3. Structure and photochemical properties of compounds 1–12 (for synthetic procedures and analytical data, see the Supporting Information). a) Re-
versible photochromism of an azobenzene molecule. b) Molecular structure of compounds 1–12. c) The content (%) of the cis isomer in the photostationary
1
state in DMSO, lirrad =365 nm, as determined by H NMR spectroscopy. d) Half-life (in the dark, at room temperature) for the thermodynamically unstable cis
isomer, as determined by UV/Vis spectroscopy at room temperature in the HDAC assay buffer with 1 vol% DMSO.
By changing the substitution pattern, we can control the
half-life of the inhibitors from minutes to hours, which is of
key importance in designing potential chemotherapeutics that
are meant, after photoactivation, to auto-inactivate in the pa-
tient’s body (Figure 3d). Very high stability was observed for
compound 7, which is in line with earlier studies on tetra-
ortho-substituted systems.[38]
of which increased from the micro- to nanomolar scale with
the introduction of a second carbon in the chain.
We tested selected compounds for the inhibition of human
recombinant class I HDACs (1–3 and 8) and HDAC6 (class IIb;
Table 1 and Figure 4). The active form of compound 1 (cis,
except for HDAC3) was less potent than SAHA, whereas com-
pounds 2 and 3 showed a remarkable potency in their trans
forms, with an IC50 value of 12 nm (15” more potent than
SAHA) in HDAC2 for compound 2 and 7.7 nm in HDAC3 for
compound 3. The same profile was observed with the visible-
light-switchable compound 7 (up to 12” more potent than
SAHA in HDAC2). The highest difference in IC50 values between
the two isomers was observed for compound 3 (10” for
HDAC3), whereas the same ratio was moderate for compounds
1 and 2 and relatively good for compound 7 (almost 6” for
HDAC3). Considerable inhibitory potency was also observed
for compound 6 (trans form), which was the shorter analogue
of 2, but the cis/trans activity difference was significant only in
the case of HDAC3. Despite promising results from the HeLa
nuclear extracts (Figure S11 in the Supporting Information), the
potency of trans-5 in HDACs1-3 was comparable to that of
SAHA and the cis/trans activity ratio was rather small (Fig-
ure 4a).
Importantly, all of the studied compounds are stable to-
wards reduction in the cellular environment, as shown by re-
peating switching cycles[42–44] in buffer in the presence of
10 mm glutathione (GSH; Figure S5 in the Supporting Informa-
tion), which is the highest concentration of GSH found in
cells.[45]
Compounds 1–12 were initially screened for their inhibitory
potency of crude enzyme activity (Figures S11 and S12 in the
Supporting Information) by using a published protocol.[41] Nu-
clear extracts from HeLa cells were used as a source of class I
HDACs.[46] Gratifyingly, for the meta-substituted compounds 2
and 5, we observed even higher potency than that of the orig-
inal drug. A significant difference in activity between the two
isomeric forms, which is crucial for photopharmacological ap-
plications, was apparent in the meta- and ortho-substituted
compounds, among which compound 3 showed the highest
trans/cis activity ratio (approximately 3”). Compounds of the
second design (8–12; Figure 3), turned out to be less potent
than those from the first design. However, in all cases, stronger
HDAC inhibition was observed for the cis isomer, the potency
In the second design, the most active form of the inhibitor
is the less stable cis isomer, which is advantageous for photo-
pharmacological applications (Figure 1). Compounds 8 and 12
proved to have lower potency than that of SAHA in HDACs1–3
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Table 1. Inhibition of HDAC activity by photoswitchable SAHA, panobinostat, and belinostat analogues.
IC50 [mm]
HDAC1
HDAC2
HDAC3
HDAC6
HDAC8
trans
cis
trans
cis
trans
cis
trans
cis
trans
cis
1
2
3
5
6
7
8
0.081Æ0.012 0.045Æ0.008 2.185Æ0.327 0.401Æ0.133 0.144Æ0.052 0.335Æ0.103 0.010Æ0.002 0.048Æ0.014 1.609Æ0.423 0.777Æ0.160
0.014Æ0.006 0.035Æ0.023 0.012Æ0.002 0.056Æ0.012 0.019Æ0.009 0.022Æ0.007 0.013Æ0.003 0.047Æ0.013 0.125Æ0.025 0.087Æ0.016
0.013Æ0.003 0.057Æ0.011 0.043Æ0.010 0.185Æ0.042 0.008Æ0.002 0.073Æ0.020 0.335Æ0.105 0.210Æ0.080 0.070Æ0.022 0.320Æ0.101
0.040Æ0.010 0.047Æ0.011 0.113Æ0.020 0.098Æ0.014 0.030Æ0.011 0.067Æ0.023 0.394Æ0.083 0.151Æ0.027 0.843Æ0.204 0.445Æ0.107
0.025Æ0.011 0.024Æ0.006 0.034Æ0.004 0.045Æ0.012 0.011Æ0.003 0.096Æ0.042 0.043Æ0.011 0.082Æ0.033 0.247Æ0.057 0.099Æ0.027
0.008Æ0.002 0.050Æ0.011 0.015Æ0.003 0.091Æ0.018 0.025Æ0.006 0.061Æ0.011 0.132Æ0.041 0.198Æ0.065 0.122Æ0.036 0.095Æ0.021
0.855Æ0.196 0.137Æ0.029 8.506Æ2.331 0.444Æ0.119 0.428Æ0.086 0.094Æ0.033 0.353Æ0.088 0.190Æ0.063 0.830Æ0.149 1.313Æ0.403
12 0.658Æ0.141 0.080Æ0.020 21.65Æ8.095 0.555Æ0.121 0.320Æ0.115 0.071Æ0.013 0.114Æ0.024 0.110Æ0.039 0.237Æ0.047 0.462Æ0.137
Figure 4. Inhibition of human recombinant class I HDACs and HDAC6 (class IIb). Inhibitory potency of trans (black) and cis (gray) forms of selected compounds
of the a) first and b) second designs. The corresponding IC50 ratios of the two isomers are also reported. The logIC50 values are presented as mean values of
three independent measurements with their respective standard deviations.
(Table 1 and Figure 4b). However, we were delighted to ob-
serve a large increase in the IC50 ratio between the trans and
cis isomers in the case of HDAC2 (19” for inhibitor 8 and
nearly 40” for 12; Figure 5b). Importantly, both compounds in
the active (cis) state showed inhibition in the same concentra-
tion range as that of SAHA (IC50 =0.44 mm for 8 and 0.56 mm
for 12 vs. 0.18 mm for SAHA).
Interestingly, opposite activity results, compared with those
for HDACs1–3, were obtained for HDAC8, since the potency of
all inhibitors (including SAHA) was dramatically reduced
(Table 1 and Figure 4a and b). This can be explained by struc-
tural and sequence alignments of class I HDACs, which reveal
a particularly malleable active site for HDAC8.[47] Compound
trans-3 was found to be more active (IC50 =70 nm, 16” more
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Figure 5. Studies on the performance of compound 12. The IC50 curves in a) HDAC1 and b) HDAC2 recombinant enzymes of trans and cis forms of the inhibi-
tor. c) Inhibition of HDAC1 activity with 50 nm of inhibitor 12 (black) and reversible photochromism (gray) after 4 isomerization cycles. d) In situ photoisomeri-
zation of 12, after 30 min of incubation of the cis form (1 mm) with HDAC2, with white light. The formation of product was monitored at the indicated times.
e) HeLa cell viability was measured after 16 h of incubation with various concentrations of each isomeric form of the inhibitor. f) On-blot luminescence detec-
tion and Coomassie Blue staining of acetylated histones (4 mg) after 16 h incubation with 1 and 2 mm of cis- and trans-12 by using the anti-acetyl lysine anti-
body. a)–e) The data are presented as mean values of three independent measurements with their respective standard deviations.
potent than SAHA) and exhibited the biggest difference in po-
tency (compared with the cis form) for HDAC8 inhibition.
Moreover, we examined the activity of selected compounds
against HDAC6, which is an isoform with a critical role in tu-
morigenesis and cancer cell metastasis.[48] A considerable inhib-
itory potency was achieved in the case of the trans isomers of
1 and 2; the cis form was comparable to the reference com-
pound (Table 1 and Figure 4a and b).
light can be used in a biological setting to change the activity
of photoswitchable HDAC inhibitors presented herein.
Finally, we elucidated the global cytotoxic activity of photo-
switchable HDACi and the extent to which it could be photo-
controlled. Chosen compounds were incubated, in both iso-
meric forms, with HeLa cells for 16 h at 378C, followed by mea-
surement of cell viability (Figure S33 in the Supporting Infor-
mation). In line with the results obtained for recombinant en-
zymes, the most promising results came from compound 12,
for which the cis form was significantly more toxic to the HeLa
cervical cancer cells than the trans form (Figure 5e). At 100 mm,
nearly full selectivity is obtained: the cis isomer kills almost all
cells, whereas the trans isomer leaves almost all cells intact.
This selectivity is remarkable, since the half-life of cis-12 at
378C is relatively short (67 min). Moreover, both isomers result-
ed in increased histone acetylation (mainly histone H4) at con-
centrations of around 2 mm (Figure 5 f), which indicated inhibi-
tion of intracellular HDAC activity. Because 12 is the only inhib-
itor prone to undergo conjugate addition (Figure 3a), possible
attack from nucleophilic residues of cellular proteins may lead
to irreversible inhibition. We observed, however, competitive
inhibition (Lineweaver–Burk analysis; Figure S19 in the Sup-
porting Information), which proved noncovalent binding to
HDACs.
The experiments described above were aimed at selecting
the optimal photocontrolled HDACi for selective chemothera-
py. Compound 12 fulfills all criteria described in the Introduc-
tion, that is, high potency of the cis state (HDAC2 IC50 =
0.56 mm) with a very large difference from the trans state. Fur-
thermore, we tested if its reversible photochromism (switching
back and forth between the isomers) was reflected in reversi-
ble changes in activity. To that end, compound 12 was tested
for HDAC1 inhibition by using cycles of trans–cis isomerization
of the same stock solution. The residual HDAC1 activity had
a reversible profile and dropped from 90 (trans) to 50% (cis)
after each isomerization (Figure 5c). This experiment excludes
the possibility that irradiation results in the irreversible forma-
tion of compounds with altered inhibitory activity against
HDACs.
Next, we evaluated the possibility of using light to change
the activity of compound 12 in situ during the HDAC2 enzy-
matic activity assay. After 30 min of incubation with the more
active cis form, white-light irradiation was applied to the enzy-
matic reaction to induce switching to the less active trans
form. In line with our expectations, the rate of product forma-
tion was considerably increased (Figure 5d) due to the poor in-
hibitory potency of trans-12. This experiment confirms that
Conclusion
We presented herein the development of potential chemother-
apeutic agents, the activity of which on their molecular targets
could be externally controlled with light. Around the azoben-
zene photoswitchable moiety, inhibitors were designed that
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were structurally related to the clinically approved HDAC inhib-
itors SAHA, panobinostat, and belinostat. The inhibitors were
optimized towards high potency and pronounced inhibitory
activity differences between the more active, thermodynami-
cally less stable cis isomer and the less active trans isomer.
Notably, the introduction of the photoswitchable moiety did
not compromise the HDAC inhibitory activity because some of
the compounds showed potencies comparable, or even superi-
or, to that of the original SAHA drug. By changing the substitu-
tion pattern of the azobenzene, we were able to strongly influ-
ence not only the potency for different HDACs, but also the
growth of HeLa cervical cancer cells. Favorably, it also enabled
the control of other important features of the photocontrolled
inhibitor, such as the PSS, lmax, and cis isomer half-life in aque-
ous medium.
ported by a VIDI grant (016.122.302) from the Netherlands Or-
ganization for Scientific Research and an ERC starting grant
(309782) from the European Union to F.J.D.
Keywords: cancer · cytotoxicity · drug design · inhibitors ·
photochemistry
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Acknowledgements
Financial support from the Netherlands Organization for Scien-
tific Research (NWO-CW), the Royal Netherlands Academy of
Arts and Sciences (KNAW), the European Research Council (ERC
advanced grant 227897), and the Ministry of Education, Culture
and Science (Gravitation Program 024.001.035) to B.L.F. is
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Received: July 17, 2015
Published online on September 29, 2015
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