Chromo-pharmacophores: Photochromic diarylmaleimide inhibitors for sirtuins

Article abstract of DOI:10.1039/c4sc01346h

Controlling the activity of sirtuins is of high biomedical relevance as the enzymes are involved in cancer, neurodegeneration and other diseases. Therefore structural elements of 3,4-bisindoylmaleimides (BIMs), which are known NAD⁺-dependent histone deacetylase (sirtuin) inhibitors, were merged with photochromic diarylmaleimides to yield photoswitchable enzyme inhibitors. The new inhibitors show excellent photophysical properties, are switchable even in polar solvents, and subtype selective against hSirt2. The inhibitory activity changes up to a factor of 22 for the two photoisomers and physiological properties can therefore be effectively toggled by irradiation with light of different wavelengths. Docking experiments using the enzyme crystal structure explain the observed activity changes based on the steric demand of the thiophene substitution and the rigidity of the molecular structure. This journal is

Full text of DOI:10.1039/c4sc01346h

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Chromo-pharmacophores: photochromic
diarylmaleimide inhibitors for sirtuins†
Cite this: DOI: 10.1039/c4sc01346h
C. Falenczyk,a M. Schiedel,b B. Karaman,c T. Rumpf,b N. Kuzmanovic,a M. Grøtli,d
c
b
a
*
W. Sippl, M. Jung and B. Konig
¨
Controlling the activity of sirtuins is of high biomedical relevance as the enzymes are involved in cancer,
neurodegeneration and other diseases. Therefore structural elements of 3,4-bisindoylmaleimides (BIMs),
which are known NAD+-dependent histone deacetylase (sirtuin) inhibitors, were merged with
photochromic diarylmaleimides to yield photoswitchable enzyme inhibitors. The new inhibitors show
excellent photophysical properties, are switchable even in polar solvents, and subtype selective against
hSirt2. The inhibitory activity changes up to a factor of 22 for the two photoisomers and physiological
properties can therefore be effectively toggled by irradiation with light of different wavelengths. Docking
experiments using the enzyme crystal structure explain the observed activity changes based on the steric
demand of the thiophene substitution and the rigidity of the molecular structure.
Received 8th May 2014
Accepted 6th August 2014
DOI: 10.1039/c4sc01346h
www.rsc.org/chemicalscience
typically a high switching fatigue resistance. In comparison to
other DTEs, the absorption of diarylmaleimides is shied
Introduction
bathochromically and therefore the isomerization is possible at
longer wavelengths, which reduces cell damage, otherwise
caused by high energy light. The optical properties and the
slightly better water solubility makes them interesting candi-
dates for photochromic inhibitors.1 However, to be useful in
biological applications, photochromic compounds should also
undergo photochromic reactions in polar solvents. One hand-
icap of diarylmaleimides is that these reactions are disfavoured
in polar solvents due to a twisted intramolecular electron charge
transfer (TICT) from the donor aryl part to the electron acceptor
maleimide moiety.11–13
There is an ongoing interest in the regulation of enzyme
activity in the eld of epigenetics, particularly with regard to
anticancer research. NAD+-dependent histone deacetylases also
termed sirtuins are enzymes, which deacetylate histones and
certain non-histone proteins. Sirtuins (Sirt1–7) represent the
class III of four histone deacetylases (HDACs). Their activity is
Photochromism has recently received increasing attention due
to a variety of potential applications in molecular optoelec-
tronics, optical data storage and as molecular switches.1–3 The
interest in photopharmacology is revealed by the increasing
number of reported photochromic enzyme inhibitors,4–6 ion
channel blockers7 and receptor ligands8,9 demonstrating the
importance of such compounds as tools for the investigation of
cellular processes and pathogeneses in life sciences.10 Two
strategies are applied for controlling enzyme activity by light:
the photochromic moiety can either be attached to the scaffold
of a known substrate or is the linker between two ligands. The
latter approach allows the light-induced change of the ligand
spacing, which is useful for targets with two binding sites.2
Various types of photochromic compounds have been devel-
oped so far, for instance spiropyranes, azobenzenes, fulgides
and diarylethenes (DTEs).2 Diarylmaleimide derivatives are well
known for their photochromic properties and their ability to
change conformational mobility reversibly upon irradiation
(Fig. 1). Both photoisomers are thermally stable and show
aInstitute of Organic Chemistry, University of Regensburg, 93040 Regensburg,
Germany. E-mail: burkhard.koenig@chemie.uni-regensburg.de
bInstitute of Pharmaceutical Sciences, University of Freiburg, 79104 Freiburg, Germany
cDepartment of Pharmaceutical Chemistry, Martin Luther University of Halle
Wittenberg, 06120 Halle/Saale, Germany
dDepartment of Chemistry and Molecular Biology, University of Gothenburg, 41296
¨
Goteborg, Sweden
† Electronic supplementary information (ESI) available: Detailed experimental
Fig. 1 Sirtuin inhibitor 1 (Ro31-8220) and by way of example the
structural related photochromic diarylmaleimide 2, which interconvert
reversible by irradiation with light of different wavelength between the
open and closed photoisomer.
procedures of the synthesis of
3 and 11, additional spectral data,
photochemical measurements, biological data and docking studies of 2, 3 and
11. See DOI: 10.1039/c4sc01346h
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dependent on nicotinamide adenine dinucleotide (NAD+),
whereas the other classes are zinc-dependent amidohy-
drolases.14–16 The biological activity of sirtuins has been linked
to the pathogenesis of cancer17 as well as viral,18 metabolic19,20
and neurological diseases.21,22 As several disorders are linked to
the perturbation of HDACs, they are a promising target for drug
discovery. Various class I and II HDACs anticancer drugs are
already in clinical trials or approved as therapeutics.23 To get
more insight into the biological activity and molecular mecha-
nism of all class III HDAC members there is a need to investi-
gate potent and isotype selective small molecule modulators for
human sirtuins. Several lead structures for small-molecule sir-
tuin inhibitors have been identied so far.16 Besides nicotin-
amide as the endogenous inhibitor, splitomicins, suramins,
indoles as well as kinase inhibitors like bisindoylmaleimides
(BIMs) are of particular interest.14–16 Diarylmaleimides are
stable photochromic compounds and structurally related to
BIMs like 1 (Ro31-8220), a potent sirtuin inhibitor (Fig. 1).14 We
envisioned that merging the structure of the DTE chromophore
and the BIM pharmacophore to new diarylmaleimides would
lead to photochromic sirtuin inhibitors. Variation of the
substitution pattern and molecular docking experiments
provides insight into the binding mode of the diarylmaleimides
and their photoisomers to the active site of the enzyme and
thereby lead to potent and isotype selective inhibitors for
sirtuins.
Scheme 1 Synthetic strategy with reagents and conditions for 11: (a)
acetyl chloride, AlCl3, CH2Cl2, 0 ꢀC, 2 h, (84%); (b) TTN, HClO4, MeOH,
RT, overnight, (83%); (c) Pd2(dba)3, XPhos, phenylboronic acid, K3PO4,
1,4-dioxane, 100 ꢀC, overnight, (52%); (d) methyl chlorooxoacetate,
AlCl3, CH2Cl2, 0 ꢀC, 2 h, (73%); (e) Pd2(dba)3, XPhos, phenylboronic
acid, K3PO4, 1,4-dioxane, 100 ꢀC, overnight, (63%); (f) aq. NH3, THF, RT,
overnight, (80%); and (g) KOtBu (1 M in THF), THF, 0 ꢀC / RT, over-
night, (17%).
Results and discussion
Synthesis
A common strategy for the synthesis of 3,4-diarylmaleimides is
the intramolecular Perkin-type condensation.24,25 The synthesis
of compound 2 (Fig. 2) was previously described by Scandola
et al.3 Adapted from this synthesis, the non-symmetric mal-
eimide 3 was prepared in a similar manner with further func-
tionalization by Suzuki coupling (see ESI†).
Unfortunately, compound 11 could not be obtained by this
strategy. Hence, we developed an alternative route for the
synthesis of 11, which is outlined in Scheme 1. Starting from 5-
chloro-2-methyl-thiophene (4) standard Friedel–Cras condi-
tions yielded ketone 5 and glyoxylate ester 8. The ester 6 was
obtained via an oxidative rearrangement from ketone 5. Suzuki
coupling yielded the functionalized esters 7 and 9. Compound
10 was prepared by aminolysis of glyoxylate ester 9, which was
subsequently used in a Perkin condensation with ester 7 to
obtain maleimide 11. Scheme 1 summarizes the synthesis of the
symmetric diarylmaleimide 11.
Photochromic properties
The photoisomerization of photochromic compounds in polar
solvents is essential for applications in biological assays. As
dimethyl sulfoxide is a standard solvent for inhibitor screening,
this solvent was used to investigate the switching ability of the
diarylmaleimides. The ring closure is suppressed in polar
solvents due to a twisted intramolecular electron transfer. This
phenomenon is well-investigated for maleimides bearing two
benzothiophenes.12 To enhance the switching ability of DTEs
the introduction of sterical hindrance either by bulky substit-
uents on the aryl moieties or on the bridge is a widely applied
concept.26–28 As seen from the perspective of the pharmacophore
only the introduction of sterically demanding substituents on
the aryl moieties are possible. Otherwise the ability to form
relevant hydrogen bonds between the maleimide and the
backbone of the enzyme is lost. We decided to replace the
benzothiophene moieties by phenyl substituted thiophenes.
Despite this change the structure of the chromophore is still
similar to the pharmacophore, but the photochromic properties
improve signicant. The exchange of the methyl-benzothio-
phenes in 2 by one or two methyl-phenyl substituted thiophenes
in 3 or 11, resp., stabilizes the antiparallel conformation, which
favors the photochemical ring closure reaction.
Fig. 2 Structures of diarylmaleimides 2 and 3.
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Enzyme inhibition
Next, the inhibitory effect of the photoresponsive compounds 2,
3 and 11 against sirtuins in a homogenous uorescent assay,
previously established by Jung et al. was determined. The
enzymatic conversion was quantied using (S)-[5-acetylamino-
1-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)-pentyl]-carbamic
acid benzyl ester also termed ZMAL29 as uorescent substrate
for deacetylation followed by a tryptic digestion step.30 1 (Ro31-
8220) has an IC50 value of 0.8 mm and is the most potent BIM
inhibitor reported so far for human Sirt2 (hSirt2).14,16 In
comparison to 1 the photoresponsive diarylmaleimides 3 and
11 showed similar inhibition strength against hSirt2. The
respective IC50 values are given in Table 2.
The scaffold of compound 2 is structurally closest related to
1. Its open isomer exhibits a lower IC50 value against hSirt2
(Table 2, entry 6) than its closed photoisomer. By the exchange
of one of the benzothiophenes in 2 by a phenyl substituted
thiophene as in compound 3 the inhibition activity increases.
However, the open and the closed photoisomer of compound 3
show the same inhibition (Table 2, entries 4 and 5). The
replacement of the second benzothiophene from the maleimide
moiety by a phenyl substituted thiophene in compound 11
yields an inhibition corresponding to an IC50 value of 4.2 mm of
the open photoisomer. The ring closed photoisomer of 11
shows a twentyfold lower enzyme inhibition with 92.3 mm
(Table 2, entries 2 and 3). Diarylmaleimide 11 gives the most
signicant change in hSirt2 inhibition activity for both photo-
isomers and therefore its isotype selectivity against human Sirt1
(hSirt1) and human Sirt3 (hSirt3) under the same assay condi-
tions30 as used for hSirt2 were determined. The isotype selec-
tivity for BIM 1 (Ro31-8220) is only small (Table 2, entry 1).
Therefore inhibitor concentrations at 20 mm and 200 mm were
used to determine the enzyme activity using nicotinamide as a
reference. Neither the open isomer nor the closed isomer of 11
inhibited hSirt1 or hSirt3 signicantly (Table 2, entries 2 and 3).
Next, the enzyme activity of hSirt2 was controlled by light
under assay conditions. Therefore, the photochemical conver-
sion from the closed isomer to the open isomer of 11 and the
corresponding inhibitory activity were measured as a function
of time (Fig. 4). Hence the less potent closed isomer of 11 was
incubated with the enzyme under assay conditions and irradi-
ated over a period of 30 min with a green LED (2.5 W, 530 nm
emission maximum). Then the cofactor NAD+ and ZMAL were
added and the assay was performed under the usual conditions.
Fig. 4 illustrates the time course of the isomerization (dotted
line) and inhibitory activity (straight line) of 11. The irradiated
samples switched from the less potent closed form of 11 to the
signicantly more potent open form.
Fig. 3 Photochromic properties of compound 11 (10 mm in DMSO):
absorption spectra changes upon continuous irradiation for 0, 2, 6, 12,
18, 40 and 60 s with light of lmax ¼ 312 nm.
The photoisomerization was monitored by absorption spec-
troscopy (Fig. 3) and the photostationary states (PSS) were
determined by HPLC measurements (see ESI†).
Upon irradiating (l ¼ 312 nm) the dissolved compounds 2, 3
and 11, a rapid color change was observed, resulting in new
absorption bands characteristic for each compound. The
photochromic properties are summarized in Table 1. Replacing
the benzothiophene moieties by phenyl-substituted thiophenes
has two consequences: (1) the absorbance maxima for the
closed isomers shi bathochromically from compound 2 to
compound 11. Therefore light of longer wavelength is sufficient
for the isomerization. (2) The photoconversion becomes more
efficient as indicated by PSS increasing from 62% to 94%. Such
high PSS is advantageous for binding investigations as the
interaction with the enzyme can be restricted to mainly one
photoisomer.
By way of example, Fig. 3 illustrates the photoisomerization
of compound 11. Upon irradiating (l ¼ 312 nm) of compound
11 in dimethyl sulfoxide solution, a rapid color change from
light green to deep purple was observed, resulting in new
absorption bands at 390 nm and 580 nm (marked with black
arrows in Fig. 3). The isosbestic points (Table 1) indicate a clean
two-component switching. Repetition of the photo-
isomerization of compound 11 shows a good fatigue resistance
over ve cycles (see ESI†).
Table 1 Photochromic properties of diarylmaleimides 2, 3 and 11 (10
mm in DMSO)
Aer 20 min complete photochemical conversion of 11 and a
constant inhibition value are observed. The determined values
correspond to the inhibition activity of the open isomer of 11
(Table 2) proving that the photoisomerization is possible in the
presence of the enzyme under assay conditions.
Additionally, the open photoisomer of 11 was tested for
cellular activity using hyperacetylation of tubulin31 as a marker.
11 showed tubulin hyperacetylation and hence target
lmax open lmax closed Isosbestic
Entry Compound [nm]
[nm]
points [nm]
PSSa
1
2
3
2
3
11
278
274
290
353, 495
329, 527
390, 580
293
291
62%
87%
316, 410, 430 94%
a
Determined by HPLC measurements.
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Table 2 IC50 values of the photochromic diarylmaleimides 2, 3 and 11 and 1 (Ro31-8220) as BIM reference
Entry
Compound
hSirt1 IC50 [mM]
hSirt2 IC50 [mM]
hSirt3 IC50 [mM]
hSirt2 IC50 closed/IC50 open
1
2
3
4
5
6
7
1 (ref. 14 and 15)
11 open
11 closed
3 open
3 closed
2 open
3.5 ꢂ 0.4
n.i.a
0.8 ꢂ 0.2
4.2 ꢂ 0.8
92.3 ꢂ 8.0
2.3 ꢂ 0.4
2.1 ꢂ 0.3
12.9 ꢂ 5.4
23.1 ꢂ 5.6
3.7 ꢂ 0.2
n.i.
n.i.
—
22
1
n.i.
b
—
—
—
—
—
—
—
2
2 closed
a
n.i.: no inhibition [<10% @ 20 mm]. b —: not tested.
engagement similar to the control inhibitor 1 (Ro31-8220)14 (see results suggest that the terminal aromatic rings and methyl
ESI†). But further studies are required as we cannot rule out groups clash with His187 and Phe235 resulting in a different
indirect effects as the compounds have not been optimized for orientation at the binding pocket compared to the closed
selectivity, e.g. against kinases.
isomers of 2 and 3 (Fig. 6). As a consequence, the distance
between the maleimide and Val233 backbone carbonyl group is
˚
increased (3.85 A) disabling the hydrogen bond formation (see
Molecular docking
ESI†). Only the closed photoisomers of 2 and 3 t well to the
binding pocket and make favourable van-der-Waals interac-
tions with the aromatic and hydrophobic residues (see ESI†).
Docking to the apo Sirt3 (PDB-ID 3GLS) substrate binding
pocket was not possible due to a different conformation of the
exible loop anking the acetyl-lysine channel. Especially, the
residues Phe157, Arg158 and Phe294 block part of the substrate
binding pocket observed in hSirt2 (see ESI†). Thus, the docking
suggests that the observed selectivity of the photochromic
inhibitor 11 arises from a different orientation of amino acid
residues located at the entrance of the acetyl-lysine channel of
hSirt3.
In order to rationalize the observed in vitro activities docking of
the open and closed form of the diarylmaleimides to hSirt2
(PDB-ID 3ZGO) was carried out. Docking of the open form of the
isomers 2, 3 and 11 to hSirt2 resulted in a similar interaction at
the acetyl-lysine channel of hSirt2 (see ESI†) favoured by a
hydrogen bond with the backbone of Val233 and van-der-Waals
interactions with hydrophobic and aromatic residues (Phe96,
Phe119, Ile169, His187, Phe234, and Phe235). Val233 represents
a conserved amino acid in sirtuins that binds the amide group
of the acetyl-lysine substrate. The open photoisomers t nicely
to the acetyl-lysine pocket of hSirt2. The angle between the
aromatic rings and the maleimide is for all open photoisomers
around ꢁ50 deg., which allows the accommodation of both
aromatic substituents in the acetyl-lysine pocket. The closed
photoisomer of 11 might be too big (distance from both ends of
In case of apo hSirt1 (PDB-ID 4IG9), which shows also an
open conformation of the substrate binding site, the docking of
the photoisomers of 11 resulted in less favourable complexes.
˚
˚
˚
the phenyl ring 14.7 A compared to 12.9 A for 3 and 10.9 A for 2)
and rigid to interact in a similar way within the binding pocket
as the open and exible isomer of 11 (Fig. 5). The docking
Fig. 5 Comparison of the binding mode of photoisomer 11 at hSirt2 in
the open (brown) and the closed form (dark green). The two residues
Fig. 4 Time course of the photochemical conversion from the closed (Phe235 and His187) that hinder the proper orientation of the closed
to the open form of 11 (dotted line) and the corresponding inhibitory photoisomer 11 are colored cyan. Water molecules are shown as red
activity against hSirt2 (straight line) upon continuous irradiation with spheres. The hydrogen bond to the backbone of Val233 is shown as
light of lmax ¼ 530 nm for 0, 2.5, 5, 7.5, 10, 20 and 30 min at an assay black line (distance between the NH group of closed 11 and the
concentration of 10 mM.
backbone carbonyl of Val233 is 3.19 A˚).
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Acknowledgements
Financial support by the Deutsche Forschungsgemeinscha
(DFG) within the GRK 1910 and within the CRC992 (MEDEP,
Medical Epigenetics) is gratefully acknowledged. We thank
Karin Schmidtkunz for western blot experiments.
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