Activation of Sirtuin 2 Inhibitors Employing Photoswitchable Geometry and Aqueous Solubility

Article abstract of DOI:10.1002/cmdc.202000148

Because isoenzymes of the experimentally and therapeutically extremely relevant sirtuin family show high similarity, addressing the unique selectivity pocket of sirtuin 2 is a promising strategy towards selective inhibitors. An unrelated approach towards selective inhibition of isoenzymes with varied tissue distribution is targeted drug delivery or spatiotemporal activation by photochemical activation. Azologization of two nicotinamide-mimicking lead structures was undertaken to combine both approaches and yielded a set of 33 azobenzenes and azopyridines that have been evaluated for their photochemical behaviour and bioactivity. For some compounds, inhibitory activity reached the sub-micromolar range in their thermodynamically favoured E form and could be decreased by photoisomerization to the metastable Z form. Besides, derivatization with long-chain fatty acids yielded potent sirtuin 2 inhibitors, featuring another intriguing aspect of azo-based photoswitches. In these compounds, switching to the Z isomer increased aqueous solubility and thereby enhanced biological activity by up to a factor of 21. The biological activity of two compounds was confirmed by hyperacetylation of sirtuin specific histone proteins in a cell-based activity assay.

Full text of DOI:10.1002/cmdc.202000148

Accepted Article
Title: Activation of sirtuin 2 inhibitors employing photoswitchable
geometry and aqueous solubility
Authors: Christoph W. Grathwol, Nathalie Wössner, Steven Behnisch-
Cornwell, Lukas Schulig, Lin Zhang, Oliver Einsle, Manfred
Jung, and Andreas Link
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000148
Link to VoR: https://doi.org/10.1002/cmdc.202000148
01/2020

10.1002/cmdc.202000148
ChemMedChem
FULL PAPER
Activation of sirtuin 2 inhibitors employing photoswitchable
geometry and aqueous solubility
Christoph W. Grathwol[a], Nathalie Wössner[b], Steven Behnisch-Cornwell[a], Lukas Schulig[a], Lin
Zhang[c], Oliver Einsle[c], Manfred Jung[b] and Andreas Link*[a]
[a]
C. W. Grathwol, Dr. S. Behnisch-Cornwell, L. Schulig, Prof. Dr. A. Link
Institute of Pharmacy
University of Greifswald
Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald, Germany
Fax: (+49) (0)3834 4204895
E-mail: link@uni-greifswald.de
[b]
[c]
N. Wössner, Prof. Dr. M. Jung
Institute of Pharmaceutical Sciences
University of Freiburg, Albertstr. 25, 79104 Freiburg, Germany
Dr. L. Zhang, Prof. Dr. O. Einsle
Institute of Biochemistry
University of Freiburg, Albertstr. 211, 79104 Freiburg, Germany
Supporting information for this article is given via a link at the end of the document.
Abstract: Because isoenzymes of the experimentally and
therapeutically extremely relevant sirtuin family show high similarity,
addressing the unique selectivity pocket of sirtuin 2 is a promising
strategy towards selective inhibitors. An unrelated approach towards
selective inhibition of isoenzymes with varied tissue distribution is
targeted drug delivery or spatiotemporal activation by photochemical
activation. Azologization of two nicotinamide mimicking lead
structures was undertaken to combine both approaches and yielded
a set of 33 azobenzenes and azopyridines that have been evaluated
for their photochemical behaviour and bioactivity. For some
compounds, inhibitory activity reached the submicromolar range in
their thermodynamically favoured E-form and could be decreased by
photoisomerization to the metastable Z-form. Besides, derivatization
with long-chain fatty acids yielded potent sirtuin 2 inhibitors, featuring
another intriguing aspect of azo-based photoswitches. In these
compounds, switching to the Z-isomer increased aqueous solubility
and thereby enhanced biological activity up to a factor of 21. The
biological activity of two compounds was confirmed via
hyperacetylation of sirtuin specific histone proteins in a cell-based
activity assay.
similarity in their amino acid sequence especially along the
catalytic cleft, which comprises the binding sites of the acyl-lysine
substrate and the cofactor NAD+.[2] Nevertheless, these three
isotypes show differences in their substrate recognition as well as
their enzymatic activity. In addition to a robust deacetylation
ability, Sirt2 efficiently binds and cleaves myristoylated peptides,
whereas in vivo histone decrotonylation has been reported for
Sirt3.[3] During sirtuin-catalyzed protein deacylation, the by-
product nicotinamide functions as an endogenous pan-sirtuin
inhibitor.[4] Hence, nicotinamide-mimicking compounds (Figure 1)
act as eligible sirtuin inhibitors demonstrated by the potently
active Sirt1 inhibitor selisistat (1), which was announced to be
intended for evaluation in a long term phase III study for the
treatment of Huntington’s disease.[5] Furthermore, 5-[(3-
amidobenzyl)oxy]nicotinamides (2) exert robust Sirt2 inhibition
evincing promising effects in the therapy of neurodegenerative
diseases or cancer.[6] In our hands, an in vitro activity screening
of a pooled kinase inhibitor library revealed 5-styrylnicotinamide 3
as a moderate inhibitor of Sirt2.[7] Based on this structure,
azologization resulted in a reversible photoswitch (4) exhibiting
comparable inhibitory activity of its stretched out E-form.[8,9]
Introduction
Sirtuins refer to a family of NAD+-dependent lysine deacylases
that are highly conserved and found throughout all domains of life.
Usually, sirtuins are classified as histone deacetylases (class III
HDACs), but also an increasing number of non-histone proteins
were identified as native substrates. In sum, sirtuin-promoted
deacylation is involved in the regulation of various cellular
processes such as DNA repair, gene transcription, aging,
metabolism, and apoptosis representing an auspicious target for
pharmacological intervention.[1] In human cells, seven sirtuin
isotypes (Sirt1–7) have been identified. Sirt1–3 are
phylogenetically closely related (class I sirtuins) exhibiting high
1
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10.1002/cmdc.202000148
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Building on our previous efforts in the design of photoswitchable
sirtuin inhibitors, we now present the synthesis of various
azopyridine-based photoswitches in analogy to compound 4.[8]
Furthermore,
another
azologization
approach
yielded
azobenzene-based photoswitches derived from recently
published 5-benzyloxynicotinamides (2).[6] The photophysical and
photochemical properties of the obtained azo dyes were studied
and adjusted to long thermal half-lives of the metastable Z-
isomers (> 300 h) under physiological conditions. The biological
activity of the compounds was determined in vitro applying a
fluorescence-based enzyme assay and could also be proven in a
urinary cancer cell line.
Figure 1. Nicotinamide-mimicking sirtuin inhibitors: Selisistat (1), 5-[(3-
amidobenzyl)oxy]nicotinamides (2) and the structurally related 5-
styrylnicotinamide 3. Exchange of the amide bond in 2 and the stilbene C,C-
double bond in 3 allows for incorporation of a photoswitchable azo moiety while
maintaining the original shape of the parent molecules.
Results and Discussion
Synthesis
Over the past decade, molecular photoswitches have permitted
light-mediated control over a vast scope of biological targets such
as ion channels, transporters, GPCRs, and various enzymes.[10]
A common strategy to achieve light-sensitivity of certain biological
target structures is the design of photochromic ligands (PCLs) by
insertion of photoswitchable moieties into known bioactive small
molecules.[11] Upon irradiation with appropriate wavelengths,
PCLs undergo reversible photoisomerization reactions,
accompanied by marked changes in shape and physicochemical
properties of the ligand. As the target affinity of a ligand is strongly
influenced by its structure and electron distribution, distinct
photoisomers often show an altered binding behavior and
consequently exert differential bioactivity.[12] As a matter of fact,
the first PCLs targeting an epigenetic regulator have been
photoswitchable sirtuin inhibitors based on a diarylmaleimide
photoswitch.[13] Utilizing an indolyl fulgimide core instead
improved the photochemical behavior of the sirtuin photoswitch
under physiological conditions.[14] Besides, azobenzene-based
photoswitches have been designed for the light-mediated
modulation of human Zn2+-dependent HDACs as well as related
bacterial amidohydrolases, offering promising perspectives
concerning highly selective antineoplastic and antimicrobial
chemotherapy.[15] Among the known molecular photoswitches,
azo dyes hold a prominent role combining a comfortable way of
synthesis with finely tunable photophysical properties. By
treatment with UV radiation, (E)-azobenzene segues in a
metastable photostationary state (PSS) comprising high amounts
of the Z-isomer followed by slow thermal relaxation of (Z)-
azobenzene back to the more stable E-isomer in the dark. Blue
light radiation accelerates Z→E isomerization, while complete
transformation is only obtained thermally.[16] Wavelength of
maximal absorption, half-life of the metastable Z-isomer, and
photoisomer distribution (PSD) are strongly influenced by
different substitutions at the azobenzene core or the incorporation
of heteroaromatic moieties affecting also other physicochemical
properties, ultimately determining absorption and distribution.[17]
Starting from commercially available methyl 5-aminonicotinate
(5), a first set of heteroaryl azo dyes was easily accessible in two
steps. Diazotization and azo coupling of 5 with phenols and
anilines gave the respective methyl 5-diazenylnicotinates (6a-f) in
moderate yields. Quantitative transformation to nicotinamides
was accomplished by successive ammonolysis, providing
phenols 7a-d and anilines 7e and 7f in satisfying overall yield
(Scheme 1). Although, ammonolysis of 5 prior to derivatization by
azo coupling might be more beneficial in the synthesis of a
compound library, we generally performed ammonolysis at the
final step because of more efficient chromatographic purification
of methyl nicotinates in comparison to 5-diazenylnicotinamides.
O
O
NH2
N
R
MeO
MeO
N
a
b
N
N
5
6a-f
O
N
R
H2N
N
6a-f
N
7a-f
R =
HO
OH
OH
6/7a
6/7b
6/7e
6/7c
OMe
NMe2
NH2
OH
OMe
6/7d
6/7f
Scheme 1. Reagents and conditions: a) 1. NaNO2, HCl, H2O, 0 °C; 2. Phenols
or anilines, NaOH, H2O, 0 °C to rt, 31–65%; b) NH3, MeOH, rt, 1–4 d, quant.
2
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10.1002/cmdc.202000148
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Aniline 6f was converted to several amides (8a-k, structures not
shown) by reaction with acyl chlorides resulting in compounds 9a-
k after final ammonolysis (Scheme 2). As nucleophilic attack of
the amino group in 6f is hampered by the two neighboring methyl
groups, amide formation required long reaction times (1–3 days)
and gave only modest yields.
A greater diversity of 5-diazenylnicotinamides was realizable
exerting Mills reaction for the synthesis of unsymmetrical azo
dyes by reaction of aromatic nitroso derivatives with anilines in
glacial acetic acid. Aromatic nitroso compounds were obtained by
oxidation of anilines with potassium peroxymonosulfate (oxone)
yielding the methyl 5-diazenylnicotinates 14a-d after
condensation with 5 (Scheme 4). In the first place, we tried to
transform the electron-deficient aminopyridine 5 to its nitroso
derivative in order use the electron-rich anilines 13a-d as
nucleophiles in the following condensation reaction. However,
transformation of 5 to its nitroso derivative by potassium
peroxymonosulfate failed, probably due to the oxidative
susceptibility of amino pyridines towards formation of N-oxides.
Consequently, we interchanged the roles of the reagents, treated
13a-d with oxone, and used the less suitable aminopyridine 5 as
nucleophile in the condensation reaction. Normally, Mills reaction
provides good yields in the synthesis of azobenzenes, but in the
case of methyl 5-diazenyl-nicotinates, yields were often poor and
required long reaction times caused by the said reduced
nucleophilicity of 5. Compounds 15a-d were obtained by
successive ammonolysis. In the case of 14a and 14c, further
derivatization was performed by acidic cleavage of the tert-butyl
based protective group, followed by amide formation and
ammonolysis to 15e and 15f. Sonogashira-type coupling of
arylbromide 14d with terminal alkynes resulted in formation of
methyl nicotinates 14g and 14h (structure not shown) in modest
yield, which were transformed to 15g and 15h as described
above.
H
N
R
a
b
O
N
O
6f
H2N
N
N
9a-k
R =
9a
n-C3
n-C
n-C
7H15
CH3
9b
9f
H7
9c
9g
5H11
9d
n-C
n-C
n-C
13H27
9e
9H19
11H23
S
F
9h
9j
9k
9i
Scheme 2. Reagents and conditions: a) acyl chloride, pyridine or DIPEA, THF,
0 °C to rt, 1–3 d, 26–58%; b) NH3, MeOH, rt, 1–4 d, quant.
Derivatization to N-alkyl nicotinamides (12a-d) was achievable by
aminolysis of ethyl nicotinate 11 using the respective primary
alkylamines (Scheme 3). On the contrary, N-alkyl nicotinamides
could not be obtained through aminolysis of the corresponding
methyl nicotinate 6f due to an insufficient reactivity of the methyl
ester with primary alkylamines.
R
O
a
N
MeO
N
5
N
-
14a d
O
NH2
O
NH2
R
a
b
EtO
O
NH2
N
a
EtO
N
b
-
N
14a d
H2N
N
N
R
N
-
N
10
11
13a d
-
15a d
c
NH2
c
O
e
b
f
b
d
b
R
N
11
N
N
14a
14c
14d
15e
15f
15g, 15h
H
N
R =
12a-d
R =
O
O
O
Br
H
3C
H
3C
12b
N
H
O
H
HO
12d
3C
12c
O
O
2
3
12a
13-15a
13-15b
13-15c
13-15d
Scheme 3. Reagents and conditions: a) 1. NaNO2, HCl, H2O, 0 °C; 2.
2,6-dimethylaniline, NaOH, H2O, 0 °C to rt, 42%; b) R-NH2, MeOH, 40–110 °C,
4 d, 88–98%.
O
O
OH
4OH
N
H
N
3
H
8
15h
15e
15f
15g
Scheme 4. Reagents and conditions: a) 1. Oxone, DCM, H2O, rt, 4–48 h; 2.
Anilines 13a-d or 5, HAc, 40 °C, 2–14 d, 7–43%; b) NH3, MeOH, rt, 4 d, quant.
c) TFA, DCM, rt, 12 h, quant. d) nBu-NH2, HATU, DIPEA, DMF, rt, 56% e)
decanoyl chloride, DIPEA, THF, rt, 80% f) alkyne, Pd(PPh3)4, CuI, NEt3, THF or
DCM, 50–85 °C, 24 h, 20%.
3
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Based on closely related 5-[(amidobenzyl)oxy]-nicotinamides (2),
we additionally synthesized a small set of analogous azobenzene
photoswitches.[6,18] Compound 18 was prepared similarly to the
original procedure and was converted to azo compounds 20a-d
applying Mills reaction and subsequent ammonolysis (Scheme 5).
assay buffer) for most of the synthesized compounds due to their
high lipophilicity. In fact, many of them were prone for aggregation
indicated by blue-shifted maxima and notably decreased
absorption in the UV-Vis spectra.[20] As
a consequence,
photoisomerization was hampered in the respective compounds.
Higher amounts of DMSO prevented aggregation and enabled
determination of Z-isomer stability in aqueous environment as
exemplified for compound 20a (Figure 2).
O
O
OH
a
b
O
MeO
MeO
O
NH2
Figure 2. Left: 20a (50 µM) in assay buffer (5% DMSO, v/v) at the thermal
N
N
O
18
16
18
N
c
d
H2N
N
N
R
-
20a d
R =
N
O
F
O
equilibrium (∆) and the PSS after 365 nm irradiation (red). Due to aggregation
of the chromophore, general light-absorption is diminished and λ ∆
appears
Bn
max
20a
20b
20c
20d
blue-shifted. Right: 20a (50 µM) in assay buffer (50% DMSO, v/v) at the thermal
equilibrium (∆), directly after 365 nm illumination (light-red) as well as additional
15 h in the dark (dark-red). The PSS after 452 nm irradiation (blue) displays
almost complete transformation to the thermal equilibrium (∆). Under these
conditions, aggregation of the compound is prevented and photoisomerization
proceeds unhindered.
Scheme 5. Reagents and conditions: a) 3-Nitrobenzyl bromide, Cs2CO3, DMF,
rt, 61%; b) Raney-Ni, H2, THF, rt, 85%; c) 1. Oxone, DCM, H2O, 2–14 h; 2.
R-NH2, HAc, 40 °C, 1–7 d, 23–42%; d) NH3, MeOH, rt, 4 d, quant.
Photochemistry
Acylation of 7f yielded compounds 9a-k, adopting PSS sufficiently
stable for acquisition (Table 1). PSD of these compounds was
approximately 90% of the respective photoisomers, thus enabling
almost perfect toggling between Z- and E-form. Regarding the
PSS half-life, amides 9h-j obtained from aromatic carboxylic acid
chlorides were inferior to amides 9a-g and 9k prepared from
aliphatic counterparts.
The design of photoswitchable drugs requires careful examination
of their photophysical and photochemical properties under
physiological conditions. With water being the solvent of choice
for all pharmaceutically relevant systems, it had to be considered
that hydrogen bonding in polar solvents strongly affects thermal
relaxation rates and may lead to rapid thermal Z→E isomerization
in certain classes of azobenzenes.[16] Though this can be
desirable in some applications, in our case slow thermal Z→E
isomerization was obligatory in order to retain the impact of short
term UV irradiation.
By inversion of the amide bond, we received compound 15a and
its derivative 15e. As reflected by strongly increased half-lives of
the thermal Z→E isomerization, inversion of the amide bond was
beneficial for the thermal stability of the Z-isomer, while PSD after
365 nm radiation was only about 60–70% of the respective Z-
isomer. Insertion of a methylene bridge instead of the amide bond
yielded 15b, 15c, and 15f exhibiting slow thermal Z→E
isomerization kinetics as well as efficient switching between both
states. Half-life of the respective Z-isomers and PSD for the
ethinyl substituted 15g and 15h were still sufficient, but not as
valid as in the methylene bridged compounds 15c and 15f.
According to their substitution pattern and spectral properties,
phenylazopyridines 7a-d (2- / 4-OH) as well as 7e and f (4-NR2)
can be classified as amino-azobenzenes (aAB). In this sort of
heteroarylic azo dyes, the rate of thermal isomerization is
intensively increased by hydrogen bonding and tautomerization.
Consequently, aAB undergo thermal Z→E isomerization within
ms to s in aqueous solution.[19] Such rapid transformations could
not be captured by our experimental set-up. Noteworthy, in aprotic
and less polar acetonitrile thermal Z→E isomerization of
compound 7a was notably decelerated, showing a half-life of 94
min in contrast to its constitutional isomer 7b, that exerted rapid
thermal isomerization. We suggest that the ortho methyl groups
in 7a impeded isomerization leading to delayed thermal
relaxation.
Azobenzene-based compounds 20a-c also elicited slow thermal
Z→E isomerization in combination with an appropriate PSD of the
respective photoisomers. Especially 20a and 20b demonstrated
almost quantitative transformation to their Z-isomers upon
365 nm irradiation. Contrarily, amino-azobenzene (aAB) 20d
showed rapid thermal Z→E isomerization due to 4-NR2
substitution.
In order to increase the thermal half-life of the Z-isomers, we
performed additional structural modifications on the
phenylazopyridine scaffold. However, investigation of thermal
Z→E isomerization kinetics could not be performed in the exact
solvent composition of the enzyme assay (i.e., 5% DMSO (v/v) in
4
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Conclusively, the half-lives of the thermal Z→E isomerization as
well as the PSD are not necessarily reflecting the situation in the
enzyme assay mixture due to the discrepancies in the solvent
compositions.[21] Nevertheless, an approximate estimation of Z-
isomer half-life in aqueous solutions was possible.
20b[b]
20c[b]
324
340
166
63
2 / 98
83 / 17
85 / 15
14 / 86
† solutions in DMSO/assay buffer (50 µM): [a] 5% DMSO (v/v), [b] 50% DMSO
(v/v), [c] 90% DMSO, (v/v); ‡ PSD determined by HPLC analysis in MeOH/water
mixtures applying isocratic conditions and evaporative light scattering detection
(ELSD).
Table 1. Absorption maxima of azo dyes 9a-k, 15a-h, and 20a-c at thermal
equilibrium (∆), half-lives of thermal Z→E isomerization and PSD after 365 nm
and 452 nm irradiation.
E/Z
@ 365 nm
(%)‡
E/Z
@ 452 nm
(%)‡
Entry
λmax∆ (nm)†
t
½
(h)†
Biochemistry
The effect of the compounds on deacetylase activity of three
9a[a]
9b[a]
9c[b]
329
332
339
345
344
346
345
337
337
330
339
325
332
329
332
326
333
355
350
352
6
8
12 / 88
10 / 90
9 / 91
90 / 10
86 / 14
87 / 13
88 / 12
90 / 10
91 / 9
human sirtuin isotypes (Sirt1–3) was investigated by
a
homogeneous fluorescence-based assay, using Z-Lys(acetyl)-
AMC (ZMAL) as substrate. In order to determine the impact of
photoisomerization, the respective compounds were irradiated
(365 nm, 5 min) in DMSO prior to incubation in the enzyme assay
mixture. The results of the irradiated probes were finally
compared to non-irradiated measurements. For compounds
displaying rapid thermal isomerization in aqueous solution (7a-7f,
11, 12a-d, 20d) only non-irradiated probes were tested.
16
21
9d[c]
9 / 91
9e[c]
22
10 / 90
10 / 90
14 / 86
14 / 86
12 / 88
29 / 71
14 / 86
31 / 69
6 / 94
As previously published, lead 3 exhibited moderate potency for
Sirt2 inhibition (IC50 = 25 µM), but at the same time represented
the least-selective hit, as Sirt3 activity was also notably affected
(Table 1).[7] Azologization of 3 yielded the direct azo analogue 7a.
As the geometry of the molecule was unaltered by this structural
modification, we expected 7a to display a biological activity
comparable to the lead. In fact, exchange of the ethylene bridge
by the azo group even improved inhibitory potency to the single
digit µM range. However, isoenzyme selectivity remained
unsatisfactory. In general, 5-diazenylnicotinamides primarily
inhibited Sirt2/Sirt3, whereas a distinct inhibition between Sirt2
and Sirt3 was harder to achieve. The existence and position of
the two methyl groups on the phenyl substructure was found to
be virtually irrelevant for biological activity as well as isoenzyme
selectivity, as both the isomers 7a/7b and compound 7e exerted
similar activity and selectivity profiles. However, exchange of the
methyl groups with methoxy ethers in 7d resulted in slightly
improved isoenzyme selectivity with decreased Sirt3 inhibition,
whereas Sirt2 inhibition was unaltered. Interestingly, aniline 7f
exhibited the highest activity with an IC50 of 6 µM against Sirt2 and
9 µM against Sirt3, whereas methylation of the amino group
provoked a slight decrease of Sirt2 inhibition in N,N-dimethyl
aniline derivative 7e indicating the relevance of the amino group
for polar target interactions.
9f[c]
21
9g[c]
26
87 / 13
91 / 9
9h[b]
9i[b]
0.7
0.6
0.6
7
91 / 9
9j[b]
93 / 7
9k[b]
89 / 11
94 / 6
15a[b]
15b[b]
15c[a]
15d[a]
15e[b]
15f[c]
15g[b]
15h[b]
20a[b]
178
23
90 / 10
86 / 14
91 / 9
319
220
254
102
50
11 / 89
9 / 91
Table 2. Influence of stilbenoid lead 3 and 6f, 7a-f, 11, 12a-d, and 20d on the
deacetylase activity of human sirtuin isotypes Sirt1–Sirt3 determined in the
fluorescence-based ZMAL activity assay.
40 / 60
5 / 95
89 / 11
79 / 21
89 / 11
92 / 8
Entry
3
Sirt1 inhibition[a]
27% @ 50 µM
n.i.
Sirt2 inhibition[a]
Sirt3 inhibition[a]
25 / 75
23 / 77
4 / 96
[b]
[b]
24.6 ± 2.8 µM
41.7 ± 2.0 µM
77
6f
n.i.
n.i.
33
80 / 20
5
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[b]
[b]
[b]
Comparable to 7f, the acetylated derivative 9a may induce an
opening of the selectivity pocket (Table S3). However, acetylation
of the amino group caused a notable loss of inhibitory potency
supporting the assumption that the amino group is involved in
polar target interactions (Table 2). Nevertheless, as Sirt2 is known
to bind and process myristoylated substrates, we anticipated a
Sirt2 selective inhibition by long-chain fatty acyl derivatives of 7f.
In fact, extension of the acyl chain (9b-g) enhanced inhibition of
Sirt2 and yielded a potent myristic acid derivative 9g. However,
with an increasing length of the fatty acyl chain, aqueous solubility
obviously became a major concern. Indeed, Sirt3 stability was
affected by 9d-g potentially due to substance precipitation during
the activity assay (Table 3).
7a
14% @ 50 µM
7.9 ± 0.6 µM
9.5 ± 0.9 µM
[b]
7b
19% @ 50 µM
10.8 ± 0.6 µM
7.9 ± 0.5 µM
7c
n.i.
55% @ 10 µM
62% 10 µM
64% @ 10 µM
39% @ 50 µM
66% @ 10 µM
7d
10% @ 10 µM
7e
17% @ 10 µM
54% @ 10 µM
[b]
[b]
7f
47% @ 100 µM
5.8 ± 0.7 µM
9.4 ± 0.7 µM
Depending on the lipophilicity, UV irradiation (365 nm) showed
varying effects on the biological activity of 9a-g. The three less
lipophilic compounds 9a-9c did not exhibit major differences in
target engagement, yet a slight decrease of affinity towards Sirt2
and Sirt3 was observable. In contrast, 9d displayed an about
fivefold increase of Sirt2 inhibition, which is most likely caused by
the improved aqueous solubility and higher abundance of the Z-
isomer in the enzyme assay mixture. Irradiation of 9g even
yielded submicromolar inhibition of Sirt2 (IC50 = 0.9 µM), yet
differential target engagement is less pronounced compared to 9d
as affinity for Sirt2 is also increased in the E-form of 9g.
11
n.i.
n.i.
n.i.,
12a
12b
12c
12d
20d
33% @ 100 µM
45% @ 100 µM
16% @ 10 µM
17% @ 10 µM
43% @ 100 µM
49% @ 10 µM
45% @ 100 µM
11% @ 10 µM
11% @ 10 µM
43% @ 100 µM
33% @ 10 µM
n.i.
n.i.
n.i.
n.i.
In order to test the influence of nonpolar, bulky substituents, we
synthesized compounds 9h-k. None of these compounds exerted
potent inhibition, neither in their non-irradiated E-form nor after
365 nm irradiation. Consequently, a certain degree of flexibility in
the hydrophobic substructure is regarded crucial for target affinity.
[a] Percent inhibition relative to controls at the indicated concentration, [b] IC50
values (μM) with statistical limits; values are mean SD of duplicate
experiments, n.i. = no inhibition detected (< 30% @ 100 µM).
±
Despite the detrimental effects of bulky hydrophobic substituents,
we found a potent inhibition of Sirt2 and Sirt3 by tert-butyl ester
15a. According to the previously mentioned low amount of Z-
isomers at the PSS after 365 nm irradiation, we found only
negligible differences in target engagement of the irradiated
probe. By insertion of a methylene bridge, we attempted to
improve the photochemical behaviour of the chromophore and at
the same time enhance flexibility of the hydrophobic acyl group.
Indeed, 15b and 15c demonstrated both a stronger inhibition and
a larger impact of irradiation compared to 15a. Boc-protected 4-
aminomethyl derivative 15c inhibited Sirt2 within the
submicromolar range (IC50 = 0.9 µM). Upon irradiation, the IC50
value is diminished three- and four-fold for Sirt2 and Sirt3,
respectively. Pharmacophore-guided docking studies indicated
binding of the voluminous tert-butyl group to the hydrophobic
acetyl-lysine channel. However, photoisomerization is not
expected to lead to substantially altered target interactions
(Figure 3).
Compounds 6f, 11 and 12a-d confirmed that the primary amide
group of the nicotinamide partial structure was essential for affinity
towards Sirt1–3, as modifications on this residue provoked a
dramatic loss of activity (Table 1). Hence, binding of compounds
7a-f was highly likely to occur in a fashion similar to nicotinamide
itself. Nicotinamide is known to bind to the enzymatic C-site,
which is part of the catalytic cleft and is contained in all sirtuin
isotypes. Thus, further interactions with specific domains were
necessary in order to improve isoenzyme selectivity.
In the case of Sirt2, a hydrophobic selectivity pocket adjacent to
the C-site presents new possibilities for specific interactions.[22]
This unique binding pocket is typically formed after binding of the
highly potent and selective sirtuin-rearranging ligands (SirReals)
by a conformational change to the so-called locked open
conformation. This conformational change is also observed upon
binding of numerous other compounds as myristoylated
substrates, for instance.[23] A recently developed fluorescence
polarization (FP)-based binding assay was utilized to determine
whether our compounds are able to prevent binding of a
fluorescently labelled SirReal to the catalytic core of Sirt2.[7]
Serendipitously, competition with the fluorescent probe could be
confirmed for 7f (Table S3). Hence, one could hypothesize that 7f
exerts a binding mode similar to that of the SirReals and possibly
is also able to introduce the same conformational change to the
locked open conformation that was observed upon SirReals
binding. Therefore, this scaffold seemed to be a promising
structure for further derivatization.
Inspired by the beneficial influence of a methylene bridge on
biological activity and photochemical properties, we synthesized
a methylene bridged derivative of 9g. In order to reduce
lipophilicity, the fatty acyl chain had to be shortened by four links.
Albeit, the resulting compound 15f exerted excellent isoenzyme
selectivity, the strength of Sirt2 inhibition was dramatically
diminished by this modification compared to 9g. However,
illumination provoked a 23-fold increase of inhibitory potency (IC50
= 1.6 µM). Besides better aqueous solubility, molecular docking
6
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10.1002/cmdc.202000148
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studies implied favourable binding of Z-15f to be responsible for
this remarkable improvement of biological activity (Figure 4).
20a showed Sirt2 inhibition with an IC50 of 0.7 µM (Table 3),
consistent with the originally published value of 0.1 µM for the
respective derivative of 2 (R = OBn). Besides, inhibition of Sirt1
and Sirt3 was almost negligible, so that 20a approximates the
excellent properties of its N-arylbenzamide counterpart. On the
contrary, inhibitory potency and isoenzyme selectivity of 20b is
significantly decreased. In fact, the IC50 of 20b regarding Sirt2
inhibition is off by a factor of 100 compared to the parent structure
(2, R = F). Also 20c and 20d did not resemble their parent
compounds. Whereas the N-arylbenzamides inhibit Sirt2 in the
nanomolar range, 20d displayed Sirt2 inhibition with an IC50 value
of about 10 µM (Table 1). In the case of 20c, unspecific
interactions were indicated by a rather linear than sigmoidal dose
response curve in the Sirt2 and Sirt3 activity assay. Since this was
not detected for irradiated probes of 20c, we assumed that
solubility issues were responsible for the observed interference.
In terms of their photochemical behaviour, 20a and 20b seemed
promising as their PSS after UV irradiation displayed high
amounts of the respective Z-isomers. Still, both compounds did
Although insertion of a methylene bridge increased target affinity,
gained flexibility at the same time reduced the overall net
structural changes provoked by photoisomerization. Insertion of a
more rigid ethinyl spacer instead was expected to enhance these
structural changes and thus increase the impact of light-
switching.[24] Furthermore, a polar hydroxyl group was integrated
in compounds 15g and 15h in order to address the excessive
lipophilicity of most compounds. However, this failed to enhance
differential target engagement: Sirt2 inhibition is diminished by a
factor of 1.6 for 15g and 2.5 for 15h. A similar influence is
indicated regarding Sirt3.
Azologization of lead 2 yielded azobenzene based photoswitches
20a-d. As in the case of lead 3, the azo group could be inserted
under full maintenance of the parent molecules geometry. Hence,
we expected 20a-d to possess comparable biological activities. In
fact, we obtained active sirtuin inhibitors also by this azologization
approach, though not in every case the azo analogues maintained
the biological activity of the respective lead structure: Compound
not exceed
illumination.
a two-fold decrease of Sirt2 inhibition after
Table 3. Influence of azo dyes 9a-k, 15a-h, and 20a-c on the deacetylase activity of human sirtuin isotypes Sirt1–Sirt3, determined by the fluorescence-based
ZMAL activity assay. Biological activity was investigated at the thermal equilibrium (∆) and the PSS after 5 min of UV irradiation (365 nm).
Entry
Sirt1 inhibition[a]
Sirt2 inhibition[a]
Sirt3 inhibition[a]
∆
PSS (365 nm)
∆
PSS (365 nm)
∆
PSS (365 nm)
9a
9b
9c
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
17% @ 10 µM
30% @ 10 µM
40% @ 10 µM
11% @ 10 µM
4% @ 10 µM
24% @ 10 µM
68% @ 100 µM
80% @ 100 µM
20% @ 10 µM
52% @ 100 µM
52% @ 100 µM
3% @ 10 µM
[b]
[b]
44.0 ± 9.5 µM
7.9 ± 0.7 µM
9d
n.i.
n.i.
u.i.
u.i.
(26% @ 10 µM)
(54% @ 10 µM)
78% @ 10 µM
65% @ 10 µM
9e
9f
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
26% @ 10 µM
46% @ 10 µM
u.i.
u.i.
n.i.
u.i.
u.i.
[b]
[b]
9g
9h
9i
n.i.
2.2 ± 0.3 µM
0.9 ± 0.2 µM
u.i.
u.i.
n.i.
27% @ 100 µM
15% @ 10 µM
80% @ 100 µM
43% @ 100 µM
52% @ 100 µM
15% @ 10 µM
59% @ 100 µM
71% @ 100 µM
n.i.
32% @ 100 µM
n.i.
n.i.
n.i.
u.i.
n.i.
u.i.
9j
9k
15a
n.i.
n.i.
n.i.
[b]
[b]
7% @ 10 µM
6.0 ± 1.1 µM
6.8 ± 0.4 µM
42% @ 10 µM
45% @ 10 µM
7
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10.1002/cmdc.202000148
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FULL PAPER
[b]
[b]
[b]
[b]
15b
15c
15d
15e
15f
35% @ 100 µM
44% @ 100 µM
6% @ 10 µM
47% @ 100 µM
10% @ 10 µM
n.i.
3.9 ± 0.2 µM
6.9 ± 0.6 µM
2.8 ± 0.1 µM
8.4 ± 0.9 µM
58% @ 10 µM
30% @ 10 µM
[b]
[b]
[b]
n.i.
0.88 ± 0.06 µM
1.1 ± 0.2 µM
4.0 ± 0.7 µM
[b]
[b]
[b]
n.i.
7.5 ± 0.5 µM
8.2 ± 1.4 µM
13.0 ± 2.2 µM
n.i.
51% @ 10 µM
61% @ 10 µM
54% @ 10 µM
n.i.
49% @ 10 µM
n.i.
[b]
[b]
n.i.
36.6 ± 9.3 µM
1.6 ± 0.2 µM
[b]
[b]
15g
15h
20a
20b
20c
60% @ 100 µM
54% @ 100 µM
n.i.
7.2 ± 0.5 µM
11.8 ± 1.0 µM
64% @ 10 µM
42% @ 10 µM
23% @ 10 µM
34% @ 10 µM
u.i.
22% @ 10 µM
18% @ 10 µM
24% @ 10 µM
47% @ 10 µM
[b]
[b]
n.i.
15.2 ± 1.8 µM
37.5 ± 5.2 µM
[b]
[b]
n.i.
n.i.
n.i.
0.70 ± 0.21 µM
1.6 ± 0.2 µM
6.4 ± 0.5 µM
6.2 ± 0.54 µM
[b]
[b]
n.i.
3.2 ± 0.5 µM
[b]
[b]
45% @ 100 µM
62% @ 100 µM
u.i.
17.2 ± 1.57 µM
[a] Percent inhibition relative to controls at the indicated concentration, [b] IC50 values (μM) with statistical limits; values are mean ± SD of duplicate experiments.
n.i. = no inhibition detected (< 30% @ 100 µM), u.i. = unspecific interactions.
Figure 3. Binding pose for compound 15c in E- (left) and Z-configuration (right) from pharmacophore-guided docking. Since compound 15c is not selective for Sirt2
and modification of the primary amide group leads to complete loss of activity, binding of the nicotinamide moiety to the residues I169 and D170 like NAD+ is
expected (C-site). For E-15c, the tert-butyl group points towards the hydrophobic acetyl-lysine channel while exposing the polar carbamate towards the solvated
entrance region. After photoisomerization, the phenyl-group shifts to the more hydrophobic binding site, leading to unfavourable interactions for the carbamate,
which might explain a decrease in activity. However, binding of the nicotinamide moiety is not expected to be affected. Conformation of Z-15c was validated by
analyses of potential energies in relation the CCNN-dihedral angles (Figure S7). The pharmacophore region for an amide group is shown as green sphere.
8
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10.1002/cmdc.202000148
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FULL PAPER
Figure 4. Binding pose for compound 15f in E- (left) and Z-configuration (right) from molecular docking. The position of the decanoyl chain is in good agreement
with the crystalized myristoylated lysine sidechain (green) which opens the selectivity pocket (PDB: 4Y6L). Through photoisomerization, hydrophilicity is increased
and the polar Z-azo group is accessible for hydrogen bond interactions with water molecules, while hiding the pyridine ring in a more hydrophobic pocket and
conceiving two strong hydrogen bonds to the charged sidechains of R97 and E116.
product was related to total target protein and to the vehicle
control. Again, we found an increase in acetylated histones
Cell-Based Activity Assay
induced by 15c and 15f which proves activity of these two
compounds in living cells.
Bioactivity towards a human urinary cancer cell line (RT-4) was
studied for the three most active inhibitors 15c, 15f (after UV
illumination) and 20a. RT-4 cells showed the highest expression
of Sirt2 in our cellular inventory and therefore have been chosen
for the cell-based activity determination. In order to prove a
general effect of the compounds inside the cells, we conducted
preliminary studies solely for irradiated probes, since the enzyme-
based activity assay indicated either comparable activities of both
photoisomers (15c, 20a), or a higher activity of the Z-isomer.
Reductive stability of 15c, 15f and 20a in the presence of the
cellular reductant glutathione (5 mM) was confirmed by HPLC
analysis (Figure S5, Table S2). For the cell-based activity assay,
we illuminated the compounds solved in DMSO with UV light
(365 nm) prior to incubation with the cells. The compiled western
blots displayed that all three compounds (20a, 15c, 15f) had no
effect on the expression of total histone H3 and total α-tubulin
protein but in contrast, a slight increase of total H4 was detectable
(Figure 5). As expected, 15c and 15f increased the levels of
acetylated lysines H3K18 and H3K56, which are both affirmed
substrates of Sirt2. In addition, we found H4K8 hyperacetylation
even though this site is usually not affected by sirtuin catalysed
deacetylation, indicating a further mode of action. In contrast,
none of the compounds influenced the acetylation level of α-
tubulin, which is frequently consulted as it is specifically
processed by Sirt2. However, also in a cell-based activity assay
of the highly potent Sirt2 inhibitor SirReal2 the degree of α-tubulin
hyperacetylation was less pronounced when examined by
western blot technique. Instead, applying an immunofluorescence
assay yielded clearer results.[22] Surprisingly, compound 20a did
not exert any activity regarding the examined substrates. In
Figure 6, we show the relative results, whereby the acetylation
Figure 5. Representative western blots for total and acetylated subunits of
histones and α-Tubulin. Compounds were irradiated by UV light (365 nm) prior
to incubation with cells.
9
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10.1002/cmdc.202000148
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FULL PAPER
in the classical sense of photoswitchable drugs. This aspect of
azo-based photoswitches has been studied recently and was
exploited in the use of photochromic azo-combrestatin A-4
analogues.[26] In the latter case, photoswitching to the bioactive Z-
isomers provoked an up to 550-fold increase of bioactivity.
Interestingly, in the same work the authors observed a longer Z-
isomer half-life of less soluble compounds in watery environment,
potentially contributing to the remarkable impact of
photoisomerization.
Even though photoisomerization of 15f and other lipophilic
compounds was hampered by chromophore aggregation in
aqueous environment, solubility of these compounds should be
enhanced in physiological media as blood, mediated by binding
to albumin proteins. In this case, switching of an albumin-bound
compound to the Z-isomer could provoke its liberation in this
specific area, thereby enabling tissue specific pharmacotherapy.
Experimental Section
Figure 6. Relative acetylated protein target after incubation with compounds at
50 µM for 24 h in urinary bladder cancer cell line RT-4 [mean of n=2].
General procedure for azo coupling of phenols: Methyl 5-
aminonicotinate (1.0 equiv.) was suspended in aqueous HCl (6 M,
3.0 equiv.) and cooled to 0 °C. An aqueous solution of NaNO2 (2.5 M,
1.0 equiv.) was added dropwise, so that the temperature did not exceed
5 °C. The resulting yellow solution was added slowly under stirring to the
appropriate phenol (1.1 equiv.) dissolved in aqueous NaOH (2 M). If
necessary, aqueous NaOH (2 M) was added to keep the resulting solution
alkaline. After complete addition, the reaction mixture was acidified with
aqueous HCl (6 M) and extracted with EtOAc. The combined organic
extracts were washed with brine, dried over MgSO4, filtrated and freed
from solvent. The crude product was purified by silica gel column
chromatography.
Conclusions
In this work, azologization of two different lead structures was
performed in order to obtain azobenzene and azopyridine-based
photoswitchable sirtuin inhibitors. Despite the high potential of
diarylmaleimides and indolylfulgides as photoswitchable sirtuin
inhibitors, we anticipated azo dyes to be more convenient, since
this class of molecular photoswitches is known to possess reliable
and highly tunable photophysical properties also under
physiological conditions. Indeed, azologization proved
successful, since we obtained biologically active compounds,
whose configuration could be reversibly toggled between E- and
Z-form by UV (365 nm) or blue (452 nm) light irradiation. However,
differential target engagement of the respective photoisomers
was not as profound as anticipated. Whereas several
photoswitchable drugs show a 10–50-fold difference in activity, in
our case differential inhibition did not exceed a factor of 3–4 for
most compounds.[25] Our docking studies implied that, despite the
considerable structural change in the azo core, flexibility of the
overall ligand structure could be responsible for the minor
differences in bioactivity of the photoisomers. Furthermore,
photoisomerization did not result in clearly unfavorable
interactions between the ligand and residues of the sirtuins active
site. Consequently, further rigidization and the introduction of
sterically demanding groups could potentially be favourable in this
regard. In contrast, pronounced effects were observed by
photoswitching of highly lipophilic derivatives: Due to poor
aqueous solubility, 15f exerted only moderate, yet selective Sirt2
inhibition (IC50 = 37 µM) in its thermally stable E-form. Since
polarity is significantly enhanced by photoisomerization, biological
activity of 15f could be raised by a factor of 23 (IC50 = 1.6 µM) for
Sirt2 inhibition. Additionally, activity of Z-15f was confirmed by
increased acetylation levels of Sirt2 specific histones. Obviously,
this effect is caused rather by a light-mediated improvement of the
aqueous solubility than by altered binding affinity, as anticipated
General procedure for azo coupling of anilines: Diazotization of methyl
5-aminonicotinate was carried out as described above. The yellow solution
containing the diazonium salt was added slowly under stirring to the
appropriate aniline (1.1 equiv.) dissolved in HCl (1 M). After complete
addition, the reaction mixture was stirred for 10 minutes, then basified
using a saturated aqueous solution of Na2CO3 and extracted with EtOAc.
The combined organic extracts were washed with brine, dried over MgSO4,
filtered and freed from solvent. The crude product was purified by silica gel
column chromatography.
General procedure for synthesis of nicotinamides from methyl
nicotinates: The respective methyl nicotinate was treated with a saturated
solution of ammonia in anhydrous MeOH (30 mL) and stirred in a sealed
vessel at 40 °C until thin layer chromatography indicated complete
conversion of the starting material (three to four days). The solvent was
evaporated under reduced pressure and the residue washed sparingly
with cold acetonitrile.
Photochemistry: All photoisomerization experiments were conducted
under ruby light of 630 nm and total exclusion of daylight. Illumination was
executed using a Bio-Link 254 Crosslinker from Vilber-Lourmat equipped
with six Vilber-Lourmat T8-L lamps (8W, 365 nm). Visible light radiation of
630 nm (ruby) and 452 nm (blue) was derived from a Paulmann FlexLED
3D strip. All compounds were irradiated in solution using
spectrophotometric grade solvents. Photoisomerization and UV/Vis-
spectra measurement was conducted in quartz glass cuvettes (114-QS,
Hellma Analytics) at room temperature.
Cloning, expression and purification of recombinant proteins:
Expression and purification of Sirt1133-747, Sirt256−356, and Sirt3118−395 was
carried out as described previously.[27] Identity and purity were verified by
10
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10.1002/cmdc.202000148
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FULL PAPER
SDS-PAGE. Protein concentration was determined by the Bradford
assay.[28] Deacylase activity of sirtuin isotypes could be inhibited with
nicotinamide and was shown to be NAD+-dependent.
[3]
[4]
[5]
Y.-B. Teng, H. Jing, P. Aramsangtienchai, B. He, S. Khan, J. Hu, H. Lin,
Q. Hao, Sci. Rep. 2015, 5, 8529.
K. J. Bitterman, R. M. Anderson, H. Y. Cohen, M. Latorre-Esteves, D. A.
Sinclair, J. Biol. Chem. 2002, 277, 45099–45107.
Fluorescence-based activity assay: The inhibitory effect of compounds
on Sirt1–3 was detected via a previously reported fluorescence-based
assay.[29] The synthetic substrate Z-Lys(acetyl)-AMC (ZMAL) is
deacetylated by sirtuins, followed by tryptic digestion and thereby release
of 7-aminomethylcumarin, leading to a fluorescent readout. Inhibition was
determined by comparing percentage substrate conversion to a DMSO
control after subtraction of the blank fluorescence signal. All compounds
were tested at 100 µM or 50 µM and 10 µM respectively. For compounds
that showed more than 70% inhibition at 10 µM an IC50 value was
determined. IC50 values were calculated with OriginPro 9.0 G using a non-
linear regression to fit the dose response curve (Figure S6). An enzyme-
free blank control and a 100% conversion control using AMC instead of
ZMAL were measured as well. Inhibition measurements were performed
in biological duplicates for all compounds.
a) AOP Orphan Pharmaceuticals AG, to be found under
https://www.aoporphan.com/global_en/our-company/newsroom/aop-
orphan-pharmaceuticals-ag-to-acquire-selisistat-a-clinical-stage-drug-
candidate-for-the-treatment-of-huntingtons-disease-hd, 2017; b) S. D.
Süssmuth et al., Br. J. Clin. Pharmacol. 2015, 79, 465–476.
[6]
[7]
[8]
[9]
T. Ai, D. J. Wilson, S. S. More, J. Xie, L. Chen, J. Med. Chem. 2016, 59,
2928–2941.
S. Swyter, M. Schiedel, D. Monaldi, S. Szunyogh, A. Lehotzky, T. Rumpf,
J. Ovádi, W. Sippl, M. Jung, Philos. Trans. R. Soc. B 2018, 373.
C. W. Grathwol, N. Wössner, S. Swyter, R. K. Hofstetter, A. Bodtke, M.
Jung, A. Link, Beilstein J. Org. Chem. 2019, 15, 2170–2183.
Molecular Modeling: All calculations were performed using the Molecular
Operating Environment (MOE) software suite (version 2019.01).[30] If not
explicitly stated otherwise, default settings and parameters were used.
C. W. Grathwol, N. Chrysochos, B. J. Elvers, A. Link, C. Schulzke, Acta
Crystallogr. Sect. E: Crystallogr. Commun. 2019, 75, 1828–1832.
[10] a) P. Leippe, J. A. Frank, Curr. Opin. Struct. Biol. 2019, 57, 23–30; b) P.
Paoletti, G. C. R. Ellis-Davies, A. Mourot, Nat. Rev. Neurosci. 2019, 20,
514–532; c) H. Cheng, J. Yoon, H. Tian, Coord. Chem. Rev. 2018, 372,
66–84; d) K. Hüll, J. Morstein, D. Trauner, Chem. Rev. 2018, 118,
10710–10747; e) I. Tochitsky, M. A. Kienzler, E. Isacoff, R. H. Kramer,
Chem. Rev. 2018, 118, 10748–10773; f) M. Zhu, H. Zhou, Org. Biomol.
Chem. 2018, 16, 8434–8445; g) M. M. Lerch, M. J. Hansen, G. M. van
Dam, W. Szymanski, B. L. Feringa, Angew. Chem. Int. Ed. 2016, 55,
10978–10999; Angew. Chem. 2016, 128, 11140–11163.
General preparation: For molecular docking of ligands to human Sirt2, a
crystal structure with open-state selectivity pocket and bound ligand was
used (PDB: 5MAT, 4RMG). Both systems were prepared by applying
AMBER14 force field parameters, adding hydrogen atoms and protonation
with Protonate3D (pH 7.4). Missing flexible protein loops were rebuilt,
followed by a restraint minimization of the protein-ligand complex.
Molecular docking: Ligands used for docking experiments were prepared
by generating three-dimensional structures from SMILES, taking into
account possible protonation states and both E/Z-isomers for azo groups.
AM1-BCC charges were applied. The binding site was defined as all
residues within 4.5 Å of the bound ligand in the crystal structure. Docking
was performed using a two-stage protocol with placement of 30 poses by
Triangle Matcher and London dG scoring, followed by an Induced Fit
refinement and more accurate MM/GBVI scoring. For each ligand, a total
of 10 final poses were obtained and visually inspected. For
pharmacophore-guided molecular docking, a sphere with 1.7 Å was
placed on the amide group of NAD+ (PDB: 4RMG), which was encoded by
the SMARTS expression “[#7X3H2][#6X3](=[#8X1])[#6]”.
[11] a) J. Broichhagen, J. A. Frank, D. Trauner, Acc. Chem. Res. 2015, 48,
1947–1960; b) W. Szymański, J. M. Beierle, H. A. V. Kistemaker, W. A.
Velema, B. L. Feringa, Chem. Rev. 2013, 113, 6114–6178.
[12] W. A. Velema, W. Szymanski, B. L. Feringa, J. Am. Chem. Soc. 2014,
136, 2178–2191.
[13] C. Falenczyk, M. Schiedel, B. Karaman, T. Rumpf, N. Kuzmanovic, M.
Grøtli, W. Sippl, M. Jung, B. König, Chem. Sci. 2014, 5, 4794–4799.
[14] N. A. Simeth, L.-M. Altmann, N. Wössner, E. Bauer, M. Jung, B. König,
J. Org. Chem. 2018, 83, 7919–7927.
[15] a) W. Szymanski, M. E. Ourailidou, W. A. Velema, F. J. Dekker, B. L.
Feringa, Chemistry 2015, 21, 16517–16524; b) S. A. Reis et al., Nat.
Chem. Biol. 2016, 12, 317–323; c) C. E. Weston, A. Krämer, F. Colin, Ö.
Yildiz, M. G. J. Baud, F.-J. Meyer-Almes, M. J. Fuchter, ACS Infect. Dis.
2017, 3, 152–161.
Acknowledgements
The Jung group thanks the Deutsche Forschungsgemeinschaft
(DFG, JU295/14-1 and 235777276/GRK1976) for funding, O.
Einsle acknowledges support by grant DFG CRC 922.
[16] H. M. D. Bandara, S. C. Burdette, Chem. Soc. Rev. 2012, 41, 1809–
1825.
Keywords: Azo dyes • Epigenetics • Photopharmacology •
[17] a) S. Crespi, N. A. Simeth, B. König, Nat. Rev. Chem. 2019, 3, 133–146;
b) D. Cameron, S. Eisler, J. Phys. Org. Chem. 2018, 31, e3858; c) M.
Dong, A. Babalhavaeji, S. Samanta, A. A. Beharry, G. A. Woolley, Acc.
Chem. Res. 2015, 48, 2662–2670; d) N. A. Simeth, A. Bellisario, S.
Crespi, M. Fagnoni, B. König, J. Org. Chem. 2019; e) J. Calbo, C. E.
Weston, A. J. P. White, H. S. Rzepa, J. Contreras-García, M. J. Fuchter,
J. Am. Chem. Soc. 2017, 139, 1261–1274.
Photoswitches • Sirtuins
References:
[1]
[2]
a) J. N. Feige, J. Auwerx, Curr. Opin. Cell Biol. 2008, 20, 303–309; b) M.
C. Haigis, L. P. Guarente, Genes Dev. 2006, 20, 2913–2921; c) E.
Verdin, M. D. Hirschey, L. W. S. Finley, M. C. Haigis, Trends Biochem.
Sci. 2010, 35, 669–675;
[18] H. Cui, Z. Kamal, T. Ai, Y. Xu, S. S. More, D. J. Wilson, L. Chen, J. Med.
Chem. 2014, 57, 8340–8357.
a) R. A. Frye, Biochem. Biophys. Res. Commun. 2000, 273, 793–798; b)
S. Greiss, A. Gartner, Mol. Cells 2009, 28, 407–415; c) M. Schiedel, D.
Robaa, T. Rumpf, W. Sippl, M. Jung, Med. Res. Rev. 2018, 38, 147–200;
[19] a) A. A. Blevins, G. J. Blanchard, J. Phys. Chem. B 2004, 108, 4962–
4968; b) W. R. Brode, J. H. Gould, G. M. Wyman, J. Am. Chem. Soc.
1952, 74, 4641–4646.
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000148
ChemMedChem
FULL PAPER
[20] a) M. Shimomura, R. Ando, T. Kunitake, Ber. Bunsen-Ges. 1983, 87,
1134–1143; b) J. M. Kuiper, J. B. F. N. Engberts, Langmuir 2004, 20,
1152–1160; c) L. H. Urner, B. Schade, M. Schulze, K. Folmert, R. Haag,
K. Pagel, ChemPhysChem 2019, 20, 1690–1697.
[21] N. A. Simeth, S. Crespi, M. Fagnoni, B. König, J. Am. Chem. Soc. 2018,
140, 2940–2946.
[22] T. Rumpf et al., Nat. Commun. 2015, 6, 6263.
[23] D. Robaa, D. Monaldi, N. Wössner, N. Kudo, T. Rumpf, M. Schiedel, M.
Yoshida, M. Jung, Chem. Rec. 2018, 18, 1701–1707.
[24] A. A. Beharry, G. A. Woolley, Chem. Soc. Rev. 2011, 40, 4422–4437.
[25] M. W. H. Hoorens, W. Szymanski, Trends Biochem. Sci. 2018, 43, 567–
575.
[26] a) S. K. Rastogi et al., Eur. J. Med. Chem. 2018, 143, 1–7; b) M.
Ishikawa, T. Ohzono, T. Yamaguchi, Y. Norikane, Sci. Rep. 2017, 7,
6909.
[27] M. Schiedel, D. Herp, S. Hammelmann, S. Swyter, A. Lehotzky, D.
Robaa, J. Oláh, J. Ovádi, W. Sippl, M. Jung, J. Med. Chem. 2018, 61,
482–491.
[28] U. K. Laemmli, Nature 1970, 227, 680–685.
[29] B. Heltweg, J. Trapp, M. Jung, Methods 2005, 36, 332–337.
[30] Molecular Operating Environment (MOE), 2019.01; Chemical Computing
Group ULC, 1010 Sherbrooke St. West, Suite #910, Montreal, QC, H3A
2R7, 2019.
12
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Entry for the Table of Contents
Switched into action! Besides fine-tuning of molecular geometry for enzyme inhibition, photoinduced isomerization of azopyridine-
based nicotinamide mimics results in improved aqueous solubility, enforcing the effect on target protein sirtuin 2. The resulting Sirt2
inhibitor could be used to study binding events in cellular systems with spatiotemporal control by two independent molecular and
supramolecular effects.
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