Structure-based development of an affinity probe for sirtuin 2

Article abstract of DOI:10.1002/anie.201509843

Sirtuins are NAD⁺-dependent protein deacylases that cleave off acetyl groups, as well as other acyl groups, from the é?-amino group of lysines in histones and other substrate proteins. Dysregulation of human Sirt2 activity has been associated w

Full text of DOI:10.1002/anie.201509843

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International Edition: DOI: 10.1002/anie.201509843
German Edition: DOI: 10.1002/ange.201509843
Enzyme Ligands
Structure-Based Development of an Affinity Probe for Sirtuin 2
Matthias Schiedel, Tobias Rumpf, Berin Karaman, Attila Lehotzky, Stefan Gerhardt,
Judit Ovµdi, Wolfgang Sippl, Oliver Einsle, and Manfred Jung*
Abstract: Sirtuins are NAD+-dependent protein deacylases
that cleave off acetyl groups, as well as other acyl groups, from
the e-amino group of lysines in histones and other substrate
proteins. Dysregulation of human Sirt2 activity has been
associated with the pathogenesis of cancer, inflammation, and
neurodegeneration, thus making Sirt2 a promising target for
pharmaceutical intervention. Here, based on a crystal structure
of Sirt2 in complex with an optimized sirtuin rearranging
ligand (SirReal) that shows improved potency, water solubility,
and cellular efficacy, we present the development of the first
Sirt2-selective affinity probe. A slow dissociation of the probe/
enzyme complex offers new applications for SirReals, such as
biophysical characterization, fragment-based screening, and
affinity pull-down assays. This possibility makes the SirReal
probe an important tool for studying sirtuin biology.
sirtuin 2 (Sirt2) is both a nuclear and a cytoplasmic protein.
By deacylating its substrate proteins, Sirt2 acts as a major
regulator of the cell cycle,[4] autophagy,[10] and peripheral
myelination,[11] as well as being a suppressor of inflammation
in the brain.[12] The involvement of Sirt2 in tumorigenesis is an
ongoing controversial debate. Generally considered as
a tumor suppressor in some types of cancer,[13] Sirt2 was
also shown to exert the opposite role of a promoter of tumor
growth.[14] Furthermore, Sirt2-mediated deacetylation of
H3K18Ac was reported to play a critical role during bacterial
infection by reprogramming the transcription of host cells.[15]
Bearing in mind the variety of Sirt2 substrates and the
downstream signaling effects, it is apparent that Sirt2
inhibitors are urgently needed to further validate the
potential of Sirt2 as a drug target to fight cancer, inflamma-
tion, and neurodegenerative diseases. Although a number of
highly potent Sirt2 inhibitors have been discovered recently
(see Figure S1 in the Supporting Information),[16] to date there
is no targeted inhibitor of Sirt2 that has reached clinical trials.
Potent, isotype-selective, and druglike modulators of Sirt2
activity with proven cellular target engagement are still
scarce. Recently, we discovered a novel class of potent and
highly selective Sirt2 inhibitors, the sirtuin rearranging ligands
(SirReals; Figure 1).[17]
N
AD+-dependent protein deacylases, also termed sirtuins,
were initially described as class III histone deacetylases
(HDACs).[1] However, a multitude of non-histone substrates
such as p53,[2] NFkB,[3] a-tubulin,[4] and BubR1[5] have been
discovered in recent years. Besides Ne-deacetylation of lysine
residues, sirtuins have been shown to catalyze further post-
translational modifications, including demyristoylation,[6]
desuccinylation,[7] and ADP-ribosylation.[8] By interacting
with their substrate proteins, sirtuins have been implicated
as influencing a wide range of cellular processes such as
apoptosis, regulation of mitosis, inflammation, ageing, and
metabolic sensing. The human genome encodes seven differ-
ent sirtuin isotypes, which differ in their subcellular local-
ization and their catalytic activity.[9] The human isotype
[*] M. Schiedel, Dr. T. Rumpf, Prof. Dr. M. Jung
Institut für Pharmazeutische Wissenschaften
Albert-Ludwigs-Universität Freiburg
Figure 1. Chemical structures of Sirt2 inhibitors SirReal1 (1) and
SirReal2 (2).
Albertstrasse 25, 79104 Freiburg im Breisgau (Germany)
E-mail: manfred.jung@pharmazie.uni-freiburg.de
Upon binding, these inhibitors induce a major rearrange-
ment of the Sirt2 active site and open up a so far unexploited
binding pocket. This unique rearrangement is the basis for the
high potency and, particularly, the excellent isotype selectivity
of the SirReals. This pocket was, therefore, termed the
“selectivity pocket”. SirReal2 (2), with an IC50 value of 0.4 mm
and remarkable isotype selectivity, is the most promising
agent among the first generation SirReals.[17] For further
characterization of this inhibitor class and to transfer the
unique features of SirReals to new applications, and allow us
to gain deeper insights into sirtuin biology, we decided to
design an affinity probe for Sirt2. This process was guided by
the structural insights obtained from Sirt2–SirReal com-
plexes. To access the Sirt2-bound SirReal without losing the
affinity and selectivity of the ligand, we aimed to exploit the
Dr. S. Gerhardt, Prof. Dr. O. Einsle
Institute für Biochemie und BIOSS Centre for Biological
Signaling Studies
Albert-Ludwigs-Universität Freiburg
Albertstrasse 21, 79104 Freiburg im Breisgau (Germany)
B. Karaman, Prof. Dr. W. Sippl
Institute für Pharmazie
Martin-Luther-Universität Halle-Wittenberg
Wolfgang-Langenbeck-Strasse 4, 06120 Halle, Saale (Germany)
Dr. A. Lehotzky, Prof. Dr. J. Ovµdi
Institute of Enzymology, Research Centre for Natural Sciences
Hungarian Academy of Sciences
Magyar Tudósok kçrffltja 2, H-1117 Budapest (Hungary)
Supporting information and ORCID(s) from the author(s) for this
article are available on the WWW under http://dx.doi.org/10.1002/
anie.201509843.
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Table 1: IC50 values of the second generation SirReals (9, 10), SirReal
probe (11), as well as SirReal1 and SirReal2 (1, 2) as references.
acyllysine substrate channel leading from the active site of
Sirt2 to the protein surface. As the methylaryl moieties of
SirReals extend towards the acyllysine channel,[17] we decided
to connect a PEG linker carrying the biotin label to this site of
the inhibitor. This would address the lysine tunnel and allow
the inhibitor to be coupled to an affinity matrix without
disturbing the inhibition. Guided by molecular docking,
which revealed that larger substituents at the naphthyl
moiety of the more potent SirReal2 (2) would lead to
a steric clash, we selected the benzyl derivative SirReal1 (1) as
the ideal starting point for the probe design (Figure S2).
To validate our probe design, two new SirReals (9, 10)
(Scheme 1) were prepared by adapting our previously de-
scribed synthesis of SirReal1 (1)[17] for available Sirt2–SirReal
structures. The first step was the propargylation of 3-amino-
phenol (3) followed by a Meerwein reaction to generate
compound 5.[18] Condensation of the resulting a-chloropro-
panal 5 with thiourea furnished the aminothiazole scaffold
6.[18b] The aminothiazole was then chloroacetylated to obtain
compound 7, and a subsequent nucleophilic substitution with
dimethylmercaptopyrimidine generated compound 8.[19]
Finally, compound 8 was functionalized through a Cu-cata-
lyzed Huisgen cycloaddition to yield 9, 10, and, eventually, the
SirReal probe (11).[20]
Compound
Sirt1 IC50 [mm]
Sirt2 IC50 [mm]
Sirt3 IC50 [mm]
1
2
9
10
11
>100
>100
>100
>100
>100
3.745Æ0.831
0.435Æ0.083
0.163Æ0.014
0.118Æ0.006
0.305Æ0.111
>100
>100
>100
>100
>100
Besides an improved potency, these second generation
SirReals have enhanced water solubility, while retaining the
unique isotype selectivity of the first generation SirReals
(Table 1). We were able to obtain a crystal structure of Sirt2 in
complex with 9, which nicely confirmed the proposed binding
mode (PDB-ID 5DY5). Triazole 9 binds to Sirt2 in a similar
fashion as the previously reported SirReal1 and SirReal2.[17]
The benzyloxy moiety is slightly twisted relative to the
conformation of the benzyl moiety of SirReal1 in complex
with Sirt2. Indeed, the triazole, as well as the terminal benzyl
group, further extend into the acyllysine binding site and
block the substrate binding site more effectively than
SirReal1 and SirReal2. Additionally, the triazole forms
hydrogen bonds with Arg97 of the cofactor binding loop
(Figure 2 and Figure S3). We could also see that the N sub-
stituent of the triazole moiety points towards the entry of the
acyllysine channel, so we could continue to pursue the
synthesis of the probe in the proposed manner.
To prove that the extended bind-
A homogeneous fluorescence-based Sirt2 activity assay[21]
led to the triazoles 9 and 10 being characterized as new Sirt2
inhibitors (Table 1) that are more than 20 times more potent
than the parent inhibitor SirReal1 (1).
ing interaction with the acyllysine
channel and its more efficient block-
age is also relevant under physio-
logical conditions, we assessed the
cellular activity of 9 and 10 by using
immunofluorescence microscopy.
Enhanced tubulin hyperacetylation
was found compared to the prior
lead structure SirReal2 (2; Fig-
ure S4), which is probably a result
of both increased potency and aque-
ous solubility. Thus, we performed
the synthesis of the SirReal probe
(11) by using a pegylated biotin with
a
terminal
azide
function
(Scheme 1). We determined an
IC50 value of 0.3 mm for our probe
by using a homogeneous fluores-
cence-based activity assay.[21] For
further biophysical characterization
we used biolayer interferometry,
which enabled us to trace the inter-
action between the surface-bound
SirReal probe and Sirt2 as a shift in
the interference pattern of the inci-
dent light. We were able to show
a dose-dependent binding of Sirt2 to
the immobilized SirReal probe and
Scheme 1. Synthesis of 9, 10, and the SirReal probe (11). Reagents and conditions: a) propargyl
bromide, NaOH, acetonitrile, 208C, 12 h, 75% yield; b) NaNO2, HCl, water, 08C, 20 min; then
acrolein, CuCl2·2H2O, acetone, 208C, 3 h; c) thiourea, ethanol, reflux, 2 h, 18% yield over two steps;
d) chloroacetyl chloride, DIPEA, acetonitrile, 208C, 2 h, 99% yield; e) 4,6-dimethyl-2-methylsulfanyl-
pyrimidine, Na2CO3, KI, DMSO, 208C, 2 h, 76% yield; f) benzyl azide, sodium ascorbate, CuSO4,
TBTA, water/tBuOH/DMF (1:1:1), 208C, 12 h, 72% yield; g) 1-azido-2-methoxyethane, sodium
ascorbate, CuSO4, TBTA, water/tBuOH/DMF (1:1:1), 208C, 12 h, 50% yield; h) azido/PEG11/biotin
conjugate, sodium ascorbate, CuSO4, TBTA, water/tBuOH/DMF (1:1:1), 208C, 12 h, 37% yield.
DIPEA=N,N-diisopropylethylamine, TBTA=tris(benzyltriazolylmethyl)amine.
determined rate constants of kon
=
6.89 Æ 0.22 ” 103 mÀ1 sÀ1 and koff
=
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Figure 2. Crystal structure of the Sirt2–9 complex. 9 is shown in yellow
sticks, the interacting residues are represented as light blue sticks.
Hydrogen bonds are shown as dashed gray lines and water molecules
are shown as blue spheres.
6.99 Æ 0.31 ” 10-4 sÀ1, which result in a KD value of 0.1 mm
(Figure 3a). The linearity of the binding (r2 = 0.9774) is
indicated in Figure S5. The previously mentioned “selectivity
pocket” is formed by two loops of the hinge region, which
connects the Rossmann fold domain with the zinc-binding
domain. During catalysis, Sirt2 undergoes a conformational
change, which brings the domains into proximity. The binding
of SirReals to the Sirt2 active site blocks this process by
locking Sirt2 in an open conformation.[17] Our kinetic
measurements, which reveal very slow dissociation kinetics,
show that Sirt2 traps the bound ligand in its active site as
a consequence of its conformational adaption upon binding
the ligand. This induces a long residence time of the ligand
and causes slow dissociation (see Figure 3a).[22] In contrast to
a previously described sirtuin affinity probe,[23] our SirReal
probe shows excellent isotype selectivity (Figure 3b). To
validate the proposed binding mode of the probe, we
preincubated Sirt2 with different potential competitors
before exposing it to the immobilized SirReal probe. We
observed competition of the SirReal probe with NAD+, an
oligopeptide containing unlabeled acetyllysine, ADPR, and
SirReal2 (Figure 3c). These results are in line with the
kinetics of SirReal1-mediated inhibition.[17] As a negative
control, we used nicotinamide, the physiological inhibitor of
sirtuin activity, which exhibited no competition with the
SirReal probe. This finding is supported by our previous
structural studies that revealed that Sirt2 can bind SirReals
and nicotinamide simultaneously. To demonstrate the suit-
ability of our SirReal probe for further applications, we
performed proof-of-concept studies for fragment-based drug
design (FBDD) and affinity pull-down experiments. As
a result of the low affinity of fragments to target enzymes,
fragment screening is routinely performed at very high
concentrations, thus limiting the application of biochemical
activity assays based on spectroscopic methods, for example,
fluorescence and UV/Vis spectroscopy.[24] Biophysical meth-
ods such as biolayer interferometry are less prone to assay
interferences and are, therefore, ideal for fragment screen-
ing.[25] To validate the suitability of the SirReal probe in
combination with biolayer interferometry for fragment
Figure 3. Biophysical characterization of the SirReal probe (11) by
using biolayer interferometry. a) Representative biolayer interferometry
sensorgrams showing different concentrations of Sirt2 binding to
immobilized 11. b) Representative biolayer interferometry sensorgrams
(only the association phase is shown) of different sirtuin isotypes
(1.7 mm) binding to immobilized 11. c) Representative biolayer inter-
ferometry sensorgrams (only the association phase is shown) of Sirt2
(110.5 mgmLÀ1) binding to immobilized 11 after preincubation with
potential competitors. d) Shift [nm] detected at the end of the
association phase after preincubation of Sirt2 (110.5 mgmLÀ1) with the
given ligands and ligand fragments (5 mm, see Figure S6 for struc-
tures). NA=nicotinamide, 4,6-DM-2-MSP=4,6-dimethyl-2-methylsulfa-
nylpyrimidine, 2-MSP=2-methylsulfanylpyrimidine, 2-(4,6-DMP)-
SAA=2-(4,6-dimethylpyrimidin-2-yl)sulfanylacetamide, MTAA =N-(5-
methylthiazol-2-yl)acetamide, 1-NAA=1-naphthylacetic acid, PTU=
propylthiouracil, ADPR=adenosine diphosphate ribose, AMP=adeno-
sine monophosphate, statistics (t-test): * pꢀ0.05; ** pꢀ0.005;
*** pꢀ0.0001, all compared pairwise to a vehicle (DMSO) control.
screening, we tested fragments of SirReal2 and ADPR to
assess their potency to prevent binding of Sirt2 to the
immobilized SirReal probe (Figure 3d and Figure S6). As
expected, fragments of SirReal2, as well as AMP as a fragment
of ADPR, significantly prevented Sirt2 from binding to the
probe. We confirmed that methylation of the pyrimidine
moiety in positions 4 and 6 is crucial for binding to Sirt2,[17]
since the dimethylated fragment (4,6-DM-2-MSP) provokes
a significant decrease in Sirt2 binding compared to the
fragment without methyl groups (2-MSP). No effect was
observed for the negative control of nicotinamide (NA) and
propylthiouracil (PTU), a pyrimidine structurally different
from 4,6-DM-2-MSP. Furthermore, we wanted to investigate
the suitability of our SirReal probe for affinity purification
and pull-down experiments. By using our SirReal probe, we
were able to extract Sirt2 selectively out of various complex
matrices, even in the presence of the similar Sirt1 or the
promiscuous ligand binding protein BSA (bovine serum
albumin). We demonstrated this by using a model protein
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Acknowledgements
We thank the Deutsche Forschungsgemeinschaft (DFG) for
funding (Ju295/8-1, Si868/6-1, SFB992: A04 & Z02), K.
Schmidtkunz for assistance with cell culture experiments,
and E. Jung for the preparation of the graphic for the Table of
Contents. J.O. was supported by the Hungarian National
Scientific Research Fund Grants OTKA T-101039 and T-
112144.
Figure 4. The SirReal probe (11) captures Sirt2 out of HL60 lysates.
a) Western blot analysis of the SirReal probe bound to Dynabeads
MyOne Streptavidin T1 after incubation with HL60 lysates. Sirt1 and
Sirt2 were detected with isotype-specific antibodies (for uncropped
blots see Figure S7d,e). b) Quantification of the Western blot analysis.
Affinity pull-down experiments lead to a significant enrichment of Sirt2
(n=3). Statistics (t-test): ** pꢀ0.005
Keywords: deacylases · drug design · protein modifications ·
proteomics · sirtuins
How to cite: Angew. Chem. Int. Ed. 2016, 55, 2252–2256
Angew. Chem. 2016, 128, 2293–2297
mixture of the three mentioned proteins (Figure S7a),
mixtures of E. coli lysates where Sirt1 or Sirt2 are overex-
pressed (Figure S7b,c), and native HL60 lysates (Figure 4 and
see Figure S7d,e). The specificity of the pull-down was shown
by blocking the Sirt2 enrichment effectively with either biotin
or SirReal2 (2, see Figure S7a). These results underline the
isotype selectivity, robustness, and broad applicability of our
SirReal probe for affinity purification and pull-down experi-
ments.
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Received: October 21, 2015
Published online: January 8, 2016
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