Identification of a cell-active non-peptide sirtuin inhibitor containing N-thioacetyl lysine

Article abstract of DOI:10.1016/j.bmcl.2009.08.028

To identify cell-active sirtuin inhibitors containing N-thioacetyl lysine, we synthesized compound 1, which was designed based on the structure of the reported N-ethoxycarbonylacetyl lysine-based sirtuin inhibitor NCS-12k. Compound 1 selectively inhibited

Full text of DOI:10.1016/j.bmcl.2009.08.028

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5670–5672
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a cell-active non-peptide sirtuin inhibitor containing
N-thioacetyl lysine
*
*
Takayoshi Suzuki , Tomomi Asaba, Erika Imai, Hiroki Tsumoto, Hidehiko Nakagawa, Naoki Miyata
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To identify cell-active sirtuin inhibitors containing N-thioacetyl lysine, we synthesized compound 1,
which was designed based on the structure of the reported N-ethoxycarbonylacetyl lysine-based sirtuin
inhibitor NCS-12k. Compound 1 selectively inhibited SIRT1 in enzyme assays. Compound 1 also caused a
dose-dependent increase in p53 acetylation in human colon cancer HCT116 cells, indicating the inhibi-
tion of SIRT1 in these cells.
Received 22 July 2009
Revised 3 August 2009
Accepted 4 August 2009
Available online 12 August 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Sirtuin inhibitor
Thioacetyl lysine
Non-peptide
Reversible protein acetylation is an important posttranslational
modification that regulates the function of histones and many non-
histone proteins.1–3 Sirtuins are enzymes that catalyze the deacet-
ylation of acetylated lysine residues of proteins using NAD+ as a
cofactor, and are involved in a diversity of cellular functions, such
as genome maintenance, longevity, and metabolism.4–6 Humans
have seven distinct sirtuin gene products (SIRT1–7). Among these,
SIRT1 and SIRT2 are reportedly associated with certain disease
states such as cancer and neurodegenerative disorders.7–9 There-
fore, SIRT inhibitors are of interest not only as tools for elucidating
the detailed biological functions of the enzyme, but also as poten-
tial therapeutic agents.10,11
To date, several classes of sirtuin inhibitors have been identified
by us and other groups.12–16 Among them, thioacetyl lysine-con-
taining peptides (Fig. 1) have been reported as mechanism-based
inhibitors of sirtuins.17–19 Mechanism-based enzyme inhibitors
are relatively inert until they are processed by the target enzyme,
which unmasks a chemically reactive warhead that leads to the
covalent modification of residues or cofactors in the active site.
This class of enzyme inhibitor has the advantage of a high specific-
ity. In the case of thioacetyl sirtuin inhibitors, the sulfur of the thi-
oamide nucleophilically attacks NAD+ at the active site of sirtuins
to enable a stable conjugation with ADP-ribose, leading to the inhi-
bition of enzyme activity.18 Although thioacetyl lysine-containing
peptides show potent inhibitory activities in enzyme assays, these
peptides are difficult to use in cellular studies because of the poor
membrane permeability resulting from their peptide structure.
This consideration led us to design a non-peptide sirtuin inhibitor
containing N-thioacetyl lysine. Herein, we report the SIRT-inhibi-
tory activity, inhibitory mechanism, and cellular activity of a
non-peptide thioacetyl lysine analogue.
Recently, we reported the N-ethoxycarbonylacetyl lysine ana-
logue NCS-12k (Fig. 1) as another mechanism-based inhibitor of
sirtuin.15 The enol form of NCS-12k nucleophilically attacks NAD+
in the active site of SIRTs to enable the creation of a stable NCS-
12k-ADP-ribose conjugate, which inhibits SIRTs. Based on these
findings, we designed a novel N-thioacetyl lysine analogue 1
(Fig. 1) in which the anilino group and the benzyloxycarbonyl
group are attached to the carbonyl and amino groups of lysine,
respectively. These substituents are recognized by amino acid res-
idues at the entrance to the N-acetylated lysine binding channel of
O
O
H
N
H
N
H2N KKGQSTSRHK
LMFKTEG OH H2N KSTGGK
APRKQ
OH
S
NH
CH3
S
NH
CH3
O
O
O
O
O
O
HN
HN
S
H
N
H
N
N
OEt
N
H
CH3
H
O
O
NCS-12k
1
* Corresponding authors. Tel./fax: +81 52 836 3407 (N.M.).
E-mail addresses: suzuki@phar.nagoya-cu.ac.jp (T. Suzuki), miyata-n@phar.nago
ya-cu.ac.jp (N. Miyata).
Figure 1. Structures of thioacetyl lysine peptides, NCS-12 k and compound 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.028

T. Suzuki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5670–5672
5671
SIRTs, similar to NCS-12k, leading to the potent inhibition of SIR-
Ts.15 In addition, the attachments of these non-peptide small
groups could overcome the membrane permeability problem of
peptides.
We synthesized compound 1 using the route outlined in
Scheme 1. 2-Benzyloxycarbonylamino-6-tert-butoxycarbonylami-
nohexanoic acid 2 was allowed to react with aniline in the pres-
ence of EDCI and HOBt to yield the corresponding anilide 3. The
removal of the Boc group of anilide 3 produced amine 4. Amine 4
was then reacted with ethyldithioacetate in the presence of
Na2CO3 in MeOH to yield the desired compound 1.20
Compound 1 was initially tested in an in vitro SIRT inhibition
assay using human recombinant SIRT1, SIRT2, and SIRT3.21 As
shown in Table 1, compound 1 inhibited SIRT1 with an IC50 of
2.7
lM, and exhibited selectivity for SIRT1 over SIRT2 and SIRT3
(IC50 for SIRT2 = 23
l
M; IC50 for SIRT3 >100 lM); this finding con-
Figure 2. Plots of product formation versus time in the absence (s) and presence of
100 (N) and 300 (d) lM of compound 1. AFU stands for arbitrary fluorescence unit.
trasts that for nicotinamide,22 a known non-selective sirtuin
inhibitor.
To examine the mechanism of SIRT1 inhibition by compound 1,
we initially determined whether inhibition by compound 1 was
time-dependent. The time course of product formation was moni-
tored in the absence and presence of compound 1. As shown in Fig-
ure 2, compound 1 was found to be a time-dependent inhibitor of
SIRT1, exhibiting nonlinear progress curves and reaching a plateau
value. These data suggest that compound 1 is an irreversible
inhibitor.
To gain further insight into the mechanism of SIRT1 inhibition
by compound 1, a mass spectroscopic analysis of a mixture of
SIRT1 incubated with compound 1 was performed.23 If compound
1 reacts with NAD+ as expected, compound 1-ADP-ribose conju-
gate 5 should be generated (Scheme 2). As depicted in Figure 3,
while the peak of NAD+ was observed at m/z 662.6, a significant
peak at m/z 953.8 was also observed. This peak corresponds to
the predicted molecular weight of compound 1-ADP-ribose conju-
gate 5. The peak was dependent on the presence of SIRT1 and com-
pound 1; that is, it was not detected in the absence of SIRT1 or
compound 1 (data not shown). These results indicate that adduct
5 was generated as a result of the SIRT1-catalyzed reaction of com-
O
NH2
O
O
HN
S
H
N
N
O
N
H
CH3
O
OH
OH
1
NAD+
O
N
NH2
SIRT
O
N
N
O
P
O
S
N
CH3
Ph
O
O
H2N
O
O
N
O
P
H
O
O
N
NH
O
N
OH
BnO
OH
OH
HO
5
Scheme 2. Proposed mechanism of SIRT inhibition by compound 1.
pound 1 with NAD+. The data from the mass spectroscopic analysis
supported the idea that compound 1 reacts with NAD+ in the active
site of SIRT1, yielding compound 1-ADP-ribose, which causes SIRT1
inhibition (Scheme 2).
O
O
O
O
a
b
HN
HN
H
N
HO
NHBoc
NHBoc
Unlike peptide inhibitors, compound 1 is a small-molecule SIRT
inhibitor that might be active in cellular assays. To test whether
this is the case, we performed a cellular assay using western blot
analysis. Since SIRT1 is known to catalyze the deacetylation of
p53 in cells with DNA damage,24 the acetylation level of p53 in hu-
man colon cancer HCT116 cells after etoposide-induced DNA dam-
age was analyzed.25 As can be seen in Figure 4, the level of
acetylated p53 was elevated depending on the dosage of com-
pound 1. These results suggest that compound 1 inhibits SIRT1 in
cells and can be used as a tool for probing the biological role of
SIRT1.
In summary, we have designed and synthesized a non-peptide
N-thioacetyl lysine analogue 1, which acts as a sirtuin inhibitor.
Compound 1 showed a potent and selective SIRT1-inhibitory activ-
ity. A kinetic analysis and mass spectroscopic analysis suggested
that the inhibitory mechanism involves the SIRT1-catalyzed syn-
thesis of compound 1-ADP-ribose conjugate 5 from thioacetyl ly-
sine analogue 1 and NAD+. Compound 1 was confirmed to inhibit
SIRT1 in a cellular study.
O
O
2
3
O
O
O
O
c
S
HN
HN
H
N
H
N
NH2
N
CH3
H
O
O
4
1
Scheme 1. Reagents and conditions: (a) aniline, 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide (EDCI), 1-hydroxybenzotriazole hydrate (HOBtÁH2O), DMF, rt,
94%; (b) HCl, AcOEt, rt, 99%; (c) ethyldithioacetate, Na2CO3, H2O, EtOH, rt, 85%.
Table 1
In vitro SIRT1-, SIRT2- and SIRT3-inhibitory activities of compound
1 and
nicotinamidea
Compound
IC50
SIRT2
2.7 0.11 23 3.1 >100
(
l
M)
Selectivity
SIRT2/SIRT1 SIRT3/SIRT1
SIRT1
SIRT3
Thus, we have identified a novel lead structure, compound 1,
from which it should be possible to develop potent and isoform-
selective sirtuin inhibitors by modifying the anilino and benzyl-
oxycarbonyl groups. This should be helpful in the development
1
8.5
>37
0.14
Nicotinamide 100 18
11 1.7 14 2.3 0.11
a
Values are the means of at least three experiments.

5672
T. Suzuki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5670–5672
O
S
N
Ph
O
N
H
%Int
.
NH
100
OH
BnO
OH
O
NAD+
conjugate 5
80
60
40
662.6
953.8
20
0
953.8
662.6
650
700
750
800
850
900
950
1000
Mass/Charge
Figure 3. Mass spectrometric detection of compound 1-ADP-ribose conjugate 5.
17. Fatkins, D. G.; Monnot, A. D.; Zheng, W. Bioorg. Med. Chem. Lett. 2006, 16, 3651.
18. Smith, B. C.; Denu, J. M. Biochemistry 2007, 46, 14478.
control
100 µM
10 µM
19. Kiviranta, P. H.; Suuronen, T.; Wallén, E. A.; Leppänen, J.; Tervonen, J.;
Kyrylenko, S.; Salminen, A.; Poso, A.; Jarho, E. M. J. Med. Chem. 2009, 52, 2153.
20. The analytical data of compound 1: mp 128–129 °C; 1H NMR (DMSO-d6,
500 MHz, d; ppm) 10.01 (1H, s), 9.96 (1H, m), 7.58 (2H, d, J = 6.1 Hz), 7.57 (1H,
d, J = 7.9 Hz), 7.37–7.29 (7H, m), 7.05 (1H, t, J = 7.5 Hz), 5.04 (2H, s), 4.13 (1H,q,
J = 8.7 Hz), 3.46 (2H, m), 2.36 (3H, s), 1.60 (2H, m), 1.41–1.34 (4H, m); 13C NMR
(DMSO-d6, 500 MHz, d; ppm) 198.80, 171.04, 156.09, 138.91, 137.00, 128.70,
128.34, 127.79, 127.70, 123.29, 119.24, 65.43, 55.36, 45.21, 32.80, 31.55, 26.92,
23.20; MS (FAB) m/z: 414 (M+); Anal. Calcd for C22H27N3O3S: C, 63.90; H, 6.58;
N, 10.16. Found: C, 63.71; H, 6.68; N, 10.25.
acetylated p53
total p53
Figure 4. Western blot detection of acetylated p53 levels in HCT116 cells after 8 h
of incubation with 20 M of etoposide and 10 or 100 M of compound 1.
l
l
21. The SIRT activity assay was performed using SIRT fluorimetric drug discovery
kits (AK-555, AK-556, and AK-557; BIOMOL Research Laboratories), according
of therapeutic agents for various diseases, as well as tools for
studying the biological roles of sirtuins.
to the supplier’s protocol. SIRT (human, recombinant) (15
(25 M), and various concentrations of the samples were incubated at 37 °C for
60 min. Fluor de Lys-SIRT substrate (25 M) was then added to the mixture.
l
L/well), NAD+
l
l
Reactions were stopped after 60 min by adding Fluor de LysTM Developer II with
nicotinamide, which stopped further deacetylation. Then, 45 min after the
addition of this developer, the fluorescence of the wells was measured using a
fluorometric reader with excitation set at 360 nm and emission detection set at
460 nm. The percent-inhibition (%-inhibition) was calculated from the
fluorescence readings of the inhibited wells relative to those of the control
wells. The compound concentration resulting in 50% inhibition was
determined by plotting log [Inh] versus the logit function of%-inhibition. IC50
values were determined using a regression analysis of the concentration/
inhibition data.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific
Research from the Japan Society for the Promotion of Science, a
Grant-in Aid for Research in Nagoya City University, and a grant
from the Ichihara International Scholarship Foundation.
References and notes
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of compound 1, 500
of NAD+,
(BIOMOL Research Laboratories), and 20
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l
L volumes containing 1 mM of DTT, 600
of SIRT1 (human recombinant)
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lM
lM
4 lM
l
7
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lM of etoposide and
1 at the indicated concentrations in 10% FBS-supplemented
compound
McCoy’s 5A medium, then collected and extracted with SDS buffer. The
protein concentrations of the lysates were determined using a Bradford protein
assay kit (Bio-Rad Laboratories); equivalent amounts of proteins from each
lysate were resolved in 15% SDS–polyacrylamide gel and were then transferred
onto nitrocellulose membranes (Bio-Rad Laboratories). After having been
blocked for 30 min with Tris-buffered saline (TBS) containing 3% skim milk, the
transblotted membrane was incubated overnight at 4 °C with acetylated p53
antibody (cell signaling; 1:1000 dilution) or p53 antibody (calbiochem; 1:1000
dilution) in TBS containing 3% skim milk. The membrane was probed with the
primary antibody, then washed twice with water, incubated with goat anti-
mouse IgG-horseradish peroxidase conjugates (diluted 1:2500) for 2 h at room
temperature, and again washed twice with water. The immunoblots were
visualized using enhanced chemiluminescence.
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