Evaluation of benzoic acid derivatives as sirtuin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.11.004

Employing a genetically modified yeast strain as a screening tool, 4-dimethylaminobenzoic acid (5) was isolated from the marine sediment-derived Streptomyces sp. CP27-53 as a weak yeast sirtuin (Sir2p) inhibitor. Using this compound as a scaffold, a series of disubstituted benzene derivatives were evaluated to elucidate the structure activity relationships for Sir2p inhibition. The results suggested that 4-alkyl or 4-alkylaminobenzoic acid is the key structure motif for Sir2p inhibitory activity. The most potent Sir2p inhibitor, 4-tert-butylbenzoic acid (20), among the tested compounds in this study turned out to be a weak but selective SIRT1 inhibitor. The calculated binding free energies between the selected compounds and the catalytic domain of SIRT1 were well correlated to their measured SIRT1 inhibitory activities.

Full text of DOI:10.1016/j.bmcl.2013.11.004

Bioorganic & Medicinal Chemistry Letters 24 (2014) 349–352
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluation of benzoic acid derivatives as sirtuin inhibitors
Yi-Pei Chen, Chad C. Catbagan, Jeannette T. Bowler, Trevor Gokey, Natalie D. M. Goodwin, Anton B. Guliaev,
⇑
Weiming Wu, Taro Amagata
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Employing a genetically modified yeast strain as a screening tool, 4-dimethylaminobenzoic acid (5) was
isolated from the marine sediment-derived Streptomyces sp. CP27-53 as a weak yeast sirtuin (Sir2p)
inhibitor. Using this compound as a scaffold, a series of disubstituted benzene derivatives were
evaluated to elucidate the structure activity relationships for Sir2p inhibition. The results suggested
that 4-alkyl or 4-alkylaminobenzoic acid is the key structure motif for Sir2p inhibitory activity. The
most potent Sir2p inhibitor, 4-tert-butylbenzoic acid (20), among the tested compounds in this study
turned out to be a weak but selective SIRT1 inhibitor. The calculated binding free energies between
the selected compounds and the catalytic domain of SIRT1 were well correlated to their measured
SIRT1 inhibitory activities.
Received 4 September 2013
Revised 1 November 2013
Accepted 5 November 2013
Available online 12 November 2013
Keywords:
Sirtuin
Class III HDAC inhibitor
Marine-derived Streptomyces sp.
4-Dimethylaminobenzoic acid
4-tert-Butylbenzoic acid
Ó 2013 Elsevier Ltd. All rights reserved.
Sirtuins are a group of NAD+-dependent histone deacetylases
(HDACs) that are evolutionally conserved from bacteria to mam-
mals.1 This group of enzymes regulates the key biological functions
including gene silencing and cell cycle by removing acetyl groups
from lysine residues in the histones or non-histone substrates. To
date, seven human isoforms (SIRT1–7) have been found and cate-
gorized as class III HDACs that are different from classical zinc-
dependent class I/II/IV HDACs.2 Recently, SIRT1 and SIRT2 have
been considered as molecular targets for cancer chemotherapy.
The physiological functions of SIRT1 with regard to tumorigenesis
include the negative regulation of the tumor suppressor gene 53,3
the positive regulation of the oncoprotein B-cell lymphoma 6 pro-
tein (BCL6),4 and induction of the FOXO-1-dependent vascular
growth factor-C (VEGF-C).5 On the other hand, SIRT2 promotes
cancer cell proliferation due to the enhanced stability of the Myc
oncoproteins.6 It has been reported that apoptosis caused by p53
accumulation in HeLa cells7 and granulocytic differentiation in
the acute promyelocytic leukemia (APL)8 can be induced based
on down-regulation and inhibition of SIRT2. Though SIRT1 and
SIRT2 are also reported to show tumor-suppressing effects9–11
and to be down-regulated in specific cancer types,12 the tumor pro-
moting effects listed above strongly suggest SIRT1/2 inhibitors
have great potential to be anticancer drugs. Encouraging evidence
includes significant cytotoxic properties of the two SIRT1/2
inhibitors, sirtinol (1: synthetic13) and toxoflavin (2: natural
product14), against the MCF-7 and A549 cancer cell lines, respec-
tively.15,16 It is interesting that the potent and selective SIRT1
inhibitor, EX-527 (3: synthetic17), required at least 100
lM to
effectively repress MCF-7 cell proliferation.18 However, the selec-
tive SIRT2 inhibitor, AC-93253 (4: synthetic), exhibited potent
cytotoxic effects (IC50 10–100 nM) against the prostate (DU-145),
lung (A549, NCI-H460) and pancreas (MiaPaCa2) cancer cell lines
with a great therapeutic window (up to 200-fold).19
We have recently reported the HDAC screening method using
the genetically modified yeast strain DMY2843 to identify SIRT1/
2 inhibitors from marine-sediment derived actinomycetes.20 The
primary screening for the extract library using the yeast assay se-
lected a total of 19 actinomycete strains that would produce yeast
sirtuin (Sir2p) inhibitors. A new polyketide tetramic acid derivative
designated as streptosetin A with weak Sir2p and SIRT1/2 inhibi-
tory activities has been isolated from one of the active strains,
Streptomyces sp. CP13-10.20 Our continued search for sirtuin inhib-
itors from the remaining active strains led to the identification of
4-dimethylaminobenzoic acid (5) produced by the Streptomyces
sp. CP27-53 strain as a Sir2p inhibitor. In this article, we report
the identification of 5 and structure activity relationships (SARs)
and SIRT inhibitory activities of benzoic acid derivatives and re-
lated analogues of 5.
⇑
Corresponding author. Tel.: +1 415 338 7713; fax: +1 415 338 2384.
E-mail address: amagata@sfsu.edu (T. Amagata).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.11.004

350
Y.-P. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 349–352
The marine sediment-derived Streptomyces sp. CP27-53 was cul-
tured in a liquid medium (15 L) containing soluble starch (1%), yeast
extract (0.4%), peptone (0.2%), CaCO3 (0.1%) and FeSO4Á7H2O
(40 mg) in artificial seawater adjusted to pH 7.4 for 10 days at
30 °C at 200 rpm. The culture was separated to broth and pellet
by centrifugation. The broth was treated with HP20 to absorb or-
ganic compounds, which were eluted with MeOH, whereas the pel-
let was extracted with MeOH three times. The combined MeOH
extract was cleaned by liquid–liquid partition between EtOAc and
H2O to give an organic extract. Yeast screening of the HPLC peak li-
brary created from the organic extract revealed that the compound
eluting at 14.3 min in the HPLC chromatogram was responsible for
the Sir2p inhibitory activity (Fig. S1). Furthermore, this active com-
pound was purified by reversed-phase HPLC and identified as 4-
dimethylaminobenzoic acid (5) based on the spectroscopic data
(see Supplementary data). The structure was further confirmed by
direct comparison of the 1H and 13C NMR data with those of an
authentic sample. This compound showed Sir2p inhibitory activity
amino group and carboxylic acid, must be on para-positions to
show Sir2p inhibitory activity. The MIC values of 400 and
800
lM for compounds 10 and 11 in Group B, respectively, indi-
cated that more methyl groups on the nitrogen enhanced the Sir2p
A
B
800 µM
400 µM
200 µM
100 µM
50 µM
25 µM
5
19 20
5
19 20
with an MIC of 200 lM after 48 h against the yeast strain.
To elucidate the SARs for Sir2p inhibition by 5, a series of substi-
tuted benzoic acid derivatives and related analogues of 5 were
evaluated using the yeast strain DMY2843 (Table 1 and Fig. 1).
The compounds in Group A (6–9) were inactive against the yeast
strain, which suggested that the two functional groups, dimethyl-
Figure 1. Yeast HDAC screening results (48 h) for 5, 19 and 20. (A) YPD media with
5-fluoro (5-FOA) and (B) YPD media. Sir2p inhibition was defined as selective
activity when the tested compound resulted in the death of yeast cells (clear) only
in the presence of 5-FOA. Cytotoxicity was observed as the death of yeast cells in
both media A and B.
Table 1
Minimum inhibition concentration (MIC) values of disubstituted benzene derivatives based on Sir2p inhibition against the yeast strain DMY2844
Structural group
A
R1
R2
Location
MIC (lM)
5
6
7
8
9
(CH3)2N–
(CH3)2N–
(CH3)2N–
(CH3)2N–
None
–COOH
–COOH
–COOH
None
p
m
o
200
NAa
NA
NA
NA
–COOH
B
C
10
11
CH3NH–
NH2–
–COOH
–COOH
p
p
400
800
12
13
14
15
16
(CH3)2N–
(CH3)2N–
(CH3)2N–
(CH3)2N–
(CH3)2N–
–COOCH3
–CONH2
–SO3H
–Br
p
p
p
p
p
400
NA
NA
NA
NA
–NO2
D
E
17
18
CH3–
(CH3)2CH–
(CH3)3N+–
(CH3)3C–
–COOH
–COOH
–COOÀ
–COOH
p
p
800
200
19
20
p
p
NA
50
a
Not active at 800 lM.

Y.-P. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 349–352
351
Figure 2. The representative conformational snapshots of compounds 5, 20 and 21 in the catalytic domain of SIRT1 produced by 0.5
ls explicit solvent MD simulations.
activities. Next, the activity data for Group C including the weaker
activity for compound 12 with methyl ester than that of compound
5 and the lack of activity for compounds 13–16 indicated that the
carboxylic acid appeared to be required for inhibition activity. It is
interesting that sulfonic acid 14 was inactive despite the similarity
between the functional groups. In Group D, compound 17, in which
the dimethylamino group was replaced by a methyl group, showed
diminished activity whereas compound 18, in which the dimethyl-
amino group was replaced by a similarly sized and shaped isopro-
pyl group, showed similar inhibitory activity to that of compound
5. The results indicated that the size of the substituent is very
important for inhibitory activity. Therefore, we evaluated the two
derivatives in Group E, compounds 19 and 20 with trimethylam-
monium and tert-butyl groups as respective substituents.
depicted in Figure 2. The more favorable binding energy for com-
pound 21 can be explained by its tighter conformational ensemble
observed for this ligand in the binding domain than those of com-
pounds 5 and 20. For compound 5, MD simulations produced two
distinct conformational families, as clearly seen by the position of
the carbonyl group. One conformational family was similar to
those of compounds 20 and 21, in which the polar functional
groups positioned towards the pocket entrance. In the second con-
formational family, compound 5 rotated by approximately 90 de-
grees. This rotation placed the carbonyl group adjacent to the
nicotinamide group of the NAD+, resulting in slight displacement
of the NAD+ from the active site and thus creating a possible escape
route from the binding pocket. This behavior was observed after
first 100 ns of simulation, and could also support weaker binding
of 5 than 20. This conformational flexibility resulted in the less
favorable binding energy of this ligand as compared to 20 and
21. The smaller size of compound 20 (as compared to 21) allowed
it more freedom to move inside the binding pocket than compound
21. Based on this conformational analysis, it is possible to propose
that increase in conformational flexibility of the compound in the
SIRT1 binding pocket leads to decrease in binding affinity. The re-
sults obtained from the MD calculations suggest that bulkier aro-
matic acid derivatives may be better SIRT1 inhibitors by
complementing the size and hydrophobic environment of the en-
zyme binding pocket, and we plan to test such derivatives in the
near future.
Compound 20 showed the best activity (MIC 50 lM) among the
derivatives tested, which supported the importance of the size of
the substituent as suggested above. The lack of activity seen
in compound 19 suggested that bulky but not charged groups
are required for enhanced inhibitory activity for benzoic acid
derivatives.
In light of their Sir2p inhibitory activities, compounds 5 and 20
were further evaluated for SIRTs inhibitory activities. Compounds 5
inhibited SIRT1 and SIRT2 with 25.3% and 30.3% at 1.6 mM whereas
compound 20 inhibited SIRT1 and SIRT2 with 54.8% and 28.0% at
1.6 mM, respectively. These results suggested that compound 20
was a weak but selective SIRT1 inhibitor (IC50 1.0 mM). Further-
more, the SIRT1 inhibitory activity was enhanced twofold by
replacing the dimethylamino group with tert-butyl group, which
supported the SAR pattern described above.
The crystal structure of the SIRT1 catalytic domain with NAD+
and an EX-527 analogue [(S)-2-chloro-5,6,7,8,9,10-hexahydrocy-
clohepta[b]indole-6-carboxamide (21)] has recently been
reported.21 Based on the SIRT1/NAD+/21 coordinates, we constructed
SIRT1/NAD+/5 and SIRT1/NAD+/20 complexes to evaluate the
In this study, 4-tert-butylbenzoic acid (20) was discovered as a new
Sir2p inhibitor based on the SAR study on the naturally occurring
weak Sir2p inhibitor, 4-dimethylaminobenzoic acid (5), isolated
from the Streptomyces sp. CP27-53. Compound 20 also showed a
weak but selective inhibitory activity against SIRT1. It is quite inter-
esting that the structure of 5 was identical to the capping group of
the potent class I/II HDAC inhibitor trichostatin A.29 This study also
demonstrated a reasonable correlation between the calculated
binding energy and potency of SIRT1 inhibition activity, suggesting
that it would be possible to establish a SIRT1 virtual screening
method by collecting more data points. The SAR study and MD
binding free energies (
DGbind) of compounds 5 and 20 relative to
compound 21. The Gbind energies were calculated by using the
D
MM-GBSA approach for post processing of explicit solvent molec-
ular dynamics (MD) trajectories. This approach has been proven
to provide absolute free energies in good agreement with experi-
mental data.22–28 The potent and selective SIRT1 inhibitor 21
(IC50 60–100 nM17) showed a binding energy of À37.5 kcal/mol
whereas compounds 5 and 20 were found to have binding energies
of À26.8 and À31.6 kcal/mol, respectively. Conformational ensem-
bles for compounds 5, 20 and 21 complexes produced by MD are

352
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This investigation was supported by the Grants from the Na-
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(A.B.G.) and SC1GM095419 (W.W.) and the Beckman Scholarship
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of Science and Engineering, San Francisco State University was
funded by the Grant (P20MD000544) from the National Center
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