Contrasting sirtuin and poly(ADP-ribose)polymerase activities of selected 2,4,6-trisubstituted benzimidazoles

Article abstract of DOI:10.1111/cbdd.13072

Both sirtuin and poly(ADP-ribose)polymerase (PARP) family of enzymes utilize NAD⁺ as co-substrate. Inhibitors of sirtuins and PARPs are important tools in drug discovery as they are reported to be linked to multiple diseases such as cancer. New potent sirtuin inhibitors (2,4,6-trisubstituted benzimidazole) were discovered from reported PARP inhibitor scaffold. Interestingly, the synthesized compounds have contrasting sirtuin and PARP-1 inhibitory activities. We showed that modification on benzimidazoles may alter their selectivity toward sirtuin or PARP-1 enzymes. This offers an opportunity for further discovery and development of new promising sirtuin inhibitors. Molecular docking studies were carried out to aid the rationalization of these observations. Preliminary antiproliferative studies of selected compounds against nasopharyngeal cancer cells also showed relatively promising results.

Full text of DOI:10.1111/cbdd.13072

DR KENG YOON YEONG (Orcid ID : 0000-0003-0433-5070)
Article type : Research Article
Contrasting sirtuin and PARP activity of selected 2,4,6-trisubstituted
benzimidazoles
Keng Yoon Yeonga,b*, Soo Choon Tanb, Chun-Wai Maic, Chee-Onn Leongc,d, Felicia Fei-
Lei Chungd, Yean Kee Leee, Chin Fei Cheee, Noorsaadah Abdul Rahmane
aSchool of Science, Monash University Malaysia Campus, Jalan Lagoon Selatan, Bandar
Sunway, 47500, Selangor, Malaysia.
bInstitute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden, 11800,
Penang, Malaysia.
cSchool of Pharmacy, International Medical University, Bukit Jalil, 57000 Kuala Lumpur,
Malaysia
dCenter for Cancer and Stem Cell Research, International Medical University, Bukit Jalil,
57000 Kuala Lumpur, Malaysia.
eDepartment of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Abstract:
Both sirtuin and poly(ADP-ribose)polymerase (PARP) family of enzymes utilize NAD+ as
co-substrate. Inhibitors of sirtuins and PARPs are important tools in drug discovery as they
are reported to be linked to multiple diseases such as cancer. New potent sirtuin inhibitors
(2,4,6-trisubstituted benzimidazole) were discovered from reported PARP inhibitor scaffold.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13072
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Interestingly, the synthesized compounds have contrasting sirtuin and PARP-1 inhibitory
activity. We showed that modification on benzimidazoles may alter their selectivity towards
sirtuin or PARP-1 enzymes. This offers an opportunity for further discovery and
development of new promising sirtuin inhibitors. Molecular docking studies were carried out
to aid the rationalization of these observations. Preliminary antiproliferative studies of
selected compounds against nasopharyngeal cancer cells also showed relatively promising
results.
Keywords: Sirtuin, PARP, Benzimidazole, Anticancer, Nasopharyngeal, Molecular docking
Yeong Keng Yoon
aSchool of Science, Monash University Malaysia Campus, Jalan Lagoon Selatan, Bandar
Sunway, 47500, Selangor, Malaysia.
bInstitute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden, 11800,
Penang, Malaysia.
Phone: +603 55145102
Fax: +603 55146184
Emails: Yeong.KengYoon@monash.edu
Introduction
The class III histone deacetylases (HDACs), or sirtuins include a group of proteins that are
homologous with the yeast SIR2 family of proteins [1,2]. Unlike other classes of HDACs, the
function of sirtuins is dependent on NAD+, which they use as a cofactor in the catalysis. To
date, seven mammalian sirtuins have been identified (SIRT1-7) [3]. Even though all sirtuins
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share a common catalytic domain, they each have unique additional N-terminal and/or C-
terminal sequences of variable length, thus displaying different catalytic activities and
substrate specificities [4]. Of the seven sirtuins, SIRT1 and SIRT2 have been linked to a
plethora of diseases such as cancer [5,6], Alzheimer’s [7] and Parkinson’s [8] disease.
Both SIRT1 and SIRT2 are believed to have a role in the development of cancer as their
functions are frequently altered in cancer cells. SIRT1 was shown to be upregulated in a
spectrum of cancers, such as in leukemia [9] as well as prostate cancer [10]. Furthermore,
since SIRT1 and SIRT2 both fulfill the same function of deacetylating p53, it has been
reported that the simultaneous inhibition of SIRT1 and SIRT2 was beneficial against cancer
by completely blocking p53 deacetylation, which led to cell death [11].
Several notable classes of small molecule sirtuin inhibitors which have been identified so far
includes indoles (EX-527) [12], α-cyanopropenamide (AGK2) [13] and tenovins (Tenovin-6)
[14]. Recently, compounds based on the benzimidazole scaffold have been demonstrated to
be potent sirtuin inhibitors [15-18].
Sirtuins and poly(ADP-ribose) polymerases (PARPs) both are strongly conserved in
eukaryotes and share the particularity of using NAD+ as a co-substrate [19,20]. Both PARPs
and sirtuins cleave NAD+ into nicotinamide and ADP-ribose. Thus, it will be interesting to
investigate if other benzimidazole motif such as 2,4-disubstituted benzimidazoles which have
been reported as potent PARP inhibitors [21] share similar inhibitory potency for sirtuin
enzymes. We hereby report on the synthesis of some 2,4-disubtituted and 2,4,6-trisubstituted
benzimidazoles and subsequently evaluated their sirtuin inhibitory activities. In addition,
antiproliferative activity of selected synthesized benzimidazole derivatives against c666-1
(nasopharyngeal) carcinoma cell line was also reported. Molecular docking was performed to
rationalize the observed changes in activity.
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1.
MATERIALS AND METHODS
All other chemicals were purchased from Sigma-Aldrich or Merck and were of analytical
grade. All melting points were taken on a Stuart melting point apparatus SMP30
(Staffordshire, UK). NMR spectra were obtained using Jeol FX-270 (270 MHz) (Jeol Ltd.,
USA) and Bruker Avance III HD (400 MHz) (Bruker Biospin Corp., Billerica, USA) NMR
spectrometers with tetramethylsilane as the internal standard. All chemical shifts are reported
in ppm. MS analysis was performed on an Agilent 6500 series accurate mass Q-TOF (Agilent
Technologies, Santa Clara, CA, USA) Analytical thin-layer chromatography (TLC) was
carried out on pre-coated aluminum silica gel sheets (Kieselgel 60 F254) from Merck
(Darmstadt, Germany). Column chromatography was performed with silica gel 60 (230–400
mesh) from Merck.
1.1
General procedure for synthesis of compounds 1-10
To a solution of carboxylic acid b (1 mmol) in pyridine (1 mL) and DMF (1 mL) was added
1,1’-carbonyldiimidazole (CDI, 1.1 mmol) and the solution was heated at 50 oC for 2 h. Then
diamine a (1 mmol) was added and the mixture was stirred at room temperature overnight.
o
Solvents were removed and the residue heated in 2 mL of acetic acid at 100 C for 1 h. After
concentration, the residue was extracted with ethyl acetate and saturated sodium bicarbonate
solution. The organic layers were washed brine, dried over MgSO4 and evaporated. The
residue was subjected to column chromatography (dichloromethane-methanol, 9:1) to afford
the title compound as free base.
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1.2
Molecular docking
The crystal structure of human SIRT2 (PDB code: 3ZGV) and PARP-1 (PDB code: 4HHZ)
were taken from the Protein Data Bank. Both enzyme and ligand were structurally optimized
prior to the actual docking simulation. After removing the co-crystallized water molecules,
hydrogen atoms were added to the protein structure. Ligand was energy minimized with
Chem 3D Pro 13.0 using the MM2 forcefield. Docking was carried out using Autodock 4.2.
For each molecule, 10 docking runs were performed. The top-ranked pose for each ligand
were retained and further analyzed with VMD 1.9.1 molecular graphics software.
1.3
Cell lines and cytotoxic assay
All human cell lines were obtained from American Type Cell Culture (ATCC) unless
specified. Human breast epithelial cells, MCF-10A were cultured in Dulbecco's modified
Eagle's medium and Ham's (DMEM/F12) supplemented with 5% horse serum, 20ng/mL
epidermal growth factor, 0.5µg/mL hydrocortisone, 10µg/mL insulin, 100IU/mL penicillin
and 100 µg/mL streptomycin. Human colon epithelial cells, CCD 841 CoN was cultured in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum,
100IU/mL penicillin and 100 µg/mL streptomycin. NP460 cells were maintained in
Keratinocyte-SFM containing epidermal growth factor and bovine pituitary extract (BPE)
(Invitrogen, USA). Human endometrial fibroblasts (T-HESC) were maintained in
DMEM/F12 supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 1.5g/L
sodium bicarbonate, 500ng/mL puromycin and 1% ITS Premix (Corning, USA). Human
nasopharyngeal carcinoma cells (c666-1) and human lung fibroblast (MRC5) were cultured
in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin
and 100 µg/mL streptomycin. All cells were kept in 37 oC incubator with 5% carbon dioxide.
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The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) cell viability assay was
used to determine the cytotoxicity of compounds on the human non-transformed cell lines as
optimized in previous studies [22,23]. All data were reported as mean ± standard deviation
(S.D) from a minimum of three independent experiments. Statistical significant difference
between negative control and treated cells were analysed using student independent t-test. A
p-value less than 0.05 (p<0.05) was considered significant different.
2.
Results
2.1
Synthesis of compounds
The benzimidazoles were synthesized using reported method outlined in Figure 1 [21].
Different functional groups were placed at the R1 and R2 position of the corresponding
diamine and carboxylic acid intermediates through de novo synthesis [24]. Direct coupling of
diamine a with carboxylic acid b in the presence of 1,1’-carbonyldiimidazole (CDI) gave
amide c. Subsequent thermal cyclization of the amide c in acetic acid afforded
benzimidazole d in two-steps (total yield = 38 ~ 75%). Compound characterization details
can be found in the Supporting Information.
2.2
Sirtuin enzymatic assays
The in vitro enzymatic screening assay for SIRT1 and SIRT2 inhibitory activity were
performed using fluorimetric sirtuin kits (AnaSpec, Fremont, CA) according to the
manufacturer’s protocol. EX-527 (SIRT1 selective inhibitor), AGK-2 (SIRT2 selective
inhibitor) and Tenovin-6 (pan-SIRT1/2 inhibitor) were used as standard control while DMSO
was used as the vehicle control. Experiments were performed in triplicates and standard
deviations obtained from all experiments are less than 20%.
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All the ten evaluated compounds demonstrated relatively higher selectivity for SIRT2 over
SIRT1 (Table 1). The trend of the results is consistent with those observed previously for
1,2,5-trisubstituted benzimidazole derivatives [15-18]. Compounds 3-6, with functional
groups other than a phenyl/pyridinyl ring at the R2 were found to have poor activity. Neither
electron donating (-OCH3) nor electron withdrawing (-CF3) showed significant activities.
However, the sirtuin inhibitory activity remarkably improved when bulkier groups such as
phenyl, phenyl(pyrrolidinyl) or pyridinyl(pyrrolidinyl) were attached to the R2 position.
These observations implied that a relatively bulkier system at the R2 position is required to
sufficiently occupy the active cavity of the sirtuin enzymes, especially the SIRT2 enzyme.
Meanwhile, incorporating a methyl group at the R1 position of the benzimidazole ring has
also greatly improved the sirtuin inhibitory activity. This was showcased by the increase in
potency for those compounds which carry the methyl group across the whole series. The
results shown here along with our earlier reports clearly demonstrated the potential of
benzimidazoles acting as potent sirtuin inhibitors.
2.3
Molecular docking
Molecular docking for both compound 9 and 10 into the active site of SIRT2 (PDB entry
code: 3ZGV, x-ray resolution = 2.30 Å) [25] and PAPR-1 (PDB entry code: 4HHZ, x-ray
resolution = 2.72 Å) [26] were performed and compared in view of their observed significant
difference in activity. The receptor and the drug candidate were optimized before actual
docking in Autodock 4.2 using standard procedure of the software.
The region which controls the activity of sirtuins can generally be divided into three binding
sites, namely the adenosine binding site (A pocket), ribose binding site (B pocket) and
nicotinamide binding site (C pocket) [27]. Analysis of the top-ranked pose of compound 10
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within the SIRT2 active binding site demonstrated several plausible molecular interactions.
Docking analysis reveals that compound 10 interacts with SIRT2 primarily due to hydrogen
bonding. Strong hydrogen bond could be observed between the N-H group from the
benzimidazole scaffold with Arg97. Meanwhile, the amide moiety from the compound could
form intra-molecular bonding with the N atom from the imidazole ring. Moreover, it can also
form hydrogen bond with Ile169 to stabilize the overall structure. Hydrogen bond interaction
can also be observed between the C=O from the phenyl(pyrrolidinyl)methanone moiety and
Glu288 (Figure 2). As shown through the in vitro assays, compound 10 (as of all the other
compounds) displayed weaker SIRT1 inhibitory activity. It was found to dock in part of the C
pocket (Ile347, Gly319 and Asp348) and loosely occupy part of the A pocket (Tyr280) and
acetyl lysine channel (Phe414) of the active site (Figure 3).
Through the acquired in vitro assay results, it was noted that changes at the R1 position
played important role towards the potency of the compounds. Therefore, docking for
compound 9 (which lacks the methyl group at R1 and showed much weaker sirtuin inhibitory
activity) was subsequently carried out to rationalize this observation. Poses for both
compounds were then compared as shown in Figure 4. The docking analysis of compound 9
showed that part of the compound (-phenyl(pyrrolidinyl)methanone) shifted out from the A
and B binding site of SIRT2 with different binding orientation, resulting in less favorable
complexes. This could explain the weaker in vitro SIRT2 inhibitory activities observed.
In comparison, both compound 9 and 10 have weaker SIRT1 activity. Similarly, the effect of
the methyl group on PARP-1 activity was investigated through molecular modeling. When
the docking poses of both compound 9 and 10 were superimposed, it was observed that both
compounds displayed different structural conformation (Figure 5). Notably, the important
interactions between amide group of 10 with Trp200, His201 and Gly202 were lost, leading
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to its weaker inhibitory activity as compared to 9. The additional methyl group at the R1
position thus have a contrary effect on the activities of sirtuins and PARP-1.
2.4
Cellular Assay
Sirtuins has been previously implicated in cancer modulation and sirtuin inhibitors has been
demonstrated to possess anticancer activity in the literature [28,29]. To investigate the
effectiveness of the synthesized compounds as anticancer agents, the three most potent sirtuin
inhibitory compounds (2, 8 and 10) were evaluated for their ability to impede the
proliferation of metastatic human nasopharyngeal carcinoma cells (c666-1). Compounds 2, 8
and 10 were screened at 50 µM using MTT assay and incubated for 72 h. Interestingly, all
three active compounds managed to significantly (p < 0.05) restrain the growth of c666-1
cancer cells (Figure 6). The tested compounds showed no significant cytotoxicity against a
panel of normal cells comprising breast (MCF-10A), colon (CCD 841 CoN), nasopharyngeal
(NP460), endometrial (T-HESC) and lung (MRC5) cells at 50 µM (with cell viability > 90%)
unlike 5-FU which demonstrated widespread non-specific cytotoxicity [30,31].
3. Discussion
Crosstalk between sirtuins and PARP enzymes have previously been reported [19,20]. There
were interest in the interplay of both classes of enzymes as they are involved in the same
biological processes (competition for the common NAD+ co-substrate, transcriptional effects
and post-translational modifications). Although sirtuins and PARPs both have NAD+ as co-
substrate and cleave NAD+ into nicotinamide and ADP-ribose, the inhibitory effects of the
synthesized benzimidazoles for sirtuin enzymes were interestingly in stark contrast to those
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observed for PARP-1. Recently, Ekblad and Schüler have reported that the activity of sirtuins
are unaffected by the most important PARP inhibitors such as rucaparib and veliparib due to
the inaccessibility of PARP inhibitors getting into sirtuin cofactor site [32]. For this series of
compounds, we found that those with methyl substitution at R1 such as 2, 8 and 10 are poor
PARP-1 inhibitors but are potent sirtuin (SIRT2) inhibitors while non-substituted compounds
at R1 such as 1, 7 and 9 are potent PARP-1 inhibitors but are poor sirtuin inhibitors.
Compounds 3-6 were found to be poor inhibitors for both enzymes. Although only 10
compounds were evaluated in this study, the fact that novel potent sirtuin inhibitors were
positively identified demonstrated the potential to similarly unearth sirtuin modulators by
minor modification from other known NAD+ targeting scaffold.
One point to note was that there has not been any literature at drug discovery level linking
nasopharyngeal cancer and SIRT2 although down-regulation of SIRT2 in nasopharyngeal
carcinoma when treated with seliciclib was reported [33]. Latent membrane protein 1 (LMP1)
is known to be expressed in the majority of nasopharyngeal carcinoma cells [34]. It was
found that LMP1 up-regulates hypoxia-inducible factor-1α (HIF-1α) transcription of
nasopharyngeal cancer cells [35]. Few studies have examined the relationship between sirtuin
and HIF, and among the very few studies carried out, it was found that SIRT2 destabilizes
HIF-1α [36]. The SIRT2 inhibitory compounds identified in this study may then have exerted
their antiproliferative effect towards nasopharyngeal carcinomas by targeting HIF-1α via
LMP1 down-regulation. This deserves to be further investigated as it may lead to a better
understanding of sirtuin mediated effects on nasopharyngeal cancer.
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Conclusion
In conclusion, we have discovered new potent sirtuin inhibitors from identified PARP
inhibitor scaffold. We demonstrated that minor modification such as the introduction of a
methyl group could alter the potency and selectivity of a compound towards either sirtuin or
PARP-1 inhibition. Thus, this new general approach could be applied to aid the search for
new promising sirtuin inhibitors from an existing class of compound. In the process, we have
also showed the potential of 2,4,6-trisubstituted benzimidazole derivatives acting as potent
sirtuin inhibitors. Moreover, these compounds possessed good anticancer activity against the
c666-1 nasopharyngeal cancer cell line and showed no significant cytotoxicity against a panel
of normal cell lines tested in this study.
Acknowledgements
The authors wish to thank Monash University and Institute for Research in Molecular
Medicine, Universiti Sains Malaysia, for supporting this work. Human nasopharyngeal
cancer cells, c666-1 was obtained from Dr. Alan Khoo, Institute of Medical Research,
Malaysia while human immortalized nasopharyngeal cells, NP460, was obtained from Dr.
George Tsao, Hong Kong University. This work was funded through Monash University
Malaysia SEED Fund and RUC Research Grant (1001/PSK/8620012).
The authors declare no conflict of interest.
Figure Legends
Figure 1. General synthesis of benzimidazoles 1-10. Reaction conditions: (i) CDI, pyridine,
DMF, (ii) AcOH, 110 oC.
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Figure 2. Compound 10 docked in the active site of SIRT2 (PDB: 3ZGV). Yellow dashed
lines show hydrogen bonds.
Figure 3. Compound 10 docked in the active site of SIRT1 (PDB: 4I5I).
Figure 4. Part of compound 9 (purple) was located out from the active site of SIRT2 when
docked while compound 10 (green) fits well into the groove (PDB: 3ZGV).
Figure 5. In the PARP-1 active site, important interactions with Gly202, His201 and Trp200
were lost with compound 10 (yellow) when the phenyl(pyrrolidinyl)methanone side chain
was oriented pointing in a different direction (PDB: 4HHZ).
Figure 6. Human nasopharyngeal cancer cells (c666-1), and a panel of human non-cancers
cells were treated with 50μM of compound 2, 8, 10 and 5-FU for 72 hours. MTT assay was
used to quantify cell viability. Results were the mean ± S.D. from at least 3 independent
experiment. Symbol “*” indicates statistical significant (p<0.05) of treated cells compared to
negative control (0.1% DMSO) using on student independent t-test.
Supporting Information contents:
1. Compound characterization details
2. NMR spectra
3. Sirt1 in vitro assay
4. Sirt2 in vitro assay
5. Cytotoxicity assay protocol
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Table 1. SIRT1 and SIRT2 inhibitory activities of benzimidazoles 1-10.
SIRT1 IC50 (µM) SIRT2 IC50 (µM) PARP-1 IC50
Compound
± S.D. or
% inhibition at
20µM
± S.D. or
% inhibition at
20µM
(µM) or
% inhibition
at 10 µM*
0.71
R1
R2
-H
19.1 %
36.5 %
1
2
-CH3
54.8 ± 2.30 µM
9.2 ± 0.75 µM
28%
-H
-CH3
-H
-OCH3
-OCH3
-CF3
10.6 %
13.8 %
6.4 %
26.3 %
29.7 %
16.8 %
10.2 %
45.0 %
NA
NA
3
4
5
6
7
NA
-CH3
-H
-CF3
8.5 %
NA
29.9 %
0.029
-CH3
-H
44.8 ± 1.91 µM
23.3 %
10.9 ± 1.10 µM
46.4 %
NA
0.012
17%
8
9
-CH3
34.6 ± 1.16 µM
6.5 ± 0.97 µM
10
-
-
-
-
-
-
0.30 ± 0.10 µM
N.D.
N.D.
N.D.
N.D.
N.D.
EX-527
AGK2
8.34 ± 2.05 µM
25.6 ± 3.08 µM
42.1 ± 3.39 µM
Tenovin-6
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N.D. Not determined; N.A. Not active (less than 15% inhibition at 10 µM). * Reported in
[24]
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.