In silico identification of poly(ADP-ribose)polymerase-1 inhibitors and their chemosensitizing effects against cisplatin-resistant human gastric cancer cells

Article abstract of DOI:10.1016/j.bmcl.2013.02.094

Poly(ADP-ribose)polymerase-1 (PARP-1) enzyme is involved in the repair of DNA damages made by certain anticancer agents. It is suggested that PARP-1 inhibitors potentiate the cytotoxic effects and circumvent the resistance of DNA-modifying anticancer agen

Full text of DOI:10.1016/j.bmcl.2013.02.094

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2642–2646
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In silico identification of poly(ADP-ribose)polymerase-1 inhibitors
and their chemosensitizing effects against cisplatin-resistant human
gastric cancer cells
Tuong Vy Thi Le a, Jee Hee Suh b, Nakjeong Kim b, Hyun-Ju Park a,
⇑
a School of Pharmacy, Sunkyunkwan University, Suwon 440-746, Republic of Korea
b Center for Metabolic Syndrome Therapeutics, Bio-organic Science Division, Korea Research Institute of Chemical Technology, PO Box 107, Yuseong-gu, Daejeon, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Poly(ADP-ribose)polymerase-1 (PARP-1) enzyme is involved in the repair of DNA damages made by cer-
tain anticancer agents. It is suggested that PARP-1 inhibitors potentiate the cytotoxic effects and circum-
vent the resistance of DNA-modifying anticancer agents such as cisplatin. In this study, we conducted
virtual screening of Korea Chemical Bank database targeting PARP-1 and identified several potent
PARP-1 inhibitors with submicromolar IC50 values (77–79 nM). We then examined the chemosensitiza-
tion of cisplatin by pre-treatment of PARP-1 inhibitors in cisplatin-resistant human gastric cancer cells.
Our results show that PARP-1 inhibitors suppress the formation of poly(ADP-ribose) and enhance the
cytotoxicity of cisplatin.
Received 5 November 2012
Revised 16 February 2013
Accepted 21 February 2013
Available online 1 March 2013
Keywords:
Cisplatin-resistance
PARP-1 inhibitor
Chemosensitization
Ó 2013 Elsevier Ltd. All rights reserved.
Poly(ADP-ribose) polymerase-1 (PARP-1) is one of the most
abundant nuclear enzymes in the eukaryote and functions as a
DNA damage sensor and signaling molecule binding to both single-
and double-stranded DNA breaks. Upon DNA damage, PARP-1 is
activated and binds to DNA breaks, which catalyses the poly(-
ADP-ribosyl)ation reactions whereby ADP-riboses are transferred
from nicotinamide dinucleotide (NAD+) to glutamic and less com-
monly to aspartic and lysine residues in PARP-1 itself and their
substrates including histones. Accumulation of negative charges
on PARP-1 and histones results in a repulsion force and subsequent
dissociation of these components from DNA. The resultant chroma-
tin relaxation facilitates for DNA damage repair.1–4 Binding of
PARP-1 to single strand breaks (SSBs) recruits components of
DNA damage repair pathways such as X-ray repair cross comple-
menting protein 1 (XRCC1) and protects them from converting into
double strand breaks (DSBs).1,5 In DSBs repair, the role of PARP-1
has been revealed through the identification of PARP-1-dependent
alternative non-homologous end joining (NHEJ) pathway.6–9 How-
ever, in response to genotoxic agents, cells express different behav-
iors dependent on stimulus intensity which triggers PARP-1
activity. PARP-1 activation by mild to moderate genotoxic stimuli
facilitates DNA repair. Thus cells survive without the risk of passing
mutated genes. More severe DNA damage induces apoptosis in
which caspase inactivates PARP-1, subsequently eliminating cells
with severe DNA damage. However, over-activation of PARP-1 by
excessive DNA damage leads to depletion of NAD+ and ATP which
prevents apoptotic cell death. Under this condition, the inhibition
of PARP-1 preserves NAD+ and ATP, therefore allow cells either to
function normally or die via apoptotic pathway.10 Based on these
observations, PARP-1 inhibitors have been suggested in single or
combination therapy of various diseases.11–18
Formation of cisplatin–DNA adducts which trigger different
downstream signaling pathways is the main cause for cytotoxic ef-
fect of cisplatin.19 Interestingly, PARP-1 showed high affinity to
the most common 1,2-d(GpG) and this affinity decreases upon auto-
modification which implicates the role of PARP-1 in repair of cis-
platin-induced DNA damage.20,21 Therefore, combination between
PARP-1 inhibitor and cisplatin enhances cytotoxicity effect of cis-
platin which has been demonstrated by recent studies.22–29 In this
study, we first reported chemosensitizing effect of PARP-1 inhibitors
on long-term cisplatin-resistant gastric cancer cells.
PARP-1 consists of three main domains: the N-terminal DNA
binding domain, the auto-modification domain and the C-terminal
catalytic domains. Most of PARP-1 inhibitors imitate interaction
between PARP-1 catalytic domain with its substrate, NAD+. Early
PARP-1 inhibitors were analogues of 3-amino benzamide because
it was observed that the benzamide moiety was crucial for the spe-
cific binding to the enzymatic site, forming three key hydrogen
bonds to the enzyme. From observations that binding affinity
would be significantly increased when the carboxamide group,
which is normally free to rotate, was restricted into lactam, many
classes of PARP-1 inhibitors have been discovered such as amino-
ethyl pyrrolo dihydroisoquinolonone, tricyclic quinoxalinone,
⇑
Corresponding author. Tel.: +82 31 290 7719; fax: +82 31 292 8800.
E-mail address: hyunju85@skku.edu (H.-J. Park).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.094

T. V. T. Le et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2642–2646
2643
Good acceptor
carbonyl group
(a)
(b)
O
N
O
X
H
At least one free
NH group
N
Electron-rich
aromatic ring
N
R
X
Non-cleavable
bond
X = H, OH, OCH3, NH2, and etc.
Figure 1. (a) Requirements for PARP-1 inhibitors. (b) Pharmacophore query.
(a)
(b)
Ser904
Gly863
Gly863
Asn868
Asn868
Arg878
Arg878
Asp770
Asp770
Tyr907
Tyr907
Asn767
Ala880
Asn767
Ala880
Tyr896
Tyr896
Leu769
Leu769
Pro881
Pro881
Figure 2. (a) FlexX-docked pose45 of D31 and D36 into active site of PARP-1. FR257517 (yellow) in the X-ray crystal structure of PARP-1 (PDB ID: 1UK0) are showed for
comparison. Hydrogen bonding interaction between D31 (atom type) and D36 (orange) are represented in yellow dotted lines. (b) Lipophilic potential surface map of the
catalytic site pocket of PARP-1 is demonstrated in the docking model of D31 and D36. Lipophilicity increases from blue (hydrophilic) to brown (lipophilic).
Table 1
quinazolinone, phthalazinone, benzimidazole, indole, and isoquin-
oline derivatives.35–43 Analysis of different classes of PARP-1 inhib-
itors had suggested three structural features which must be taken
into design of PARP-1 inhibitors: (i) electron-rich aromatic ring, (ii)
a non-cleavable bond in the 3 position relative to carboxamide
group, (iii) carboxamide moiety which is free to rotate or restricted
to ring system.2 Based on the model, we created a pharmacophore
query (Fig. 1b) in which the quinazolinone core makes a sand-
PARP-1 inhibitors identified from Korean Chemical Bank
44
ID
Chembank
ID
Hit Structure
IC50
M)
(
l
F
O
D04 6338MJ0004
D07 2139SI0007
D30 6254SO0030
0.304
8.886
0.189
NH
N
N
O
wiched hydrophobic interaction, including
the phenyl ring of tyrosine residues Tyr907 and CH–
with Cb of Tyr869, the oxygen of carbonyl group will form hydro-
gen bonds with Ser904O and Gly863NH whereas NH moiety
p–
p
interaction with
p
interaction
NH
NH
NH
c
N
O
forms hydrogen bond with Gly863C@O. The side chain consists
of at least two carbon units allows the R group to reach the deep
pocket located in the auto-modification domain of PARP-1. The
pharmacophore-based virtual screening was performed against
Korea Chemical Bank (http://www.chembank.org/) chemical data-
base containing about 5 million chemicals using the Unity program
in Sybyl 8.1. Only 27 compounds satisfied the pharmacophore
query (Fig. 1b), and they were subjected to PARP-1 inhibitory as-
say.34 Seven hit compounds were identified and the two most po-
tent compounds showed nanomolar IC50 values (Table 1).
However, these two compounds, D31 and D36, were registered
for patent by KuDo pharmaceutical company for PARP-1 inhibitory
activity. The other compounds except D30, have not been patented
but they have already been reported in the literatures.40–43
Drug resistance is one of the greatest obstacles in cancer ther-
apy. Due to their ability to transform necrotic cell death into apop-
totic cell death, PARP-1 inhibitors enhance the effect of different
DNA-alkylating agents such as topoisomerase I inhibitors, doxoru-
bicin, cisplatin, oxaliplatin, gemcitabine, etc.24–31 To examine
whether PARP-1 inhibitors enhance the effect of cisplatin in resis-
tant cell lines, we conducted chemosensitizing experiments of
PARP-1 inhibitor against cisplatin-resistant cell line. For this exper-
iment, cisplatin-resistant human gastric cancer cell line (YCC-3/D)
O
N
N
N
O
F
D31 6255SO0031
D32 6256SO0032
0.077
1.008
N
N
N
N
O
Cl
NH
NH
N
N
N
O
D36 6355SO0036
D38 6357SO0038
0.079
0.388
N
O
Cl
NH
N
N

2644
T. V. T. Le et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2642–2646
Table 2
IC50 values of cisplatin against YCC-3 versus YCC-3/D after a 36-h treatment46
Cell line
IC50
(
lg/mL)
RF
YCC-3
12.3
2.137
YCC-3/D
26.29
was established by treating parent cell line (YCC-3) with cisplatin
0.5 g/mL for 18 months and then increasing cisplatin concentra-
tion at each 0.5 g/mL increment and fixed at 2.5 g/mL for an-
l
l
l
other 6 months. Dose-dependent analysis against cisplatin of
YCC-3/D showed that the YCC-3/D was less sensitive to cisplatin
comparable to the parent cell line YCC-3 with RF (resistance factor)
value around 2 (RF value26 was calculated by ratio between IC50
value of YCC-3/D and IC50 value of YCC-3 against cisplatin) (Ta-
ble 2). PARP-1 inhibitor D31 and D36 were re-synthesized for che-
mo-sensitization experiments (see Supplementary data for the
details of synthetic methods and structural characterization of
compounds). The treatment of D31 or D36 alone was not cytotoxic
in YCC-3/D. The cytotoxicity IC50 values of D31 and D36 against
Figure 5. Western blot50 with PAR antibodies showing the reduction of PAR
formation by PARP-1 inhibitors in YCC-3/D cells. The PARP-1 antibody detects two
bands, 113 kDa PARP-1 and 89 kDa apoptosis-induced cleavage fragment of PARP-1.
but it did not improve the cytotoxicity of cisplatin in YCC-3/D cells.
To verify whether D31 and D36 inhibit the PARP-1 enzymatic
activity in YCC-3/D cells, Western blot analysis was conducted. In
the Western blot (Fig. 5), the cleavage of PARP-1 into 89-kD and
24-kD fragments by caspase-3 was detected in YCC-3/D cells,
which is a characteristic of apoptosis.48,49 In response to cisplatin
treatment, PARP-1 is activated and results in the synthesis of
poly(ADP-ribose) polymer (PAR) on acceptor proteins.1–4 Our
results revealed the decrease in PAR formation in the presence of
PARP-1 inhibitors, D31 and D36, in YCC-3/D cells.
YCC-3/D are above 200
the concentration of 50
sensitization experiments. Pre-treatment of 50
tors, D31 and D36, significantly decreased IC50 values of cisplatin,
from 26.29 to 17.14 g/mL and 24.59–15.19 g/mL, respectively,
l
l
M and around 100
M was chosen for the following chemo-
M PARP-1 inhibi-
lM (Fig. 3). Therefore,
l
l
l
in YCC-3/D cells (Fig. 4). In contrast, no significant sensitization ef-
fects were obtained by pre-treatment of PARP-1 inhibitors into the
parent YCC-3 cells (Supplementary data). We also tried co-treat-
ment of the same concentration of D31 and D36 with cisplatin,
In summary, we reported here an identification of potent
PARP-1 inhibitors through pharmacophore-based virtual screening
of Korean Chemical Database. The two most potent PARP-1 inhibi-
tors, D31 and D36, effectively improved the sensitivity of
Figure 3. Cytotoxic effect of PARP-1 inhibitors against cisplatin-resistant human gastric cancer cell line YCC-3/D.46 IC50 values were determined after exposure of YCC-3/D to
PARP-1 inhibitors for 36 h.
Figure 4. Chemosensitizing effect47 of PARP-1 inhibitors, D31 and D36 against YCC-3/D. YCC-3/D cells were pre-treated with PARP-1 inhibitors for 8 h, and then treated with
cisplatin for another 36 h to determine IC50 values.

T. V. T. Le et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2642–2646
2645
37. Penning, T. D.; Zhu, G. D.; Gandhi, V. B.; Gong, J.; Liu, X.; Shi, Y.; Klinghofer, V.;
Johnson, E. F.; Donawho, C. K.; Frost, D. J.; Bontcheva-Diaz, V.; Bouska, J. J.;
Osterling, D. J.; Olson, A. M.; Marsh, K. C.; Luo, Y.; Giranda, V. L. J. Med. Chem.
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ing PARP-1 catalytic activity.
38. Menear, K. A.; Adcock, C.; Alonso, F. C.; Blackburn, K.; Copsey, L.; Drzewiecki, J.;
Fundo, A.; Le Gall, A.; Gomez, S.; Javaid, H.; Lence, C. F.; Martin, N. M.;
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Acknowledgments
This study was supported by Basic Science Research Program
through the National Research Foundation of Korea(NRF) funded
by the Ministry of Education, Science and Technology (Grant
2010-0029358), and in part by a grant of the Korea Healthcare
technology R&D Project, Ministry for Health & Welfare, Republic
of Korea (Grant A092006).
40. KuDos Pharmaceuticals, WO 02/48117, 2002.
41. Hattori, K.; Kido, Y.; Yamamoto, H.; Ishida, J.; Kamijo, K.; Murano, K.; Ohkubo,
M.; Kinoshita, T.; Iwashita, A.; Mihara, K.; Yamazaki, S.; Matsuoka, N.;
Teramura, Y.; Miyake, H. J. Med. Chem. 2004, 47, 4151.
42. Hattori, K.; Kido, Y.; Yamamoto, H.; Ishida, J.; Iwashita, A.; Mihara, K. Bioorg.
Med. Chem. Lett. 2007, 17, 5577.
43. Ishida, J.; Yamamoto, H.; Kido, Y.; Kamijo, K.; Murano, K.; Miyake, H.; Ohkubo,
M.; Kinoshita, T.; Warizaya, M.; Iwashita, A.; Mihara, A.; Matsuoka, N.; Hattori,
K. Bioorg. Med. Chem. Lett. 2006, 14, 1378.
Supplementary data
44. PARP-1 inhibitory assay34: Into the wells of a 96-well plate, 20
l
L of a 250 nM
solution of NAD+ in PARP assay buffer (pH 8.00) consisted of 50 mM Tris and
2 mM MgCl2, 10 L of activated DNA at a concentration of 50 g/mL dissolved
in PARP assay buffer, and 10 L of the inhibitors at different concentrations in
PARP assay buffer were added. The reaction was initiated by adding PARP-1 at
a concentration of 2 g/mL, 10
g/mL DNA, and 100 nM NAD+ with various
concentrations of inhibitors in a total volume of 50 L. The plate was incubated
for 15 min at room temperature and the amount of NAD+ remaining was
determined by adding 20 L of KOH 2 N and 20 L of acetophenone 20%. After
being incubated at 4 °C for 10 min, 90 L of formic acid (88%) was added to the
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
02.094.
l
l
l
l
l
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l
reaction mixture and heated at 100 °C for 10 min. The plate was allowed to
cool to room temperature and fluorescent intensity was read on a SPECTRAmax
GEMINI XS microplate spectrofluorometer at excitation wavelength of 400 nm
and emission wavelength of 445 nm.
45. Virtual screening and docking experiments: Protein modeling and docking
experiments have been performed with the Sybyl 8.1 software package
(Tripos, Inc., St. Louis, MO, USA) based on Linux CentOS 4.0. To select the
quinazoline-based candidate PARP-1 inhibitos, the pharmacophore search was
conducted against Korea Chemical Bank database using the Unity program in
Sybyl 8.1. The structures of D31 and D36 were prepared in MOL2 format using
the sketcher module and Gasteiger–Huckel charges were assigned to the ligand
atoms. The structure of D31 and D36 were optimized by energy minimization
until a convergence value of 0.001 kcal/(Å mol).The X-ray coordinate of PARP-1
complexed with FR257517 inhibitor (PDB ID: 1UK0)33 was retrieved from the
PDB, one monomer and all crystallographic water molecules were removed.
After the hydrogen atoms and atomic charges were added to the receptor, side
chain amides of PARP-1 were fixed. The active site was defined as all the amino
acid residues enclosed within 6.5 Å radius sphere centered by the bound
inhibitor FR257517. The docking was performed using the default parameters
of the FlexX programs implanted in the sybyl 8.1, and subsequent scoring for
FlexX solution was conducted by a consensus scoring function (CScore). One of
the conformers of D31 and D36 having the highest consensus score
(CScore = 5) were selected and complexed with PARP-1, resulting in a final
model (Fig. 2).
46. Cytotoxicity effect of cisplatin and PARP-1 inhibitors: YCC-3 and YCC-3/D cells32
were seeded into 96-well plate at a density of 5000 cells/well and incubated at
37 °C/5% CO2 overnight. PARP-1 inhibitors or cisplatin were treated to cells and
incubated for 36 h at 37 °C/5% CO2. To determine the proportion of cell death,
the cisplatin-containing media was removed and 100
10 L of a 1.90 mg/mL MTS solution (Promega CellTiter 96AQueous One Solution
Cell Proliferation assay) was added to each well, which was followed by
incubation for 2 h at 37 °C. The absorbance was determined using
lL of medium containing
l
a
microculture plate reader (ELISA reader) at 490 nm. Each assay was
performed in triplicate. The drug concentration required to inhibit cell
proliferation by 50% (IC50) was determined by plotting the percentage of cell
growth inhibition vs. the drug concentration.
47. Chemosensitizing effect of PARP-1 inhibitors: YCC-3/D cells32 were seeded into
96-well plate at a density of 2500 cells/well and incubated at 37 °C/5% CO2
overnight. PARP-1 inhibitors were treated to cells for 8 h. After removing
media containing PARP-1 inhibitors, cisplatin was added to cells at
indicated concentration and incubated for 36 h at 37 °C/5% CO2. To
determine the proportion of cell death, the cisplatin-containing media
was removed and 100 lL of medium containing 10 lL of a 1.90 mg/mL MTS
solution (Promega CellTiter 96AQueous One Solution Cell Proliferation assay)
was added to each well, which was followed by incubation for 2 h at 37 °C.
The absorbance was determined using a microculture plate reader (ELISA
reader) at 490 nm. Each assay was performed in triplicate. The drug
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concentration required to inhibit cell proliferation by 50% (IC50
) was
determined by plotting the percentage of cell growth inhibition versus the
chemotherapeutic drug concentration.
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50. Western blot analysis: The cell lysates were boiled in a Laemmli sample buffer
for 5 min at 95 °C, and 30 lg of the protein lysates from each sample were
electrophoresed on an 8% SDS–polyacrylamide gel and transferred to a PVDF
membrane (Bio-Rad) with a constant 80 V for 90 min. The membrane was pre-
blocked in TBST containing 5% skim milk powder for 1 h at room temperature.
The blot was incubated for 3 h at room temperature with a 1:1000-diluted

2646
T. V. T. Le et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2642–2646
monoclonal antibody to human PARP-1 (BD Biosciences, Becton Dickinson
anti-mouse IgG whole antibody (Amersharm Bio). Then, the blot was washed
three times for 10 min each time with TBST. Protein was detected using an
enhanced chemiluminescence Western blot analysis system (Amersharm Bio).
Korea), 1:1000-diluted PAR antibody (Trevigen, Kormed Corp.), and beta actin
(Abcam, Inc.) in TBST. The blot was washed three times for 10 min each time
with TBST. The membrane was incubated for 1 h with peroxidase-conjugated