Structure-based design of new poly (ADP-ribose) polymerase (PARP-1) inhibitors

Article abstract of DOI:10.1007/s11030-017-9754-7

Poly (ADP-ribose) polymerase (PARP-1) is a well-established nuclear protein with prominent role in signaling and DNA repair. Various clinical candidates have been identified with the role in PARP-1 inhibition. Based on the pharmacophoric features identified from previous studies and molecular docking interactions, thiazolidine-2,4-dione derivatives have been evaluated for their PARP inhibitory activity. From an in vitro assay, 5-((1-(4-isopropylbenzyl)-1H-indol-3-yl)methylene)thiazolidine-2,4-dione (16) was identified as a potent inhibitor having low micromolar inhibitory activity (IC50=0.74±0.25μM). Thus, a structure-based design approach utilized in the present study helped to identify thiazolidine-2,4-dione as a novel scaffold against PARP-1 for potential development of potent anticancer therapeutics.

Full text of DOI:10.1007/s11030-017-9754-7

Mol Divers
DOI 10.1007/s11030-017-9754-7
ORIGINAL ARTICLE
Structure-based design of new poly (ADP-ribose) polymerase
(PARP-1) inhibitors
Navriti Chadha1 · Ameteshar Singh Jaggi2 · Om Silakari1
Received: 14 October 2016 / Accepted: 27 May 2017
© Springer International Publishing Switzerland 2017
Abstract Poly (ADP-ribose) polymerase (PARP-1) is a
well-established nuclear protein with prominent role in sig-
naling and DNA repair. Various clinical candidates have been
identified with the role in PARP-1 inhibition. Based on the
pharmacophoric features identified from previous studies
and molecular docking interactions, thiazolidine-2,4-dione
derivatives have been evaluated for their PARP inhibitory
activity. From an in vitro assay, 5-((1-(4-isopropylbenzyl)-
1H-indol-3-yl)methylene)thiazolidine-2,4-dione (16) was
identified as a potent inhibitor having low micromolar
Introduction
Poly (ADP-ribose) polymerase 1 (PARP-1) is the most
abundant enzyme of the PARP family and is a key player
for posttranslational modification of proteins in response
to DNA damage and genotoxic stress [1]. The modular
architecture of PARP-1 consists of three domains: (1) a
DNA binding domain (DBD) or N-terminal region con-
sisting of three zinc motifs with a role in the recognition
of DNA damage and direct PARP-1 binding to damaged
inhibitoryactivity(IC50 = 0.74 0.25 μM). Thus, astructure- DNA, (2) an automodification domain (AMD) with a role
based design approach utilized in the present study helped
to identify thiazolidine-2,4-dione as a novel scaffold against
PARP-1 for potential development of potent anticancer ther-
apeutics.
in the interaction between PARP-1 and its partner proteins
and (3) a catalytic domain or C-terminal region consisting
of a NAD+ binding domain responsible for the catalytic
function of PARP-1 [2]. Elevated levels of DNA damage
stimulate PARP-1-induced poly (ADP-ribose) production,
thereby exhausting the energy pools of NAD+ and ATP
leading to cellular energy crisis, irreversible cytotoxicity
and necrotic cell death [3]. The dramatic consequences of
PARP-1 hyperactivation are frequently observed in vari-
ous cancerous conditions, myocardial reperfusion injury,
cardiac ischemia, stroke and diabetes-associated anomalies
[4–6].
Keywords Structure-based drug design · Docking ·
Thiazolidine-2,4-dione · Indole · Molecular modeling ·
PARP
An investigation of the X-ray crystallographic structure
of PARP-1 in complex with various inhibitors as well as
NAD+ indicates the importance of three hydrogen bond-
ing interactions between a carboxamide moiety and critical
amino acid residues Gly202 (N–H to Gly C=O and C=O
to Gly N–H) and Ser243 (C=O to Ser O–H). In addition,
Electronic supplementary material The online version of this
article (doi:10.1007/s11030-017-9754-7) contains supplementary
material, which is available to authorized users.
Om Silakari
omsilakari@gmail.com
B
π
π
stacking of the aromatic ring system of the inhibitor
−
moiety with Tyr246 was found in all available crystal struc-
tures [7]. Several PARP-1 inhibitors are being investigated
for their effectiveness and some are undergoing clini-
cal investigation, including indazole-7-carboxamide (nira-
parib, MK-4827) [8], phthalazinone (talazoparib, BMN-673;
1
Molecular Modeling Lab (MML), Department of
Pharmaceutical Sciences and Drug Research,
Punjabi University, Patiala 147002 Punjab, India
2
Department of Pharmaceutical Sciences and Drug Research,
Punjabi University, Patiala 147002, India
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Fig. 1 Structures of PARP-1 inhibitors under clinical evaluation
olaparib, AZD-2281) [9,10], benzimidazole-4-carboxamide
(veliparib, ABT-888) [11] and azepinoindolone (rucaparib,
RG014699) [12] (Fig. 1). These compounds are bicyclic or
tricyclic structures with a crucial carboxamide group either
rotationally constrained substituted on the ring or incorpo-
rated into the ring system to form a lactam.
[14], herein we report the design, synthesis and in vitro
activity of new thiazolidine-2,4-dione derivatives against
PARP-1 enzyme. The TZD ring was chosen to provide
the crucial for hydrogen bonding interaction with Gly202
and Ser243 of the PARP-1 active site. The TZD ring sys-
tem was substituted with an indole ring to provide a ring
π
π
stacking with Tyr246. The hydrophobic
Thiazolidine-2,4-dione (TZD) is a well-established het-
erocyclic ring system exhibiting a wide range of phar-
macological activities including anti-hyperglycemic, anti-
cancer, anti-arthritic and antimicrobial. The scaffold has
shown its effectiveness against a broad spectrum of protein
targets including peroxisome proliferator-activated recep-
system for
−
pocket was observed to be fairly large; thus, the indole moi-
ety was further substituted with different benzyl groups.
The general structure of compounds 11–18 is displayed in
Scheme 1.
γ
tor (PPAR ), aldose reductase (ALR2), phosphoinositide
γ
α
3-kinase (PI3K ), phosphoinositide 3-kinase (PI3K )/
mitogen-activated protein kinase kinase (MEK), PIM kinase
and protein tyrosine phosphatase 1B (PTP1B). The chemical
structure of TZD and various substitutions on the scaffold
provide pharmacophore guidance for targeting various pro-
tein targets [13].
Results and discussion
PARP-1 inhibitor design
An alanine scanning mutagenesis and pharmacophore anal-
ysis of PARP-1 inhibitors previously carried out by our
group led to the identification of common pharmacophore
required for activity characterized by the presence of a ben-
Based on the pharmacophore analysis and active site
fingerprinting of PARP-1 reported in our previous studies
Scheme 1 Synthesis of thiazolidine-2,4-dione analogs
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DMF. The intermediates formed were purified by column
chromatography using silica gel (100–200 mesh) and ethyl
acetate and petroleum ether as solvent system. The presence
of two singlets corresponding to the aliphatic benzyl proton
(∼5 ppm) and aldehyde proton (∼10 ppm) in the 1H-NMR
spectrum of the intermediates confirms the formation of the
desired products. Intermediates 3–8 are reported in the lit-
erature [19–22], and their physicochemical properties (e.g.,
melting point) were found to be slightly different likely due to
environmental fluctuations and manual errors. Subsequently,
the substituted indole aldehydes were condensed via a Kno-
evenagel condensation reaction with thiazolidine-2,4-dione
in the presence of β-alanine using acetic acid as solvent. The
synthesis of the final compounds was confirmed by 1H-NMR
based on the presence of a broad singlet at ∼12 ppm corre-
sponding to the NH proton and a sharp singlet at ∼5.5 ppm
corresponding to the aliphatic benzyl protons. 13C spectrum
recorded two low-intensity signals at ∼167 ppm correspond-
ing to two carbonyl groups and one signal at ∼49 ppm for
the alkyl carbon. The benzyl chlorides considered for the
reaction were substituted with different hydrophobic groups,
including isopropyl, trifluoromethyl, methyl, chloro and flu-
oro.
Fig. 2 Pharmacophoric and interaction features required for the design
of TZD-based PARP inhibitors
zamide group and an aromatic ring system [14]. In this
context, thiazolidine-2,4-dione was chosen to provide the
amide group required to form three hydrogen bond inter-
actions with Gly202 and Ser243 amino acid residues. This
TZD ring was condensed with an indole nucleus to pro-
π
π
stacking interaction with the aromatic amino
vide a
−
acid Tyr246 (Fig. 2). Further, a benzyl ring with varied sub-
stituents was attached to the indole nitrogen and the impact of
these substituents was assessed in in vitro assay results. The
designed molecules were subjected to docking simulation
using CDOCKER software in Discovery Studio (DS); the
docking poses were first scrutinized for essential interactions
and further optimized using the Prime MM-GBSA algorithm
[15–18]. The CDOCKER binding energy and MM-GBSA
scores of the synthesized molecules are listed in Table 1.
Biochemical activity
A PARP-1 enzyme assay indicates that compounds 12, 13,
15, 16, 17 and 18 have inhibitory activities comparable
to 3-aminobenzamide (reference) (Table 1). Comparison of
the docking results with the observed experimental PARP-
1 inhibition showed that embedding hydrophobic groups
(e.g., ethyl, isopropyl, tert-butyl) improved PARP inhibi-
tion. Substitutions at the R position of the TZD scaffold
showed that ethyl, isopropyl, tert-butyl, trifluoromethyl and
chloro groups improved inhibitory activity according to their
respective lipophilic character. Moreover, the docked con-
Chemistry
Compounds 11–18 were synthesized using a two-step reac-
tion process depicted in Scheme 1. In the first step, indole-
3-carboxaldehyde was benzylated using substituted benzyl
chlorides at the N-H position in the presence of the nucle-
ophilic agent 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in
Table 1 Inhibitory activities of
compounds 11–18 against
PARP-1 enzyme
Compound
R
CDOCKER
interaction energy
MM-GBSA
score
IC50 (μM)
11
H
−39.29
−43.67
−41.90
−43.77
−44.72
−47.04
−50.21
−45.62
−67.48
−69.14
−66.48
−71.64
−72.07
−77.48
−87.62
−79.86
>100
12
3-Cl
4.81 2.31
5.03 2.19
13.95 6.59
3.07 2.62
0.74 0.25
0.82 0.11
1.56 0.54
4.78
13
4-F
14
4-CH3
3-CF3
15
16
4-CH(CH3)2
4-(CH3)3
4-CH2CH3
17
18
3-Aminobenzamide
IC50 represents the dose of a compound in mole required to produce 50% inhibition of poly (ADP-
ribose)polymerase enzyme. IC50 values expressed as mean SD, n = 2. MM-GBSA score shows the binding
energy of the inhibitor to the protein expressed as kcal/mol
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Fig. 3 Docking interactions of hit compound 16 at the PARP-1 active site: a 3D view, b 2D view, c surface view
formations obtained for these structures confirmed that the
larger groups could be accommodated well into the PARP-1
pocket. Docking of compound 16 (IC50 = 0.74 0.25 μM)
indicates that this analog fits well in the pocket (interac-
tions shown in Fig. 3). However, while tert-butyl-containing
compound 17 (IC50 = 0.82 0.11 μM) exhibited similar
inhibition to compound 16 (IC50 = 0.74 0.25 μM), it did
not improve PARP inhibition. This may be due to the steric
clashes of the tert-butyl group within the active site of PARP-
1. Thus, biochemical activity results show that a substitution
of the hydrophobic group improves potency to submicromo-
lar range and an isopropyl substituent is optimum for activity
(IC50value = 0.74 μM).
Material and methods
Chemistry
Chemicals and solvents utilized were of analytical grade
or dry distilled. Reactions were monitored using thin-layer
chromatography (TLC) with n-hexane/ethyl acetate (7:3) as
mobile phase using pre-coated Merck silica gel aluminum
sheets (Merck, India) and were visualized in an UV chamber.
Synthesized compounds were purified using column chro-
matography using silica gel (mesh 100–200). Melting points
were determined using open tubes in a paraffin bath and are
uncorrected. 1H-NMR and 13C-NMR spectra were recorded
on a Bruker AC 300 NMR Spectrometer (400 and 100 MHz,
respectively), and chemical shift ( ) values were recorded as
δ
parts per million (ppm) using tetramethylsilane as internal
standard. NMR abbreviations used are: singlet (s), doublet
(d), triplet (t), quartet (q), quintet (quin.) and multiplet (m).
Coupling constants are proved in Hertz (Hz). FTIR spectra
were recorded on an FTIR Perkin-Elmer 1710 series as KBr
pellets and signals recorded at cm−1 scale. Mass spectra were
recorded on a Micromass Q-TOF spectrometer (Waters, MA,
USA) in positive electrospray ionization (+ESI) mode. Melt-
Conclusion
The investigation of the PARP-1 active site inspired us to
design several new small thiazolidine-2,4-dione analogs for
PARP inhibition using structure-based as well as knowledge-
based approach. Compound 16, with an isopropyl group,
emerged as an attractive lead compound with an IC50 value
of 0.74 0.25 μM.
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ing points were determined using open tube method and the
values were uncorrected.
mass spectrum of compound 16 showed quasi-molecular ion
signal for [M+H]+ at m/z 377.21 forming the base signal
(100%). The spectral data of compounds 11–18 are listed in
the Supplementary material.
Synthesis of compounds 3–10
For the synthesis of intermediates 3–10, indole-3-
carboxylaldehyde (5 mmol) was dissolved in appropriate
quantity of dimethylformamide (DMF) followed by addi-
tion of 2.4 mL of 1,8-diazabicycloundec-7-ene and 6 mmol
benzyl chloride. The mixture was refluxed for >20 h and the
product was extracted by fractionation from ethyl acetate and
water. The organic layer was concentrated to give crude prod-
uct which was then purified using column chromatography
(petroleum ether/ethyl acetate, 9:1). These compounds were
characterized by 1H NMR spectroscopy where the presence
of a singlet at ∼9 ppm confirms the presence of aldehyde
group of indole while a sharp singlet appeared at ∼5.5 ppm
for CH2 of benzyl group. The spectral data of compounds
3–10 are listed in Supplementary material.
Biochemical activity
IC50 values for PARP inhibition were determined by using
a Trevigen HT Colorimetric PARP ELISA Assay kit (Cat#
4677-0960K). The assay was performed in 96-well histone-
coated strip wells, according to the protocol provided by
Trevigen. The strip wells were first rehydrated using the
provided PARP buffer solution and incubated for 30 min.
This was followed by adding a serial dilution of inhibitors,
diluted PARP enzyme and a PARP cocktail (containing acti-
vated DNA, biotinylated NAD). The reaction was allowed
to proceed for 60 min at room temperature. The strip wells
were then washed with PBS and 0.1% Triton X-100 (2 times)
and PBS (2 times). Thereafter, diluted streptavidin-linked
peroxidase (Strep-HRP) was added to detect the extent of
ribosylation and the mixture was incubated for 60 min. The
plates were washed with PBS, followed by the addition of
the TACS-Sapphire colorimetric substrate and allowed to
develop color for 15 min in the dark. The reaction was
stopped by adding 0.2N HCl, and the optical densities were
read at 450 nm using an ELISA plate reader. All the data
points were determined in triplicate, and the IC50 value was
obtained by plotting the % inhibition against log concentra-
tion values.
Synthesis of compounds 11–18
For the synthesis of final compounds, compounds 3–10
obtained from the previous step (0.3 mmol), β-alanine
(6.0 mmol), thiazolidine-2,4-dione (6.0 mmol) and 10 mL
glacial acetic acid were refluxed in a round-bottom flask
for around 4 h. A small portion of water was added, the
precipitate was collected by vacuum filtration, washed with
glacial acetic acid, distilled water and ether. The solids
were then dried at 40 ◦C. Further, the final compounds
11–18 were synthesized via Knoevenagel condensation in
which the nucleophilic addition of the active methylene
hydrogen of thiazolidine-2,4-dione to the carbonyl group
of substituted indole-3-carbaldehyde is carried out in the
presence of a catalytic amount of β-alanine. The most
active compound 16 (5-((1-(4-isopropylbenzyl)-1H-indol-
3-yl)methylene)thiazolidine-2,4-dione) was obtained as a
yellow solid (81% yield) and a melting point in the range of
228–230 ◦C. The compound’s IR spectrum showed the pres-
ence of band at ∼ 3400 cm−1 and strong absorption bands
at ∼ 1730 and ∼ 1680 cm−1 which were assigned to the
two carbonyl groups. The 1H NMR spectra of the compound
showed that the isopropyl protons appear as a doublet at 1.17
ppm for (CH3)2 and multiplet at 2.84 ppm for their adjacent
CH proton. The aliphatic benzyl protons appear at 5.51 ppm,
aromatic protons in the range of 7.18–8.18 ppm, while the
thiazolidine-2,4-dione NH appears at 12.24 ppm. The 13C
NMR spectrum of compound 16 showed the presence of 22
carbons. The signals at 167.35 and 167.23 ppm correspond
to the two carbonyl carbons of TZD ring, the aliphatic ben-
Acknowledgements Authors thank Council of Scientific and Indus-
trial Research (CSIR), New Delhi, for providing funds for carrying
out the research work (No. 02(0111)/12/EMR-II) and Department of
Science and Technology (DST), New Delhi, for providing INSPIRE
fellowship (No. IF150415). Authors also extend thanks to Sophisticated
Analytical Instrumentation Facility, Punjab University, Chandigarh, for
performing spectroscopic analysis and Apsara Innovations, Bengaluru,
for extending technical support for performing in silico studies.
Compliance with ethical standards
Conflict of interest Authors have no conflict of interests.
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