3-Diarylethyne quinazolinones: A new class of senescence inducers

Article abstract of DOI:10.1039/c2md20302b

A series of 3-diarylethyne quinazolinone compounds (4a-ad) were synthesized and evaluated for their cytotoxicity. They exhibited significant anticancer activity against MDA-MB-231, A549, MIAPaCa-2 and Colo-205 cancer cell lines and were highly effective in the MCF-7 cancer cell line with IC₅₀ values ranging from 2.27 to 3.88 μM. FACS analyses and BrdU assays revealed that there is G1 cell cycle arrest. Moreover, cells treated with these compounds show up-regulation of p53, p21, p16, TRF1 and POT1 and down-regulation of SKP2, TRF2 and tankyrase protein levels. To assess the senescence inducing ability of these compounds further, a senescence associated β-gal assay was performed. Interestingly, two of these compounds (4s and 4w) exhibited significant telomerase inhibition activity and inhibited tankyrase-1 protein, a protein partner associated with the sheltrin complexes of telomeres.

Full text of DOI:10.1039/c2md20302b

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CONCISE ARTICLE
3-Diarylethyne quinazolinones: a new class of
senescence inducers†
Cite this: Med. Chem. Commun., 2013,
4, 575
a
Ahmed Kamal, Farheen Sultana,a M. Janaki Ramaiah,b Y. V. V. Srikanth,a
*
A. Viswanath,a E. Vijaya Bharathi,a Ranjita Nayak,a S. N. C. V. L. Pushpavalli,b
b
b
*
Chatla Srinivas and Manika Pal-Bhadra
A series of 3-diarylethyne quinazolinone compounds (4a–ad) were synthesized and evaluated for their
cytotoxicity. They exhibited significant anticancer activity against MDA-MB-231, A549, MIAPaCa-2 and
Colo-205 cancer cell lines and were highly effective in the MCF-7 cancer cell line with IC50 values
ranging from 2.27 to 3.88 mM. FACS analyses and BrdU assays revealed that there is G1 cell cycle arrest.
Moreover, cells treated with these compounds show up-regulation of p53, p21, p16, TRF1 and POT1
and down-regulation of SKP2, TRF2 and tankyrase protein levels. To assess the senescence inducing
ability of these compounds further, a senescence associated b-gal assay was performed. Interestingly,
two of these compounds (4s and 4w) exhibited significant telomerase inhibition activity and inhibited
tankyrase-1 protein, a protein partner associated with the sheltrin complexes of telomeres.
Received 5th October 2012
Accepted 23rd December 2012
DOI: 10.1039/c2md20302b
www.rsc.org/medchemcomm
inhibitory function.10 In this context there is a great demand for
small molecules that could cause senescence and increase CDKI
Introduction
(i.e. p16, p21 and p27) protein levels.11 These ndings relating to
senescence-induced cell death through pro-senescence therapy
could provide a very useful target for the development of small
molecule inhibitors.
Cellular senescence stops the growth of cells, which limits the
lifespan of mammalian cells and prevents abnormal cell
proliferation.1 Recent studies on the molecular analysis of
senescence in cancer revealed oncogene induced senescence
(OIS) as well as tumour-suppressor-dependent senescence
(PICS). Previously, considerable attention has been focused on
the role of DNA damaging agents that usually cause apoptosis.
However, recently, pre-mature senescence has emerged as an
excellent strategy for the development of anticancer therapeu-
tics.2–4 The process of senescence takes place by the shortening
of telomeres (i.e., replicative senescence) or by stress. Replica-
tive senescence in human cells occurs as a consequence of
progressive shortening of TTAGGG repeat tracts at chromo-
somal ends. Proteins such as TRF1, TRF2 and POT1 bind to the
telomere and protect telomeres, wherein TRF2 and POT1
mainly function in the elongation of telomeres,5,6 in which loss
of TRF2 would lead to DNA damage, cell cycle arrest and p53
activation.7,8 The p53 tumor-suppressor protein has a key role in
the induction of cellular senescence. p53 can safe guard the
cells from the formation of a tumour phenotype,9 whereas
malignant cells will proliferate rapidly due to the lack of CDK
Telomere maintenance by telomerase allows human cancer
cells to divide indenitely. In most of human somatic tissues
telomerase activity is not detectable and is highly activated in
germ and cancer cells.12,13 Shortened telomeres directly elicit a
DNA damage check point response and result in replicative
senescence.14 In telomerase positive cells telomere length is
maintained at a constant average value. Long telomeres recruit
a greater number of telomeric proteins. Over expression of
TRF1, a duplex telomeric repeat binding protein in telomere
positive cells, results in telomere shortening, whereas the
dominant negative mutant version of TRF1 induces elonga-
tion.15 Poly(ADP-ribosylation) of TRF1 by a telomeric poly(ADP-
ribose) polymerase (PARP), tankyrase1, leads to loss of its DNA
binding activity and subsequent degradation of TRF1.16,17
Tankyrase-1 diminishes the effect of telomerase inhibitors in
telomerase-positive cells. PARP inhibitors that could inhibit
tankyrase-1 and enhance telomere shortening and arrest the
cell cycle are in high demand.
Quinazolinone (1, Fig. 1) is a naturally occurring alkaloid
and is also a core structural subunit in many bioactive natural
products as well as synthetic compounds and is known to
possess antibacterial, antimicrobial, antituberculosis, antihy-
pertensive and anticancer properties.18,19 A diverse class of
quinazolinones were discovered that could target different
cellular proteins. Among them some of the compounds were
aDivision of Organic Chemistry, Tarnaka, Hyderabad 500 607, India. E-mail:
ahmedkamal@iict.res.in; Fax: +91-40-27193189; Tel: +91-40-27193157
bCentre for Chemical Biology Indian Institute of Chemical Technology, Tarnaka,
Hyderabad 500 607, India. E-mail: manika@iict.res.in; Fax: +91-40-27160387; Tel:
+91-40-27191814
† Electronic supplementary information (ESI) available: Spectral data of
compounds 7a–c, 9, 10a–c and 4a–ad and experimental procedures for
syntheses and biological evaluations. See DOI: 10.1039/c2md20302b
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Scheme 2 Reagents and conditions: (i) Ac2O, 150 ꢀC, 30 min; (ii) AcOH, reflux,
8h; (iii) aromatic aldehydes, AcOH, reflux, 8 h.
Fig. 1 Chemical structures of quinazolinone (1), 4(3H)-quinazolinone 2-styryl
substituted derivatives (2), 3-ethynyl-1H-indazoles (3), 3-diarylethyne quinazoli-
nones (4a–ad).
quinazolinone moiety with a variety of structural modications
at the C-2 position. These compounds were evaluated for their
cytotoxicity. Moreover, they were investigated for cell cycle
effects and activation of different proteins that are known to
play an important role in the regulation and execution of
senescence.
identied as dual inhibitors of P-glycoprotein (Pgp) and multi-
drug resistance associated proteins (MRP1) belonging to a
family of ABC (ATP-binding cassette) transport proteins
involved in drug resistance.20 Similarly 2-styryl substituted
4(3H)-quinazolinones (2) are an important class of pharmaco-
logically active compounds that exhibit anticancer activity by
inhibition of tubulin polymerization.21,22 A series of 2-methyl-
quinazolinones and 2-arylquinazolinones have been reported as
inhibitors of DNA repair enzyme poly(ADP-ribose) polymerase.23
Recently a new series of 3-ethynyl-1H-indazoles (3) have been
shown to exhibit signicant antiproliferative activity both in
monolayer human cancer cell cultures and in three dimen-
sional tumour models.24 Structurally similar 3-arylethynyl
substituted quinoline-8-carboxamides were identied as
human recombinant PARP-1 inhibitors.25 Structure–activity
relationship (SAR) studies in both these series revealed that the
ethynyl substituent plays an important role in cytotoxicity.
Moreover, various other reports have shown that a rigid triple
bond functionality in biologically active molecules also
contributes to the observed antiviral and antiproliferative
activities which might be due to the appended p-conjugated
systems that provides the required conformation to bind with
the corresponding biological targets.26
Table 1 Structural modifications on diarylethyne quinazolinones
Compound
R1
R
4a
4b
4c
2-Methylphenyl
4-Fluorophenyl
4-Chlorophenyl
Phenyl
Phenyl
Phenyl
4d
4e
4f
4g
4h
4i
4-Hydroxyphenyl
4-Methoxyphenyl
3,5-Dihydroxyphenyl
4-Hydroxy-3-nitro-phenyl
3,5-Dimethoxyphenyl
3,4,5-Trimethoxyphenyl
2-Naphthyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Our earlier efforts towards the synthesis of a variety of novel
compounds led to the development of efficient anticancer
agents.27–31 In continuation of these attempts and considering
the biological importance of the quinazolinone core skeleton
and the signicant role of various ethynyl substituents in
various active compounds, we herein report the synthesis of 3-
diarylethyne quinazolinone compounds (4a–ad) by incorpo-
rating various diarylethyne moieties at the N-3 position of the
4j
4k
4l
5-Nitro-2-furyl
4-Chlorophenyl
Phenyl
4-tert-Butyl phenyl
4-tert-Butyl phenyl
4-tert-Butyl phenyl
4-tert-Butyl phenyl
4-tert-Butyl phenyl
4-tert-Butyl phenyl
4-tert-Butyl phenyl
4-tert-Butyl phenyl
4-tert-Butyl phenyl
4-tert-Butyl phenyl
4-tert-Butyl phenyl
4-tert-Butyl phenyl
6-Methoxy-2-naphthyl
6-Methoxy-2-naphthyl
6-Methoxy-2-naphthyl
6-Methoxy-2-naphthyl
6-Methoxy-2-naphthyl
6-Methoxy-2-naphthyl
6-Methoxy-2-naphthyl
4m
4n
40
4p
4q
4r
4s
4t
4u
4v
4w
4x
4y
4z
4aa
4ab
4ac
4ad
4-Hydroxyphenyl
3,5-Dihydroxyphenyl
5-Nitro-2-thiophenyl
3-Hydroxy-4-methoxyphenyl
3,4-Dimethoxyphenyl
Benzo[d](1,3)dioxole
2,6-Dichlorophenyl
3,4,5-Trimethoxyphenyl
2-Naphthyl
5-Nitro-2-furyl
4-Quinolinyl
4-Fluorophenyl
2,6-Dichlorophenyl
3,4-Dimethoxyphenyl
3,4,5-Trimethoxyphenyl
2-Naphthyl
5-Nitro-2-furyl
4-Quinolinyl
Scheme 1 Reagents and conditions: (i) Pd(PPh3)4, Cul, BuNH2, ether, rt, 6 h.
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Table 2 In vitro cytotoxicity IC50a (mM) values of compounds 4i, 4p, 4q, 4s and 4wg
Comp.
MCF7b
MDA-MB-231b
A549c
MIAPaCa2d
Colo-205e
MCF-10Af
SI
3.25
3.09
4.77
6.66
4i
4p
4q
4s
4w
Doxo
2.46 Æ 0.11
3.88 Æ 0.30
2.72 Æ 0.09
2.27 Æ 0.11
2.45 Æ 0.15
0.10 Æ 0.01
5.56 Æ 0.29
5.69 Æ 0.40
5.84 Æ 0.33
5.44 Æ 0.51
5.44 Æ 0.26
0.16 Æ 0.01
6.54 Æ 0.07
5.74 Æ 0.08
6.15 Æ 0.10
5.60 Æ 0.31
5.87 Æ 0.52
0.73 Æ 0.01
6.0 1 Æ 0.52
6.80 Æ 0.33
5.89 Æ 0.13
5.29 Æ 0.07
5.21 Æ 0.08
0.14 Æ 0.01
6.24 Æ 0.21
5.19 Æ 0.09
5.68 Æ 0.30
4.85 Æ 0.34
5.44 Æ 0.42
0.37 Æ 0.01
8 Æ 0.51
12 Æ 0.23
13 Æ 0.10
15 Æ 1.23
28 Æ 2.01
31 Æ 1.05
11.4
13.0
a
b
c
IC50 values are indicated as the mean Æ SD of three independent experiments. Human breast adenocarcinoma cell line. Human lung
d
e
f
adenocarcinoma epithelial cell line. Human pancreatic cancer cell line. Colorectal adenocarcinoma. Mammary epithelial cell line (normal
cell line). g Doxo: doxorubicin.
Biological evaluation
Results and discussion
(A) In vitro cytotoxicity. Preliminary screening of all the
synthesized quinazolinones (4a–ad) was carried out to evaluate
their cytotoxicity. In this study, MCF-7 cells were treated with
compounds 4a–ad at 2 and 4 mM concentrations and tested for
cell viability. Concentrations below 2 mM have negligible effects
on the cytotoxicity (ESI, Table 1†). All these compounds
exhibited signicant cytotoxicity and some of them (4i, 4p, 4q,
4s and 4w) showed promising activity. These potential
compounds were further screened against a panel of different
human cancer cell lines (MCF-7, Colo-205, A549 [with wild-type
p53] and MDA-MB-231, MIAPaCa [with p53 mutation]).32,33
Compounds 4i, 4p, 4q, 4s and 4w exhibited strong cytotoxicity
in all ve cell lines tested with slightly higher cytotoxicity in
MCF-7 with IC50 values in the range of 2.27 to 3.88 mM.
Compound 4s with 4-tert-butyl-substitution at the 3-aryl ethynyl
moiety was found to be the most potent in the series with IC50
value of 2.27 mM against MCF-7 cancer cell lines. Similarly,
compounds 4p, 4q and 4w showed a more signicant activity
Chemistry
3-Diarylethyne quinazolinones (4a–ad) were prepared as shown
in Schemes 1 and 2. Treatment of 4-iodo-2-methylbenzenamine
(5) with various phenylacetylenes (6a–c) by employing Sonoga-
shira coupling using tetrakis(triphenylphosphine)palladium as
catalyst, CuI as cocatalyst, butyl amine as a base and ether as
solvent afforded the corresponding 2-methyl-4-(phenylethynyl)-
benzenamine intermediates (7a–c) in quantitative yields
(Scheme 1). Anthranilic acid (8) on reaction with acetic anhy-
dride at 150 ꢀC afforded the cyclized product 2-methyl-4H-benzo
[d][1,3]oxazin-4-one (9). This, upon reaction with different
2-methyl-4-(arylethynyl)benzenamines (7a–c) in acetic acid
under reux conditions, provided the corresponding 2-methyl-
3-(2-methyl-4-arylethynyl)quinazolin-4(3H)-ones (10a–c). These,
upon condensation with various substituted aromatic alde-
hydes in acetic acid at room temperature, afforded the desired
quinazolinones (4a–ad) (Table 1).
Fig. 2 Fluorescent activated cell sorting (FACS) analysis of compounds 4i, 4p, 4q, 4s and 4w. The effect of tested compounds on cell cycle distribution of MCF-7 cell line
was examined by staining the cells with propidium iodide (PI). Cells were treated with the test compounds at 4 mM for 24 h. Histograms were analyzed using Summit
Software. Doxorubicin was used as a positive control. Control cells work as negative control.
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functionalities did not result in any signicant difference in
cytotoxicity. Overall, substitutions at the 3-position of the ary-
lethynyl group can signicantly affect the cytotoxicity of these
quinazolinone compounds. The anticancer activity of these
compounds was conrmed by comparing the effect of the
compounds in MCF-7 and MCF-10A (normal cells) and the
Selectivity Index (SI) was deduced (Table 2). Previous studies
indicated that the presence of p53 is responsible for drug
sensitivity.34 Contrary to this, a recent study by Lam and co-
workers35 showed that activation of p53 is not essential for loss
of cell viability. Further studies by Cyr and co-workers36 have
clearly demonstrated that the cytotoxic effect caused by staur-
osporine and 5-uorouracil is higher in cells with mutant p53
Fig. 3 Effect of compounds 4i, 4p, 4q, 4s, 4w and Doxo on cell proliferation.
MCF-7 cells were seeded at a density of ꢁ104 cells per well and were grown for
24 h. After 24 h, the compounds 4i, 4p, 4q, 4s, 4w and Doxo were added to the than wild-type. Thus, our data clearly reveals the cytotoxicity
respective wells at a final concentration of 4 mM and left for 24 h. The colorimetry
inducing nature of the compounds.
(B) Effect on cell cycle. In order to understand the cell cycle
regulatory nature of these compounds, FACS was performed.
intensity at l 450 nm was measured and is proportional to the amount of BrdU
incorporated in cells.*** indicates p < 0.001, ** indicates p < 0.05 when compared
to control (C) untreated cells. 4s and 4w showed lower BrdU incorporation rates.
MCF-7 cells were treated with 4 mM aliquots of compounds
(4a–ad) using doxorubicin (Doxo) as the positive control for
Doxorubicin (Doxo) works as a positive control and control cells without any
treatment work as negative control.
than unsubstituted phenyl ring (4i) and 4-methoxynapthalene
containing compounds. However, structural modications on
the 2-styryl ring with phenyl and other heteroaromatic
Fig. 4 (a) Effect of compounds on senescence. MCF-7 cells were treated with
compounds 4s and 4w at 4 mM concentrations for 72 h and an SA-b-gal assay was
conducted. The morphological changes were monitored under microscope
(magnification, 40Â) for the visualization of blue colour formation, which is the
indication of cells undergoing senescence. Compounds 4s and 4w treated cells
assumed a blue coloration, which reveals that the cells are undergoing senes-
cence whereas control untreated cells which serve as negative control did not
show blue colour. Doxorubicin was used as the positive control. (b) Comparison of
p53 expression in MCF-7 (p53 WT) and MDA-MB-231 (p53mutant) cell lines. The
respective cell lines were treated with compounds 4s, 4w and Doxo at 4 mM
concentrations for 24 h. Cell lysates were collected and subjected to Western blot
analysis. b-actin was used as the loading control and doxorubicin (Doxo) as
positive control.
Fig. 5 (a) Effect of compounds 4i, 4p, 4q, 4s, 4w and Doxo on p53 expression.
MCF-7 cells were treated with compounds 4i, 4p, 4q, 4s, 4w and Doxo at 4 mM
concentrations for 24 h. Following the termination of the incubation, p53 ELISA
was conducted. Data were representing the mean Æ SD of three independent
experiments. *** indicates p < 0.001, ** indicates p < 0.01, * indicates p < 0.05
when compared to control untreated cells (C) respectively. Doxorubicin (Doxo)
was used as a positive control and untreated control cells (C) work as negative
control. (b) Effect of compounds 4i, 4p, 4q, 4s, 4w and Doxo on various proteins.
MCF-7 cells were treated with compounds 4i, 4p, 4q, 4s, 4w and Doxo at 4 mM
concentrations for 24 h. The cell lysates were collected, subjected to Western blot
analysis and the levels of protein (p53, p21, p16, SKP2) were observed using
specific antibodies. b-Actin was used as loading control and doxorubicin (Doxo)
as a positive control. Untreated control cells (C) work as negative control.
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24 h. Although all the compounds in the series caused G1 cell
cycle arrest (ESI, Table 2 and Fig. 1†), compounds 4i, 4p, 4q, 4s
and 4w displayed a strong G1 phase cell cycle arrest (around
90% cells accumulate in the G1 phase) while the control cells
treated with DMSO showed only 70% G1 phase cells (Fig. 2). To
further conrm the G1 cell cycle arrest properties of these
compounds, a BrdU cell proliferation assay was conducted with
the above representative compounds at 4 mM concentrations for
24 h and the optical density (OD) was measured for the incor-
porated BrdU content. Among them, 4s and 4w treated cells
exhibited lower BrdU incorporation rates (Fig. 3). Thus, these
two compounds were considered as highly effective.
(C) Inducers of senescence. The strong irreversible G1 cell
cycle arrest prompted us to examine the senescence inducing
nature of the compounds. Thus the MCF-7 cells were incubated
with effective compounds 4s and 4w for 72 h at 4 mM concen-
trations and the senescence associated b-gal assay,37
a
biomarker assay, was conducted. Compound treated cells
exhibited blue coloration and adopted a attened morphology
with spread out cytoplasm (Fig. 4a) which are typical morpho-
logical changes associated with cellular senescence. Untreated
cells do not turn blue. In order to prove the requirement of p53
for the senescence activity, we used K562 cells in which p53 is
absent (K562) wherein low levels of senescence were observed in
doxorubicin (Doxo) and 4s as well as 4w treated cells. (ESI,
Fig. 2†). p53 induction was further compared in p53 positive
(MCF-7) and p53 mutant (MDA-MB-231) cells using Doxo, 4s
and 4w. It was observed that p53 induction is much higher in
cells expressing wild-type p53 rather than mutant cells (Fig. 4b).
(D) Effect on CDK inhibitors and p53. The senescence
program is controlled by major proteins such as p53, p21 and
p16 (INK4a) and contributes to the outcome of chemotherapy.
p16 protein serves as a marker for senescence.38 Mammalian
cells telomere shortening initiates senescence by activating p53,
the central regulator of cellular stress.39 Moreover the expres-
sion of p21, p16 and p27 has been implicated in chemotherapy
induced cell cycle arrest.40,41 Interestingly, we observed G1 cell
cycle arrest as well as senescence for these compounds. This
prompted us to further examine the expression pattern of p53,
p21, p16 and SKP2 proteins that are implicated in G1 cell cycle
arrest and senescence. We observed an increase in the expres-
sion of p53, p21 and p16 and a decrease in the expression of
SKP2. These observations were in accordance with the previous
studies on ordonin which induces senescence in colorectal
cancer cells (HCT-116, SW-116) by up-regulating p21 and p1642
(Fig. 5a and b).
Fig. 6 (a) Effect of compounds on DNA damage at telomeres. MCF-7 cells were
incubated with compounds 4i, 4p, 4q, 4s, 4w and Doxo at 4 mM concentrations
for 24 h. Cell lysates were harvested and subjected to Western blot analysis using
antibodies for TRF1, TRF2, POT1, tankyrase-1, g H2AX and b-actin. Doxorubicin
(Doxo) was used as positive control and untreated control cells (C) work as
negative control. (b) Mode of action of 3-diarylethyne quinazolinone compounds.
Compounds 4i, 4p, 4q, 4s and 4w inhibit tankyrase-1 (i.e. PARP-5a inhibition)
which causes an increase in the level of TRF1 binding to telomeres, which ulti-
mately ends in telomere shortening, which further results into a senescence
phenotype due to an increase in the protein levels of p16 and p21. (c) Effect of
compounds 4i, 4p, 4q, 4s, 4w and Doxo on telomerase activity. MCF-7 cells were
treated with compounds 4i, 4p, 4q, 4s, 4w and Doxo at 4 mM concentrations for
24 h. Cell lysates were harvested and subjected to telomerase assay (TRAP eze XL
telomerase assay). The fluorescence emission/excitation at 495/516 nm (F) and
600/620 nm (R) was measured. The F/R ratio gives telomerase activity. Data
represent the mean Æ SD of three independent experiments. The compounds (4s
(E) DNA damage at telomeres
The dsDNA portion of telomeres is bound directly by two
proteins, TRF1 and TRF2 and telomeric ssDNA is bound directly
and 4w) were found to decrease telomerase enzymatic activity; *** indicates p <
0.001, ** indicates p < 0.01, * indicates p < 0.05 when compared to control
untreated cells (C). Doxorubicin (Doxo) was used as a positive control and
untreated control cells (C) work as negative control. The lysate from control cells
which have been heat treated (C + heat), thereby losing their telomerase activity,
also works as positive control.
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by the protein POT1.43 In general, low g H2AX signals were
observed at telomeres of proliferating cells, whereas upon DNA
damage response (DDR) activation of g H2AX signals were
observed which led to the recruitment of DNA damage response
proteins. Thus we conducted Western blot analyses to observe
the level of DNA damage at the telomeres by measuring the
protein levels of g H2AX, which is an indicator of DNA damage
at the telomere. An increased expression of g H2AX signals was
observed, which revealed DNA damage at the telomeres. Thus,
in this scenario, we became interested in examining the effect of
these compounds (4i, 4p, 4q, 4s and 4w) on telomere associated
proteins such as TRF1, TRF2 and POT1. An increase in the level
of TRF1, POT1 with concomitant decrease in TRF2 was
observed. Tankyrase-1 is a protein involved in regulating the
access of telomerase to telomeres by poly ADP-ribosylating TRF-
1. Thus, we examined the expression level of tankyrase-1 in
compound treated cells and we observed a decrease in the level
of tankyrase-1 (PARP-5a) revealing the telomerase inhibitory
nature of the compounds.44 Thus, we can conclude that the
inhibitory activity on tankyrase-1 and expression of g H2AX at
the telomere ends may lead to un-repairable DNA damage
leading to cell cycle arrest and cytotoxicity (Fig. 6a). In cells, the
maintenance of telomeres is essential for continuous growth of
tumor cells. Sheltrin is a multi-protein complex anchored to the
double stranded region of mammalian telomeres by the telo-
meric-repeat binding factor (TRF-1 and TRF2). Tankyrase-1 or
TNKS or TRF1 interact with ankyrin-related ADP-ribose poly-
merase or PARP-5 which is a bonade PARP protein that poly
(ADP-ribosylates) TRF1, thereby releasing TRF1 from the telo-
mere and causing telomere elongation. Thus, tankyrases func-
tion as positive regulators of telomere length.45–48 It was also
reported that PARP inhibitory agents block tankyrase-1 in intact
cells.46,49 Therefore, we propose that our compounds inhibit
PARP-1 and PARP-5 (tankyrase-1) activity and stabilize TRF1,
which results in the inhibition of telomerase access to telo-
meres (shortened telomeres). This can lead to senescence by up-
regulation of p16 (Fig. 6b). Thus, these compounds induce
senescence by affecting PARP proteins. Similarly to our results,
a known PARP inhibitor, 3-aminobenzamide (3-AB), enhances
TRF1 protein expression as well as binding to telomeres
and causes telomere shortening.46 There were also reports
that tankyrase-1 (TNKS-1) is equally sensitive to inhibition of
PARP. The inhibition of TRF1 by tankyrase-1, or the
inhibition of tankyrase-1, which results in increased TRF1,
also involves the TIN2 protein that interconnects TRF1 and
TRF2.
Conclusion
In conclusion, we have synthesized a new series of 3-diary-
lethyne quinazolinone compounds (4a–ad) and screened them
for their cytotoxicity, among which two compounds, 4s and 4w,
were found to be potential cytotoxic agents against MCF-7
cancer cells. These compounds lead to G1 cell cycle arrest and
inhibit tankyrase-1 to induce DNA damage by controlling telo-
merase activity. Apart from this, they induced the activation of
p53, p21, p16, TRF1 and POT1 proteins that play vital role in the
regulation and execution of senescence. These compounds are
working towards the inhibition of PARP-5 activity (tankyrase-1)
and cause increased binding of TRF1 proteins to telomeres,
thereby causing the shortening of telomeres. Thus, we can
conclude that these compounds affect mainly PARP activity as
well as other proteins that are involved in senescence. Consid-
ering the importance of these compounds to induce senescence
in anticancer therapy, the present investigation offers an
important insight in this aspect. The above two compounds (4s
and 4w) could be considered as potential leads towards the
development of a new class of anticancer therapeutics. Further
biological evaluations are currently underway and will be
reported in due course.
Acknowledgements
The authors F. S., A.V. and E. V. B. are thankful to CSIR; Y. V. V.
S. is thankful to UGC and R. N. is thankful to DST, New Delhi,
India for the award of research fellowships.
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