Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5541
ladder. However, higher ligand concentrations can lead to non-
G-quadruplex mediated effects involving inhibition of telom-
erase, primer dimerization, or PCR amplification through Taq
polymerase inhibition.24 Our data suggest that both ligands
inhibit telomerase activity considerably by stabilizing G-
quadruplex structure, but higher ligand concentrations produce
non-G-quadruplex related telomerase inhibition. Similar results
were also reported in the literature.24
Recently, Jantos et al. have reported binding properties of
oxazole-based peptide macrocycles with the c-kit and human
telomeric quadruplex.17 The structural diversity between the
above-mentioned reported molecules and the ligands studied
here is not only the different building blocks (oxazole vs furan)
used to build the molecules but also the placement and the
chemistry of the side chains. Placing the side chains on the
heterocyclic rings rather than on the carbon atom adjacent to
amino groups makes the synthesis easier and will not affect
the planarity of the molecules. In spite of having such diversity,
newly synthesized molecules have similar binding affinities as
reported for the oxazole-based peptide macrocycles. The toxicity
and biological activities of the oxazole-based peptide macro-
cycles have not been reported and thus cannot be compared
with ligands reported in the current study. But the reported
thermodynamic preference of the oxazole-based peptide mac-
rocycles for binding to a parallel G-quadruplex structure (c-kit
quadruplex) compared to an antiparallel conformation (human
telomeric quadruplex) is worth testing for the molecules studied
here, and work is in progress.
Figure 4. Inhibition of telomerase extension activity by PCR based
TRAP assay: (lane 1) -ve control, TRAP without telomerase extract;
(lane 2) +ve control, telomerase extract with no added ligands; (lanes
3-6) ligand 3; (lanes 7-10) ligand 4. Inhibition of telomeric ladder
started at 25 µM (lanes 5, 9) for ligands 3 and 4 and is achievable at
50 µM (lanes 6, 10). TS+CXext is the minimal dimer representing
telomerase activity. ITAS represents internal control.
any conformational changes in the telomeric quadruplex struc-
ture in the presence of 100 mM KCl.
The promising biophysical results prompted us to perform
biological assays for these ligands. We carried out MTT assay
to check the cytotoxicity of the ligands in A549 cells. The IC50
values obtained from the MTT assay were found to be 500 µM
for ligands 3 and 4 (Supporting Information, Figure 3). They
were found to be considerably less toxic compared to the other
well-studied ligands. Low cytotoxicity of these ligands raises
the issue of cellular uptake. Direct evidence demonstrating
cellular uptake would involve tagging the ligand with a
fluorescent probe and observing its localization through fluo-
rescence microscopy. However, attachment of a fluorescent tag
to ligands 3 and 4 would affect the entire physical chemistry of
interaction. We performed cell cycle analysis,23 which would
provide the effect of the ligands on cell cycle progression in
different phases of growth and hence provide indirect evidence
of cellular uptake. Cell cycle study shows an increase in the
population of cells in the G1 phase (growth phase) of cell cycle
upon treatment with ligand 3 and 4 in comparison to the control
(untreated cells, Supporting Information Figure 5). This obser-
vation clearly indicates the cellular uptake of these ligands.
Next we performed the PCR based telomeric repeat ampli-
fication protocol (TRAP) assay, which was followed by
photometric enzyme immunoassay for the detection of telom-
erase activity.24 G-quadruplex interactive agents block telom-
erase activity through the stabilization of G-quadruplex struc-
tures, prevent the primer from binding to the hTR RNA template
of telomerase, and hamper its extension. We performed PCR
based TRAP at different concentrations of ligand and observed
inhibition of the characteristic telomeric ladder suggestive of
inhibition of telomere extension and telomerase activity. The
ligands used in the study stabilize the quadruplex structure as
observed in fluorescence and DSC studies and prevent the
telomerase activity leading to the disappearance of the charac-
teristic ladder (Figure 4). Internal control (ITAS), a 36 bp
product, allows discrimination between telomerase inhibition
and Taq polymerase inhibition. Figure 4 shows no inhibition
of ITAS, indicating no polymerase inhibition by ligands 3 and
4. An ELISA based TRAP assay was carried out to quantify
the telomerase activity in the presence of increasing concentra-
tions of ligands 3 and 4. The data quantified from this assay
are in concordance with the data presented in Figure 4. The
relative units of TRAP activity are found to be maximum for
positive and minimum for negative control, respectively, as
shown in Supporting Information, Figure 5. Quadruplex inter-
acting ligands display dose-dependent inhibition of the telomere
In conclusion, we show that 18- and 24-membered cyclic
oligopeptides developed from a novel furan amino acid,
5-(aminomethyl)-2-furancarboxylic acid, having one and two
cationic side chains are able to interact with G-quadruplex
structures selectively and thus stabilize the structures. These
ligands have low cytotoxicity and are able to efficiently inhibit
the activity of telomerase, which makes them promising ligands
for exploration for anticancer therapy.
Acknowledgment. We thank CSIR and DST, New Delhi,
for financial support and CSIR (S.R., N.K.) and UGC (A.A.),
New Delhi, for research fellowships.
Supporting Information Available: Experimental section,
NMR, mass and elemental analysis data, and cell cycle analysis.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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