The reactivation of telomerase activity in most cancer cells
supports the concept that telomerase is a relevant target in oncology,
and telomerase inhibitors have been proposed as new potential
anticancer agents. The telomeric G-rich single-stranded DNA can adopt
in vitro
an intramolecular quadruplex structure, which
has been shown to inhibit telomerase activity. We used a fluorescence
assay to identify molecules that stabilize G-quadruplexes.
Intramolecular folding of an oligonucleotide with four repeats of the
human telomeric sequence into a G-quadruplex structure led to
fluorescence excitation energy transfer between a donor (fluorescein)
and an acceptor (tetramethylrhodamine) covalently attached to the 5′
and 3′ ends of the oligonucleotide, respectively. The melting of
the G-quadruplex was monitored in the presence of putative
G-quadruplex-binding molecules by measuring the fluorescence emission
of the donor. A series of compounds (pentacyclic crescent-shaped
dibenzophenanthroline derivatives) was shown to increase the melting
temperature of the G-quadruplex by 2–20°C at 1 μM dye
concentration. This increase in
T
m
value was
well correlated with an increase in the efficiency of telomerase
inhibition
in vitro
. The best telomerase inhibitor
showed an IC
50
value of 28 nM in a standard telomerase
repeat amplification protocol assay. Fluorescence energy transfer can
thus be used to reveal the formation of four-stranded DNA structures,
and its stabilization by quadruplex-binding agents, in an effort to
discover new potent telomerase inhibitors.
