Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay

Abstract

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 M dye concentration. This increase in Tm 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.telomere ͉ DNA structure ͉ G-quartet T elomerase was first identified in ciliates (1). This enzyme is an essential factor in immortalization and tumorigenesis (2-4). Furthermore, telomerase is active in most human tumor cells and inactive in most somatic cells and is therefore an attractive target for the design of anticancer agents. Most human telomeric DNA is double-stranded and contains (TTAGGG͞CCCTAA) n repeats except for the extreme terminal part where the 3Ј region of the G-rich strand is singlestranded (5). For human cells, these 3Ј overhangs are surprisingly long (averaging 130-210 bases in length), exist during most of the cell cycle, and are present on all chromosomal ends. The G-rich single-stranded DNA can adopt an unusual four-stranded DNA structure involving G-quartets (6-8) (see Fig. 1) or it might fold back and displace one strand of a telomeric duplex to form a so-called T-loop (9). Optimal telomerase activity requires the nonfolded single-stranded form of the primer and G-quartet formation has been shown to directly inhibit telomerase elongation in vitro (10). Therefore, a drug that stabilizes quadruplexes could interfere with telomerase and telomere replication (11-13).The peculiar geometry of the quadruplex structure should allow its specific recognition by small synthetic ligands, and previous experiments have shown that this assumption is correct (14). Many of the G4 ligands were shown to have antitelomerase activity in vitro, with IC 50 in the low micromo...

Interactions of cryptolepine and neocryptolepine with unusual DNA structures

“…However, its binding affinity is very modest. Cryptolepine stabilizes the F21MB quadruplex by 3 °C at 3 µM, whereas the best G4 ligands stabilize by 20 °C or more at 1 µM compound [44]. It is not a potent telomerase inhibitor (IC 50 = 9.4 µM); the best inhibitors described so far have an IC 50 of 50 nM or lower [37,44,[48][49][50].…”

“…The melting temperature of a G4-forming fluorescent oligonucleotide may be recorded in the presence of various concentrations of the ligand [35,44]. At 1 µM cryptolepine, there is no significant stabilization of the Gquadruplex, but a weak stabilization (ΔT m = 3 °C) is observed at 3 µM (data not shown).…”

Ascididemin and meridine stabilise G-quadruplexes and inhibit telomerase in vitro

“…The number of G4 ligands has grown rapidly over a few years: a range of molecules has been shown to inhibit telomerase through binding to its substrate [14,26,27,36,[44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59]. On the other hand, few natural products have been reported as G-quadruplex-mediated telomerase inhibitors, although one, telomestatin, is exceptionally potent with an IC50 of 5 nM against telomerase [25].…”

“…LiCl is added in order to approach physiological ionic strength without stabilising the quadruplex (lithium is a monocation which does not interact with G-quartets). The melting of the G-quadruplex was monitored in the presence and or in absence of the dye by measuring the fluorescence of fluorescein [36]. Several concentrations of each compound (1, 3, 5 or 10 µM) were tested, alone or in the presence of a double-stranded competitor (a self-complementary, 26 bp-long oligodeoxynucleotide "ds26", sequence 5' d-CAATCGGATCGAATTCGATCCGATTG 3').…”

“…Fluorescence measurements with the F21MB oligonucleotide (0.2 µM) were initially performed on a Spex Fluoromax 3 instrument with 600 µL of solution in a 0.1 M lithium chloride, 10 mM sodium cacodylate pH 7.2 buffer [36]. In the experiments presented here, a real time PCR apparatus (MX3000P, Stratagene) was used, allowing the simultaneous recording of independent 96 samples.…”