Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay

The reactivation of telomerase activity in most cancer cells supports the c oncept that telomerase is a relevant target in oncology, and telomerase inh ibitors have been proposed as new potential anticancer agents. The telomeri c G-rich single-stranded DNA can adopt in vitro an intramolecular quadruple x structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. In tramolecular folding of an oligonucleotide with four repeats of the human t elomeric sequence into a G-quadruplex structure led to fluorescence excitat ion energy transfer between a donor (fluorescein) and an acceptor (tetramet hylrhodamine) covalently attached to the 5' and 3' ends of the oligonucleot ide, respectively. The melting of the C-quadruplex was monitored in the pre sence of putative G-quadruplex-binding molecules by measuring the fluoresce nce emission of the donor. A series of compounds (pentacyclic crescent-shap ed dibenzophenanthroline derivatives) was shown to increase the melting tem perature of the C-quadruplex by 2-20 degreesC at 1 muM dye concentration. T his increase in T-m value was well correlated with an increase in the effic iency of telomerase inhibition in vitro. The best telomerase inhibitor show ed an IC50 value of 28 nM in a standard telomerase repeat amplification pro tocol assay. Fluorescence energy transfer can thus be used to reveal the fo rmation of four-stranded DNA structures, and its stabilization by quadruple x-binding agents, in an effort to discover new potent telomerase inhibitors .