NMR-Based Model of a Telomerase-Inhibiting Compound Bound to G-Quadruplex DNA

Abstract

The single-stranded (TTAGGG)n tail of human telomeric DNA is known to form stable G-quadruplex structures. Optimal telomerase activity requires the nonfolded single-stranded form of the primer, and stabilization of the G-quadruplex form is known to interfere with telomerase binding. We have identified 3,4,9, 10-perylenetetracarboxylic diimide-based ligands as potent inhibitors of human telomerase by using a primer extension assay that does not use PCR-based amplification of the telomerase primer extension products. A set of NMR titrations of the ligand into solutions of G-quadruplexes using various oligonucleotides related to human telomeric DNA showed strong and specific binding of the ligand to the G-quadruplex. The exchange rate between bound and free DNA forms is slow on the NMR time scale and allows the unequivocal determination of the binding site and mode of binding. In the case of the 5'-TTAGGG sequence, the ligand-DNA complex consists of two quadruplexes oriented in a tail-to-tail manner with the ligand sandwiched between terminal G4 planes. Longer telomeric sequences, such as TTAGGGTT, TTAGGGTTA, and TAGGGTTA, form 1:1 ligand-quadruplex complexes with the ligand bound at the GT step by a threading intercalation mode. On the basis of 2D NOESY data, a model of the latter complex has been derived that is consistent with the available experimental data. The determination of the solution structure of this telomerase inhibitor bound to telomeric quadruplex DNA should help in the design of new anticancer agents with a unique and novel mechanism of action.

Telomere maintenance mechanisms as a target for drug development

“…The NMR structure of a PIPER ± G-quadruplex complex (Figure 4), the ®rst de®nitive structure of a ligand ± Gquadruplex complex, showed that the binding mode of PIPER to G-quadruplexes is similar to that proposed by us for the porphyrins, i.e. external stacking to Gtetrads (Fedoro et al, 1998).…”

“…As with other G-quadruplex-interactive compounds, PIPER showed very good telomerase and DNA polymerase inhibitory activities (Fedoro et al, 1998).…”

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].…”

A model for triple helix formation on human telomerase reverse transcriptase (hTERT) promoter and stabilization by specific interactions with the water soluble perylene derivative, DAPER

“…Studying G-quadruplex stabilizing drugs [35,36], we have previously shown that DAPER is able to selectively bind to G-quadruplex structures with respect to duplex, on account of G-quadruplex larger aromatic area and higher charge density [37][38][39]. Both these features characterize also triplex structure with respect to duplex, suggesting us to investigate DAPER for its ability to bind and stabilize the anti-parallel triplexes [40].…”