Reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action

Abstract

Quadruplex ligands are often considered as telomerase inhibitors. Given the fact that some of these molecules are present in the clinical setting, it is important to establish the validity of this assertion. To analyze the effects of these compounds, we used a direct assay with telomerase-enriched extracts. The comparison of potent ligands from various chemical families revealed important differences in terms of effects on telomerase initiation and processivity. Although most quadruplex ligands may lock a quadruplex-prone sequence into a quadruplex structure that inhibits the initiation of elongation by telomerase, the analysis of telomerase-elongation steps revealed that only a few molecules interfered with the processivity of telomerase (i.e., inhibit elongation once one or more repeats have been incorporated). The demonstration that these molecules are actually more effective inhibitors of telomeric DNA amplification than extension by telomerase contributes to the already growing suspicion that quadruplex ligands are not simple telomerase inhibitors but, rather, constitute a different class of biologically active molecules. We also demonstrate that the popular telomeric repeat amplification protocol is completely inappropriate for the determination of telomerase inhibition by quadruplex ligands, even when PCR controls are included. As a consequence, the inhibitory effect of many quadruplex ligands has been overestimated.G-quadruplex ͉ telomere ͉ telomeric repeat amplification protocol assay ͉ direct assay T he unlimited proliferative potential of cancer cells depends on telomere maintenance (1). As a consequence, several classes of telomerase inhibitors have been developed for anticancer purposes. Optimal telomerase activity requires an unfolded single-stranded telomeric overhang (2-4). This overhang may adopt an unusual DNA structure called a G-quadruplex, composed of G-quartets and formed in the presence of cations (5). Therefore, ligands that selectively bind to and stabilize telomeric G-quadruplex structures could act as indirect telomerase inhibitors (Fig. 1A) (6). The number of identified G-quadruplex ligands has grown rapidly over the past few years (for a recent review, see ref. 7); some of these molecules exhibit potent in vivo antitumor activity, and clinical trials started recently.Although various methods are available to measure telomerase activity, the telomeric repeat amplification protocol (TRAP) is most widely used (8). With a few notable exceptions (6, 9-12), TRAP [or a variant called TRAP-G4 (13)] has been the standard method to measure telomerase inhibition by G-quadruplex ligands (reviewed in ref. 7). The original method was improved by the use of an internal control (often called ITAS for internal telomerase assay standard) that coamplifies with the telomerase products and allows the detection of nonspecific Taq polymerase inhibitors. Notably, the ITAS product does not contain any telomeric sequence and therefore is not able to form a G-quadruplex (5,14). We demonstrate here that TRAP is...

ESI-MS Investigation of an Equilibrium between a Bimolecular Quadruplex DNA and a Duplex DNA/RNA Hybrid

“…The ribonucleic acid component of telomerase, TR, containing the template, associates with the complementary 3 single-stranded overhang of telomeric DNA. This is followed by processive repeat addition synthesis of DNA in which a telomeric repeat DNA sequence is synthesised, followed by dissociation of the singlestranded product from the template, and translocation of the template (RNA) to enable synthesis of the next telomeric repeat [20][21][22][23][24]. The mechanism by which translocation occurs has not been fully elucidated, and the role(s), if any, of secondary DNA structures (e.g.…”

G-quadruplexes: selective DNA targeting for cancer therapeutics?

“…G-quadruplex structures are present in telomeres and are inhibitory to telomerase activity in vitro, although in vivo evidence has been difficult to obtain [10]. However, in ciliates, the formation of telomeric G-quadruplexes have been demonstrated in vivo using a G-quadruplex structure-specific antibody [11].…”

Targeting G-Quadruplex DNA with Small Molecules

“…[105] Telomestatin (SOT-095), a natural product isolated from Streptomyces anulatus 3533-SV4, [106] is one of the strongest and most specific inhibitors of telomerase reported to date (IC 50 ≅ 1 µM). [95,96] Telomestatin has molecular dimensions similar to those of G-tetrad DNA and can bind to various G-quadruplexes with modest affinity (K d ≅ 30 nM). [107] Telomestatin exhibits good selectivity for intramolecular versus intermolecular G-quadruplex structures, [108] and it has a 70-fold lower affinity for duplex DNA.…”

“…5) is the most extensively studied to date. TMPyP4 inhibits both telomerase (IC 50 ≅ 0.7-10 µM) [95,96] and Taq DNA polymerase (IC 50 ≅ 2 µM). [57] Telomerase inhibition by TMPyP4 is relatively insensitive to metal coordination by the porphyrin, but is highly dependent on the groups at the meso positions.…”