G-quadruplex ligands

Haider SM, Parkinson GN, Neidle S (2003) Structime of a G-quadruplex-ligand complex. J Mol Biol 326(1) 117-125... 

This relatively small DCL yielded the first peptidic ligand for G-quadruplex DNA, providing a lead compound for the further development of novel G-quadruplex ligands with possible therapeutic utility. 

Nielsen, M. C. Ulven, T. Selective extraction of G-quadruplex ligands from a rationally designed scaffold-based dynamic combinatorial library. Chem. Ear. J. 2008,14, 9487-9490. 

Figure 6 A small G-quadruplex ligand accelerates quadruplex formation, (a) Gel electrophoresis experiment. 100 [iM of TG3T was incubated at 4°C for 4.5 h in a pH 7.2 10 mM lithium cacodylate buffer supplemented with 110 mM KCl, in the presence of various concentrations (0-100 tM) of a specific quadruplex ligand, 360A. Samples were then loaded on a 20% acrylamide TBE IX gel supplemented with 20 mM KCl. Nucleic acids were revealed by UV-shadow using a 254 nm UV lamp. A retarded band reveals the formation of a quadruplex the presence of the quadruplex ligand induces a further retardation. (b) Isothermal (4°C) absorbance measurements at 295 nm of 31 vM TG4T in the absence (circle, left Y-scale) or presence (squares, right Y-square) of 10 iM 360A in a pH 7.2 10 mM lithium cacodylate buffer supplemented with 110 mM NaCl...

The objective of this chapter is to present the latest view on the cellular mechanism(s) of action of G-quadruplex ligands and to discuss their potential use in therapy. 

Figure 2 Chemical formula of some G-quadruplex ligands...

G-quadruplex ligands behave as telomerase inhibitors. (A) Long-term treatment of A549 human cell line with a sub-toxic concentration of 12459 (0.04 i.M) leads to the cell-culture growth arrest after several weeks. (B) TRF decrease induced by 12459 in A549 cells. (C) Senescence induced by 12459, revealed by SA-b galactosidase activity at PD 50... 

Eor BRACO-19, an important decrease in the nuclear hTERT, together with the formation of cytoplasmic hTERT bound to ubiquitin may explain the telomerase activity down-regulation. Other antitumor agents have been also found to down-regulate telomerase activity and hTERT expression has been reported as a marker of cell proliferation. Thus, the simplest explanation for the effect of the G-quadruplex ligands to down-regulate telomerase activity is related to their antiproliferative activity. 

It was also observed earlier that G-quadruplex ligands indueed more rapid effects on cell growth than that initially expected for telomerase inhibition alone. Apoptosis and short-term response were observed with triazine derivatives (12459, 115405), telomestatin, and more recently with the pyridine dicarboxamide derivatives (307A, 360A). " Telomestatin induced the activation of ATM and Chk2 that corresponded to an activation of the DNA damage response. 12459 induced apoptosis through the mitochondrial pathway and also provoked the early activation of P53. ... 

Short-term and massive apoptosis were also observed from the interference of the telomere-capping function of telomerase when hTERT or hTR are modified by mutations.The observation that BRACO-19 causes chromosome end-to-end fusions associated with the appearance of pl6-associated senescence led to the proposal that G-quadruplex ligands mostly act to disrupt the telomere structure.Such telomeric dysfunction was also observed in cell lines treated with RHSP4 or with 307A and in cell lines resistant to 12459 with typical images of telophase bridges" (Figure 4). 

The evidence that the antiproliferative effect of G-quadruplex ligands is independent of the presence of telomerase activity also comes from two series of experiments. 

As any potential therapeutic agents, the biological effect of G-quadruplex ligands may be overcome by the appearance of acquired-resistance phenotypes or to be lowered by the intrinsic expression of proteins already known to be associated with resistance to anticancer agents. The selection as well as the analysis of resistant cells is a well-known strategy to obtain information on the mechanisms of drug inactivation and on their target proteins, if a modification of their expression, or a mutation can be related to the resistance phenotype. 

G-quadruplex ligands induced a telomere dysfunction that is characterized by the appearance of anaphase bridges and telomere end-to-end fusions in treated cells. The induction of short-term apoptosis is also frequently observed. The telomere dysfunction may appear before any evidence of telomere shortening by... 

Figure 6 Proposed role of hTERT overexpression in resistant JFD18 cell line to control telomere cappingf The telomere length increase and hTERT overexpression stabilize telomere-capping alterations induced by 12459 in JED 18 resistant cells. Transfection of JFD18 by DN-hTERT reduces the telomere length and restores sensitivity to the short-term effects of 12459. In parental cells, DN-hTERT transfection does not change the sensitivity to 12459, indicating that the short effect of the G-quadruplex ligand is independent of telomerase activity...

As a conclusion to this part, the modest selectivity of 12459 for G4 over duplex DNA may render questionable some of these findings, that is, the relationship between short-term effects and its molecular action against telomeres. Future work to obtain resistant cell lines from other sources and with more selective G-quadruplex ligands, such as telomestatin or pyridine dicarboxamide derivatives will aim to confirm some of the views presented here. 

By using these two techniques our group has analyzed the effect of several series of G-quadruplex ligands on the behavior of the G-overhang in human cell lines. 

In vivo protection experiments with DMS that is able to transfer a methyl group to the N7 position of guanines enabled us to demonstrate that telomestatin engages the telomeric overhang in vivo in a DNA structure resistant to action of DMS. These experiments represent the first proof of the existence of G-quadruplexes at the telomeric G-overhang under the effect of the treatment by a G-quadruplex ligand. 

Other G-quadruplex ligands, such as the triazine derivative 12459 and the pyridine dicarboxamide derivatives 360A and 307A showed different effects, as compared to telomestatin, on the conformation and the integrity of the telomeric G-overhang. °... 

Future works will aim to determine the effect of G-quadruplex ligands on other components of the shelterin complex and associated factors, such as WRN or BLM helicases that cooperate with POTl for telomere sequence unwinding. ... 

One of the most interesting demonstration that telomere is a significant target for these ligands has been provided by a study using a radiolabelled G-quadruplex ligand. The localization of the chromosomal binding sites of a... 

G-quadruplex ligands from the trisubstituted acridine series were also found to inhibit in vitro the unwinding by the RecQ helicases, BLM and WRN. Therefore, because of their additional effects on RecQ helicases, these ligands should also disrupt telomere synthesis (Figure 11). In addition, their action against other G-quadruplex resolvases during replication might provoke DNA... 

Figure 11 Schematic model presenting the possible effect of a G-quadruplex ligand on G-quadruplex forming elements during replication. Stabilization of G-quadruplex might block G4 resolvases and further lead to a replication block with deleterious consequences...

It would be interesting to determine whether such action will be a benefit for antitumor activity. A possible way to determine this is to examine the effect of the ligands in cells defective for these resolvases. A replication block at telomere would be expected to rapidly induce larger deletions of telomeric sequence than in wild-type cells. These results may orient the strategy to select future G-quadruplex ligand or to combine the actual ligands with specific inhibitors of these resolvases. 

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