No abstract
The protection and maintenance of the genome in human cells is critical. The cell uses signaling pathways that can down regulate the cell cycle when DNA-damage occurs, giving an opportunity for efficient repair before division.1 However, this raises the question of how cells manage to distinguish the telomeres from DNA double-strand breaks. A six-protein complex named "telosome" or "shelterin" (Figure 1) binds to the double-stranded telomeric DNA and single-stranded telomeric G-overhang at the chromosome ends.2,3 The complex shelters telomeres from the DNA-damage response machinery and protects chromosomes from shortening, nonhomologous end-joining, and homology-directed repair. Moreover, the shelterin component POT1 (protection of telomeres 1)4 has been shown to modulate the activity of telomerase,5 the enzyme capable of conferring infinite proliferative capacity on cells by extension of the G-overhang.6 It has been shown that the telomeric G-overhang, when folded into G-quadruplexes, is resistant to extension by telomerase,7 and that synthetic small molecules that stabilize these structures can decrease the enzyme efficiency.8-10 Gomez et al. showed that the potent G-quadruplex binding natural product telomestatin induces apoptosis of cancer cells via a mechanism proposed to involve the uncapping of POT1 from telomeres.11 Herein, we describe a novel synthetic small molecule (compound 1, Figure 1), which exhibits unprecedented G-quadruplex stabilization leading to an alteration of shelterin at the telomeres of human cancer cells.Compound 1 was designed following intensive research on the biology of G-quadruplex nucleic acids.12 The design rationale comprises certain structural features shared by known quadruplex binding small molecules, with particular emphasis on an electron rich aromatic surface, the potential for a flat conformation, and an ability to participate in hydrogen bonding.13 The small molecule is readily accessible in six synthetic steps that are easily scalable and amenable to molecular diversity (see Supporting Information).We first evaluated the potential for 1 to stabilize the telomeric G-quadruplex by FRETmelting experiments.14 Compound 1 stabilized the human telomeric G-quadruplex with a maximum ΔT m of 35 K in 60 mM K + and 44 K in 100 mM Na + at 0.18 and 0.34 μM compound, respectively. In contrast, the ligand-induced double-stranded DNA stabilization was negligible with a ΔT m of 0.5 K in 60 mM K + at 1 μM compound. It is noteworthy that the G-quadruplex melting profile was almost unaffected by the presence of 25 mol equiv of © 2008 American Chemical Society riou@mnhn.fr; sb10031@cam.ac.uk. Supporting Information Available: Experimental details for the synthesis of 1, FRET-melting, direct telomerase extension assay, in vitro POT1 uncapping assay, POT1 and γH2AX in cellulo experiments, growth inhibition assay. This material is available free of charge via the Internet at http://pubs.acs.org. The data recorded for 1 represent the highest induced shifts in melting temperature for the telomeri...
Telomeres of human chromosomes contain a G-rich 3-overhang that adopts an intramolecular G-quadruplex structure in vitro which blocks the catalytic reaction of telomerase. Agents that stabilize G-quadruplexes have the potential to interfere with telomere replication by blocking the elongation step catalyzed by telomerase and can therefore act as antitumor agents. We have identified by Fluorescence Resonance Energy Transfer a new series of quinoline-based G-quadruplex ligands that also exhibit potent and specific anti-telomerase activity with IC 50 in the nanomolar concentration range. Long term treatment of tumor cells at subapoptotic dosage induces a delayed growth arrest that depends on the initial telomere length. This growth arrest is associated with telomere erosion and the appearance of the senescent cell phenotype (large size and expression of -galactosidase activity). Our data show that a G-quadruplex interacting agent is able to impair telomerase function in a tumor cell thus providing a basis for the development of new anticancer agents.telomerase inhibitor ͉ tetraplex ͉ drug-DNA recognition
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