Abstract C159: Specific structural changes in telomeric DNA induced by triazoloacridone compound C-1305 prevent the formation of TRF1/2 shelterin complexes

Abstract

Abstract C-1305 is a triazoloacridone derivative with potent activity in lung and colon cancer models. We reported previously that compound C-1305 binds to DNA by intercalation and induces structural distortions in DNA regions containing guanine triplets GGG, at concentrations as low as 0.1 µM. This is a unique property of C-1305 since similar results were not observed for any of the structurally related triazoloacridone derivatives tested or other DNA binding compounds. Human telomeres consist of tandem repeats of the TTAGGG fragments and form specialized DNA-protein complexes consisting of telomere binding proteins (shelterins), localized at the ends of the linear chromosomes. Most of telomeric DNA is double-stranded except the most extreme part where 3′ region of the G strand is single-stranded and may form the so-called G-quadruplex structures. Two telomere-binding proteins, TRF1 and TRF2, are the key components of the shelterin complex that bind double-stranded telomeric DNA. Both TRF1 and TRF2 contain the so-called Myb-like domain, which is responsible for sequence-specific binding of these proteins to DNA. Previous studies have shown that the absence or non-functionality of either of TRF proteins results in increased telomere length and chromosomal fusions that ultimately leads to cell death. In this study, we wanted to clarify whether compound C-1305 and its structural analogs bind telomeric DNA and stabilize G-quadruplexes. The binding affinities of studied compounds to telomeric DNA were tested against different DNA substrates using microdialysis and DNA melting temperature assays and stabilization of G-quadruplexes by FRET assay. Given that telomeric repeats contain GGG triplets, we also wanted to assess whether C-1305 induces structural distortions within telomeric sequences and whether drug-DNA binding changes the affinity of TRF1 and TRF2 to telomeric DNA. We show here that from all studied compounds C-1305 showed the highest affinity and specificity to double-stranded oligonucleotides containing telomeric TTAGGG repeats and to a oligonucleotide folded into intramolecular G-quadruplex structure. Based on the molecular modeling studies, we also propose a model of C-1305/G-quadruplex complex. We recently postulated, that TRF1/2-telomeric DNA complexes may be excellent targets for anticancer drugs, which by specific binding to telomeric DNA disturb the formation of shelterin-DNA complexes. In the second part of this work, we characterized interactions of recombinant TRF1 and TRF2 proteins and Myb-like domain fragments, with telomeric DNA, in the absence or presence of compound C-1305 and its analogs. Using chemical probing, we found that C-1305 induces structural changes within GGG triplets present in the telomeric sequences in double-stranded DNA substrate. We also showed by the EMSA assay that compound C-1305 is disturbs the binding of TRF1/2 proteins and Myb-domain peptide with telomeric DNA probes, containing consensus binding sequences for both studied shelterins. Together, our results show that compound C-1305 specifically binds both G-quadruplex and double-stranded telomeric DNA. Moreover, this compound induces structural changes in double stranded telomeric DNA, specifically within GGG triplets, that leads to greatly decreased affinity of both TRF1 and TRF2 to telomeric DNA substrates. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C159.