Human telomeric DNA is now known to be transcribed into noncoding RNA sequences, termed TERRA. These sequences, which are believed to play roles in the regulation of telomere function, can form higher-order quadruplex structures and may themselves be the target of therapeutic intervention. The crystal structure of a TERRA quadruplex-acridine small-molecule complex at a resolution of 2.60 Å, is reported here and contrasts remarkably with the structure of the analogous DNA quadruplex complex. The bimolecular RNA complex has a parallel-stranded topology with propeller-like UUA loops. These loops are held in particular conformations by multiple hydrogen bonds involving the O2' hydroxyl groups of the ribonucleotide sugars and play an active role in binding the acridine molecules to the RNA quadruplex. By contrast, the analogous DNA quadruplex complex has simpler 1:1 acridine binding, with no loop involvement. There are significant loop conformational changes in the RNA quadruplex compared to the native TERRA quadruplex (Collie, G. W.; Haider, S. M.; Neidle, S.; Parkinson, G. N. Nucleic Acids Res. 2010, 38, 5569 - 5580), which have implications for the future design of small molecules targeting TERRA quadruplexes, and RNA quadruplexes more generally.

Structure-based modeling methods have been used to design a series of disubstituted triazole-linked acridine compounds with selectivity for human telomeric quadruplex DNAs. A focused library of these compounds was prepared using click chemistry and the selectivity concept was validated against two promoter quadruplexes from the c-kit gene with known molecular structures, as well as with duplex DNA using a FRET-based melting method. Lead compounds were found to have reduced effects on the thermal stability of the c-kit quadruplexes and duplex DNA structures. These effects were further explored with a series of competition experiments, which confirmed that binding to duplex DNA is very low even at high duplex:telomeric quadruplex ratios. Selectivity to the c-kit quadruplexes is more complex, with some evidence of their stabilization at increasing excess over human telomeric quadruplex DNA. Selectivity is a result of the dimensions of the triazole-acridine compounds, and in particular the separation of the two alkyl-amino terminal groups. Both lead compounds also have selective inhibitory effects on the proliferation of cancer cell lines compared to a normal cell line, and one has been shown to inhibit the activity of the telomerase enzyme, which is selectively expressed in tumor cells, where it plays a role in maintaining telomere integrity and cellular immortalization.

The good results obtained with pyrimido[5,6,1-de]acridines 7 and with pyrazolo[3,4,5-kl]acridinecarboxamides 8 prompted us to the synthesis of two new series of bis acridine derivatives: the bis(pyrimidoacridines) 5 and the bis(pyrazoloacridinecarboxamides) 6. Compounds 5 can be regarded also as cyclized derivatives of bis(acridine-4-carboxamides) 3 and compounds 6 as cyclized derivatives of bis(acridine-4-carboxamides) 4. The noncovalent DNA-binding properties of these compounds have been examined using fluorometric techniques. The results indicate that (i) the target compounds are excellent DNA ligands; (ii) the bis derivatives 5 and 6 are more DNA-affinic than corresponding monomers 7 and 8; (iii) the new bis 5 and 6 result always less efficient in binding than related bis(acridine-4-carboxamides) 3 and 4; and (iv) in both series 5 and 6 a clear, remarkable in some cases, preference for binding to AT rich duplexes can be noted. In vitro cytotoxic potency of these derivatives toward the human colon adenocarcinoma cell line (HT29) is described and compared to that of reference drugs. Structure-activity relationships are discussed. We could identify six very potent cytotoxic compounds for further in vitro studies: a cytotoxic screening against six human cancer cell lines and the National Cancer Institute (NCI) screening on 60 human tumor cell lines. Finally, compound 6a was selected for evaluation in a NCI in vivo hollow fiber assay.

Selective recognition of DNA folding is central to multiple biological and pharmacological applications aimed at precise targeting of distinct genomic regions. Here, we focused on the recognition of physiologically relevant G-quadruplex (G-4) structures by bis-phenanthroline (bis-Phen) ligands containing two Phen moieties covalently linked through an amine or thioether bond. The transition metal ions Mn(2+), Ni(2+), Cu(2+), and the biologically relevant Mg(2+) and Zn(2+) efficiently form 1 : 1 bis-Phen complexes characterised by a large planar structure fit to successfully recognise G-quartet arrangements.Interestingly, metal ion complexation dramatically affects ligand-stabilising effects on G-quadruplex, the melting temperature of the folded structure being increased up to 30 degrees C at ligand concentrations as low as 1 microM in the presence of Ni(2+) and Cu(2+). In addition, the test complexes were able to induce G-4 formation from essentially unfolded G-rich sequences even in the absence of K(+) ions as shown by gel shift and circular dichroism experiments. In line with their G-4 stabilising properties bis-Phen complexes are effective inhibitors of telomerase activity, Ni(II) complexes being effective in the sub-micromolar range. This is combined with lack of unselective DNA-damaging activity and short-term cellular toxicity, which makes the novel compounds (above all their Ni(II) complexes) interesting antiproliferative drug leads.

The good results obtained in the past decade with various types of potential bisintercalating agents, e.g., LU 79553, DMP 840, BisBFI, MCI3335, WMC-26, BisAC, BisPA, and the asymmetrical derivative WMC-79 (Chart 1), prompted us to investigate a new series of asymmetrical bisintercalators, compounds 1a-t (Chart 2), which can combine the potentiality of bisintercalation with a possible different mechanism of action due to two diverse chromophores. The DNA-binding properties of these compounds have been examined using fluorometric techniques: target compounds are excellent DNA ligands, with a clear preference for binding to AT-rich duplexes. In vitro cytotoxicity of these derivatives toward human hormone-refractory prostate adenocarcinoma cell line (PC-3) is described. Apoptosis assays of four selected compounds are also reported. Very potent cytotoxic compounds, some of them capable of inducing early apoptosis, have been identified.