Telomeric ends of chromosomes, which comprise noncoding repeat sequences of guanine-rich DNA, are fundamental in protecting the cell from recombination and degradation. Disruption of telomere maintenance leads to eventual cell death, which can be exploited for therapeutic intervention in cancer. Telomeric DNA sequences can form four-stranded (quadruplex) structures, which may be involved in the structure of telomere ends. Here we describe the crystal structure of a quadruplex formed from four consecutive human telomeric DNA repeats and grown at a K(+) concentration that approximates its intracellular concentration. K(+) ions are observed in the structure. The folding and appearance of the DNA in this intramolecular quadruplex is fundamentally different from the published Na(+)-containing quadruplex structures. All four DNA strands are parallel, with the three linking trinucleotide loops positioned on the exterior of the quadruplex core, in a propeller-like arrangement. The adenine in each TTA linking trinucleotide loop is swung back so that it intercalates between the two thymines. This DNA structure suggests a straightforward path for telomere folding and unfolding, as well as ways in which it can recognize telomere-associated proteins.

Crystal structure of parallel quadruplexes from human telomeric DNA.

Pd -catalyzed oxidative coupling with organometallic reagents via C–H activation

Over the past decade, nucleic acid chemists have seen the spectacular emergence of molecules designed to interact efficiently and selectively with a peculiar DNA structure named G-quadruplex. Initially derived from classical DNA intercalators, these G-quadruplex ligands progressively became the focal point of new excitement since they appear to inhibit selectively the growth of cancer cells thereby opening interesting perspectives towards the development of novel anti-cancer drugs. The present article aims to help researchers enter this exciting research field, and to highlight recent advances in the design of G-quadruplex ligands.

A hitchhiker's guide to G-quadruplex ligands

Maintenance of telomeres--specialized complexes that protect the ends of chromosomes--is undertaken by the enzyme complex telomerase, which is a key factor that is activated in more than 80% of cancer cells that have been examined so far, but is absent in most normal cells. So, targeting telomere-maintenance mechanisms could potentially half tumour growth across a broad spectrum of tumour types, with little cytotoxic effect outside tumours. Here, we describe the current understanding of telomere biology, and the application of this knowledge to the development of anticancer drugs.

Telomere maintenance as a target for anticancer drug discovery.

Targeting telomeres and telomerase

Telomerase is a major new target for the rational design of novel anticancer agents. We have previously identified anthraquinone-based molecules capable of inhibiting telomerase by stabilizing G-quadruplex structures formed by the folding of telomeric DNA. In the present study we describe the synthesis and biological evaluation of a series of analogous fluorenone-based compounds with the specific aims of, first, determining if the anthraquinone chromophore is a prerequisite for activity and, second, whether the conventional cytotoxicity inherent to anthraquinone-based molecules may be reduced by rational design. This fluorenone series of compounds exhibits a broad range of telomerase inhibitory activity, with the most potent inhibitors displaying levels of activity (8-12 microM) comparable with other classes of G-quadruplex-interactive agents. Comparisons with analogous anthraquinone-based compounds reveal a general reduction in the level of cellular cytotoxicity. Molecular modeling techniques have been used to compare the interaction of fluorenone- and analogous anthraquinone-based inhibitors with a human G-quadruplex structure and to rationalize their observed biological activities.

2,7-Disubstituted amidofluorenone derivatives as inhibitors of human telomerase.

Telomerase is an attractive target for the design of new anticancer drugs. We have previously described a series of 1,4- and 2,6-difunctionalized amidoanthracene-9,10-diones that inhibit human telomerase via stabilization of telomeric G-quadruplex structures. The present study details the preparation of three further, distinct series of regioisomeric difunctionalized amidoanthracene-9,10-diones substituted at the 1,5-, 1,8-, and 2,7-positions, respectively. Their in vitro cytotoxicity and Taq DNA polymerase and human telomerase inhibition properties are reported and compared with those of their 1,4- and 2,6-isomers. Potent telomerase inhibition (telIC50 values 1.3−17.3 μM) is exhibited within each isomeric series. In addition, biophysical and molecular modeling studies have been conducted to examine binding to the target G-quadruplex structure formed by the folding of telomeric DNA. These studies indicate that the isomeric diamidoanthracene-9,10-diones bind to the human telomeric G-quadruplex structure wi...

Human Telomerase Inhibition by Regioisomeric Disubstituted Amidoanthracene-9,10-diones

Inhibition of the ability of the enzyme telomerase to add telomeric repeats to the end of chromosomes is a novel target for potential anticancer therapy. This paper examines the hypothesis that compounds possessing a planar aromatic chromophore inhibit telomerase via stabilization of, and binding to, a folded guanine quadruplex structure. Two series of telomerase inhibitors have been designed based on the 2,6-disubstituted amidoanthracene-9,10-dione and 3,6-disubstituted acridine chromophores in order to investigate structure-activity relationships between biological activity and substituent group size. The relative binding energies between these compounds and the folded human telomere DNA quadruplex were determined using molecular simulation methods, involving explicitly solvated structures. The results obtained are in excellent agreement with the biological activity as measured in vitro using a modified TRAP assay and in general agreement with the ranking order of binding enthalpies found in isothermal titration calorimetry studies. This broad agreement provides strong support for the hypothesis that guanine quadruplexes are the primary target for telomerase inhibitors with extended planar chromophores.

Molecular Modeling Studies on G-Quadruplex Complexes of Telomerase Inhibitors: Structure−Activity Relationships

An analogue of the DNA-binding compound Hoechst 33258, in which the piperazine ring has been replaced by an imidazoline group, has been cocrystallized with the dodecanucleotide sequence d(CGCGAATTCGCG)2. The structure has been solved by X-ray diffraction analysis and has been refined to an R-factor of 19.7% at a resolution of 2.0 A. The ligand is found to bind in the minor groove, at the central four AATT base pairs of the B-DNA double helix, with the involvement of a number of van der Waals contacts and hydrogen bonds. There are significant differences in minor groove width for the two compounds, along much of the AATT region. In particular this structure shows a narrower groove at the 3' end of the binding site consistent with the narrower cross-section of the imidazole group compared with the piperazine ring of Hoechst 33258 and therefore a smaller perturbation in groove width. The higher binding affinity to DNA shown by this analogue compared with Hoechst 33258 itself, has been rationalised in terms of these differences.

Variability in DNA minor groove width recognised by ligand binding: the crystal structure of a bis-benzimidazole compound bound to the DNA duplex d(CGCGAATTCGCG)2

Alexis A. Wood

Papers

2,7-Disubstituted amidofluorenone derivatives as inhibitors of human telomerase.

Human Telomerase Inhibition by Regioisomeric Disubstituted Amidoanthracene-9,10-diones

Molecular Modeling Studies on G-Quadruplex Complexes of Telomerase Inhibitors: Structure−Activity Relationships

Variability in DNA minor groove width recognised by ligand binding: the crystal structure of a bis-benzimidazole compound bound to the DNA duplex d(CGCGAATTCGCG)2

Sequence-dependent crossed helix packing in the crystal structure of a B-DNA decamer yields a detailed model for the holliday junction