A crystallographic and modelling study of a human telomeric RNA (TERRA) quadruplex

DNA telomeric repeats in mammalian cells are transcribed to guanine-rich RNA sequences, which adopt parallel-stranded G-quadruplexes with a propeller-like fold. The successful crystallization and structure analysis of a bimolecular human telomeric RNA G-quadruplex, folded into the same crystalline environment as an equivalent DNA oligonucleotide sequence, is reported here. The structural basis of the increased stability of RNA telomeric quadruplexes over DNA ones and their preference for parallel topologies is described here. Our findings suggest that the 2'-OH hydroxyl groups in the RNA quadruplex play a significant role in redefining hydration structure in the grooves and the hydrogen bonding networks. The preference for specific nucleotides to populate the C3'-endo sugar pucker domain is accommodated by alterations in the phosphate backbone, which leads to greater stability through enhanced hydrogen bonding networks. Molecular dynamics simulations on the DNA and RNA quadruplexes are consistent with these findings. The computations, based on the native crystal structure, provide an explanation for RNA G-quadruplex ligand binding selectivity for a group of naphthalene diimide ligands as compared to the DNA G-quadruplex.

Bis-guanylhydrazone diimidazo[1,2-a:1,2-c]pyrimidine as a novel and specific G-quadruplex binding motif

A bis-guanylhydrazone derivative of diimidazo[1,2-a:1,2-c]pyrimidine has unexpectedly been found to be a potent stabiliser of several quadruplex DNAs, whereas there is no significant interaction with duplex DNA. Molecular modeling suggests that the guanylhydrazone groups play an active role in quadruplex binding.

Biaryl polyamides as a new class of DNA quadruplex-binding ligands

We report a novel class of biaryl polyamides highly selective for G-quadruplex DNA, and with significant cytotoxicity in several cancer cell lines; they form planar U-shaped structures that match the surface area dimensions of a terminal G-quartet in quadruplex structures rather than the grooves of duplex DNA.

Targeting Human Gastrointestinal Stromal Tumor Cells with a Quadruplex-Binding Small Molecule

Most of human gastrointestinal stromal tumors (GIST) are driven by activating mutations in the protooncogene KIT, a tyrosine kinase receptor. Clinical treatment with imatinib targets the kinase domain of KIT, but tumor regrowth occurs as a result of them development of resistant mutations in the kinase active site. An alternative small-molecule approach to GIST therapy is described, in which the KIT gene is directly targeted, and thus, kinase resistance may be circumvented. A naphthalene diimide derivative has been used to demonstrate the concept of dual quadruplex targeting. This compound strongly stabilizes both telomeric quadruplex DNA and quadruplex sites in the KIT promoter in vitro. It is shown here that the compound is a potent inducer of growth arrest in a patient-derived GIST cell line at a concentration (similar to 1 mu M) that also results in effective inhibition of telomerase activity and almost complete suppression of KIT mRNA and KIT protein expression. Molecular modeling studies with a telomeric quadruplex have been used to rationalize aspects of the experimental quadruplex melting data.

Structure-based design of selective high-affinity telomeric quadruplex-binding ligands.

A library of triazole-based telomeric quadruplex-selective ligands has been developed that mimic an established family of tri-substituted acridine-based ligands, using crystal structure data as a starting-point for computer-based design. Binding affinities, estimated by electrospray mass spectrometry, are in accord with the design concept.

A click chemistry approach to C3 symmetric, G-quadruplex stabilising ligands.

Described is the structure-based design and synthesis of a series of tris-triazole G-quadruplex binding ligands utilising the copper catalysed azide–alkyne ‘click’ reaction. The results of G-quadruplex stabilisation by the ligands are reported and discussed.

Targeting telomerase and telomeres: a click chemistry approach towards highly selective 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, but is absent in most normal cells. Targeting telomere maintenance mechanisms could potentially halt tumour growth across a broad spectrum of cancer types, with little cytotoxic effect outside cancer cells. Here, we describe in detail a new class of G-quadruplex binding ligands synthesized using a click chemistry approach. These ligands comprise a 1,3-di(1,2,3-triazol-4-yl)benzene pharmacophore, and display high levels of selectivity for interaction with G-quadruplex DNA vs. duplex DNA. The ability of these ligands to inhibit the enzymatic activity of telomerase correlates with their ability to stabilize quadruplex DNA, and with estimates of affinity calculated by molecular modeling.

Molecular dynamics and principal components analysis of human telomeric quadruplex multimers.

Guanine-rich DNA repeat sequences located at the terminal ends of chromosomal DNA can fold in a sequence-dependent manner into G-quadruplex structures, notably the terminal 150–200 nucleotides at the 3' end, which occur as a single-stranded DNA overhang. The crystal structures of quadruplexes with two and four human telomeric repeats show an all-parallel-stranded topology that is readily capable of forming extended stacks of such quadruplex structures, with external TTA loops positioned to potentially interact with other macromolecules. This study reports on possible arrangements for these quadruplex dimers and tetramers, which can be formed from 8 or 16 telomeric DNA repeats, and on a methodology for modeling their interactions with small molecules. A series of computational methods including molecular dynamics, free energy calculations, and principal components analysis have been used to characterize the properties of these higher-order G-quadruplex dimers and tetramers with parallel-stranded topology. The results confirm the stability of the central G-tetrads, the individual quadruplexes, and the resulting multimers. Principal components analysis has been carried out to highlight the dominant motions in these G-quadruplex dimer and multimer structures. The TTA loop is the most flexible part of the model and the overall multimer quadruplex becoming more stable with the addition of further G-tetrads. The addition of a ligand to the model confirms the hypothesis that flat planar chromophores stabilize G-quadruplex structures by making them less flexible.

Tri- and tetra-substituted naphthalene diimides as potent G-quadruplex ligands.

A series of tri- and tetra-substituted naphthalene diimides have been designed and synthesized. Several compounds show exceptional affinity for telomeric G-quadruplex DNA in classical and competition FRET assays and SPR studies. They inhibit telomerase in the TRAP assay, and show potent senescence-based short-term anti-proliferative effects on MCF7 and A549 cancer cell lines, and localize in the nucleus and particularly the nucleolus of MCF7 cells.

The triazatruxene derivative azatrux binds to the parallel form of the human telomeric G-quadruplex under molecular crowding conditions: Biophysical and molecular modeling studies

The present study has employed a combination of spectroscopic, calorimetric and computational methods to explore the binding of the three side-chained triazatruxene derivative, termed azatrux, to a human telomeric G-quadruplex sequence, under conditions of molecular crowding. The binding of azatrux to the tetramolecular parallel [d(TGGGGT)](4) quadruplex in the presence and absence of crowding conditions, was also characterized. The data indicate that azatrux binds in an end-stacking mode to the parallel G-quadruplex scaffold and highlights the key structural elements involved in the binding. The selectivity of azatrux for the human telomeric G-quadruplex relative to another biologically relevant G-quadruplex (c-Kit87up) and to duplex DNA was also investigated under molecular crowding conditions, showing that azatrux has good selectivity for the human telomeric G-quadruplex over the other investigated DNA structures. (C) 2011 Elsevier Masson SAS. All rights reserved.

A structural analysis of G-quadruplex/ligand interactions

This focused review article discusses in detail, all available high-resolution small molecule ligand/G-quadruplex structural data derived from crystallographic and NMR based techniques, in an attempt to understand key factors in ligand binding and to highlight the biological importance of these complexes. In contrast to duplex DNA, G-quadruplexes are four-stranded nucleic acid structures folded from guanine rich repeat sequences stabilized by the stacking of guanine G-quartets and extensive Watson-Crick/Hoogsteen hydrogen bonding. Thermally stable, these topologies can play a role in telomere regulation and gene expression. The core structures of G-quadruplexes form stable scaffolds while the loops have been shown, by the addition of small molecule ligands, to be sufficiently adaptable to generate new and extended binding platforms for ligands to associate, either by extending G-quartet surfaces or by forming additional planar dinucleotide pairings. Many of these structurally characterised loop rearrangements were totally unexpected opening up new opportunities for the design of selective ligands. However these rearrangements do significantly complicate attempts to rationally design ligands against well defined but unbound topologies, as seen for the series of napthalene diimides complexes. Drawing together previous findings and with the introduction of two new crystallographic quadruplex/ligand structures we aim to expand the understanding of possible structural adaptations available to quadruplexes in the presence of ligands, thereby aiding in the design of new selective entities. (C) 2011 Elsevier Masson SAS. All rights reserved.