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G-quadruplex (G4) sequences, which can fold into higher-order G4 structures, are abundant in the human genome and are over-represented in the promoter regions of many genes involved in human cancer initiation, progression, and metastasis. They are plausible targets for G4-binding small molecules, which would, in the case of promoter G4s, result in the transcriptional downregulation of these genes. However, structural information is currently available on only a very small number of G4s and their ligand complexes. This limitation, coupled with the currently restricted information on the G4-containing genes involved in most complex human cancers, has led to the development of a phenotypic-led approach to G4 ligand drug discovery. This approach was illustrated by the discovery of several generations of tri- and tetra-substituted naphthalene diimide (ND) ligands that were found to show potent growth inhibition in pancreatic cancer cell lines and are active in in vivo models for this hard-to-treat disease. The cycles of discovery have culminated in a highly potent tetra-substituted ND derivative, QN-302, which is currently being evaluated in a Phase 1 clinical trial. The major genes whose expression has been down-regulated by QN-302 are presented here: all contain G4 propensity and have been found to be up-regulated in human pancreatic cancer. Some of these genes are also upregulated in other human cancers, supporting the hypothesis that QN-302 is a pan-G4 drug of potential utility beyond pancreatic cancer.

The role of G-quadruplexes in human cancers is increasingly well-defined. Accordingly, G-quadruplexes can be suitable drug targets and many small molecules have been identified to date as G-quadruplex binders, some using computer-based design methods and co-crystal structures. The role of bound water molecules in the crystal structures of G-quadruplex-small molecule complexes has been analyzed in this study, focusing on the water arrangements in several G-quadruplex ligand complexes. One is the complex between the tetrasubstituted naphthalene diimide compound MM41 and a human intramolecular telomeric DNA G-quadruplex, and the others are in substituted acridine bimolecular G-quadruplex complexes. Bridging water molecules form most of the hydrogen-bond contacts between ligands and DNA in the parallel G-quadruplex structures examined here. Clusters of structured water molecules play essential roles in mediating between ligand side chain groups/chromophore core and G-quadruplex. These clusters tend to be conserved between complex and native G-quadruplex structures, suggesting that they more generally serve as platforms for ligand binding, and should be taken into account in docking and in silico studies.

This unique and practical resource provides the most complete and concise summary of underlying principles and approaches to studying nucleic acid structure, including discussion of x-ray crystallography, NMR, molecular modelling, and databases. Its focus is on a survey of structures especially important for biomedical research and pharmacological applications. To aid novices, the book includes an introduction to technical lingo used to describe nucleic acid structure and conformations (roll, slide, twist, buckle, etc.). This completely updated edition features expanded coverage of the latest advances relevant to recognition of DNA and RNA by small molecules and proteins. In particular, the reader will find extensive new discussions on: RNA folding, ribosome structure and antibiotic interactions, DNA quadruplexes, DNA and RNA protein complexes, and short interfering RNA (siRNA). This handy guide ends with a complete list of resources, including relevant online databases and software. * Completely updated with expanded discussion of topics such as RNA folding, ribosome structure and antibiotic interactions, DNA quadruplexes, DNA and RNA protein complexes, and short interfering RNA (siRNA)* Includes a complete list of resources, including relevant online databases and software* Defines technical lingo for novices

The stabilisation of G-quadruplexes (G4s) by small-molecule compounds is an effective approach for causing cell growth arrest, followed by cell death. Some of these compounds are currently being developed for the treatment of human cancers. We have previously developed a substituted naphthalene diimide G4-binding molecule (CM03) with selective potency for pancreatic cancer cells, including gemcitabine-resistant cells. We report here that CM03 and the histone deacetylase (HDAC) inhibitor SAHA (suberanilohydroxamic acid) have synergistic effects at concentrations close to and below their individual GI50 values, in both gemcitabine-sensitive and resistant pancreatic cancer cell lines. Immunoblot analysis showed elevated levels of γ-H2AX and cleaved PARP proteins upon drug combination treatment, indicating increased levels of DNA damage (double-strand break events: DSBs) and apoptosis induction, respectively. We propose that the mechanism of synergy involves SAHA relaxing condensed chromatin, resulting in higher levels of G4 formation. In turn, CM03 can stabilise a greater number of G4s, leading to the downregulation of more G4-containing genes as well as a higher incidence of DSBs due to torsional strain on DNA and chromatin structure.

DNA requires hydration to maintain its structural integrity. Crystallographic analyses have enabled patterns of water arrangements to be visualized. We survey these water motifs in this review, focusing on left- and right-handed duplex and quadruplex DNAs, together with the i-motif. Common patterns of linear spines of water organization in grooves have been identified and are widely prevalent in right-handed duplexes and quadruplexes. By contrast, a left-handed quadruplex has a distinctive wheel of hydration populating the almost completely circular single groove in this structure.

The tetrasubstituted naphthalene diimide compound QN-302 binds to G-quadruplex (G4) DNA structures. It shows high potency in pancreatic ductal adenocarcinoma (PDAC) cells and inhibits the transcription of cancer-related genes in these cells and in PDAC animal models. It is currently in Phase 1a clinical evaluation as an anticancer drug. A study of structure–activity relationships of QN-302 and two related analogues (CM03 and SOP1247) is reported here. These have been probed using comparisons of transcriptional profiles from whole-genome RNA-seq analyses, together with molecular modelling and molecular dynamics simulations. Compounds CM03 and SOP1247 differ by the presence of a methoxy substituent in the latter: these two compounds have closely similar transcriptional profiles. Whereas QN-302 (with an additional benzyl-pyrrolidine group), although also showing down-regulatory effects in the same cancer-related pathways, has effects on distinct genes, for example in the hedgehog pathway. This distinctive pattern of genes affected by QN-302 is hypothesized to contribute to its superior potency compared to CM03 and SOP1247. Its enhanced ability to stabilize G4 structures has been attributed to its benzyl-pyrrolidine substituent fitting into and filling most of the space in a G4 groove compared to the hydrogen atom in CM03 or the methoxy group substituent in SOP1247.

Quadruplex nucleic acids — helical four-stranded structures known to form from guanine-rich nucleic acid sequences through Hoogsteen-type hydrogen bonding — were once just laboratory curiosities. However, they are now emerging from this rather obscure status to become important targets for small-molecule drugs, which can stabilize the quadruplex structures and thereby promote selective downregulation of gene expression and telomerase inhibition, and also activate DNA damage responses. Most of these quadruplex-binding small molecules can stabilize a range of cellular quadruplexes, as well as clusters of quadruplexes within a single gene or telomere, which could be an advantage. This widespread stabilization can generate a polygene response, and thus is able to simultaneously affect several key driver genes in human cancers, with potential therapeutic benefit. Quadruplex DNA structures were first observed more than 50 years ago, but have only relatively recently attracted interest for their role in the development of cancer. This Perspective considers attempts to selectively bind and stabilize these structures using small molecules, with the aim of developing anticancer therapeutics.

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.

Intronic GGGGCC repeat expansions in C9orf72 are the most common known cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), which are characterised by degeneration of cortical and motor neurons, respectively. Repeat expansions have been proposed to cause disease by both the repeat RNA forming foci that sequester RNA‐binding proteins and through toxic dipeptide repeat proteins generated by repeat‐associated non‐ATG translation. GGGGCC repeat RNA folds into a G‐quadruplex secondary structure, and we investigated whether targeting this structure is a potential therapeutic strategy. We performed a screen that identified three structurally related small molecules that specifically stabilise GGGGCC repeat G‐quadruplex RNA. We investigated their effect in C9orf72 patient iPSC‐derived motor and cortical neurons and show that they significantly reduce RNA foci burden and the levels of dipeptide repeat proteins. Furthermore, they also reduce dipeptide repeat proteins and improve survival in vivo , in GGGGCC repeat‐expressing Drosophila . Therefore, small molecules that target GGGGCC repeat G‐quadruplexes can ameliorate the two key pathologies associated with C9orf72 FTD/ALS. These data provide proof of principle that targeting GGGGCC repeat G‐quadruplexes has therapeutic potential. Synopsis Small molecules targeting G‐quadruplex GGGGCC repeat RNA are effective at ameliorating disease phenotypes in C9orf72 patient neurons, and in vivo phenotypes in C9orf72 flies. Therefore, targeting expanded GGGGCC RNA could be an effective therapeutic strategy for C9orf72 ALS and FTD. FRET based screen identifies small molecules that specifically bind to C9orf72 repeat RNA G‐quadruplexes. Small molecules reduce RNA foci and dipeptide repeat proteins (DPRs) in C9orf72 patient neurons. G‐quadruplex GGGGCC binding small molecule improves survival and reduces levels of the toxic DPR poly‐GR in C9orf72 flies. Provides proof of principle for targeting GGGGCC RNA G‐quadruplexes in C9orf72 FTD/ALS. Graphical Abstract Small molecules targeting G‐quadruplex GGGGCC repeat RNA are effective at ameliorating disease phenotypes in C9orf72 patient neurons, and in vivo phenotypes in C9orf72 flies. Therefore, targeting expanded GGGGCC RNA could be an effective therapeutic strategy for C9orf72 ALS and FTD.