Gary N. Parkinson

Quadruplex DNA: sequence, topology and structure

G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential quadruplex sequences have been identified in G-rich eukaryotic telomeres, and more recently in non-telomeric genomic DNA, e.g. in nuclease-hypersensitive promoter regions. The natural role and biological validation of these structures is starting to be explored, and there is particular interest in them as targets for therapeutic intervention. This survey focuses on the folding and structural features on quadruplexes formed from telomeric and non-telomeric DNA sequences, and examines fundamental aspects of topology and the emerging relationships with sequence. Emphasis is placed on information from the high-resolution methods of X-ray crystallography and NMR, and their scope and current limitations are discussed. Such information, together with biological insights, will be important for the discovery of drugs targeting quadruplexes from particular genes.

Structural Basis of DNA Quadruplex Recognition by an Acridine Drug

The crystal structure of a complex between the bimolecular human telomeric quadruplex d(TAGGGTTAGGGT)2 and the experimental anticancer drug BRACO-19, has been determined, to 2.5 A resolution. The binding site for the BRACO-19 molecule is at the interface of two parallel-folded quadruplexes, sandwiched between a G-tetrad surface and a TATA tetrad, and held in the site by networks of water molecules. The structure rationalizes the existing structure-activity data and provides a starting-point for the structure-based design of quadruplex-binding ligands

Selectivity in Ligand Recognition of G-Quadruplex Loops.

A series of disubstituted acridine ligands have been cocrystallized with a bimolecular DNA G-quadruplex. The ligands have a range of cyclic amino end groups of varying size. The crystal structures show that the diagonal loop in this quadruplex results in a large cavity for these groups, in contrast to the steric constraints imposed by propeller loops in human telomeric quadruplexes. We conclude that the nature of the loop has a significant influence on ligand selectivity for particular quadruplex folds.

Abstract 2326: The first crystal structure of a telomeric quadruplex DNA with a metal-containing ligand

The inhibition of telomerase function in cancer cells via induction of a higher-order four-stranded quadruplex arrangement in the telomeric DNA substrate, requires the participation of a quadruplex-stabilizing ligand. We showed several years ago that certain metal complexes could fulfil this role and not only have selectivity for a quadruplex arrangement but had significant biological activity. We now report on the crystal structures of square-planar nickel (II) and copper (II) complexes containing salphen ligands with a human telomeric quadruplex. The structures show that the metal complexes are bound on an external G-quartet face of the quadruplex, and that the metal itself is positioned above the potassium-containing ion channel of the quadruplex, providing important stabilization. The planar groups of the ligand are stacked onto guanine bases, and the charged amino side-chains are positioned in the grooves of the quadruplex. These features are enabling the available structure-activity data to be rationalized and will be discussed in terms of the design of future complexes with enhanced activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2326. doi:10.1158/1538-7445.AM2011-2326

Abstract 2310: Down-regulating BCL-2 gene expression in a resistant GIST cell line by a RNA G-quadruplex interacting small molecule ligand

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL We have previously shown that KIT protein expression in the gastrointestinal tumour (GIST) cell line GIST882 can be down-regulated using a small molecule approach that targets expression at the transcriptional level. The small-molecule naphthalene diimide derivative ND1 developed as a G-quadruplex telomere targeting agent can inhibit telomerase activity and can also reduce c-kit mRNA levels, consistent with a dual G-quadruplex mechanism of action in this cell line. An Imatinib (Gleevec) resistant patient-derived cell line GIST48 expresses high levels of the anti-apoptotic protein bcl-2. The compound ND1 down-regulates the expression of bcl-2 in this resistant cell line, while having no discernable effect on c-kit protein expression levels or telomerase activity, both associated with DNA quadruplex stabilization. Instead we observe that translation of the apoptosis-related protein is impeded, while mRNA levels remain unaffected. The identification of a G-rich quadruplex-forming sequence in the 5’-UTR region of the bcl-2 gene, upstream of the translation start site, lead us to the development of a dual luciferase reporter assay. Utilizing a psiCHECK-2 vector we inserted the G-rich 67 base pair bcl-2 5’-UTR gene sequence upstream of the Renilla luciferase start codon. In vitro experiments have shown that the G-rich sequence can form a stable RNA G-quadruplex and interfere with translation. Our in vivo transfection experiments with ND1 in a GIST cell line shows concentration-dependent inhibition of bcl-2 protein expression. RNA G-quadruplex formation and ligand stabilization may thus be a novel anti-cancer route for the down-regulation of the anti-apoptotic protein bcl-2 in chemo-resistant cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2310. doi:10.1158/1538-7445.AM2011-2310

Abstract 3679: Targeting drug-resistant GIST cells with a quadruplex-binding small molecule

Gastrointestinal tumours (GIST) originate, at least in part, from disregulation and consequent aberrant expression of the c-kit oncogene. Imatinib is an effective inhibitor of the ATP-binding activity of the kinase domain in the c-kit protein, and is used clinically to treat GIST patients. However acquired resistance to Imatinib is a severe problem hindering long-term patient survival. We have previously reported on an alternative approach to c-kit therapy that involves targeting and stabilising quadruplex DNA sequences with a small molecule, (i) in two sequences within the c-kit promoter so that c-kit expression is down-regulated, and (ii) in the single-stranded overhang of telomeric DNA, inhibiting telomerase activity in GIST cells. We have previously reported that both of these are targeted by a potent (IC 50 ≪ 1μM) tetra-substituted naphthalene diimide compound in the Imatinib-sensitive GIST882 patient-derived cell line. This compound is equally potent in the patient-derived GIST48 Imatinib-resistant line; we report here that it has no effect on c-kit expression, telomerase activity, or the expression of other oncogenes in which promoter quadruplexes have been identified. Instead we find that the translation of the apoptosis-related bcl-2 gene is profoundly affected, but not its transcription. The bcl-2 gene has a potential quadruplex-forming region in the 5′-UTR sequence, and we report here that this forms a stable RNA quadruplex structure, which is further stabilised by the binding of the naphthalene diimide ligand. A reporter assay approach using a dual luciferase psiCHECK-2 vector, containing the bcl-2 5′-UTR sequence, is being used to validate the key concept that this G-rich sequence is involved in modulating translation in vitro, and in vivo through transfection into mammalian cells. Our current experiments in an Imatinib-resistant GIST cell line in the presence of the compound are enabling us to substantiate this cellular target and characterize the mode of action. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3679.