Lombardo, Caterina Maria; (2011) The development of new G-quadruplex stabilising ligands using click chemistry. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

G-quadruplexes are highly specific nucleic acid organisations characteristic of G-rich sequences. They are polymorphic quadruple helices with a hydrophobic core of sets of G-quartets each formed by four guanines interacting via Hoogsteen hydrogen bonds, and four negatively charged grooves formed by the phosphate backbone. A considerable number of genes containing G-rich genomic sequences are believed to play a key role in tumour development, e.g. the proto-oncogenes Ckit-1 and Ckit-2, the hypoxia inducible factor 1α promoter HIF-1α and telomeres, therefore these putative G-quadruplexes are an appealing target for cancer therapy. Many different G-quadruplex ligands have been synthesized to date, but the specificity and the low in vitro/in vivo activity of the majority have not been sufficient for them to progress further. The approach described here towards the discovery of G-quadruplex targeting drugs focuses on the synthesis of libraries derived from combinations of different building blocks. "Click-chemistry" Cu(I) catalysed Huisgen 1,3-dipolar cycloaddition has been an essential tool to achieve this objective. Two classes of building blocks have been synthesized, azides and alkynes, which include an aromatic core and basic side chains that is positively charged under physiological conditions. With these fragments, three libraries of G-quadruplex ligands have been generated, extending the aromatic surface by the click reaction, which results in 1,4-triazole ring formation. The aromatic cores and triazole rings in the final compounds are appropriate for interactions with the G-quartets in a quadruplex, and the positively charged side chains interact with the grooves. The diversity introduced in each library enabled me to investigate the binding influence of the core structure and of the number of side chains. The affinity and selectivity of these compounds for G-quadruplexes has been evaluated by binding experiments using FRET, circular dichroism and ESI-MS. Short and long term cytotoxicity studies have been performed on a panel of cancer cells either expressing telomerase, or genes which could be regulated by G-quadruplex sequences; telomerase inhibition has been studied with the cell free TRAP assay. Two lead ligands have been identified N,N'-((4,4'-(Naphthalene-2,7-diyl)bis(1H-l,2,3-triazole- 4,l-diyl))bis(3,1-phenylene))bis(2-(diethylamino)acetamide) (compound 33) and N,N',N''-((4,4',4"-(Benzene-1,3,5-triyl)tris(1H-1,2,3-triazole-4,1-diyl))tris(benzene-3,1-diyl))tris(3-(pyrrolidin-1-yl)propanamide) (compound 46). Compound 46 showed telomerase inhibitory activity in vitro, probably due to stabilisation of the parallel G-quadruplex structure on the telomeres, as suggested by biophysical studies. Furthermore, compound 33 displayed selective cytotoxicity for renal cancer cells RCC4 in vitro, which could be ascribed to G-quadruplex formation in the promoter region of HIF-1α gene, which is up-regulated in renal cancers.

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