Schultes, Christoph Michael; (2005) Studies on the biophysical and biochemical properties of telomere-targeting agents. Doctoral thesis (Ph.D.), University College London.

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Abstract

The activation of the holoenzyme telomerase is one of the key events in the oncological transformation of human cells. Inhibition of this important DNA- interacting complex has thus become established as a promising potential strategy for anti-cancer drug development. The single-stranded, G-rich telomeric DNA substrate of telomerase can fold in a variety of ways to form higher-order G-quadruplexes. Detailed characterisation of these structures has enabled the application of rational drug design approaches to the development of small molecules that will have specificity for the unique features of these structures, thereby making possible the selective targeting of telomeric DNA. The central purpose of the thesis is an investigation of the ability of a series of acridine-based compounds to interact with telomeric DNA via quadruplex- ligand complexes, with the aim of inhibiting telomerase-mediated telomere maintenance or influencing other telomere-associated processes. The work comprises two main sections. Initial screening and biophysical characterisation of a compound library using a fluorescence-based (fluorescence resonance energy transfer, FRET) assay allowed compounds with promising characteristics to be identified. Strength of binding via quadruplex stabilisation was assessed by the measurement of increases in the melting temperature of the DNA in complex with the ligand. Further data from separate ligand-DNA binding experiments (using surface plasmon resonance, SPR) allowed more detailed examination of these interactions. Together with computational techniques, including molecular dynamics simulations and free energy calculations, this enabled the construction of an in silico model to rationalise the observed results. Studies to investigate the biological effects of cellular exposure to the compounds were also undertaken. In vitro biochemical techniques to measure telomerase enzyme inhibition (telomere repeat amplification protocol, TRAP assay) and cell culture experiments to examine the effect on cancer cell growth were conducted to this effect. This has been translated into structure-activity relationships indicating the relative contributions of specific chemical moieties to the ligand-DNA interaction, while further elucidating the cellular mode of action of the compounds and the biochemical consequences of exposure.

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