Drewe, William Charles; (2008) The synthesis and biological evaluation of novel urea-based ligands as G-quadruplex interacting agents. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In recent years the formation of G-quadruplex DNA structures has received much attention due to its anti-cancer therapeutic potential. The proposed anti-cancer properties of G-quadruplex DNA arise from the fact that guanine rich DNA sequences with a predisposition for forming these higher-order DNA structures have been identified in the human telomere, as well as in the promoter regions of many proto-oncogenes. Human telomeric DNA consists of a tandem repeated sequence 5'- (TTAGGG)n-3' and is single stranded for its final ~200 base pairs. While this single stranded region of the genome is the substrate of the enzyme telomerase which is up regulated in ~90% of human cancer cells and leads directly to immortality, it is also able to form a G-quadruplex. Hence, upon introduction of a G-quadruplex stabilising ligand, the single stranded telomerase substrate is sequestered as a G-quadruplex, resulting in telomerase inhibition, telomere dysfunction and specific loss of cancer cell viability. This thesis describes the synthesis and initial biological evaluation of four series of novel non-polycyclic urea-based ligands rationally designed to target G-quadruplex DNA structures. Chemical modifications including structural substitution patterns, linker, side chain, and thiourea for urea have been used to investigate the aspects of ligand structure which lead to optimal biological properties. Assessment of the stabilisation of the G-quadruplex formed by the human telomere, as well as those in the promoter regions of the c-kit and c-myc proto-oncogenes was achieved by biophysical assay based upon fluorescent resonance energy transfer (FRET) and surface plasmon resonance (SPR). These methodologies were also used to assess the G-quadruplex selectivity of these ligands relative to duplex DNA. These experiments show that these ligands are less potent than BRACO-19 and TMPyP4, however are significantly more G-quadruplex selective in vitro. An explanation of selectivity as well as the factors which lead to enhanced G-quadruplex interaction was assessed by molecular modelling. Analysis of short-term cancer cell cytotoxicity assessed by the sulforhodamine-B (SRB) assay, combined with the biophysical assay results afforded lead structures whose in vitro telomeric mechanism of action was investigated by a modified telomerase repeat amplification protocol (TRAP-LIG), and by the induction of cellular senescence and telomere end - end fusion.

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