Characterisation of the interactions and biological impacts of cisplatin analogues and metallo-drug complexes with DNA

Abstract

The work described in this thesis develops and applies several methodologies to determine the potential of cisplatin analogues and novel metallo-drugs as anti-cancer compounds. These methodologies centred around three approaches that elucidate the DNA binding properties of these metallo-drugs. The first approach characterises the mode of DNA binding and measures the metallo-drug effect on modifying DNA structure. The second approach analysed both the ability of the metallo-drugs to inhibit DNA replication and determined the DNA binding sequence specificity. The third approach compared the binding affinity of metallo-drugs for GSH or DNA. The metallo-drugs tested include cisplatin, which served as a positive control since its interactions with DNA have been well studied. The cisplatin analogues tested included carboplatin, oxaliplatin, DCBP, DCRP, DACH, and metallo-intercalators 56MESS and 3478MEEN. Other metallo-drugs such as the copper(II) 56MESS analogue Cu56MESS and the ruthenium(II) compounds RuP1 and RuP2 were also studied. Finally, the well defined DNA strand breaker phleomycin was included to serve as a positive control in for compounds that may exhibit endonuclease activity. Data presented in this thesis demonstrated that the novel application of free solution CE-CC was effective to determine metallo-drug induced structure modifications on a linear dsDNA substrate. Furthermore, metallo-drugs were able to be differentiated into their DNA binding mode by combining CE-CC data with plasmid mobility data on a 2D scatter plot analysis. The 2D scatter plot analysis of metallo-drug/DNA structure modification showed that the ruthenium(II) groove binder RuP2, with is additional pyrrole ligand compared to the RuP1 analogue, could substantially increase the molecular weight of a linear dsDNA substrate. Furthermore, the 2D scatter plot DNA structure analysis revealed the platinum(II) metallo-intercalator 3478MEEN adopted exhibited duel covalent binding and π-π base stacking intercalation with DNA. The covalent binding activity associated with 3478MEEN was also evident in an interstrand crosslink assay which involved a novel application of a urea-based denaturing agarose slab gel electrophoresis and 5"FAM linear dsDNA substrate. The Linear Amplification (LA) reaction was employed to characterise metallo-drug induced inhibition of DNA replication in addition to identifying the test compound's sequence binding specificity. A novel application of Rhodamine 6G labelled nucleotides was used to quantify metallo-drug induced inhibition of DNA replication by a DNA polymerase. Furthermore, the application of a 5"FAM labelled primer in a LA reaction together with separation of the products by Capillary Electrophoresis with Laser Induced Fluorescence (CE-LIF) was performed. From these data, sites of adduct formation were identified that corresponded to the known sequence specificity of cisplatin, in addition the binding specificity of other metallo-drugs were determined. A kinetic study was employed to measure metallo-drug thiol bond reactivity with GSH. It was demonstrated that the thiol bond reactivity with the control platinum(II) compounds cisplatin, oxaliplatin and carboplatin were in agreement with published data, in addition novel reactivity data were obtained for compounds not previously studied. Finally, an in vitro competitive assay and analysis protocol was developed to quantitatively analyse the binding preference of the metallo-drug between DNA and GSH. A 2D scatter plot analysis of metallo-drug reactivity with GSH and DNA revealed that Cu56MESS and the DNA strand breaker, phleomycin, exhibit a similar DNA and GSH reaction profiles in the absence of a reducing agent which warrants further investigation. Overall, developed methodologies produced data that showed that the platinum(II) metallo-intercalator 3478MEEN adopted dual covalent binder and π-π base stacking DNA binding modes. The copper(II) metallo-drug Cu56MESS showed negligible DNA strand breaking activity in the absence of a reducing agent like H2O2, however, it did exhibit GSH reactivity suggesting that the need for a reducing agent may not be necessary for reactions with other biological substrates. Finally, the ruthenium(II) groove binder RuP2 with its additional pyrrole ligand compared to RuP1 could substantially increase the molecular weight of a linear dsDNA substrate.

Date of Award	2013
Original language	English