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Molecular recognition, gene modulation and biological activity of fluorescent sequence specific DNA-interacting polyamides

Pett, LC; (2017) Molecular recognition, gene modulation and biological activity of fluorescent sequence specific DNA-interacting polyamides. Doctoral thesis , UCL (University College London). Green open access

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Abstract

DNA-binding polyamides can be programmed to target critical genomic sequences to inhibit specific protein-DNA interactions and modulate gene expression. Inherently non-genotoxic, these non-covalent binding small molecules exert their biological activity without inflicting DNA damage and could provide the basis for potentially less toxic DNA-targeting anti-cancer therapeutics. In this thesis, polyamide design strategies were explored to enhance the biological effects of these small molecules through increasing DNA binding affinity, sequence selectivity and nuclear uptake. In addition, whole transcriptome analysis was used to gain a greater understanding of their genome-wide effects on gene expression. The introduction of the fluorescent p-anisylbenzimidazole (Hx) DNA recognition element provided an intrinsic probe to monitor cellular uptake and a new framework for the design of fluorescent sequence selective DNA-binding molecules, capable of efficient nuclear localisation and in vitro gene regulation. Hx-polyamide HxIP, targeted to the 5’-TACGAT-3’ sequence overlapping the inverted CCAAT Box 2 (ICB2) on the topoisomerase II (topo II) promoter, inhibited the repressive binding of transcription factor NF-Y, causing re-stimulation of gene expression and increased cellular sensitivity to etoposide. Subsequent, polyamide functionalisation at the N1 position of the heterocyclic rings through the introduction of a cationic alkyl amino group further enhanced polyamide biological activity and offers a strategy to improve the drug-like properties of these small molecules. N-terminal modification of the HxIP polyamide structure through substitution of the p-anisyl component of the Hx moiety with heterocyclic functional groups of differing shape, size and polarity was also investigated. The introduction of the N-terminus functionalities negatively affected cellular uptake, resulting in a reduced biological response relative to the parent polyamide. Furthermore, confirmation of the G・C specific recognition properties of the p-anisyl-aza-4-benzimidazole (AzaHx) moiety expanded both the number of biological relevant sequences that can be targeted using the Hx-based generation of polyamides and the repertoire of inherently fluorescent DNA recognition heterocycles. Genome-wide transcriptome profiling of MDA-MB-231 breast cancer cells revealed that polyamides AzaHxPI and HxIP generated distinct gene expression profiles and stimulated polyamide-specific biological responses through targeting different DNA sequences. AzaHxPI inhibited cell growth and induced apoptosis, and in contrast HxIP showed limited cytotoxicity. Mechanistic studies showed AzaHxPI induced anti-proliferative and pro-apoptotic effects through the modulation of multiple signalling pathways, without evidence of DNA damage. These results emphasised the potential of polyamides as apoptosis-inducing DNA-targeting agents benefiting from enhanced sequence selectivity, which in turn confers polyamide-specific biological responses.

Type: Thesis (Doctoral)
Title: Molecular recognition, gene modulation and biological activity of fluorescent sequence specific DNA-interacting polyamides
Event: UCL (University College London
Open access status: An open access version is available from UCL Discovery
Language: English
Keywords: Polyamides, Sequence specific DNA-binding, Molecular recognition, Non-covalent, Modulation of gene expression, Chemosensitisation, Apoptosis-inducing agent
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/1566579
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