Abstract:
An emerging anti-cancer strategy is the development of cancer prodrugs for targeted therapies. Nitroaromatic prodrugs have shown great promise in this regard due to the ability of the strongly electronegative nitro group to act as a “molecular switch”, which upon reduction can activate the cytotoxic properties of other substituent groups in the molecule. A variety of nitroaromatic prodrugs in combination with oxygen-independent nitroreductase (NTR) enzymes derived from bacteria have been evaluated in various Gene Directed Enzyme Prodrug Therapy (GDEPT) models to develop next-generation cancer therapies, but further work is required to effectively study and optimise these strategies.
A key limitation in GDEPT is inefficient gene delivery. To improve gene delivery it is important to be able to effectively monitor vector distribution and gene expression. Standard invasive methods involving biopsy are both cumbersome and invasive so other reporter methods such as the use of fluorescence aim to improve on these, at least in a preclinical setting. In this study we aimed to test and screen novel masked fluorophores for their ability to report on NTR activity in a GDEPT context, as well as other biological applications including identification of novel NTRs and their potential as cellular reporters of gene expression.
Initially a panel of novel masked fluorophores with a blue light emission profile were screened to identify optimal probes to report on the activity of the Escherichia coli NTR NfsB. The panel was then increased to include masked fluorophores with a range of emission spectra. With the aim of creating a multiplex genetic reporter system these were screened against an extended NTR library derived from 19 different bacterial species.
Certain masked fluorophores that were structural analogues of promising nitroaromatic prodrugs were also used to screen environmental DNA libraries to recover novel NTR enzymes. A high throughput flow cytometry method was developed and used to discover several NTRs with prodrug activity.
In a final application, a masked fluorophore was incorporated into a targeted ablation system where activating NTRs had been placed under the control of a stem cell specific promoter, SOX2. The masked fluorophore was used to report on cellular SOX2 expression and viability prior to the addition of a cell-ablating prodrug, metronidazole.
The research within this thesis has shown that masked fluorophores have the ability to accurately report NTR activity and that they have the sensitivity to differentiate between different NTRs. This imaging capability could greatly benefit pre-clinical GDEPT research. The research undertaken in this thesis has also shown that there are several other biological applications for masked fluorophore/NTR pairings beyond their use in GDEPT.
