The characterisation and application of bacterial nitroreductase enzymes

<p>Cancer is an increasing global concern, with the number of people diagnosed growing rapidly each year. Gene directed enzyme prodrug therapy (GDEPT) is emerging as a front-runner of new technologies that seek to combat the growing number of cases. One developing approach to GDEPT involves the use of bacterial nitroreductase enzymes to reduce prodrug substrates, which, upon reduction to their active form, are toxic to cancer cells through DNA crosslinking. Nitroreductases have the ability to activate a variety of nitro-quenched compounds, not only anti-cancer prodrugs, but also nil bystander antibiotics and masked fluorophores, through the reduction of strongly electron-withdrawing nitro substituents on aromatic rings. My research initially sought to exploit this capability by partnering nitroreductases with nil bystander antibiotics for targeted cell ablation, as a component of a larger gene directed enzyme prodrug therapy project. This has potential to provide important safety features for removal of viral and bacterial vectors following anti-cancer gene therapy. From this, the main focus evolved into utilising nitroreductase enzymes for targeted cell ablation for applications in developmental and regenerative biology. This exploited the ability of nitroreductases to activate nil bystander antibiotics in partnership with masked fluorophores for imaging purposes. It has previously been shown that antibiotics can be applied to a nitroreductase under control of a tissue-specific promoter in a transgenic model organism, enabling controlled ablation of that tissue at precise stages of development. However, direct imaging of the nitroreductase location and activity, by application of masked fluorophore probes prior to ablation, has not previously been explored. During the course of this work, several promising combinations of nitroreductases that exhibit opposing specificities for certain combinations of masked fluorophores and nil-bystander antibiotics were identified through screening in bacterial systems. In general, these results were found to translate effectively into eukaryotic cell lines. Pairs of nitroreductases that have opposite specificities for two different antibiotic substrates offer potential for the multiplexed ablation of either (or both) of two different labelled tissues in the same transgenic model organism, according to the substrate(s) administered to that organism. Throughout this screening process, a nitroaromatic substrate (niclosamide) was identified that is, uniquely, initially toxic to Escherichia coli but becomes non-toxic upon reduction of the nitro substituent. Using niclosamide, a novel strategy with potential for identification of new nitroreductases, as well as selection-based directed evolution to improve desired activities, was explored.</p>

The characterisation and application of bacterial nitroreductase enzymes

<p>Cancer is an increasing global concern, with the number of people diagnosed growing rapidly each year. Gene directed enzyme prodrug therapy (GDEPT) is emerging as a front-runner of new technologies that seek to combat the growing number of cases. One developing approach to GDEPT involves the use of bacterial nitroreductase enzymes to reduce prodrug substrates, which, upon reduction to their active form, are toxic to cancer cells through DNA crosslinking. Nitroreductases have the ability to activate a variety of nitro-quenched compounds, not only anti-cancer prodrugs, but also nil bystander antibiotics and masked fluorophores, through the reduction of strongly electron-withdrawing nitro substituents on aromatic rings. My research initially sought to exploit this capability by partnering nitroreductases with nil bystander antibiotics for targeted cell ablation, as a component of a larger gene directed enzyme prodrug therapy project. This has potential to provide important safety features for removal of viral and bacterial vectors following anti-cancer gene therapy. From this, the main focus evolved into utilising nitroreductase enzymes for targeted cell ablation for applications in developmental and regenerative biology. This exploited the ability of nitroreductases to activate nil bystander antibiotics in partnership with masked fluorophores for imaging purposes. It has previously been shown that antibiotics can be applied to a nitroreductase under control of a tissue-specific promoter in a transgenic model organism, enabling controlled ablation of that tissue at precise stages of development. However, direct imaging of the nitroreductase location and activity, by application of masked fluorophore probes prior to ablation, has not previously been explored. During the course of this work, several promising combinations of nitroreductases that exhibit opposing specificities for certain combinations of masked fluorophores and nil-bystander antibiotics were identified through screening in bacterial systems. In general, these results were found to translate effectively into eukaryotic cell lines. Pairs of nitroreductases that have opposite specificities for two different antibiotic substrates offer potential for the multiplexed ablation of either (or both) of two different labelled tissues in the same transgenic model organism, according to the substrate(s) administered to that organism. Throughout this screening process, a nitroaromatic substrate (niclosamide) was identified that is, uniquely, initially toxic to Escherichia coli but becomes non-toxic upon reduction of the nitro substituent. Using niclosamide, a novel strategy with potential for identification of new nitroreductases, as well as selection-based directed evolution to improve desired activities, was explored.</p>

Synaptic Alterations in a Transgenic Model of Tuberous Sclerosis Complex: Relevance to Autism Spectrum Disorders

Tuberous sclerosis complex (TSC) is a rare, multi-system genetic disease with serious neurological and mental symptoms, including autism. Mutations in the TSC1/TSC2 genes lead to the overactivation of mTOR signalling, which is also linked to nonsyndromic autism. Our aim was to analyse synaptic pathology in a transgenic model of TSC: two-month-old male B6;129S4-Tsc2tm1Djk/J mice with Tsc2 haploinsufficiency. Significant brain-region-dependent alterations in the expression of several synaptic proteins were identified. The most prominent changes were observed in the immunoreactivity of presynaptic VAMP1/2 (ca. 50% increase) and phospho-synapsin-1 (Ser62/67) (ca. 80% increase). Transmission electron microscopy demonstrated serious ultrastructural abnormalities in synapses such as a blurred structure of synaptic density and a significantly increased number of synaptic vesicles. The impairment of synaptic mitochondrial ultrastructure was represented by excessive elongation, swelling, and blurred crista contours. Polyribosomes in the cytoplasm and swollen Golgi apparatus suggest possible impairment of protein metabolism. Moreover, the delamination of myelin and the presence of vacuolar structures in the cell nucleus were observed. We also report that Tsc2+/− mice displayed increased brain weights and sizes. The behavioural analysis demonstrated the impairment of memory function, as established in the novel object recognition test. To summarise, our data indicate serious synaptic impairment in the brains of male Tsc2+/− mice.