NTR Mechanism of Action
Bacterial nitroreductases are members of a diverse family of oxidoreductase enzymes that can catalyse the bioreductive activation of nitroaromatic compounds, including anti-cancer prodrugs, antibiotic prodrugs and PET imaging probes. These enzymes have diverse applications in medicine and research, including the anti-cancer strategy 'gene‑directed enzyme-prodrug therapy' and targeted ablation of nitroreductase‑expressing cells in transgenic zebrafish to model degenerative disease.
Nitroreductase-mediated catalysis is governed by a ping pong bi-bi redox reaction mechanism involving two redox half reactions; an oxidative half reaction consisting of initial electron transfer from a nicotinamide cofactor (NAD(P)H) to the bound FMN moiety, and a reductive half reaction consisting of subsequent electron transfer from the FMN to a terminal electron acceptor substrate (e.g., the nitro group of an aromatic ring). Nitroreduction can be considered an ‘electronic switch’ that results in one of the largest shifts in electronic effect achievable through catalysis mediated by a single enzyme. As such, the addition of one or more strongly electron‑withdrawing nitro substituents to an aromatic ring is a strategy that has been exploited widely in prodrug design, whereby addition of an electronegative nitro group draws electron density away from other highly reactive substituents, rendering them inert. Nitroreductase‑mediated reduction of the nitro group effectively pushes electrons back into the ring, unleashing the reactive substituents and resulting in, for example, activation of cytotoxic DNA damaging metabolites such as nitro-CBI-DEI, CB1954 and metronidazole.
When localised to and expressed in a cell, a nitroreductase enzyme can ‘flick a molecular switch’, reducing otherwise innocuous prodrugs to cytotoxic forms that induce cell death in a targeted manner. Cells that researchers may wish to target for ablation include cancerous tissues in humans, or cells which are theorised to play a role in developmental or regenerative processes in model organisms. Controlled ablation of the latter in a regenerative species, such as the zebrafish, can aid researchers in understanding the processes by which these cells are regenerated, and compounds that promote this . This can in turn provide insight into how we might better treat human degenerative diseases, such as Diabetes and Retinitis Pigmentosa. The Ackerley lab has a core wild type nitroreductase library comprised of over 58 oxidoreductases from a variety of culturable and unculturable bacterial species. In addition, we have generated multiple large directed evolution libraries based on a number of these wild type enzymes.
Engineering nitroreductase enzymes for improved activation of cancer prodrugs and imaging molecules
A very useful property of nitroreductase enzymes is their ability to activate a variety of nitro-masked chemotherapeutic drugs and positron emission tomography (PET) imaging probes. Non-invasive fluorescent or PET imaging enables visualisation of the cells or tissues in a patient in which the enzyme is being expressed, allowing one to confirm localisation of the enzyme before delivering the prodrug and inducing cell death. As such, this project aims to identify and engineer nitroreductase enzymes for optimised activation of both next-generation cancer prodrugs and PET imaging probes.
Engineering nitroredutase enzymes for targeted cell ablation in zebrafish
Inducible and targeted ablation of specific cell populations within an organism is a powerful way to study the physiological roles of cells during development and generate models of human degenerative disease states. Zebrafish are ideal candidates for these studies due to their innate capacity to regenerate tissues and their translucent appearance, which allows developmental and regenerative processes to be easily monitored. Targeted ablation in zebrafish can be achieved by using a cell-specific promoter to express a nitroreductase gene in target cells, followed by administration of a nil-bystander prodrug, such as metronidazole.
Upon nitroreduction, metronidazole is converted to a cytotoxic, cell-entrapped form which induces death in nitroreductase-expressing cells. In this research we identify and engineer improved nitroreductases to achieve cell-specific ablation at dramatically reduced prodrug concentrations. Faster ablation kinetics and lower prodrug doses permits enhanced interrogations of cell function, ablation of recalcitrant cell types and extended duration of ablation experiments to enable modelling of chronic disease states.
Detoxification and bioremediation of xenobiotics
Many xenobiotic nitroaromatic compounds are generated directly or indirectly during industrial processes such as the production of explosives (2,4,6-TNT and 2,4-DNT), pesticides (parathion), plastics (mono- and di-nitrophenols) and dyes. The presence of these compounds in the environment has been associated with carcinogenic and mutagenic effects and degradation or removal of these compounds is of high importance. The Ackerley lab has identified Type I nitroreductases from a variety of bacterial species that are capable of reducing such compounds to less toxic/bioavailable derivatives.These enzymes may be used for bioremediation purposes, whereby nitroreductase‑expressing bacteria could be incorporated into composting and bioslurry processes to achieve decontamination in situ. In a complementary approach, plant species such as Arabidopsis and Nicotiana could be engineered to express these nitroreductase enzymes, enabling phytoremediation of contaminated waters and soils. This project aims to use metagenomic functional screening strategies to identify novel degradative enzymes followed by enzyme engineering to maximise these bioremediative activities.