Nitroreductase gene-directed enzyme prodrug therapy: insights and advances toward clinical utility

Abstract

This review examines the vast catalytic and therapeutic potential offered by type I (i.e. oxygen-insensitive) nitroreductase enzymes in partnership with nitroaromatic prodrugs, with particular focus on gene-directed enzyme prodrug therapy (GDEPT; a form of cancer gene therapy). Important first indications of this potential were demonstrated over 20 years ago, for the enzyme-prodrug pairing of Escherichia coli NfsB and CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, it has become apparent that both the enzyme and the prodrug in this prototypical pairing have limitations that have impeded their clinical progression. Recently, substantial advances have been made in the biodiscovery and engineering of superior nitroreductase variants, in particular development of elegant high-throughput screening capabilities to enable optimization of desirable activities via directed evolution. These advances in enzymology have been paralleled by advances in medicinal chemistry, leading to the development of second- and third-generation nitroaromatic prodrugs that offer substantial advantages over CB1954 for nitroreductase GDEPT, including greater dose-potency and enhanced ability of the activated metabolite(s) to exhibit a local bystander effect. In addition to forging substantial progress towards future clinical trials, this research is supporting other fields, most notably the development and improvement of targeted cellular ablation capabilities in small animal models, such as zebrafish, to enable cell-specific physiology or regeneration studies.

Targeting Phenotypically TolerantMycobacterium tuberculosis

“…The strongly electronegative nitro moiety has varying degrees of reactivity that depend on its local environment within a molecule. Mycobacterial nitroreductases may catalyze the conversion of the nitro group (-NO 2 ), by first reducing it to a nitroso (-NO), then to a hydroxylamine (-NHOH), and finally to an amine (-NH 2 ) (255, 256). Nitrofurans and nitroimidazoles undergo intrabacterial bioactivation to metabolites that can redox-cycle and cause oxidative damage, and/or dismutate to an electrophilic nitroso intermediate that reacts with intracellular thiols (257, 258).…”

Enzyme/Prodrug Systems for Cancer Gene Therapy

“…The two classes of enzymes are categorized based on their sensitivity to oxygen. Type I NTR can produce nitroso, hydroxylamine and/or amine terminated products in the presence of molecular oxygen, while type II, on the other hand, can only produce these products when oxygen is absent (Williams et al 2015). The four classes of prodrugs include dinitroaziridinyl benzamides, dinitrobenzamide mustards, 4-nitrobenzyl carbamates, and nitroindolines.…”

“…However, the CB1954 low conversion rates, and dose-dependent hepatotoxicity are remained as major challenges for clinical application of this GDEPT method (Mitchell, Minchin 2008; Kestell et al 2000). For additional and more in depth information related to the nitroreductase/CB1954 system, readers are referred to an excellent review by (Williams et al 2015). …”

Targeting assay of a fusion protein applied in enzyme prodrug therapy

“…The enzymes are directed to the tumor sites by targeted molecules, including antibodies (5), mesenchymal stem cells (6), polymers (7) and genes (8,9). Nontoxic prodrugs, which are substrates of the enzymes, are hydrolyzed by these enzymes in order to release the cytotoxic agents (3,10).…”