Abstract
Bacterial nitroreductase enzymes that can efficiently convert nitroaromatic prodrugs to a cytotoxic form have numerous applications in targeted cellular ablation. For example, the generation of cytotoxic metabolites that have low bystander potential (i.e., are largely confined to the activating cell) has been exploited for precise ablation of specific cell types in animal and cell-culture models; while enzyme-prodrug combinations that generate high levels of bystander cell killing are useful for anti-cancer strategies such as gene-directed enzyme-prodrug therapy (GDEPT). Despite receiving substantial attention for such applications, the canonical nitroreductase NfsB from Escherichia coli has flaws that limit its utility, in particular a low efficiency of conversion of most prodrugs. Here, we sought to engineer a superior broad-range nitroreductase, E. coli NfsA, for improved activity with three therapeutically-relevant prodrugs: the duocarmycin analogue nitro-CBI-DEI, the dinitrobenzamide aziridine CB1954 and the 5-nitroimidazole metronidazole. The former two prodrugs have applications in GDEPT, while the latter has been employed for targeted ablation experiments and as a precise ‘off-switch’ in GDEPT models to eliminate nitroreductase-expressing cells. Our lead engineered NfsA (variant 11_78, with the residue substitutions S41Y, L103M, K222E and R225A) generated reduced metabolites of CB1954 and nitro-CBI-DEI that exhibited high bystander efficiencies in both bacterial and 2D HEK-293 cell culture models, while no cell-to-cell transfer was evident for the reduced metronidazole metabolite. We showed that the high bystander efficiency for CB1954 could be attributed to near-exclusive generation of the 2-hydroxylamine reduction product, which has been shown in 3D cell culture to cause significantly greater bystander killing than the 4-hydroxylamine species that is also produced by NfsB. We similarly observed a high bystander effect for nitro-CBI-DEI in HCT-116 tumor spheroids in which only a small proportion of cells were expressing variant 11_78. Collectively, our data identify variant 11_78 as a broadly improved prodrug-activating nitroreductase that offers advantages for both targeted cellular ablation and suicide gene therapy applications.
Highlights
Bacterial nitroreductase enzymes are members of a diverse family of oxidoreductase enzymes that can catalyze the bioreductive activation of nitroaromatic compounds, including anti-cancer prodrugs such as nitro-CBI-DEI ((1-(chloromethyl)-5-nitro-1,2dihydro-3H-benzo[e]indol-3-yl)(5-(2-(dimethylamino)ethoxy)1H-indol-2-yl)methanone) and CB1954 (5-(aziridin-1-yl)-2,4dinitrobenzamide), and antibiotic prodrugs such as metronidazole (2-methyl-5-nitroimidazole-1-ethanol) (Williams et al, 2015) (Figure 1)
To discover improved prodrug-converting nitroreductases we screened two previously-generated nfsA gene libraries: 1) a randomized codon mutagenesis library (“7RCM”) that uses NDT or NNK degenerate codons to randomize seven key residues in the active site of NfsA (S41, F42, F83, S224, R225, F227 and K222; Copp et al, 2020); and 2) a site-directed mutagenesis library (“10SDM”) that encodes all possible combinations of ten NfsA substitutions previously found to improve activation of the dinitrobenzamide mustard prodrug PR-104A (I5T, S41Y, E99G, L103M, K222E, R225A, R225G, R225P, F227S, L229V; Copp et al, 2017)
Was 11_78 the most generally-improved of the variants tested in this study (Table 1), on the basis of the IC50 values we measured in stably-transfected HEK-293 cells this is one of the most active nitroreductases to have been reported to date, with each of the three prodrugs
Summary
Bacterial nitroreductase enzymes are members of a diverse family of oxidoreductase enzymes that can catalyze the bioreductive activation of nitroaromatic compounds, including anti-cancer prodrugs such as nitro-CBI-DEI ((1-(chloromethyl)-5-nitro-1,2dihydro-3H-benzo[e]indol-3-yl)(5-(2-(dimethylamino)ethoxy)1H-indol-2-yl)methanone) and CB1954 (5-(aziridin-1-yl)-2,4dinitrobenzamide), and antibiotic prodrugs such as metronidazole (2-methyl-5-nitroimidazole-1-ethanol) (Williams et al, 2015) (Figure 1). Low-bystander prodrugs have potential to provide an ‘off-switch’ for nitroreductase-based cellular therapies, enabling contained self-sterilization of nitroreductase-expressing vector cells. Such an ‘off-switch’ would improve the safety and controllability of gene therapy as it could be triggered at any required point upon administration of the low-bystander prodrug, and addresses specific concerns associated with the growing number of gene therapy technologies that are limited by a lack of therapeutic control following vector delivery (Goverdhana et al, 2005; Curtin et al, 2008; Das et al, 2016; Goswami et al, 2019)
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