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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

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Engineering the Escherichia coli Nitroreductase NfsA to Create a Flexible Enzyme-Prodrug Activation System

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with nitroreductase (NTR) as keyword