NTR 2.0: a rationally-engineered prodrug converting enzyme with substantially enhanced efficacy for targeted cell ablation

Sharrock, Abigail V.; Mulligan, Timothy S.; Hall, Kelsi R; Williams, Elizabeth; White, David T.; Zhang, Liyun; Matthews, Fiona E.; Nimmagadda, Saumya; Washington, Selena; Le, Katherine; Meir-Levi, Danielle; Cox, Olivia L.; Saxena, Mohit; Calof, Anne L.; Lopez-Burks, Martha E.; Lander, Arthur D.; Ding, Ding; Ackerley, David F.; Mumm, Jeff S

doi:10.1101/2020.05.22.111427

Cited by 9 publications

(12 citation statements)

References 49 publications

(76 reference statements)

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“…For example, the IC 50 for this cell line with nitro-CBI-DEI is nearly 2-fold improved over HCT-116 cells expressing E. coli NfsB ( Wilson et al, 2009 ; the only previous report of a human cell line IC 50 for nitro-CBI-DEI), while the IC 50 with CB1954 is comparable to those of the top five nitroreductase-expressing HCT-116 lines examined in a broad survey of 47 promising nitroreductase candidates ( Prosser et al, 2013 ). Most notably, the IC 50 with metronidazole is nearly three logs better than the IC 50 we previously measured for E. coli NfsB expressed in the same HEK-293 cell line ( Sharrock et al, 2020 ). Escherichia coli NfsB has been widely used in transgenic animal models, in particular zebrafish, to ablate promoter-defined cell types; however, these models have frequently been confounded by a requirement to apply near-toxic (or even toxic) doses of metronidazole to achieve effective cell ablation ( Mathias et al, 2014 ).…”

Section: Discussioncontrasting

confidence: 57%

“…Escherichia coli NfsB has been widely used in transgenic animal models, in particular zebrafish, to ablate promoter-defined cell types; however, these models have frequently been confounded by a requirement to apply near-toxic (or even toxic) doses of metronidazole to achieve effective cell ablation ( Mathias et al, 2014 ). We recently addressed this limitation by developing “NTR 2.0” - a rationally engineered variant of Vibrio vulnificus that achieves effective cell ablation in transgenic zebrafish at 100-fold lower doses of metronidazole ( Sharrock et al, 2020 ). In HEK-293 cells, variant 11_78 confers a 1.5-fold lower IC 50 than even NTR 2.0, suggesting it will also be highly effective for targeted ablation applications.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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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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Section: Discussioncontrasting

confidence: 57%

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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“…In this line, rods co-express membrane-tagged YFP and an improved NTR ("NTR 2.0"). The membrane-tagged YFP facilitates improved imaging of rod outer segments, while NTR 2.0 enables cell ablation at a reduced concentration of Mtz (e.g., 10 µM versus 2.5 mM; (Sharrock et al, 2020)). To assess if lead compounds preserved outer segment morphologies, intravital confocal imaging was performed as above but using the ).…”

Section: Zebrafish Model Validation Iv: Confocal Intravital Microscopymentioning

confidence: 99%

Large-scale phenotypic drug screen identifies neuroprotectants in zebrafish and mouse models of retinitis pigmentosa

Zhang

Chen

et al. 2021

eLife

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Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in multiple zebrafish and mouse RP models, reasoning drugs effective across species and/or independent of disease mutation may translate better clinically. We first performed a large-scale phenotypic drug screen for compounds promoting rod cell survival in a larval zebrafish model of inducible RP. We tested 2,934 compounds, mostly human-approved drugs, across six concentrations, resulting in 113 compounds being identified as hits. Secondary tests of 42 high-priority hits confirmed eleven lead candidates. Leads were then evaluated in a series of mouse RP models in an effort to identify compounds effective across species and RP models, i.e., potential pan-disease therapeutics. Nine of eleven leads exhibited neuroprotective effects in mouse primary photoreceptor cultures, and three promoted photoreceptor survival in mouse rd1 retinal explants. Both shared and complementary mechanisms of action were implicated across leads. Shared target tests implicated parp1-dependent cell death in our zebrafish RP model. Complementation tests revealed enhanced and additive/synergistic neuroprotective effects of paired drug combinations in mouse photoreceptor cultures and zebrafish, respectively. These results highlight the value of cross-species/multi-model phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for RP patients.

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“…It is for example the case of the pancreas and the liver where the zebrafish NTR model is almost exclusively employed in contrast to the various mice models of injury that are used to cover regeneration from different cellular sources ( Table 1 ) [ 31 , 33 , 67 ]. The NTR model is also exploited to study regeneration in the heart and the brain and is continuously improving [ 94 , 95 , 96 , 97 ]. Although this relatively simple model provides valuable clues about regeneration, zebrafish models recapitulating more closely the disease will determine how zebrafish regenerate in such settings, therefore further increasing our understanding of regeneration and the success of transposition to mammals.…”

Section: Discussionmentioning

confidence: 99%

Zebra-Fishing for Regenerative Awakening in Mammals

2021

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Regeneration is defined as the ability to regrow an organ or a tissue destroyed by degeneration or injury. Many human degenerative diseases and pathologies, currently incurable, could be cured if functional tissues or cells could be restored. Unfortunately, humans and more generally mammals have limited regenerative capabilities, capacities that are even further declining with age, contrary to simpler organisms. Initially thought to be lost during evolution, several studies have revealed that regenerative mechanisms are still present in mammals but are latent and thus they could be stimulated. To do so there is a pressing need to identify the fundamental mechanisms of regeneration in species able to efficiently regenerate. Thanks to its ability to regenerate most of its organs and tissues, the zebrafish has become a powerful model organism in regenerative biology and has recently engendered a number of studies attesting the validity of awakening the regenerative potential in mammals. In this review we highlight studies, particularly in the liver, pancreas, retina, heart, brain and spinal cord, which have identified conserved regenerative molecular events that proved to be beneficial to restore murine and even human cells and which helped clarify the real clinical translation potential of zebrafish research to mammals.

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NTR 2.0: a rationally-engineered prodrug converting enzyme with substantially enhanced efficacy for targeted cell ablation

Cited by 9 publications

References 49 publications

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

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

Large-scale phenotypic drug screen identifies neuroprotectants in zebrafish and mouse models of retinitis pigmentosa

Zebra-Fishing for Regenerative Awakening in Mammals

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