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

Abstract

Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here, we used rational engineering and cross-species screening to develop a NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community.

Construction of a Reduction‐responsive DNA Microsphere using a Reduction‐cleavable Spacer based on a Nitrobenzene Scaffold

“…After the reduction of nitro group in the RCS to amino and/or aminoxy groups, [6a] a cleavage reaction via β‐1,6‐elimination could occur, as depicted in Figure 1B. Also, the p‐ nitrobenzene scaffold including RCS can show the nicotinamide‐adenine dinucleotide (phosphate) (NA(P)DH)‐dependent reduction with the aid of enzymes, such as a nitroreductase, which should result in the similar cleavage reaction [6a,15,16] . In contrast, no response toward thiol‐reducing agents including reduced glutathione (GSH), which cannot reduce the nitro groups, is expected [8] .…”

Insights into the control and consequences of breathing adjustments in fishes-from larvae to adults

“…Once achieved, NECs could be activated specifically with light or capsaicin by expressing channel rhodopsin ( Antinucci et al, 2020 ) or rat TRPV1 channels ( Matty et al, 2016 ). Similarly, NECs could be inhibited with light by expressing transient receptor potential cation channels ( Antinucci et al, 2020 ) or ablated with metronidazole treatment in fish with NECs expressing nitroreductase ( Sharrock et al, 2022 ). With such experiments, it would be possible to establish a direct link between NEC activity and ventilation.…”

The Role of Transposable Elements in Sexual Development

“…To name a few, prime editing and piRNA-mediated interference can modulate or silence specific epigenetic-related genes (including TEs), respectively ( Wannier et al, 2021 ; Priyadarshini et al, 2022 ). In addition, the role of specific cell types could be disentangled by cell ablation through bacterial nitroreductase systems ( Sharrock et al, 2022 ). Subsequently, the effects of these manipulations can be investigated with high throughput techniques such as BOLARAMIS which allows spatial resolution of the transcriptome ( Liu et al, 2021 ).…”