The NTR/prodrug revolution: Tools for controlling cell loss and regeneration.

Abstract

Here, we review the history, advancements, and broad utility of the NTR/prodrug system, and suggest future strategies for developing versatile ablation models. As a chemogenetic tool, the nitroreductase (NTR)/prodrug system enables precise spatiotemporal control over cell ablation. The technology leverages bacterial NTR enzymes (e.g. <i>nfsB</i>) to convert inert prodrugs into cytotoxic agents, thereby allowing researchers to induce targeted cell death. Although the NTR/prodrug approach was first implemented in transgenic mice, it was subsequently adapted to zebrafish, where it has been extensively optimized and applied. Consequently, zebrafish remain the primary focus of this review. Nevertheless, the utility of the NTR/prodrug system has expanded to other important model organisms, including <i>Drosophila</i>, <i>Nematostella</i>, <i>Xenopus</i>, medaka, and rats, enabling detailed studies of tissue damage and regeneration. This review highlights how the NTR system has been deployed to model a spectrum of human diseases, including Parkinson's disease, retinal degeneration, demyelinating disorders, and kidney disease. These models provide valuable platforms to study pathogenesis in vivo. Furthermore, the precise and controllable nature of NTR ablation makes it an ideal tool for high-throughput chemical and genetic screens aimed at discovering pro-regenerative and protective compounds. The development of NTR2.0, an enzyme variant with over 100-fold greater activity, along with more potent prodrugs such as ronidazole (RNZ), has dramatically broadened experimental possibilities. These improvements permit chronic ablation and long-term disease modeling at well-tolerated drug concentrations. Here, we present some key considerations, including transgenic design for optimal cell-type specificity, calibrating expression levels for desired ablation kinetics, and suitable controls to allow interpretation. These best practices will allow the researcher to develop a precise, reproducible, and versatile platform for either modeling human disease or dissecting regenerative mechanisms.