Chemical Constructions, Mechanism and Biological Interpretation of Nitroreductase-Responsive Systems: From Hypoxia-Associated Imaging to Activatable Therapy

Nitroreductase (NTR)-responsive systems convert the enzymatic reduction of nitroaromatic substrates into detectable signals, molecular retention, supramolecular assembly, or therapeutic activation. Their interpretation is nevertheless complicated by the distinct catalytic properties of bacterial NTRs, experimentally introduced or engineered NTR systems, and endogenous mammalian nitro-reducing enzymes. In particular, an NTR-responsive signal in mammalian tissues should not automatically be interpreted as a direct measurement of oxygen concentration or as evidence for the activity of a single endogenous “NTR.” This review analyzes NTR-responsive systems through an integrated framework that connects catalytic source, chemical event, signal-transduction mechanism, and biological application. We first distinguish defined bacterial NTRs, engineered or experimentally introduced NTR systems, and endogenous mammalian nitro-reducing activity. We then describe the recognition, linker, and output modules used in molecular design and compare three major reaction architectures: direct reduction, reduction-triggered cleavage, and reduction-triggered retention or assembly. Photoinduced electron transfer (PET), intramolecular charge transfer (ICT), fluorescence resonance energy transfer (FRET), aggregation-induced emission (AIE), chemiluminescence, photoacoustic imaging, and fluorine nuclear magnetic resonance (19F NMR) are discussed as signal-transduction modalities rather than as independent activation mechanisms. Multi-input sensing, activatable therapy, and the validation of these systems in complex biological models are subsequently evaluated. Particular emphasis is placed on product-level confirmation, enzyme attribution, oxygen-dependent interpretation, calibration, pharmacokinetics, biodistribution, and safety. By linking chemical activation to the biochemical source of reduction and to the limitations of biological inference, this review provides criteria for selecting, validating, and translating NTR-responsive systems for hypoxia-associated imaging and activatable therapy.