Live-Cell Fluorescent Imaging of Pyridine Nucleotide Coenzymes: Sensor Platforms, Biological Applications, and Pharmacological Opportunities

Abstract

Pyridine nucleotide coenzymes, including NAD+/NADH and NADP+/NADPH redox couples, form a highly interconnected metabolic and signaling network that links cellular bioenergetics, biosynthesis, redox homeostasis, and stress responses. These cofactors are compartmentalized across the cytosol, mitochondria, and nucleus and are continuously remodeled by multiple biosynthetic, recycling, interconversion, and consumption pathways. Their spatiotemporal dynamics are further rewired in cancer, aging, neurodegeneration, and inflammation, making them informative reporters of pathway activity and attractive pharmacological targets. In this review, we discuss fluorescent biosensors for pyridine nucleotide imaging, including small-molecule probes, genetically encoded indicators, and chemigenetic platforms. We compare these approaches in terms of analyte definition, selectivity, dynamic range, reversibility, spatiotemporal resolution, and suitability for subcellular imaging. We then summarize what these tools have revealed about the networked regulation of pyridine nucleotide coenzymes in physiology and disease and highlight their growing utility in drug discovery, mechanism-of-action studies, and phenotypic screening. Finally, we outline the technical challenges that must be addressed to make pyridine nucleotide imaging more quantitative, interoperable, and predictive in chemical biology and biomedicine.