Nicotinamide Riboside\nand CD38: Covalent Inhibition\nand Live-Cell Labeling

Nicotinamide adenine dinucleotide (NAD<sup>+</sup>) is\nrequired\nfor a myriad of metabolic, signaling, and post-translational events\nin cells. Its levels in tissues and organs are closely associated\nwith health conditions. The homeostasis of NAD<sup>+</sup> is regulated\nby biosynthetic pathways and consuming enzymes. As a membrane-bound\nprotein with robust NAD<sup>+</sup> hydrolase activity, cluster of\ndifferentiation 38 (CD38) is a major degrader of NAD<sup>+</sup>.\nDeficiency or inhibition of CD38 enhances NAD<sup>+</sup> levels in\nvivo, resulting in various therapeutic benefits. As a metabolic precursor\nof NAD<sup>+</sup>, nicotinamide mononucleotide can be rapidly hydrolyzed\nby CD38, whereas nicotinamide riboside (NR) lacks CD38 substrate activity.\nGiven their structural similarities, we explored the inhibition potential\nof NR. To our surprise, NR exhibits marked inhibitory activity against\nCD38 by forming a stable ribosyl–ester bond with the glutamate\nresidue 226 at the active site. Inspired by this discovery, we designed\nand synthesized a clickable NR featuring an azido substitution at\nthe 5′-OH position. This cell-permeable NR analogue enables\ncovalent labeling and imaging of both extracellular and intracellular\nCD38 in live cells. Our work discovers an unrecognized molecular function\nof NR and generates a covalent probe for health-related CD38. These\nfindings offer new insights into the role of NR in modulating NAD<sup>+</sup> metabolism and CD38-mediated signaling as well as an innovative\ntool for in-depth studies of CD38 in physiology and pathophysiology.