CD38 is a signaling enzyme responsible for catalyzing the synthesis of cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate; both are universal Ca(2+) messenger molecules. Ablation of the CD38 gene in mice causes multiple physiological defects, including impaired oxytocin release, that result in altered social behavior. A series of catalysis-based inhibitors of CD38 were designed and synthesized, starting with arabinosyl-2'-fluoro-2'-deoxynicotinamide mononucleotide. Structure-function relationships were analyzed to assess the structural determinants important for inhibiting the NADase activity of CD38. X-ray crystallography was used to reveal the covalent intermediates that were formed with the catalytic residue, Glu226. Metabolically stable analogues that were resistant to inactivation by phosphatase and esterase were synthesized and shown to be effective in inhibiting intracellular cADPR production in human HL-60 cells during induction of differentiation by retinoic acid. The inhibition was species-independent, and the analogues were similarly effective in blocking the cyclization reaction of CD38 in rat ventricular tissue extracts, as well as inhibiting the α-agonist-induced constriction in rat mesentery arteries. These compounds thus represent the first generally applicable and catalysis-based inhibitors of the Ca(2+) signaling function of CD38.

CD38 catalyzes the synthesis of cyclic ADP-ribose (cADPR), a Ca 2؉ messenger responsible for regulating a wide range of physiological functions. It is generally regarded as an ectoenzyme, but its intracellular localization has also been well documented. It is not known if internal CD38 is enzymatically active and contributes to the Ca 2؉ signaling function. In this study, we engineered a novel soluble form of CD38 that can be efficiently expressed in the cytosol and use cytosolic NAD as a substrate to produce cADPR intracellularly. The activity of the engineered CD38 could be decreased by mutating the catalytic residue Glu-226 and increased by the double mutation E146A/T221F, which increased its cADPR synthesis activity by >11-fold. Remarkably, the engineered CD38 exhibited the ability to form the critical disulfide linkages required for its enzymatic activity. This was verified by using a monoclonal antibody generated against a critical disulfide, Cys-254 -Cys-275. The specificity of the antibody was established by x-ray crystallography and site-directed mutagenesis. The engineered CD38 is thus a novel example challenging the general belief that cytosolic proteins do not possess disulfides. As a further refinement of this approach, the engineered CD38 was placed under the control of tetracycline using an autoregulated construct. This study has set the stage for in vivo manipulation of cADPR metabolism. Mobilization of Ca2ϩ from intracellular stores is of fundamental importance in virtually all aspects of cellular activity. The major intracellular Ca 2ϩ stores are in the endoplasmic reticulum (ER) 2 and are mobilized by specific messenger molecules, inositol trisphosphate and cyclic ADP-ribose (cADPR). The latter is a novel cyclic nucleotide derived from NAD. It was first described in sea urchin eggs (1, 2) but has since been established as a second messenger molecule responsible for regulating a wide range of physiological functions as diverse as abscisic acid signaling in plants (3) and sponges (4) and social behavior in mice (Ref. 5; reviewed in Refs. 6 and 7). It targets the ryanodine receptor of the endoplasmic Ca 2ϩ stores. The synthesis and hydrolysis of cADPR in mammalian cells are catalyzed by CD38 (8), a transmembrane protein ubiquitously expressed in virtually all tissues (reviewed in Ref. 9). Gene knock-out studies have established that CD38 plays a critical role in a wide range of physiological functions, including insulin secretion (10), susceptibility to bacterial infection (11), and social behavior of mice through modulating neuronal oxytocin secretion (5).CD38 is a membrane protein with a short N-terminal tail, a single transmembrane segment, and a large C-terminal domain containing all the enzymatic activities (8, 12, 13). The crystal structure of the catalytic domain of CD38 has been solved, and the mechanism of how it catalyzes the multiple reactions responsible for metabolizing cADPR has also been elucidated to atomic resolution by x-ray crystallography (14 -16). It is established that the s...