Research into the biological role of the Ca2+-releasing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate) has been hampered by a lack of chemical probes. To find new chemical probes for exploring NAADP signaling, we turned to virtual screening, which can evaluate millions of molecules rapidly and inexpensively. We used NAADP as the query ligand to screen the chemical library ZINC for compounds with 3D-shape and electrostatic similarity. We tested the top-ranking hits in a sea urchin egg bioassay and found that one hit, Ned-19, blocks NAADP signaling at nanomolar concentrations. In intact cells, Ned-19 blocked NAADP signaling and fluorescently labeled NAADP receptors. Moreover, we show the utility of Ned-19 as a chemical probe by using it to demonstrate that NAADP is a key causal link between glucose sensing and Ca2+ increases in mouse pancreatic beta cells.

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Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2؉ -releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca 2؉ release and NAADP binding. A fluorometry bioassay was used to assess NAADPmediated Ca 2؉ release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca 2؉ release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca 2؉ release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high-and one lowaffinity) on the NAADP receptor. Ca2ϩ is an extremely versatile and powerful second messenger (1). Three accepted Ca 2ϩ -releasing second messengers that cells utilize to control the spatiotemporal properties of these Ca 2ϩ signals are inositol 1,4,5-trisphosphate (2), cyclic ADPribose (3), and nicotinic acid adenine dinucleotide phosphate (NAADP) 2 (4). Of these three messengers, little is known about the mechanism of action of NAADP. NAADP is the most potent of the three Ca 2ϩ -releasing messengers (5), and its actions are pharmacologically distinct from inositol 1,4,5-trisphosphate and cyclic ADP-ribose (6 -9). Controversy surrounds both the identity of the NAADP-activated channel and its organellar location. There is evidence for the NAADP receptor being the transient receptor potential mucolipin 1 channel (10), the ryanodine receptor (11, 12), or the two-pore channel (Refs. 13-15; for review, see Ref. 16). There is evidence for the Ca 2ϩ store targeted by NAADP being the endoplasmic reticulum (11) or physically distinct (17), acidic, lysosome-related organelles (18).One of the most intriguing features of NAADP is the relationship between concentration, binding, and response (5,9,19). In sea urchin eggs, a subthreshold concentration of NAADP that does not itself release Ca 2ϩ is able to prevent Ca 2ϩ release in response to a second addition of NAADP that would normally be able to release Ca 2ϩ (20,21). This unusual desensitization may relate to the formation of a spatial and temporal "memory" (22,23). This effect is thought to be due to a second high-affinity inhibitory site present on the receptor (24); thus, at low concentrations, NAADP only binds to this site and is able to prevent Ca 2ϩ release via this receptor. In contrast to sea urchin eggs, in mammalian tissue, NAADP has a bell-shaped concentration-r...

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