Synthesis and characterization of antagonists of cyclic-ADP-ribose-induced Ca²⁺ release

Cyclic ADP-ribose (cADPR) is a naturally-occurring metabolite of NAD⁺ that is as effective as inositol trisphosphate in mobilizing intracellular Ca²⁺. A series of analogs modified at the 8-position of the adenine group were synthesized for the investigation of the relationship between the structure of the metabolite and its Ca²⁺-mobilizing activity. Substitution with an amino group at the 8-position of the adenine ring produced an antagonist. The ¹H-NMR spectrum of 8-amino-cADPR showed characteristics of that of cADPR and confirmed the replacement of the 8-proton. By itself, 8-amino-cADPR (150 nM) did not induce Ca²⁺ release from sea-urchin-egg homogenates but totally blocked cADPR (135 nM) from doing so. The effect was reversible, since high concentrations of cADPR could overcome the inhibition. Addition of 8-amino-cADPR to egg homogenates during the cADPR-induced Ca²⁺ release blocked the release immediately, demonstrating the effectiveness of the antagonist. Measurements of [³²P]cADPR binding to its microsomal binding site showed that 8-amino-cADPR was as effective as cADPR itself in competing for the binding site. In addition to blocking cADPR from releasing Ca²⁺, 8-amino-cADPR also inhibited cADPR from potentiating Ca²⁺-release induced by either divalent cations or by caffeine. Two other 8-substituted analogs were also synthesized. Both 8-Br- and 8-azido-cADPR were also antagonists, although with less potency than 8-amino-cADPR. These results show that alterations at the 8-position of the adenine group do not inhibit cADPR from binding to its receptor but do eliminate the ability of the metabolite to activate the Ca²⁺-release mechanism.