ROLES FOR ADENOSINE RIBOSE HYDROXYL-GROUPS IN CYCLIC ADENOSINE 5'-DIPHOSPHATE RIBOSE-MEDIATED CA2+ RELEASE

Cyclic adenosine diphosphate ribose (cADPR) is a naturally occurring a nd potent Ca2+. mobilizing agent. Structural analogues are currently r equired as pharmacological tools for the investigation of this topical molecule, but modifications to date have concentrated primarily upon the purine ring. Two novel dehydroxylated analogues of cADPR have now been prepared from chemically synthesized nicotinamide adenine dinucle otide (NAD(+)) precursors modified in the ribose moiety linked to aden ine, ADP-ribosyl cyclase of Aplysia californica catalyzed the conversi on of 2'(A)-deoxy-NAD(+) and 3'(A)-deoxy NAD(+) into the corresponding 2'(A)-deoxy-cADPR and 3'(A)-deoxy-cADPR analogues, respectively. Thes e analogues were used to assess the effect of 2'- and 3'-hydroxyl grou p deletion in the adenosine ribose moiety of cADPR on the Ca2+-releasi ng potential of cADPR. These compounds were found to have comparativel y markedly different activities as agonists for Ca2+ mobilization in s ea urchin egg homogenate. 2'(A)-Deoxy-cADPR is similar to cADPR, where as 3'(A)-deoxy-cADPR is at least 100-fold less potent, indicating that the 3'(A)-hydroxyl group, but not the 2'(A)-hydroxyl group, is essent ial for calcium releasing activity. EC50 values recorded were 32 nM, 5 8 nM, and 5 mu M for cADPR, 2'(A)-deoxy-cADPR, and 3'(A)-deoxy-cADPR, respectively, Moreover, 200 nM 2'(A)-deoxy-cADPR was required to desen sitize the cADPR-sensitive Ca2+ channel to a subsequent addition of 10 0 nM cADPR, but 20 mu M 3'(A)-deoxy-cADPR was required to produce the same desensitizing effect. This is in accordance with the 100-fold low er potency exhibited by the latter analogue. To further investigate th e importance of the 3'-hydroxyl group, we have also synthesized 3'(A)- O-methyl-cADPR, in which the 3'-hydroxyl group of adenosine has been m ethylated and its ability potentially to donate a hydrogen atom in a h ydrogen bond has been removed, Although inactive in releasing Ca2+, 3' (A)-O-methyl-cADPR inhibited cADPR-induced Ca2+ release in a dose-depe ndent manner with an approximate IC50 value of 5 mu M, whereas 3'-O-me thyladenosine had no effect. This further supports the requirement of a 3'-OH group for Ca2+ releasing activity. In addition, however, it su ggests that this group may not be crucial for ligand-receptor recognit ion. Thus, replacement of the hydrogen atom of the hydroxyl with a met hyl group effects a change of activity from an agonist to an antagonis t of cADPR-induced Ca2+ release. Two other analogues with modification s in the 2' and/or 3' positions, 3'-cADPR phosphate and 2',3'-cyclic-c ADPR phosphate, were synthesized and tested for their Ca2+-mobilizing activity in sea urchin egg homogenates. Both analogues were inactive w ith respect to both agonistic and antagonistic activities on the cADPR -sensitive Ca2+ release mechanism. These are the first steps toward a wider structure-activity relationship for cADPR, and this is the first study to implicate a crucial role for the adenosine ribose hydroxyl g roups of cADPR in the biological activity of this cyclic nucleotide. A dditionally, this is the first report of a cADPR receptor antagonist t hat is not modified at the 8-position of the purine ring.