Roles for Adenosine Ribose Hydroxyl Groups in Cyclic Adenosine 5‘-Diphosphate Ribose-Mediated Ca²⁺ Release

Abstract

Cyclic adenosine diphosphate ribose (cADPR) is a naturally occurring and potent Ca2+-mobilizing agent. Structural analogues are currently required 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 dinucleotide (NAD+) precursors modified in the ribose moiety linked to adenine. ADP-ribosyl cyclase of Aplysia californica catalyzed the conversion of 2'A-deoxy-NAD+ and 3'A-deoxy-NAD+ into the corresponding 2'A-deoxy-cADPR and 3'A-deoxy-cADPR analogues, respectively. These analogues were used to assess the effect of 2'- and 3'-hydroxyl group deletion in the adenosine ribose moiety of cADPR on the Ca2+-releasing potential of cADPR. These compounds were found to have comparatively markedly different activities as agonists for Ca2+ mobilization in sea urchin egg homogenate. 2'A-Deoxy-cADPR is similar to cADPR, whereas 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 essential for calcium releasing activity. EC50 values recorded were 32 nM, 58 nM, and 5 microM for cADPR, 2'A-deoxy-cADPR, and 3'A-deoxy-cADPR, respectively. Moreover, 200 nM 2'A-deoxy-cADPR was required to desensitize the cADPR-sensitive Ca2+ channel to a subsequent addition of 100 nM cADPR, but 20 microM 3'A-deoxy-cADPR was required to produce the same desensitizing effect. This is in accordance with the 100-fold lower potency exhibited by the latter analogue. To further investigate the 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 methylated and its ability potentially to donate a hydrogen atom in a hydrogen bond has been removed. Although inactive in releasing Ca2+, 3'A-O-methyl-cADPR inhibited cADPR-induced Ca2+ release in a dose-dependent manner with an approximate IC50 value of 5 microM, whereas 3'-O-methyladenosine had no effect. This further supports the requirement of a 3'-OH group for Ca2+ releasing activity. In addition, however, it suggests that this group may not be crucial for ligand-receptor recognition. Thus, replacement of the hydrogen atom of the hydroxyl with a methyl group effects a change of activity from an agonist to an antagonist of cADPR-induced Ca2+ release. Two other analogues with modifications in the 2' and/or 3' positions, 3'-cADPR phosphate and 2',3'-cyclic-cADPR phosphate, were synthesized and tested for their Ca2+-mobilizing activity in sea urchin egg homogenates. Both analogues were inactive with 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 groups of cADPR in the biological activity of this cyclic nucleotide. Additionally, this...

2′-Deoxy Cyclic Adenosine 5′-Diphosphate Ribose Derivatives: Importance of the 2′-Hydroxyl Motif for the Antagonistic Activity of 8-Substituted cADPR Derivatives

“…17 In sea urchin homogenates, it was found that 3′-OMe-cADPR blocked the cADPR-induced Ca 2+ release in a concentration-dependent manner, while 3′-deoxy-cADPR was inactive, suggesting the 3′-OH as an important motif for binding to the receptor. 17 The amino substitutent at position 8 together with the 2′-hydroxyl group may be cooperative in interacting with the receptor by hydrogen bonding, and importantly, both groups are probably required for the outstanding antagonistic activity of 2. Thus, the deletion of the 2′-OH consequently results in the breakdown of the cooperative hydrogen bonding, and this probably explains the differential bioactivities for analogues 2 and 8.…”

“…Among southern ribose-modified analogues, 2′-deoxy-cADPR was first synthesized by Ashamu et al 17 and is almost as potent as cADPR in mediating Ca 2+ release from cADPR-sensitive stores in sea urchin egg homogenates, indicating that 2′-OH deletion has no or little effect on the agonistic activity of cADPR, at least in the invertebrate assay system. It was not demonstrated, however, whether such a deletion might also potentially affect antagonistic activity.…”

Pharmacological Modulation of Sarcoplasmic Reticulum Function in Smooth Muscle

“…CD38 is a bifunctional enzyme that catalyzes both their synthesis and degradation of cADPR. Adapted with permission from Ashamu et al (1997). 466 cADPR hydrolase (cADPRH) (Guse, 2002) (Fig.…”

A Minimal Structural Analogue of Cyclic ADP-ribose

“…Concerning the southern ribose moiety of cADPR, it was found that the 3Ј-hydroxyl group was essential for Ca 2ϩ release in the sea urchin egg system, whereas the 2Ј-hydroxyl was not (12). O-Methylation of the 3Ј-hydroxyl group generated an antagonist (12).…”

“…O-Methylation of the 3Ј-hydroxyl group generated an antagonist (12). The stable carbocyclic derivative cyclic aristeromycin diphosphoribose was an agonist in both sea urchin eggs and T lymphocytes (13,14).…”