Roles for Adenosine Ribose Hydroxyl Groups in Cyclic Adenosine 5′-Diphosphate Ribose-Mediated Ca 2+ Release ² Gloria A. Ashamu, ‡ Jaswinder K. Sethi, § Antony Galione, § and Barry V. L. Potter* ,‡ School of Pharmacy and Pharmacology, UniVersity of Bath, ClaVerton Down, Bath BA2 7AY, U.K., and UniVersity Department of Pharmacology, Oxford UniVersity, Mansfield Road, Oxford OX1 3QT, U.K. ReceiVed March 12, 1997; ReVised Manuscript ReceiVed May 19, 1997 X ABSTRACT: Cyclic adenosine diphosphate ribose (cADPR) is a naturally occurring and potent Ca 2+ - 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 Ca 2+ -releasing potential of cADPR. These compounds were found to have comparatively markedly different activities as agonists for Ca 2+ 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. EC 50 values recorded were 32 nM, 58 nM, and 5 µM 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 Ca 2+ channel to a subsequent addition of 100 nM cADPR, but 20 µM3′ 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 Ca 2+ ,3′ A -O-methyl-cADPR inhibited cADPR-induced Ca 2+ release in a dose-dependent manner with an approximate IC 50 value of 5 µM, whereas 3′-O-methyladenosine had no effect. This further supports the requirement of a 3′-OH group for Ca 2+ 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 Ca 2+ 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 Ca 2+ -mobilizing activity in sea urchin egg homogenates. Both analogues were inactive with respect to both agonistic and antagonistic activities on the cADPR-sensitive Ca 2+ 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 is the first report of a cADPR receptor antagonist that is not modified at the 8-position of the purine ring. Cyclic adenosine diphosphate ribose (cADPR; 1 structure 1, Figure 1), a metabolite of NAD + , was first discovered during investigations of the Ins(1,4,5)P 3 -independent Ca 2+ mobilization mechanisms in sea urchin egg preparations (Clapper et al., 1987; Dargie et al., 1989). It has been suggested to be the physiological modulator of the ryanodine receptor (Galione et al., 1993a), although there is increasing evidence that cADPR may not act directly on the ryanodine channel but that its agonistic action is mediated by an accessory protein (Walseth et al., 1993), which has recently been suggested to be calmodulin (Lee et al., 1994a). Current evidence, therefore, is indicative of a binding site distinct from that of ryanodine. cADPR releases Ca 2+ by a pathway independent of D-myo-inositol 1,4,5-trisphosphate [Ins- (1,4,5)P 3 ]. The widespread occurrence of cADPR in mam- malian cells and the ubiquitous presence of the enzymes that ² We thank the Medical Research Council and the Wellcome Trust (Program Grant 045491) for financial support and the University of Bath for a Research Bursary (G.A.A.). A.G. is a Wellcome Trust Fellow and B.V.L.P. is a Lister Institute Research Professor. * To whom correspondence should be addressed: Tel 1225 826639; Fax 1225 826114; E-mail B.V.L.Potter@bath.ac.uk. ‡ University of Bath. § Oxford University. X Abstract published in AdVance ACS Abstracts, July 1, 1997. 1 Abbreviations: cADPR, cyclic adenosine 5′-diphosphate ribose; NAD + , nicotinamide adenine dinucleotide; DCC, N,N-dicyclohexyl- carbodiimide; DCU, dicyclohexylurea; AMP, adenosine 5′-monophos- phate; KH2PO4, potassium dihydrogen phosphate; H3PO4, orthophos- phoric acid; NMR, nuclear magnetic resonance; HPLC, high- performance liquid chromatography; Rt, high-performance liquid chromatography retention time; PO(OEt)3, triethyl phosphate; POCl3, phosphorus oxychloride; RFU, relative fluorescence units; TEAB, triethylammonium bicarbonate; ES, electrospray. 9509 Biochemistry 1997, 36, 9509-9517 S0006-2960(97)00567-9 CCC: $14.00 © 1997 American Chemical Society