Medicinal chemistry and pharmacology of cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is a signaling molecule that has been shown to regulate calcium mobilization from intracellular stores in a wide variety of biological systems. Synthesis of structural analogs of cADPR has provided insights into structure-activity relationships as well as produced pharmacological research tools with useful properties such as, hydrolysis-resistance and cell permeability. The first generation of cADPR analogs was synthesized by a chemo-enzymatic approach that took advantage of the broad substrate specificity of Aplysia ADP-ribosyl cyclase. Analogs synthesized by this approach provided useful structure-activity information, including the importance of the 8-position of the adenine in determining agonistic or antagonistic activity and of the 3'-hydroxyl group of the southern ribose for activity. Hydrolysis resistant analogs were generated by replacing the southern ribose with a carbocyclic structure or by replacing the adenine ring with 7-deaza- or 3-deaza-adenine. Approaches to synthesize cADPR analogs by total chemical approaches have been recently reported. These approaches allow the synthesis of analogs with stable linkages between N1 of adenine and the northern ribose (or surrogate) that are not possible with the enzymatic strategy. This review will focus on the synthesis and properties of analogs that have been shown to have utility in dissecting the role of cADPR in calcium signaling.     

Calcium signaling by cyclic ADP-ribose and NAADP

Ca²⁺ mobilization as a signaling mechanism has been placed on center stage with the discovery of the first Ca²⁺ messenger, inositol trisphosphate (IP₃). This article focuses on two new Ca²⁺ release activators, which mobilize internal Ca²⁺ stores via mechanisms totally independent of IP₃. They are cyclic ADP-ribose (cADPR) and nicotinic acid dinucleotide phosphate (NAADP), metabolites derived respectively from NAD and NADP. Major advances in the past decade in the understanding of these two novel signaling mechanisms are chronologically summarized.     

Vasodilation by the calcium-mobilizing messenger cyclic ADP-ribose

In artery smooth muscle, adenylyl cyclase-coupled receptors such as beta-adrenoceptors evoke Ca(2+) signals, which open Ca(2+)-activated potassium (BK(Ca)) channels in the plasma membrane. Thus, blood pressure may be lowered, in part, through vasodilation due to membrane hyperpolarization. The Ca(2+) signal is evoked via ryanodine receptors (RyRs) in sarcoplasmic reticulum proximal to the plasma membrane. We show here that cyclic adenosine diphosphate-ribose (cADPR), by activating RyRs, mediates, in part, hyperpolarization and vasodilation by beta-adrenoceptors. Thus, intracellular dialysis of cADPR increased the cytoplasmic Ca(2+) concentration proximal to the plasma membrane in isolated arterial smooth muscle cells and induced a concomitant membrane hyperpolarization. Smooth muscle hyperpolarization mediated by cADPR, by beta-adrenoceptors, and by cAMP, respectively, was abolished by chelating intracellular Ca(2+) and by blocking RyRs, cADPR, and BK(Ca) channels with ryanodine, 8-amino-cADPR, and iberiotoxin, respectively. The cAMP-dependent protein kinase A antagonist N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked hyperpolarization by isoprenaline and cAMP, respectively, but not hyperpolarization by cADPR. Thus, cADPR acts as a downstream element in this signaling cascade. Importantly, antagonists of cADPR and BK(Ca) channels, respectively, inhibited beta-adrenoreceptor-induced artery dilation. We conclude, therefore, that relaxation of arterial smooth muscle by adenylyl cyclase-coupled receptors results, in part, from a cAMP-dependent and protein kinase A-dependent increase in cADPR synthesis, and subsequent activation of sarcoplasmic reticulum Ca(2+) release via RyRs, which leads to activation of BK(Ca) channels and membrane hyperpolarization.     

Connexin-43 hemichannels mediate cyclic ADP-ribose generation and its Ca2+-mobilizing activity by NAD+/cyclic ADP-ribose transport

The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD(+). cADPR increases intracellular Ca(2+) through the intracellular ryanodine receptor/Ca(2+) release channel (RyR). It has been known that intracellular NAD(+) approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD(+). Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fcγ receptor (FcγR) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD(+) export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as FcγR stimulation. FcγR stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between FcγR stimulation and NAD(+)/cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD(+) and importing cADPR into the cell to activate intracellular calcium mobilization.     

Bioorganic chemistry of cyclic ADP-ribose (cADPR).

The objective of this brief review is to present an overview of the bioorganic chemistry of cyclic-ADP-ribose (cADPR) with special emphasis on the methodology used for the synthesis of analogues of cADPR. New structural analogues of cADPR can be prepared using either the biomimetic method or ADP-ribosyl cyclase from Aplysia californica. For the most part, both procedures give similar product profiles, but higher yields are generally obtained with the enzymatic method. These synthetic methodologies have allowed the transformation of a variety of structurally modified analogues of NAD+ into their corresponding cyclic nucleotides. Several of these novel analogues are more potent than cADPR in inducing calcium release and are also more stable towards degradative enzymes. They could serve as valuable affinity probes for the isolation of cADPR-binding proteins.     

NMR studies and semi-empirical energy calculations for cyclic ADP-ribose

A possible pH-dependent conformational switch was investigated for cyclic ADP-ribose. NMR signals for the exchangeable protons were observed in H2O at low temperature, but there was no direct evidence for the protonation of N-3 at neutral pH that has previously been postulated. MNDO calculations indicated that pH dependent 31P chemical shift changes are attributable to protonation of the phosphate adjacent to the N-1 of adenine, and not due to trans-annular hydrogen bonding with a protonated N-3.     

Adp-ribosyl cyclase and cyclic ADP-ribose hydrolase act as a redox sensor. a primary role for cyclic ADP-ribose in hypoxic pulmonary vasoconstriction

Hypoxic pulmonary vasoconstriction is unique to pulmonary arteries and serves to match lung perfusion to ventilation. However, in disease states this process can promote hypoxic pulmonary hypertension. Hypoxic pulmonary vasoconstriction is associated with increased NADH levels in pulmonary artery smooth muscle and with intracellular Ca(2+) release from ryanodine-sensitive stores. Because cyclic ADP-ribose (cADPR) regulates ryanodine receptors and is synthesized from beta-NAD(+), we investigated the regulation by beta-NADH of cADPR synthesis and metabolism and the role of cADPR in hypoxic pulmonary vasoconstriction. Significantly higher rates of cADPR synthesis occurred in smooth muscle homogenates of pulmonary arteries, compared with homogenates of systemic arteries. When the beta-NAD(+):beta-NADH ratio was reduced, the net amount of cADPR accumulated increased. This was due, at least in part, to the inhibition of cADPR hydrolase by beta-NADH. Furthermore, hypoxia induced a 10-fold increase in cADPR levels in pulmonary artery smooth muscle, and a membrane-permeant cADPR antagonist, 8-bromo-cADPR, abolished hypoxic pulmonary vasoconstriction in pulmonary artery rings. We propose that the cellular redox state may be coupled via an increase in beta-NADH levels to enhanced cADPR synthesis, activation of ryanodine receptors, and sarcoplasmic reticulum Ca(2+) release. This redox-sensing pathway may offer new therapeutic targets for hypoxic pulmonary hypertension.     

Hydrolysis of the phosphoanhydride linkage of cyclic ADP-ribose by the Mn-dependent ADP-ribose/CDP-alcohol pyrophosphatase

Cyclic ADP-ribose (cADPR) metabolism in mammals is catalyzed by NAD glycohydrolases (NADases) that, besides forming ADP-ribose, form and hydrolyze the N¹-glycosidic linkage of cADPR. Thus far, no cADPR phosphohydrolase was known. We tested rat ADP-ribose/CDP-alcohol pyrophosphatase (ADPRibase-Mn) and found that cADPR is an ADPRibase-Mn ligand and substrate. ADPRibase-Mn activity on cADPR was 65-fold less efficient than on ADP-ribose, the best substrate. This is similar to the ADP-ribose/cADPR formation ratio by NADases. The product of cADPR phosphohydrolysis by ADPRibase-Mn was N¹-(5-phosphoribosyl)-AMP, suggesting a novel route for cADPR turnover.     

Ca2+ release induced by cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is the most potent Ca²⁺-mobilizing agent known. It has been found in many different cell types, where it is synthesized from its precursor NAD⁺ by ADP-ribosyl cyclases. cADPR binds to Ca²⁺ channels in the endoplasmic reticulum membrane to activate a Ca²⁺-release mechanism. This release is itself potentiated by elevated cytoplasmic Ca²⁺ concentrations. Thus, cADPR may function as an endogenous regulator of Ca²⁺-induced Ca²⁺ release, and there is excitement that it may also function as a Ca²⁺-mobilizing second messenger.     

Abscisic acid signaling through cyclic ADP-ribose in hydroid regeneration

Cyclic ADP-ribose (cADPR) is an intracellular calcium (Ca(2+)(i)) mobilizer involved in fundamental cell functions from protists to higher plants and mammals. Biochemical similarities between the drought-signaling cascade in plants and the temperature-sensing pathway in marine sponges suggest an ancient evolutionary origin of a signaling cascade involving the phytohormone abscisic acid (ABA), cADPR, and Ca(2+)(i). In Eudendrium racemosum (Hydrozoa, Cnidaria), exogenously added ABA stimulated ADP-ribosyl cyclase activity via a protein kinase A (PKA)-mediated phosphorylation and increased regeneration in the dark to levels observed under light conditions. Light stimulated endogenous ABA synthesis, which was conversely inhibited by the inhibitor of plant ABA synthesis Fluridone. The signal cascade of light-induced regeneration uncovered in E. racemosum: light --> increasing ABA --> PKA --> cyclase activation --> increasing [cADPR](i) --> increasing [Ca(2+)](i) --> regeneration is the first report of a complete signaling pathway in Eumetazoa involving a phytohormone.     

Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of beta-NAD(+) to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5'-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS-PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K(m) values of 30.4+/-1.5 and 695. 3+/-171.2 microM and V(max) values of 330.4+/-90 and 102.8+/-17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca(2+)](i) in porcine TSM cells.     

A novel fluorescent cell membrane-permeable caged cyclic ADP-ribose analogue

Cyclic adenosine diphosphate ribose is an endogenous Ca(2+) mobilizer involved in diverse cellular processes. A cell membrane-permeable cyclic adenosine diphosphate ribose analogue, cyclic inosine diphosphoribose ether (cIDPRE), can induce Ca(2+) increase in intact human Jurkat T-lymphocytes. Here we synthesized a coumarin-caged analogue of cIDPRE (Co-i-cIDPRE), aiming to have a precisely temporal and spatial control of bioactive cIDPRE release inside the cell using UV uncaging. We showed that Co-i-cIDPRE accumulated inside Jurkat cells quickly and efficiently. Uncaging of Co-i-cIDPRE evoked Ca(2+) release from endoplasmic reticulum, with concomitant Ca(2+) influx in Jurkat cells. Ca(2+) release evoked by uncaged Co-i-cIDPRE was blocked by knockdown of ryanodine receptors (RyRs) 2 and 3 in Jurkat cells. The associated Ca(2+) influx, on the other hand, was abolished by double knockdown of Stim1 and TRPM2 in Jurkat cells. Furthermore, Ca(2+) release or influx evoked by uncaged Co-i-cIDPRE was recapitulated in HEK293 cells that overexpress RyRs or TRPM2, respectively, but not in wild-type cells lacking these channels. In summary, our results indicate that uncaging of Co-i-cIDPRE incites Ca(2+) release from endoplasmic reticulum via RyRs and triggers Ca(2+) influx via TRPM2.     

Sympathetic potentiation of cyclic ADP-ribose formation in rat cardiac myocytes

We examined the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of adrenergic receptors in the heart after excitation of sympathetic neurons. To address this question, ADP-ribosyl cyclase activity was measured as the rate of [(3)H]cADP-ribose formation from [(3)H]NAD(+) in a crude membrane fraction of rat ventricular myocytes. Isoproterenol at 1 microM increased ADP-ribosyl cyclase activity by 1.7-fold in ventricular muscle; this increase was inhibited by propranolol. The stimulatory effect on the cyclase was mimicked by 10 nM GTP and 10 microM guanosine 5'-3-O-(thio)triphosphate, whereas 10 microM GTP inhibited the cyclase. Cholera toxin blocked the activation of the cyclase by isoproterenol and GTP. The above effects of isoproterenol and GTP in ventricular membranes were confirmed by cyclic GDP-ribose formation fluorometrically. These results demonstrate the existence of a signal pathway from beta-adrenergic receptors to membrane-bound ADP-ribosyl cyclase via G protein in the ventricular muscle cells and suggest that increased cADP-ribose synthesis is involved in up-regulation of cardiac function by sympathetic stimulation.     

Determination of endogenous levels of cyclic ADP-ribose in rat tissues

Cyclic ADP-ribose (cADPR) is a potent mediator of calcium mobilization in sea urchin eggs. The cADPR synthesizing enzyme is present not only in the eggs but also in various mammalian tissue extracts. The purpose of this study was to ascertain whether cADPR is a naturally occurring nucleotide in mammalian tissues. Rat tissues were frozen and powdered in liquid N2, followed by extraction with perchloric acid at -10 degrees C. [32P]cADPR was prepared and used as a tracer. The acid extracts were chromatographed on a Mono-Q column and cADPR in the fractions were determined by its ability to release Ca2+ from egg homogenates. That the release was mediated by cADPR and not inositol trisphosphate (IP3) in the extracts was shown by the fact that the homogenates, subsequent to Ca2+ release induced by active fractions, were desensitized to authentic cADPR but not to IP3. Furthermore, the Ca2+ release activity was shown to co-elute with [32P]cADPR. The endogenous level of cADPR determined in rat liver is 3.37 +/- 0.64 pmol/mg, in heart is 1.04 +/- 0.08 pmol/mg and in brain is 2.75 +/- 0.35 pmol/mg. These results indicate cADPR is a naturally occurring nucleotide and suggest that it may be a general second messenger for mobilizing intracellular Ca2+.     

Mechanism-based inhibitors of CD38: a mammalian cyclic ADP-ribose synthetase

The soluble domain of human CD38 catalyzes the conversion of NAD(+) to cyclic ADP-ribose and to ADP-ribose via a common covalent intermediate [Sauve, A. A., Deng, H. T., Angelletti, R. H., and Schramm, V. L. (2000) J. Am. Chem. Soc. 122, 7855-7859]. Here we establish that mechanism-based inhibitors can be produced by chemical stabilization of this intermediate. The compounds nicotinamide 2'-deoxyriboside (1), 5-methylnicotinamide 2'-deoxyriboside (2), and pyridyl 2'-deoxyriboside (3) were synthesized and evaluated as inhibitors for human CD38. The nicotinamide derivatives 1 and 2 were inhibitors of the enzyme as determined by competitive behavior in CD38-catalyzed conversion of nicotinamide guanine dinucleotide (NGD(+)) to cyclic GDP-ribose. The K(i) values for competitive inhibition were 1.2 and 4.0 microM for 1 and 2, respectively. Slow-onset characteristics of reaction progress curves indicated a second higher affinity state of these two inhibitors. Inhibitor off-rates were slow with rate constants k(off) of 1.5 x 10(-5) s(-1) for 1 and 2.5 x 10(-5) s(-1) for 2. Apparent dissociation constants K(i(total)) for 1 and 2 were calculated to be 4.5 and 12.5 nM, respectively. The similar values for k(off) are consistent with the hydrolysis of common enzymatic intermediates formed by the reaction of 1 and 2 with the enzyme. Both form covalently attached deoxyribose groups to the catalytic site nucleophile. Chemical evidence for this intermediate is the ability of nicotinamide to rescue enzyme activity after inactivation by either 1 or 2. A covalent intermediate is also indicated by the ability of CD38 to catalyze base exchange, as observed by conversion of 2 to 1 in the presence of nicotinamide. The deoxynucleosides 1 and 2 demonstrate that the chemical determinants for mechanism-based inhibition of CD38 can be satisfied by nucleosides that lack the 5'-phosphate, the adenylate group, and the 2'-hydroxyl moiety. In addition, these compounds reveal the mechanism of CD38 catalysis to proceed by the formation of a covalent intermediate during normal catalytic turnover with faster substrates. The covalent 2'-deoxynucleoside inactivators of CD38 are powerful inhibitors by acting as good substrates for formation of the covalent intermediate but are poor leaving groups from the intermediate complex because hydrolytic assistance of the 2'-hydroxyl group is lacking. The removal of the adenylate nucleophile required for the cyclization reaction provides slow hydrolysis as the only exit from the covalent complex.     

Cyclic 3-deaza-adenosine diphosphoribose: a potent and stable analog of cyclic ADP-ribose

Cyclic 3-deaza-adenosine diphosphoribose (3-deaza-cADPR), an analog of cyclic adenosine diphosphoribose (cADPR) was synthesized. 3-deaza-cADPR differs from cADPR by only the substitution of carbon for nitrogen at the 3-position of the purine ring. Similar to cADPR, the analog has potent calcium releasing activity in sea urchin egg homogenates and was able to induce calcium release at concentrations as low as 0.3 nM. The EC(50) value for 3-deaza-cADPR-induced calcium release was 1 nM, which is about 70 times more potent than cADPR. The properties of calcium release induced by 3-deaza-cADPR in all other respects were similar to those of cADPR. Thus, 3-deaza-cADPR and cADPR were capable of cross-desensitizing each other and their calcium releasing activities were potentiated by Sr(2+) as well as caffeine. 8-amino-cADPR, a selective antagonist of cADPR, was also able to inhibit 3-deaza-cADPR induced calcium release. Taken together, these data suggest that 3-deaza-cADPR releases calcium through the same mechanism as cADPR. 3-deaza-cADPR was found to be resistant to both heat and enzymatic hydrolysis. Only 15% of 3-deaza-cADPR was destroyed after boiling this compound for 2 h. No loss of 3-deaza-cADPR was observed when treated with CD38 under conditions where cADPR was completely hydrolyzed. Thus, 3-deaza-cADPR is a potent and stable analog of cADPR. These properties should make 3-deaza-cADPR a useful probe in studies focused on the mechanism of cADPR action.