Cyclic ADP ribose isomers: Production, chemical structures, and immune signaling.

Cyclic adenosine diphosphate (ADP)-ribose (cADPR) isomers are signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via nicotinamide adenine dinucleotide (oxidized form) (NAD+) hydrolysis. We show that v-cADPR (2'cADPR) and v2-cADPR (3'cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of 2'cADPR-producing TIR domains reveal conformational changes that lead to an active assembly that resembles those of Toll-like receptor adaptor TIR domains. Mutagenesis reveals a conserved tryptophan that is essential for cyclization. We show that 3'cADPR is an activator of ThsA effector proteins from the bacterial antiphage defense system termed Thoeris and a suppressor of plant immunity when produced by the effector HopAM1. Collectively, our results reveal the molecular basis of cADPR isomer production and establish 3'cADPR in bacteria as an antiviral and plant immunity-suppressing signaling molecule.

On a Magical Mystery Tour with 8-Bromo-Cyclic ADP-Ribose: From All-or-None Block to Nanojunctions and the Cell-Wide Web.

A plethora of cellular functions are controlled by calcium signals, that are greatly coordinated by calcium release from intracellular stores, the principal component of which is the sarco/endooplasmic reticulum (S/ER). In 1997 it was generally accepted that activation of various G protein-coupled receptors facilitated inositol-1,4,5-trisphosphate (IP3) production, activation of IP3 receptors and thus calcium release from S/ER. Adding to this, it was evident that S/ER resident ryanodine receptors (RyRs) could support two opposing cellular functions by delivering either highly localised calcium signals, such as calcium sparks, or by carrying propagating, global calcium waves. Coincidentally, it was reported that RyRs in mammalian cardiac myocytes might be regulated by a novel calcium mobilising messenger, cyclic adenosine diphosphate-ribose (cADPR), that had recently been discovered by HC Lee in sea urchin eggs. A reputedly selective and competitive cADPR antagonist, 8-bromo-cADPR, had been developed and was made available to us. We used 8-bromo-cADPR to further explore our observation that S/ER calcium release via RyRs could mediate two opposing functions, namely pulmonary artery dilation and constriction, in a manner seemingly independent of IP3Rs or calcium influx pathways. Importantly, the work of others had shown that, unlike skeletal and cardiac muscles, smooth muscles might express all three RyR subtypes. If this were the case in our experimental system and cADPR played a role, then 8-bromo-cADPR would surely block one of the opposing RyR-dependent functions identified, or the other, but certainly not both. The latter seemingly implausible scenario was confirmed. How could this be, do cells hold multiple, segregated SR stores that incorporate different RyR subtypes in receipt of spatially segregated signals carried by cADPR? The pharmacological profile of 8-bromo-cADPR action supported not only this, but also indicated that intracellular calcium signals were delivered across intracellular junctions formed by the S/ER. Not just one, at least two. This article retraces the steps along this journey, from the curious pharmacological profile of 8-bromo-cADPR to the discovery of the cell-wide web, a diverse network of cytoplasmic nanocourses demarcated by S/ER nanojunctions, which direct site-specific calcium flux and may thus coordinate the full panoply of cellular processes.

[Medicinal Chemical Studies Based on the Theoretical Design of Bioactive Compounds].

I have engaged in medicinal chemical studies based on the theoretical design of bioactive compounds. First, I present a three-dimensional structural diversity-oriented conformational restriction strategy for developing bioactive compounds based on the characteristic steric and stereoelectronic features of cyclopropane. Using this strategy, various biologically active small molecule compounds, such as receptor agonists/antagonists and enzyme inhibitors, were effectively developed. The strategy was also applied to develop versatile peptidomimetics and membrane-permeable cyclic peptides. Next, studies on Ca2+-mobilizing second messengers, cyclic ADP-ribose (cADPR) and myo-inositol trisphosphates (IP3), are described. In these studies, stable equivalents of cADPR were developed, since biological studies of cADPR have been limited due to its instability. Various potent IP3 receptor ligands, which were designed using the d-glucose structure as a bioisostere of the myo-inositol moiety of IP3, have been identified. Organic chemistry studies have also been extensively performed, because excellent organic chemistry is essential for promoting high-level medicinal chemical studies. For examples, new methods for the synthesis of chiral cyclopropanes, new radical reactions with silicon tethers, and kinetic anomeric effect-dependent stereoselective glycosidations have been developed.

Probing Ca2+ release mechanisms using sea urchin egg homogenates.

Sea urchin eggs have been extensively used to study Ca2+ release through intracellular Ca2+-permeable channels. Their amenability to homogenization yields a robust, cell-free preparation that was central to establishing the Ca2+ mobilizing actions of cyclic ADP-ribose and NAADP. Egg homogenates have continued to provide insight into the basic properties and pharmacology of intracellular Ca2+ release channels. In this chapter, we describe methods for the preparation of egg homogenates and monitoring Ca2+ release using fluorimetry and radiotracer flux.

Synthetic cADPR analogues may form only one of two possible conformational diastereoisomers.

Cyclic adenosine 5'-diphosphate ribose (cADPR) is an emerging Ca2+-mobilising second messenger. cADPR analogues have been generated as chemical biology tools via both chemo-enzymatic and total synthetic routes. Both routes rely on the cyclisation of a linear precursor to close an 18-membered macrocyclic ring. We show here that, after cyclisation, there are two possible macrocyclic product conformers that may be formed, depending on whether cyclisation occurs to the "right" or the "left" of the adenine base (as viewed along the H-8 → C-8 base axis). Molecular modelling demonstrates that these two conformers are distinct and cannot interconvert. The two conformers would present a different spatial layout of binding partners to the cADPR receptor/binding site. For chemo-enzymatically generated analogues Aplysia californica ADP-ribosyl cyclase acts as a template to generate solely the "right-handed" conformer and this corresponds to that of the natural messenger, as originally explored using crystallography. However, for a total synthetic analogue it is theoretically possible to generate either product, or a mixture, from a given linear precursor. Cyclisation on either face of the adenine base is broadly illustrated by the first chemical synthesis of the two enantiomers of a "southern" ribose-simplified cIDPR analogue 8-Br-N9-butyl-cIDPR, a cADPR analogue containing only one chiral sugar in the "northern" ribose, i.e. 8-Br-D- and its mirror image 8-Br-L-N9-butyl-cIDPR. By replacing the D-ribose with the unnatural L-ribose sugar, cyclisation of the linear precursor with pyrophosphate closure generates a cyclised product spectroscopically identical, but displaying equal and opposite specific rotation. These findings have implications for cADPR analogue design, synthesis and activity.

Synthesis and Biological Evaluation of a New Structural Simplified Analogue of cADPR, a Calcium-Mobilizing Secondary Messenger Firstly Isolated from Sea Urchin Eggs.

Herein, we reported on the synthesis of cpIPP, which is a new structurally-reduced analogue of cyclic ADP-ribose (cADPR), a potent Ca2+-releasing secondary messenger that was firstly isolated from sea urchin eggs extracts. To obtain cpIPP the "northern" ribose of cADPR was replaced by a pentyl chain and the pyrophosphate moiety by a phophono-phosphate anhydride. The effect of the presence of the new phosphono-phosphate bridge on the intracellular Ca2+ release induced by cpIPP was assessed in PC12 neuronal cells in comparison with the effect of the pyrophosphate bridge of the structurally related cyclic N1-butylinosine diphosphate analogue (cbIDP), which was previously synthesized in our laboratories, and with that of the linear precursor of cpIPP, which, unexpectedly, revealed to be the only one provided with Ca2+ release properties.

Specific cyclic ADP-ribose phosphohydrolase obtained by mutagenic engineering of Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase.

Cyclic ADP-ribose (cADPR) is a messenger for Ca2+ mobilization. Its turnover is believed to occur by glycohydrolysis to ADP-ribose. However, ADP-ribose/CDP-alcohol diphosphatase (ADPRibase-Mn) acts as cADPR phosphohydrolase with much lower efficiency than on its major substrates. Recently, we showed that mutagenesis of human ADPRibase-Mn at Phe37, Leu196 and Cys253 alters its specificity: the best substrate of the mutant F37A + L196F + C253A is cADPR by a short difference, Cys253 mutation being essential for cADPR preference. Its proximity to the 'northern' ribose of cADPR in docking models indicates Cys253 is a steric constraint for cADPR positioning. Aiming to obtain a specific cADPR phosphohydrolase, new mutations were tested at Asp250, Val252, Cys253 and Thr279, all near the 'northern' ribose. First, the mutant F37A + L196F + C253G, with a smaller residue 253 (Ala > Gly), showed increased cADPR specificity. Then, the mutant F37A + L196F + V252A + C253G, with another residue made smaller (Val > Ala), displayed the desired specificity, with cADPR kcat/KM ≈20-200-fold larger than for any other substrate. When tested in nucleotide mixtures, cADPR was exhausted while others remained unaltered. We suggest that the specific cADPR phosphohydrolase, by cell or organism transgenesis, or the designed mutations, by genome editing, provide opportunities to study the effect of cADPR depletion on the many systems where it intervenes.

Variable-Temperature NMR Spectroscopy, Conformational Analysis, and Thermodynamic Parameters of Cyclic Adenosine 5'-Diphosphate Ribose Agonists and Antagonists.

Cyclic adenosine 5'-diphosphate ribose (cADPR) is a ubiquitous Ca2+-releasing second messenger. Knowledge of its conformational landscape is an essential tool for unraveling the structure-activity relationship (SAR) in cADPR. Variable-temperature 1H NMR spectroscopy, in conjunction with PSEUROT and population analyses, allowed us to determine the conformations and thermodynamic parameters of the furanose rings, γ-bonds (C4'-C5'), and ß-bonds (C5'-O5') in the cADPR analogues 2'-deoxy-cADPR, 7-deaza-cADPR, and 8-bromo-cADPR. A significant finding was that, although the analogues are similar to each other and to cADPR itself in terms of overall conformation and population (ΔG°), there were subtle yet important differences in some of thermodynamic properties (ΔH°, ΔS°) associated with each of the conformational equilibria. These differences prompted us to propose a model for cADPR in which the interactions between the A2'-N3, A5″-N3, and H2-R5' atoms serve to fine-tune the N-glycosidic torsion angles (χ).

A combined variable temperature 600 MHz NMR/MD study of the calcium release agent cyclic adenosine diphosphate ribose (cADPR): Structure, conformational analysis, and thermodynamics of the conformational equilibria.

A combined variable temperature 600 MHz NMR/molecular dynamics study of the Ca2+-release agent cyclic adenosine 5'-diphosphate ribose (cADPR) was conducted. In addition to elucidating the major and minor orientations of the conformationally flexible furanose rings, γ- (C4'-C5'), and ß- (C5'-O5') bonds, the thermodynamics (ΔHo, ΔSo) associated with each of these conformational equilibria were determined. Both furanose rings were biased towards a south conformation (64-74%) and both ß-bonds heavily favored trans conformations. The R-ring γ-bond was found to exist almost exclusively as the γ+ conformer, whereas the A-ring γ-bond was a mixture of the γ+ and γt conformers, with the trans conformer being slightly favored. Enthalpic factors accounted for most of the observed conformational preferences, although the R-ring furanose exists as its major conformation based solely on entropic factors. There was excellent agreement between the NMR and MD results, particularly with regard to the conformer identities, but the MD showed a bias towards γ+ conformers. The MD results showed that both N-glycosidic χ-bonds are exclusively syn. Collectively the data allowed for the construction of a model for cADPR in which many of the conformationally flexible units in fact effectively adopt single orientations and where most of the conformational diversity resides in its A-ring furanose and γ-bond.

Synthesis of 8-Substituted Analogues of Cyclic ADP-4-Thioribose and Their Unexpected Identification as Ca2+-Mobilizing Full Agonists.

A series of 8-substituted analogues of cyclic ADP-4-thioribose (cADPtR, 3), which is a stable equivalent of Ca2+-mobilizing second messenger cyclic ADP-ribose (cADPR, 1), were designed as potential pharmacological tools for studies on cADPR-modulated Ca2+ signaling pathways. These 8-amino analogue (8-NH2-cADPtR, 4), 8-azido analogue (8-N3-cADPtR, 5), and 8-chloro analogue (8-Cl-cADPtR, 6) were efficiently synthesized, where the stereoselective N1-ß-thioribosyladenine ring closure reaction via an α/ß-equilibrium of the 1-aminothioribose derivative and construction of the characteristic 18-membered pyrophosphate ring by Ag+-promoted activation of a phenyl phosphorothioate type substrate were the two key steps. Although 8-NH2-cADPR (2) is a well-known potent antagonist against cADPR-inducing Ca2+-release, the 4-thioribose congener 8-NH2-cADPtR turned out unexpectedly to be a full agonist in sea urchin egg homogenate evaluation system. This important finding suggested that the ring-oxygen in the N1-ribose of cADPR analogues is essential for the antagonistic activity in the Ca2+-signaling pathway, which can contribute to clarify the structure-agonist/antagonist activity relationship.

Design, Synthesis, and Identification of 4″α-Azidoethyl-cyclic ADP-Carbocyclic-ribose as a Highly Potent Analogue of Cyclic ADP-Ribose, a Ca(2+)-Mobilizing Second Messenger.

Cyclic adenosine diphosphate-carbocyclic-ribose (cADPcR, 2) is a stable equivalent of cyclic adenosine diphosphate-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. On the basis of the structure-activity relationship of cADPR-related compounds and three-dimensional structural modeling of cADPcR, we designed and synthesized cyclic-ADP-4″α-azidoethyl carbocyclic-ribose (N3-cADPcR, 3) to demonstrate that it has a highly potent Ca(2+)-mobilizing activity (EC50 = 24 nM). N3-cADPcR will be a useful precursor for the preparation of biological tools effective to investigate cADPR-mediated signaling pathways.

Synthesis of 7-Deaza-cyclic Adenosine-5'-diphosphate-carbocyclic-ribose and Its 7-Bromo Derivative as Intracellular Ca(2+)-Mobilizing Agents.

Cyclic ADP-carbocyclic-ribose (cADPcR, 3) is a biologically and chemically stable equivalent of cyclic ADP-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. We became interested in the biological activity of the 7-deaza analogues of cADPcR, i.e., 7-deaza-cADPcR (7) and its 7-bromo derivative, i.e., 7-deaza-7-Br-cADPcR (8), because 7-deazaadenosine is an efficient bioisostere of adenosine. The synthesis of 7 and 8 required us to construct the key N1-carbocyclic-ribosyl-7-deazaadenosine structure. Therefore, we developed a general method for preparing N1-substituted 7-deazaadenosines by condensing a 2,3-disubstituted pyrrole nucleoside with amines. Using this method, we prepared the N1-carbocyclic ribosyl 7-deazaadenosine derivative 10a, from which we then synthesized the target 7-deaza-cADPcR (7) via an Ag(+)-promoted intramolecular condensation to construct the 18-membered pyrophosphate ring structure. The corresponding 7-bromo derivative 8, which was the first analogue of cADPR with a substitution at the 7-position, was similarly synthesized. Biological evaluation for Ca(2+)-mobilizing activity in the sea urchin egg homogenate system indicated that 7-deaza-cADPcR (7) and 7-deaza-7-Br-cADPcR (8) acted as a full agonist and a partial agonist, respectively.

Cyclic adenosine 5'-diphosphoribose (cADPR) mimics used as molecular probes in cell signaling.

Cyclic adenosine 5'-diphosphate ribose (cADPR) is a second messenger in the Ca(2+) signaling pathway. To elucidate its molecular mechanism in calcium release, a series of cADPR analogues with modification on ribose, nucleobase, and pyrophosphate have been investigated. Among them, the analogue with the modification of the northern ribose by ether linkage substitution (cIDPRE) exhibits membrane-permeate Ca(2+) agonistic activity in intact HeLa cells, human T cells, mouse cardiac myocytes and neurosecretory PC12 cell lines; thus, cIDPRE and coumarin-caged cIDPRE are valuable probes to investigate the cADPR-mediated Ca(2+) signal pathway.

Cyclic adenosine 5'-diphosphate ribose analogs without a "southern" ribose inhibit ADP-ribosyl cyclase-hydrolase CD38.

Cyclic adenosine 5'-diphosphate ribose (cADPR) analogs based on the cyclic inosine 5'-diphosphate ribose (cIDPR) template were synthesized by recently developed stereo- and regioselective N1-ribosylation. Replacing the base N9-ribose with a butyl chain generates inhibitors of cADPR hydrolysis by the human ADP-ribosyl cyclase CD38 catalytic domain (shCD38), illustrating the nonessential nature of the "southern" ribose for binding. Butyl substitution generally improves potency relative to the parent cIDPRs, and 8-amino-N9-butyl-cIDPR is comparable to the best noncovalent CD38 inhibitors to date (IC50 = 3.3 µM). Crystallographic analysis of the shCD38:8-amino-N9-butyl-cIDPR complex to a 2.05 Å resolution unexpectedly reveals an N1-hydrolyzed ligand in the active site, suggesting that it is the N6-imino form of cADPR that is hydrolyzed by CD38. While HPLC studies confirm ligand cleavage at very high protein concentrations, they indicate that hydrolysis does not occur under physiological concentrations. Taken together, these analogs confirm that the "northern" ribose is critical for CD38 activity and inhibition, provide new insight into the mechanism of cADPR hydrolysis by CD38, and may aid future inhibitor design.

'Click cyclic ADP-ribose': a neutral second messenger mimic.

Analogues of the potent Ca(2+) releasing second messenger cyclic ADP-ribose (cADPR) with a 1,2,3-triazole pyrophosphate bioisostere were synthesised by click-mediated macrocyclisation. The ability to activate Ca(2+) release was surprisingly retained, and hydrolysis of cADPR by CD38 could also be inhibited, illustrating the potential of this approach to design drug-like signalling pathway modulators.

CD38 Structure-Based Inhibitor Design Using the N1-Cyclic Inosine 5'-Diphosphate Ribose Template.

Few inhibitors exist for CD38, a multifunctional enzyme catalyzing the formation and metabolism of the Ca(2+)-mobilizing second messenger cyclic adenosine 5'-diphosphoribose (cADPR). Synthetic, non-hydrolyzable ligands can facilitate structure-based inhibitor design. Molecular docking was used to reproduce the crystallographic binding mode of cyclic inosine 5'-diphosphoribose (N1-cIDPR) with CD38, revealing an exploitable pocket and predicting the potential to introduce an extra hydrogen bond interaction with Asp-155. The purine C-8 position of N1-cIDPR (IC50 276 µM) was extended with an amino or diaminobutane group and the 8-modified compounds were evaluated against CD38-catalyzed cADPR hydrolysis. Crystallography of an 8-amino N1-cIDPR:CD38 complex confirmed the predicted interaction with Asp-155, together with a second H-bond from a realigned Glu-146, rationalizing the improved inhibition (IC50 56 µM). Crystallography of a complex of cyclic ADP-carbocyclic ribose (cADPcR, IC50 129 µM) with CD38 illustrated that Glu-146 hydrogen bonds with the ligand N6-amino group. Both 8-amino N1-cIDPR and cADPcR bind deep in the active site reaching the catalytic residue Glu-226, and mimicking the likely location of cADPR during catalysis. Substantial overlap of the N1-cIDPR "northern" ribose monophosphate and the cADPcR carbocyclic ribose monophosphate regions suggests that this area is crucial for inhibitor design, leading to a new compound series of N1-inosine 5'-monophosphates (N1-IMPs). These small fragments inhibit hydrolysis of cADPR more efficiently than the parent cyclic compounds, with the best in the series demonstrating potent inhibition (IC50 = 7.6 µM). The lower molecular weight and relative simplicity of these compounds compared to cADPR make them attractive as a starting point for further inhibitor design.

Cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP) as messengers for calcium mobilization.

Cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate were discovered >2 decades ago. That they are second messengers for mobilizing Ca(2+) stores has since been firmly established. Separate stores and distinct Ca(2+) channels are targeted, with cyclic ADP-ribose acting on the ryanodine receptors in the endoplasmic reticulum, whereas nicotinic acid adenine dinucleotide phosphate mobilizes the endolysosomes via the two-pore channels. Despite the structural and functional differences, both messengers are synthesized by a ubiquitous enzyme, CD38, whose crystal structure and catalytic mechanism have now been well elucidated. How this novel signaling enzyme is regulated remains largely unknown and is the focus of this minireview.

Synthesis and calcium mobilization activity of cADPR analogues which integrate nucleobase, northern and southern ribose modifications.

Novel cADPR mimics, which integrate nucleobase, northern and southern ribose modifications were synthesized. The key steps of the synthesis were a Cu(I)-catalyzed Hüisgen [3+2] cycloaddition and a microwave-assisted intramolecular pyrophosphorylation. Preliminary biological investigations showed that these cADPR mimics are membrane-permeating agonists of the calcium signaling pathway. The introduction of chlorine or fluorine at the 2'-position of the southern riboses led to a decrease of activity. The existence of a hydrophobic group on the 3'-OH of the southern riboses does not obviously alter the agonistic activity.

Cyclic ADP-ribose and NAADP: fraternal twin messengers for calcium signaling.

The concept advanced by Berridge and colleagues that intracellular Ca(2+)-stores can be mobilized in an agonist-dependent and messenger (IP(3))-mediated manner has put Ca(2+)-mobilization at the center stage of signal transduction mechanisms. During the late 1980s, we showed that Ca(2+)-stores can be mobilized by two other messengers unrelated to inositol trisphosphate (IP(3)) and identified them as cyclic ADP-ribose (cADPR), a novel cyclic nucleotide from NAD, and nicotinic acid adenine dinucleotide phosphate (NAADP), a linear metabolite of NADP. Their messenger functions have now been documented in a wide range of systems spanning three biological kingdoms. Accumulated evidence indicates that the target of cADPR is the ryanodine receptor in the sarco/endoplasmic reticulum, while that of NAADP is the two pore channel in endolysosomes.As cADPR and NAADP are structurally and functionally distinct, it is remarkable that they are synthesized by the same enzyme. They are thus fraternal twin messengers. We first identified the Aplysia ADP-ribosyl cyclase as one such enzyme and, through homology, found its mammalian homolog, CD38. Gene knockout in mice confirms the important roles of CD38 in diverse physiological functions from insulin secretion, susceptibility to bacterial infection, to social behavior of mice through modulating neuronal oxytocin secretion. We have elucidated the catalytic mechanisms of the Aplysia cyclase and CD38 to atomic resolution by crystallography and site-directed mutagenesis. This article gives a historical account of the cADPR/NAADP/CD38-signaling pathway and describes current efforts in elucidating the structure and function of its components.