cADPR Analogues Research Articles

Identification of a small chemical as a lysosomal calcium mobilizer and characterization of its ability to inhibit autophagy and viral infection.

We previously identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as one of the cyclic adenosine diphosphoribose (cADPR)'s binding proteins and found that GAPDH participates in cADPR-mediated Ca2+ release from endoplasmic reticulum via ryanodine receptors (RyRs). Here, we aimed to chemically synthesise and pharmacologically characterise novel cADPR analogues. Based on the simulated cADPR-GAPDH complex structure, we performed the structure-based drug screening, identified several small chemicals with high docking scores to cADPR's binding pocket in GAPDH and showed that two of these compounds, C244 and C346, are potential cADPR antagonists. We further synthesised several analogues of C346 and found that its analogue, G42, also mobilised Ca2+ release from lysosomes. G42 alkalised lysosomal pH and inhibited autophagosome-lysosome fusion. Moreover, G42 markedly inhibited Zika virus (ZIKV, a flavivirus) or murine hepatitis virus (MHV, a β-coronavirus) infections of host cells. These results suggest that G42 inhibits virus infection, likely by triggering lysosomal Ca2+ mobilisation and inhibiting autophagy.

Chemical Biology Tools to Elucidate the Solution Conformation and Cellular Interactions of Cyclic Adenosine 5′‐Diphosphate Ribose

The movement of calcium ions (Ca2+) is intimately involved in regulating many physiological processes, and so there is great interest in the structure of molecules, especially second messengers, that regulate this flow. One such messenger is cyclic adenosine 5′‐diphosphate ribose (cADPR), which is suspected to play a role in Ca2+ release in cardiomyocytes. The speculated mechanism of action involves the binding of cADPR to FKBP12.6, releasing FKBP12.6 from RyR2 channels, through which Ca2+ enters the sarcoplasm from the sarcoplasmic reticulum during ventricular systole. Guided by the principle that structure determines function, we aim to advance the investigation of regulatory cADPR interactions by synthesizing and studying cADPR and its analogs using the combined tools of liquid chromatography‐mass spectrometry (LCMS) and nuclear magnetic resonance (NMR) spectroscopy. The endogenous cyclization of nicotinamide adenine dinucleotide (NAD+) to cADPR by CD38 is mimicked in vitro using ADP‐ribosylcyclase (ADP‐RC, sourced from Aplysia californica). The low substrate specificity of ADP‐RC conveniently permits the cyclization of various cADPR analogs. Kinetic assays were prompted by an observed feedback inhibition of ADP‐RC by nicotinamide (IC50 ~ 1mM), and performed using LCMS. Following synthesis and purification, NMR experimental data provided insight into favored structural conformations of cADPR (Saatori et al., 2018) Photoaffinity labeling (PAL) was aimed at identifying the cognate binding partners of cADPR, some of which have been previously described by Walseth et al. (1993). Conformational analyses of cADPR analogs revealed structural trends with correlations to the biological activity of the analogs.Support or Funding InformationDepartment of Chemistry, Saint John’s University

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.

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 β-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).

Cyclic ADP-Carbocyclic-Ribose and -4-Thioribose, as Stable Mimics of Cyclic ADP-Ribose, a Ca2+-Mobilizing Second Messenger.

Cyclic ADP-ribose (cADPR), a general mediator involved in Ca2+ signaling, has the characteristic 18-membered ring consisting of an adenine, two riboses and a pyrophosphate, in which the two primary hydroxy groups of the riboses are linked by a pyrophosphate unit. This review focuses on chemical synthetic studies of cADPR analogues of biological importance. Although cADPR analogues can be synthesized by enzymatic and chemo-enzymatic methods using ADP-ribosyl cyclase, the analogues obtained by these methods are limited due to the substrate-specificity of the enzymes. Consequently, chemical synthetic methods providing a greater variety of cADPR analogues are required. Although early chemical synthetic studies demonstrated that construction of the large 18-membered ring structure is difficult, the construction was achieved using the phenylthiophosphate-type substrates by treating with AgNO3 or I2. This is now a general method for synthesizing these types of biologically important cyclic nucleotides. Using this method as the key step, the chemically and biologically stable cADPR mimic, cADP-carbocyclic-ribose (cADPcR) and -4-thioribose (cADPtR), were synthesized.

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-β-thioribosyladenine ring closure reaction via an α/β-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.

Synthesis of cyclic N (1)-pentylinosine phosphate, a new structurally reduced cADPR analogue with calcium-mobilizing activity on PC12 cells.

Cyclic N1-pentylinosine monophosphate (cpIMP), a novel simplified inosine derivative of cyclic ADP-ribose (cADPR) in which the N1-pentyl chain and the monophosphate group replace the northern ribose and the pyrophosphate moieties, respectively, was synthesized. The role played by the position of the phosphate group in the key cyclization step, which consists in the formation of a phosphodiester bond, was thoroughly investigated. We have also examined the influence of the phosphate bridge on the ability of cpIMP to mobilize Ca2+ in PC12 neuronal cells in comparison with the pyrophosphate bridge present in the cyclic N1-pentylinosine diphosphate analogue (cpIDP) previously synthesized in our laboratories. The preliminary biological tests indicated that cpIMP and cpIDP induce a rapid increase of intracellular Ca2+ concentration in PC12 neuronal cells.

CD38-mediated Ca2+ signaling contributes to glucagon-induced hepatic gluconeogenesis

CD38 is a multifunctional enzyme for the synthesis of Ca2+ second messengers. Glucagon promotes hepatic glucose production through Ca2+ signaling in the fasting condition. In this study, we investigated the role of CD38 in the glucagon signaling of hepatocytes. Here, we show that glucagon induces cyclic ADP-ribose (cADPR) production and sustained Ca2+ increases via CD38 in hepatocytes. 8-Br-cADPR, an antagonistic cADPR analog, completely blocked glucagon-induced Ca2+ increases and phosphorylation of cAMP response element-binding protein (CREB). Moreover, glucagon-induced sustained Ca2+ signals and translocation of CREB-regulated transcription coactivator 2 to the nucleus were absent and glucagon-induced glucose production and expression of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (Pck1) are remarkably reduced in hepatocytes from CD38−/− mice. Furthermore, in the fasting condition, CD38−/− mice have decreased blood glucose and hepatic expression of G6Pase and Pck1 compared to wild type mice. Our data suggest that CD38/cADPR-mediated Ca2+ signals play a key role in glucagon-induced gluconeogenesis in hepatocytes, and that the signal pathway has significant clinical implications in metabolic diseases, including type 2 diabetes.

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.

A novel method for the synthesis of sulfur substituted-cyclopyrophosphate of cADPR analogs

Abstract A facile and efficient protocol for the synthesis of sulfur substituted-cyclopyrophosphate of cIDPRE ( P S 1 -cIDPRE ) was developed. The key step was the cyclization process which was completed by the sulfur substituted cyclization precursor 1b via the one-pot phosphoramidite strategy.

Design and Synthesis of Cyclic ADP‐4‐Thioribose as a Stable Equivalent of Cyclic ADP‐Ribose, a Calcium Ion‐Mobilizing Second Messenger

Design and Synthesis of Cyclic ADP‐4‐Thioribose as a Stable Equivalent of Cyclic ADP‐Ribose, a Calcium Ion‐Mobilizing Second Messenger

Identification of a Novel Pathway of Transforming Growth Factor-β1 Regulation by Extracellular NAD+ in Mouse Macrophages: IN VITRO AND IN SILICO STUDIES

Extracellular β-nicotinamide adenine dinucleotide (NAD(+)) is anti-inflammatory. We hypothesized that NAD(+) would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-β1. Indeed, NAD(+) led to increases in both active and latent cell-associated TGF-β1 in RAW 264.7 mouse macrophages as well as in primary peritoneal macrophages isolated from both C3H/HeJ (TLR4-mutant) and C3H/HeOuJ (wild-type controls for C3H/HeJ) mice. NAD(+) acts partially via cyclic ADP-ribose (cADPR) and subsequent release of Ca(2+). Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca(2+) ionophores recapitulated the effects of NAD(+) on TGF-β1, whereas the cADPR antagonist 8-Br-cADPR, Ca(2+) chelation, and antagonism of L-type Ca(2+) channels suppressed these effects. The time and dose effects of NAD(+) on TGF-β1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-β1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-β1 by NAD(+). Thus, in vitro and in silico evidence points to NAD(+) as a novel modulator of TGF-β1.

Design and synthesis of cADPR analogues with simplified ribose and nucleobase

Design and synthesis of cADPR analogues with simplified ribose and nucleobase

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.

Aberrant Cyclization Affords a C-6 Modified Cyclic Adenosine 5′-Diphosphoribose Analogue with Biological Activity in Jurkat T Cells

Two nicotinamide adenine dinucleotide (NAD+) analogues modified at the 6 position of the purine ring were synthesized, and their substrate properties toward Aplysia californica ADP-ribosyl cyclase were investigated. 6-N-Methyl NAD+ (6-N-methyl nicotinamide adenosine 5′-dinucleotide 10) hydrolyzes to give the linear 6-N-methyl ADPR (adenosine 5′-diphosphoribose, 11), whereas 6-thio NHD+ (nicotinamide 6-mercaptopurine 5′-dinucleotide, 17) generates a cyclic dinucleotide. Surprisingly, NMR correlation spectra confirm this compound to be the N1 cyclic product 6-thio N1-cIDPR (6-thio cyclic inosine 5′-diphosphoribose, 3), although the corresponding 6-oxo analogue is well-known to cyclize at N7. In Jurkat T cells, unlike the parent cyclic inosine 5′-diphosphoribose N1-cIDPR 2, 6-thio N1-cIDPR antagonizes both cADPR- and N1-cIDPR-induced Ca2+ release but possesses weak agonist activity at higher concentration. 3 is thus identified as the first C-6 modified cADPR (cyclic adenosine 5′-diphosphoribose) analogue antagonist; it represents the first example of a fluorescent N1-cyclized cADPR analogue and is a new pharmacological tool for intervention in the cADPR pathway of cellular signaling.

Synthesis and use of cell-permeant cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is a second messenger that acts on ryanodine receptors to mobilize Ca2+. cADPR has a net negative charge at physiological pH making it not passively membrane permeant thereby requiring it to be injected, electroporated or loaded via liposomes. Such membrane impermeance of other charged intracellular messengers (including cyclic AMP, inositol 1,4,5-trisphosphate and nicotinic acid adenine dinucleotide phosphate) and fluorescent dyes (including fura-2 and fluorescein) has been overcome by synthesizing masked analogs (prodrugs), which are passively permeant and hydrolyzed to the parent compound inside cells. We now report the synthesis and biological activity of acetoxymethyl (AM) and butoxymethyl (BM) analogs of cADPR. Extracellular addition of cADPR-AM or cADPR-BM to neuronal cells in primary culture or PC12 neuroblastoma cells induced increases in cytosolic Ca2+. Pre-incubation of PC12 cells with thapsigargin, ryanodine or caffeine eliminated the response to cADPR-AM, whereas the response still occurred in the absence of extracellular Ca2+. Combined, these data demonstrate that masked cADPR analogs are cell-permeant and biologically active. We hope these cell-permeant tools will facilitate cADPR research and reveal its diverse physiological functions.

Concise syntheses of trifluoromethylated cyclic and acyclic analogues of cADPR.

A novel trifluoromethylated analogue of cADPR, 8-CF3-cIDPDE (5) was designed and synthesized via construction of N1,N9-disubstituted hypoxanthine, trifluoromethylation and intramolecular condensation. A series of acyclic analogues of cADPR were also designed and synthesized. These compounds could be useful molecules for studying the structure-activity relationship of cADPR analogues and exploring the cADPR/RyR Ca2+ signalling system.

Role of CD38, a Cyclic ADP-Ribosylcyclase, in Morphine Antinociception and Tolerance

Our previous studies have demonstrated that an increase in intracellular levels of Ca(2+) in neurons is an important component of both the antinociception produced by morphine and morphine's tolerance. The present study tested the hypothesis that the Ca(2+) signaling second messenger, cyclic ADP-ribose (cADPR), derived from CD38 activation participates in morphine antinociception and tolerance. We first showed that morphine's antinociceptive potency was increased by the intracerebroventricular injection of CD38 substrate beta-NAD(+) in mice. Furthermore, morphine tolerance was reversed by intracerebroventricular administration of each of three different inhibitors of the CD38-cADPR-ryanodine receptor Ca(2+) signaling pathway. These inhibitors were the ADP-ribosylcyclase inhibitor nicotinamide, cADPR analog 8-bromo-cADPR, and a large dose of ryanodine (>50 muM) that blocks the ryanodine receptor. In CD38 gene knockout [CD38(-/-)] mice, the antinociceptive action of morphine was found to be less potent compared with wild-type (WT) mice, as measured by tail-flick response, hypothermia assay, and observations of straub tail. However, there was no difference in locomotor activation between CD38(-/-) and WT animals. It was also found that less tolerance to morphine developed in CD38(-/-) mice compared with WT animals. These results indicate that cADRP-ryanodine receptor Ca(2+) signaling associated with CD38 plays an important role in morphine tolerance.

Novel nucleobase-simplified cyclic ADP-ribose analogue: A concise synthesis and Ca2+-mobilizing activity in T-lymphocytes

A purine nucleobase-simplified cyclic ADP ribose (cADPR) analogue was synthesized, in which a 1,2,3-triazole-4-amide was constructed, instead of a purine moiety, and the northern ribose was replaced by an ether strand. Compound exhibits calcium release activity in intact T-lymphocytes and indicates that it is a membrane-permeable cADPR mimic. Thus, the cADPR analogue containing 1,2,3-triazole-4-amide provides a novel template for further designing cADPR analogues and elucidating their structure-activity relationships.

Concise synthesis of novel acyclic analogues of cADPR with an ether chain as the northern moiety

To study the properties of hydrolysates of cyclic adenosine diphosphate ribose (cADPR), a series of novel acyclic analogues of cADPR with an ether chain as the northern moiety and 8-substituted adenine or hypoxanthine as the base moiety were synthesized via an N1 substitution construction, followed by bisphosphorylation, phosphoramidition or pyrophosphorylation. These compounds also provide various precursors for synthesizing cADPR analogues.