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Takayoshi Tsuzuki,
Natsumi Sakaguchi,
Takashi Kudoh,
Satoshi Takano,
Masato Uehara,
Takashi Murayama,
Takashi Sakurai,
Minako Hashii,
Haruhiro Higashida, Karin Weber,
Andreas H Guse,
Tomoshi Kameda,
Takatsugu Hirokawa,
Yasuhiro Kumaki,
Barry V L Potter,
Hayato Fukuda,
Mitsuhiro Arisawa,
Satoshi Shuto
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ABSTRACT: Oh, what a difference an S makes: A thioribose analogue (cADPtR, see scheme) of cyclic ADP-ribose (cADPR) was synthesized that is stable and has structural and electrostatic features similar to those of cADPR. cADPtR is the first stable equivalent of cADPR that is as active as cADPR in various cellular systems, making it useful for investigating Ca(2+) ion-release signaling pathways.
Angewandte Chemie International Edition 05/2013; · 13.45 Impact Factor
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ABSTRACT: 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 Ca(2+) 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.
Journal of Medicinal Chemistry 02/2012; 55(4):1478-89. · 4.80 Impact Factor
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ABSTRACT: The role of the multifunctional enzyme CD38 in formation of the Ca(2+)-mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) was investigated. Gene silencing of CD38 did neither inhibit NAADP synthesis in intact Jurkat T cells nor in thymus or spleen obtained from CD38 knock out mice. In vitro, both NAADP formation by base-exchange and degradation to 2-phospho adenosine diphosphoribose were efficiently decreased. Thus in vivo CD38 appears to be a NAADP degrading rather than a NAADP forming enzyme, perhaps avoiding desensitizing NAADP levels in intact cells.
FEBS letters 11/2011; 585(22):3544-8. · 3.54 Impact Factor
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ABSTRACT: The structural features needed for antagonism at the cyclic ADP-ribose (cADPR) receptor are unclear. Chemoenzymatic syntheses of novel 8-substituted 2'-deoxy-cADPR analogues, including 8-bromo-2'-deoxy-cADPR 7, 8-amino-2'-deoxy-cADPR 8, 8- O-methyl-2'-deoxy-cADPR 9, 8-phenyl-2'-deoxy-cADPR 10 and its ribose counterpart 8-phenyl-cADPR 5 are reported, including improved syntheses of established antagonists 8-amino-cADPR 2 and 8-bromo-cADPR 3. Aplysia californica ADP-ribosyl cyclase tolerates even the bulky 8-phenyl-nicotinamide adenine 5'-dinucleotide as a substrate. Structure-activity relationships of 8-substituted cADPR analogues in both Jurkat T-lymphocytes and sea urchin egg homogenate (SUH) were investigated. 2'-OH Deletion decreased antagonistic activity (at least for the 8-amino series), showing it to be an important motif. Some 8-substituted 2'-deoxy analogues showed agonist activity at higher concentrations, among which 8-bromo-2'-deoxy-cADPR 7 was, unexpectedly, a weak but almost full agonist in SUH and was membrane-permeant in whole eggs. Classical antagonists 2 and 3 also showed previously unobserved agonist activity at higher concentrations in both systems. The 2'-OH group, without effect on the Ca (2+)-mobilizing ability of cADPR itself, is an important motif for the antagonistic activities of 8-substituted cADPR analogues.
Journal of Medicinal Chemistry 04/2008; 51(6):1623-36. · 5.25 Impact Factor
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Takashi Kudoh,
Takashi Murayama,
Minako Hashii,
Haruhiro Higashida,
Takashi Sakurai,
Clarisse Maechling,
Bernard Spiess, Karin Weber,
Andreas H. Guse,
Barry V.L. Potter,
Mitsuhiro Arisawa,
Akira Matsuda,
Satoshi Shuto
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ABSTRACT: We previously developed cyclic ADP-carbocyclic-ribose (cADPcR, 3a) as a stable mimic of cyclic ADP-ribose (cADPR, 1), a Ca2+-mobilizing second messenger. The unsaturated carbocyclic-ribose analogs of cADPR, i.e., 4″,6″-didehydro-cADPcR (8a) and its inosine congener 4″,6″-didehydro-cIDPcR (8b) were newly designed and successfully synthesized using the key intramolecular condensation reaction with S-phenyl phosphorothioate-type substrates. The Ca2+-mobilizing potency of the compounds was examined in sea urchin egg homogenates, NG108-15 neuronal cells, and permeabilized Jurkat T-lymphocytes, which may indicate that 4″,6″-didehydro-cADPcR is the first cADPR analog selectively active in T cells. Acid–base behavior and conformation of 8a were also investigated and compared with those of cADPcR.Graphical abstract
Tetrahedron. 01/2008; 64(41):9754-9765.
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Tetrahedron Letters 01/2008; · 2.68 Impact Factor
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ABSTRACT: A series of nicotinamide hypoxanthine 5'-dinucleotide (NHD+) analogues modified at C-8 (2-5) and 7-deaza-NHD+ were synthesized, and cyclization in the presence of Aplysia ADP-ribosyl cyclase was studied. All 8-substituted NHD+ analogues were converted into their N1-cyclic forms by the enzyme, while in contrast, 7-deaza-NHD+ 17 was hydrolyzed into 7-deazainosine 5'-diphosphoribose (7-deaza-IDPR) 25. Correlations are made showing that the conformation of the NHD+ substrate is the key to successful cyclization. The pharmacological activities of these novel cIDPR derivatives were evaluated in both permeabilized and intact Jurkat T-lymphocytes. The results show that in permeabilized cells both 8-iodo 1g and 8-N3-N1-cIDPR 1d have an activity comparable to that of cADPR, while 8-iodo 1g and 8-phenyl-N1-cIDPR 1c have a small but significant effect in intact cells and can therefore be regarded as membrane-permeant; thus, cIDPR derivatives are emerging as important novel biological tools to study cADPR-mediated Ca2+ release in T-cells.
Journal of Medicinal Chemistry 09/2006; 49(17):5162-76. · 5.25 Impact Factor
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Andreas Gasser,
Günter Glassmeier,
Ralf Fliegert,
Matthias F Langhorst,
Stephan Meinke,
Dörte Hein,
Sylvia Krüger, Karin Weber,
Inka Heiner,
Norman Oppenheimer,
Jürgen R Schwarz,
Andreas H Guse
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ABSTRACT: Stimulation of Jurkat T cells by high concentrations of concanavalin A (ConA) induced an elevation of the endogenous adenosine diphosphoribose (ADPR) concentration and an inward current significantly different from the Ca2+ release-activated Ca2+ current (I(CRAC)). Electrophysiological characterization and activation of a similar current by infusion of ADPR indicated that the ConA-induced current is carried by TRPM2. Expression of TRPM2 in the plasma membrane of Jurkat T cells was demonstrated by reverse transcription-PCR, Western blot, and immunofluorescence. Inhibition of ADPR formation reduced ConA-mediated, but not store-operated, Ca2+ entry and prevented ConA-induced cell death of Jurkat cells. Moreover, gene silencing of TRPM2 abolished the ADPR- and ConA-mediated inward current. Thus, ADPR is a novel second messenger significantly involved in ConA-mediated cell death in T cells.
Journal of Biological Chemistry 03/2006; 281(5):2489-96. · 4.77 Impact Factor
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Takashi Kudoh,
Masayoshi Fukuoka,
Satoshi Ichikawa,
Takashi Murayama,
Yasuo Ogawa,
Minako Hashii,
Haruhiro Higashida,
Svenja Kunerth, Karin Weber,
Andreas H Guse,
Barry V L Potter,
Akira Matsuda,
Satoshi Shuto
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ABSTRACT: We previously developed cyclic ADP-carbocyclic ribose (cADPcR, 2) as a stable mimic of cyclic ADP-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. A series of the N1-ribose modified cADPcR analogues, designed as novel stable mimics of cADPR, which were the 2"-deoxy analogue 3, the 3"-deoxy analogue 4, the 3"-deoxy-2"-O-(methoxymethyl) analogue 5, the 3"-O-methyl analogue 6, the 2",3"-dideoxy analogue 7, and the 2",3"-dideoxydidehydro analogue 8, were successfully synthesized using the key intramolecular condensation reaction with phenylthiophosphate-type substrates. We investigated the conformations of these analogues and of cADPR and found that steric repulsion between both the adenine and N9-ribose moieties and between the adenine and N1-ribose moieties was a determinant of the conformation. The Ca(2+)-mobilizing effects were evaluated systematically using three different biological systems, i.e., sea urchin eggs, NG108-15 neuronal cells, and Jurkat T-lymphocytes. The relative potency of Ca(2+)-mobilization by these cADPR analogues varies depending on the cell-type used: e.g., 3"-deoxy-cADPcR (4) > cADPcR (2) > cADPR (1) in sea urchin eggs; cADPR (1) > cADPcR (2) approximately 3"-deoxy-cADPcR (4) in T-cells; and cADPcR (2) > cADPR (1) > 3"-deoxy-cADPcR (4) in neuronal cells, respectively. These indicated that the target proteins and/or the mechanism of action of cADPR in sea urchin eggs, T-cells, and neuronal cells are different. Thus, this study represents an entry to cell-type selective cADPR analogues, which can be used as biological tools and/or novel drug leads.
Journal of the American Chemical Society 06/2005; 127(24):8846-55. · 9.91 Impact Factor
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ABSTRACT: Cyclic ADP-ribose (cADPR) is an endogenous Ca(2+)-mobilizing second messenger in many cell types and organisms. Although the biological activity of several modified analogues of cADPR has been analyzed, most of these structures were still very similar to the original molecule. Recently, we have introduced simplified analogues in which the northern ribose (N(1)-linked ribose) was replaced by an ether strand. Here we also demonstrate that the southern ribose (N(9)-linked ribose) can be replaced by an ether strand resulting in N(1)-[(phosphoryl-O-ethoxy)-methyl]-N(9)-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthinecyclic pyrophosphate (cIDP-DE). This minimal structural analogue of cyclic ADP-ribose released Ca(2+) from intracellular stores of permeabilized Jurkat T lymphocytes. In intact T lymphocytes initial subcellular Ca(2+) release events, global Ca(2+) release, and subsequent global Ca(2+) entry were observed. Cardiac myocytes freshly prepared from mice responded to cIDP-DE by increased recruitment of localized Ca(2+) signals and by global Ca(2+) waves.
Journal of Biological Chemistry 05/2005; 280(16):15952-9. · 4.77 Impact Factor
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ABSTRACT: Cyclic ADP-ribose (cADPR) is an endogenous Ca2+-mobilizing second messenger in many cell types and organisms. Although the biological activity of several modified analogues
of cADPR has been analyzed, most of these structures were still very similar to the original molecule. Recently, we have introduced
simplified analogues in which the northern ribose (N1-linked ribose) was replaced by an ether strand (Gu, X., Yang, Z., Zhang, L., Kunerth, S., Fliegert, R., Weber, K., Guse,
A. H., and Zhang, L. (2004) J. Med. Chem. 47, 5674–5682). Here we also demonstrate that the southern ribose (N9-linked ribose) can be replaced by an ether strand resulting in N1-[(phosphoryl-O-ethoxy)-methyl]-N9-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthinecyclic pyrophosphate (cIDP-DE). This minimal structural analogue of cyclic ADP-ribose released
Ca2+ from intracellular stores of permeabilized Jurkat T lymphocytes. In intact T lymphocytes initial subcellular Ca2+ release events, global Ca2+ release, and subsequent global Ca2+ entry were observed. Cardiac myocytes freshly prepared from mice responded to cIDP-DE by increased recruitment of localized
Ca2+ signals and by global Ca2+ waves.
Journal of Biological Chemistry 04/2005; 280(16):15952-15959. · 4.77 Impact Factor
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ABSTRACT: N1-[(5' '-O-Phosphorylethoxy)methyl]-5'-O-phosphorylinosine 5',5''-cyclicpyrophosphate (cIDPRE 2a) and the 8-substituted derivatives 8-bromo-, 8-azido-, 8-amino-, and 8-Cl-cIDPRE (2b-e) were synthesized from N1-[(5''-acetoxyethoxy)methyl]-2',3'-O-isopropylideneinosine (5) in good yields. The pharmacological activities of cIDPRE and the 8-substituted derivatives (2a-e) were analyzed in intact and permeabilized human Jurkat T-lymphocytes. The results indicate that cIDPRE permeates the plasma membrane, releases Ca2+ from an intracellular, cADPR-sensitive Ca2+ store, and subsequently initiates Ca2+ release-activated Ca2+ entry. The Ca(2+)-releasing activity of cIDPRE was confirmed directly in permeabilized cells. Using time-resolved confocal Ca2+ imaging at the single cell level, the development of global Ca2+ signals starting from local small Ca2+ signals evoked by cIDPRE was observed. 8-N3-cIDPRE 2c and 8-NH2-cIDPRE 2d were similarly effective in their agonistic activity as compared to cIDPRE 2a, showing almost indistinguishable concentration-response curves for 2a, 2c, and 2d and very similar kinetics of Ca2+ signaling. In contrast, the halogenated derivatives 8-Br- and 8-Cl-cIDPRE (2b and 2e) did not significantly elevate [Ca2+]i. Therefore, cIDPRE 2a, 8-N3-cIDPRE 2c, and 8-NH2-cIDPRE 2d are novel membrane permeant cADPR mimic and may provide important novel tools to study cADPR-mediated Ca2+ signaling in intact cells.
Journal of Medicinal Chemistry 12/2004; 47(23):5674-82. · 5.25 Impact Factor
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ABSTRACT: In Jurkat T cells, the type 3 ryanodine receptor (RyR) was knocked-down by stable integration of plasmid expressing type 3 ryanodine receptor antisense RNA. Stable integration of the antisense plasmid in individual clones was demonstrated by PCR of genomic DNA, expression of antisense RNA by reverse transcriptase PCR, and efficiently reduced expression of type 3 ryanodine receptor protein by Western blot. Selected clones were successfully used to analyze T cell receptor/CD3 complex-mediated Ca(2+) signaling. Reduced expression of the type 3 RyR resulted in (i) significantly decreased Ca(2+) signaling in the sustained phase and (ii) in permeabilized cells in a significantly impaired response toward cyclic ADP-ribose but not to d-myo-inositol 1,4,5-trisphosphate. For the first time, the role of the type 3 RyR in sustained Ca(2+) signaling was directly visualized by confocal Ca(2+) imaging as a significant contribution to the number and the magnitude of subcellular Ca(2+) signals. These data suggest that the type 3 ryanodine receptor is essential in the sustained Ca(2+) response in T cells.
Journal of Biological Chemistry 01/2003; 277(52):50636-42. · 4.77 Impact Factor
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Andreas H Guse,
Céline Cakir-Kiefer,
Masayoshi Fukuoka,
Satoshi Shuto, Karin Weber,
Victoria C Bailey,
Akira Matsuda,
Georg W Mayr,
Norman Oppenheimer,
Francis Schuber,
Barry V L Potter
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ABSTRACT: Three novel analogues modified in the "northern" ribose (ribose linked to N1 of adenine) of the Ca(2+) mobilizing second messenger cyclic adenosine diphosphoribose, termed 2"-NH(2)-cyclic adenosine diphosphoribose, cyclic adenosine diphospho-carbocyclic-ribose, and 8-NH(2)-cyclic adenosine diphospho-carbocyclic-ribose, were synthesized (chemoenzymatically and by total synthesis) and spectroscopically characterized, and the pK(a) values for the 6-amino/imino transition were determined in two cases. The biological activity of these analogues was determined in permeabilized human Jurkat T-lymphocytes. 2"-NH(2)-cyclic adenosine diphosphoribose mediated Ca(2+) release was slightly more potent than that of the endogenous cyclic adenosine diphosphoribose in terms of the concentration-reponse relationship. Both compounds released Ca(2+) from the same intracellular Ca(2+) pool. In addition, the control compound 2"-NH(2)-adenosine diphosphoribose was almost without effect. In contrast, only at much higher concentrations (> or =50 microM) did the "northern" carbocyclic analogue, cyclic adenosine diphospho-carbocyclic-ribose, significantly release Ca(2+) from permeabilized T cells, whereas the previously reported "southern" carbocyclic analogue, cyclic aristeromycin diphosphoribose, was slightly more active than the endogenous cyclic adenosine diphosphoribose. Likewise, 8-NH(2)-cyclic adenosine diphospho-carbocyclic-ribose, expected to antagonize Ca(2+) release as demonstrated previously for 8-NH(2)-cyclic adenosine diphosphoribose, did not inhibit cyclic adenosine diphosphoribose mediated Ca(2+) release. This indicates that the 2"-NH(2)-group substitutes well for the 2"-OH-group it replaces; it may be oriented toward the outside of the putative cyclic adenosine diphosphoribose receptor binding domain and/or it can potentially also engage in H bonding interactions with residues of that domain. In sharp contrast to this, replacement of the endocyclic furanose oxygen atom by CH(2) in a carbocyclic system obviously interferes with a crucial element of interaction between cyclic adenosine diphosphoribose and its receptor in T-lymphocytes.
Biochemistry 06/2002; 41(21):6744-51. · 3.42 Impact Factor
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ABSTRACT: cADP-ribose (cADPr) has recently been shown to release Ca2+ from an intracellular store of permeabilized T lymphocyte cell lines (Guse, A. H., da Silva, C. P., Emmrich, F., Ashamu,
G. A., Potter, B. V. L., and Mayr, G. W. (1995) J. Immunol. 155, 3353-3359). Using permeabilized Jurkat and HPB.ALL T lymphocytes, the effects of varying concentrations of inorganic
phosphate and Mg2+ on cADPr-induced Ca2+ release were investigated. cADPr-induced Ca2+ release was dependent on the concentration of inorganic phosphate, showing very low Ca2+ release activity between 0.5 and 2 mM inorganic phosphate. At 4 to 5 mM inorganic phosphate, the cADPr-induced Ca2+ release was much more pronounced, reaching maximal values at 10 mM inorganic phosphate. The underlying mechanism for this stimulatory effect was an increased loading of the cADPr-sensitive
Ca2+ store, which was demonstrated by enhanced resequestration of Ca2+ selectively into the cADPr-sensitive Ca2+ store. The free Mg2+ concentration also influenced cADPr-induced Ca2+ release in permeabilized cells: at 0 and 8.58 mM the release was nearly completely abolished, whereas at 1.06 mM maximal Ca2+ release by cADPr was observed. High performance liquid chromatographic analysis of exogenously added cADPr revealed that
the catabolism of cADPr at varying Mg2+ and Pi concentrations had only minor relevance for the modulatory effects observed.
To correlate the effects of inorganic phosphate and Mg2+ on cADPr-induced Ca2+ release observed in the permeabilized cell preparations, measurements of these ions in intact Jurkat T lymphocytes were carried
out. Intact Jurkat T cells stimulated via the T cell receptor·CD3 complex did not respond with significant elevation of the
free intracellular Mg2+ concentration. In contrast, stimulation via the T cell receptor·CD3 complex resulted in an increase in the intracellular
inorganic phosphate concentration. These data indicate a role for the intracellular inorganic phosphate concentration in the
regulation of cADPr-mediated Ca2+ release in T lymphocytes.
Journal of Biological Chemistry 09/1996; 271(39):23946-23953. · 4.77 Impact Factor
