Simplification of adenophostin A defines a minimal structure for potent glucopyranoside-based mimics of D-myo-inositol 1,4,5-trisphosphate

The synthesis of 1-O-[(3S,4R)-3-hydroxytetrahydrofuran-4-yl]-alpha-D-glucopyranosid e 3,4,3'-trisphosphate (7), a novel Ca2+ mobilising agonist at the Ins(1,4,5)P3 receptor, designed by excision of two motifs of adenophostin A is reported, defining a potential minimal structure for potent glucopyranoside-based agonists of Ins(1,4,5)P3 receptors.     

Inframolecular studies of the protonation of adenophostin A: comparison with 1-D-myo-inositol 1,4,5-trisphosphate

Adenophostin A is a glyconucleotide natural product with the highest known potency for the D-myo-inositol 1,4,5-trisphosphate receptor. Using synthetic adenophostin A we have investigated the macroscopic and microscopic protonation process of this compound by performing (31)P NMR, (1)H NMR, and potentiometric titration experiments. The logarithms of the first to the fourth stepwise protonation constants are, respectively, log K(1) = 8.48, log K(2) = 6.20, log K(3) = 4.96, and log K(4) = 3.80. The latter constant refers to the protonation equilibrium involving the N1 adenine nitrogen. From the microconstants the protonation fractions of each individual phosphate group can be calculated. Remarkably, the ionization state of the phosphates of adenophostin A at near physiological pH is very similar to those of inositol 1,4,5-trisphosphate, indicating that differences in phosphate charge cannot account for the high potency of this molecule. The analysis of the (1)H chemical shifts vs pH provided complementary conformational information. In particular, a slight "wrongway shift" of H1" can be related to the protonation of P2, thus indicating a short H1"-P2 distance. Our results are in line with a recently published model in which, however, a certain degree of constraint would keep the ribose 2'-phosphate moiety close to the glucose ring phosphates.     

Xylopyranoside-based agonists of D-myo-inositol 1,4,5-trisphosphate receptors: synthesis and effect of stereochemistry on biological activity

The synthesis of a series of tetrahydrofuranyl alpha- and beta-xylopyranoside trisphosphates, designed by excision of three motifs of adenophostin A is reported. The synthetic route features improved preparations of allyl alpha-D-xylopyranoside and its 2-O-benzyl ether, and gives access to four diastereoisomeric trisphosphates, which show a range of abilities to mobilise Ca2+ from the intracellular stores of hepatocytes. A comparison of the potencies of the four trisphosphates provides useful information relating to the effects of stereochemical variation on the recognition of carbohydrate-based trisphosphates by D-myo-inositol 1,4,5-trisphosphate receptors. 1-O-[(3'S,4'R)-3-hydroxytetrahydrofuran-4-yl] alpha-D-xylopyranoside 3,4,3'-trisphosphate (8) is the most active member of the series with a potency close to Ins(1,4,5)P3; a beta-linked analogue, 1-O-[(3'R,4'S)-3-hydroxytetrahydrofuran-4-yl] beta-D-xylopyranoside 3,4,3'-trisphosphate, is ca. 20-fold weaker than Ins(1,4,5)P3, and the other compounds are much less active. While no compound attained a potency close to that of adenophostin A, we believe that 8 represents the minimal structure for potent Ca2+-releasing activity in this type of carbohydrate-based analogue.     

Formation and biological action of inositol 1,4,5-trisphosphate

A wide variety of receptors appear to be coupled to a phospholipase C (EC 3.1.4.3) that hydrolyzes inositol lipids. This reaction is believed to provide a link between receptor activation and cellular Ca2+ mobilization. The mechanisms by which this occurs are believed to involve inositol 1,4,5-trisphosphate (1,4,5-IP3), which signals release of Ca2+ from the endoplasmic reticulum. In rat parotid acinar cells made permeable with saponin, 1,4,5-IP3 induced rapid release of sequestered Ca2+. In intact parotid cells, the concentration-response relationship for methacholine-induced IP3 formation was similar to the relationship for muscarinic receptor occupancy by methacholine. About 10-fold lower concentrations of methacholine were sufficient to increase cytosolic [Ca2+] and to activate secretion, indicating an excess IP3 forming capacity for the muscarinic receptor. The mechanisms for the coupling of receptors to IP3 formation were studied in pancreatic acinar cells made permeable electrically. In this preparation, nonhydrolyzable derivatives of GTP potentiated agonist-induced IP3 production, which suggests the involvement of a guanine nucleotide-dependent regulatory protein. The effects of agonists and guanine nucleotides were not altered by pretreating the acinar cells with cholera or pertussis toxins, which indicated that the regulatory protein linking receptors to IP3 formation is distinct from the ones involved in the regulation of adenylate cyclase.     

Molecular recognition of adenophostin, a very potent Ca2+ inducer, at the D-myo-inositol 1,4,5-trisphosphate receptor.

The recognition mode of adenophostin A at the D-myo-inositol 1,4, 5-trisphosphate [Ins(1,4,5)P(3)] receptor was investigated. Comparison of conformations of Ins(1,4,5)P(3) and adenophostin A by using the combination of NMR and molecular mechanics (MM) calculations demonstrated that adenophostin A adopted a moderately extended conformation regarding the distance between the 2'-phosphoryl group and the 3' ',4' '-bisphosphate motif, as suggested previously [Wilcox, R. A. et al. (1995) Mol. Pharmacol. 47, 1204-1211]. Based on the nuclear Overhauser effect (NOE) observed between 3'-H and 1' '-H and on MM calculations, the molecular shape of adenophostin A proved to be an extended form at least in solution, in contrast to Wilcox's compactly folded, preliminary hairpin model. GlcdR(2,3',4')P(3), an adenophostin analogue without adenine moiety, was found to be less potent than adenophostin A and almost equipotent to Ins(1,4,5)P(3). We propose the possibility that (i) the optimal spatial arrangement of the three phosphoryl groups and/or (ii) the interaction of the adenine moiety of adenophostin A with the putative binding site, if it exists in the vicinity of the Ins(1,4,5)P(3)-binding site, might account for the exceptional potency of adenophostin A.     

ATP-dependent adenophostin activation of inositol 1,4,5-trisphosphate receptor channel gating: kinetic implications for the durations of calcium puffs in cells

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) is a ligand-gated intracellular Ca(2+) release channel that plays a central role in modulating cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)). The fungal metabolite adenophostin A (AdA) is a potent agonist of the InsP(3)R that is structurally different from InsP(3) and elicits distinct calcium signals in cells. We have investigated the effects of AdA and its analogues on single-channel activities of the InsP(3)R in the outer membrane of isolated Xenopus laevis oocyte nuclei. InsP(3)R activated by either AdA or InsP(3) have identical channel conductance properties. Furthermore, AdA, like InsP(3), activates the channel by tuning Ca(2+) inhibition of gating. However, gating of the AdA-liganded InsP(3)R has a critical dependence on cytoplasmic ATP free acid concentration not observed for InsP(3)-liganded channels. Channel gating activated by AdA is indistinguishable from that elicited by InsP(3) in the presence of 0.5 mM ATP, although the functional affinity of the channel is 60-fold higher for AdA. However, in the absence of ATP, gating kinetics of AdA-liganded InsP(3)R were very different. Channel open time was reduced by 50%, resulting in substantially lower maximum open probability than channels activated by AdA in the presence of ATP, or by InsP(3) in the presence or absence of ATP. Also, the higher functional affinity of InsP(3)R for AdA than for InsP(3) is nearly abolished in the absence of ATP. Low affinity AdA analogues furanophostin and ribophostin activated InsP(3)R channels with gating properties similar to those of AdA. These results provide novel insights for interpretations of observed effects of AdA on calcium signaling, including the mechanisms that determine the durations of elementary Ca(2+) release events in cells. Comparisons of single-channel gating kinetics of the InsP(3)R activated by InsP(3), AdA, and its analogues also identify molecular elements in InsP(3)R ligands that contribute to binding and activation of channel gating.     

Adenine-nucleotide binding sites on the inositol 1,4,5-trisphosphate receptor bind caffeine, but not adenophostin A or cyclic ADP-ribose.

Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP3R) results in a more pronounced Ca2+ release in the presence of inositol 1,4,5-trisphosphate (IP3). We have expressed the cDNAs encoding two putative adenine-nucleotide binding sites of the neuronal form of IP3R-1 as glutathione S-transferase (GST)-fusion proteins in bacteria. Specific [alpha-32P]ATP binding was observed for the two GST-fusion proteins, representing aa 1710-1850 and aa 1944-2040 of IP3R-1. The ATP-binding sites in both fusion proteins had the same nucleotide specificity as found for the intact IP3R (ATP > ADP > AMP > GTP). Smaller GST-fusion proteins (aa 1745-1792 and aa 2005-2023) displayed a much weaker ATP-binding activity. CoA, which also potentiated IP3-induced Ca2+ release in A7r5 cells, interacted with the ATP-binding sites on the fusion proteins. Such interaction was not observed for 1,N6-etheno CoA and 3'-dephospho-CoA, which are much less effective in potentiating IP3-induced Ca2+ release. Since the adenine-containing compounds adenophostin A, caffeine and cyclic ADP-ribose modulate IP3-induced Ca2+ release, a possible effect of these compounds on the ATP-binding sites was examined. ATP stimulated adenophostin A- and IP3-induced Ca2+ release in A7r5 cells with an EC50 of respectively 21 and 20 microM. Also the threshold concentration of ATP for stimulating the release was similar for the two agonists. Adenophostin A (100 microM) and cyclic ADP-ribose (100 microM) were ineffective in displacing [alpha-32P]ATP from the binding sites of both GST-fusion proteins. Caffeine (50 mM), however, inhibited [alpha-32P]ATP binding to both fusion proteins by more than 50%. These data provide evidence for a direct interaction of caffeine but not of adenophostin A or cyclic ADP-ribose with the adenine-nucleotide binding sites of the IP3R.     

Characterization of a biological detector cell for quantitation of inositol 1,4,5-trisphosphate

Prior strategies to measure inositol 1,4,5-trisphosphate (IP(3)) in single cells either have been qualitative or have had a limited spatial resolution. Capillary electrophoresis combined with a biological detector cell has been used to quantitate IP(3) in small regions of a Xenopus oocyte. To improve the detection limits of this method, we elucidated the experimental parameters which influenced the sensitivity and reliability of the IP(3)-detector cell coupled to capillary electrophoresis. The variables which influenced the detector cell were the magnitude of the voltage drop across the detector cell, the duration of this voltage drop, the direction of fluid flow in the capillary, the concentration of free Ca(2+) around the detector cell, and the presence of protease inhibitors during permeabilization of the detector cell. For the sample volumes imposed by the capillary diameter, the detector cell acted primarily as an IP(3) mass detector rather than a concentration detector. Characterization of the experimental variables influencing the sensitivity and reliability of this detector cell has the potential to enhance other analyte measurements performed by mating capillary electrophoresis with a biological detector cell.     

Comparative studies of D-myoinositol 1,4,5-trisphosphate and synthetic 6-deoxy D-myoinositol 1,4,5-trisphosphate: binding and calcium release activity in rat parotid microsomes.

In this study, we report our data on the binding of D-myoinositol(1,4,5)P3 and of 6-deoxy D-myoinositol(1,4,5)P3 to a rat parotid microsomal fraction and their effect on Ca2+ release. The binding affinity and the potency of 6-deoxy Ins(1,4,5)P3 to induce Ca2+ release are about 100 times lower than those of Ins(1,4,5)P3. However, maximal concentrations of both inositol trisphosphates induce similar calcium efflux and present comparable displacement of radioligand binding. Experiments were performed to exclude that the microsomal preparations used display rapid metabolism of Ins(1,4,5)P3 or 6-deoxy Ins(1,4,5)P3 during binding and Ca2+ release. We also report that, in permeabilized rat parotid acini preparations, 6-deoxy Ins(1,4,5)P3 is about 100 times less potent than Ins(1,4,5)P3 in inducing Ca2+ release. These data indicate that removal of the hydroxyl group in position 6 of the Ins(1,4,5)P3 molecule severely reduces its binding affinity which seems, in a large part at least, responsible for the reported loss of potency in mobilizing Ca2+. Nevertheless, 6-deoxy Ins(1,4,5)P3 seems to be a full agonist for the release of Ca2+.     

Curcumin: a new cell-permeant inhibitor of the inositol 1,4,5-trisphosphate receptor

Curcumin (diferuoylmethane or 1,7-bis (4-hydroxy-3-methoxyphenol)-1,6-hepatadiene-3,5-dione) is the active ingredient of the spice turmeric. Curcumin has been shown to have a number of pharmacological and therapeutic uses. This study shows that curcumin is a potent inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca2+ channel (InsP3 receptor). In porcine cerebellar microsomes, the extent of InsP3-induced Ca2+ release (IICR) is almost completely inhibited by 50 microM curcumin (IC50 = 10 microM). As the extent of IICR cannot be restored back to control levels by the addition of excess InsP3 and since it has little effect on [3H]InsP3 binding to cerebellar microsomes, this inhibition is likely to be non-competitive in nature. IICR in cerebellar microsomes is biphasic consisting of a fast and slow component. The rate constants for the two components are both reduced by curcumin to similar extents (by about 70% of control values at 40 microM curcumin). In addition, curcumin also reduces agonist (ATP)-stimulated Ca2+ mobilization from intact HL-60 cells, indicating that curcumin is cell permeant. However, since it also affects intracellular Ca2+ pumps and possibly ryanodine receptors, it may lead to complex Ca2+ transient responses within cells, which may well explain some of its putative therapeutic properties.     

Calcium and inositol 1,4,5-trisphosphate receptors: a complex relationship.

Increases in intracellular free Ca2+ concentration ([Ca2+]i), whether initiated by changes in plasma membrane potential or receptor-stimulated polyphosphoinositide hydrolysis, can be astonishingly complex, often occurring as repetitive Ca2+ spikes and regenerative Ca2+ waves that propagate through the cell and sometimes into neighbouring cells. The key to understanding these complex Ca2+ signals lies in understanding the interactions between the different pools from which Ca2+ can rapidly enter the cytosol and the activities of the various Ca(2+)-transporting systems that reverse the process.     

Synthesis of D- and L-myo-inositol 2,4,5-trisphosphate and trisphosphorothioate: structural analogues of D-myo-inositol 1,4,5-trisphosphate

The preparation of D- and L-myo-inositol 2,4,5-trisphosphate is described, together with the phosphorothioate counterparts. The known chiral diols D- and L-1,4-di-O-benzyl-5,6-bis-O-p-methoxybenzyl-myo-inositol were regioselectively protected at the 3-position using a benzyl group via a 2,3-O-stannylene acetal. Removal of the p-methoxybenzyl groups of each enantiomer gave D- and L-1,3,6-tri-O-benzyl-myo-inositol. Phosphitylation with bis(benzyloxy)diisoproplyaminophosphine and 1H-tetrazole gave the trisphosphite intermediate for each enantiomer. Oxidation with 3-chloroperoxybenzoic acid gave the fully protected D- and L-myo-inositol 2,4,5-trisphosphates. Sulphoxidation of the D- and L-2,4,5-trisphosphite intermediates gave the fully protected D- and L-myo-inositol 2,4,5-trisphosphorothioate compounds. The fully protected trisphosphates were deblocked using hydrogenolysis and the phosphorothioates were deprotected using sodium in liquid ammonia. The individual compounds were then purified using ion exchange chromatography to afford pure D- and L-myo-inositol 2,4,5-trisphosphates together with the corresponding phosphorothioates.     

Isoforms of the inositol 1,4,5-trisphosphate receptor are expressed in bovine oocytes and ovaries: the type-1 isoform is down-regulated by fertilization and by injection of adenophostin A.

Mammalian fertilization is characterized by the presence of long-lasting intracellular calcium ([Ca2+]i) oscillations that are required to induce oocyte activation. One of the Ca2+ channels that may mediate this Ca2+ release is the inositol 1,4, 5-trisphosphate receptor (IP(3)R). Three isoforms of the receptor have been described, but their expression in oocytes and possible roles in mammalian fertilization are not well known. Using isoform-specific antibodies against IP(3)R types 1, 2, and 3 and Western analysis, we determined the isoforms that are expressed in bovine metaphase II oocytes and ovaries. In oocytes, all isoforms are expressed, but type 1 is present in overwhelmingly larger amounts and is likely responsible for the majority of Ca2+ release at fertilization. In ovarian microsomes, all three isoforms appear well expressed, suggesting the participation of all IP(3)R isoforms in ovarian Ca2+ signaling. We then investigated whether the reported cessation/reduction in amplitude of fertilization-associated [Ca2+]i oscillations, which is observed as pronuclear formation approaches, corresponded with down-regulation of the IP(3)R-1 isoform. Fertilization resulted in approximately 40% reduction in the amount of receptor by 16 h postinsemination. In addition, injection of adenophostin A, a potent IP(3)R agonist that elicits high-frequency [Ca2+]i oscillations in mammalian oocytes, induced similar reduction in receptor numbers. Together, these data show that 1) the three IP(3)R isoforms are expressed in bovine oocytes; 2) IP(3)R-1 is likely to mediate most of the Ca2+ release during fertilization; 3) its down-regulation may explain the decline in amplitude of sperm-induced [Ca2+]i rises as fertilization progresses toward pronuclear formation; and 4) agonists of the IP(3)R induce down-regulation of the type-1 receptor in oocytes similar to that evoked by fertilization.     

Expression of a truncated form of inositol 1,4,5-trisphosphate receptor type III in the cytosol of DT40 triple inositol 1,4,5-trisphosphate receptor-knockout cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel playing a major role in Ca2+ signaling. Three isoforms of IP3R have been identified and most cell types express different proportions of each isoform. The DT40 B lymphocyte cell line lacking all three IP3R isoforms (DT40IP3R-KO cells) represents an excellent model to re-express any recombinant IP3R and analyze its specific properties. In the study presented here, we confirmed that DT40IP3R-KO cells do not express any IP3-sensitive Ca2+ release channel. However, with an immunoblot approach and a [3H]IP3 binding approach we demonstrated the presence of a C-terminally truncated form of IP3R type III in the cytosolic fraction of DT40IP3R-KO cells. We further showed that this truncated IP3R retained the ability to couple to the Ca2+ entry channel TRPC6. Therefore, a word of caution is offered about the interpretation of results obtained in using DT40IP3R-KO cells to study the cellular mechanisms of Ca2+ entry.     

Comparison of and chromogranin effect on inositol 1,4,5-trisphosphate sensitivity of cytoplasmic and nucleoplasmic inositol 1,4,5-trisphosphate receptor/Ca2+ channels

The nucleus also contains the inositol 1,4,5-trisphosphate receptor (IP3R)/Ca2+ channels in the nucleoplasm proper independent of the nuclear envelope or the cytoplasm. The nuclear IP3R/Ca2+ channels were shown to be present in small IP3-dependent nucleoplasmic Ca2+ store vesicles, yet no information is available regarding the IP3 sensitivity of nuclear IP3R/Ca2+ channels. Here, we show that nuclear IP3R/Ca2+ channels are 3-4-fold more sensitive to IP3 than cytoplasmic ones in both neuroendocrine PC12 cells and nonneuroendocrine NIH3T3 cells. Given the presence of phosphoinositides and phospholipase C and the importance of IP3-mediated Ca2+ signaling in the nucleus, the high IP3 sensitivity of nuclear IP3R/Ca2+ channels seemed to reflect the physiological needs of the nucleus to finely control the IP3-dependent Ca2+ concentrations. It was further shown that the IP3R/Ca2+ channels of secretory cells are 7-8-fold more sensitive to IP3 than those of nonsecretory cells. This difference appeared to result from the presence of secretory cell marker protein chromogranins (thus secretory granules) in secretory cells; expression of chromogranins in NIH3T3 cells increased the IP3 sensitivity of both nuclear and cytoplasmic IP3R/Ca2+ channels by approximately 4-6-fold. In contrast, suppression of chromogranin A expression in PC12 cells changed the EC50 of IP3 sensitivity for cytoplasmic IP3R/Ca2+ channels from 17 to 47 nM, whereas suppression of chromogranin B expression changed the EC50 of cytoplasmic IP3R/Ca2+ channels from 17 to 102 nM and the nuclear ones from 4.3 to 35 nM. Given that secretion is the major function of secretory cells and is under a tight control of intracellular Ca2+ concentrations, the high IP3 sensitivity appears to reflect the physiological roles of secretory cells.     

Structure and expression of the rat inositol 1,4,5-trisphosphate receptor

The complete primary structure of the inositol 1,4,5-trisphosphate receptor from rat brain was elucidated using a series of overlapping cDNA clones. Two different sets of clones that either contain or lack a 45-nucleotide sequence in the amino-terminal third of the protein were isolated, suggesting a differential splicing event that results in the biosynthesis of either a 2734- or 2749-amino acid receptor protein. Hydrophobicity analysis demonstrates the presence of a cluster of hydrophobic sequences in the carboxyl-terminal third of the protein that probably comprise eight transmembrane regions and that may form the calcium channel intrinsic to the receptor. The receptor was universally expressed at low levels in all tissues and cultured cells tested. Transfection of a full-length expression construct of the inositol 1,4,5-trisphosphate receptor into COS cells resulted in the biosynthesis of a 260-kDa protein that bound inositol 1,4,5-trisphosphate and formed high molecular weight complexes similar to the native receptor as analyzed by sucrose gradient centrifugations. On the other hand, the protein product synthesized by a mutant receptor construct in which the amino-terminal 418 amino acids were deleted failed to bind inositol 1,4,5-trisphosphate. The mutant receptor still formed high molecular weight complexes, suggesting that it folded normally and that the amino-terminal sequences of the receptor are part of the ligand binding domain.     

Synthesis and application of photoaffinity analogues of inositol 1,4,5-trisphosphate selectively substituted at the 1-phosphate group.

We have synthesized two photolabile arylazido-analogues of Ins(1,4,5)P3 selectively substituted at the 1-phosphate group for determination of Ins(1,4,5)P3-binding proteins. These two photoaffinity derivatives, namely N-(4-azidobenzoyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AbaIP3) and N-(4-azidosalicyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AsaIP3), bind to high affinity Ins(1,4,5)P3-specific binding sites at a 9-fold lower affinity (Kd = 66 and 70 nM) than Ins(1,4,5)P3 (Kd = 7.15 nM) in a fraction from rat pancreatic acinar cells enriched in endoplasmic reticulum (ER). Other inositol phosphates tested showed comparable (DL-myo-inositol 1,4,5-trisphosphothioate, Kd = 81 nM) or much lower affinities for the binding sites [Ins(1,3,4,5)P4, Kd = 4 microM; Ins(1,4)P2, Kd = 80 microM]. Binding of AbaIP3 was also tested on a microsomal preparation of rat cerebellum [Kd = 300 nM as compared with Ins(1,4,5)P3, Kd = 45 nM]. Ca2+ release activity of the inositol derivatives was tested with AbaIP3. It induced a rapid and concentration-dependent Ca2+ release from the ER fraction [EC50 (dose producing half-maximal effect) = 3.1 microM] being only 10-fold less potent than Ins(1,4,5)P3 (EC50 = 0.3 microM). From the two radioactive labelled analogues ([3H]AbaIP3 and 125I-AsIP3) synthesized, the radioiodinated derivative was used for photoaffinity labelling. It specifically labelled three proteins with apparent molecular masses of 49, 37 and 31 kDa in the ER-enriched fraction. By subfractionation of this ER-enriched fraction on a Percoll gradient the 37 kDa Ins(1,4,5)P3 binding protein was obtained in a membrane fraction which showed the highest effect in Ins(1,4,5)P3-inducible Ca2+ release (fraction P1). The other two Ins(1,4,5)P3-binding proteins, of 49 and 31 kDa, were obtained in fraction P2, in which Ins(1,4,5)P3-induced Ca2+ release was half of that obtained in fraction P1. We conclude from these data that the 37 kDa and/or the 49 and 31 kDa proteins are involved in Ins(1,4,5)P3-induced Ca2+ release from the ER of rat pancreatic acinar cells.     

Total synthesis of L-2,2-difluoro-2-deoxy-MYO-inositol 1,4,5-trisphosphate, a potent inhibitor of the enzymes of d-MYO-inositol 1,4,5-trisphosphate metabolism

The synthesis of the enantiomers of 2,2-difluoro-2-deoxy-myo-inositol 1,4,5-trisphosphate is reported. L-2,2-difluoro-2-deoxy-myo-inositol 1,4,5-trisphosphate is a potent inhibitor of 3-kinase and 5-phosphatase.     

The inositol 1,4,5-trisphosphate receptor (Itpr) gene family in Xenopus: identification of type 2 and type 3 inositol 1,4,5-trisphosphate receptor subtypes

Studies in the Xenopus model system have provided considerable insight into the developmental role of intracellular Ca2+ signals produced by activation of IP3Rs (inositol 1,4,5-trisphosphate receptors). However, unlike mammalian systems where three IP3R subtypes have been well characterized, our molecular understanding of the IP3Rs that underpin Ca2+ signalling during Xenopus embryogenesis relate solely to the original characterization of the 'Xenopus IP3R' cloned and purified from Xenopus laevis oocytes several years ago. In the present study, we have identified Xenopus type 2 and type 3 IP3Rs and report the full-length sequence, genomic architecture and developmental expression profile of these additional IP3R subtypes. In the light of the emerging genomic resources and opportunities for genetic manipulation in the diploid frog Xenopus tropicalis, these data will facilitate manipulations to resolve the contribution of IP3R diversity in Ca2+ signalling events observed during vertebrate development.     

Synthesis of three enantiomeric pairs of scyllo-inositol phosphate and molecular interactions between all possible regioisomers of scyllo-inositol phosphate and inositol 1,4,5-trisphosphate 3-kinase

scyllo-Inositol phosphates, which are among the stereoisomers of myo-inositol phosphate, can have 15 possible regioisomers including three enantiomeric pairs: scyllo-I(1,2)P(2), scyllo-I(1,2,4)P(3), scyllo-I(1,2,3,4)P(4). We herein describe the facile synthetic routes to the three enantiomeric pairs of scyllo-inositol phosphate and the molecular interactions between 15 regioisomers of scyllo-inositol phosphate and inositol 1,4,5-trisphosphate 3-kinase. Geometry of the enzyme binding site is discussed.