Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027819 (neuroblastoma)
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The ability of two enantiomeric fluoro-analogues of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to mobilize intracellular Ca2+ stores in SH-SY5Y neuroblastoma cells has been investigated. (-)-D-2,2-difluoro-2-deoxy-myo-Ins(1,4,5)P3 [D-2,2-F2-Ins(1,4,5)P3] was a full agonist [EC50 0.21 microM] and slightly less potent than D-Ins(1,4,5)P3 [EC50 0.13 microM]. (+)-L-2,2-F2Ins(1,4,5)P3 was a very poor agonist, confirming the stereospecificity of the Ins(1,4,5)P3 receptor. D-2,2-F2-Ins(1,4,5)P3 mobilized Ca2+ with broadly similar kinetics to Ins(1,4,5)P3 and was a substrate for Ins(1,4,5)P3 3-kinase inhibiting Ins(1,4,5)P3 phosphorylation (apparent Ki = 10.2 microM) but was recognised less well than Ins(1,4,5)P3. L-2,2-F2-Ins(1,4,5)P3 was a potent competitive inhibitor of 3-kinase (Ki = 11.9 microM). Whereas D-2,2-F2-Ins(1,4,5)P3 was a good substrate for Ins(1,4,5)P3 5-phosphatase, L-2,2-F2Ins(1,4,5)P3 was a relatively potent inhibitor (Ki = 19.0 microM).
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PMID:Synthetic D- and L-enantiomers of 2,2-difluoro-2-deoxy-myo-inositol 1,4,5-trisphosphate interact differently with myo-inositol 1,4,5-trisphosphate binding proteins: identification of a potent small molecule 3-kinase inhibitor. 133 23

Intracellular Ca2+ stores in permeabilized SH-SY5Y neuroblastoma cells were mobilized by D-myo-inositol 1,4,5-trisphosphate [D-Ins(1,4,5)P3] and two of its synthetic analogues, DL-myo-inositol 1,4-bisphosphate 5-phosphorothioate (DL-InsP3-5S) and DL-myo-inositol 1,4,5-trisphosphorothioate (DL-InsP3S3). The concentrations of D-Ins(1,4,5)P3, DL-InsP3-5S, and DL-InsP3S3 required for half-maximal release were 0.11, 0.8, and 2.5 microM, respectively. All agents were full agonists, releasing 55-60% of sequestered 45Ca2+. D-Ins(1,4,5)P3-induced mobilization of Ca2+ was transient, and Ca2+ reuptake followed D-Ins(1,4,5)P3 metabolism closely. DL-InsP3S3-induced mobilization was persistent, consistent with the resistance of this analogue to metabolic enzymes. In contrast, DL-InsP3-5S-induced Ca2+ mobilization was followed by reuptake of Ca2+, albeit at a slower rate than that seen with D-Ins(1,4,5)P3. DL-InsP3-5S and DL-InsP3S3 were resistant to D-Ins(1,4,5)P3 5-phosphatase and potently inhibited the enzyme, with Ki values of 6.8 and 1.7 microM, respectively. DL-InsP3S3 was resistant to D-Ins(1,4,5)P3 3-kinase and was a very weak inhibitor of the enzyme (Ki = 230 microM). The ability of DL-InsP3-5S to inhibit D-Ins(1,4,5)P3 phosphorylation (apparent Ki = 5 microM) and its loss of Ca(2+)-releasing ability on incubation with D-Ins(1,4,5)P3 3-kinase suggest that this analogue may undergo phosphorylation to inositol 1,3,4-trisphosphate 5-phosphorothioate. These differential and complementary properties of DL-InsP3-5S and DL-InsP3S3 may be useful in dissecting the roles of D-Ins(1,4,5)P3 and D-myo-inositol 1,3,4,5-tetrakisphosphate in Ca2+ homeostasis.
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PMID:Synthetic phosphorothioate-containing analogues of inositol 1,4,5-trisphosphate mobilize intracellular Ca2+ stores and interact differentially with inositol 1,4,5-trisphosphate 5-phosphatase and 3-kinase. 164 49

The ability of two fluoro-analogues of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) to mobilize intracellular Ca2+ stores in SH-SY5Y neuroblastoma cells has been investigated. DL-2-deoxy-2-fluoro-scyllo-Ins(1,4,5)P3 (2F-Ins(1,4,5)P3) and DL-2,2-difluoro-2-deoxy-myo-Ins(1,4,5)P3 (2,2-F2-Ins(1,4,5)P3) were full agonists (EC50s 0.77 and 0.41 microM respectively) and slightly less potent than D-Ins(1,4,5)P3 (EC50 0.13 microM), indicating that the axial 2-hydroxyl group of Ins(1,4,5)P3 is relatively unimportant in receptor binding and stimulation of Ca2+ release. Both analogues mobilized Ca2+ with broadly similar kinetics and were substrates for Ins(1,4,5)P3 3-kinase but, qualitatively, were slightly poorer than Ins(1,4,5)P3. 2F-Ins(1,4,5)P3 was a weak substrate for Ins(1,4,5)P3 5-phosphatase but 2,2-F2-Ins(1,4,5)P3 was apparently not hydrolysed by this enzyme, although it inhibited its activity potently (Ki = 26 microM).
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PMID:Ca2(+)-mobilising properties of synthetic fluoro-analogues of myo-inositol 1,4,5-trisphosphate and their interaction with myo-inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase. 217 65

Inositol-polyphosphate-induced Ca2+ mobilization was investigated in saponin-permeabilized SH-SY5Y human neuroblastoma cells. Ins(1,4,5)P3 induced a dose-related release from intracellular Ca2+ stores with an EC50 (concn. giving half-maximal effect) of 0.1 microM and a maximal release of 70%. Ins(1,3,4)P3, DL-Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5 did not evoke Ca2+ mobilization in these cells when used at concentrations up to 10 microM. However, Ins(1,3,4,5)P4 was found to release Ca2+ in a dose-related manner, but the response was dependent on the source of Ins(1,3,4,5)P4 used. When commercially available D-Ins(1,3,4,5)P4 was used, the EC50 and maximal response values were 1 microM and 50% respectively, compared with values for chemically synthesized DL-Ins(1,3,4,5)P4 of 2 microM and 25%. The enhanced maximal response of commercial D-Ins(1,3,4,5)P4 was decreased by pretreatment with rat brain crude Ins(1,4,5)P3 3-kinase and was therefore concluded to be indicative of initial Ins(1,4,5)P3 contamination of the Ins(1,3,4,5)P4 preparation. When metabolism of DL-Ins(1,3,4,5)P4 (10 microM) in these cells at 25 degrees C was investigated by h.p.l.c., substantial amounts of Ins(1,4,5)P3 (0.2 microM) and Ins(1,3,4)P3 (0.8 microM) were found to be produced within 3 min. Analysis of DL-Ins(1,3,4,5)P4 incubation with cells at 4 degrees C, however, indicated that metabolism had been arrested ([3H]Ins(1,4,5)P3 detection limits were estimated to be approx. 0.01 microM). When chemically synthesized DL-Ins(1,3,4,5)P4 and incubation conditions of low temperature were used, the Ca2(+)-releasing properties of this compound were established to be 1 microM and 19% for the EC50 and maximal response values respectively. The results obtained strongly suggest that Ins(1,3,4,5)P4 alone has the ability to release intracellular Ca2+. However, in the presence of sub-maximal concentrations of Ins(1,4,5)P3, Ca2+ release appears to be synergistic with Ins(1,3,4,5)P4, but at supramaximal concentrations not even additive effects are observed.
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PMID:Inositol 1,3,4,5-tetrakisphosphate-induced release of intracellular Ca2+ in SH-SY5Y neuroblastoma cells. 217 65

Electrically permeabilised [3H]inositol-labelled SH-SY5Y human neuroblastoma cells were employed to examine the effects of two synthetic, phosphatase-resistant analogues of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] on the metabolism of cell membrane-derived [3H]Ins(1,4,5)P3 or exogenous [5-32P]Ins(1,4,4)P3. Incubation of permeabilised SH-SY5Y cells for 5 min at 37 degrees C with carbachol and guanosine 5'-[gamma-thio]triphosphate caused a decrease in [3H]phosphoinositol phospholipid levels and an increase in [3H]inositol phosphate accumulation with inositol 4-phosphate, inositol 1,4-bisphosphate, Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate comprising approximately 79%, 16%, 3% and 2%, respectively, of the increase. Inositol 1-phosphate levels did not increase upon stimulation, nor was inositol 4-phosphate converted rapidly to inositol. In parallel incubations, the analogues, DL-inositol 1,4,5-trisphosphorothioate (DL-InsP3S3) and DL-inositol 1,4-bisphosphate 5-phosphorothioate (DL-InsP3S), and synthetic racemic Ins(1,4,5)P3 (DL-InsP3), altered the profile of the [3H]inositol phosphates recovered and led, at millimolar concentrations, to a 10-15-fold increase in [3H]Ins(1,4,5)P3. The extent of inhibition of [3H]Ins(1,4,5)P3 metabolism was, however, greatest in the presence of synthetic D-Ins(1,4,5)P3 (greater than or equal to 5 mM), when [3H]Ins(1,4,5)P3 comprised approximately 50% of the increase in total [3H]inositol phosphates. Thus, under these conditions, at least 50% of [3H]inositol phosphates were derived from [3H]phosphatidylinositol 4,5-bisphosphate. [32P]Pi release from exogenous [5-32P]Ins(1,4,5)P3 was also inhibited by DL-InsP3S3, DL-InsP3S and DL-InsP3, with half-maximal inhibition at approximately 50 microM, 160 microM and 240 microM respectively. These actions were approximately ten times more potent than the effects of these compounds on [3H]inositol phosphate accumulation, indicating that homogenous mixing of exogenous and cell-membrane-derived Ins(1,4,5)P3 does not occur. These findings indicate that DL-InsP3S3 and DL-InsP3S inhibit Ins(1,4,5)P3 5-phosphatase. In contrast, the effects of synthetic DL-InsP3 and D-Ins(1,4,5)P3 are due to isotopic dilution. Whilst DL-InsP3S3 was the most potent inhibitor of dephosphorylation of exogenous or cell-membrane-derived Ins(1,4,5)P3, it was the weakest inhibitor of 3-kinase-catalysed Ins(1,4,5)P3 phosphorylation. Similarly, although approximately 50 times less potent than DL-InsP3S3, 2,3-diphosphoglycerate inhibited Ins(1,4,5)P3 5-phosphatase activity and was apparently without effect of Ins(1,4,5)P3 3-kinase activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Inhibition of inositol 1,4,5-trisphosphate metabolism in permeabilised SH-SY5Y human neuroblastoma cells by a phosphorothioate-containing analogue of inositol 1,4,5-trisphosphate. 220 1

The novel, synthetic, adenophostin A analogue 2-hydroxyethyl-alpha-D-glucopyranoside-2,3',4'-trisphosphate [Glu(2,3',4')P3] was synthesized to probe the structure-activity relationship at the D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R]. This study was stimulated by the recent observation that the fungal isolates adenophostins A and B were very potent, metabolically resistant, Ins(1,4,5)P3R agonists [J. Biol. Chem. 269:369-372 (1994)]. Gluc(2,3',4')P3 can be visualized as a truncated version of adenophostin A, in which the 2'- and 3'-carbons of the ribose ring, with their terminal phosphate groups, are retained and the remainder of the adenosine residue is excised. Gluc(2,3',4')P3 specifically displaced [3H]Ins(1,4,5)P3 from pig cerebellar Ins(1,4,5)P3 binding sites, with an affinity (IC50 = 130 nM) only 5-fold weaker than that of Ins(1,4,5)P3 (IC50 = 27 nM). Gluc(2,3',4')P3 was also a full agonist for Ca2+ release, being only 10-12-fold less potent than Ins(1,4,5)P3 in saponin-permeabilized SH-SY5Y neuroblastoma cells [EC50 = 647 nM; Ins(1,4,5)P3 EC50 = 52 nM] and Madin-Darby canine kidney cells [EC50 = 2484 nM; Ins(1,4,5)P3 EC50 = 247 nM]. Gluc(2,3',4')P3 did not significantly interact with recombinant Ins(1,4,5)P3 3-kinase and 5-phosphatase enzymes and was also poorly metabolized by saponin-permeabilized SH-SY5Y cells. However, Gluc(2,3',4')P3 was a considerably weaker ligand (approximately 500-fold) and agonist (approximately 1000-fold) than adenophostin A, suggesting that the partial excision of the adenosine residue compromised structural motifs that have favorable interactions with the Ins(1,4,5)P3R. Indeed, molecular dynamics simulations revealed that the potencies of the three compounds show a correlation with the relative distance of the two vicinal ring phosphates from the remaining phosphate. Gluc(2,3',4')P3, with its alpha-glucoside ring, is the first synthetic Ins(1,4,5)P3 analogue that is not structurally based on a phosphorylated inositol isomer and that exhibits potent activity at the Ins(1,4,5)P3R. This, combined with the metabolic resistance of Gluc(2,3',4')P3, thus affords a novel approach for the investigation of the cellular role of Ins(1,4,5)P3 and its receptor.
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PMID:2-Hydroxyethyl-alpha-D-glucopyranoside-2,3',4'-trisphosphate, a novel, metabolically resistant, adenophostin A and myo-inositol-1,4,5-trisphosphate analogue, potently interacts with the myo-inositol-1,4,5-trisphosphate receptor. 760 61