Abstract
Adenophostins A and B, which are metabolic products of the fungusPenicillium brevicompactum, are potent agonists at thed-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] receptor. In the current study, adenophostin A was ∼50-fold more potent than Ins(1,4,5)P3at both releasing Ca2+ from the intracellular stores of permeabilized platelets and displacing [3H]Ins(1,4,5)P3 from its receptor on rat cerebellar membranes. Various analogues bearing structural features found in the adenophostins and/or Ins(1,4,5)P3 were examined to elucidate the molecular basis for the observed enhanced potency. 2-AMP did not induce Ca2+ release from permeabilized platelets or have any effect on Ins(1,4,5)P3-induced Ca2+ release. Two carbohydrate-based analogues, (2-hydroxyethyl)-α-d-glucopyranoside-2′,3,4-trisphosphate and α,α′-trehalose-3,4,3′,4′-tetrakisphosphate, could induce release of Ca2+ and displace [3H]Ins(1,4,5)P3 from its binding site on rat cerebellar membranes, although both were less potent than Ins(1,4,5)P3. In common with adenophostin A, release of Ca2+ from the intracellular stores could be inhibited by heparin, and both analogues were metabolically resistant. This study is the first to demonstrate the activity of a synthetic disaccharide at the Ins(1,4,5)P3 receptor and that the Ins(1,4,5)P3 receptor is capable of accommodating an increased steric bulk. The minimal importance of the 2-hydroxyl group of Ins(1,4,5)P3 (occupied by the pyranoside oxygen in adenophostin) was confirmed by comparing the activity ofdl-scyllo-Ins(1,2,4)P3 [which differs from Ins(1,4,5)P3 solely by the orientation of this hydroxyl group] with that of Ins(1,4,5)P3. An analogue of this compound, namely,dl-6-CH2OH-scyllo-Ins(1,2,4)P3, which possesses an equatorial hydroxymethyl group analogous to the 5′-hydroxymethyl group of adenophostin, was found to be equipotent to Ins(1,4,5)P3, demonstrating the tolerance of the Ins(1,4,5)P3 receptor to additional steric bulk at this position.
An elevation in the intracellular levels of Ca2+ is known to be a key signaling event coupling cell activation by a wide range of extracellular stimuli to characteristic physiological responses. The ligation of plasma membrane receptors coupled to heterotrimeric G proteins or associated with cytosolic tyrosine kinases causes activation of members of the phospholipase C family of enzymes. Activated phospholipase C hydrolyzes phosphatidylinositol-4,5-bisphosphate to generate the two signaling molecules sn-1,2-diacylglycerol and Ins(1,4,5)P3. sn-1,2-Diacylglycerol is the endogenous activator of the serine/threonine-specific family of protein kinases, termed protein kinase C (1), and Ins(1,4,5)P3 is responsible for mediating the release of Ca2+ by binding to specific receptors on specialized intracellular storage sites (2,3). Three Ins(1,4,5)P3-R subtypes, together with splice variants of each of these, have been identified, and the genes have been cloned (4, 5). The Ins(1,4,5)P3-gated Ca2+ channel has been demonstrated to exist as a complex of Ins(1,4,5)P3-R subunits (6) and may be homotetrameric or heterotetrameric (7)
In non-voltage-excitable cells, release of intracellular Ca2+ by Ins(1,4,5)P3 is followed by entry of Ca2+ into the cells across the plasma membrane by a mechanism termed “store-operated” Ca2+ entry that is dependent on the filling state of the intracellular Ca2+ store (8); therefore not only is Ins(1,4,5)P3 directly responsible for the release of Ca2+ from the intracellular stores, but through depletion of these stores, it is indirectly responsible for Ca2+ entry (9, 10).
Due to the pivotal role of Ins(1,4,5)P3 in intracellular signal transduction pathways, there has been much attention directed at determining the structural motifs of Ins(1,4,5)P3 responsible for its receptor binding capability and Ca2+-releasing activity (11). The structure-activity studies performed with Ins(1,4,5)P3 analogues have indicated a key role for the vicinal diequatorial 4,5-bisphosphate system in mediating Ca2+ release (12-14), whereas an equatorial 6-OH is thought to be responsible for enhanced binding (15, 16). Both adenophostin A (Fig. 1) and its 6"-O-acetylated homologue adenophostin B possess equivalent features in the form of the glucose-3,4-bisphosphate and the adjacent 2-hydroxyl group, with the pyranoside oxygen acting as a surrogate of C-2 in Ins(1,4,5)P3. A direct equivalent to the third phosphate group at position 1 of Ins(1,4,5)P3 is not present in the adenophostins, but they both bear a phosphate group at position 2′ of ribose. Removal of this phosphate group has been demonstrated to cause a 1000-fold reduction in binding affinity.(17)
To investigate the molecular basis for the high potency of adenophostin A, we examined the biological activity of several molecules baring different structural relationships to adenophostin A and/or Ins(1,4,5)P3 and have compared these with both adenophostin A and Ins(1,4,5)P3 (Fig. 1). In this study, we report the Ca2+-releasing activity of Ins(1,4,5)P3, adenophostin A, and these structurally related compounds in permeabilized rabbit platelets together with their ability to displace [3H]Ins(1,4,5)P3 from its receptor in rat cerebellum.
Experimental Procedures
Materials
Chemically synthesized Ins(1,4,5)P3 was purchased from the Rhode Island Chemical Group (Kingston, RI). Fura-2 (pentapotassium salt) was from Molecular Probes (Eugene, OR). [3H]Ins(1,4,5)P3 (20–60 Ci/mmol, 10 μCi/ml) and45Ca2+ (5–50 mCi/mg Ca2+, 2 mCi/ml) were purchased from Amersham International (Buckinghamshire, UK). FP100 filters were purchased from Whatman (Maidstone, UK). Heparin, oligomycin, creatine phosphokinase, phosphocreatine, saponin A, leupeptin, pepstatin, and ATP were obtained from Sigma Chemical (Poole, Dorset, UK). Ionomycin was purchased from Calbiochem (San Diego, CA). Adenophostin A was a generous gift from Dr. M. Takahashi (Sankyo, Tokyo, Japan). 2′-AMP-free acid purchased from Sigma was converted into the triethylammonium salt to increase its aqueous solubility, and its purity was confirmed by31P NMR and high performance liquid chromatography. Glc(2′,3,4)P3 was synthesized as previously described (18), and Trehal(3,4,3′,4′)P4 was synthesized in a similar fashion (19). Racemic 6-CH2OH-scyllo-Ins(1,2,4)P3(13) and scyllo-Ins(1,2,4)P3 (20) were synthesized as previously described.
Methods
Preparation of platelets.
Washed rabbit platelets were prepared as previously described (21). The resulting platelet pellet from this preparation was resuspended in HEPES-buffered Tyrode’s solution (consisting of 10 mm HEPES, 145 mmNaCl, 5 mm KCl, 1 mmMgCl2, 0.5 mmNa2HPO4, 5.5 mmglucose, 0.25% bovine serum albumin, pH 7.4) before performing the following procedures.
45Ca2+ release from intracellular stores.
Platelets were washed in high-K+buffer A [consisting of 120 mm KCl, 2 mmKH2PO4, 5 mm(CH2COONa)2O · 6H2O, 6 mm MgCl2, 20 mm HEPES, in MilliQ water; 5 mm ATP was added, pH adjusted to 6.9, and free Ca2+ concentration adjusted to <150 nm] and then suspended to 3 × 109/ml. The platelets were then permeabilized with 40 μg/ml saponin A, which was removed by further washing in buffer A. The intracellular Ca2+ stores were loaded with 45Ca2+ (2 μCi/ml) for 1 hr in the presence of 10 μg/ml oligomycin. Total release of 45Ca2+ from the stores was determined by a 3-min incubation with 75 μmionomycin. Release of45Ca2+ from the intracellular stores was determined at 4°, 3 min after the addition of either Ins(1,4,5)P3, adenophostin A or the structural analogues by separation of free and retained45Ca2+ through filtration of cells using Whatman FP100 filters.45Ca2+ release was determined by liquid scintillation spectroscopy (22).
Ins(1,4,5)P3-induced Ca2+ release from permeabilized platelets monitored by spectrophotofluorimetry.
Platelets were isolated and washed as above and then resuspended in high K+ buffer B (consisting of 100 mm KCl, 20 mm NaCl, 5 mmMgCl2, 20 mm HEPES, 2 mmEGTA, pH 7.2) at a concentration of 3 × 109/ml. After permeabilization with 40 μg/ml Saponin A (1 min, 20°), the platelets were washed again in buffer B in the absence of EGTA but in the presence of 20 units/ml creatine phosphokinase and 10 μg/ml oligomycin according to a modification of a previously described method (23). Ca2+ uptake into stores was initiated by the addition of 3 mm ATP and 50 mm phosphocreatine. Ca2+ release from the stores was monitored using Fura-2 (free acid, 0.5 μm) in the extracellular buffer. Changes in fluorescence were measured using a PTI dual-wavelength spectrophotofluorimeter (excitation, 340 and 380 nm; emission, 510 nm; slit width, 4 nm). Experiments were performed at 20°. The traces shown in the figures represent an increase in fluorescence of Fura-2 that is due to the transient release of Ca2+ from the intracellular stores followed by a decrease in fluorescence that is due to Ca2+ resequestration. The Ca2+/Fura-2-fluorescence was calibrated as previously described (24).
Displacement of [3H]Ins(1,4,5)P3binding to specific Ins(1,4,5)P3-Rs on rat cerebellar membranes.
The preparation of rat cerebellar membranes and displacement of [3H]Ins(1,4,5)P3 bound to the Ins(1,4,5)P3-Rs on the membranes were performed as previously described (25). Briefly, the cerebella were removed from six rats (200–250 g) and homogenized (twice at 10 sec at 4°) in buffer C (consisting of 20 mm Tris·HCl, 20 mm NaCl, 100 mm KCl, 1 mm EDTA, 1 mg/ml bovine serum albumin, pH 7.7) containing the protease inhibitors 10 μm leupeptin and 10 μm pepstatin. After centrifugation (50,000 × g for 13 min at 4°), the pellet was resuspended in buffer C and homogenized as described above, and the protein content was adjusted to 5 mg/ml. The cerebellar membranes were either used immediately or frozen (−80°) until use. The binding assay mixture in a total volume of 250 μl contained 1 nm[3H]Ins(1,4,5)P3 and structural analogues diluted in buffer C at appropriate concentrations. Binding was initiated by the addition of 250 μg of the cerebellar membrane preparation. The assay tubes were incubated (4°) for 10 min before termination of the reaction by centrifugation (10,000 ×g for 4 min at 4°). Nonspecific binding of [3H]Ins(1,4,5)P3 was assessed as the counts remaining on inclusion of 10 μmnonradiolabeled Ins(1,4,5)P3 in the assay mixture. After centrifugation, the supernatant was carefully removed, the pellet was resuspended, and radioactivity bound to the cerebellar membrane was determined by liquid scintillation counting.
Results
Ca2+ release from permeabilized platelets.
Rabbit platelets permeabilized with saponin and in the presence of oligomycin displayed ATP-dependent45Ca2+ uptake into their nonmitochondrial stores. Uptake reached a steady state by 45 min and was monitored throughout the time course of the experiment and found to remain essentially unchanged. The ionomycin-releasable component of accumulated 45Ca2+ was found to be >92%; again, this was not found to change significantly throughout the time course of any of the45Ca2+-release experiments undertaken.
Treatment of permeabilized platelets with Ins(1,4,5)P3 (0.003–30 μm) for 3 min (4°) caused a dose-dependent release of45Ca2+ from preloaded intracellular stores (Fig. 2). A time of 3 min was chosen because45Ca2+ release had reached a maximal plateau at this time (results not shown). Adenophostin A (0.0001–0.3 μm) also caused a dose-dependent release of45Ca2+ from the stores of permeabilized platelets; however, it displayed a ∼55-fold lower EC50 value than Ins(1,4,5)P3 (Table1). Synthetic carbohydrate-based analogues Glc(2′,3,4)P3 and Trehal(3,4,3′,4′)P4 were also examined for their ability to release 45Ca2+from permeabilized platelets. Glc(2′,3,4)P3(0.01–10 μm) dose-dependently released45Ca2+ from the intracellular stores of permeabilized platelets (Fig. 1) with an EC50 value of 2.05 ± 0.35 μm, ∼5-fold higher than Ins(1,4,5)P3 (Table 1). Trehal (3,4,3′,4′)P4 (0.1–300 μm) was also able to dose-dependently release45Ca2+ from the intracellular stores of permeabilized platelets (Fig. 2), with an EC50 value of 100 μm, ∼250-fold higher than Ins(1,4,5)P3 (Table 1).
The above findings were extended through examination of the kinetics of Ca2+ release of Ins(1,4,5)P3, adenophostin A, Glc(2′,3,4)P3, Trehal(3,4,3′,4′)P4, and 2′-AMP. Ca2+ release was monitored in the presence of the fluorescent dye Fura-2 (free acid) by spectrophotofluorimetry. The addition of 1 μm Ins(1,4,5)P3caused release of Ca2+ from the intracellular stores of permeabilized platelets detected as a rapid increase in the fluorescence of Fura-2 free acid (Fig.3). The increase in fluorescence was transient, presumably due to the metabolism of Ins(1,4,5)P3 to give inactive products, resulting in resequestration of Ca2+ back into the intracellular stores by Ca2+-ATPase activity. The addition of 200 μm 2′-AMP did not stimulate release of Ca2+ from the intracellular stores of permeabilized platelets and had no effect on Ins(1,4,5)P3 binding to its receptor, as determined by the lack of effect on Ca2+ release by the subsequent addition of Ins(1,4,5)P3 (Fig.3, top left).
The addition of adenophostin A to permeabilized platelets caused a dose-dependent release of Ca2+ from the intracellular stores. However, unlike the Ca2+release by Ins(1,4,5)P3, which was transient, Ca2+ release by adenophostin A reached a maximum and was then maintained at a plateau phase over the time course of the experiment (Fig. 3, top right). Heparin, which is able to compete with Ins(1,4,5)P3 for its binding site and inhibit Ca2+ release, was also found to inhibit release of Ca2+ induced by a submaximal concentration of adenophostin A (Fig. 3, second from top left). Glc(2′,3,4)P3 was also observed to release Ca2+ from the intracellular stores of platelets, detected as an increase in fluorescence of Fura-2 (Fig. 3,second from top right). As in the case of adenophostin A, there was a sustained elevation of the fluorescence signal, suggesting that Glc(2′,3,4)P3 is also poorly metabolized in this permeabilized platelet system. Again, Glc(2′,3,4)P3-induced Ca2+release was inhibited by the Ins(1,4,5)P3-R antagonist heparin (Fig. 3, second from bottom left). Trehal(3,4,3′,4′)P4 also released Ca2+ from permeabilized platelets, and in correlation with the findings for45Ca2+ release, no increase in fluorescence (which indicates indicating Ca2+mobilization) was detected until concentrations of Trehal(3,4,3′,4′)P4 of >1 μm were applied to the cells (Fig. 3, second from bottom right). Trehal(3,4,3′,4′)P4 also caused a sustained increase in the fluorescence signal, which, again, indicated poor metabolism of this compound in this permeabilized platelet system compared with Ins(1,4,5)P3. Trehal(3,4,3′,4′)P4-induced Ca2+ release was also inhibited by the Ins(1,4,5)P3-R antagonist heparin (Fig. 3,bottom left).
Two racemic scyllo-inositol-based analogues,dl-scyllo-Ins(1,2,4)P3 and its 6-deoxy-6-hydroxymethyl homologue,dl-6-CH2OH-scyllo-Ins(1,2,4)P3, were also evaluated in the45Ca2+-release assay (Fig.4). Both compounds were relatively potent mobilizers of Ca2+, with EC50 values 4-fold higher than and approximately equal to those of Ins(1,4,5)P3, respectively (Table 1).
Displacement of specific [3H]Ins(1,4,5)P3binding to rat cerebellar membranes
[3H]Ins(1,4,5)P3 was readily displaced from its specific binding site on rat cerebellar membranes by nonradiolabeled Ins(1,4,5)P3 with an IC50 of 0.038 ± 0.005 μm(Fig. 5, Table 1). Adenophostin A also displaced specifically bound [3H]Ins(1,4,5)P3 from rat cerebellar membranes, although adenophostin A was around 50 times more potent than Ins(1,4,5)P3 with an IC50 value of 0.00074 ± 0.00042 μm (Fig. 5, Table 1).
Displacement of [3H]Ins(1,4,5)P3 from the binding site on rat cerebellar membranes by Glc(2′,3,4)P3 was ∼5-fold less effective than displacement by Ins(1,4,5)P3 and ∼280-fold less effective than by adenophostin A, whereas Trehal(3,4,3′,4′)P4 was 10-fold less effective than Ins(1,4,5)P3 and 500-fold less effective than adenophostin A. In agreement with the45Ca2+-release data,dl-6-CH2OH-scyllo-Ins(1,2,4)P3seemed to be equipotent to Ins(1,4,5)P3 in displacement of [3H]Ins(1,4,5)P3, whereasdl-scyllo-Ins(1,2,4)P3 was ∼4-fold less potent (Fig. 6 and Table1).
Discussion
There have been few studies in which the biological activity of the adenophostins has been examined (17, 26-28), and as a consequence, Ca2+ release by the adenophostins has been reported in only a small number of tissue types (26, 28). Takahashiet al. (26) first demonstrated the high potency of adenophostins A and B at the Ins(1,4,5)P3-R and showed them to be equipotent at displacing [3H]Ins(1,4,5)P3 from purified rat cerebellar Ins(1,4,5)P3-Rs with an IC50 value of 1.3 nm and more potent than Ins(1,4,5)P3 with an IC50 value of 23 nm. We have also demonstrated, using a rat cerebellar membrane preparation (25), that adenophostin A is more potent than Ins(1,4,5)P3at displacing [3H]Ins(1,4,5)P3 with similar IC50 values of 0.74 and 38 nm, respectively. In rat cerebellar microsomes, Takahashiet al. demonstrated the ED50 values for Ca2+ release to be 1.4 and 170 nmfor adenophostin A and Ins(1,4,5)P3, respectively, whereas in permeabilized NG108–15 cells, the ED50 values were 53 and 2400 nm, respectively (26). Therefore, in a cell-free system, adenophostin A was ∼100-fold more potent at releasing Ca2+ than Ins(1,4,5)P3, whereas in a permeabilized whole-cell system, adenophostin A was found to be ∼45-fold more potent at releasing Ca2+. In agreement, we found adenophostin A to be ∼55-fold more potent than Ins(1,4,5)P3 at releasing Ca2+ from the intracellular stores of permeabilized rabbit platelets. Moreover, in a later study using purified type 1 Ins(1,4,5)P3-Rs, adenophostin had only a 10-fold higher potency than Ins(1,4,5)P3. Release of 45Ca2+ from permeabilized rabbit platelets by adenophostin was inhibited by the Ins(1,4,5)P3-R antagonist heparin, indicating interaction of adenophostin with the Ins(1,4,5)P3-R. Unlike Ins(1,4,5)P3, however, adenophostin A caused a sustained, rather than transient, release of Ca2+when added to permeabilized platelets, indicating that adenophostin A is resistant to the metabolizing enzymes located in permeabilized platelets.
To further elucidate the structural features responsible for the high potency of adenophostin A, we investigated the biological activity of several compounds whose structures represent different aspects of the construction of adenophostin A. Two of the major differences between adenophostin A and Ins(1,4,5)P3 are the adenosine component and the hydroxymethyl substituent, and compounds were prepared in an attempt to examine the contribution of each of these moieties.
The finding that 2′-AMP alone was inactive at releasing Ca2+ from the intracellular stores of permeabilized platelets supports the assumption that the activity of the adenophostins originates in the phosphorylated glucose component, with the 3,4-bisphosphate/2-hydroxyl on the glucopyranose ring mimicking the key structures of Ins(1,4,5)P3responsible for Ca2+ release. However, the fact that the adenophostins are more potent than Ins(1,4,5)P3 suggests not only that the Ins(1,4,5)P3-R is able to accommodate the bulk of the adenosine component at the 1′′ position of the glucopyranose ring but also that this structure is necessary for the high potency of the adenophostins.
The 1-phosphate group of Ins(1,4,5)P3, although not essential for its activity, is thought to enhance its potency (15). It has been suggested that the 2′-phosphate on the ribose ring of the adenophostins may be positioned to fit the Ins(1,4,5)P3-R more effectively than the 1-phosphate of Ins(1,4,5)P3 and therefore further enhance the affinity of the adenophostins for the Ins(1,4,5)P3-R (26). Indeed, the importance of this phosphate group at the 2′-position of the ribose ring was confirmed when its removal caused a 1000-fold reduction in the activity of adenophostin A (17). If a binding pocket for the adenosine moiety of the adenophostins exists at the Ins(1,4,5)P3-R, it is possible that 2′-AMP alone might bind to the Ins(1,4,5)P3-R, interfere with Ins(1,4,5)P3 binding, and thus inhibit Ins(1,4,5)P3-induced Ca2+release. However, 2′-AMP had no effect on Ins(1,4,5)P3-induced Ca2+release in permeabilized platelets, indicating that when separated from the glucopyranose bisphosphate motif, 2′-AMP alone has no significant affinity for the Ins(1,4,5)P3-R or, if able to bind, it does not interfere with Ins(1,4,5)P3binding or Ca2+-release activity.
Given that features from both the glucopyranose ring and the adenosine moiety of adenophostin A are necessary for its potent binding and Ca2+-releasing activity, it remains to be established which of these features are sufficient for such activity. Glc(2′,3,4)P3 possesses the glucopyranose ring of adenophostin, whereas all except C2 and C3 of the ribose ring and the 2′-phosphate group of the adenosine component have been removed (18). In common with adenophostin, Glc(2′,3,4)P3 was metabolically resistant when added to a permeabilized cell preparation, and Ca2+ release from the intracellular stores of permeabilized platelets was inhibited by the Ins(1,4,5)P3-R antagonist heparin. However, although Glc(2′,3,4)P3 was found to both release Ca2+ from the intracellular stores of permeabilized rabbit platelets (EC50 = 2.05 μm) and displace [3H]Ins(1,4,5)P3 from the Ins(1,4,5)P3 binding sites of rat cerebellar membranes (IC50 = 0.21 μm), it was ∼5-fold lower in potency than Ins(1,4,5)P3 and ∼280-fold weaker than adenophostin in both binding and Ca2+-release studies. Similarly, Wilcox et al. (29) reported a 5-fold lower affinity for Glc(2′,3,4)P3 compared with Ins(1,4,5)P3 in binding studies using pig cerebellar membranes and a 10–12-fold lower potency for Glc(2′,3,4)P3 in Ca2+-release studies using SH-SY5Y and MKCK cells compared with Ins(1,4,5)P3.
These findings initially suggested that the excised region of adenosine was important for conferring the extreme potency of the adenophostins. Alternatively, however, it may be that because the terminal 2′-position phosphate group of Glc(2′,3,4)P3 is not as spatially constrained as the 2′-phosphate on the ribose ring of adenophostin, it is not able to confer the same increased potency to Glc(2′,3,4)P3 (29); the 2′-position phosphate may be required to be held in a precise position with respect to the glucopyranose ring to increase its potency (26).
The tetrakisphosphate Trehal(3,4,3′,4′)P4 is larger and conformationally more rigid than Glc(2′,3,4)P3 and consists of two copies of the phosphorylated glucopyranose component of adenophostin in aC 2 symmetrical molecule. This molecule possesses all the key features of Glc(2′,3,4)P3 believed to be important for Ca2+ release but, in addition, holds the phosphates of the second glucopyranose ring in a more rigid conformation. Again, like Glc(2′,3,4)P3 and adenophostin, Trehal(3,4,3′,4′)P4 was demonstrated to be metabolically resistant, and release of Ca2+ was inhibited by heparin. Trehal(3,4,3′,4′)P4 was found to be ∼10-fold less potent than Ins(1,4,5)P3 at displacing [3H]Ins(1,4,5)P3 from its receptor on rat cerebellar membrane but ∼250-fold less potent at releasing 45Ca2+ from the stores of permeabilized platelets, a finding confirmed using dynamic measurements of Ca2+ release monitored in the presence of the Ca2+-specific fluorescent dye Fura-2 by spectrophotofluorimetry. This study is the first to demonstrate the activity of a synthetic disaccharide derivative at the Ins(1,4,5)P3-R and to demonstrate that as predicted, the Ins(1,4,5)P3-R is capable of accommodating the steric bulk of the second glucose residue of Trehal(3,4,3′,4′)P4. A preliminary molecular modeling study of Trehal(3,4,3′,4′)P4 and adenophostin A suggested that although either one of the two equivalent glucopyranose-3,4-bisphosphate components of this molecule would be a good mimic of the equivalent structure in adenophostin A, neither phosphate group on the second ring would occupy a position in space equivalent to that of the 2′-phosphate in the adenophostins (19). The reason for the disparity in the Ca2+-release activity and [3H]Ins(1,4,5)P3displacement is not immediately clear, but it seems unlikely that it is due to the assay conditions because Glc(2′,3,4)P3and adenophostin A gave the same relative potency in binding and Ca2+-release assays compared with Ins(1,4,5)P3. Thus, it may be that one or both of the phosphate groups on the second ring of Trehal(3,4,3′,4′)P4 somehow reduces the ability of this analogue to cause opening of the integral ion channel. This unusual aspect of the activity of Trehal(3,4,3′,4′)P4 may have implications for the design of Ins(1,4,5)P3-R antagonists.
Finally, the differences in activity betweendl-scyllo-Ins(1,2,4)P3 anddl-6-CH2OH-scyllo-Ins(1,2,4)P3 may have implications for the effect of the 5′′-hydroxymethyl group in adenophostin A. The observation thatdl-6-CH2OH-scyllo-Ins(1,2,4)P3is equipotent with Ins(1,4,5)P3 implies that the CH2OH component, which is not present in Ins(1,4,5)P3 itself, is tolerated by the Ins(1,4,5)P3-R despite the additional steric bulk. The presence of an analogous structure in adenophostin A is in accordance with this finding. This observation is significant because previous studies of position 3-substituted Ins(1,4,5)P3 analogues seemed to show that large substituents at this position were not tolerated by the receptor, and this led some researchers to suggest that adenophostin A must bind in a different orientation to Ins(1,4,5)P3 to overcome the sterically handicapping CH2OH group.(29). Our results also imply that at least in scyllo-analogues of Ins(1,4,5)P3, replacement of the secondary hydroxyl group at this position with an hydroxymethyl group enhances potency at the Ins(1,4,5)P3-R. This motif could therefore be of interest in the design of Ins(1,4,5)P3-R ligands.
On the basis of the activities of the analogues examined in the current study, we were able to draw several conclusions regarding the structural basis for the activity of the adenophostins. As we and others have previously suggested (18, 26, 29), the Ca2+-releasing activity of adenophostin A does indeed seem to reside in the 3,4-phosphorylated glucopyranose motif, which mimics the most important parts of Ins(1,4,5)P3. This activity is somehow augmented by the 2′-AMP component, although this structure, in isolation, cannot cause Ca2+ release or antagonize Ins(1,4,5)P3-mediated Ca2+-release. The addition of a third phosphate group, as in Glc(2′,3,4)P3, which is theoretically able to access the area of the receptor available to the 2′-phosphate of the adenophostins, was not sufficient to confer adenophostin-like potency when this group was conformationally highly mobile. A phosphorylated disaccharide such as Trehal(3,4,3′,4′)P4 can be accommodated by the receptor binding site, but again, it is likely that accurate placement of a phosphate group on the accessory residue would be required for high potency. Whether optimal placing of this third phosphate is all that is required for potency greater than that of Ins(1,4,5)P3 remains to be seen. The presence of a 5′′-CH2OH component in adenophostin A did not require the glucopyranose component to interact with the receptor in a different way to Ins(1,4,5)P3. Indeed, a molecule closely related to Ins(1,4,5)P3 yet possessing this component [6-CH2OH-scyllo-Ins(1,2,4)P3] showed Ins(1,4,5)P3-like potency; therefore, at least in scyllo-inositol analogues, a CH2OH group at this position may give rise to a modest increase in potency, and a similar effect may apply to adenophostin A. Interestingly, in adenophostin B, the 5′′-CH2OH group is acetylated, giving even greater steric bulk yet no decrease in potency. It is not clear whether adenophostin-like potency can be attained by a simple disaccharide framework with appropriately placed phosphate and hydroxyl groups or whether something resembling the adenine ring system is also required. It may be that this structure serves to orient the third phosphate in a particular way at the receptor or that the adenine itself has favorable interactions with a region close to the Ins(1,4,5)P3-binding site. Given the current state of knowledge, either or both of these alternatives are possible. A definitive resolution of this last point may be attainable by the synthesis and evaluation of disaccharide-like adenophostin analogues lacking the adenine structure.
Acknowledgments
We thank Dr. M. Takahashi for an authentic sample of adenophostin A.
Footnotes
- Received March 28, 1997.
- Accepted June 20, 1997.
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Send reprint requests to: Dr. C. T. Murphy, Department of Pharmacology, School of Pharmacy & Pharmacology, University of Bath, Bath, Avon BA2 7AY, United Kingdom. E-mail:c.t.murphy{at}bath.ac.uk
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This work was supported by the British Heart Foundation (J.W., C.T.M.) and The Wellcome Trust with project (J.W., B.V.L.P.) and program grant (BVLP) support and the Biotechnology and Biological Research Council, Intracellular Signaling Programme (B.V.L.P.).
Abbreviations
- Ins(1
- 4,5)P3,d-myo-inositol-1,4,5-trisphosphate
- Glc(2′
- 3,4)P3, (2-hydroxyethyl)-α-d-glucopyranoside-2′,3,4-trisphosphate
- Trehal(3
- 4,3′,4′)P4, α,α′-trehalose-3,4,3′,4′-tetrakisphosphate
- dl-scyllo-Ins(1
- 2,4)P3,dl-scyllo-inositol-1,2,4-trisphosphate
- dl-6-CH2OH-scyllo-Ins(1
- 2,4)P3,dl-6-deoxy-6-hydroxymethyl-scyllo-inositol-1,2,4-trisphosphate
- Ins(1
- 4,5)P3-R, inositol-1,4,5-trisphosphate receptor
- The American Society for Pharmacology and Experimental Therapeutics