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Departments of Pharmacology (C.T.M., C.J.L., J.W.) and Medicinal Chemistry (A.M.R., D.J.J., B.V.L.P.), School of Pharmacy and Pharmacology, University of Bath, Bath, Avon, BA2 7AY, UK
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Summary |
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Summary
Introduction
Procedures
Results
Discussion
References
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Adenophostins A and B, which are metabolic products of the fungus
Penicillium brevicompactum, are potent agonists at the
D-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)P3
at 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)-
-D-glucopyranoside-2
,3,4-trisphosphate and ,-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 of
DL-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.
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Introduction |
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Summary
Introduction
Procedures
Results
Discussion
References
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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)
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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.
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Experimental Procedures |
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Summary
Introduction
Procedures
Results
Discussion
References
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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) and
45Ca2+ (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 by
31P 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 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.5 mM Na2HPO4, 5.5 mM glucose, 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 mM KH2PO4, 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 µM ionomycin. Release of 45Ca2+ 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 retained 45Ca2+ 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 mM MgCl2, 20 mM HEPES, 2 mM EGTA, 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)P3
binding 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 µM
nonradiolabeled 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.
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Results |
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Summary
Introduction
Procedures
Results
Discussion
References
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Ca2+ release from permeabilized platelets. Rabbit platelets permeabilized with saponin and in the presence of oligomycin displayed ATP-dependent 45Ca2+ 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 the 45Ca2+-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 of 45Ca2+ from preloaded intracellular stores (Fig. 2). A time of 3 min was chosen because 45Ca2+ 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 of 45Ca2+ from the stores of permeabilized platelets; however, it displayed a ~55-fold lower EC50 value than Ins(1,4,5)P3 (Table 1). 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 released
45Ca2+ 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 release
45Ca2+ 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).
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,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)P3
caused 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).
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,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 for
45Ca2+ 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 the
45Ca2+-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).
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Displacement of specific [3H]Ins(1,4,5)P3 binding 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).
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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 the
45Ca2+-release data,
DL-6-CH2OH-scyllo-Ins(1,2,4)P3
seemed to be equipotent to Ins(1,4,5)P3 in
displacement of
[3H]Ins(1,4,5)P3, whereas
DL-scyllo-Ins(1,2,4)P3 was
~4-fold less potent (Fig. 6 and Table
1).
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Discussion |
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Summary
Introduction
Procedures
Results
Discussion
References
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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). Takahashi et 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)P3 at displacing [3H]Ins(1,4,5)P3 with similar IC50 values of 0.74 and 38 nM, respectively. In rat cerebellar microsomes, Takahashi et al. demonstrated the ED50 values for Ca2+ release to be 1.4 and 170 nM for 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)P3 responsible 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)P3
binding 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 a
C2 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)P3
displacement is not immediately clear, but it seems unlikely that it is
due to the assay conditions because Glc(2,3,4)P3 and 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 between
DL-scyllo-Ins(1,2,4)P3 and
DL-6-CH2OH-
scyllo-Ins(1,2,4)P3 may have
implications for the effect of the 5-hydroxymethyl group in
adenophostin A. The observation that
DL-6-CH2OH-scyllo-Ins(1,2,4)P3
is 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
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.).
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
| |
Abbreviations |
|---|
Ins(1, 4,5)P3,
D-myo-inositol-1,4,5-trisphosphate;
Glc(2, 3,4)P3,
(2-hydroxyethyl)--D-glucopyranoside-2,3,4-trisphosphate;
Trehal(3, 4,3,4)P4,
,-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.
| |
References |
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|
Summary
Introduction
Procedures
Results
Discussion
References
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