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Vol. 59, Issue 5, 1206-1215, May 2001
Department of Pharmacology, University of Cambridge, Cambridge, CB2 1QJ, United Kingdom (V.C., E.P.N., C.W.T.); and Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom (A.M.R., S.S., G.H., R.D.M., B.V.L.P.)
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results and Discussion
Conclusions
References
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Adenophostin A is the most potent known agonist of inositol
1,4,5-trisphosphate (InsP3) receptors. Ca2+
release from permeabilized hepatocytes was 9.9 ± 1.6-fold more sensitive to adenophostin A (EC50, 14.7 ± 2.4 nM)
than to InsP3 (145 ± 10 nM), consistent with the
greater affinity of adenophostin A for hepatic InsP3
receptors (Kd = 0.48 ± 0.06 and
3.09 ± 0.33 nM, respectively). Here, we systematically modify the
structures of the glucose, ribose, and adenine moieties of adenophostin
A and use Ca2+ release and binding assays to define their
contributions to high-affinity binding. Progressive trimming of the
adenine of adenophostin A reduced potency, but it fell below that of
InsP3 only after complete removal of the adenine. Even
after substantial modifications of the adenine (to uracil or even
unrelated aromatic rings, retaining the 
-orientation), the analogs
were more potent than InsP3. The only analog with an
-ribosyl linkage had massively decreased potency. The 2'-phosphate
on the ribose ring of adenophostin A was essential and optimally active
when present on a five-membered ring in a position stereochemically
equivalent to its location in adenophostin A. Xylo-adenophostin, where xylose replaces the glucose
ring of adenophostin A, was only slightly less potent than adenophostin
A, whereas manno-adenophostin (mannose replacing glucose) had similar potency to InsP3. These results are
consistent with the relatively minor role of the 3-hydroxyl of
InsP3 (the equivalent is absent from
xylo-adenophostin) and greater role of the equatorial
6-hydroxyl (the equivalent is axial in
manno-adenophostin). This is the first comprehensive
analysis of all the key structural elements of adenophostin A, and it
provides a working model for the design of related high-affinity
ligands of InsP3 receptors.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
Conclusions
References
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Inositol
1,4,5-trisphosphate (InsP3) receptors are
intracellular Ca2+ channels that are expressed in
most mammalian cells, where they are largely responsible for mediating
the release of Ca2+ from intracellular stores
evoked by many cell-surface receptors. Functional
InsP3 receptors are homo- or hetero-tetrameric
assemblies of subunits encoded by three related mammalian genes and
their splice variants (Taylor et al., 1999
). Each of these assemblies is capable of forming a Ca2+ channel that is
regulated by InsP3 and cytosolic
Ca2+ (Miyakawa et al., 1999), but the subtypes
differ in their distributions and in some aspects of their regulation
(for review, see Taylor et al., 1999).
Exhaustive analyses of both naturally occurring and synthetic inositol
phosphates and their analogs have so far identified only agonists of
InsP3 receptors and none has substantially
exceeded the affinity of the naturally occurring ligand
InsP3. A few, generally low-affinity, partial
agonists have been identified (Marchant et al., 1997b; Wilcox et al.,
1998; Murphy et al., 2000), but no antagonists. Indeed, the only
effective antagonists of InsP3 binding to its
receptor are heparin and decavanadate, neither of which has either high
affinity or adequate selectivity for InsP3
receptors (Taylor and Richardson, 1991). Caffeine, the xestospongins (Gafni et al., 1997), and 2-aminoethoxydiphenyl borate (Maruyama et
al., 1997) also block InsP3 receptors, but at
sites distinct from the InsP3-binding site and
they too lack either specificity or high affinity (Wilcox et al., 1998;
Short and Taylor, 2000).
All known high-affinity agonists of InsP3
receptors include structures analogous to the equatorial
4,5-bisphosphate groups and equatorial 6-hydroxyl of
InsP3 (Potter and Lampe, 1995; Wilcox et al.,
1998) (Fig. 1). The binding site itself
lies within the N-terminal portion of each InsP3
receptor subunit and includes several basic residues, which are
conserved between all three receptor subtypes and are likely to
interact with the negatively charged phosphate groups of
InsP3 (Yoshikawa et al., 1996). More recent
studies have shown that the InsP3-binding site of
the type 1 InsP3 receptor is formed by two
distinct domains of about 120 and 250 residues linked by a loop that
includes the SI splice site (Yoshikawa et al., 1999).
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The demonstration that adenophostins, products of the fungus
Penicillium brevicompactum, are the most potent known
agonists of InsP3 receptors (Takahashi et al.,
1994a) introduced fresh opportunities to develop high-affinity ligands
of InsP3 receptors. Adenophostins A and B are not
metabolized by the enzymes that degrade InsP3,
they do not bind to InsP4 receptors (Takahashi et al.,
1994a), and in both functional and radioligand binding assays of
all three InsP3 receptor subtypes they bind with
about 10-fold greater affinity than InsP3
(Takahashi et al., 1994b; Hirota et al., 1995; Marchant et al., 1997a;
Murphy et al., 1997; Missiaen et al., 1998; Shuto et al., 1998; Bird et
al., 1999; Marwood et al., 1999).
The structure of adenophostin A suggests that its glucose
3",4"-bisphosphate structure and adjacent 2"-hydroxyl may mimic the
critical 4,5-bisphosphate and 6-hydroxyl of InsP3
(Fig. 1). The adenine group may increase the strength of the binding
either by improving the positioning of the 2'-phosphate of adenophostin A (analogous to the 1-phosphate of InsP3) or
through a more direct interaction with a residue (possibly aromatic)
close to the InsP3-binding site of the receptor
(Takahashi et al., 1994a; Marchant et al., 1997a; Hotoda et al., 1999).
The charge distribution on the phosphate groups of adenophostin A at
physiological pH is virtually identical with that in the equivalent
phosphate groups of InsP3 (Felemez et al., 1999).
In the present study, we examine the effects of a range of synthetic adenophostin A analogs on 45Ca2+ release from the intracellular stores of permeabilized hepatocytes. We provide a comprehensive and systematic analysis of the roles of the ribose and purine rings, the glucose ring, and the stereochemistry of the links between these structures in mediating the high-affinity interaction between adenophostin A and InsP3 receptors.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
Conclusions
References
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Materials.
InsP3 (1) was
from American Radiolabeled Chemicals Inc. (St. Louis, MO). Thapsigargin
was from Alamone Laboratories (Jerusalem, Israel), and ionomycin was
from Calbiochem (Nottingham, UK). The analogs of adenophostin A were
synthesized as follows: adenophostin A (2) (Marwood et al.,
2000a,c); purinophostin (3) and imidophostin (5)
(Marwood et al., 2000b); ribophostin (6) (Jenkins et al.,
1997); Glc(2',3,4)P3 (8) (Jenkins and Potter,
1996); 27-29 (Marchant et al., 1997a);
furanophostin (7) (Marwood et al., 1999); 17, 18, and 26 (Shuto et al., 1998); 9 and
10 (Rosenberg et al., 2000); acyclophostin (19)
and 25 (Van Straten et al., 1997);
20-23 (Rosenberg et al., 2001);
xylo-adenophostin (11) and
manno-adenophostin (12) (Marwood et al., 2000c);
uridophostin (13); and 4, 14, and
30 (Marwood et al., 2000d). The syntheses of 15,
16, and furanophostin 3,4-bisphosphorothioate (24, furanophostin-PS2) will be
reported elsewhere. The structures of the analogs used, together with
their abbreviations, are shown in Fig. 2.
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45Ca2+ Release from the Intracellular
Stores of Permeabilized Cells.
Hepatocytes were isolated from the
livers of male Wistar rats (200-300 g) as described previously
(Marchant et al., 1997a) and permeabilized in
Ca2+-free cytosol-like medium (140 mM KCl,
20 mM NaCl, 2 mM MgCl2, 1 mM EGTA, 20 mM PIPES,
pH 7.0) by incubation with saponin (10 µg/ml) at 37°C for 8 min.
The cells were resuspended (2.2 × 106
cells/ml) in cytosol-like medium supplemented with 300 µM
CaCl2 (free [Ca2+] = 200 nM), ATP (1.5 mM), creatine phosphate (5 mM), creatine phosphokinase (1 unit/ml), the mitochondrial inhibitor
p-trifluoromethoxyphenylhydrazone (10 µM), and
45CaCl2 (5 µCi/ml). After
5 min at 37°C, during which the intracellular stores loaded to steady
state with 45Ca2+,
InsP3, adenophostin, A or an analog were added,
together with thapisgargin (1 µM) to inhibit further
Ca2+ uptake. After a further 1 min, the
45Ca2+ contents of the
stores were determined by filtration through Whatman GF/C filters
followed by washing with ice-cold sucrose (310 mM) and sodium citrate
(1 mM). The actively accumulated
45Ca2+ content of the
stores was defined as that which could be released by ionomycin (10 µM).
[3H]InsP3 Binding to Hepatic
Membranes.
Membranes were prepared from perfused rat livers using
Percoll-gradient centrifugation as described previously (Beecroft et al., 1999). Briefly, after perfusion in situ with cold buffered saline,
the liver was removed, homogenized with a Dounce homogenizer in cold
buffered sucrose (25 ml; 250 mM sucrose, 5 mM Hepes, 1 mM EGTA, pH
7.4), filtered through gauze, and then centrifuged (25,000g,
10 min). The pellet was resuspended in buffered sucrose (48 ml)
containing Percoll (11.8% final v/v; Amersham Pharmacia Biotech,
Uppsala, Sweden), recentrifuged (35,000g, 30 min), and the
membranes harvested as a fluffy band just beneath the fatty layer at
the top of each tube. The membranes in hypo-osmotic medium (1 mM EGTA,
5 mM HEPES, pH 7.4, 2°C) were centrifuged (48,000g, 10 min) and the pellet resuspended in binding medium (BM: 50 mM Tris, 1 mM
EDTA, pH 8.3, 2°C) at ~20 mg of protein per millilter, before
freezing in liquid nitrogen and storage at 
80°C.
), bound
and free ligand were separated by centrifugation (20,000g, 5 min, 4°C). Total binding was typically 3000 dpm/tube, of which
~30% was specific.
Analysis.
Equilibrium-competition binding curves were fitted
to logistic equations using nonlinear curve-fitting routines
(Kaleidagraph; Synergy Software, Reading, PA):
![B=<FR><NU>(T−N)</NU><DE>1+<FENCE><FR><NU>[L]</NU><DE>IC<SUB>50</SUB></DE></FR></FENCE><SUP>n<SUB><UP>H</UP></SUB></SUP></DE></FR>+N](/content/vol59/issue5/fulltext/1206/img001.gif)
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Results and Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
Conclusions
References
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Adenophostin A Is a Potent Agonist of Hepatic InsP3
Receptors.
The results shown in Fig.
3 confirm previous work (Marchant et al.,
1997a) by demonstrating that adenophostin A is a full agonist of
hepatic InsP3 receptors with about 10-fold
greater affinity than InsP3. In assays of
Ca2+ mobilization, adenophostin A was 9.9 ± 1.6-fold more potent than InsP3, and in
radioligand binding assays it had 6.4 ± 1.1-fold greater affinity
(Table 1). Similar results have been
obtained with other tissues (see above). Whereas our previous
work (Marchant et al., 1997a) used adenophostin A purified from
P. brevicompactum (Takahashi et al., 1994a), the present
results were obtained with synthetic adenophostin A (Marwood et al.,
2000a). However, in parallel comparisons synthetic
(EC50 = 9.6 ± 1.0 nM,
nH = 2.46 ± 0.19, n = 3) and natural (EC50 = 9.2 ± 1.7 nM,
nH = 2.99 ± 0.32, n = 3) adenophostin A had indistinguishable effects on
Ca2+ release from permeabilized hepatocytes
(Marwood et al., 2000a). The similarity is important in providing the
justification for subsequent analyses of the effects of synthetic
analogs of adenophostin A.
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10 µM released the same fraction of the intracellular stores as a
maximal concentration of InsP3 (~50%).
Furthermore, when a maximally effective concentration of any one of the
analogs was combined with 10 µM InsP3, the
response was no greater than that evoked by the
InsP3 alone (data not shown). Although the fraction of the stores released by a maximally effective concentration of InsP3 varied somewhat between experiments
(29-55%), when averaged across all the experiments reported here,
50 ± 4% of the actively accumulated
45Ca2+ was released by 10 µM InsP3 (Table 1). An indistinguishable response was evoked by a maximal concentration (1 µM) of adenophostin A (Table 1). The responses to InsP3, adenophostin
A, and each of the active analogs were positively cooperative (Hill
coefficients, nH > 1) (Table
2). None of the inactive
(18, 23) or very weakly active (10,
22, 25, 26) analogs behaved as
competitive antagonists: the response to a submaximal concentration of
InsP3 (200 nM) was undiminished in the presence
of 10 µM of any of these analogs (data not shown).
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Role of the Adenosine Moiety in High-Affinity Binding of
Adenophostin A.
Adenophostin A (2, Fig. 1) can be
viewed as a phosphorylated glucose glycosidically linked at its
1"-position to an adenosine phosphorylated at the 2'-position. In the
first series of experiments, we examined the functional consequences of
systematically trimming the adenosine (Fig.
4). The results show that as elements of
the adenine (2-6, 17) and then of the ribose (7-10) ring were successively removed,
there was a progressive decrease in the potency of the analog to evoke Ca2+ mobilization. Analogs (ribophostin,
6; furanophostin, 7; and 17) without
an adenine moiety were only slightly less potent than
InsP3, but after complete removal of the adenine and opening of the five-membered ring
[Glc(2',3,4)P3, 8] the potency fell
to 12 ± 1-fold less than that of InsP3.
Shortening of the flexible O-C-C chain in 8 by two atoms to
give the -C-glycoside 9 caused a further
slight reduction in potency. Interestingly, the isomeric
-C-glycoside 10 was much weaker, despite its
apparent resemblance to InsP3. Also, in relation
to 6 (ribophostin), some of us have recently reported that elaboration of the methyl group in 6 to propyl or
phenylpropyl groups did not appreciably affect potency (De Kort et al.,
2000).
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), and removal of the 2'-phosphate from adenophostin A reduced affinity by about 100-fold (Takahashi et al., 1994b). There
is a similar 100- to 300-fold difference in the affinities of
Ins(4,5)P2 and
Ins(1,4,5)P3 for InsP3
receptors (Nerou et al., 2001), in keeping with the idea that the
2'-phosphate of adenophostin A mimics the 1-phosphate of
Ins(1,4,5)P3 (Fig. 1). The conclusion gains
further support from the 159 ± 19-fold lesser potency of 25 relative to acyclophostin (19) (Fig. 5B); the major difference between them being the loss of the 2'-phosphate from
25 (Table 2).
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Effects of Modifying the Nucleoside and Its Links with the Glucose
Ring.
The effects of modifying the base (adenine) of adenophostin
A on biological activity are shown in Fig. 5A. Removal of the amino
group from the adenine (to give purinophostin, 3) had a
minimal effect. Replacement of the pyrimidine ring of the adenine by a
benzene ring (4) reduced potency by 4.0 ± 1.0-fold,
and its complete removal reduced the potency of the analog
(imidophostin, 5) to almost that of
InsP3 (Fig. 5A). Even replacement of the purine
moiety (adenine) of adenophostin A with a much smaller pyrimidine
(uracil, to give uridophostin, 13) caused the potency to
decrease by only 2.31 ± 0.40-fold. Finally, replacement of the
entire adenine moiety of adenophostin A with unrelated single
(14) or double (15, 16) aromatic rings
with a -C-ribosyl glycosidic linkage produced ligands
with activities comparable with that of 4 and
significantly greater than that of InsP3 (Fig.
5A). As expected, changing the stereochemistry of the
C-glycosidic linkage of 14 from to (30) massively decreased potency (by 29 ± 11-fold relative to 14, and to 11.8 ± 2.8-fold less than that
of InsP3). Hence, although large aromatic groups
attached to the 1'-position in a -orientation improve potency
(14 is 10.2 ± 1.9-fold more potent than
17), they have the opposite effect when attached in the
-orientation (30 is 2.8 ± 0.7-fold less
potent than 17). The effect may be caused by steric
hindrance around the important 2"-OH and/or the 2'-phosphate group,
which may occur with the - but not the -oriented rings.
-glycosidic linkage gives
the most potent agonist (adenophostin A), even radical changes to the
structure of the attached group are tolerated. Each of the analogs with
a -glycosidically linked aromatic system (3-5 and 13-16) is significantly more potent than
17 in which there is no substitution at the 1'-position.
In the adenophostins, the 2'-phosphate group is separated from the
-glucopyranosyl ring by a three-atom -O-C-C- chain. It seems that
some conformational restraint of this chain by incorporation of its two
C atoms into a five-membered ring of appropriate stereochemistry (e.g.,
ribose) is necessary for optimal activity. However, removal of part of
the ribose ring to give an apparently flexible structure (acyclophostin, 19) produces an analog with significant activity, providing the adenine is retained. Acyclophostin is unusual
in that its efficacy is affected by pH (Beecroft et al., 1999), but
under the conditions used to compare analogs in this study, it was only
1.37 ± 0.13-fold less potent than InsP3.
Acyclophostin (19) certainly has much greater potency than
8 (which lacks the adenine moiety of 19) or
25, (which lacks the 2'-phosphate) (Fig. 5B). These results
suggest that even when the critical 2'-phosphate is positioned on a
flexible link between the adenine and glucose ring, it can still
contribute significantly to binding affinity.
Other ring structures can effectively replace the ribose of
adenophostin A to give analogs (27-29) (Fig. 2) more potent than 18. However, even the most active of these analogs (28), which is more potent than 8, is
still 6.0 ± 0.3-fold less potent than ribophostin (6),
where the 2'-phosphate is attached to a ribose ring (Fig. 5C).
It is interesting that analog 26, which is a regioisomer of
the relatively potent 17, shows very low activity. The
likely reason is that the stereochemical differences between 26 and 17 at the positions corresponding to C-2' and -3' of adenophostin A result in a different three-dimensional positioning of the 2'-phosphate group at the receptor binding site.
Steric hindrance from the differently orientated hydroxymethyl group
and five-membered ring may also be involved. Similar factors may
account for the low activity of 27, the fructose component of which retains the required stereochemistry, but has a different type
of glycosidic linkage to glucose and an extra hydroxymethyl group.
We conclude that of the analogs examined so far, a ribose ring (or
close relative) between the pyranosyl and adenine moieties positions
the 2'-phosphate most effectively for optimal binding.
Modifications to the Glucose Ring of Adenophostin A.
Whereas
InsP3 is based on a myo-inositol ring
structure, the analogous 4,5-bisphosphate and 6-hydroxyl groups in
adenophostin A are attached to a glucose ring. The functional
consequences of modifying this sugar are shown in Fig.
6A.
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; Nerou et al., 2001).
Manno-adenophostin (12) in which mannose replaces
the glucose of adenophostin A, is substantially less potent (12.2 ± 2.3-fold) than the parent compound, although still only 1.51 ± 0.15-fold less potent than InsP3 (Fig. 6A). The
potency of 12 is surprising because although it differs from
adenophostin only in the orientation of the hydroxyl equivalent to the
6-hydroxyl of InsP3 (Fig. 1), inversion or
removal of the 6-hydroxyl in inositol phosphates invariably reduces
potency by at least 100-fold (Kozikowski et al., 1993; Wilcox et al.,
1994; Nerou et al., 2001). There are no other known high-affinity
agonists of InsP3 receptors lacking a hydroxyl
equivalent to the equatorial 6-hydroxyl of InsP3.
It has been suggested that the 6-OH group of
InsP3 may donate an H-bond to the receptor
(Kozikowski et al., 1993), an interaction that is presumably disrupted
when this group is axial. However, an additional role for the
equatorial 6-OH has been proposed, in which it may influence the
behavior of the crucial bisphosphate by mediating its interaction with
the 1-phosphate (Felemez et al., 2000). Changing the orientation of the
6-OH to axial (InsP3 to
epi-InsP3) alters the communication
between the 4,5-bisphosphate and 1-phosphate so that they behave
effectively as if isolated from one another. This "inframolecular"
effect may partly underlie the greatly decreased activity of
epi-InsP3 (Felemez et al., 2000). In
adenophostin A, the analogous 3",4"-bisphosphate and 2'-phosphate group
are located on separate rings. The effect of modifying the mediating
hydroxyl group (2"-OH) may therefore be different from that in
InsP3.
Stereochemistry of the O-Glycosidic Linkage. Because xylo-adenophostin (11) is almost as potent as adenophostin A (2), and xylose-based analogs are both simpler and require fewer chemical steps for their synthesis than glucose-based analogs, we chose to explore the functional consequences of modifying the stereochemistry of the O-glycosidic linkage between the pyranosyl and furanosyl moieties using xylose-based analogs (Fig. 6B).
As shown above, 11 is only 1.9 ± 0.6-fold less potent than 2. Just as furanophostin (7), which lacks the adenine and 4'-CH2OH of adenophostin A, is 22 ± 5-fold less potent than its parent (2), so 20, the xylose-based analog of furanophostin, is 17 ± 3-fold less potent than its parent (11). The similarities establish that modifications to the adenosine moiety have similar consequences for glucose- and xylose-based analogs. These results provide the justification for using 20 to explore the functional consequences of changing the stereochemistry of the O-glycosidic linkage between the furanosyl and pyranosyl rings (Fig. 6B). Changing the stereochemistry of the O-glycosidic linkage to xylose from (20) to (23) massively
decreased potency, so that 23 was effectively inactive. It
is likely that in low-energy conformations of 23, the
three-dimensional location of the third phosphate group is very
different from that in 20, and that it is therefore unable
to mimic the 1-phosphate of InsP3 or the
2'-phosphate of adenophostin A. Analog 22 is
stereochemically similar to the glucose-based 26, and they
show similar low potency. Unlike 26, however, 22 has no hydroxymethyl groups and so it is likely that its weak activity
(and by implication that of 26) is again simply attributable
to incorrect spatial positioning of its nonvicinal phosphate group. It
is interesting that analog 21, which combines the
stereochemical changes to the tetrahydrofuranyl ring of 22 with a change from an - to a -O-glycosidic linkage shows significant activity (19 ± 2-fold weaker than
InsP3). Molecular models suggest that the
combined effect of the stereochemical changes in 21 is to
return the nonvicinal phosphate group to a spatial orientation more
closely approaching that in 20 than in 22,
23, or 26 (Rosenberg et al., 2001).
Phosphorothioate Modifications to the Phosphate Groups.
Substitution of phosphates by phosphorothioate groups in certain
inositol phosphates can give partial agonists (Potter and Lampe, 1995;
Murphy et al., 2000), and might thereby provide a step toward the
development of antagonists. However, the strategy has so far been
limited by the relatively low affinity of these phosphorothioate
analogs. It seems that partial agonists of this type can be generated
by combining a slight structural perturbation of the
InsP3 molecule (particularly at position 3) with
phosphorothioate substitution (Potter and Lampe, 1995). Replacement of
the 4- and 5-phosphate groups of
3-deoxy-3-fluoro-Ins(1,4,5)P3 with
phosphorothioates, for example, produced a partial agonist with an
affinity only 10-fold less than InsP3 for type 1 InsP3 receptors (Wilcox et al., 1997). Thus, it
seems that higher affinity partial agonists may be created by limiting
phosphorothioate substitution to the bisphosphate, leaving the
1-phosphate group, with its ability to enhance affinity, unchanged.
). However, maximal concentrations of InsP3
and furanophostin-PS2 caused similar amounts of
Ca2+ to be released from the intracellular stores
(41 ± 2%, n = 3 for each). Furthermore, in both
functional and binding assays furanophostin-PS2
was about 12-fold less potent than InsP3 (Tables 2 and 3). Although rapid measurements of
rates of Ca2+ release would be required to
unequivocally establish the efficacy of
furanophostin-PS2 (Marchant et al., 1997b), our
results are certainly consistent with the conclusion that
furanophostin-PS2 is a full agonist of hepatic
InsP3 receptors. A possible explanation for the
unexpectedly high efficacy of furanophostin-PS2
may be that its 5"-hydroxymethyl group rather closely mimics the
3-hydroxyl of InsP3 and therefore fails to
provide sufficient structural perturbation around the pseudo-3 position
to significantly reduce the efficacy of
furanophostin-PS2. Future attempts to develop high-affinity ligands with low intrinsic activity may therefore require
more substantial modifications to the 5"-position of adenophostin analogs.
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Binding of Adenophostin A Analogs to Hepatic InsP3 Receptors. Because InsP3-binding sites are present at low density in hepatocytes and the only available radioligand ([3H]InsP3) has relatively low specific activity, radioligand binding analyses are more demanding and costly than functional assays; they must also be performed in a medium (BM) with high pH and low ionic strength. For these reasons, our radioligand binding analyses of adenophostin A analogs have focused on only the key ligands.
The results are summarized in Table 3. In Fig. 7 the affinity of each analog is expressed relative to the affinity of InsP3 measured in parallel experiments (under Experimental Procedures); the analogous comparisons from functional assays are shown alongside. Other than acyclophostin (19), which we previously suggested was a partial agonist at pH 8.3 (the pH used for binding assays) but a full agonist at pH7 (Beecroft et al., 1999),
there were no substantial disparities between the binding and
functional assays. The rank orders of the analogs shown, which included
modifications to each of the major elements of adenophostin A, were
similar in binding and functional assays (Fig. 7). The results are
therefore consistent with each of the analogs
(1-3, 5, 6, 12,
13, and 24) being a full agonist, differing from
adenophostin A only in its affinity for hepatic InsP3 receptors.
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Conclusions |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
Conclusions
References
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Adenophostin A is the most potent known agonist of
InsP3 receptors. None of the modifications we
have made to the structure of adenophostin A has succeeded in either
increasing its affinity for InsP3 receptors or in
changing its efficacy. We have, however, designed new ligands based
upon adenophostin A with potencies higher than that of
InsP3. Using both these ligands and simplified analogs of adenophostin A, we have established which structural elements of adenophostin A determine its high affinity for
InsP3 receptors (Fig.
8) in hepatocytes, which express
predominantly type 2 InsP3 receptors (Taylor et
al., 1999).
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The glucose 3",4"-bisphosphate and adjacent 2"-hydroxyl of adenophostin
A are thought to mimic the critical 4,5-bisphosphate and 6-hydroxyl of
InsP3 (Fig. 1) (Takahashi et al., 1994a). The 2'-phosphate of adenophostin A is thought to mimic the 1-phosphate of
InsP3 and for both ligands removal of this
phosphate causes the affinity to decrease by at least 100-fold (Fig. 4)
(Shuto et al., 1998). The 3-hydroxyl of InsP3
contributes little to binding (Kozikowski et al., 1993; Nerou et al.,
2001), and likewise removal of the analogous hydroxyl from adenophostin
A (to give xylo-adenophostin, 11) has minimal
effect. These results suggest that structure-activity relationships
derived from inositol phosphates can be used to predict the activity of
adenophostin analogs, at least for some substituents of the pyranosyl
ring. However, the unexpectedly high potency of
manno-adenophostin (12) suggests that the roles
of the 2'-hydroxyl of adenophostin A and the 6-hydroxyl of
InsP3 are not exactly analogous: this hydroxyl
group is clearly more important for InsP3 than
adenophostin A binding.
We have explored in detail the contribution of the adenine group to the
high-affinity binding of adenophostin A to InsP3
receptors. Progressive removal of elements of the adenine cause
progressive decreases in binding affinity (Fig. 4), but the adenine can
be replaced by completely unrelated aromatic ring systems
(13, 14, and 16) with only modest
decreases in potency (Fig. 5A). The stereochemistry of the link between
the furanosyl ring and the aromatic rings is, however, crucial:
-1'-aromatic substituent (as occurs in adenophostin A) enhances
affinity, whereas a similar group in the -orientation has the
opposite effect (Fig. 5A). A phosphate group at the position equivalent
to the 1-position of InsP3 (2' in adenophostin A)
is essential (see above) and optimally active when attached to a
furanosyl ring (Fig. 5). The stereochemistry of the
O-glycosidic link between the pyranosyl and furanosyl rings is crucial, with an -glycosidic linkage (as occurs in adenophostin A) optimally positioning the pseudo 1-phosphate.
As the most potent stable agonist of InsP3 receptors, adenophostin A is already widely used to examine the mechanisms underlying intracellular Ca2+ regulation. Our analyses of the key determinants of the high-affinity interaction between adenophostin A and InsP3 receptors should facilitate development of further related analogs with properties tailored to meet specific biological requirements.
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Acknowledgments |
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We thank Professor J. H. Van Boom (Leiden, the Netherlands) for gifts of acyclophostin and compound 25 and Dr. D. J. Jenkins and H. J. Rosenberg for useful discussions.
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Footnotes |
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Received December 6, 2000; Accepted January 29, 2001
This study was supported by program Grants from The Wellcome Trust to C.W.T. (039662) and B.V.L.P. (060554) and by a Wellcome Trust Prize Studentship (to R.D.M.).
Send reprint requests to: Dr. C. W. Taylor, Department of Pharmacology, University of Cambridge, Tennis Court Rd., Cambridge, CB2 1QJ, UK. E-mail: cwt1000{at}cam.ac.uk
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Abbreviations |
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InsP3, D-myo-inositol 1,4,5-trisphosphate; BM, binding medium; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid).
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
Conclusions
References
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-D-glucopyranoside-2',3.4-trisphosphate.
Carbohydr Res
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)
and adenophostin A (
). B, effects of the indicated concentrations of
InsP3 (
