Activation of IP3 receptors by synthetic bisphosphate ligands

Article abstract of DOI:10.1039/b819328b

Ca²⁺ release by d-myo-inositol 1,4,5-trisphosphate receptors (IP₃Rs) is widely considered to require the vicinal 4,5-bisphosphate motif of IP₃, with P-5 and P-4 engaging the α and β domains of the binding site; using synth

Full text of DOI:10.1039/b819328b

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Activation of IP receptors by synthetic bisphosphate ligandsw
3
a
a
b
b
Kana M. Sureshan, Andrew M. Riley, Ana M. Rossi, Stephen C. Tovey,
b
b
a
Skarlatos G. Dedos, Colin W. Taylor and Barry V. L. Potter*
Received (in Cambridge, UK) 30th October 2008, Accepted 20th January 2009
First published as an Advance Article on the web 4th February 2009
DOI: 10.1039/b819328b
2
+
Ca
IP
motif of IP
release by D-myo-inositol 1,4,5-trisphosphate receptors
Rs) is widely considered to require the vicinal 4,5-bisphosphate
, with P-5 and P-4 engaging the a and b domains of
2
fact that the bisphosphate Ins(4,5)P is a full agonist with low
1
affinity supports this suggestion. But the inability of
1
11
(
3
2
+
3
Ins(1,4)P
2
to mobilise Ca
3
or bind to IP R suggests that
the binding site; using synthesis and mutagenesis we show that
the adenine of synthetic glyconucleotides, through an interaction
with Arg504, can replace the interaction of either P-1 or P-5
with the a-domain producing, respectively, the most potent
bisphosphate agonist yet synthesised and the first agonist of
interaction of P-1 alone with the a-domain, where it directly
contacts only Arg568 (R568, Fig. 2), is too weak to stabilise
1
binding. Ins(1,5)P is also inactive presumably because it has
2
2
insufficient interactions with the b-domain. Hence, the pre-
vailing view is that the vicinal bisphosphate moiety of IP
3
is
IP
3
R without a vicinal bisphosphate motif; this will stimulate
R ligand design.
essential for activity.
Our molecular docking experiments have suggested that
1
3
new approaches to IP
3
0
the 2 -AMP motif of AdA interacts with the a-domain of
the IBC, perhaps more strongly than P-1 of IP . We therefore
D-myo-Inositol 1,4,5-trisphosphate (IP , 1, Fig. 1) is an intra-
3
3
2
+
00
cellular messenger that evokes Ca
release from the intra-
receptors
-gated Ca channels. Extensive struc-
ture–activity studies suggest that the vicinal 4,5-bisphosphate
structure of IP is essential, while the 1-phosphate has an
reasoned that it may be possible for 3 -dephospho-AdA
(3, Scheme 1) to bridge the domains effectively and release
cellular stores of most animal cells by binding to IP
3
2+
1,2
2+
Ca . Alternatively, if the 2 -AMP interacts with the
0
(
IP
3
R), which are IP
3
0
0
b-domain, it may allow 4 -dephospho-AdA (4) to activate
IP R. We therefore developed methods to synthesise 3 and 4
3
3
3
ancillary role that substantially increases affinity. For more
than 20 years, this enduring conclusion has guided design of
new ligands for the IP R. Other inositol phosphate regioisomers
(Scheme 1).z Briefly, thioglycosides 5a and 5b were used
to glycosylate a ribofuranose acceptor leading to disaccha-
rides 6a and 6b, which were individually subjected to
3
4
are active through different binding modes, but they all have
the vicinal bisphosphate structure. The fungal metabolite,
5
adenophostin A (AdA, 2, Fig. 1), which is at least ten times
¨
Vorbruggen condensations with silylated 6-chloropurine to
give 7a and 7b, the precursors for 3 and 4, respectively.
Ammonolysis of each installed the N-6 amino group and
exposed the required pairs of hydroxyl groups for phosphory-
lation. Phosphitylation in the presence of imidazolinium
triflate and in situ oxidation followed by removal of benzyl
protecting groups by transfer hydrogenolysis gave 3 and 4,
which were purified to homogeneity.
more potent than IP
explore the mechanisms of IP
nucleotide trisphosphate structurally related to IP
site that substantially overlaps the IP -binding site, and struc-
3
, provides additional opportunities to
R activation. AdA is a glyco-
. It binds to a
3
3
6
3
ture–activity studies with synthetic analogues of AdA have
established that the adenine (or another aromatic group) is
1
4
Using methods reported previously, we show that 4 does
2+
6,7
essential for its enhanced affinity.
The IP -binding core (IBC) of the IP R forms a clam-like
not evoke Ca
effective, albeit at high concentrations (Fig. 3). These results
3
release via recombinant IP R1, but 3 is
3
3
0
0
00
0
structure with IP held between two domains (a and b) linked
support the idea that P-3 and P-4 of AdA mimic P-5 and P-4
3
8
by a flexible hinge. The 4-phosphate group (P-4) of IP3
of IP3, respectively, and that the 2 -AMP motif of AdA is able
interacts mainly with residues in the b-domain, while the
to bind the a-domain of the IBC more effectively than P-1 of
1
-phosphate (P-1) and 5-phosphate (P-5) interact predo-
minantly with the a-domain (Fig. 2). We therefore speculated
that binding of IP might pull the two domains together in
IP
together and activate IP
bisphosphate. Bisphosphate 3, the AdA equivalent of
Ins(1,4)P , is the first agonist of IP R without a vicinal
bisphosphate motif.
3
. This allows bisphosphate 3 to pull the two domains
3
R1, even though it lacks a vicinal
3
a manner reminiscent of glutamate binding to ionotropic
9
glutamate receptors, and so cause the IBC to adopt a more
2
3
1
0
constrained structure. Because both P-1 and P-5 interact
with the a-domain, it may be possible to bridge the two
domains using a ligand with only one of these groups. The
a
Wolfson Laboratory of Medicinal Chemistry, Department of
Pharmacy and Pharmacology, University of Bath, Claverton Down,
UK Bath BA2 7AY. E-mail: B.V.L.Potter@bath.ac.uk;
Fax: +44 (0)1225386114; Tel: +44 (0)1225386639
Department of Pharmacology, University of Cambridge,
UK CB2 1PD
b
w Dedicated to Dr Melanie N. Trusselle (1973–2008).
3 3
Fig. 1 IP receptor ligands; IP (1) and adenophostin A (2).
1
204 | Chem. Commun., 2009, 1204–1206
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Fig. 2 Interactions of IP
3
with the a (blue) and b (green) domains of
the IBC of IP R1 (PDB code 1n4k). Water molecules are omitted and
3
only direct interactions between IP
shown.
3
and residues in the IBC are
0
Scheme 2 Synthesis of 2 -dephospho-AdA and structures of glucose
,4-bisphosphate and Ins(4,5)P : (a) K CO , BnOH, 70 1C, 93%;
b) Pd(OH) /C, cyclohexene, MeOH, H O, 80 1C, 92%. Bn = benzyl.
3
2
2
3
(
2
2
Phosphate groups are coloured according to putative interactions with
the a (blue) or b (green) domains of the IBC.
interactions with the IBC. To distinguish between these two
0
possible roles, we synthesised 2 -dephospho-AdA, the adeno-
phostin equivalent of Ins(4,5)P (8, Scheme 2). Interestingly,
2
1
treatment of a benzyl-protected version of AdA (9) with
5
0
sodium benzoxide in BnOH led to removal of the 2 -dibenzyl-
phosphate group giving bisphosphate 10 as the major product
with minor amounts of other by-products. Use of the milder
base potassium carbonate resulted in clean and highly selective
conversion into 10. Deprotection and purification as before
0
gave 8 in excellent yield. The 2 -phosphate triester in 9 is
comparatively less crowded and hence more prone to nucleo-
0
0
00
philic attack than those in the 3 ,4 vicinal positions and this is
likely to be the reason for this interesting high selectivity.
We also synthesised the related glucose 3,4-bisphosphate
(
11, Scheme 2). Because 11 can be viewed as AdA without
0
the 2 -AMP motif, any difference in the activities of 8 and 11
illustrates the role of the adenosine moiety. In functional
assays, 8 was 100 times more potent than 11 (Fig. 3), confirm-
ing that the adenosine motif directly contributes to AdA
00
00
Scheme 1 Structures and syntheses of 3 - and 4 -dephospho-AdA.
Phosphate groups are coloured according to putative interactions with
the a (blue) or b (green) domains of the IBC. Bn = benzyl.
0
binding even when it lacks the 2 -P group. Compound 8 is
the first potent synthetic bisphosphate agonist of the IP
3
R;
it is 430-fold more potent than Ins(4,5)P (Fig. 3, Table 1).
2
Our molecular modelling suggests a key role for arginine
04 (R504) in recognising the adenine of AdA, perhaps via a
5
1
3
cation–p interaction. Using targeted mutagenesis, we esta-
blished a cell line expressing only IP R1 with R504 mutated to
3
glutamine, to determine whether R504 selectively contributes
to the activity of AdA analogues. For the mutant IP R, the
3
EC50 for IP
3 2
and Ins(4,5)P was reduced by 57- and 8.8-fold,
respectively. For AdA and 8, the reductions were 434- and
2
+
Table 1 Ca release via IP
show the EC50 values for each agonist in DT40 cells stably expressing
IP R1
3
R1. Results (means ꢁ SEM, n = 3–8)
2+
3
Fig. 3 Concentration-dependent effects of ligands on Ca
via IP
R1 stably expressed in DT40 cells. Results show means ꢁ SEM;
n = 8.
release
3
Agonist
IP
Ins(4,5)P
AdA
8
EC50
3
35 ꢁ 5 nM
2
7.59 ꢁ 2.63 mM
2.9 ꢁ 0. 9 nM
240 ꢁ 5 nM
0
0
The 2 -AMP motif of AdA may allow P-2 to bind more
effectively than P-1 of IP , or the adenosine itself may have
3
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Notes and references
z All new compounds were thoroughly characterised and exhibited
satisfactory parameters using standard spectroscopic techniques.
A typical example follows.
0
00 00
Synthesis of 3 -O-(a-D-glucopyranosyl)-adenosine-3 ,4 -bisphosphate
8).
A suspension of bisphosphate 10 (45 mg, 0.037 mmol) and 20%
Pd(OH) on carbon (120 mg) in a mixture of cyclohexene (1.6 mL),
MeOH (3 mL) and H O (0.22 mL) was heated at 80 1C overnight. The
(
2
2
mixture was filtered through a membrane filter and solvents evaporated
in vacuo. Purification on an AG ion-exchange column (0–100% gradient
elution, with 150 mM TFA and water) afforded pure bisphosphate 8
1
00
00
(
20 mg, 92%). H NMR (400 MHz, D O): 3.74–3.90 (m, 6H, H-2 , H-5 ,
2
0
0
00
00
H-5A , H-5
9
B
H-6
A
, H-6
B
), 4.10 (ddd, like a q, 1H, 9.66 Hz, 9.66 Hz,
0
0
0
.66 Hz, H-4 ), 4.42 (dd, 1H, 7.24 Hz, 3.86 Hz, H-4 ), 4.47–4.55 (m, 2H,
0
00
0
H-3 , H-3 ), 4.87 (dd, like a t, 1H, 5.80 Hz, 5.31 Hz, H-2 ), 5.19 (d, 1H,
00 0
.38 Hz, H-1 ), 6.19 (d, 1H, 5.80 Hz, H-1 ), 8.40 (s, 1H, H-2), 8.50
13
3
(
7
Fig. 4 Suggested interactions of 8 with the IBC of IP
3
R1, based on
00
0
s, 1H, H-8). C NMR (100 MHz, D
2
O): 60.08 (C-6 ), 60.79 (C-5 ),
13
00 31 00 31
0.48 (C-2 , P coupled), 71.60 (d, 3.83 Hz, C-5 , P coupled), 72.83
31
molecular docking experiments using AdA. The side chain of R504
is shown in pale blue.
0
0
0
0
00 31
(C-4 , P coupled), 73.48 (C-2 ), 76.37 (C-3 ), 77.71 (C-3 , P coupled),
0 0 00
3.95 (C-4 ), 88.23 (C-1 ), 99.02 (C-1 ), 118.91 (C-5), 142.81 (C-8), 144.43
31
8
(
C-2), 148.16 (C-4), 149.91 (C-6). P NMR (161.94 MHz, D
2
O with
107-fold, and the response to 3 (r3 mM) was abolished. The
greater effect of this mutation on the potency of AdA and 8 is
+
excess of TEA): 4.50, 3.62. m/z (ES+) = 590.2 [(M + H) , 100%]; 612.1
+
+
[
(M + Na) , 100%]; m/z (ESꢂ) 588.2 [(M ꢂ H) , 100%]; HRMS: mass
+
H O N P [M + H] , 590.0895; found, 590.0895.
consistent with the proposed interaction between adenine and
calcd for C16
26 15 5 2
R504 (Fig. 4). Indeed, the potency of AdA and 8 at IP R1
3
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both depend largely on R504 because, at the mutated receptor,
AdA is only equipotent with IP
potent than Ins(4,5)P
3
, and 8 is less than 3-fold more
3
4
5
6
B. V. L. Potter and D. Lampe, Angew. Chem., Int. Ed. Engl., 1995,
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2
.
3
We conclude that, while the three phosphate groups and
adenine of AdA most likely make incremental contributions to
IP
IP
3
R binding, a vicinal bisphosphate moiety is not essential for
R activation. P-4 of IP , which contacts the b-domain of
1
994, 269, 369.
3
3
V. A. Correa, A. M. Riley, S. Shuto, G. Horne, E. P. Nerou,
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Pharmacol., 2001, 59, 1206.
the IBC, is required, but P-5, which contacts the a-domain,
can (as in 3) make an interaction that can be substantially
compensated by a cation–p interaction between the adenine of
AdA and R504 in the a-domain. The same interaction can also
serve to mitigate the loss of P-1 to provide a potent agonist (8)
with only two phosphate groups. Aside from challenging the
long-standing dogma that a vicinal bisphosphate is essential
7
H. Hotoda, K. Murayama, S. Miyamoto, Y. Iwata, M. Takahashi,
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9
C. W. Taylor, P. C. A. da Fonseca and E. P. Morris, Trends
Biochem. Sci., 2004, 29, 210.
for agonists of IP
3
R, these results illustrate the potential to
R ligands. At their simplest, these might
design less polar IP
comprise two motifs that interact with the IBC domains linked
by a suitable spacer.
3
10 J. Chan, A. E. Whitten, C. M. Jeffries, I. Bosanac, T. K. Mal,
J. Ito, H. Porumb, T. Michikawa, K. Mikoshiba, J. Trewhella and
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1
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and Lys residues, replacing interactions with a polar phos-
phate by cation–p interactions should have more general
applications in the chemical biology of inositol phosphate
signalling and probably elsewhere.
14 A. J. Laude, S. C. Tovey, S. G. Dedos, B. V. L. Potter,
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We acknowledge the support by Programme Grants from
the Wellcome Trust [082837 to AMR (Bath) and BVLP,
1
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0
72084 to CWT].
1
206 | Chem. Commun., 2009, 1204–1206
This journal is ꢀc The Royal Society of Chemistry 2009