Table 1 Ca2+ release via IP3R evoked by guanophostin Aa
EC50/nM
Hill coefficient Ca2+ release (%)
N-terminus of helix a6 is consistent with a stabilizing interaction
with the helix dipole. This observation adds an extra element to
our original model.4a
Ins(1,4,5)P3
Adenophostin A
Guanophostin A
a
23.7 ¡ 3.7 1.30 ¡ 0.14
0.9 ¡ 0.1 1.20 ¡ 0.06
1.3 ¡ 0.3 0.99 ¡ 0.08
81 ¡ 1
81 ¡ 2
83 ¡ 1
In conclusion, for the first time we have achieved the synthesis
of a full agonist of the IP3R that is equipotent to adenophostin A.
This study reveals that the 2-position of a purine base is worthy of
exploration for further design to the enhance affinity of
Ins(1,4,5)P3R ligands. Further work in this area is underway.
We thank Dr Andrew Riley for useful discussions and the
Wellcome Trust for Programme Grant support (060544 to BVLP
and 072084 to CWT).
Results (means ¡ SEM, n = 3–6) show the concentration of each
ligand required to cause the half-maximal response (EC50), the Hill
coefficient, and the % of the intracellular stores released by a
maximal concentration of each ligand.
between the guanidinium side chain of Arg504 and the guanine
base of guanophostin was observed. An ab initio study of cation-p
interactions in protein-DNA complexes revealed that in general
those involving arginine and guanine are more stable than
arginine–adenine pairs.16 In addition to the base-protein interac-
tions observed in binding mode B of adenophostin A, which
include an N3–Arg269 hydrogen bond, additional potential
hydrogen bonding interactions exist between the guanine 2-amino
group and the binding core amino acid side chain of Glu505. The
only 2-substituted adenophostin derivative so far synthesized on an
adenine base is 2-methoxy-N6-methyl-adenophostin.4a When
evaluated for Ca2+ mobilization activity, this compound was ca.
six-fold less potent than adenophostin A and only two-fold more
potent than Ins(1,4,5)P3.Thus, (with the caveat that the IP3R
binding core is not the whole, much larger, receptor) the higher
potency of guanophostin A may illustrate the advantage of having
a H-bond donor motif at the C-2 position of the purine.
In the X-ray structure17 of the mouse IP3R1 binding core, the
1-phosphate group of Ins(1,4,5)P3 approaches the N-terminal of
alpha helix 6 (a6), comprising of residues 568–585 (a9 in ref. 17). It
is known that the dipole of an alpha helix can stabilise negatively
charged groups such as phosphate at its N-terminal.18 Thus, this is
a likely interaction for the analogous 29-phosphate of adenophos-
tin and its analogues, which may even be better aligned with the
axis of this alpha helix (Fig. 2). The fact that our model of
guanophostin A binding places the 29-phosphate closer to the
Notes and references
{ Data for 15; 1H NMR (400 MHz, D2O): d 3.67–3.85 (m, 6H, H-50,
H-6A0, H-6B0, H-5A9, H-5B9, H-20), 4.04 (dd, 1H, 16.0, 9.11 Hz, H-40), 4.37
(m, 1H, H-49), 4.40-4.45 (m, 1H, H-30), 4.52 (m, 1H, H-39), 5.16 (d, 1H,
3.73 Hz, H-10), 5.20–5.26 (m, 1H, H-29), 6.17 (d, 1H, 6.18 Hz, H-19), 9.00
(s, 1H, H-8). 13C NMR (100 MHz, D2O): d 60.03 (C-59), 60.47 (C-60), 70.21
(C-20, 31P coupled), 71.36 (C-50, 31P coupled), 72.95 (C-40, 31P coupled),
73.42 (C-39, 31P coupled), 75.39 (C-29, 31P coupled), 77.90 (C-30, 31P
coupled), 84.46 (C-49), 88.36 (C-19, 31P coupled), 98.04 (C-10), 108.31 (C-5),
136.53 (C-8), 149.38 (C-4), 154.84 (C-6), 155.18 (C-2). 31P NMR
(161.94 MHz, D2O with excess of TEA): d 3.278, 3.54, 4.297. m/z (ES) =
684.1 [(M 2 H), 100%]; HRMS: Mass calcd for C16H25N5O19P3 [M 2 H],
684.0362; Found, 684.0379.
1 M. J. Berridge, P. Lipp and M. D. Bootman, Nat. Rev. Mol. Cell Biol.,
2000, 1, 11.
2 M. Takahashi, T. Kagasaki, T. Hosoya and S. Takahashi, J. Antibiot.,
1993, 46, 1643.
3 M. Takahashi, K. Tanzawa and S. Takahashi, J. Biol. Chem., 1994, 269,
369.
4 (a) H. J. Rosenberg, A. M. Riley, A. J. Laude, C. W. Taylor and
B. V. L. Potter, J. Med. Chem., 2003, 46, 4860; (b) F. Chretien,
N. Moitessier, F. Roussel, J.-P. Mauger and Y. Chapleur, Current Org.
Chem., 2000, 4, 513.
5 V. A. Correa, A. M. Riley, S. Shuto, G. Horne, E. P. Nerou,
R. Marwood, B. V. L. Potter and C. W. Taylor, Mol. Pharmacol., 2001,
59, 1206.
6 R. A. Wilcox, C. Erneux, W. U. Primrose, R. Gigg and S. R. Nahorski,
Mol. Pharmacol., 1995, 47, 1204.
7 A. M. Riley, V. Correa, M. F. Mahon, C. W. Taylor and B. V. L.
Potter, J. Med. Chem., 2001, 44, 2108.
8 (a) A. A. Koshkin, J. Fensholdt, H. M. Pfundheller and C. Lomholt,
J. Org. Chem., 2001, 66, 8504; (b) M. J. Robins, R. Zou, Z. Guo and
S. F. Wnuk, J. Org. Chem., 1996, 61, 9207; (c) M. Zhong and
M. J. Robins, Tetrahedron Lett., 2003, 44, 9327.
9 C. Rosenbohm, D. S. Pedersen, M. Frieden, F. R. Jensen, S. Arent,
S. Larsen and T. Koch, Bioorg. Med. Chem., 2004, 12, 2385.
10 Y. Hayakawa and M. Kataoka, J. Am. Chem. Soc., 1998, 120, 12395.
11 M. Ashwell, C. Bleasdale, B. T. Golding and I. K. O’Neill, J. Chem.
Soc., Chem. Commun., 1990, 955.
12 X. Chen, E. R. Kern, J. C. Drach, E. Gullen, Y.-C. Cheng and
J. Zemlicka, J. Med. Chem., 2003, 46, 1531.
13 During treatment of sodium benzoxide with 2-amino-6-chlorpurine, in
addition to the expected formation 6-O-benzyl-guanine, the formation
of 2-N-benzyl guanine analogue have been reported. C. R. Frihart and
N. J. Leonard, J. Am. Chem. Soc., 1973, 95, 7174.
14 A. J. Laude, S. C. Tovey, S. G. Dedos, B. V. L. Potter, S. C. R. Lummis
and C. W. Taylor, Cell. Calcium, 2005, 38, 45.
15 G. Jones, P. Willett, R. C. Glen, A. R. Leach and R. Taylor, J. Mol.
Biol., 1997, 267, 727.
16 R. Wintjens, J. Lievin, M. Rooman and E. Buisine, J. Mol. Biol., 2000,
302, 395.
Fig. 2 The highest scored GOLD docked binding mode of guanophostin
A with the 29 endo conformation of the ribose ring shows the potential of
this molecule to interact with Arg269 and Glu505 in addition to the
previously proposed cation-p interaction between Arg504 and the purine
ring. A new 29-phosphate helix–dipole interaction is also illustrated.
17 I. Bosanac, J.-R. Alattia, T. K. Mal, J. Chan, S. Talarico, F. K. Tong,
K. I. Tong, F. Yoshikawa, T. Furuichi, M. Iwai, T. Michikawa,
K. Mikoshiba and I. Mitsuhiko, Nature, 2002, 420, 696.
18 W. G. J. Hol, P. T. Vanduijnen and H. J. C. Berendsen, Nature, 1978,
273, 443.
This journal is ß The Royal Society of Chemistry 2006
Chem. Commun., 2006, 2015–2017 | 2017