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WO1998043992A1 - Cyclic adenosine diphosphate ribose analogues - Google Patents

Cyclic adenosine diphosphate ribose analogues Download PDF

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Publication number
WO1998043992A1
WO1998043992A1 PCT/GB1998/000921 GB9800921W WO9843992A1 WO 1998043992 A1 WO1998043992 A1 WO 1998043992A1 GB 9800921 W GB9800921 W GB 9800921W WO 9843992 A1 WO9843992 A1 WO 9843992A1
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cadpr
bromo
deaza
induced
compound
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Antony Galione
Barry Potter
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University of Bath
Oxford University Innovation Ltd
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University of Bath
Oxford University Innovation Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • Cyclic adenosine.diphosphate ribose is a ubiquitous Ca 2+ mobilizing metabolite of ⁇ -NAD + (1 , 2). It is reported to mediate Ca 2+ release via ryanodine sensitive channels in many cell types, in both animal and plant kingdoms (1 , 3-6). Endogenous levels of cADPR have been detected and reported to be equally widespread (7). This finding has led to the postulation that cADPR may be the endogenous/physiological regulator of ryanodine receptors (5, 8-10).
  • cADPR metabolising enzymes are also present which can modulate its levels (11).
  • the synthetic activity of ADP-ribosyl cyclase and catabolic activity of cADPR hydrolase are often co-localised on the same polypeptide. In these cases, the hydrolase activity often exceeds that of cyclase (1).
  • Aplysia ADP-ribosyl cyclase which is isolated and purified from soluble ovotestis extracts of the sea hare. Aplysia californica.
  • the first series of pharmacologically useful cADPR analogues to be synthesised were the 8-substituted analogues (13). These differ from cADPR by a substitution at the 8 position of the adenine ring. This single modification abolishes the agonistic activity of these compounds and instead produces specific competitive antagonists of cADPR-sensitive Ca 2+ release (13).
  • 8-NH 2 -cADPR has been used successfully to demonstrate the involvement of cADPR - mediated Ca 2+ signalling in sea urchin eggs during fertilisation (14) and NO and cGMP-induced Ca 2+ release (15), in Purkinje neurons (16), in hippocampal synaptic plasticity, in permeabilized Jurkat T cells (6), in intestinal smooth muscle during cholecystokinin-induced contractions (5), in PC12 cells (17) and in excitation contraction coupling in cardiac myocytes ( 8).
  • the 8- substituted analogues are prone to hydrolysis by endogenous enzymes (13).
  • ABBREVIATIONS cADPR cyclic adenosine 5'-diphosphate ribose
  • IP 3 myo-inositol-( 1 ,4,5)-trisphosphate; cAMP cyclic adenosine 3', 5'-monophosphate; cGMP cyclic guanosine 3', ⁇ '-monophosphate;
  • the invention provides compounds having the formula (1)
  • Y is halo, C1 - C20 hydrocarbon, NR 2 , OR, SR, nitro or carboxyl,
  • R is H or C1 - C20 hydrocarbon and R groups can be the same or different, and one or each Z represents H or one Z is a cageing group.
  • X 7 is CH and X 3 is N.
  • the preparation of such a compound is described below. Preparation of compounds where X 7 is not CH, or where X 3 is not N can readily be achieved by known chemistry using a different starting compound 6 (see the reaction scheme below).
  • Y is preferably halo, particularly bromo. This is the compound whose preparation is described in detail below. Alternatively Y may be amino. Alternatively Y may be iodo, either a natural or a radioisotope such as 125 l. Radiolabelled compound may be useful in an assay, eg. a competitive binding assay, to screen for other compounds with more potent binding characteristics for a cADPR receptor. Compounds where Y is other than bromo may readily be prepared inter alia using known chemistry with a different n-succinimide derivative in reaction (i).
  • R is H or C1 - C20 hydrocarbon e.g. alkyl, aryl, aralkyl, alkaryl, alkenyl or alkynyl and which may be substituted or unsubstituted.
  • R is H or C1 - C4 alkyl.
  • Either both groups Z represent hydrogen atoms (or negative charges) as in a conventional diphosphate system.
  • one group Z may be a "cageing" group e.g. 1-(o-nitrophenyl)ethyl, whereby the compound of the invention is a caged analogue adapted to be converted into an active compound in situ e.g. by light irradiation.
  • the chemistry of such caged analogues is well known, see Aarhaus R et al, J. Biol. Chem., 1995, 270, 7745; McCray J A and Trentham D R, Ann. Rev. Biophys., Biophys. Chem, 1989, 18, 239-270 and such compounds can be prepared by standard methods.
  • the two sugar rings have been shown as ribose for simplicity, it is envisaged that other groups may be present as in known nucleotide chemistry.
  • the 5'- oxygen atom may be replaced by S, CHF, CF 2 , SO, NR or CH 2 .
  • the 2'- and 3'-hydroxyl groups may be replaced by H, F, NH 2 , N 3 or O-hydrocarbyl.
  • Such compounds may be made by known chemical methods analogous to those described below.
  • 7-deaza-8-bromo-cADPR The synthesis of 7-deaza-8-bromo-cADPR. Briefly, 7-deaza- adenosine (Tubercidin) was brominated and selectively phosphorylated to yield 7-deaza-8-bromo-adenosine ⁇ '-monophosphate (7-deaza-8-bromo- AMP). This was then coupled to nicotinamide mononucleotide (NMN) to form 7-deaza-8-bromo- ⁇ -NAD + as has previously been described for 7- deaza- ⁇ -NAD + (20).
  • NPN nicotinamide mononucleotide
  • the material was purified by ion exchange chromatography and the fractions containing the product, which eluted between 90 and 120mM TEAB, were combined, the solvent removed in vacuo and excess TEAB removed by coevaporation with MeOH to yield the title compound in 27.7% yield.
  • Lytechinus pictus sea urchins were from Marinus Inc. Long Beach, CA. USA. Fluo-3 and Fura 2 was purchased from Cambridge Bioscience (Molecular Probes). All other chemicals were from Sigma inc. (London). Cyclic ADP-ribose, 8-bromo-cADPR and 7 deaza-cADPR were synthesised as previously described (12, 20).
  • Figure 1 A shows the chemical structure of 7-deaza-8-bromo-cADPR as compared with that of cADPR and two previously reported analogues 8-bromo-cADPR, a specific cADPR antagonist (10) and 7-deaza cADPR, a hydrolysis resistant, partial agonist (20). Whereas the latter two compounds have a single modification made at either the 7 or 8 position of the adenosine ring, 7-deaza-8-bromo- cADPR possesses both these modifications. The effect of each modification on the Ca 2+ mobilizing (agonistic) ability was first tested using sea urchin egg homogenates (2.5%).
  • Figure 1 B shows the Ca 2+ releasing action of 2 ⁇ M applications of 7-deaza-8-bromo-cADPR and related cyclic nucleotides.
  • 7-deaza-8- bromo-cADPR did not induce Ca 2+ release from sea urchin egg microsomes, even at concentrations up to 20 ⁇ M (a supra-maximal agonist concentration).
  • 7-deaza-8-bromocADPR resembles the antagonist 8-bromo-cADPR.
  • 7-deaza-8-bromo-cADPR like 8-bromo-cADPR, was also an antagonist of cADPR-sensitive Ca 2+ release was investigated next.
  • FIG. 2 shows that this was indeed the case.
  • Sea urchin egg homogenates pre-treated with either 8-bromo-cADPR or 7-deaza-8-bromo- cADPR were markedly less responsive to 100 nM cADPR (Figure 2A). These inhibitory actions were dependent on the antagonist concentration ( Figure 2B).
  • 7-deaza-8-bromo-cADPR should have greater hydrophobic character than any previously synthesised.
  • Eggs were co-injected with the 1 P 3 receptor antagonist, heparin (250 ⁇ g/ml) and Ca 2+ sensitive fluorochrome fura-2 (2 ⁇ M).
  • Figure 4A shows that upon sperm addition to control heparinized eggs, a propagating Ca 2+ wave was produced (Figure 4A, open squares).
  • Figure 1 (A) Structural formulae of cADPR 7-deaza-cADPR, 8-bromo- cADPR and 7-deaza-8-bromo-cADPR. Modifications were made to cADPR at the 7 and 8 positions of the adenosine ring. The N 7 nitrogen atom is replaced with a carbon (and associated proton) to form 7-deaza cADPR whilst 8-bromo-cADPR is formed by substituting the hydrogen on the C-8 position with a bromine atom. 7-Deaza-8-bromo-cADPR has both these modifications.
  • FIG. 2 Concentration dependent inhibition of cADPR-induced Ca 2+ release by 8-bromo-cADPR and 7-deaza-8-bromo-cADPR.
  • Lytechinus pictus egg homogenates (2.5 %, v/v) containing the Ca 2+ sensitive dye, fluo-3 (3 ⁇ M) were prepared as described in Materials and Methods.
  • Their sensitivity to cADPR-induced Ca 2+ release was used to test the antagonistic action of 8-bromo-cADPR (A) as compared to that of 7-deaza-8-bromo-cADPR (B).
  • Figure 3 Effect of heat treatment on antagonist activity of 8-bromo- cADPR and 7-deaza-8-bromo-cADPR.
  • FIG. 4 Antagonistic actions of extracellularly applied 7-deaza-8- bromo-cADPR and 8-bromo-cADPR on fertilisation-induced Ca 2+ transients in intact sea urchin eggs.

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Abstract

A compound having formula (1) where at least one of X?3 and X7¿ is CR and any remaining X3 or X7 is N; Y is halo, C1-C20 hydrocarbon, NR¿2?, OR, SR, nitro or carboxyl; R is H or C1-C20 hydrocarbon and R groups can be the same or different; and one or each Z represents H or one Z is a cageing group. The compound 7-deaza-8-bromo-cyclic adenosine 5'-diphosphate ribose has been shown to be a stable hydrolysis-resistant cADPR antagonist.

Description

CYCLIC ADENOSINE DIPHOSPHATE RIBOSE ANALOGUES
INTRODUCTION
Cyclic adenosine.diphosphate ribose (cADPR) is a ubiquitous Ca2+ mobilizing metabolite of β-NAD+ (1 , 2). It is reported to mediate Ca2+ release via ryanodine sensitive channels in many cell types, in both animal and plant kingdoms (1 , 3-6). Endogenous levels of cADPR have been detected and reported to be equally widespread (7). This finding has led to the postulation that cADPR may be the endogenous/physiological regulator of ryanodine receptors (5, 8-10).
As is the case for the more established intracellular messengers (i.e. IP3 cAMP and cGMP), cADPR metabolising enzymes are also present which can modulate its levels (11). The synthetic activity of ADP-ribosyl cyclase and catabolic activity of cADPR hydrolase are often co-localised on the same polypeptide. In these cases, the hydrolase activity often exceeds that of cyclase (1). However, one notable exception is Aplysia ADP-ribosyl cyclase which is isolated and purified from soluble ovotestis extracts of the sea hare. Aplysia californica. The exceptionally high level of cvclase activity exhibited by this enzyme (1) has been well exploited to synthesise large quantities of cADPR. In addition, the finding that this cyclase exhibits loose substrate specificity, has allowed the development of a chemo-enzymatic synthesis of a number of cADPR analogues ( 12).
The first series of pharmacologically useful cADPR analogues to be synthesised were the 8-substituted analogues (13). These differ from cADPR by a substitution at the 8 position of the adenine ring. This single modification abolishes the agonistic activity of these compounds and instead produces specific competitive antagonists of cADPR-sensitive Ca2+ release (13). Since its discovery, 8-NH2-cADPR has been used successfully to demonstrate the involvement of cADPR - mediated Ca2+ signalling in sea urchin eggs during fertilisation (14) and NO and cGMP-induced Ca2+ release (15), in Purkinje neurons (16), in hippocampal synaptic plasticity, in permeabilized Jurkat T cells (6), in intestinal smooth muscle during cholecystokinin-induced contractions (5), in PC12 cells (17) and in excitation contraction coupling in cardiac myocytes ( 8). However, like the parent compound, cADPR, the 8- substituted analogues are prone to hydrolysis by endogenous enzymes (13). Indeed, this may explain the absence of inhibitory effect on secretogue-induced Ca2+ release in rat pancreatic beta-cells (19) and during induction of LTD in Purkinje neurons (16), where a role for cADPR- mediated Ca2+ signalling remains controversial.
These observations underscore the need for a stable, hydrolysis-resistant cADPR antagonist. Recently, we reported on the synthesis of another analogue of cADPR, 7-deaza-cADPR. and demonstrated that it is more stable to heat-induced hydrolysis and is also a poor substrate for cADPR hydrolase (20). These changes in stability were also brought about by a single modification, that is a replacement of the 7 position nitrogen with carbon (Figure 1A). These findings then raised an intriguing question: what would be the biological activity of a compound modified at both the 7 and 8 positions of the adenosine ring? A hybrid' analogue, was successfully synthesised namely, 7-deaza-8-bromo-cADPR (Figure 1A). Its biological properties were examined and are reported herein. Our findings show that 7-deaza-8-bromo-cADPR retains both useful pharmacological properties i.e. it is a hydrolysis resistant, antagonist of cADPR-induced Ca2+ release. Furthermore, owing to the lipophilic nature of both the bromo- and CH- moieties, we have explored its potential as a membrane permeable analogue of cADPR, as has been established for 8-bromo-cGMP (Br-cGMP) c.f. cGMP (21). A single molecular species exhibiting all three properties would be a very powerful pharmacological tool for investigations of cADPR-mediated Ca2+ signalling in intact cells. We report here that 7-deaza-8-bromo-cADPR is such a tool.
ABBREVIATIONS: cADPR cyclic adenosine 5'-diphosphate ribose;
ADPR Adenosine 5'-diphosphate ribose;
IP3 myo-inositol-( 1 ,4,5)-trisphosphate; cAMP cyclic adenosine 3', 5'-monophosphate; cGMP cyclic guanosine 3', δ'-monophosphate;
CICR calcium-induced calcium release
ASW artificial sea water
IM intracellular medium.
The invention provides compounds having the formula (1)
Figure imgf000005_0001
where at least one of X3 and X7 is CR and any remaining X3 or X7 is N,
Y is halo, C1 - C20 hydrocarbon, NR2, OR, SR, nitro or carboxyl,
R is H or C1 - C20 hydrocarbon and R groups can be the same or different, and one or each Z represents H or one Z is a cageing group. Preferably X7 is CH and X3 is N. The preparation of such a compound is described below. Preparation of compounds where X7 is not CH, or where X3 is not N can readily be achieved by known chemistry using a different starting compound 6 (see the reaction scheme below).
Y is preferably halo, particularly bromo. This is the compound whose preparation is described in detail below. Alternatively Y may be amino. Alternatively Y may be iodo, either a natural or a radioisotope such as 125l. Radiolabelled compound may be useful in an assay, eg. a competitive binding assay, to screen for other compounds with more potent binding characteristics for a cADPR receptor. Compounds where Y is other than bromo may readily be prepared inter alia using known chemistry with a different n-succinimide derivative in reaction (i).
R is H or C1 - C20 hydrocarbon e.g. alkyl, aryl, aralkyl, alkaryl, alkenyl or alkynyl and which may be substituted or unsubstituted. Preferably R is H or C1 - C4 alkyl.
Either both groups Z represent hydrogen atoms (or negative charges) as in a conventional diphosphate system. Or one group Z may be a "cageing" group e.g. 1-(o-nitrophenyl)ethyl, whereby the compound of the invention is a caged analogue adapted to be converted into an active compound in situ e.g. by light irradiation. The chemistry of such caged analogues is well known, see Aarhaus R et al, J. Biol. Chem., 1995, 270, 7745; McCray J A and Trentham D R, Ann. Rev. Biophys., Biophys. Chem, 1989, 18, 239-270 and such compounds can be prepared by standard methods.
Although the two sugar rings have been shown as ribose for simplicity, it is envisaged that other groups may be present as in known nucleotide chemistry. Thus, on either ribose, the 5'- oxygen atom may be replaced by S, CHF, CF2, SO, NR or CH2. Also the 2'- and 3'-hydroxyl groups may be replaced by H, F, NH2, N3 or O-hydrocarbyl. Such compounds may be made by known chemical methods analogous to those described below. MATERIALS AND METHODS
Chemo-enzymatic synthesis of 7-deaza-8-bromo-cADPR.
The synthesis of 7-deaza-8-bromo-cADPR. Briefly, 7-deaza- adenosine (Tubercidin) was brominated and selectively phosphorylated to yield 7-deaza-8-bromo-adenosine δ'-monophosphate (7-deaza-8-bromo- AMP). This was then coupled to nicotinamide mononucleotide (NMN) to form 7-deaza-8-bromo-β-NAD+ as has previously been described for 7- deaza-β-NAD+(20). The enzymatic cyclization of 7-deaza-8-bromo-NAD+ to 7-deaza-8-bromo-cADPR was catalysed by crude Aplysia ADP-ribosyl cyclase and the product was purified by ion-exchange chromatography (12, 20). The extinction coefficient for 7-deaza-8-bromo-cADPR was determined by total phosphate analysis to be 10.85 x 103 M~ cm~1 (277 nm) and this value was used to determine the concentrations of 7-deaza-8- bromo-cADPR used in the rest of the study.
The reaction scheme below shows synthesis of 7-deaza-8- bromo-cADPR 5. Reagents and Conditions: (i) N-bromosuccinimide, K+ acetate, DMF, 10 min, 40%; (ii) (a) PO(Oet)3 50°C, 15 min; (b) POCI3 0°C, 2h; (c) H20, 44%; (iii) DCC, pyridine:H20 4:1 , 7d, 28%; (iv) 1.5mM solution in 25 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid, ADP-ribosyl cyclase, 30 min, 31 %. Preparation of 7-Deza-8-Bromo-Adenosine (7)
To a stirred solution of dry 7 deazaadenosine (Tubercidin, 235mgs, O.δδmmol) and dry potassium acetate (185mgs, 1.88 mmol) in DMF (3.8ml) was added dropwise a solution of freshly recrystallised N-bromosuccinimide (335mgs, 1.88mmol) in DMF (1.9ml). The reaction mixture immediately turned dark red and was stirred for a further 10 min. The DMF was removed by evaporation in vacuo and the residue dissolved in methanol and adsorbed onto silica gel. Flash chromatography was used to purify the product with elution, using a gradient of 0-10% MeOH/CHCI3. Combination of the fractions containing material with a Rf of 0.07
(10%MeOH/CHCI3) yielded a yellow solid product (57) in 40% yield. This was used in the next step without further purification.
NMR δH (400 MHz, d6DMS0, ppm); 3.48 (1 H. m H5'; D20 ex, ABX, JAB=12.2, .^=3 9Hz) 3.66 (1 H, m, H5'; D20 ex, ABX, JAB=12.2, JAX=3.9Hz), 3-92 (1 H, dt, J=3.9, 2.4Hz, H4'), 4.14 (1 H, m, H3'; D20 ex, dd, J=5.7, 2.4Hz), 5.13 (2H, m, H2' and 3'OH; D20 ex, dd, J=6.8, 5.7Hz, 1 H), 5.31 (1 H, d, J=6.6Hz, D20 ex, 2'OH), 5.70 (1 H, dd, J=5.6, 3.8Hz D20 ex, 5'OH), 5.84 (1 H, d, J=6.8Hz) H1 '), 6.80 (1 H. s, H7), 7.29 (2H, br s. D20 ex, NH2), 8.02 (1 H, s, H2). NMR δc (100.4 MHz, d6DMSO, ppm); 62.7 (C5'), 71.3 (C3'),
71 .6 (C2'), 86.5 (C4'), 90.4 (C1 '), 103.3 (C7), 104.3 (C5), 109.7 (C8), 149.9 (C4), 151 9 (C2), 156.9 (C6), m/z (FAB+) 345, 347 (M++1 ), 213, 215 (M++1 -ribose).
Preparation of 7-Deaza-8-Bromo~Adenosine 5*-Monophosphate (8)
To a cooled solution of dry 7-deaza-8-bromo-adenosine (100 mg 290 μmol) suspended in triethyl phosphate (2ml) was added POCI3 (100 μl, 500 μmol) and the reaction was monitored by HPLC (starting material 1.74min, product 2.15min). After 90min cooled 1 :9 pyridine: water (10ml) was added to the above followed by stirring for a further 30min. This product was purified by ion exchange chromatography and the fractions containing the product, which eluted between 310 and
370mM TEAB, were pooled evaporated in vacuo to give the pure product in 44% yield. NMR δH (400 MHz, D20) 4.08-4.15 (3H. m, H4' and H5'), 4.53
(1 H, dd, J=5.7, 5.8Hz, H3'), 5 07 (1 H, dd, J=5.3, 5.8Hz, H2'), 6.04 (1 H, d,
J=5.3Hz, H1'), 6.42 (1 H, s, H7), 7.92 (1 H, s, H2), δP (161.7MHz, D20); 2.35ppm, broadens when proton coupled, δc (100.4MHz, D20); 65.2 (d. JCP=3.7Hz, C5'), 70.3 (C31), 72.1
(C2'), 83.6 (d, JCP=7.4Hz, C4'), 89.8 (C1'), 104.0 (C5), 105.2 (C7), 110.4
(C8), 149.9 (C4). 150.0 (C2), 154.5 (C6),
UV: λmax276 nm, ε=13.34x103 " 1- HPLC retention time: product 2.151min, m/z (FAB+) 425, 427 (M++l); (FAB') 423, 425 (M+-2), 341 (M+-Br-3).
Preparation of 7-Deaza-8-Bromo-Nicotinamide Adenine Dinucleotide (7-Deaza-8-Bromo-NAD ) (9)
To a solution of 7-deaza-8-bromo-adenosine 5'- monophosphate (94μmol) and NMN (20mg, 60μmol) in MilliQ water (1ml) was added pyridine (4ml) and an excess of DCC (0.5g) and the reaction was stirred at room temperature for 7 days. The reaction was quenched by the addition of MilliQ water (50ml), the mixture kept at 4°C for 2h, filtered and extracted with CHCI3 (3x20ml). The material was purified by ion exchange chromatography and the fractions containing the product, which eluted between 90 and 120mM TEAB, were combined, the solvent removed in vacuo and excess TEAB removed by coevaporation with MeOH to yield the title compound in 27.7% yield. NMR: δH (400MHz, D20); 4.03-4.32 (8H, m, HN2\ HN3\ HA4', HN4', 2HA5' 2HN5'), 4.56 (1 H, dd, J=5.8, 5-3Hz, HA3') 5 16 (1 H, dd, J=5.9, 5.8HZ, HA2'), 5 99 (1 H, d, J=4.4Hz, HN1 '), 6.06 (1 H, d, J=5.9HZ, HA1 '), 6.60 (1 H, s, HA7), 8.05 (1 H, s. HA2), 8.14 (1 H, dd, J=8.3, 6.4HZ, HN5), 8.73 (1 H, d, J=8.3Hz, HN4), 9.09 (1 H. d, J=6.4HZ, HN6), 9.25 (1 H, s, HN2). δP (161 7MHZ, D20); -10.5 -10.9ppm (2d, JPP= 20.1 HZ), δc (100.4 Hz, D20); 65 7 (d, J=3.7Hz, CA5'), 66.5 (CN5*) 70.0 (CA3') 71 4 (CN3'), 7 1.9 (CA2' ), 78.5 (CN2' ), 83.5 (d, J=9.2Hz, CA4'), 87.8 (d, J=9.2Hz, CN4' ), 89.8 (CA1'), 100.9 (CN1'), 104.3 (CA5), 105.0 (CA7), 110.5 (CA8), 129 6 (CN4), 134.2 (CN3), 140.6 (CN5), 143.1 (CN6), 146.3 (CN2), 150.4 (CA2), 150.7 (CA4), 155.5 (CA6), 166.1 (CO),
UV: λmax=270nm, ε=13.3x10'M' 1,
HPLC retention time 2.1 6min, m/z (electrospray+) 741 , 743 (M++l); (electrospray) 739, 741 (M+-1).
Preparation of 7-Deaza-8-Bromo Cyclic Adenosine Diphosphate Ribose (7-Deaza-8-bromo-cADPR) (5)
7-Deaza-8-bromo NAD+ (triethylammonium salt, 12μmol)) was cyclised as described and the product 7-deaza-8-bromo-cADPR which eluted between 130 and HδOrnM TEAB, was evaporated to dryness in vacuo keeping the water bath temperature below 30°C. Excess TEAB was removed by evaporation with MeOH and the cyclic compound was isolated as the glassy triethylammonium salt in 31% yield as quantified by UV and stored at -70°C.
NMR δH (400MHz, D20); 3.89-3.93 (1 H, m, HA5'), 3.98-4.03 (1 H, m, HR5'), 4 1-4.20 (1 H, m, HA4*),4.23-4.27 (1 H, m, HR5'), 4.30-4.40 (2H, m, HA5', HR3'), 5.44 (1 H, dd, J=5.2, 6.1 Hz, HA2'), 5.97 (1 H, d, J=4.0Hz, HR1'), 6.03 (1 H, d, J=6.1 Hz, HA1'). 6 91 (1 H, s, HA7), 8 75 (1 H, s, HA2), all other protons are obscured by the water peak at 4 δppm δp (161. 7MHz, D20); -10.8, -1 1.6ppm (2d, JPP=18HZ), UV: λmax =277Dm, ε=10-85x103M'1Cm-1, HPLC retention time 4.8min, m/z (electrospray+) 619, 621 (M++1 ); (electrospray") 617, 619 (M+-1 ), 79, 81 (Br).
In vitro Ca2+ release assays
In vitro Ca2+ release assays were performed on sea urchin egg homogenates (2.5%, v/v) prepared from unfertilised Lytechinus pictus eggs according to the method of Clapper and Lee (22) with modifications as previously described (23). Extramicrosomal Ca2+ was thus measured by monitoring changes in fluo-3 (3 μm) fluorescence (excitation 490 nm and emission 530 nm) in a Perkin-Elmer LS-SOB fluorimeter. All additions (not exceeding 5 μl) were made to cuvettes (containing 500 μl homogenate) in intracellular medium (1 M) (containing 10 μm EGTA).
Intracellular free Ca2+ measurements in intact eggs
Ca2+ imaging of intact cells was performed using unfertilised Lytechinus pictus eggs micro-injected with 2 μM fura-2 and 250 μg/ml heparin as previously described (23, 24).
Materials
Lytechinus pictus sea urchins were from Marinus Inc. Long Beach, CA. USA. Fluo-3 and Fura 2 was purchased from Cambridge Bioscience (Molecular Probes). All other chemicals were from Sigma inc. (London). Cyclic ADP-ribose, 8-bromo-cADPR and 7 deaza-cADPR were synthesised as previously described (12, 20).
RESULTS & DISCUSSION Since the elucidation of the structure of cADPR (25, 26) and the development of a chemoenzymatic synthesis for cADPR and its analogues (12), interest has mounted concerning the structure-function relationships between the ligand and its endogenous receptor(s). To further aid these diagnostic studies, we synthesised 7-deaza-8-bromo- cADPR. a novel analogue of cADPR. Figure 1 A shows the chemical structure of 7-deaza-8-bromo-cADPR as compared with that of cADPR and two previously reported analogues 8-bromo-cADPR, a specific cADPR antagonist (10) and 7-deaza cADPR, a hydrolysis resistant, partial agonist (20). Whereas the latter two compounds have a single modification made at either the 7 or 8 position of the adenosine ring, 7-deaza-8-bromo- cADPR possesses both these modifications. The effect of each modification on the Ca2+ mobilizing (agonistic) ability was first tested using sea urchin egg homogenates (2.5%). Figure 1 B shows the Ca2+ releasing action of 2 μM applications of 7-deaza-8-bromo-cADPR and related cyclic nucleotides. Unlike the agonists cADPR and 7-deaza cADPR, 7-deaza-8- bromo-cADPR did not induce Ca2+ release from sea urchin egg microsomes, even at concentrations up to 20μM (a supra-maximal agonist concentration). In this respect 7-deaza-8-bromocADPR resembles the antagonist 8-bromo-cADPR. Whether 7-deaza-8-bromo-cADPR, like 8-bromo-cADPR, was also an antagonist of cADPR-sensitive Ca2+ release was investigated next. Figure 2 shows that this was indeed the case. Sea urchin egg homogenates pre-treated with either 8-bromo-cADPR or 7-deaza-8-bromo- cADPR were markedly less responsive to 100 nM cADPR (Figure 2A). These inhibitory actions were dependent on the antagonist concentration (Figure 2B). Both 8-bromo-cADPR and 7-deaza-8-bromo-cADPR exhibited similar inhibition potencies with comparable IC50 values (IC50 = 0.97 ± 0.04μm (S.E. n=3) for 8-bromo-cADPR: IC50= 0.73 ± 0.05 μM (S.E. n=3) for 7-deaza-8-bromo-cADPR.). Lower concentrations (31 mM) of either antagonist were also significant in preventing Ca2+ release by 100 nM cADPR which did not exceed 85% of control values. Whether this is due to the presence of more than one population of receptors which exhibit different binding affinities and/or sensitising properties remains to be investigated. Nonetheless, both 8-substituted analogues appeared to behave similarly with respect to these actions on the cADPR-induced Ca2+ release channel in sea urchin egg homogenates. As previous studies have shown that 8-substituted cADPR analogue (7,13, [new 14] and 7-deaza-cADPR (20) are able to displace cADPR binding, it is likely that 7-deaza-8-bromo- cADPR also interacts at the cADPR receptor in the same specific manner. Since a modification on the 8-position does not alter the stability of the molecule (13) but a substitution of N7with a carbon has been shown to render the cyclic compound more resistant to hydrolysis (20), we investigated whether, 7-deaza-8-bromo-cADPR could differ from 8-bromo- cADPR, but resemble 7-deaza-cADPR in this respect. We subjected standard solutions of both antagonists to heat-induced hydrolysis. This treatment has previously been shown to strip unstable cyclic compounds such as cADPR and 8 NH2-cADPR, of their biological activity (13, 18, 20). Figure 3 shows the effect of heat treatment on the antagonistic actions of both 7-deaza-8-bromo-cADPR and 8-bromo-cADPR. Whereas 8-bromo- cADPR is stripped of its antagonistic activity, 7-deaza-8-bromo-cADPR remained an effective antagonist of cADPR-induced Ca2+ release (Figure 3B compared to A). HPLC analysis of the same samples confirmed that this loss of activity was due to degradation of 8-bromo-cADPR whilst 7-deaza-8-bromo-cADPR remained intact (data not shown). These data are in line with the chemical stability of 7-deaza adenosine c.f. adenosine (27) and suggests that 7-deaza-8-bromo-cADPR is the first stable cADPR antagonist. This new compound therefore has potential for application in intact cells/tissues such as Jurkat T cells and neuronal tissue which express high hydrolase activities but may still have a functional cADPR- mediated Ca2+ signalling pathway (1).
When these antagonists were tested for resistance to enzyme mediated hydrolysis (by cADPR hydrolases) a similar resistance emerged. Egg homogenates (2.5%. containing fluo-3), were incubated overnight (at 17°C) with 20 μM 8-bromo-cADPR or 7-deaza-8-bromo- cADPR. Samples (50 μl) were then taken and tested for antagonistic activity on cADPR-induced Ca2+ release (as described for figure 2 but where the cuvette concentration of the antagonists was initially 2 μM). Whereas the antagonistic activity of 8-bromo-cADPR, in response to cADPR (100 nM), had dramatically reduced following the prolonged incubation with L. pictus cADPR hydrolase (65.8 ± 8.3% of control cADPR- induced Ca2+ release, n=3), the levels of 7-deaza-8-bromo-cADPR remained high thereby producing a greater antagonistic effect on cADPR- induced Ca2+ release (28.5 ± 11.4% of control cADPR-induced Ca2+ release, n=3). This was also confirmed by HPLC analysis of the same samples (data not shown).
It has been demonstrated that the presence of a lipophilic bromine moiety in cGMP affords greater membrane permeability to Br- cGMP (21) and the replacement of a nitrogen with CH- group also offers greater hydrophobicity (28). Therefore, the novel cADPR analogue,
7-deaza-8-bromo-cADPR, should have greater hydrophobic character than any previously synthesised. We investigated this by testing the effect of extracellular applications of 7-deaza-8-bromo-cADPR on fertilisation- induced Ca2+ mobilisation in intact sea urchin eggs. Eggs were co-injected with the 1 P3 receptor antagonist, heparin (250 μg/ml) and Ca2+ sensitive fluorochrome fura-2 (2 μM). Figure 4A shows that upon sperm addition to control heparinized eggs, a propagating Ca2+ wave was produced (Figure 4A, open squares). This Ca2+ wave had an average amplitude of 1705 ± 119 nM Ca2+ (S.E.M, n = 11) and took 41.9 ± 5.8 s (S.E.M, n = 11 ) to reach this peak. These data are consistent with previously reported observations of sperm-induced Ca2+ mobilization from IP3-insensitive Ca2+ stores (14, 29). When heparinized eggs were pre-incubated (5 minutes) with bathing solution containing 7-deaza-8-bromo-cADPR (100 μM). the sperm-induced intracellular Ca2+ transients were completely abolished (figure 4A, filled circles) suggesting inhibition of the redundant cADPR- sensitive Ca2+ release mechanism (14., 29). In contrast, when 8-bromo- cADPR was present in the bathing medium, a Ca2+ transient was still observed, albeit dramatically reduced (figure 4A, filled triangles). The wave property of the Ca2+ response was also abolished by both treatments where the Ca2+ rises occurred slowly (if at all) and over the whole egg. Although the inhibition of fertilisation-induced Ca2+ transients was dependent on antagonist concentration (figure 4B. filled symbols), 7-deaza-8-bromo-cADPR appeared to be the more effective antagonist. Neither antagonist released Ca2+ in the eggs during the 5 minute pre-incubation period which is consistent with the absence of agonistic activity observed In vitro (figure 1 B).
Since intracellular Ca2+ mobilization is a pre-requisite for the cortical reaction (30, 31), we also monitored the formation of activation envelopes following fecundation. Treatment of eggs with either 7-deaza-8- bromo-cADPR or 8-bromo-cADPR in the bathing medium, was found to prevent the cortical reaction (in heparinized eggs) in a concentration dependent manner (Figure 4B, open symbols). This is in keeping with previous reports which demonstrate inhibition of the cortical reaction only once both redundant mechanisms have been blocked (14, 29). Eggs that were treated with antagonist only (i.e. not micro-injected with heparin), were also scored for activation envelopes in the same experiments. These consistently showed >95% activation. This suggested that neither 7-deaza-8-bromo-cADPR nor 8-bromo-cADPR acted as spermicides, rather they are able to permeate the sea urchin egg plasma membrane and specifically compete for endogenous cADPR-binding sites and inhibit agonist-induced Ca2+ mobilisation. Presumably, since the net charge on 7-deaza-8-bromo-cADPR is only -1 at physiological pH (figure 1A), this compares well with 8-bromo-cGMP which has the same net charge and where 8-substitution confers membrane permeability. This property now eliminates the need for potentially disruptive protocols such cell permeabilization or micro-injection as methods for introducing cADPR antagonists into whole cells. This advancement should greatly aid investigations of the role of c.ADPR in physiological responses to extracellular stimuli. In conclusion, we have demonstrated that, unlike 8-bromo- cADPR. 7-deaza-8-bromo-cADPR is a stable hydrolysis-resistant cADPR antagonist. This is the first report of such a compound. In addition, we have exploited the lipophilic nature of the bromo-moiety to produce a compound that is also sufficiently membrane permeable. In all this makes 7-deaza-8-bromo-cADPR a very powerful pharmacological tool for investigations of cAD PR-mediated Ca2+ signalling in intact cells.
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London.
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7. Walseth, T.F., Aarhus. R., Zeleznikar, R. and Lee. H.C. (1991) Biochim. Biophys. Ada 1094, 113-120. 8. Galione, A. and Summerhill. R.J. (1996) in Ryanodine
Receptors (Sorrentino. V., ed.), pp. 51-70. CRC Press Inc., London.
9. Sitsapesan, R., McGarry. S.J. and Williams, A.J. (1995) Trends Pharmacol. Sci. 16. 386-391.
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12. Ashamu, G.A., Galione. A. and Potter, B.V.L. (1995) J Chem. Soc, Chem. Commun., 1359-1360. 13. Walseth, T.F. and Lee, H.C. (1993) Biochim. Biophys. Ada.
1178, 235-242.
14. Lee, H.C, Aarhus, R. and Walseth, T.F. (1993) Science 261 , 352-355.
15. Willmott, N., Sethi, J.K., Walseth, T.F., Lee, H.C, White, A.M. and Galione, A. (1996) J: Biol. Chem. 271 , 3699-3705.
16. Linden, D.J., Dawson, T.M. and Dawson, V.L (1995) J Neurosci 15, 5098-5105.
17. Clementi, E., Riccio, M., Sciorati, C, Nistico, G. and Meldolesi, J. (1996) J Biol.Chem. 271 , 17739-17745. 18. Rakovic. S., Galione. A., Ashamu, G.A., Potter, B.V.L. and
Terrar. D.A. (1996) Curr. Biol. 6, 989-996.
19. Willmott, N.J., Galione, A. and Smith. P.A. (1995) Cell Calcium 18, 411-419.
20. Bailey, V.C, Sethi, J.K., Fortt, S.M., Galione, A. and Potter, B.V.L. (1997) Chem.& Biol., 4, 51-61. 21. Butt, E., Nolte. C, Schulz, S., Beltman, J., Beavo, J.A., Jastorff. B. and Walter, U. (1992) Biochem. Pharmacol. 43, 2591-2600.
22. Clapper, D.L., Walseth, T.F., Dargie. P.J. and Lee, H.C. (1987) J Biol. Chem. 262, 9561 -9568. 23. Sethi. J.K., Empson, R.M. and Galione, A. (1996) Biochem. J.
319, 613-617.
24. Genazzani. A.A., Empson. R.,M. and Galione. A. (1996) J. Biol. Chem. 271 , 11599-11602.
25. Kim, H., Jacobson, E.L. and Jacobson. M.K. (1993) Biochem. Biophvs. Res. Commun. 194, 1143-1147.
26. Lee, H.C, Walseth, T.F., Bratt. G.T., Hayes, R.N. and Clapper. D.L. (1989) J Biol. Chem. 264, 1608- 1615.
27. Pike. J.E., Slechta. L. and Wilev. P.F. (1964) J.Heterocyclic Chem. 1 , 109-161. 28. Ji, L.N., Corfu, N.A. and Sigel. H. (1991)J. Chem. Soc. -Dalton
Trans., 1367-1375.
29. Galione, A., McDougall. A., Busa, W.B., Willmott, N., Gillot, I. and Whitaker, M. (1993) Science 261, 348-352.
30. Sasaki, H. (1984) Dev. Biol. 101 , 125-135. 31. Sardet, C. and Chang, P. (1987) Cell Differ. 21, 1-19.
FIGURE LEGENDS
Figure 1 : (A) Structural formulae of cADPR 7-deaza-cADPR, 8-bromo- cADPR and 7-deaza-8-bromo-cADPR. Modifications were made to cADPR at the 7 and 8 positions of the adenosine ring. The N7 nitrogen atom is replaced with a carbon (and associated proton) to form 7-deaza cADPR whilst 8-bromo-cADPR is formed by substituting the hydrogen on the C-8 position with a bromine atom. 7-Deaza-8-bromo-cADPR has both these modifications. (B) Calcium releasing action of cADPR, 7-deaza- cADPR, 8-bromo-cADPR and 7-deaza-8-bromo-cADPR in sea urchin egg homogenates. Lytechinus pictus egg homogenates (2.5%, v/v) containing the Ca2+ sensitive dye. fluo-3 (3 μM) was prepared as described in Materials and Methods. Aliquots (500 μl) of these were challenged with 5 μl of cyclic compounds to give a final cuvette concentration of 2 μM. The Ca2+ release profiles observed are shown and are representative of three separate experiments.
Figure 2: Concentration dependent inhibition of cADPR-induced Ca2+ release by 8-bromo-cADPR and 7-deaza-8-bromo-cADPR. Lytechinus pictus egg homogenates (2.5 %, v/v) containing the Ca2+ sensitive dye, fluo-3 (3 μM) were prepared as described in Materials and Methods. Their sensitivity to cADPR-induced Ca2+ release was used to test the antagonistic action of 8-bromo-cADPR (A) as compared to that of 7-deaza-8-bromo-cADPR (B). This was done by pre- treating homogenates with increasing concentrations of antagonist (in 5μl Glu IM + EGTA), 3 minutes prior to challenge with 100 nM cADPR. Representative traces are shown in (A) and (B) where the addition artefact has been removed for clarity (gap in traces). The amount of Ca2+ released by cADPR application was determined and expressed as a percentage of control cADPR release, i.e. the amount of Ca2+ released in homogenates treated with vehicle. The inhibition curves that were obtained for each antagonist are shown in (C). Each value represents the mean ± SE of triplicates. The IC50 values were estimated to 0.97 ± 0.04 μM for 8-bromo- cADPR and 0.73 ± 0.05 μM for 7-deaza-8-bromo-cADPR (S.E. n=3).
Figure 3: Effect of heat treatment on antagonist activity of 8-bromo- cADPR and 7-deaza-8-bromo-cADPR.
Experimental conditions were as for figure 2. Complete inhibition of cADPR-induced release was seen when homogenates were pre-treated with 10 μM of either 8-bromo-cADPR or 7-deaza-8-bromo- cADPR (A). The stock solutions (1 mM) of either 8-bromo-cADPR or 7-deaza-8-bromo-cADPR were also incubated at 85°C for 90 min in a water bath. After heat-induced hydrolysis, the antagonistic action of 8-bromo-cADPR was abolished (B). In contrast, heat-treated 7-deaza-8- bromo-cADPR remained an effective antagonist to cADPR-induced Ca + release. Fluorimetric traces are representative of three similar experiments.
Figure 4 . Antagonistic actions of extracellularly applied 7-deaza-8- bromo-cADPR and 8-bromo-cADPR on fertilisation-induced Ca2+ transients in intact sea urchin eggs.
(A). Control response (open squares) showing a typical fertilisation-induced Ca2+ transient in intact L. pictus eggs pre-injected with heparin and fura-2 (final concentrations in egg were approximately 250 μg/ml and 2 μM respectively). Sperm were added at time = 0. Despite the presence of heparin, an IP3 receptor antagonist, the wave properties of the sperm-induced Ca2+ rise were intact and activation envelopes were formed (14, 29). The peak Ca2+ rise following the addition of sperm was 1705 + 119 nM Ca2+ (SEM, n=11). Addition of 100 μM 7-deaza-8-bromo- cADPR in the bathing medium (5 minutes prior to sperm addition), completely abolished the fertilisation-induced Ca2+ response (filled circles). However, the same concentration, 100 μM, of 8-bromo-cADPR only reduced the Ca2+ transient (filled triangles). The reduction of the fertilisation transient with 100 μM 8-bromo-cADPR was similar to that seen using 50 μM 7-deaza-8-bromo-cADPR (988 ± 81 nM Ca2+; SEM, n=6) supporting the greater effectiveness of the 7-deaza-8-bromo-cADPR compound. Neither antagonist mobilized Ca2+ in the eggs during the 5 minute pre-incubation period. (B). Dose-dependent action of 7-deaza-8-bromo-cADPR and 8-bromo- cADPR on peak fertilisation-induced Ca2+ rise and egg activation.
For each concentration of 7-deaza-8-bromo-cADPR (circles) and 8-bromo-cADPR (triangles), 4-9 eggs in a single dish were co-injected with heparin (250 μg/ml) and Fura (2 μM). Following incubation in artificial sea water containing 7-deaza-8-bromo-cADPR or 8-bromo-cADPR for 5 minutes sperm were added and the maximum change in intracellular free Ca2+ (primary Y-axis, filled symbols) was monitored on one of the pre- injected eggs. Following recovery of the Ca2+ transient we scored the presence or absence of an activation envelope in all injected eggs. The results (secondary Y-axis, open symbols) show that in heparinized eggs bathed in artificial sea water containing either 7-deaza-8-bromo-cADPR or 8-bromo-cADPR, there was a dose dependent reduction in the % of the eggs activated. 7-deaza-8-bromo-cADPR appeared to be more effective at all concentrations. Remaining non-heparinized eggs were all consistently activated, indicating that sperm activity was unaffected by increasing concentrations of either antagonist.

Claims

1. A compound having the formula (1 )
Figure imgf000023_0001
where at least one of X3 and X7 is CR and any remaining X3 or X7 is N, Y is halo, C1 - C20 hydrocarbon, NR2, OR, SR, nitro or carboxyl,
R is H or C1 - C20 hydrocarbon and R groups can be the same or different, and one or each Z represents H or one Z is a cageing group.
2. 7-Deaza-8-bromo-cyclic adenosine δ'-diphosphate ribose.
3. A method of screening for a compound which binds to a cADPR receptor, which method comprises performing a competitive binding assay in which the compound is caused to compete with a compound according to claim 1 or claim 2 for binding to the cADPR receptor.
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V.C.BAILEY ET AL.: "7-Deaza Cyclic Adenosine 5'-Diphosphate Ribose : First Example of a Ca2+-Mobilizing Partial Agonist Related to Cyclic Adenosine 5'-Diphosphate Ribose.", CHEMISTRY AND BIOLOGY, vol. 4, 1997, pages 51 - 61, XP002068815 *
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