KF26777 (2-(4-bromophenyl)-7,8-dihydro-4-propyl-1H-imidazo[2,1-i]purin-5(4H)-one dihydrochloride), a new potent and selective adenosine A3 receptor antagonist
Introduction
Adenosine modulates many cellular functions via G protein-coupled receptors. Adenosine receptors are classified into A1, A2A, A2B and A3 receptors, which is based on cDNA cloning, affinities for agonists and antagonists and the mechanism of signal transduction Linden et al., 1991, Zhou et al., 1992, Palmer and Stiles, 1995. Activation of adenosine A3 receptors inhibits adenylyl cyclase and stimulates phospholipase C (Abbracchio et al., 1995) and D (Ali et al., 1996). The adenosine A3 receptor cDNAs have been cloned from multiple species. The homology of amino acid sequence in adenosine A3 receptors between rat (Zhou et al., 1992) and human (Salvatore et al., 1993) is only 74%. The difference between rat and human receptors seems to contribute to a marked difference in the affinity of ligands to these adenosine A3 receptors (Linden, 1994). Also, the tissue distribution of the adenosine A3 receptor varies across species: the rat mRNAs are detected in testis, lung, heart, kidney, brain and circulating inflammatory cells (Zhou et al., 1992); human mRNAs are widespread but the most abundant expression is in the lung, liver (Salvatore et al., 1993) and eosinophils (Walker et al., 1997).
Compared with adenosine A1 and A2A receptors, activation of adenosine A3 receptors requires a relatively high concentration of adenosine, which occurs during hypoxic stress and cellular damage such as ischemia and inflammation. The effect of the adenosine A3 receptor on brain ischemia is complicated: chronic preadministration of an adenosine A3 receptor agonist had a highly neuroprotective postischemic effect, but acute administration of the same agonist during ischemia exacerbated histological and functional damage (von Lubitz et al., 1994). With respect to inflammation, function of adenosine A3 receptors is controversial. In lung of patients with airway inflammation, the mRNA level of adenosine A3 receptors increased (Walker et al., 1997). In addition, stimulation of adenosine A3 receptors induces bronchospasm (Meade et al., 1996), enhances antigen-dependent degranulation (Ramkumar et al., 1993) and protects rat basophilic leukemia RBL-2H3 cells from apoptosis (Gao et al., 2001). Conversely, adenosine A3 receptor activation inhibits production of tumor necrosis factor-α (TNF-α) and inflammatory cytokines Hasko et al., 1998, McWhinney et al., 1996, Szabo et al., 1998, chemotaxis (Walker et al., 1997), degranulation and superoxide anion release (Ezeamuzie and Philips, 1999). In adenosine A3 receptor knockout mice, an adenosine A3 receptor selective agonist, 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA) exhibited no potentiation of antigen-dependent degranulation of bone marrow-derived mast cells, and also the ability of Cl-IB-MECA to inhibit lipopolysaccharide-induced TNF-α production in vivo was decreased remarkably in contrast to wild type mice (Salvatore et al., 2000). These data support the finding that adenosine A3 receptors play a role in both anti- and proinflammatory responses. Thus, it is not clear whether an adenosine A3 receptor agonist or antagonist is a potential target for the treatment of brain ischemia and inflammatory diseases. In order to elucidate the physiological function of adenosine A3 receptors and to clarify the therapeutic benefit of drugs that act on the receptor, potent and selective adenosine A3 receptor antagonists are required.
Although several classes of adenosine A3 receptor antagonists have been reported including dihydropyridines, triazoloquinazolines, flavonoids, pyridines, isoquinolines and triazolonaphthyridines (Baraldi et al., 2000), the majority of these antagonists are not characterized sufficiently. We have identified a novel imidazopurine derivative, KF26777, 2-(4-bromophenyl)-7,8-dihydro-4-propyl-1H-imidazo[2,1-i]purin-5(4H)-one dihydrochloride (Fig. 1), as a potent and selective adenosine A3 receptor antagonist, and we report here the biochemical and pharmacological characterization of KF26777.
Section snippets
Materials
N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide ([125I]AB-MECA, specific activity, 74 TBq/mmol) was purchased from Amersham (Buckinghamshire, England). [3H]N6-cyclohexyladenosine ([3H]CHA, 0.925–1.85 TBq/mmol), [3H]-2-[p-(2-carboxyethyl)phenylamino]-5′-N-ethylcarboxamidoadenosine ([3H]CGS21680, 1.11–2.22 TBq/mmol), 8-[dipropyl-2,3,4,5-3H(N)]cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX, 2.96–4.44 TBq/mmol) and [35S]guanosine 5′-O-(3-thiotriphosphate) ([35S]GTPγS, 46.2 TBq/mmol) were from
High affinity and selectivity to human adenosine A3 receptors of KF26777
As reported previously (Salvatore et al., 1993), [125I]AB-MECA bound with high affinity to membranes prepared from HEK293 cells expressing cloned human adenosine A3 receptors (clone HS-21a). KF26777 potently inhibited the [125I]AB-MECA binding to human adenosine A3 receptors in a dose dependent manner. The Ki value was 0.20±0.038 nM. Affinities of binding to other human adenosine receptor subtypes by KF26777 was much lower than that to the adenosine A3 receptors. The Ki values of KF26777 for
Discussion
Compound KF26777 is a potent and selective antagonist for human adenosine A3 receptors as evidenced by following. First, KF26777 showed a high affinity binding to human adenosine A3 receptors with a Ki value of 0.20 nM (Table 1). Scatchard analysis indicated that the binding mode of KF26777 was competitive, while that of I-ABOPX is noncompetitive (Fig. 2). Second, it was shown that KF26777 possessed about 9000-, 2350- and 3100-fold selectivity for the adenosine A3 receptors over the adenosine A1
Acknowledgements
We are grateful to Dr. Joel Linden for the supply of cell lines expressing each adenosine receptor subtypes, Dr. John A. Salmon for helpful discussion and Ms. Taeko Suzuki and Ms. Mayumi Ono for their excellent technical assistance.
References (51)
- et al.
Binding characteristics of [3H]prazosin to rat brain α-adrenergic receptors
Eur. J. Pharmacol.
(1979) - et al.
A new generation of Ca2+ indicators with greatly improved fluorescence properties
J. Biol. Chem.
(1985) - et al.
An agonist of adenosine A3 receptors decreases interleukin-12 and interferon-γ production and prevents lethality in endotoxemic mice
Eur. J. Pharmacol.
(1998) - et al.
Pharmacological characterization of novel A3 adenosine receptor-selective antagonists
Neuropharmacology
(1997) - et al.
Induction of apoptosis in HL-60 human promyelocytic leukemia cells by adenosine A3 receptor agonists
Biochem. Biophys. Res. Commun.
(1996) Cloned adenosine A3 receptors: pharmacological properties, species differences and receptor functions
Trends Pharmacol. Sci.
(1994)- et al.
Molecular cloning of adenosine A1 and A2 receptors
Trends Pharmacol. Sci.
(1991) - et al.
Protein measurements with the Folin phenol reagents
J. Biol. Chem.
(1951) - et al.
Activation of adenosine A3 receptors on macrophages inhibits tumor necrosis factor-α
Eur. J. Pharmacol.
(1996) - et al.
Ligand: a versatile computerized approach for characterization of ligand-binding systems
Anal. Biochem.
(1980)
Adenosine receptors
Neuropharmacology
The A3 adenosine receptor is the unique adenosine receptor which facilitates release of allergic mediators in mast cells
J. Biol. Chem.
Disruption of the A3 adenosine receptor gene in mice and its effect on stimulated inflammatory cells
J. Biol. Chem.
Inhibitory effect of adenosine on degranulation of human cultured mast cells upon cross-linking Fc epsilon RI
Biochem. Biophys. Res. Commun.
Interaction of mianserin, amitriptyline and haloperidol with guinea pig cerebral histamine H2 receptors studied with [125I]iodoaminopotentidine
Eur. J. Pharmacol.
(±)-[3H]epinephrine and (−)[3H]dihydroalprenolol binding to β1- and β2-noradrenergic receptors in brain, heart, and lung membranes
J. Biol. Chem.
Adenosine A3 receptor stimulation and cerebral ischemia
Eur. J. Pharmacol.
G protein-dependent activation of phospholipase C by adenosine A3 receptors in rat brain
Mol. Pharmacol.
Sustained activation of phospholipase D via adenosine A3 receptors is associated with enhancement of antigen- and Ca2+-ionophore-induced secretion in a rat mast cell line
J. Pharmacol. Exp. Ther.
Some quantitative uses of drug antagonists
Br. J. Pharmacol. Chemother.
Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine derivatives as highly potent and selective human A3 adenosine receptor antagonists
J. Med. Chem.
A3 adenosine receptor ligands: history and perspectives
Med. Res. Rev.
Pharmacological evidence for α2-adrenoceptor heterogeneity; differential binding properties of [3H]rauwolscine and [3H]idazoxan in rat brain
J. Pharmacol. Exp. Ther.
Antagonist binding properties of five cloned muscarinic receptors expressed in CHO-K1 cells
Mol. Pharmacol.
Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50% inhibition (IC50) of an enzymatic reaction
Biochem. Pharmacol.
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Recent developments in adenosine receptor ligands and their potential as novel drugs
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2009, Neurochemistry InternationalRecent advances in adenosine receptor (AR) ligands in pulmonary diseases
2009, Annual Reports in Medicinal ChemistryCitation Excerpt :Compound 13 inhibited the binding of [35S]GTPγS, which was stimulated by 2-chloro-N6-(3-iodobenzyl)adenosine-5-N-methyluronamide (Cl-IB-MECA) with an IC50 value of 270±85 nM. It also antagonized the [Ca2+]I mobilization, induced by Cl-IB-MECA, with a KB value of 0.42±0.14 nM, suggesting that it is a highly potent and selective A3 AR antagonist [68]. A tricyclic series, illustrated by lead compound 14 (MRE-3008-F20), displayed high affinity (0.28 nM) and selectivity (>30,000-fold) for the A3 AR [69].
Generation of adenosine A<inf>3</inf> receptor functionally humanized mice for the evaluation of the human antagonists
2006, Biochemical PharmacologyCitation Excerpt :However, unexpectedly, the human A3AR did not lead to the phosphorylation of PKB and ERK1/2, the potentiation of IgE/antigen-dependent mast cell degranulation, or the A3AR internalization in A3ARh/h mice, probably due to the uncoupling of member(s) of the mouse G proteins for the activation of PI3Kγ. Although KF26777, a highly potent and selective antagonist for the human A3AR [30], bound to the human A3AR (Ki value, 0.2 nM) and completely antagonized the [Ca2+]i elevation in A3ARh/h mice, the pharmacological effects of KF26777 as an anti-inflammatory agent could not be evaluated because the mast cell degranulation was not potentiated. To overcome the uncoupling, we here generated A3AR functionally humanized mice (A3ARc/c mice) by replacing the mouse A3AR gene with a human/mouse chimeric A3AR sequence in which whole intracellular regions of the human A3AR were substituted for the corresponding regions of the mouse A3AR.
Human adenosine A<inf>3</inf> receptor leads to intracellular Ca <sup>2+</sup> mobilization but is insufficient to activate the signaling pathway via phosphoinositide 3-kinase γ in mice
2005, Biochemical PharmacologyCitation Excerpt :However, the Kd value for the human A3AR on BMMCs from A3ARh/h mice was 1.42 ± 0.27 nM (Fig. 3A), which was two-fold lower than that in human A3AR-expressing human embryonic kidney 293 (HEK293) cells, as measured previously according to the same methods used here (0.67 ± 0.03 nM [24]). The Ki value of KF26777 for the human A3AR on BMMCs from A3ARh/h mice was 0.27 ± 0.11 nM (Fig. 3B), which was equivalent to that on the A3AR-expressing HEK293 cells (0.20 ± 0.038 nM [24]). The A3AR elicits a Ca2+ response via heterotrimeric G proteins of the Gi/o family [4–6].