JNJ16259685, a highly potent, selective and systemically active mGlu1 receptor antagonist
Introduction
Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS) of vertebrates. Glutamate elicits and modulates synaptic responses in the CNS by activating two families of receptors, ligand-gated cation channels termed ionotropic glutamate receptors and G protein-coupled glutamate receptors termed metabotropic glutamate (mGlu) receptors. To date, eight mGlu receptor subtypes and multiple splice variants have been cloned and classified into three groups based upon sequence homology, pharmacological profile and preferential signal transduction pathway. Group I mGlu receptors (mGlu1 and -5) are coupled to the stimulation of phospholipase C, whereas group II (mGlu2 and -3) and III receptors (mGlu4, -6, -7 and -8) are negatively coupled to adenylyl cyclase activation (Conn and Pin, 1997). mGlu1 receptors are perceived as important targets for a variety of neuropathologies, including stroke, pain and epilepsy (Bordi and Ugolini, 1999, Gasparini et al., 2002).
The precise assessment of the physiological or pathological role of the mGlu1 receptor has been impeded by the lack of potent, selective and systemically active mGlu1 receptor antagonists. Initially, the search for such compounds was limited to amino acid derivatives acting at the glutamate binding site (reviewed in Schoepp et al., 1999). Although these compounds were useful pharmacological tools for examination of group I mGlu receptors, their lack of selectivity, low potency and poor blood–brain-barrier penetration rendered them inappropriate for investigating the potential role of these receptors in behavior and disease models. The identification of the first non-competitive agent, 7-(hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), showed that compounds structurally unrelated to glutamate could also inhibit mGlu1 receptor function, presumably via an allosteric site at the receptor protein (Litschig et al., 1999). Later, (3aS,6aS)-6a-naphtalan-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[c]furan-1-on (BAY 36-7620), another non-competitive mGlu1 receptor antagonist with a higher potency than CPCCOEt, was discovered (Carroll et al., 2001). We have described the pharmacological profile of the new non-competitive mGlu1 receptor selective antagonist 1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone (R214127) (Lavreysen et al., 2003). Non-amino acid-like agents that potently inhibit the function of the rat but not the human mGlu1 receptor have also been identified (Malherbe et al., 2003). Recently, Micheli et al. (2003) reported a series of 2,4-dicarboxy-pyrroles acting as potent non-competitive mGlu1 receptor antagonists. In contrast to competitive mGlu1 receptor antagonists, all these so-called negative allosteric modulators have been shown to bind within the seven transmembrane (7TM) domain of the mGlu1 receptor (Lavreysen et al., 2003, Micheli et al., 2003, Pin et al., 2003). Several features make them attractive tools for studying the mGlu1 receptor under normal and pathological conditions. Besides increased potency, non-competitive antagonists exhibit a higher degree of selectivity for the mGlu1 receptor, probably because the 7TM domain is less conserved than the glutamate binding pocket (Parmentier et al., 2000). Furthermore, their non-amino acid structure makes them penetrate the brain more easily. Finally, their non-competitive mechanism of action makes them less susceptible to the influence of high concentrations of glutamate, which may be present in diseases states (Pin and Acher, 2002).
So far, only BAY 36-7620 and the 2,4-dicarboxy-pyrroles described by Micheli et al. (2003) have been shown to be centrally active, by behavioral in vivo studies and/or ex vivo receptor occupancy assays (De Vry et al., 2001, Micheli et al., 2003, Lavreysen et al., 2004). The potency with which BAY 36-7620 occupies the receptor after subcutaneous administration is very low, however (Lavreysen et al., 2004). To scrutinize mGlu1 receptor function in the CNS and to provide further information on how this receptor can be exploited for therapeutic use, there thus remains a need for highly potent, subtype-selective mGlu1 receptor antagonists that show good central availability.
In this paper, we report on a new non-amino acid-like mGlu1 receptor antagonist, (3,4-dihydro-2H-pyrano[2,3]b quinolin-7-yl) (cis-4-methoxycyclohexyl) methanone (JNJ16259685), a structural analogue of R214127 which was discovered by a functional screening approach. We demonstrate that JNJ16259685 acts selectively upon the mGlu1 receptor in a non-competitive fashion and that it is highly potent, both in recombinant and in native systems. We further show that JNJ16259685 occupies central mGlu1 receptors with high potency after systemic administration in rats.
Section snippets
Materials
All cell culture and transfection reagents were obtained from Invitrogen (Carlsbad, USA), except for soybean trypsin inhibitor, DNAse I and cytosine-β-d-arabinofuranoside, which came from Sigma-Aldrich (Steinheim, Germany), recombinant murine (rm) interferon (IFN)-β, which was kindly provided by Dr. P. Vanhoenacker (University of Ghent, Belgium) and FuGENE 6, which came from Roche Molecular Biochemicals (Mannheim, Germany). JNJ16259685 (see Fig. 1 for structure), R214127 and
Effect of JNJ16259685 on recombinant mGlu receptors
JNJ16259685 was tested for agonist and antagonist activity at cloned mGlu1 and -5 receptors in a Ca2+ mobilization assay. JNJ16259685 potently and completely inhibited the glutamate (30 μM)-induced increase in intracellular Ca2+ concentrations at the rat mGlu1a receptor with an IC50 value of 3.24±1.00 nM (n=4, Fig. 2). IC50 values for CPCCOEt and BAY 36-7620 were 17.8±10.3 μM (n=6) and 161±38 nM (n=3), respectively. The potency of JNJ16259685 in blocking glutamate (30 μM)-induced Ca2+
Discussion
JNJ16259685 is a non-amino acid, non-competitive mGlu1 receptor antagonist, exhibiting low-nanomolar potency. JNJ16259685 is about 6000 times more potent than CPCCOEt and 50 times more potent than BAY 36-7620 in a Ca2+ mobilization assay using CHO-dhfr− cells. JNJ16259685 exhibits a much lower potency at the rat and human mGlu5a receptor compared to the rat and human mGlu1a receptor, and shows no appreciable agonist or antagonist activity in [35S]GTPγS assays at recombinant mGlu2, -3, -4, or -6
Acknowledgements
We thank Prof. Dr. Fred Tilders and Prof Dr. Josée Leysen for helpful discussions and careful reading of the manuscript. The technical assistance of Tineke Willemoens, Cindy Wintmolders and Arjan Buist is greatly appreciated. We sincerely thank Dr. David Ashton for his consistent support.
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