Elsevier

Neuropharmacology

Volume 38, Issue 10, October 1999, Pages 1493-1503
Neuropharmacology

2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist

https://doi.org/10.1016/S0028-3908(99)00082-9Get rights and content

Abstract

In the present paper we describe 2-methyl-6-(phenylethynyl)-pyridine (MPEP) as a potent, selective and systemically active antagonist for the metabotropic glutamate receptor subtype 5 (mGlu5). At the human mGlu5a receptor expressed in recombinant cells, MPEP completely inhibited quisqualate-stimulated phosphoinositide (PI) hydrolysis with an IC50 value of 36 nM while having no agonist or antagonist activities at cells expressing the human mGlu1b receptor at concentrations up to 30 μM. When tested at group II and III receptors, MPEP did not show agonist or antagonist activity at 100 μM on human mGlu2, -3, -4a, -7b, and -8a receptors nor at 10 μM on the human mGlu6 receptor. Electrophysiological recordings in Xenopus laevis oocytes demonstrated no significant effect at 100 μM on human NMDA (NMDA1A/2A), rat AMPA (Glu3-(flop)) and human kainate (Glu6-(IYQ)) receptor subtypes nor at 10 μM on the human NMDA1A/2B receptor. In rat neonatal brain slices, MPEP inhibited DHPG-stimulated PI hydrolysis with a potency and selectivity similar to that observed on human mGlu receptors. Furthermore, in extracellular recordings in the CA1 area of the hippocampus in anesthetized rats, the microiontophoretic application of DHPG induced neuronal firing that was blocked when MPEP was administered by iontophoretic or intravenous routes. Excitations induced by microiontophoretic application of AMPA were not affected.

Introduction

Metabotropic glutamate (mGlu) receptors are a heterogeneous family of G-protein-coupled receptors linked to multiple second messengers and modulation of ion channel functions in the nervous system (Knöpfel et al., 1995a, Conn and Pin, 1997). Molecular cloning has revealed the existence of eight distinct subtypes, termed mGlu1–mGlu8 receptors, which fall into three groups based on sequence similarities, agonist profiles and preferential signal transduction pathways activated in heterologous systems. Further diversity in this receptor family is generated by splice variants in the C-terminal cytoplasmic domain. Group I receptors (mGlu1 and -5) couple to phospholipase C and up or down regulate neuronal excitability (Gereau and Conn, 1995) whereas group II (mGlu2 and -3) and group III (mGlu4, -6, -7, and -8) receptors inhibit adenylyl cyclase and reduce synaptic transmission.

Metabotropic glutamate receptors have been implicated as potentially important therapeutic targets for a number of neurological and psychiatric disorders, although these studies are largely based on compounds that do not discriminate between mGlu receptor subtypes (for review see Knöpfel et al., 1995a, Conn and Pin, 1997, Nicoletti et al., 1997). The elucidation of the role of each individual mGlu receptor subtype is hampered by the lack of potent subtype-selective and systemically active compounds. In an effort to identify novel compounds we have used microfluorimetric measurements of intracellular calcium concentration and PI (phosphoinositide) hydrolysis assays as functional read-outs for human mGlu5a receptor expressing cell lines (Daggett et al., 1995, Veliçelebi et al., 1998). This approach has the capacity to detect not only competitive agonists and antagonists but also modulators acting at undiscovered sites of the mGlu receptors. The first selective and structurally novel mGlu5 receptor antagonists identified were 6-methyl-2-(phenylazo)-pyridin-3-ol (SIB-1757, Fig. 1) and (E)-2-methyl-6-styryl-pyridine (SIB-1893, Fig. 1) with IC50 values of 3.7 μM and 3.5 μM, respectively, in the PI hydrolysis assay (Varney et al., 1999).

Here we report on the in vitro pharmacology of 2-methyl-6-(phenylethynyl)-pyridine (MPEP, Fig. 1), a significantly more potent antagonist derived by structural derivatization around SIB-1757 and SIB-1893. MPEP was characterized for selectivity at a panel of recombinant mGlu, NMDA (N-methyl-d-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate) and kainate receptor subtypes expressed in non-neuronal cells. To address activity at native receptors, the effect of MPEP on DHPG-((R,S)-3,5-dihydroxyphenylglycine)-induced PI hydrolysis in neonatal rat brain slices was investigated. Using in vivo electrophysiology in the hippocampus of adult anesthetized rats we studied central effects of MPEP following iontophoretic application and systemic administration. These studies indicate that MPEP is a valuable pharmacological tool to explore the function of the mGlu5 receptor in the nervous system under normal and pathophysiological conditions.

Parts of this work were presented on January 6–8 1999 at the British Pharmacological Society, Winter Meeting in Brighton, UK (Gasparini et al., 1999b).

Section snippets

Chemicals

MPEP was synthesized in one step using a palladium-catalyzed coupling reaction between 2-bromo-6-methyl-pyridine and phenylacetylene as described by Sashida et al. (1988). MPEP was used in all experiments as its hydrochloride salt. Glutamate, DHPG, quisqualate, (1S,3R)-ACPD ((1S,3R)-1-amino-cyclopentane-1,3-dicarboxylate) and l-AP4 (l-2-amino-4-phosphonobutyrate) were obtained from Tocris (Anawa Trading SA, Zurich, Switzerland). Tissue culture reagents were purchased from Life Technologies

Effects on recombinant group I mGlu receptors

MPEP was tested for agonist and antagonist activity at cloned human mGlu5a and mGlu1b receptors in a PI hydrolysis assay. MPEP potently and completely inhibited quisqualate-induced PI hydrolysis (N=3) with an IC50 value of 36 nM (25, 51 nM; 90% confidence interval), a Hill coefficient of 0.85 (0.64, 1.07; 90% confidence interval) and a maximal inhibition of 96.7% (88.9, 104.5; 90% confidence interval) at the human mGlu5a receptor expressed in L(tk) cells (Fig. 2A). No inhibition by MPEP was

Discussion

The elucidation of the physiological roles of mGlu receptors in the nervous system has been limited primarily by the lack of high-affinity subtype-selective and systemically active compounds. Although group I receptors were the first to be cloned, very few group I selective compounds are known. Initially, the search for novel agonists and antagonists was focused on phenylglycine derivatives which were found to possess a wide spectrum of agonist, partial agonist and antagonist activity depending

Acknowledgements

We thank Richard Brom, Thomas Ferrat, Werner Inderbitzin, Therese Leonhardt, Snezana Lukic, Hans Peter Müller, Anne-Catherine Pasche, Doris Rüegg, Christine Stierlin and Peter Wicki for excellent technical assistance.

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