Elsevier

Neuropharmacology

Volume 57, Issue 4, September 2009, Pages 438-445
Neuropharmacology

Identification of a novel transmembrane domain involved in the negative modulation of mGluR1 using a newly discovered allosteric mGluR1 antagonist, 3-cyclohexyl-5-fluoro-6-methyl-7-(2-morpholin-4-ylethoxy)-4H-chromen-4-one

https://doi.org/10.1016/j.neuropharm.2009.06.017Get rights and content

Abstract

Currently tested allosteric modulators for metabotropic glutamate receptor 1 (mGluR1) are known to regulate the activity of mGluR1 mainly through transmembrane (TM) domain 6 and/or 7. We identified a novel interaction site, N760 in TM5, which negatively regulates activation of mGluR1 with a newly discovered selective mGluR1 antagonist, 3-cyclohexyl-5-fluoro-6-methyl-7-(2-morpholin-4-ylethoxy)-4H-chromen-4-one (CFMMC). CFMMC inhibited l-glutamate-induced intracellular Ca2+ mobilization ([Ca2+]i) in Chinese hamster ovary (CHO) cells expressing recombinant human mGluR1a with IC50 value of 50 nM, whereas it did not inhibit [Ca2+]i in CHO cells expressing human mGluR5a (IC50; >10 μM). To identify the amino acid residues critical for antagonism of CFMMC, we constructed various point mutants of human mGluR1 and evaluated them in [Ca2+]i assays. The inhibitory effects of CFMMC were significantly affected in point mutations of either I725 in TM4 or N760 in TM5, as well as mutations of W798, F801 and Y805 in TM6 or T815 in TM7. Further studies revealed that antagonistic activities of not only CFMMC but also other, structurally unrelated, mGluR1 antagonists such as 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-298198) and Compound 1 were reduced in N760 mutated mGluR1a. These results indicate that some mGluR1 allosteric antagonists require N760 in TM5 to demonstrate negative modulation of mGluR1 in addition to the reported amino acid residues in TM6 and TM7.

Introduction

Metabotropic glutamate receptors (mGluRs) which belong to family C G-protein-coupled receptors (GPCRs) have been reported to be involved in various central nervous system (CNS) disorders such as schizophrenia, epilepsy, anxiety, pain, cognitive dysfunction and drug abuse (Conn and Pin, 1997, Gasparini et al., 2002, Spooren et al., 2003, Kew, 2004). To date, eight mGluR subtypes (mGluR1-8) have been identified and classified into three groups based on sequence homology and pharmacological profiles. Group I consists of mGluR1 and mGluR5, and are coupled to intracellular calcium mobilization through Gq protein. On the other hand, both group II mGluRs (mGluR2 and mGluR3) and group III mGluRs (mGluR4, mGluR6, mGluR7 and mGluR8) are negatively coupled to adenylate cyclase via Gi/o protein (Conn and Pin, 1997, Kew, 2004).

Various allosteric modulators for mGluRs have been reported to date. Several negative allosteric modulators for group I have been reported including BAY36-7620, CPCCOEt, EM-TBPC, FTIDC, JNJ16259685, LY456066, R214127 and YM-298198 for mGluR1, and MPEP for mGluR5 (Litschig et al., 1999, Carroll et al., 2001, Lavreysen et al., 2003, Lavreysen et al., 2004, Malherbe et al., 2003a, Malherbe et al., 2003b, Kohara et al., 2005, Ritzen et al., 2005, Suzuki et al., 2007). Point mutagenesis studies revealed which amino acid residues are critical for the allosteric modulation of mGluR1 and mGluR5 by some of these compounds. W798, F801 and Y805 in hmGluR1a in TM6 are conserved within group I mGluRs and were reported to be necessary for the interaction of an mGluR1 antagonist, EM-TBPC, with mGluR1 and of an mGluR5 antagonist, MPEP, with mGluR5 (Malherbe et al., 2003a, Malherbe et al., 2003b). In addition, two residues located in TM7 of mGluR1 (T815 and A818) and three residues of mGluR5 (P655 and S658 in TM3, and A810 in TM7) were reported to be responsible for the selective action of CPCCOEt at mGluR1 or MPEP at mGluR5, respectively (Litschig et al., 1999, Pagano et al., 2000). However, our previous study with FTIDC indicated that W798 and Y805 may not be crucial for antagonistic activity of FTIDC (Suzuki et al., 2007). Thus, it remains to be answered if these amino acids in TM6 and TM7 were critical for all mGluR1 negative allosteric modulators and if other interaction sites in mGluR1 existed.

In this report we describe the characteristics of a recently identified mGluR1 antagonist, 3-cyclohexyl-5-fluoro-6-methyl-7-(2-morpholin-4-ylethoxy)-4H-chromen-4-one (CFMMC). CFMMC inhibited mGluR1-mediated intracellular Ca2+ mobilization ([Ca2+]i) with good potency in a non-competitive manner. Point mutagenesis studies on mGluR1 revealed that two new interaction sites, I725 in TM4 and N760 in TM5 in addition to F801 and Y805 in TM6 and T815 in TM7. Furthermore, we tested CFMMC as well as other structurally unrelated mGluR1 negative allosteric modulators with six selected point mutated mGluR1as in order to reveal which amino acids are commonly important in binding mode of structurally diverse mGluR1 antagonists.

Section snippets

Materials

CFMMC, FTIDC and Compound 1 were identified in-house; a detailed synthetic protocol for FTIDC and Compound 1 has been reported (Ito et al., 2008). l-glutamate was purchased from Sigma–Aldrich (St. Louis, MO). l-proline was purchased from Wako Pure Chemical Industries (Osaka, Japan). Dialyzed fetal bovine serum, culture media and other reagents used for cell culture were purchased from Invitrogen (Carlsbad, CA). JNJ16259685 was purchased from Tocris Bioscience (Bristol, UK). YM-298198 and

Activity of CFMMC on recombinant group I mGluRs and mode of action of CFMMC toward mGluR1

We identified 3-cyclohexyl-5-fluoro-6-methyl-7-(2-morpholin-4-ylethoxy)-4H-chromen-4-one (CFMMC) (Fig. 1A) as a potent and selective mGluR1 antagonist by chemical modification using CHO cells expressing hmGluR1a and the FLIPR assay. In CHO cells expressing hmGluR1a, CFMMC inhibited l-glutamate-induced increases in [Ca2+]i, with IC50 value of 50 ± 6.5 nM (n = 12). In contrast, CFMMC (up to a concentration of 10 μM) had little effect on l-glutamate-induced increases in [Ca2+]i in CHO cells

Discussion

We have identified a potent and selective mGluR1 antagonist, CFMMC, from a new chromenone class of chemicals exhibiting more than 200-fold selectivity to hmGluR1a over hmGluR5a. CFMMC decreased the maximal l-glutamate-induced [Ca2+]i in CHO cells expressing hmGluR1a in a non-competitive manner, unlike a competitive antagonist, LY367385 (Suzuki et al., 2007). The inhibitory activity of CFMMC toward mGluR1a was dramatically reduced in the chimera mGluR1(693)5a, but not in the chimera

Acknowledgements

We thank Dr. Naohiro Tsukamoto, Mr. Takaharu Maruyama and Ms. Aki Kawagishi for developing the assays used in this study, and Dr. Akio Ohno for his useful discussions.

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