In Vitro Pharmacological Characterization of Novel Isoxazolopyridone Derivatives as Allosteric Metabotropic Glutamate Receptor 7 Antagonists

Abstract

Novel isoxazolopyridone derivatives that are metabotropic glutamate receptor (mGluR) 7 antagonists were discovered and pharmacologically characterized. 5-Methyl-3,6-diphenylisoxazolo [4,5-c]pyridin-4(5H)-one (MDIP) was identified by random screening, and 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo [4,5-c] In CHO cells coexpressing human mGluR7 with G␣ 15 , MDIP and MMPIP also inhibited the L-AP4-induced cAMP response. The maximal degree of inhibition by MMPIP was higher than that by MDIP in a cAMP assay. MMPIP was able to antagonize an allosteric agonist, the N,NЈ-dibenzhydryl-ethane-1,2-diamine dihydrochloride (AMN082)-induced inhibition of cAMP accumulation. In the absence of these agonists, MMPIP caused a further increase in forskolin-stimulated cAMP levels in CHO cells expressing mGluR7, whereas a competitive antagonist, LY341495, did not. This result indicates that MMPIP has an inverse agonistic activity. The intrinsic activity of MMPIP was pertussis toxin-sensitive and mGluR7-dependent. MMPIP at concentrations of at least 1 M had no significant effect on mGluR1, mGluR2, mGluR3, mGluR4, mGluR5, and mGluR8. MMPIP is the first allosteric mGluR7-selective antagonist that could potentially be useful as a pharmacological tool for elucidating the roles of mGluR7 on central nervous system functions.Metabotropic glutamate receptors (mGluRs) belong to a family of G protein-coupled receptors thought to contribute to the modulation of neuronal excitability and neurotransmitter release. Eight mGluR subtypes (mGluR1-mGluR8) have been cloned, and they have been classified into three groups based on sequence homology, pharmacological profile, and signal transduction pathway. mGluR1 and mGluR5 belong to group I mGluRs, and they are coupled to phospholipase C and subsequent intracellular calcium release via G q protein. mGluR2 and mGluR3 belong to group II mGluRs, whereas mGluR4, mGluR6, mGluR7, and mGluR8 belong to group III mGluRs. The subtypes of group II and group III mGluRs are negatively coupled to adenylate cyclase via G i protein (Conn and Pin, 1997 , 5-methyl-3,6-diphenylisoxazolo[4,5-c

Group III mGluR regulation of synaptic transmission at the SC-CA1 synapse is developmentally regulated

“…A recent study also reported that AMN082 does not induce calcium mobilization downstream of mGluR7 in a cell line coexpressing the promiscuous G protein Gα15 (Suzuki et al, 2007). This is in contrast to previous reports demonstrating activity of AMN082 in reduction of cAMP and GTPγ[ 35 S] binding assays (Mitsukawa et al, 2005;Suzuki et al, 2007). These data suggest that the effects of AMN082 may be context-specific.…”

“…AMN802 has not been rigorously characterized at established mGluR7-mediated responses in native systems or across multiple cell populations and signaling pathways. Furthermore, it was recently reported that AMN082 does not activate mGluR7 in CHO cells as assessed by measuring mGluR7-mediated calcium responses in cells expressing the promiscuous G protein, Gα15 (Suzuki et al, 2007). This suggests that the pharmacology of AMN082 may be complex, and that this compound may not serve as a general agonist of mGluR7 across multiple responses and cell populations.…”

“…In this previous paper, AMN082 elicited a full agonist response with nanomolar potency when measuring inhibition of cAMP accumulation or GTPγ[ 35 S] binding in cells expressing mGluR7 (Mitsukawa et al, 2005;Suzuki et al, 2007). Previous studies suggest that allosteric modulators such as AMN802 may differentially modulate coupling of a receptor to different signaling pathways or in distinct cellular contexts.…”

Metabotropic Glutamate Receptors: Potential Drug Targets for Psychiatric Disorders

“…In a cAMP assay, MDIP and MMPIP had IC 50 values of 99 nM and 220 nM, respectively. The inhibitory modes of these compounds are noncompetitive and allosteric, and MMPIP appears to have an inverse agonist activity [173].…”

Development of a β-Lactamase Reporter Gene Assay for Metabotropic Glutamate Receptor 1 by Using Coexpression of Glutamate Transporter

“…Real-time PCR was carried out using a modification of a previously described method. 18 TaqMan Gene Expression Assays (Assay ID; Rn00570130_m1) and TaqMan Rodent GAPDH Control Reagents (Applied Biosystems, Foster City, CA) were used to quantify mRNA expression of GLAST and GAPDH, respectively. Reverse transcription of total RNA (1 μg) was performed in a total volume of 20 μL using random hexamers and a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol.…”