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]pyridin-4(5H)-one (MMPIP) was produced by chemical modification of MDIP. MDIP and MMPIP inhibited l-(+)-2-amino-4-phosphonobutyric acid (l-AP4)-induced intracellular Ca2+ mobilization in Chinese hamster ovary (CHO) cells coexpressing rat mGluR7 with Gα15 (IC50 = 20 and 26 nM). The maximal response in agonist concentration-response curves was reduced in the presence of MMPIP, and its antagonism is reversible. MMPIP did not displace [3H](2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495) bound to mGluR7. These results suggested that these isoxazolopyridone derivatives are allosteric antagonists. In CHO cells expressing rat mGluR7, MDIP and MMPIP inhibited l-AP4-induced inhibition of forskolin-stimulated cAMP accumulation (IC50 = 99 and 220 nM). In CHO cells coexpressing human mGluR7 with Gα15, 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,N′-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 μM 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.
Footnotes
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.124701.
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ABBREVIATIONS: mGluR, metabotropic glutamate receptor; CNS, central nervous system; AMN082, N,N′-dibenzhydryl-ethane-1,2-diamine dihydrochloride; hCT, human calcitonin; CHO, Chinese hamster ovary; CHO-rat mGluR7, Chinese hamster ovary cells expressing rat mGluR7; CHO-rat mGluR7/Gα15, Chinese hamster ovary cells coexpressing rat mGluR7 with Gα15; CHO-human mGluR7/Gα15, Chinese hamster ovary cells coexpressing human mGluR7 with Gα15; FLIPR, fluorometric imaging plate reader; MDIP, 5-methyl-3,6-diphenylisoxazolo[4,5-c]pyridin-4(5H)-one; MMPIP, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one; l-AP4, l-(+)-2-amino-4-phosphonobutyric acid; l-CCG-I, (2S, 1′S,2′S)-2-(carboxycyclopropyl)glycine; MPEP, 2-methyl-6-(phenylethynyl)pyridine; CPPG, (R,S)-α-cyclopropyl-4-phosphonophenylglycine; LY341495 (LY), (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid; 5-HT, 5-hydroxytryptamine; IBMX, 3-isobutyl-1-methylxanthine; PTX, pertussis toxin; FTIDC, 4-[1-(2-fluoropyridin-3-yl)-5-methyl-1H-1,2,3-triazol-4-yl]-N-isopropyl-N-methyl-3,6-dihydropyridine-1(2H)-carboxamide; NFAT, nuclear factor of activated T cells; dhfr, dihydrofolate reductase; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received April 20, 2007.
- Accepted July 2, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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