Methoxy-PEPy

[3H]-methoxymethyl-MTEP and [3H]-methoxy-PEPy: potent and selective radioligands for the metabotropic glutamate subtype 5 (mGlu5) receptor

The design, synthesis, and characterization of two potent, non-competitive radioligands, [3H]-methoxymethyl-MTEP and [3H]-methoxy-PEPy, that are selective for the mGlu5 receptor are described.

In vivo receptor occupancy of mGlu5 receptor antagonists using the novel radioligand [3H]3-methoxy-5-(pyridin-2-ylethynyl)pyridine)

In vivo receptor occupancy of mGlu5 receptor antagonists was quantified in rat and mouse brain using the mGlu5 receptor selective antagonist [3H]3-methoxy-5-(pyridin-2-ylethynyl)pyridine) ([3H]methoxy-PEPy). Administration of [3H]methoxy-PEPy (50 microCi/kg i.v.) to mGlu5 receptor-deficient mice revealed binding at background levels in forebrain, whereas wild-type mice exhibited 14-fold higher binding in forebrain relative to cerebellum. Systemic administration of the mGlu5 receptor antagonists 2-methyl-6-(phenylethynyl)pyridine (MPEP) and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) reduced the binding of [3H]methoxy-PEPy in rats and mice, reflecting mGlu5 receptor occupancy by these compounds. MPEP (10 mg/kg i.p.) and MTEP (3 mg/kg i.p.) maintained >75% receptor occupancy for 2 h in rats, while in mice MPEP and MTEP achieved >75% occupancy for only 30 and 15 min, respectively. Compound levels in plasma were substantially lower in mice suggesting species differences in receptor occupancy result from differences in absorption or metabolism of the compounds. These findings demonstrate that [3H]methoxy-PEPy is useful for determining the occupancy of mGlu5 receptors in the brain.

"Selective" Class C G Protein-Coupled Receptor Modulators Are Neutral or Biased mGlu5 Allosteric Ligands

Numerous positive and negative allosteric modulators (PAMs and NAMs) of class C G protein-coupled receptors (GPCRs) have been developed as valuable preclinical pharmacologic tools and therapeutic agents. Although many class C GPCR allosteric modulators have undergone subtype selectivity screening, most assay paradigms have failed to perform rigorous pharmacologic assessment. Using mGlu₅ as a representative class C GPCR, we tested the hypothesis that allosteric modulator selectivity was based on cooperativity rather than affinity. Specifically, we aimed to identify ligands that bound to mGlu₅ but exhibited neutral cooperativity with mGlu₅ agonists. We additionally evaluated the potential for these ligands to exhibit biased pharmacology. Radioligand binding, intracellular calcium (iCa²⁺) mobilization, and inositol monophosphate (IP₁) accumulation assays were undertaken in human embryonic kidney cells expressing low levels of rat mGlu₅ (HEK293A-mGlu₅-low) for diverse allosteric chemotypes. Numerous "non-mGlu₅" class C GPCR allosteric modulators incompletely displaced allosteric mGlu₅ radioligand [³H]methoxy-PEPy binding, consistent with a negative allosteric interaction. Affinity estimates for CPCCOEt (mGlu₁ ligand), PHCCC (mGlu₄ ligand), GS39783 (GABA_B ligand), AZ12216052 (mGlu₈ ligand), and CGP7930 (GABA_B ligand) at mGlu₅ were within 10-fold of their target receptor. Most class C GPCR allosteric modulators had neutral cooperativity with both orthosteric and allosteric mGlu₅ agonists in functional assays; however, NPS2143 (calcium-sensing receptor (CaSR) NAM), cinacalcet (CaSR PAM), CGP7930, and AZ12216052 were partial mGlu₅ agonists for IP₁ accumulation, but not iCa²⁺ mobilization. By using mGlu₅ as a model class C GPCR, we find that for many class C GPCR allosteric modulators, subtype selectivity is driven by cooperativity and misinterpreted owing to unappreciated bias.

Kinetic and system bias as drivers of metabotropic glutamate receptor 5 allosteric modulator pharmacology

Allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGlu₅) have been proposed as potential therapies for various CNS disorders. These ligands bind to sites distinct from the orthosteric (or endogenous) ligand, often with improved subtype selectivity and spatio-temporal control over receptor responses. We recently revealed that mGlu₅ allosteric agonists and positive allosteric modulators exhibit biased agonism and/or modulation. To establish whether negative allosteric modulators (NAMs) engender similar bias, we rigorously characterized the pharmacology of eight diverse mGlu₅ NAMs. Radioligand inhibition binding studies revealed novel modes of interaction with mGlu₅ for select NAMs, with biphasic or incomplete inhibition of the radiolabeled NAM, [³H]methoxy-PEPy. We assessed mGlu₅-mediated intracellular Ca²⁺ (iCa²⁺) mobilization and inositol phosphate (IP₁) accumulation in HEK293A cells stably expressing low levels of mGlu₅ (HEK293A-rat mGlu₅-low) and mouse embryonic cortical neurons. The apparent affinity of acetylenic NAMs, MPEP, MTEP and dipraglurant, was dependent on the signaling pathway measured, agonist used, and cell type (HEK293A-rat mGlu₅-low versus mouse cortical neurons). In contrast, the acetylenic partial NAM, M-5MPEP, and structurally distinct NAMs (VU0366248, VU0366058, fenobam), had similar affinity estimates irrespective of the assay or cellular background. Biased modulation was evident for VU0366248 in mouse cortical neurons where it was a NAM for DHPG-mediated iCa²⁺ mobilization, but neutral with DHPG in IP₁ accumulation assays. Overall, this study highlights the inherent complexity in mGlu₅ NAM pharmacology that we hypothesize may influence interpretation when translating into preclinical models and beyond in the design and development of novel therapeutics for neuropsychiatric and neurological disorders.

Inducible expression and pharmacological characterization of the mouse metabotropic glutamate 5b receptor

The metabotropic glutamate receptor subtype 5 (mGlu5) and glutamatergic neurotransmission are associated with the pathophysiology of disorders such as anxiety, depression or chronic pain. Human and rat mGlu5 receptors have been cloned and characterized previously. We now describe the cloning of the mouse mGlu5b receptor gene from adult mouse brain and its expression using an ecdysone-inducible system. This subtype has an extra 96 bp sequence which is inserted to the cytoplasmic tail and is identical to the insert present in human and rat mGlu5b. Mouse mGlu5b receptor expression was induced in HEK-293EcR cells by incubation with ponasterone A, an analogue of the insect hormone ecdysone. A fluorometric calcium transient assay system was used to characterize the basic pharmacologic profile of an isolated stable cell line. Quisqualic acid was the most potent receptor agonist (EC(50) approximately 7 nM) although the cells also responded to l-glutamic acid and the Group I-selective receptor agonist, 3,5-dihydroxyphenylglycine (3,5-DHPG). The calcium transients stimulated by these agonists were potently inhibited by reference allosteric mGlu5 antagonists - 2-methyl-6-(phenylethynyl)pyridine (MPEP), 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) and 3-methoxy-5-(pyridine-2-ylethynyl)pyridine (methoxy-PEPy) (IC(50) ranges: 0.8-66 nM). The availability of this mouse mGlu5b receptor-expressing cell line will facilitate in vitro characterization of mGlu5 receptor-selective agonists or antagonists prior to in vivo pharmacologic testing.