Allosteric modulators of metabotropic glutamate receptor 5 (mGlu(5)) are a promising therapeutic strategy for a number of neurological disorders. Multiple mGlu(5)-positive allosteric modulator (PAM) chemotypes have been discovered that act as either pure PAMs or as PAM-agonists in recombinant and native cells. While these compounds have been tested in paradigms of receptor activation, their effects on receptor regulatory processes are largely unknown. In this study, acute desensitization of mGlu(5) mediated intracellular calcium mobilization by structurally diverse mGlu(5) orthosteric and allosteric ligands was assessed in human embryonic kidney 293 cells and primary murine neuronal cultures from both striatum and cortex. We aimed to determine the intrinsic efficacy and modulatory capacity of diverse mGlu(5) PAMs [(R)-5-((3-fluorophenyl)ethynyl)-N-(3-hydroxy-3-methylbutan-2-yl)picolinamide (VU0424465), N-cyclobutyl-6-((3-fluorophenyl)ethynyl)picolinamide (VU0360172), 1-(4-(2,4-difluorophenyl)piperazin-1-yl)-2-((4-fluorobenzyl)oxy)ethanone (DPFE), ((4-fluorophenyl) (2-(phenoxymethyl)-6,7-dihydrooxazolo[5,4-c]pyridin-5(4H)-yl)methanone) (VU0409551), 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB)] on receptor desensitization and whether cellular context influences receptor regulatory processes. Only VU0424465 and VU0409551 induced desensitization alone in human embryonic kidney 293-mGlu(5) cells, while all PAMs enhanced (S)-3,5-dihydroxyphenylglycine (DHPG)-induced desensitization. All mGlu(5) PAMs induced receptor desensitization alone and enhanced DHPG-induced desensitization in striatal neurons. VU0424465 and VU0360172 were the only PAMs that induced desensitization alone in cortical neurons. With the exception of (CDPPB), PAMs enhanced DHPG-induced desensitization in cortical neurons. Moreover, differential apparent affinities, efficacies, and cooperativities with DHPG were observed for VU0360172, VU0409551, and VU0424465 when comparing receptor activation and desensitization in a cell type-dependent manner. These data indicate that biased mGlu(5) allosteric modulator pharmacology extends to receptor regulatory processes in a tissue dependent manner, adding yet another layer of complexity to rational mGlu(5) drug discovery.

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.

G protein-coupled receptors (GPCRs) are highly 'druggable' proteins and represent the largest class of current therapeutic targets. Two distinct neuromodulatory GPCRs, the adenosine A1 receptor (A1AR) and the metabotropic glutamate receptor subtype 5 (mGlu5), have been implicated in Alzheimer's disease (AD) pathology and as potential targets to treat cognitive impairments and disease progression. Both GPCRs are co-located in the same brain regions and CNS-resident cell types, e.g. neurons and astrocytes, and implicated in AD pathology. GPCR discovery programs generally only consider GPCR activity in isolation, without factoring in the influence of other GPCRs or stimuli present. Of note, both glutamate and adenosine are often present within culture medium and/or released from cultured cells. GPCR cross-talk and/or heteromerization can introduce pharmacological heterogeneity and offer new avenues for targeted drug development. We sought to test the hypothesis that coincident activation of co-located GPCRs modulates signalling in primary neuron and astrocyte cultures. Primary neuronal cultures were derived from striatum and cortices of E16 mice and astrocyte cultures derived from cortices of rat pups (p1-4). High-throughput signaling assays were performed (iCa2+ mobilization, inositol phosphate (IP1) and cAMP accumulation) to confirm functional mGlu5 and A1AR in the cultures by assessing the ability of: 1) A1AR selective agonist (MeCCPA) to inhibit forskolin stimulation of cAMP accumulation; and 2) mGlu5 selective allosteric agonist (VU0424465) to stimulate iCa2+ and IP1 accumulation. Coincident activation of A1AR enhanced mGlu5-mediated iCa2+ mobilization in response to both orthosteric and allosteric agonists in striatal and cortical neurons. However, A1AR activation had no effect on mGlu5-mediated IP1 accumulation in cortical neurons. Conversely, coincident activation of mGlu5 had little influence on A1AR-mediated inhibition of cAMP accumulation. Collectively, our data demonstrate that coincident activation of mGlu5 and A1AR differentially modulates intracellular signalling pathways in primary neurons. Future work exploring the underlying mechanisms may reveal new strategies for targeting these GPCRs to treat AD and cognitive disorders.

G protein‐coupled receptors (GPCRs) represent the largest class of targets for current therapeutics. Two distinct modulatory GPCRs within the CNS, the adenosine A1 receptor (A1AR) and the metabotropic glutamate receptor subtype 5 (mGlu5), have been implicated in Alzheimer's disease (AD) pathology and as potential targets to treat cognitive impairments and disease progression. Both GPCRs are present on the same CNS‐resident cell types, e.g. neurons and astrocytes, and are expressed in brain regions implicated in AD pathology. GPCR discovery programs generally only consider GPCR activity in isolation, without factoring in the influence of other GPCRs or stimuli present. Of note, both glutamate and adenosine are often present within culture medium and/or released from cultured cells. GPCR cross‐talk and/or heteromerization can introduce pharmacological heterogeneity and offer new avenues for targeted drug development. We sought to test the hypothesis that coincident activation of co‐located GPCRs modulates signaling in primary cultures of CNS resident cells. Primary cultures were derived from striatum and cortices of E16 mice and cultured for 6–8 days. High‐throughput signaling assays (iCa2+ mobilization, inositol phosphate (IP1) and cAMP accumulation) and radioligand binding studies were performed to confirm expression and function of mGlu5 and A1AR in the cultures by assessing the ability of: 1) A1AR selective (MeCCPA) and non‐selective (NECA) agonists to inhibit forskolin stimulation of cAMP accumulation; and 2) mGlu5 selective allosteric agonist (VU0424465) and group I mGlu selective orthosteric agonist (DHPG) to stimulate iCa2+ and IP1 accumulation. Experiments performed in the presence and absence of adenosine deaminase revealed that ambient adenosine was not influencing mGlu5 signalling. Coincident activation of A1AR enhanced mGlu5‐mediated iCa2+ mobilization in response to both orthosteric and allosteric agonists in primary striatal and cortical neuronal cultures. However, A1AR activation had no effect on mGlu5‐mediated IP1 accumulation in cortical neurons. Conversely, coincident activation of mGlu5 had little influence on A1AR‐mediated inhibition of cAMP accumulation, but inhibited AR‐stimulation of ERK1/2 phosphorylation. Collectively, our data demonstrate that coincident activation of mGlu5 and A1AR differentially modulates intracellular signaling pathways. Future work exploring the underlying mechanisms may reveal new strategies for targeting these GPCRs to treat AD and cognitive disorders.Support or Funding InformationThis work was supported by National Health and Medical Research Council of Australia (APP1123722 to KJG and LTM) and Australian Research Council Future Fellowships to KJG and KL.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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 (GABAB ligand), AZ12216052 (mGlu8 ligand), and CGP7930 (GABAB 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.

Significance The orthosteric binding sites of the five muscarinic acetylcholine receptor (mAChR) subtypes are highly conserved, making the development of selective antagonists challenging. The allosteric sites of these receptors are more variable, allowing one to imagine allosteric modulators that confer subtype selectivity, which would reduce the major off-target effects of muscarinic antagonists. Accordingly, a large library docking campaign was prosecuted seeking unique positive allosteric modulators (PAMs) for antagonists, ultimately revealing a PAM that substantially potentiates antagonist binding leading to subtype selectivity at the M 2 mAChR. This study supports the feasibility of discovering PAMs that can convert an armamentarium of potent but nonselective G-protein–coupled receptor (GPCR) antagonist drugs into subtype-selective reagents.

Muscarinic receptor agonists are characterized by apparently strict restraints on their tertiary or quaternary amine and their distance to an ester or related center. On the basis of the active state crystal structure of the muscarinic M2 receptor in complex with iperoxo, we explored potential agonists that lacked the highly conserved functionalities of previously known ligands. Using structure-guided pharmacophore design followed by docking, we found two agonists (compounds 3 and 17), out of 19 docked and synthesized compounds, that fit the receptor well and were predicted to form a hydrogen-bond conserved among known agonists. Structural optimization led to compound 28, which was 4-fold more potent than its parent 3. Fortified by the discovery of this new scaffold, we sought a broader range of chemotypes by docking 2.2 million fragments, which revealed another three micromolar agonists unrelated either to 28 or known muscarinics. Even pockets as tightly defined and as deeply studied as that of the muscarinic reveal opportunities for the structure-based design and the discovery of new chemotypes.

Chronic stress accelerates metastasis - the main cause of death in cancer patients - through the activation of beta-adrenoceptors (beta ARs). We have previously shown that beta(2)AR signaling in MDA-MB-231(HM) breast cancer cells, facilitates invadopodia formation and invasion in vitro. However, in the tumor microenvironment where many stromal cells also express beta AR, the role of beta(2)AR signaling in tumor cells in metastasis is unclear. Therefore, to investigate the contribution of beta(2)AR signaling in tumor cells to metastasis in vivo, we used RNA interference to generate MDA-MB-231(HM) breast cancer cells that are deficient in beta(2)AR. beta(2)AR knockdown in tumor cells reduced the proportion of cells with a mesenchymal-like morphology and, as expected, reduced tumor cell invasion in vitro. Conversely, overexpression of beta(2)AR in low metastatic MCF-7 breast cancer cells induced an invasive phenotype. Importantly, we found that knockdown of beta(2)AR in tumor cells significantly reduced the impact of stress on metastasis in vivo. These findings highlight a crucial role for beta(2)AR tumor cell signaling in the adverse effects of stress on metastasis, and indicate that it may be necessary to block beta(2)AR on tumor cells to fully control metastatic progression. (C) 2016 The Authors. Published by Elsevier Inc.

Introduction For efficient metastatic dissemination, tumor cells form invadopodia to degrade and move through three-dimensional extracellular matrix. However, little is known about the conditions that favor invadopodia formation. Here, we investigated the effect of β-adrenoceptor signaling - which allows cells to respond to stress neurotransmitters - on the formation of invadopodia and examined the effect on tumor cell invasion. Methods To characterize the molecular and cellular mechanisms of β-adrenergic signaling on the invasive properties of breast cancer cells, we used functional cellular assays to quantify invadopodia formation and to evaluate cell invasion in two-dimensional and three-dimensional environments. The functional significance of β-adrenergic regulation of invadopodia was investigated in an orthotopic mouse model of spontaneous breast cancer metastasis. Results β-adrenoceptor activation increased the frequency of invadopodia-positive tumor cells and the number of invadopodia per cell. The effects were selectively mediated by the β 2 -adrenoceptor subtype, which signaled through the canonical Src pathway to regulate invadopodia formation. Increased invadopodia occurred at the expense of focal adhesion formation, resulting in a switch to increased tumor cell invasion through three-dimensional extracellular matrix. β 2 -adrenoceptor signaling increased invasion of tumor cells from explanted primary tumors through surrounding extracellular matrix, suggesting a possible mechanism for the observed increased spontaneous tumor cell dissemination in vivo . Selective antagonism of β 2 -adrenoceptors blocked invadopodia formation, suggesting a pharmacological strategy to prevent tumor cell dissemination. Conclusion These findings provide insight into conditions that control tumor cell invasion by identifying signaling through β 2 -adrenoceptors as a regulator of invadopodia formation. These findings suggest novel pharmacological strategies for intervention, by using β-blockers to target β 2 -adrenoceptors to limit tumor cell dissemination and metastasis.