Preclinical profile of a novel metabotropic glutamate receptor 5 positive allosteric modulator

doi:10.1016/j.ejphar.2011.02.003

European Journal of Pharmacology

Volume 659, Issues 2–3, 1 June 2011, Pages 146-154

https://doi.org/10.1016/j.ejphar.2011.02.003 Get rights and content

Abstract

Recent reports have indicated that patients with schizophrenia have a profound hypo-functionality of glutamatergic signaling pathways. Positive allosteric modulation of mGlu₅ receptor has been postulated to augment NMDA function and thereby alleviate the glutamatergic hypo-function observed in schizophrenic patients. Here we report the in vitro and in vivo characterization of CPPZ (1-(4-(2-chloro-4-fluorophenyl)piperazin-1-yl)-2-(pyridin-4-ylmethoxy)ethanone), a structurally novel positive allosteric modulator selective for mGlu₅ receptor. In HEK293 cells stably over-expressing human mGlu₅ receptor, CPPZ potentiates the intracellular calcium response elicited by a suboptimal concentration of the endogenous agonist glutamate. CPPZ does not have any intrinsic agonist activity and behaves functionally as a positive allosteric modulator. This is further supported by binding data, which demonstrate that CPPZ is able to displace the negative allosteric modulator MPEP but does not compete with the orthosteric ligand quisqualic acid. Instead, CPPZ enhances the binding of the orthosteric ligand. In native preparations, CPPZ potentiates calcium flux in rat cortical neurons stimulated with the group I agonist dihydroxyphenylglycine (DHPG). In addition, CPPZ modulates long-term potentiation in rat hippocampal slices, a process known to be NMDA dependent. In vivo, CPPZ reverses hyper locomotion triggered by the NMDA open channel blocker MK801 in CD1 mice. CPPZ was also able to reduce rat conditioned avoidance responding to electric shock. Both in vitro and in vivo data demonstrate that this novel compound acts as an mGlu₅ receptor positive allosteric modulator, which modulates NMDA dependent responses and suggests that the enhancement of mGlu₅ receptor activity may prove useful in the treatment of schizophrenia.

Introduction

Current therapies for schizophrenia are successful mainly in treating positive symptoms and do not significantly affect negative and cognitive symptoms. Through pharmacological and genetic research, investigators have identified possible deficiencies in glutamatergic dependent pathways and thereby identified novel potential targets (Olney et al., 1999). The glutamatergic hypothesis is supported by the clinical observation that phencyclidine (PCP) and ketamine, two use-dependent N-methyl-d-aspartate (NMDA) receptor antagonists, are able to induce psychotic symptoms in normal volunteers as well as exacerbate psychotic symptoms in schizophrenic patients (Javitt and Zukin, 1991). According to this hypothesis one might alleviate symptoms of schizophrenia by augmenting NMDA receptor signaling. Several different mechanisms are currently under evaluation among them is activation of mGlu₅ receptors (Fitzjohn et al., 1996). The mGlu₅ receptor is coupled to G_αq/11 and glutamate binding results in the activation of phospholipase C, increase in inositol phosphate hydrolysis and the release of intracellular Ca⁺⁺ stores (Hermans and Challiss, 2001, Pin and Duvoisin, 1995). This leads to the phosphorylation of NMDA receptor and enhancement of its function (Benquet et al., 2002, Salter, 1998). Many reports have shown that the activation of mGlu₅ receptors can regulate NMDA receptor function in neuronal circuits located in the frontal cortex and hippocampus which are thought to be affected in schizophrenic patients (Aniksztejn et al., 1991, Awad et al., 2000, Mannaioni et al., 2001). The orthosteric binding site of mGlu₅ receptor is an unfavorable target because of the difficulty to obtain subtype specific agonists. Therefore, much effort has been placed in identifying compounds that bind to an allosteric site which is located in the 7-transmembrane domain and positively modulate the receptor response. These allosteric sites are more heterogeneous than the orthosteric site which should allow for greater subtype selectivity (Raddatz et al., 2007). In addition to selectivity, positive allosteric modulators would have the advantage that receptor activation will be controlled by endogenous glutamate release and native neural circuit activity thereby selectively enhancing mGlu₅ receptor-related neurotransmission and reducing possible adverse affects of tonic agonist activation. Several research groups have attempted to discover mGlu₅ receptor positive allosteric modulators and these efforts have resulted in the identification of several novel chemical entities with allosteric modulator activity (de Paulis et al., 2006, Kinney et al., 2005, O'Brien et al., 2003). All of these compounds bind to an allosteric site that is shared with the negative allosteric modulator MPEP (Kuhn et al., 2002). In addition, several compounds have been tested in various in vivo animal models for antipsychotic activity and were active in these assays (Kinney et al., 2005, Liu et al., 2008). Here we describe the identification of a structurally novel mGlu₅ receptor positive allosteric modulator, which was able to enhance mGlu₅ receptor activity, modulate NMDA receptor dependent responses and demonstrate antipsychotic activity.

Section snippets

Calcium Mobilization Assay in HEK293 Cell Line

A Ca⁺⁺ influx assay similar to that described by Andreas Ritzén et al. (Ritzen et al., 2009) was used to detect mGlu₅ receptor positive allosteric modulator activity in an HEK293 cell line stably expressing the d-isoform of human mGlu₅ receptor. Positive allosteric modulator activity was assessed by measuring potentiation of the EC₂₀ response to l-glutamate in the presence of a test compound. The measurements were made using the fluorometric imaging plate reader (FLIPR) 384 (Molecular Devices,

Functional Potency and Selectivity of CPPZ in Cells Stably Expressing Human Recombinant mGlu₅ Receptor

The positive modulator activity of CPPZ (Fig.1) was determined in a functional assay that monitored release of intracellular calcium in a HEK293 cell line stably expressing the d-isoform of human mGlu₅ receptor (Fig. 2A–C). As shown in Fig. 2A, CPPZ concentration-dependently potentiated the calcium response elicited by the endogenous ligand glutamate. The pEC₅₀ of glutamate (6.22 ± 0.02) was significantly potentiated by preincubation with 12.5 μM CPPZ (pEC₅₀ = 6.91 ± 0.04) with a slight suppression of

Discussion

Many different findings support the hypothesis that there is a hypo-function of the NMDA receptor in schizophrenic patients. Therefore, enhancing NMDA receptor function is one way to treat the symptoms of this disease. We have focused on identifying compounds that can increase mGlu₅ receptor activity and thereby augment NMDA receptor function. Instead of developing an orthosteric agonist that would potentially cause adverse side effects through excessive mGlu₅ receptor activation or has

Conclusions

In summary, CPPZ was characterized as a positive modulator of mGlu₅ receptor in both human recombinant and primary cell culture Ca⁺⁺ release assays. It binds to the same allosteric site as MPEP and potentiates binding of ligands to the orthosteric site. The effects on NMDA receptor dependent response seen in vitro and in vivo indicate that mGlu₅ receptor positive allosteric modulators may be able to reverse the hypo-function of the NMDA receptor that is thought to be associated with

Acknowledgments

The authors would like to thank Linda A. Sygowski, Robert Manning, Gregory D. Yasay and Julie M. Wilson for technical assistance. In addition, we would like to thank Dan Widzowski, Thomas Hudzik, Clay W. Scott, Dean Brown, Megan M. King, Todd A. Brugel, Donna L. Maier, Will Heaton, Becky Brockel, Thomas M. Stormann and Edwin Johnson for their scientific. Last but not least, we would like to thank the staff of the AstraZeneca Wilmington veterinary medicine department for their support in animal

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Neuropharmacology and AnalgesiaPreclinical profile of a novel metabotropic glutamate receptor 5 positive allosteric modulator

Abstract

Introduction

Section snippets

Calcium Mobilization Assay in HEK293 Cell Line

Functional Potency and Selectivity of CPPZ in Cells Stably Expressing Human Recombinant mGlu5 Receptor

Discussion

Conclusions

Acknowledgments

Neurobiol. Dis.

Eur. J. Pharmacol.

J. Psychiatr. Res.

Neuropharmacology

Biochem. Pharmacol.

Bioorg. Med. Chem. Lett.

J. Biol. Chem.

Biochem. Pharmacol.

Adv. Drug Deliv. Rev.

J. Biol. Chem.

Activation of metabotropic glutamate receptor 5 has direct excitatory effects and potentiates NMDA receptor currents in neurons of the subthalamic nucleus

J. Neurosci.

mGluR5 positive allosteric modulators facilitate both hippocampal LTP and

Neuropsychopharmacology

Two distinct signaling pathways upregulate NMDA receptor responses via two distinct metabotropic glutamate receptor subtypes

J. Neurosci.

Neuropharmacology and Analgesia
Preclinical profile of a novel metabotropic glutamate receptor 5 positive allosteric modulator

Functional Potency and Selectivity of CPPZ in Cells Stably Expressing Human Recombinant mGlu₅ Receptor