In Vitro Functional Characterization of GET73 as Possible Negative Allosteric Modulator of Metabotropic Glutamate Receptor 5.

Sarah Beggiato,Andrea C Borelli,Nicholas Pintori,Luca Ferraro,Antonella Loche,Maria C Tomasini,Roberto Cacciaglia,M Paola Castelli

doi:10.3389/fphar.2018.00327

Abstract

The present study was aimed to further characterize the pharmacological profile of N-[4-(trifluoromethyl) benzyl]-4-methoxybutyramide (GET73), a putative negative allosteric modulator (NAM) of metabotropic glutamate subtype 5 receptor (mGluR5) under development as a novel medication for the treatment of alcohol dependence. This aim has been accomplished by means of a series of in vitro functional assays. These assays include the measure of several down-stream signaling [intracellular Ca++ levels, inositol phosphate (IP) formation and CREB phosphorylation (pCREB)] which are generally affected by mGluR5 ligands. In particular, GET73 (0.1 nM–10 μM) was explored for its ability to displace the concentration-response curve of some mGluR5 agonists/probes (glutamate, L-quisqualate, CHPG) in different native preparations. GET73 produced a rightward shift of concentration-response curves of glutamate- and CHPG-induced intracellular Ca++ levels in primary cultures of rat cortical astrocytes. The compound also induced a rightward shift of concentration response curve of glutamate- and L-quisqualate-induced increase in IP turnover in rat hippocampus slices, along with a reduction of CHPG (10 mM)-induced increase in IP formation. Moreover, GET73 produced a rightward shift of concentration-response curve of glutamate-, CHPG- and L-quisqualate-induced pCREB levels in rat cerebral cortex neurons. Although the engagement of other targets cannot be definitively ruled out, these data support the view that GET73 acts as an mGluR5 NAM and support the significance of further investigating the possible mechanism of action of the compound.

Highlights

Alcohol dependence is a chronic relapsing disorder, which continues to be a concerning health and socio-economic issue worldwide (World Health Organization [WHO], 2014)
The effects of higher GET73 concentrations were counteracted by methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP), suggesting that the compound might exert a positive modulation at mGluR5 (Ferraro et al, 2011, 2013; Beggiato et al, 2013)
Taking into account these issues, the possible ability of GET73 to shift the concentration-response curves of different agonists/probes on different intracellular pathways associated with mGluR5 activation (i.e., Ca++ levels, inositol phosphate (IP) turnover and CREB phosphorylation (pCREB) levels; Wang et al, 2007) and in different in vitro preparations, has been evaluated

Summary

Introduction

Alcohol dependence is a chronic relapsing disorder, which continues to be a concerning health and socio-economic issue worldwide (World Health Organization [WHO], 2014). The development of N-[4-(trifluoromethyl) benzyl]4-methoxybutyramide (GET73) falls within this context In preclinical studies this compound has shown the ability to reduce alcohol intake along with anxiolytic-like properties (Loche et al, 2012; Ferraro et al, 2013). Its possible mechanism of action has been related to the modulation of glutamate neurotransmission through the metabotropic glutamate subtype 5 receptor (mGluR5) (Ferraro et al, 2011, 2013; Beggiato et al, 2013), a promising target for the development of pharmacological alcohol dependence treatments (Olive, 2009; Duncan and Lawrence, 2012; Holmes et al, 2013; Goodwani et al, 2017), and for many other psychiatric conditions, such as anxiety and depressive states (Tatarczynska et al, 2001). The results of several Phase 1 clinical studies indicate that GET73 is safe and well-tolerated both in healthy volunteers (Haass-Koffler et al, 2017a,b), and in alcohol-dependent patients (ongoing study NCT01842503)

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Discussion

Conclusion