**Metabotropic Receptors for Glutamate and GABA**

Gregory Stewart, Julie Kniazeff, Laurent Prézeau, Philippe Rondard, Jean-Philippe Pin and Cyril Goudet *Institut de Génomique Fonctionnelle, CNRS UMR5203 - INSERM U661 - Universités Montpellier 1&2 France*

## **1. Introduction**

G protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and due to their ubiquitous expression and vast array of functions they present attractive targets for the treatment of a wide number of diseases and disorders. Accordingly, they represent up to 30% of targets of current therapeutics (Overington et al., 2006). Despite the capacity of GPCRs to modulate many (patho-)physiological functions there is a high attrition rate with regard to new compounds entering clinical trials. There are many reasons for the number of failed drug-like compounds such as non-specificity, unfavourable pharmacokinetic profile and lack of clinical efficacy. In this regard, molecules targeting neurotransmitter receptors in the CNS traditionally have poor side-effect profiles due to the high concentrations required to pass the blood-brain barrier. There remain many specific challenges in drug discovery such as promiscuous GPCR-effector coupling; differential celland tissue-specific effects; ligand-induced changes in receptor trafficking; and proteinprotein interactions and receptor oligomerisation (Galandrin et al., 2007; Hanyaloglu and von Zastrow, 2008; Kniazeff et al., 2011; Wettschureck and Offermanns, 2005).

GPCRs are divided into three main classes (A-C) based on structural homology; however all GPCRs possess a 7-alpha-helical transmembrane-spanning (7TM) domain, which facilitates the transduction of extracellular signals into intracellular responses. GPCRs recognise a myriad of different stimuli from photons, amino acids and biogenic amines to large peptides and proteins. Class A (rhodopsin-like) GPCRs are among the best characterised and consist of a relatively short N-terminal domain, a 7TM domain connected by extracellular and intracellular loops, and an intracellular C-terminal domain (Fredriksson et al., 2003). Class B (secretin-like) GPCRs have comparatively long N-terminal domains with similar 7TM and C-terminal topography as Class A receptors. By far and away, Class C (glutamate-like) GPCRs have the most distinct topography compared the other GPCRs; they possess large, structured N-terminal domains, which form a venus-fly trap-like structure known as the venus-fly trap (VFT) domain. The VFT domain is often (with exceptions) connected to the 7TM domain via a cysteine-rich domain, and further to this the C-terminal domain is often comparatively longer than those of Class A GPCRs. Structurally, all GPCRs are similar in their 7TM domains, yet the activation mechanisms, at least by the endogenous ligand varies

Metabotropic Receptors for Glutamate and GABA 5

concentration- and/or oligomerisation-dependent event (Sato et al., 2007; Scholten et al.,

Taken together, the ligand-receptor-effector combinations, receptor oligomerisation and allosteric modulation of GPCRs furnish a mode of fine-tuning functional outputs and

This chapter will focus on two major receptor types of Class C GPCRs, the metabotropic glutamate and metabotropic -amino-butyric acid (GABA) receptors, which are the GPCRs of the major excitatory and inhibitory neurotransmitters in the adult brain, respectively. These receptors represent major targets for many CNS disorders such as schizophrenia, Parkinson's disease, Alzheimer's disease, epilepsy and diseases of addiction (Conn et al.,

Metabotropic glutamate (mGlu) receptors are widely expressed in the CNS and are activated by the excitatory neurotransmitter, glutamate. These receptors play a vital role in the regulation on neuronal excitability and synaptic transmission (Conn and Pin, 1997). Consequently, these receptors are valuable targets for treating neurological disorders such as schizophrenia, Parkinson's disease and neuropathic pain, either by correcting neurological imbalances in non-glutamatergic systems or through treating disregulation of

The members of the mGlu receptor family are obligate dimers and long thought of as obligate homodimers, but have recently been demonstrated to selectively form heterodimers amongst other mGluR subtypes in HEK cells (Doumazane et al., 2011). This propensity may be of utility in texturing the glutamatergic response across diverse brain regions. mGlu receptors consist of 8 subtypes that are divided into three subgroups (I-III) based on sequence homology, function and pharmacological profile (Pin and Acher, 2002). Group I mGluRs (mGlu1 and mGlu1) are Gq/11-coupled thereby signalling through the phospholipase C-IP3-Ca2+ axis; whereas Group II (mGlu2 and mGlu3) and Group III (mGlu4, mGlu6, mGlu7 and mGlu8) signal through inhibitory G proteins (Gi/o), which most likely serve as intermediaries between the receptor and ligand-gated ion channels, such as voltageoperated potassium channels (Kv2 channels) and voltage-operated calcium channels (Cav2

In drug discovery the understanding of the molecular mechanisms of ligand binding and receptor activation are paramount in order to investigate novel and improved methods for targeting these receptors therapeutically. In this regard, it is important to determine the overall receptor activation event by breaking it down into its fundamental component. Furthermore, to gather information about mGlu receptors, we must also use information gained from studies of other Class C GPCRs to form a global conformational image. Ligand binding in a VFT structure has been described with the periplasmic binding protein, which appears to be similar in class C receptors (O'Hara et al., 1993). The VFT remains in a state of equilibrium between two main conformations: open (o) and closed (c), known as the resting state. The orthosteric ligands bind primarily to the open VFT in lobe 1 and subsequently

2011; Urizar et al., 2011).

2009a; Tyacke et al., 2010).

glutamatergic signalling.

potentially, therefore, clinical outcomes.

**2. Metabotropic glutamate receptors**

**2.1 Phylogeny and structure/function of mGlu receptors**

channels) (Doupnik, 2008; Herlitze et al., 1996; Peleg et al., 2002).

greatly across the classes. The orthosteric (endogenous ligand) binding site in Class A GPCRs lies in the 7TM helical bundle (with exceptions, e.g. CXCR4 chemokine receptor and relaxin family receptors (Allen et al., 2007; Sudo et al., 2003)); class B receptor ligands tend to bind in the large N-terminal domain and have been postulated to possess a bimodal receptor activation mechanism, whereby after the ligand binding event the ligand-Nterminal complex inserts into the 7TM helical bundle to elicit receptor activation (Hoare, 2005); class C receptor orthosteric ligands bind in the VFT domain and, through a series of conformational changes, are able to induce receptor activation via the 7TM domain (Pin et al., 2004)(Figure 1).

Fig. 1. Canonical orthosteric ligand-binding domains of the three classes of GPCRs. Highlighted in yellow are the typical binding regions of orthosteric ligands, in addition to the general architecture of the three major classes of GPCRs.

One large hindrance to drug discovery is the high degree of protein sequence and structural conservation between orthosteric sites of receptors of the same family, increasing the difficulty to specifically and selectively target a single receptor subtype. However, by their very nature GPCRs are highly dynamic proteins that are able to adopt a spectrum of conformational arrangements and it is this characteristic that allows GPCRs to be modulated by, not only a range of orthosteric ligands, but also ligands that bind in a topographically distinct region to the orthosteric binding pocket. These ligands are known as allosteric ligands and are able to modulate the affinity and/or efficacy of the orthosteric ligand, and indeed, possess their own efficacy in the absence of orthosteric ligand (Christopoulos and Kenakin, 2002; Conn et al., 2009a). This phenomenon presents a unique opportunity to exploit GPCRs as drug targets through offering novel and often less-conserved ligand binding sites across receptor subtypes.

Despite the best-characterised coupling partners of GPCRs being heterotrimeric G proteins, they are also well known to couple to a host of other intracellular proteins (e.g. arrestins and small G proteins (Burridge and Wennerberg, 2004; Lefkowitz, 1998)), thus adding an extra degree of complexity to the pluri-dimensional response of ligand-GPCR interactions. Furthermore, promiscuous coupling has been shown, in some cases, to be a

greatly across the classes. The orthosteric (endogenous ligand) binding site in Class A GPCRs lies in the 7TM helical bundle (with exceptions, e.g. CXCR4 chemokine receptor and relaxin family receptors (Allen et al., 2007; Sudo et al., 2003)); class B receptor ligands tend to bind in the large N-terminal domain and have been postulated to possess a bimodal receptor activation mechanism, whereby after the ligand binding event the ligand-Nterminal complex inserts into the 7TM helical bundle to elicit receptor activation (Hoare, 2005); class C receptor orthosteric ligands bind in the VFT domain and, through a series of conformational changes, are able to induce receptor activation via the 7TM domain (Pin et

Fig. 1. Canonical orthosteric ligand-binding domains of the three classes of GPCRs. Highlighted in yellow are the typical binding regions of orthosteric ligands, in addition to

One large hindrance to drug discovery is the high degree of protein sequence and structural conservation between orthosteric sites of receptors of the same family, increasing the difficulty to specifically and selectively target a single receptor subtype. However, by their very nature GPCRs are highly dynamic proteins that are able to adopt a spectrum of conformational arrangements and it is this characteristic that allows GPCRs to be modulated by, not only a range of orthosteric ligands, but also ligands that bind in a topographically distinct region to the orthosteric binding pocket. These ligands are known as allosteric ligands and are able to modulate the affinity and/or efficacy of the orthosteric ligand, and indeed, possess their own efficacy in the absence of orthosteric ligand (Christopoulos and Kenakin, 2002; Conn et al., 2009a). This phenomenon presents a unique opportunity to exploit GPCRs as drug targets through offering novel and often less-conserved ligand

Despite the best-characterised coupling partners of GPCRs being heterotrimeric G proteins, they are also well known to couple to a host of other intracellular proteins (e.g. arrestins and small G proteins (Burridge and Wennerberg, 2004; Lefkowitz, 1998)), thus adding an extra degree of complexity to the pluri-dimensional response of ligand-GPCR interactions. Furthermore, promiscuous coupling has been shown, in some cases, to be a

the general architecture of the three major classes of GPCRs.

binding sites across receptor subtypes.

al., 2004)(Figure 1).

concentration- and/or oligomerisation-dependent event (Sato et al., 2007; Scholten et al., 2011; Urizar et al., 2011).

Taken together, the ligand-receptor-effector combinations, receptor oligomerisation and allosteric modulation of GPCRs furnish a mode of fine-tuning functional outputs and potentially, therefore, clinical outcomes.

This chapter will focus on two major receptor types of Class C GPCRs, the metabotropic glutamate and metabotropic -amino-butyric acid (GABA) receptors, which are the GPCRs of the major excitatory and inhibitory neurotransmitters in the adult brain, respectively. These receptors represent major targets for many CNS disorders such as schizophrenia, Parkinson's disease, Alzheimer's disease, epilepsy and diseases of addiction (Conn et al., 2009a; Tyacke et al., 2010).

#### **2. Metabotropic glutamate receptors**

#### **2.1 Phylogeny and structure/function of mGlu receptors**

Metabotropic glutamate (mGlu) receptors are widely expressed in the CNS and are activated by the excitatory neurotransmitter, glutamate. These receptors play a vital role in the regulation on neuronal excitability and synaptic transmission (Conn and Pin, 1997). Consequently, these receptors are valuable targets for treating neurological disorders such as schizophrenia, Parkinson's disease and neuropathic pain, either by correcting neurological imbalances in non-glutamatergic systems or through treating disregulation of glutamatergic signalling.

The members of the mGlu receptor family are obligate dimers and long thought of as obligate homodimers, but have recently been demonstrated to selectively form heterodimers amongst other mGluR subtypes in HEK cells (Doumazane et al., 2011). This propensity may be of utility in texturing the glutamatergic response across diverse brain regions. mGlu receptors consist of 8 subtypes that are divided into three subgroups (I-III) based on sequence homology, function and pharmacological profile (Pin and Acher, 2002). Group I mGluRs (mGlu1 and mGlu1) are Gq/11-coupled thereby signalling through the phospholipase C-IP3-Ca2+ axis; whereas Group II (mGlu2 and mGlu3) and Group III (mGlu4, mGlu6, mGlu7 and mGlu8) signal through inhibitory G proteins (Gi/o), which most likely serve as intermediaries between the receptor and ligand-gated ion channels, such as voltageoperated potassium channels (Kv2 channels) and voltage-operated calcium channels (Cav2 channels) (Doupnik, 2008; Herlitze et al., 1996; Peleg et al., 2002).

In drug discovery the understanding of the molecular mechanisms of ligand binding and receptor activation are paramount in order to investigate novel and improved methods for targeting these receptors therapeutically. In this regard, it is important to determine the overall receptor activation event by breaking it down into its fundamental component. Furthermore, to gather information about mGlu receptors, we must also use information gained from studies of other Class C GPCRs to form a global conformational image. Ligand binding in a VFT structure has been described with the periplasmic binding protein, which appears to be similar in class C receptors (O'Hara et al., 1993). The VFT remains in a state of equilibrium between two main conformations: open (o) and closed (c), known as the resting state. The orthosteric ligands bind primarily to the open VFT in lobe 1 and subsequently

Metabotropic Receptors for Glutamate and GABA 7

As we have described above, an allosteric modulator binding in the 7TM affects both the G protein activation and agonist affinity for the VFT. Together with the fact that a conformational change in the VFT dimer activates the 7TM, this indicates that VFT and 7TM converse in both ways. The question that remains is how the stimulus is transduced through

In most of class C GPCRs, VFT and 7TM are connected with the CRD. The CRD is an 80 residues long domain containing 9 cysteines. This domain is present in mGlu, CaS, GPRC6A and T1R receptors, but not in GABAB receptors. The structure of this domain has been solved for mGlu3 (Muto et al., 2007), and this domain appears to be a rigid 40Å long structure, which is most likely to form a physical gearing system between the VFT and 7TM domains. In agreement with these physical findings, both deletion of the CRD in mGlu or CaS receptors and mutations of T1R3 CRD abolish the agonist-induced receptor activation (Hu et al., 2000; Jiang et al., 2004). Furthermore, we have shown that the VFT and CRD domains in mGlu2 are linked by a disulphide bridge between a cysteine at the bottom of the VFT and the only cysteine that is not engaged in intradomain disulphide bond within the CRD (Rondard et al., 2006). Rondard et al., had shown that the mutation of the residues involved in this interaction abolished agonist-mediated activation of the receptor. This supports the idea of a central role for the CRD in the transduction of the conformational

The exact mechanisms of 7TM activation Class C and indeed, mGlu receptors remain to be solved. This notwithstanding, there are approaches that can be employed in an attempt to determine the molecular mechanisms involved in the conformational changes that the 7TM domains undergoes upon activation. One of these approaches is entails the use of both positive and negative allosteric modulators. The first allosteric modulators of class C GPCRs to be described were found to be non-competitive antagonists or inverse agonists (Carroll et al., 2001; Litschig et al., 1999; Pagano et al., 2000). Other compounds have been described that potentiated the effect of the agonists (increased affinity and efficacy) (Felts et al., 2010; Hammond et al., 2010; Urwyler et al., 2001). These molecules are structurally distinct from the orthosteric agonists and antagonists, as reflected in their binding within the 7TM, in a binding region that is reminiscent of the orthosteric binding pocket in Class A receptors (Brauner-Osborne et al., 2007; Goudet et al., 2004). So far, no endogenous PAM or NAM binding in the 7TM pocket has been described. Selective pressure in the evolution of a site/pocket is often indicative of a biological function, but there is no conserved pocket located within the 7TM domain of mGluRs, making it less likely that there is an endogenous allosteric ligand that acts in that region. The absence of conservation allowed the discovery of molecules specific for a single subtype of mGlu receptor, as opposed to a ligand acting at the well conserved orthosteric binding site. If both PAM and NAM act at the 7TM, then their opposite effects are likely due to differences in the residues that the ligands are in contact with in the 7TM. Specifically, several studies indicate that PAM and NAM bind to overlapping but not identical sites (Miedlich et al., 2004; Petrel et al., 2004). Some of these interaction networks should stabilize the active conformation of the 7TM, whilst some others should lock the receptor in its inactive conformation. However, it was shown that structurally different molecules bind essentially at the same position in the 7TM, only the precise identity of the residues contacting the molecule may differ. It appears that the position of PAM/NAM binding site is largely conserved in the whole family and includes residues from TM3, 5, 6 and 7 (Hu et al., 2000; Miedlich et al., 2004; Pagano et al., 2000). However, in some cases, two distinct sites have been

the VFT region to the 7TM domain?

changes from the VFT dimer to the 7TM in these receptors.

promote the closed conformation as interactions with lobe 2 stabilises this state. This suggests that, if agonists induce the closure of the VFT, orthosteric antagonists act to prevent the closure of the VFT, thereby blocking the appropriate mechanisms leading to 7TM activation (Bessis et al., 2000; Bessis et al., 2002; Kunishima et al., 2000; Tsuchiya et al., 2002). For a number of years, the question on how ligand binding in the VFT results in 7TM activation remained to be elucidated. The breakthrough came from the rst crystal structures of a class C VFT dimer, from the mGlu1 receptor, crystallised in the presence and absence of glutamate (Kunishima et al., 2000). These structures confirmed the overall structure of the domain and, perhaps more importantly, the agonist binding mode in a single VFT domain. It also revealed large, structural rearrangements of the VFT dimer resulting in a change of the relative orientation of the two protomers. A general mechanism for VFT dimer conformational changes was proposed by the authors: two orientations of the VFT dimer exist and are in equilibrium: a resting (R) and an active (A) orientation. In the R orientation, the VFTs interact via lobe-I only, leaving the lobes-II separate from each other. In the A orientation, there is a reorganization of the VFTs relative orientation such that they also interact via each lobe-II. This large reorientation from R to A was proposed to induce the conformational changes required for 7TM activation. Resting and active designations were given to the different orientations as glutamate was proposed to stabilize the A form. The active and inactive property of the A and R orientations are further supported by mGlu1 structures obtained in the presence of an antagonist (MCPG) or in the presence of a potentiator (Gd3+) in which the dimer orientation is R and A, respectively (Tsuchiya et al., 2002).

When considering the various conformations for the VFT and the VFT dimer, there are a total of six theoretical conformations that are possible: Roo, Rco and Rcc and Aoo, Aco and Acc, where A and R are indicative for the VFT dimer orientation and c and o for the VFT conformation. It is assumed that agonist binding to at least one of the VFT stabilizes the c form, which is the driving force leading to the VFT dimer reorientation from R to A. In agreement, only Roo, Rco, Aco and Acc are likely to exist. However, new crystal structures of the isolated VFT dimer in the 'forbidden' conformation Rcc (Muto et al., 2007) and Aoo (PDB accession number, 3KS9) were recently deposited in the protein data bank (PDB). In particular, the Aoo conformation appears to be highly unlikely to occur within a dynamic equilibrium as many residues of the same polarity from lobe 2 would be in close proximity to one another, so much so that this would likely destabilise this conformation through the repulsive forces exerted within lobe 2 (Tsuchiya et al., 2002). Whilst explanations for these surprising observations have not been provided, the absence of 7TM may have alleviated some conformational constraints that may otherwise be exerted on the VFT from the 7TM, acting as a structural tether that inhibits certain conformations.

A question arising upon closer analysis of the crystal structure is the number of agonists needed to activate a class C GPCR dimer. When considering the reorientation of the VFT from R to A as the sole mechanism responsible for 7TM activation, one may wonder whether there is a functional difference between Aco and Acc conformation. In other words, what would be the difference in binding one or two agonists? It was shown that in class C heterodimers a single subunit was responsible in binding the endogenous ligand (GABAB1 in GABAB receptor and T1R1 or T1R2 in the taste receptors)(Kniazeff et al., 2002; Nelson et al., 2001). This suggests that a single agonist molecule is sufcient to fully activate heterodimeric receptors in these cases.

promote the closed conformation as interactions with lobe 2 stabilises this state. This suggests that, if agonists induce the closure of the VFT, orthosteric antagonists act to prevent the closure of the VFT, thereby blocking the appropriate mechanisms leading to 7TM activation (Bessis et al., 2000; Bessis et al., 2002; Kunishima et al., 2000; Tsuchiya et al., 2002). For a number of years, the question on how ligand binding in the VFT results in 7TM activation remained to be elucidated. The breakthrough came from the rst crystal structures of a class C VFT dimer, from the mGlu1 receptor, crystallised in the presence and absence of glutamate (Kunishima et al., 2000). These structures confirmed the overall structure of the domain and, perhaps more importantly, the agonist binding mode in a single VFT domain. It also revealed large, structural rearrangements of the VFT dimer resulting in a change of the relative orientation of the two protomers. A general mechanism for VFT dimer conformational changes was proposed by the authors: two orientations of the VFT dimer exist and are in equilibrium: a resting (R) and an active (A) orientation. In the R orientation, the VFTs interact via lobe-I only, leaving the lobes-II separate from each other. In the A orientation, there is a reorganization of the VFTs relative orientation such that they also interact via each lobe-II. This large reorientation from R to A was proposed to induce the conformational changes required for 7TM activation. Resting and active designations were given to the different orientations as glutamate was proposed to stabilize the A form. The active and inactive property of the A and R orientations are further supported by mGlu1 structures obtained in the presence of an antagonist (MCPG) or in the presence of a potentiator (Gd3+) in which the dimer orientation is

When considering the various conformations for the VFT and the VFT dimer, there are a total of six theoretical conformations that are possible: Roo, Rco and Rcc and Aoo, Aco and Acc, where A and R are indicative for the VFT dimer orientation and c and o for the VFT conformation. It is assumed that agonist binding to at least one of the VFT stabilizes the c form, which is the driving force leading to the VFT dimer reorientation from R to A. In agreement, only Roo, Rco, Aco and Acc are likely to exist. However, new crystal structures of the isolated VFT dimer in the 'forbidden' conformation Rcc (Muto et al., 2007) and Aoo (PDB accession number, 3KS9) were recently deposited in the protein data bank (PDB). In particular, the Aoo conformation appears to be highly unlikely to occur within a dynamic equilibrium as many residues of the same polarity from lobe 2 would be in close proximity to one another, so much so that this would likely destabilise this conformation through the repulsive forces exerted within lobe 2 (Tsuchiya et al., 2002). Whilst explanations for these surprising observations have not been provided, the absence of 7TM may have alleviated some conformational constraints that may otherwise be exerted on the VFT from the 7TM,

A question arising upon closer analysis of the crystal structure is the number of agonists needed to activate a class C GPCR dimer. When considering the reorientation of the VFT from R to A as the sole mechanism responsible for 7TM activation, one may wonder whether there is a functional difference between Aco and Acc conformation. In other words, what would be the difference in binding one or two agonists? It was shown that in class C heterodimers a single subunit was responsible in binding the endogenous ligand (GABAB1 in GABAB receptor and T1R1 or T1R2 in the taste receptors)(Kniazeff et al., 2002; Nelson et al., 2001). This suggests that a single agonist molecule is sufcient to fully activate

R and A, respectively (Tsuchiya et al., 2002).

heterodimeric receptors in these cases.

acting as a structural tether that inhibits certain conformations.

As we have described above, an allosteric modulator binding in the 7TM affects both the G protein activation and agonist affinity for the VFT. Together with the fact that a conformational change in the VFT dimer activates the 7TM, this indicates that VFT and 7TM converse in both ways. The question that remains is how the stimulus is transduced through the VFT region to the 7TM domain?

In most of class C GPCRs, VFT and 7TM are connected with the CRD. The CRD is an 80 residues long domain containing 9 cysteines. This domain is present in mGlu, CaS, GPRC6A and T1R receptors, but not in GABAB receptors. The structure of this domain has been solved for mGlu3 (Muto et al., 2007), and this domain appears to be a rigid 40Å long structure, which is most likely to form a physical gearing system between the VFT and 7TM domains. In agreement with these physical findings, both deletion of the CRD in mGlu or CaS receptors and mutations of T1R3 CRD abolish the agonist-induced receptor activation (Hu et al., 2000; Jiang et al., 2004). Furthermore, we have shown that the VFT and CRD domains in mGlu2 are linked by a disulphide bridge between a cysteine at the bottom of the VFT and the only cysteine that is not engaged in intradomain disulphide bond within the CRD (Rondard et al., 2006). Rondard et al., had shown that the mutation of the residues involved in this interaction abolished agonist-mediated activation of the receptor. This supports the idea of a central role for the CRD in the transduction of the conformational changes from the VFT dimer to the 7TM in these receptors.

The exact mechanisms of 7TM activation Class C and indeed, mGlu receptors remain to be solved. This notwithstanding, there are approaches that can be employed in an attempt to determine the molecular mechanisms involved in the conformational changes that the 7TM domains undergoes upon activation. One of these approaches is entails the use of both positive and negative allosteric modulators. The first allosteric modulators of class C GPCRs to be described were found to be non-competitive antagonists or inverse agonists (Carroll et al., 2001; Litschig et al., 1999; Pagano et al., 2000). Other compounds have been described that potentiated the effect of the agonists (increased affinity and efficacy) (Felts et al., 2010; Hammond et al., 2010; Urwyler et al., 2001). These molecules are structurally distinct from the orthosteric agonists and antagonists, as reflected in their binding within the 7TM, in a binding region that is reminiscent of the orthosteric binding pocket in Class A receptors (Brauner-Osborne et al., 2007; Goudet et al., 2004). So far, no endogenous PAM or NAM binding in the 7TM pocket has been described. Selective pressure in the evolution of a site/pocket is often indicative of a biological function, but there is no conserved pocket located within the 7TM domain of mGluRs, making it less likely that there is an endogenous allosteric ligand that acts in that region. The absence of conservation allowed the discovery of molecules specific for a single subtype of mGlu receptor, as opposed to a ligand acting at the well conserved orthosteric binding site. If both PAM and NAM act at the 7TM, then their opposite effects are likely due to differences in the residues that the ligands are in contact with in the 7TM. Specifically, several studies indicate that PAM and NAM bind to overlapping but not identical sites (Miedlich et al., 2004; Petrel et al., 2004). Some of these interaction networks should stabilize the active conformation of the 7TM, whilst some others should lock the receptor in its inactive conformation. However, it was shown that structurally different molecules bind essentially at the same position in the 7TM, only the precise identity of the residues contacting the molecule may differ. It appears that the position of PAM/NAM binding site is largely conserved in the whole family and includes residues from TM3, 5, 6 and 7 (Hu et al., 2000; Miedlich et al., 2004; Pagano et al., 2000). However, in some cases, two distinct sites have been

Metabotropic Receptors for Glutamate and GABA 9

Studying molecular mechanisms and pharmacology of GPCRs in heterologous cells systems can be exceptionally useful due to the eradication of confounding factors such as multiple receptor subtypes; in addition the capacity to modulate receptor expression and function of specific signalling pathways with relative ease. However, these systems are rarely indicative of native systems and it needs to be recognised that various GPCR interactions exist *in vivo* that do not exist in heterologous cell systems for a myriad of reasons. One such interaction is that of protein-protein interactions, whereby the physical or functional interaction of a number of proteins can greatly alter its behaviour. An example of this occurrence is a fundamental component of some Class C GPCR pharmacology, such that receptor activitymodifying proteins (RAMPs) modulate the pharmacology of receptors such as the calcitonin and calcitonin receptor-like receptor (Sexton et al., 2006). mGlu receptors are also a family

In cortical neurons, the simultaneous activation of adenosine A1 and mGlu1 receptors has been shown to synergistically decrease the neuronal toxicity due to application of NMDA (Ciruela et al., 2001). In astrocytes or in co-transfected HEK293 cells, activation of A1 receptors elicits an increased mGlu1 response via Gi/o proteins (Ciruela et al., 2001; Toms and Roberts, 1999). That effect could be indicative of cross-talk and priming of the intracellular Ca2+ response; however, Hirono et al. (2001) did not observe any potentiation of the mGlu1 response upon co-activation of the A1 receptor in in cerebellar Purkinje cells, supporting the hypothesis of cooperativity (physical or otherwise) rather than cross-talk of the signalling pathways. Although both receptors are co-localized and coimmunoprecipitated from neurons and transfected HEK293 cells, the existence and the requirement of a direct physical interaction is yet to be clearly

The mGlu5, adenosine A2A and dopamine D2 receptors are highly expressed in the striatum. These receptors have been proposed to play vital roles in the dysregulation of the motor coordination observed in the Parkinson's disease. Indeed, antagonists of both mGlu5 and A2A display anti-parkinsonian effects, while the dopamine D2 receptor is the target of L-DOPA, which is used to treat parkinsonian symptoms. It has been suggested that these three receptors may act in concert in pairs or as a triplet via signalling cross-talk or otherwise, to influence the striatal function in motor coordination (Agnati et al., 2003; Cabello et al., 2009). Indeed, this cross-regulation was observed *in vivo*, where mGlu5 antagonist-induced motor effects were augmented by A2A receptor antagonists; and conversely these effects were diminished in A2A-D2 receptor double knock-out mice (Kachroo et al., 2005). The exact molecular mechanisms of the cross-regulation are not well understood, but DARPP-32 (dopamine- and cAMP-regulated neuronal phosphoprotein) may play a pivotal role. Adenosine A2A receptors have been show to increase DARP-32 phosphorylation via the Gs signaling axis, whilst D2 receptors counteract this effect via the Gi/o pathway (Agnati et al., 2003); Furthermore, the co-activation of adenosine A2A and dopamine D2 receptors synergistically potentiated DARPP-32 phosphorylation *ex vivo* studies in striatum tissues. Notwithstanding, the regulation of intracellular Ca2+ and cAMP signals underpins other

that are capable of interacting with non-mGluR proteins to form complexes.

**2.2 Protein-protein interactions of mGlu receptors**

**2.2.1 mGlu1–A1 receptors**

established (Ciruela et al., 2001).

**2.2.2 mGlu5–A2A–D2 receptors**

identified for PAMs, as exemplified at mGlu5 (Chen et al., 2008). See Figure 2 for a schematic overview of mGlu receptor architecture and binding domains.

Fig. 2. Architecture, binding domains and dimerisation states of mGlu and GABAB receptors.

identified for PAMs, as exemplified at mGlu5 (Chen et al., 2008). See Figure 2 for a schematic

Fig. 2. Architecture, binding domains and dimerisation states of mGlu and GABAB receptors.

overview of mGlu receptor architecture and binding domains.

#### **2.2 Protein-protein interactions of mGlu receptors**

Studying molecular mechanisms and pharmacology of GPCRs in heterologous cells systems can be exceptionally useful due to the eradication of confounding factors such as multiple receptor subtypes; in addition the capacity to modulate receptor expression and function of specific signalling pathways with relative ease. However, these systems are rarely indicative of native systems and it needs to be recognised that various GPCR interactions exist *in vivo* that do not exist in heterologous cell systems for a myriad of reasons. One such interaction is that of protein-protein interactions, whereby the physical or functional interaction of a number of proteins can greatly alter its behaviour. An example of this occurrence is a fundamental component of some Class C GPCR pharmacology, such that receptor activitymodifying proteins (RAMPs) modulate the pharmacology of receptors such as the calcitonin and calcitonin receptor-like receptor (Sexton et al., 2006). mGlu receptors are also a family that are capable of interacting with non-mGluR proteins to form complexes.

#### **2.2.1 mGlu1–A1 receptors**

In cortical neurons, the simultaneous activation of adenosine A1 and mGlu1 receptors has been shown to synergistically decrease the neuronal toxicity due to application of NMDA (Ciruela et al., 2001). In astrocytes or in co-transfected HEK293 cells, activation of A1 receptors elicits an increased mGlu1 response via Gi/o proteins (Ciruela et al., 2001; Toms and Roberts, 1999). That effect could be indicative of cross-talk and priming of the intracellular Ca2+ response; however, Hirono et al. (2001) did not observe any potentiation of the mGlu1 response upon co-activation of the A1 receptor in in cerebellar Purkinje cells, supporting the hypothesis of cooperativity (physical or otherwise) rather than cross-talk of the signalling pathways. Although both receptors are co-localized and coimmunoprecipitated from neurons and transfected HEK293 cells, the existence and the requirement of a direct physical interaction is yet to be clearly established (Ciruela et al., 2001).

#### **2.2.2 mGlu5–A2A–D2 receptors**

The mGlu5, adenosine A2A and dopamine D2 receptors are highly expressed in the striatum. These receptors have been proposed to play vital roles in the dysregulation of the motor coordination observed in the Parkinson's disease. Indeed, antagonists of both mGlu5 and A2A display anti-parkinsonian effects, while the dopamine D2 receptor is the target of L-DOPA, which is used to treat parkinsonian symptoms. It has been suggested that these three receptors may act in concert in pairs or as a triplet via signalling cross-talk or otherwise, to influence the striatal function in motor coordination (Agnati et al., 2003; Cabello et al., 2009). Indeed, this cross-regulation was observed *in vivo*, where mGlu5 antagonist-induced motor effects were augmented by A2A receptor antagonists; and conversely these effects were diminished in A2A-D2 receptor double knock-out mice (Kachroo et al., 2005). The exact molecular mechanisms of the cross-regulation are not well understood, but DARPP-32 (dopamine- and cAMP-regulated neuronal phosphoprotein) may play a pivotal role. Adenosine A2A receptors have been show to increase DARP-32 phosphorylation via the Gs signaling axis, whilst D2 receptors counteract this effect via the Gi/o pathway (Agnati et al., 2003); Furthermore, the co-activation of adenosine A2A and dopamine D2 receptors synergistically potentiated DARPP-32 phosphorylation *ex vivo* studies in striatum tissues. Notwithstanding, the regulation of intracellular Ca2+ and cAMP signals underpins other

Metabotropic Receptors for Glutamate and GABA 11

relationship between receptor oligomerisation and functional cross-talk. The study of the precise mechanism of this phenomenon is still ongoing, and can perhaps furnish novel approaches for targeting these receptors for the treatment of schizophrenia and other

Another important interaction that further implicates the role of the glutamatergic system in schizophrenia is the interaction of the *N-*methly-D-aspartate (NMDA) receptor and mGlu5. This GPCR-ion channel interaction has been relatively well characterised from a functional stand point, but the molecular mechanisms of the interaction are only beginning to be

Indeed, in hippocampal neurons, mGlu5a co-localises with NMDA receptors, which mediates a slow excitatory postsynaptic current (Collingridge et al., 1983; Oliet et al., 1997). The activation of mGlu5 receptors enhances the NMDA-evoked responses in different regions of the brain, such as the hippocampus, the striatum, the cortex, or the spinal cord (Aniksztejn et al., 1992; Harvey and Collingridge, 1993). Recently, Perroy et al., (2008) have shown that both receptors, indeed, interact via the C-terminal domain of mGlu5a. Through use of the bioluminescence resonance energy transfer (BRET) approach, they demonstrated that a significant and specific BRET signal can be measured between the two receptors, and moreover that this signal was transiently increased by activation of either the mGlu5a receptor or the NMDA receptor; this suggests an allosteric interaction and ligand-dependent conformational rearrangement of the opposite protomer in the hetero-oligomer. Interestingly however, when co-expressed, the functional response of the either receptor was reduced, compared to the response when either receptor was expressed in isolation. Thus suggesting a reciprocal and constitutive suppression of the signalling between NMDA and mGlu5a receptors, which was suggested to be independent of the G protein coupling of mGlu5a. The inhibitory reciprocal effect was dependent on the physical interaction between these receptors, given that the inhibition was abolished upon suppression of the C-terminal

Group I mGlu receptors (mGlu1 and mGlu5) are extensively expressed throughout neurons in the CNS and, in addition, mGlu5 is expressed in glial cells. mGlu1 is most abundantly expressed in Purkinje cells of the cerebellar cortex and in the olfactory bulb, in addition to strong expression in the hippocampus, substantia nigra and globus pallidus (Baude et al., 1993; Martin et al., 1992); and mGlu5 is greatly expressed in corticolimbic regions, such as the striatum, hippocampus and cerebral cortex (Ferraguti and Shigemoto, 2006). For example in the hippocampus, mGlu1 has been demonstrated to be involved in synaptic transmission and plasticity, in addition to neuronal excitability (Bortolotto et al., 1999), whilst in both mGlu1 and mGlu5 are required for the induction of long-term depression (LTD) in corticostriatal synapses (Sung et al., 2001). Through the use of knockout (KO) mice the putative function of mGluRs can be elucidated and, indeed, mGlu1 and mGlu5 KO mice have been studied. In mGlu1 KO animals is a marked deficits in long-term potentiation (LTP) in hippocampal slices and in context-dependent fear conditioning task (Aiba et al., 1994a); suggesting reduced hippocampal-mediated learning and memory. Furthermore,

domain involved in receptor hetero-oligomerisation (Perroy et al., 2008).

**2.3 Localisation and physiological function**

neuronal disorders.

unfolded.

**2.2.4 mGlu5-NMDA receptors**

signalling interactions between these receptors (Ferre et al., 2002). Not only may this phenomenon be due to signalling cross talk amongst these receptors, but may be a result of physical interactions and allosteric regulation across heteromers. It A2A–D2 hetero-oligomers are mediated by electrostatic interactions between a basic-rich motif in the third intracellular loop of the D2 receptor and an acidic/serine residue-containing motif in the C-terminus of the adenosine A2A receptor (Azdad et al., 2009; Ciruela et al., 2004; Ferre et al., 2007). Additionally, are postulated to not only be co-expressed, but also to form hetero-oligomers in striatal neurons and in heterologous cells systems (Ferre et al., 2002). Recently, Cabello et al. (2009) demonstrated that mGlu5, dopamine D2 and adenosines A2A receptors are localised within the same dendritic spines in glutamatergic striatal synapses, which led them to hypothesise that there may be hetero-oligomeric triplets of A2A, mGlu5 and D2 receptors; this association was then investigated through the employment of various fluorescence techniques. Their data supported the formation of heterooligomers containing all three receptors and thus allosterically interacting with one another to influence either efficacy or affinity or both. It is noteworthy that additional cross-regulation between A2A and mGlu5 receptors has been reported in hippocampal neurons, where the inhibition of A2A receptors decreased the mGlu5-mediated potentiation of NMDA receptor responses (Tebano et al., 2006). However, the molecular mechanisms involved are yet to be elucidated.

#### **2.2.3 mGlu2–5-HT2A receptors**

One of the best-characterized receptor complex involving a Class C GPCR is the complex between mGlu2 and the serotonin 5-HT2A receptor. It is well documented that these receptors are both targeted by antipsychotic drugs such as 5-HT2A receptor inverse agonists and mGlu2 receptor agonists and PAMs (Benneyworth et al., 2008; Benneyworth et al., 2007). Furthermore, 5HT2A receptors are the target of hallucinogenic substances, for example LSD and psilocybin, which induce hallucinogenic episodes that are thought to be similar to some of the symptoms in schizophrenics (Aghajanian and Marek, 1999). Indeed, nonhallucinogenic 5HT2A agonists (5-HT included) activate the Gq signalling axis, whilst hallucinogenic compounds are proposed to additionally activate Gi/o and Src tyrosine kinase pathways, in cortical neurons (Gonzalez-Maeso et al., 2007; Gonzalez-Maeso et al., 2003). Activation of mGlu2 receptors in the prefrontal cortex by the mGlu2 PAM, biphenylindanone A (BINA), abrogated the hallucinogenic effects of compounds such as (-)2,5 dimethoxy-4-bromoamphetamine, [(-)DOB] (Benneyworth et al., 2007); suggesting functional antagonism between mGlu2 and 5HT2A receptors in prefrontal cortex, an interaction that is possibly altered in schizophrenics (Gonzalez-Maeso et al., 2007). In fact, co-expression of both receptors revealed that the hallucinogen-induced Gi coupling of 5- HT2A is ameliorated by mGlu2 in basal conditions, but abolished when mGlu2 is activated. The mechanism of this complex cross-talk remains to be fully unraveled, but it has been proposed to be the result of mGlu2–5-HT2A receptor oligomerisation. In cortical neurons, these receptors co-localise and co-immunoprecipitate (Gonzalez-Maeso et al., 2008). Indeed, biophysical approaches have been employed to demonstrate that these GPCRs are in fact in close enough proximity to be compatible with a physical association (Gonzalez-Maeso et al., 2008). Moreover, by adopting a chimeric approach between mGlu2 and mGlu3 (TM4 and TM5 substitution), the authors were able to demonstrate that mGlu3 receptors with substituted TM domains were able to oligomerise with the 5-HT2A receptor, further to exhibiting functional cross-talk (Gonzalez-Maeso et al., 2008). This supports the potential relationship between receptor oligomerisation and functional cross-talk. The study of the precise mechanism of this phenomenon is still ongoing, and can perhaps furnish novel approaches for targeting these receptors for the treatment of schizophrenia and other neuronal disorders.

## **2.2.4 mGlu5-NMDA receptors**

10 Pharmacology

signalling interactions between these receptors (Ferre et al., 2002). Not only may this phenomenon be due to signalling cross talk amongst these receptors, but may be a result of physical interactions and allosteric regulation across heteromers. It A2A–D2 hetero-oligomers are mediated by electrostatic interactions between a basic-rich motif in the third intracellular loop of the D2 receptor and an acidic/serine residue-containing motif in the C-terminus of the adenosine A2A receptor (Azdad et al., 2009; Ciruela et al., 2004; Ferre et al., 2007). Additionally, are postulated to not only be co-expressed, but also to form hetero-oligomers in striatal neurons and in heterologous cells systems (Ferre et al., 2002). Recently, Cabello et al. (2009) demonstrated that mGlu5, dopamine D2 and adenosines A2A receptors are localised within the same dendritic spines in glutamatergic striatal synapses, which led them to hypothesise that there may be hetero-oligomeric triplets of A2A, mGlu5 and D2 receptors; this association was then investigated through the employment of various fluorescence techniques. Their data supported the formation of heterooligomers containing all three receptors and thus allosterically interacting with one another to influence either efficacy or affinity or both. It is noteworthy that additional cross-regulation between A2A and mGlu5 receptors has been reported in hippocampal neurons, where the inhibition of A2A receptors decreased the mGlu5-mediated potentiation of NMDA receptor responses (Tebano et al.,

2006). However, the molecular mechanisms involved are yet to be elucidated.

One of the best-characterized receptor complex involving a Class C GPCR is the complex between mGlu2 and the serotonin 5-HT2A receptor. It is well documented that these receptors are both targeted by antipsychotic drugs such as 5-HT2A receptor inverse agonists and mGlu2 receptor agonists and PAMs (Benneyworth et al., 2008; Benneyworth et al., 2007). Furthermore, 5HT2A receptors are the target of hallucinogenic substances, for example LSD and psilocybin, which induce hallucinogenic episodes that are thought to be similar to some of the symptoms in schizophrenics (Aghajanian and Marek, 1999). Indeed, nonhallucinogenic 5HT2A agonists (5-HT included) activate the Gq signalling axis, whilst hallucinogenic compounds are proposed to additionally activate Gi/o and Src tyrosine kinase pathways, in cortical neurons (Gonzalez-Maeso et al., 2007; Gonzalez-Maeso et al., 2003). Activation of mGlu2 receptors in the prefrontal cortex by the mGlu2 PAM, biphenylindanone A (BINA), abrogated the hallucinogenic effects of compounds such as (-)2,5 dimethoxy-4-bromoamphetamine, [(-)DOB] (Benneyworth et al., 2007); suggesting functional antagonism between mGlu2 and 5HT2A receptors in prefrontal cortex, an interaction that is possibly altered in schizophrenics (Gonzalez-Maeso et al., 2007). In fact, co-expression of both receptors revealed that the hallucinogen-induced Gi coupling of 5- HT2A is ameliorated by mGlu2 in basal conditions, but abolished when mGlu2 is activated. The mechanism of this complex cross-talk remains to be fully unraveled, but it has been proposed to be the result of mGlu2–5-HT2A receptor oligomerisation. In cortical neurons, these receptors co-localise and co-immunoprecipitate (Gonzalez-Maeso et al., 2008). Indeed, biophysical approaches have been employed to demonstrate that these GPCRs are in fact in close enough proximity to be compatible with a physical association (Gonzalez-Maeso et al., 2008). Moreover, by adopting a chimeric approach between mGlu2 and mGlu3 (TM4 and TM5 substitution), the authors were able to demonstrate that mGlu3 receptors with substituted TM domains were able to oligomerise with the 5-HT2A receptor, further to exhibiting functional cross-talk (Gonzalez-Maeso et al., 2008). This supports the potential

**2.2.3 mGlu2–5-HT2A receptors**

Another important interaction that further implicates the role of the glutamatergic system in schizophrenia is the interaction of the *N-*methly-D-aspartate (NMDA) receptor and mGlu5. This GPCR-ion channel interaction has been relatively well characterised from a functional stand point, but the molecular mechanisms of the interaction are only beginning to be unfolded.

Indeed, in hippocampal neurons, mGlu5a co-localises with NMDA receptors, which mediates a slow excitatory postsynaptic current (Collingridge et al., 1983; Oliet et al., 1997). The activation of mGlu5 receptors enhances the NMDA-evoked responses in different regions of the brain, such as the hippocampus, the striatum, the cortex, or the spinal cord (Aniksztejn et al., 1992; Harvey and Collingridge, 1993). Recently, Perroy et al., (2008) have shown that both receptors, indeed, interact via the C-terminal domain of mGlu5a. Through use of the bioluminescence resonance energy transfer (BRET) approach, they demonstrated that a significant and specific BRET signal can be measured between the two receptors, and moreover that this signal was transiently increased by activation of either the mGlu5a receptor or the NMDA receptor; this suggests an allosteric interaction and ligand-dependent conformational rearrangement of the opposite protomer in the hetero-oligomer. Interestingly however, when co-expressed, the functional response of the either receptor was reduced, compared to the response when either receptor was expressed in isolation. Thus suggesting a reciprocal and constitutive suppression of the signalling between NMDA and mGlu5a receptors, which was suggested to be independent of the G protein coupling of mGlu5a. The inhibitory reciprocal effect was dependent on the physical interaction between these receptors, given that the inhibition was abolished upon suppression of the C-terminal domain involved in receptor hetero-oligomerisation (Perroy et al., 2008).

#### **2.3 Localisation and physiological function**

Group I mGlu receptors (mGlu1 and mGlu5) are extensively expressed throughout neurons in the CNS and, in addition, mGlu5 is expressed in glial cells. mGlu1 is most abundantly expressed in Purkinje cells of the cerebellar cortex and in the olfactory bulb, in addition to strong expression in the hippocampus, substantia nigra and globus pallidus (Baude et al., 1993; Martin et al., 1992); and mGlu5 is greatly expressed in corticolimbic regions, such as the striatum, hippocampus and cerebral cortex (Ferraguti and Shigemoto, 2006). For example in the hippocampus, mGlu1 has been demonstrated to be involved in synaptic transmission and plasticity, in addition to neuronal excitability (Bortolotto et al., 1999), whilst in both mGlu1 and mGlu5 are required for the induction of long-term depression (LTD) in corticostriatal synapses (Sung et al., 2001). Through the use of knockout (KO) mice the putative function of mGluRs can be elucidated and, indeed, mGlu1 and mGlu5 KO mice have been studied. In mGlu1 KO animals is a marked deficits in long-term potentiation (LTP) in hippocampal slices and in context-dependent fear conditioning task (Aiba et al., 1994a); suggesting reduced hippocampal-mediated learning and memory. Furthermore,

Metabotropic Receptors for Glutamate and GABA 13

as quisqualate and [(1*S*,3*R*)-ACPD] also bind to ionotropic glutamate and other mGluR subtypes, respectively (Niswender and Conn, 2010). A range of other orthosteric ligands have been generated, but have limited use due to their low affinity and/or potency. As previously discussed, mGlu receptor subtype selectivity is difficult to obtain due to the high

Therefore, one approach is to target non-canonical ligand-binding sites; from this strategy a major breakthrough in Group I mGlu receptor pharmacology was made, with the discovery of CPCCOEt, which was the first mGlu1 negative allosteric modulator (NAM)(Annoura et al. 1996). CPCCOEt was later discovered to bind to an allosteric domain and this highlighted the capacity of ligands to bind in allosteric binding modes, thereby modulating orthosteric ligand function (Litschig et al., 1999). Thereafter, structurally distinct NAMs for mGlu1 were also discovered such as BAY36-7620 and FTIDC (Carroll et al., 2001; Suzuki et al., 2007). mGlu5 selective NAMs were also identified of which the two flagship molecules were MPEP and MTEP, both providing good potency and selectivity (Anderson et al., 2002; Gasparini et

In addition to NAMs, a wide variety of PAMs have also been identified and characterised. Two of these PAMs, Ro 67-4853 and Ro 01-6128 both potentiated DHPG-mediated VOCC inhibition responses in CA3 neurons, but did not exhibit any agonist activity of their own, suggesting their main characteristic is the allosteric potentiation of orthosteric ligand binding and/or efficacy (Knoflach et al., 2001). Interestingly, these PAMs were found to bind to a topographically distinct domain to the NAM binding region, when they failed to displace the well-characterised allosteric antagonist, R214127 (Hemstapat et al., 2006). These data suggest that mGlu1 possesses multiple allosteric binding sites, in addition to its orthosteric ligand-binding site. Similar to mGlu1, mGlu5 PAMs have also been discovered, such as DFB, CPPHA, CDPPB, VU29, and ADX47273, with CDPPB also having some PAM

Anxiety and depression are two of the most common mental disorders, with a lifetime prevalence of approximately 17% and 12%, respectively (Andrade et al., 2003; Depping et al., 2010). It has now been well documented that mGlu1 receptors and the glutamatergic system represent tractable targets for treating these common disorders (Bittencourt et al.,

Anxiety results from an imbalance between GABAergic and glutamatergic systems, either from overactive glutamatergic neurotransmission or inadequate GABAergic activity in hypothalamus, periaqueductal gray, hippocampus and prefrontal cortex (Engin and Treit, 2008). It is hypothesised that the antagonism of mGlu1 receptors is capable of augmenting the GABAergic response, whilst concomitantly decreasing the NMDA receptor-mediated glutamatergic response in key brain regions involved in anxiety. It has been demonstrated that intraperitoneal administration of the mGlu1 antagonist, 1-aminoindan-1,5-dicarboxylic acid (AIDA), rats exhibited anxiolytic-like behaviours in the conflict drinking test and in elevated plus maze tests (Klodzinska et al., 2004). This reinforces the results seen by Chojnacka-Wojcik et al., (1997) where intrahippocampal injection of the Group I mGlu receptor antagonist, (S)-4-carboxy-3-hydroxyphenyl-glycine (S-4C3H-PG), reduced anxiety-

degree of sequence and structural homology between subtypes.

activity at mGlu1 (Conn et al., 2009b; Hemstapat et al., 2006).

**2.4.2 mGlu1 in anxiety and depression**

2004; Paul and Skolnick, 2003).

al., 1999).

these mice are also cerebellar-LTD deficient, suggesting that mGlu1 receptors are important for LTD induction in the cerebellum and subsequently motor learning, as demonstrated by the ataxic gait of the mGlu1 KO mice (Aiba et al., 1994b). Recently, mice have been generated whereby the mGlu5 gene can be selectively disrupted in the central nucleus of the amygdala; these mice exhibited a lack of mechanical hypersensitivity induced by peripheral inflammation (Kolber et al., 2010), strongly suggesting a role of mGlu5 in the regulation of inflammatory pain transmission. Both mGlu1 and mGlu5 KO mice exhibit deficiencies in prepulse inhibition of the startle reflex, which is an indicator of sensorimotor gating that is impaired in schizophrenic patients, a trait that can be reversed through treatment with antipsychotics (Brody et al., 2003; Brody et al., 2004).

mGlu2 and mGlu3 (Group II) are widely expressed in the CNS, of which mGlu2 is more limited in expression compared to mGlu3. mGlu2 expression has been observed in Golgi cells of the cerebellar cortex and in mitral cells of the accessory olfactory bulb (Ohishi et al., 1998; Ohishi et al., 1994). mGlu3 receptors have been observed in the olfactory tubercle, neocortex, limbic cortex, and is also present in Golgi cells of the cerebellar cortex (Tamaru et al., 2001). Similar to Group I mGlu receptors, KO mice have also been generated for Group II mGluRs, with both mGlu2 and mGlu3 KO mice exhibiting a loss of mGlu2/3 agonist, LY354740-induced anxiolytic behaviour in an elevated plus maze test (Linden et al., 2005). Further to this, mGlu2, but not mGlu3 KO mice displayed a loss of Group II agonistmediated antipsychotic behaviour (Fell et al., 2008; Woolley et al., 2008), highlighting the role of mGlu2 in anxiety and psychotic behaviours. Interestingly, in addition to these functions, Group II mGlu receptors have also been demonstrated to modulate the release of other neurotransmitters, for example, LY354740 reduced KCl-induced [3H]-GABA release in rat primary cortical cultures, this effect was then reversed with the mGlu2/3 antagonist, LY341495 (Schaffhauser et al., 1998).

Group III mGluRs (consisting of mGlu4, mGlu6, mGlu7 and mGlu8) are mainly expressed on presynaptic neurons throughout the CNS, with the exception of mGlu6, which is expressed postsynaptically on retinal ON bipolar cells (Nakajima et al., 1993). mGlu4 is highly expressed in the cerebellum and consequently, mGlu4 KO mice experience deficits in spatial memory (Gerlai et al., 1998) and learning of complex motor tasks (Pekhletski et al., 1996). mGlu6 KO display deficits in ON response to light stimulation, yet the OFF response remained unchanged (Masu et al., 1995), highlighting the importance of mGlu6 in synaptic neurotransmission in retinal ON bipolar cells. mGlu7 deficient mice display learning and memory deficits, in addition to exhibiting an epileptic phenotype (Bushell et al., 2002; Sansig et al., 2001). Both mGlu7 and mGlu8 KO animals display increase anxiety (Cryan et al., 2003; Duvoisin et al., 2005).

As previously mentioned, the mGluR family of receptors are expressed widely through the CNS and exhibit a wide number of functions; moreover through KO studies, we can deduce the key roles played by each mGluR subtype and subsequently tailor our pharmacological armamentarium accordingly.

#### **2.4 Pharmacology and clinical relevance**

#### **2.4.1 Ligands for group I mGlu receptors**

The first selective orthosteric agonist at mGlu1 and mGlu5 receptors is (S)-3,5 dihydroxyphenylglycine, [(S)-3,5-DHPG], and this remains the case given that ligands such

these mice are also cerebellar-LTD deficient, suggesting that mGlu1 receptors are important for LTD induction in the cerebellum and subsequently motor learning, as demonstrated by the ataxic gait of the mGlu1 KO mice (Aiba et al., 1994b). Recently, mice have been generated whereby the mGlu5 gene can be selectively disrupted in the central nucleus of the amygdala; these mice exhibited a lack of mechanical hypersensitivity induced by peripheral inflammation (Kolber et al., 2010), strongly suggesting a role of mGlu5 in the regulation of inflammatory pain transmission. Both mGlu1 and mGlu5 KO mice exhibit deficiencies in prepulse inhibition of the startle reflex, which is an indicator of sensorimotor gating that is impaired in schizophrenic patients, a trait that can be reversed through treatment with

mGlu2 and mGlu3 (Group II) are widely expressed in the CNS, of which mGlu2 is more limited in expression compared to mGlu3. mGlu2 expression has been observed in Golgi cells of the cerebellar cortex and in mitral cells of the accessory olfactory bulb (Ohishi et al., 1998; Ohishi et al., 1994). mGlu3 receptors have been observed in the olfactory tubercle, neocortex, limbic cortex, and is also present in Golgi cells of the cerebellar cortex (Tamaru et al., 2001). Similar to Group I mGlu receptors, KO mice have also been generated for Group II mGluRs, with both mGlu2 and mGlu3 KO mice exhibiting a loss of mGlu2/3 agonist, LY354740-induced anxiolytic behaviour in an elevated plus maze test (Linden et al., 2005). Further to this, mGlu2, but not mGlu3 KO mice displayed a loss of Group II agonistmediated antipsychotic behaviour (Fell et al., 2008; Woolley et al., 2008), highlighting the role of mGlu2 in anxiety and psychotic behaviours. Interestingly, in addition to these functions, Group II mGlu receptors have also been demonstrated to modulate the release of other neurotransmitters, for example, LY354740 reduced KCl-induced [3H]-GABA release in rat primary cortical cultures, this effect was then reversed with the mGlu2/3 antagonist,

Group III mGluRs (consisting of mGlu4, mGlu6, mGlu7 and mGlu8) are mainly expressed on presynaptic neurons throughout the CNS, with the exception of mGlu6, which is expressed postsynaptically on retinal ON bipolar cells (Nakajima et al., 1993). mGlu4 is highly expressed in the cerebellum and consequently, mGlu4 KO mice experience deficits in spatial memory (Gerlai et al., 1998) and learning of complex motor tasks (Pekhletski et al., 1996). mGlu6 KO display deficits in ON response to light stimulation, yet the OFF response remained unchanged (Masu et al., 1995), highlighting the importance of mGlu6 in synaptic neurotransmission in retinal ON bipolar cells. mGlu7 deficient mice display learning and memory deficits, in addition to exhibiting an epileptic phenotype (Bushell et al., 2002; Sansig et al., 2001). Both mGlu7 and mGlu8 KO animals display increase anxiety (Cryan et al., 2003;

As previously mentioned, the mGluR family of receptors are expressed widely through the CNS and exhibit a wide number of functions; moreover through KO studies, we can deduce the key roles played by each mGluR subtype and subsequently tailor our pharmacological

The first selective orthosteric agonist at mGlu1 and mGlu5 receptors is (S)-3,5 dihydroxyphenylglycine, [(S)-3,5-DHPG], and this remains the case given that ligands such

antipsychotics (Brody et al., 2003; Brody et al., 2004).

LY341495 (Schaffhauser et al., 1998).

Duvoisin et al., 2005).

armamentarium accordingly.

**2.4 Pharmacology and clinical relevance 2.4.1 Ligands for group I mGlu receptors**  as quisqualate and [(1*S*,3*R*)-ACPD] also bind to ionotropic glutamate and other mGluR subtypes, respectively (Niswender and Conn, 2010). A range of other orthosteric ligands have been generated, but have limited use due to their low affinity and/or potency. As previously discussed, mGlu receptor subtype selectivity is difficult to obtain due to the high degree of sequence and structural homology between subtypes.

Therefore, one approach is to target non-canonical ligand-binding sites; from this strategy a major breakthrough in Group I mGlu receptor pharmacology was made, with the discovery of CPCCOEt, which was the first mGlu1 negative allosteric modulator (NAM)(Annoura et al. 1996). CPCCOEt was later discovered to bind to an allosteric domain and this highlighted the capacity of ligands to bind in allosteric binding modes, thereby modulating orthosteric ligand function (Litschig et al., 1999). Thereafter, structurally distinct NAMs for mGlu1 were also discovered such as BAY36-7620 and FTIDC (Carroll et al., 2001; Suzuki et al., 2007). mGlu5 selective NAMs were also identified of which the two flagship molecules were MPEP and MTEP, both providing good potency and selectivity (Anderson et al., 2002; Gasparini et al., 1999).

In addition to NAMs, a wide variety of PAMs have also been identified and characterised. Two of these PAMs, Ro 67-4853 and Ro 01-6128 both potentiated DHPG-mediated VOCC inhibition responses in CA3 neurons, but did not exhibit any agonist activity of their own, suggesting their main characteristic is the allosteric potentiation of orthosteric ligand binding and/or efficacy (Knoflach et al., 2001). Interestingly, these PAMs were found to bind to a topographically distinct domain to the NAM binding region, when they failed to displace the well-characterised allosteric antagonist, R214127 (Hemstapat et al., 2006). These data suggest that mGlu1 possesses multiple allosteric binding sites, in addition to its orthosteric ligand-binding site. Similar to mGlu1, mGlu5 PAMs have also been discovered, such as DFB, CPPHA, CDPPB, VU29, and ADX47273, with CDPPB also having some PAM activity at mGlu1 (Conn et al., 2009b; Hemstapat et al., 2006).

#### **2.4.2 mGlu1 in anxiety and depression**

Anxiety and depression are two of the most common mental disorders, with a lifetime prevalence of approximately 17% and 12%, respectively (Andrade et al., 2003; Depping et al., 2010). It has now been well documented that mGlu1 receptors and the glutamatergic system represent tractable targets for treating these common disorders (Bittencourt et al., 2004; Paul and Skolnick, 2003).

Anxiety results from an imbalance between GABAergic and glutamatergic systems, either from overactive glutamatergic neurotransmission or inadequate GABAergic activity in hypothalamus, periaqueductal gray, hippocampus and prefrontal cortex (Engin and Treit, 2008). It is hypothesised that the antagonism of mGlu1 receptors is capable of augmenting the GABAergic response, whilst concomitantly decreasing the NMDA receptor-mediated glutamatergic response in key brain regions involved in anxiety. It has been demonstrated that intraperitoneal administration of the mGlu1 antagonist, 1-aminoindan-1,5-dicarboxylic acid (AIDA), rats exhibited anxiolytic-like behaviours in the conflict drinking test and in elevated plus maze tests (Klodzinska et al., 2004). This reinforces the results seen by Chojnacka-Wojcik et al., (1997) where intrahippocampal injection of the Group I mGlu receptor antagonist, (S)-4-carboxy-3-hydroxyphenyl-glycine (S-4C3H-PG), reduced anxiety-

Metabotropic Receptors for Glutamate and GABA 15

in NMDA-dependent cognitive and learning deficits (Lu et al., 1997). Therefore, adopting an mGlu5 agonist or PAM could alleviate the cognitive symptoms in schizophrenic patients; moreover, the use of a PAM will allow relatively specific mGlu5 in the afflicted region whilst maintaining the spatio-temporal regulation of other mGlu5-containing neurons. Indeed, the abovementioned mGlu5 PAM, CDPPB, which has a suitable potency and solubility profile for *in vivo* studies, has been demonstrated to decrease amphetamine-induced disruption of prepulse inhibition (PPI) startle response and locomotor activity (Kinney et al., 2005); and to increase hippocampal synaptic plasticity, an important feature in cognition (Ayala et al.,

Group II mGlu receptors (mGlu2 and mGlu3) are generally localised presynaptically and negatively regulate cAMP signalling, and moreover, VOCCs. As with nearly all orthosteric mGlu pharmacological agents there is the underlying issue of selectivity. DCG-IV and LY379268 are reference Group II mGlu agonists, BINA and LY487379 are highly potent PAMs and the recently discovered MNI series of compounds (MNI-135, MNI-136 and MNI-137) are potent negative allosteric modulators (Galici et al., 2006; Hemstapat et al., 2006; Johnson et al., 2003; Linden et al., 2005; Schweitzer et al., 2000). Despite the large array of pharmacological tools available for Group II mGlu receptors, there remains a paucity of ligands that selectively differentiate between mGlu2 and mGlu3, which is due to the high degree of sequence homology between the two. Of lesser therapeutic relevance, there are also Group II mGlu receptor antagonists, such as 2*S*-2-amino-2-(1*S*,2*S*-2 carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionic acid (LY341495) and (1*R*,2*R*,3*R*,5*R*,6*R*)-2 amino-3-(3, 4-dichlorobenzyloxy)-6-fluorobicyclo[3.1.0] hexane-2,6-dicarboxylic acid (MGS0039), which have been suggested to have some anti-depressant and anti-obsessivecompulsive characteristics; however they are mostly used and pharmacological tools (Palucha and Pilc, 2005; Shimazaki et al., 2004). Given the lack of selectivity across Group II mGlu receptors it is difficult to pharmacologically distinguish the roles of each receptor in

various animal models of disease states without the use of knockout animals.

Addiction is a unique disorder in that it is not only a physiological dependence, but is also a psychological dependence on, canonically, drugs of abuse. It is believed that mGlu2/mGlu3 receptor ligands could be capable of treating addiction to such substances as cocaine and nicotine. In fact, not only is it that mGlu2/mGlu3 receptor activation is involved in recovery of a dysfunctional system in the corticolimbic system, but it has been shown that the function of Group II mGlu receptors is impaired, either by receptor downregulation or dampening of the G protein-mediated signalling, after acute and chronic stimulation by nicotine, cocaine and ethanol (Bowers et al., 2004; Kenny and Markou, 2004; Neugebauer et al., 2000). Indeed, mechanistically, the decrease in function is hypothesised to be due to an alteration in expression of the activator of G protein signalling 3 (AGS3), whereby AGS3 is overexpressed during withdrawal of repeated dosing of cocaine (Bowers et al., 2004). The authors went on to postulate that AGS3 gates expression of cocaine-induced plasticity in prefrontal cortex, via the regulation of G protein signalling. Furthermore, the downregulation of mGlu2/mGlu3 receptors has been observed during cocaine withdrawal

2008; Conn et al., 2009b).

**2.4.4 Group II mGlu receptor pharmacology**

**2.4.5 Group II mGlu receptors in addiction**

like behaviours in rats. The anxiolytic actions of mGlu1 blockade were further confirmed through the study of the mGlu1-selective antagonist, JNJ16259685 (Steckler et al., 2005). This study demonstrated that treatment with JNJ16259685 alleviated the suppression of the licking response in a conflict drinking test, which is consistent with other well characterized anxiolytic drugs (Petersen and Lassen, 1981). However, JNJ16259685 treatment did not induce anxiolytic-type behaviour in elevated plus maze tests, the authors thus postulating that the effects of JNJ16259685 be context specific (Steckler et al., 2005).

Depression is a complex disorder involving the interplay between different neurotransmitters, including noradrenaline, serotonin, dopamine and glutamate (Paul and Skolnick, 2003). Drugs for the treatment for depression are generally based on increasing the lifetime of biogenic amines, such as noradrenaline and serotonin, in the synaptic cleft, for example fluoxetine and escitalopram, which are inhibitors of serotonin- and serotonin and noradrenaline-reuptake transporters, respectively. Over the past decade, it has become more recognised that the glutamatergic system may also play a vital role in the regulation of depression, specifically NMDA receptors, where NMDA receptor expression was reduced in post-mortem depressive brains (Feyissa et al., 2009). This theory was retrospectively reinforced by evidence that NMDA receptor antagonists produce anti-depressant effects, whereby competitive and noncompetitive antagonists of NMDA receptors, 2-amino-7-phosphonoheptanoic acid (AP-7) and Dizolcipine (MK-801) emulated anti-depressant effects of gold standard anti-depressants (Trullas and Skolnick, 1990). Given the regulatory link between mGlu1 and NMDA receptors it was postulated that mGlu1 receptor antagonists or NAMs could mimic the anti-depressant effect of NMDA receptor inhibitors. The mGlu1 antagonist, JNJ-16567083 has been shown to be efficacious in despair-based animal models of depression, specifically forced swim test and tail suspension test (Belozertseva et al., 2007; Molina-Hernandez et al., 2008).

#### **2.4.3 mGlu5 and schizophrenia**

Schizophrenia is a complex multi-faceted disease that manifests itself as a host of symptoms such as paranoia, social withdrawal and delusions, along with a number of cognitive deficits. Given that there is no single causative factor, there is some difficulty in finding a suitable target. Current first-line treatment involves broad-spectrum biogenic amine (e.g. dopamine, serotonin, acetylcholine) receptor antagonists, but these to not satisfactorily treat the cognitive symptoms. The underlying rationale of this approach is to decrease dopaminergic neurotransmission in thalamocortical and limbic circuits. One potential mode of treating schizophrenia lies within targeting GABAergic and glutamateric interneuons in pivotal cortical and limbic regions, specifically, the disregulation of the disinhibition of glutamatergic neurotransmission (Chavez-Noriega et al., 2002; Coyle, 2006). The blockade of *N-*methly-D-aspartate (NMDA) receptors on these interneurons results in a glutamatergic disinhibition, which in turn leads to an overexcitability of thalamocortical neurons, which is mostly mediated by DL-a-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA) receptors in thalamocortical synapses. Within these regions NMDA and mGlu5 receptors have been demonstrated to functionally and physically interact, i.e. the activation of mGlu5 receptors increases the activity of NMDA receptors on GABAergic and glutamatergic neurons (Conn et al., 2009b); it is thus postulated that the activation of mGlu5 can be employed as a means to decrease neuronal excitability in thalamocortical regions. This hypothesis is reinforced through knockout studies, whereby the knockout of mGlu5 resulted

like behaviours in rats. The anxiolytic actions of mGlu1 blockade were further confirmed through the study of the mGlu1-selective antagonist, JNJ16259685 (Steckler et al., 2005). This study demonstrated that treatment with JNJ16259685 alleviated the suppression of the licking response in a conflict drinking test, which is consistent with other well characterized anxiolytic drugs (Petersen and Lassen, 1981). However, JNJ16259685 treatment did not induce anxiolytic-type behaviour in elevated plus maze tests, the authors thus postulating

Depression is a complex disorder involving the interplay between different neurotransmitters, including noradrenaline, serotonin, dopamine and glutamate (Paul and Skolnick, 2003). Drugs for the treatment for depression are generally based on increasing the lifetime of biogenic amines, such as noradrenaline and serotonin, in the synaptic cleft, for example fluoxetine and escitalopram, which are inhibitors of serotonin- and serotonin and noradrenaline-reuptake transporters, respectively. Over the past decade, it has become more recognised that the glutamatergic system may also play a vital role in the regulation of depression, specifically NMDA receptors, where NMDA receptor expression was reduced in post-mortem depressive brains (Feyissa et al., 2009). This theory was retrospectively reinforced by evidence that NMDA receptor antagonists produce anti-depressant effects, whereby competitive and noncompetitive antagonists of NMDA receptors, 2-amino-7-phosphonoheptanoic acid (AP-7) and Dizolcipine (MK-801) emulated anti-depressant effects of gold standard anti-depressants (Trullas and Skolnick, 1990). Given the regulatory link between mGlu1 and NMDA receptors it was postulated that mGlu1 receptor antagonists or NAMs could mimic the anti-depressant effect of NMDA receptor inhibitors. The mGlu1 antagonist, JNJ-16567083 has been shown to be efficacious in despair-based animal models of depression, specifically forced swim test and tail

Schizophrenia is a complex multi-faceted disease that manifests itself as a host of symptoms such as paranoia, social withdrawal and delusions, along with a number of cognitive deficits. Given that there is no single causative factor, there is some difficulty in finding a suitable target. Current first-line treatment involves broad-spectrum biogenic amine (e.g. dopamine, serotonin, acetylcholine) receptor antagonists, but these to not satisfactorily treat the cognitive symptoms. The underlying rationale of this approach is to decrease dopaminergic neurotransmission in thalamocortical and limbic circuits. One potential mode of treating schizophrenia lies within targeting GABAergic and glutamateric interneuons in pivotal cortical and limbic regions, specifically, the disregulation of the disinhibition of glutamatergic neurotransmission (Chavez-Noriega et al., 2002; Coyle, 2006). The blockade of *N-*methly-D-aspartate (NMDA) receptors on these interneurons results in a glutamatergic disinhibition, which in turn leads to an overexcitability of thalamocortical neurons, which is mostly mediated by DL-a-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA) receptors in thalamocortical synapses. Within these regions NMDA and mGlu5 receptors have been demonstrated to functionally and physically interact, i.e. the activation of mGlu5 receptors increases the activity of NMDA receptors on GABAergic and glutamatergic neurons (Conn et al., 2009b); it is thus postulated that the activation of mGlu5 can be employed as a means to decrease neuronal excitability in thalamocortical regions. This hypothesis is reinforced through knockout studies, whereby the knockout of mGlu5 resulted

that the effects of JNJ16259685 be context specific (Steckler et al., 2005).

suspension test (Belozertseva et al., 2007; Molina-Hernandez et al., 2008).

**2.4.3 mGlu5 and schizophrenia**

in NMDA-dependent cognitive and learning deficits (Lu et al., 1997). Therefore, adopting an mGlu5 agonist or PAM could alleviate the cognitive symptoms in schizophrenic patients; moreover, the use of a PAM will allow relatively specific mGlu5 in the afflicted region whilst maintaining the spatio-temporal regulation of other mGlu5-containing neurons. Indeed, the abovementioned mGlu5 PAM, CDPPB, which has a suitable potency and solubility profile for *in vivo* studies, has been demonstrated to decrease amphetamine-induced disruption of prepulse inhibition (PPI) startle response and locomotor activity (Kinney et al., 2005); and to increase hippocampal synaptic plasticity, an important feature in cognition (Ayala et al., 2008; Conn et al., 2009b).

#### **2.4.4 Group II mGlu receptor pharmacology**

Group II mGlu receptors (mGlu2 and mGlu3) are generally localised presynaptically and negatively regulate cAMP signalling, and moreover, VOCCs. As with nearly all orthosteric mGlu pharmacological agents there is the underlying issue of selectivity. DCG-IV and LY379268 are reference Group II mGlu agonists, BINA and LY487379 are highly potent PAMs and the recently discovered MNI series of compounds (MNI-135, MNI-136 and MNI-137) are potent negative allosteric modulators (Galici et al., 2006; Hemstapat et al., 2006; Johnson et al., 2003; Linden et al., 2005; Schweitzer et al., 2000). Despite the large array of pharmacological tools available for Group II mGlu receptors, there remains a paucity of ligands that selectively differentiate between mGlu2 and mGlu3, which is due to the high degree of sequence homology between the two. Of lesser therapeutic relevance, there are also Group II mGlu receptor antagonists, such as 2*S*-2-amino-2-(1*S*,2*S*-2 carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionic acid (LY341495) and (1*R*,2*R*,3*R*,5*R*,6*R*)-2 amino-3-(3, 4-dichlorobenzyloxy)-6-fluorobicyclo[3.1.0] hexane-2,6-dicarboxylic acid (MGS0039), which have been suggested to have some anti-depressant and anti-obsessivecompulsive characteristics; however they are mostly used and pharmacological tools (Palucha and Pilc, 2005; Shimazaki et al., 2004). Given the lack of selectivity across Group II mGlu receptors it is difficult to pharmacologically distinguish the roles of each receptor in various animal models of disease states without the use of knockout animals.

#### **2.4.5 Group II mGlu receptors in addiction**

Addiction is a unique disorder in that it is not only a physiological dependence, but is also a psychological dependence on, canonically, drugs of abuse. It is believed that mGlu2/mGlu3 receptor ligands could be capable of treating addiction to such substances as cocaine and nicotine. In fact, not only is it that mGlu2/mGlu3 receptor activation is involved in recovery of a dysfunctional system in the corticolimbic system, but it has been shown that the function of Group II mGlu receptors is impaired, either by receptor downregulation or dampening of the G protein-mediated signalling, after acute and chronic stimulation by nicotine, cocaine and ethanol (Bowers et al., 2004; Kenny and Markou, 2004; Neugebauer et al., 2000). Indeed, mechanistically, the decrease in function is hypothesised to be due to an alteration in expression of the activator of G protein signalling 3 (AGS3), whereby AGS3 is overexpressed during withdrawal of repeated dosing of cocaine (Bowers et al., 2004). The authors went on to postulate that AGS3 gates expression of cocaine-induced plasticity in prefrontal cortex, via the regulation of G protein signalling. Furthermore, the downregulation of mGlu2/mGlu3 receptors has been observed during cocaine withdrawal

Metabotropic Receptors for Glutamate and GABA 17

One pharmacological avenue that is only beginning to be explored at Class C GPCRs is that of extracellular domain allosteric modulators. For the umami taste receptors, it has been long known that purinergic ribonucleotides, such as inosine- and guanine-monophosphate molecules (IMP and GMP) were potent positive allosteric modulators of the L-glutamate action at the umami receptor (Yamaguchi and Ninomiya, 2000). Interestingly, mutants that altered the effects of glutamate effect were also enhanced by IMP and GMP (Zhang et al., 2008). By employing a chimeric approach along with mutagenesis and molecular modelling, sweet-umami receptors were analysed and the mode of binding and action of IMP was postulated; specifically, the residues lining the IMP binding pocket at the sweet-umami taste receptor, T1R1, were determined (Zhang et al., 2008). It was demonstrated that IMP binds to a novel site that is adjacent to the glutamate binding pocket, the authors thus proposed a model for ligand cooperativity for the mechanism of action of IMP in the T1R1 VFT. The binding of L-glutamate close to the hinge region of the VFT would stabilize the closed conformation of the domain; moreover, binding of 5' ribonucleotides to an adjacent site closer to the putative entrance of the VFT would further stabilize the closed conformation, thereby potentiating the affinity and/or efficacy of L-glutamate. At mGlu receptors, the glutamate-binding pocket is well conserved across the mGlu subtypes, encumbering the discovery selective orthosteric agonists and antagonists (Brauner-Osborne et al., 2007). However, recently, long alkyl chain containing derivatives of (R)-PCEP, a molecule discovered by virtual screening on the VFT of mGlu receptors, revealed a new binding pocket in mGlu4 (Selvam et al., 2010). Indeed, these compounds not only bind in the glutamate-binding pocket itself, but may also interact with a novel, putative binding pocket adjacent to the glutamate-binding site. Given this new interacting region is formed with residues that are less conserved across the eight mGlu subtypes, this mode of targeting mGlu receptors may furnish compounds with greater selectivity. One such compound may already exist in LSP1-2111, with its L-AP4-like moiety and a 4-hydroxy-3-methoxy-5-nitrophenyl moiety, it is possible that this molecule bridges across two distinct binding domains, in a similar fashion to bitopic ligands at muscarinic receptors (Antony et al., 2009; Valant et al., 2008; Valant et al., 2009). Accordingly, this ligand has superior selectivity at mGlu4 and

For an overview of chemical structures of a small range of classical orthosteric mGlu

Parkinson's disease is one of the most common of neurological disorders, which is largely characterised by its effects on motor function, such as bradykinesia and dyskinesia; further to other non-motor symptoms, for example pain and gastrointestinal dysfunction. Parkinson's disease arises mostly due to a progressive degeneration of dopaminergic neurons in the substantia nigra, leading to excessive cholinergic neurotransmission in the striatum (Pisani et al., 2003). Subsequently, the inhibitory effect that dopamine provides in these circuits augments GABAergic firing in the striatopallidal pathway leading to excessive inhibition of GABAergic neurons in the subthalamic nucleus, in turn leading to the abnormal enhancement of glutamatergic neurons (Hirsch, 2000). Currently, the frontline treatment is levo-dopa, which compensates for the diminished dopaminergic function. However, the activation of presynaptic mGlu4 specifically, may result in the diminution of

mGlu6 over mGlu7 and mGlu8 (Beurrier et al., 2009).

**2.4.7 Group III mGlu receptors and Parkinson's disease**

receptor ligands, refer to Figure 3 below.

periods, specifically these receptors were downregulated in the shell and core of the nucleus accumbens (Ghasemzadeh et al., 2009). These alterations in expression and function in turn results in an impairment of long-term depression (LTD) in nucleus accumbens and prefrontal cortex in response to chronic morphine and cocaine exposure, respectively (Moussawi and Kalivas, 2010); similarly, a reduced activation of mGlu2/mGlu3 receptors resulted in a decrease in long-term potentiation (LTP) after self-administered cocaine withdrawal (Moussawi et al., 2009). Indeed, it is well documented that mGlu2/mGlu3 function is altered in the case of substance withdrawal, however the system is regulated in a manner of ways. Explicitly, Group II mGlu receptors are involved in the circuitry that leads to reward processing and addictive behaviour. The activation of mGlu2/mGlu3 receptors with the orthosteric agonist, LY379268 resulted in the attenuation of the reinstatement of cocaine-seeking behaviour after exposure, compared to a conventional reinforcer (in this case, sweetened condensed milk) (Baptista et al., 2004). The authors proposed that this was a cocaine-specific effect and was most likely related to the mechanism of action of cocaine itself. Functionally, this regulation may lie in the pre-activation of mGlu2 receptors, whereby in mGlu2 knockout mice there was an increased release of glutamate and dopamine in response to cocaine, in the nucleus accumbens (Morishima et al., 2005). Whilst this does provide some evidence on how glutamate is involved in reward circuitry, one must remain circumspect on their conclusions given any compensatory mechanisms are not accounted for.

#### **2.4.6 Group III mGlu receptors and their ligands**

For many years, much of the drug discovery efforts have been directed towards Group I and II receptors to exploit their roles in central nervous disorders such as schizophrenia and neuropathic pain. However, of late, efforts have been turned to developing selective ligands for Group III as novel targets for disorders, for example, Parkinson's disease. The prototypical Group III-selective orthosteric agonist is L-amino-4-phosphonobutyrate (L-AP4), yet this ligand is only selective for Group III mGlu receptors, not within the group. In an attempt to ameliorate the affinity and potency, a series of constrained cyclic forms of glutamate were generated and so was created aminocyclopentane-1,3,4-tricarboxylate (ACPT-I), which showed mildly enhanced potency at mGlu4 and mGlu8 compared to mGlu5 and mGlu6 (Acher et al., 1997; Schann et al., 2006). Similar to the agonists, there are only selective antagonists for Group III mGlu receptors, but not within the group. For example, there are the -methyl analogues of L-AP4 and L-SOP, specifically MAP4 and MSOP, respectively, with affinity in the micromolar range (Wright et al., 2000). In addition to these, there are the hallmark antagonists of mGlu receptors such as DCG-IV and LY341495, which both have reasonable affinity for Group III mGlu receptors, but also have strong affinity at Group I and Group II receptors, respectively; notably, DCG-IV is also a Group II mGlu receptor agonist (Brabet et al., 1998). Allosteric modulators that act in the 7TM domain Group III mGlu receptors have also been characterised, specifically N-Phenyl-7- (hydroxyimino)cyclopropa[b]chromen-1acarboxamide (PHCCC) and cis-2-([(3,5- Dichlorophenyl)amino]carbonyl)cyclohexanecarboxylic acid (VU0155041), which are both PAMs at mGlu4 (Niswender et al., 2008); 6-(4-Methoxyphenyl)-5-methyl-3-(4-pyridinyl) isoxazolo[4,5-c]pyridine-4(5H)-one hydrochloride (MMPIP), a NAM for mGlu7 (Niswender et al., 2010); however there remains a relative paucity of allosteric modulators for mGlu6 and mGlu8.

periods, specifically these receptors were downregulated in the shell and core of the nucleus accumbens (Ghasemzadeh et al., 2009). These alterations in expression and function in turn results in an impairment of long-term depression (LTD) in nucleus accumbens and prefrontal cortex in response to chronic morphine and cocaine exposure, respectively (Moussawi and Kalivas, 2010); similarly, a reduced activation of mGlu2/mGlu3 receptors resulted in a decrease in long-term potentiation (LTP) after self-administered cocaine withdrawal (Moussawi et al., 2009). Indeed, it is well documented that mGlu2/mGlu3 function is altered in the case of substance withdrawal, however the system is regulated in a manner of ways. Explicitly, Group II mGlu receptors are involved in the circuitry that leads to reward processing and addictive behaviour. The activation of mGlu2/mGlu3 receptors with the orthosteric agonist, LY379268 resulted in the attenuation of the reinstatement of cocaine-seeking behaviour after exposure, compared to a conventional reinforcer (in this case, sweetened condensed milk) (Baptista et al., 2004). The authors proposed that this was a cocaine-specific effect and was most likely related to the mechanism of action of cocaine itself. Functionally, this regulation may lie in the pre-activation of mGlu2 receptors, whereby in mGlu2 knockout mice there was an increased release of glutamate and dopamine in response to cocaine, in the nucleus accumbens (Morishima et al., 2005). Whilst this does provide some evidence on how glutamate is involved in reward circuitry, one must remain circumspect on their conclusions given any compensatory mechanisms are not

For many years, much of the drug discovery efforts have been directed towards Group I and II receptors to exploit their roles in central nervous disorders such as schizophrenia and neuropathic pain. However, of late, efforts have been turned to developing selective ligands for Group III as novel targets for disorders, for example, Parkinson's disease. The prototypical Group III-selective orthosteric agonist is L-amino-4-phosphonobutyrate (L-AP4), yet this ligand is only selective for Group III mGlu receptors, not within the group. In an attempt to ameliorate the affinity and potency, a series of constrained cyclic forms of glutamate were generated and so was created aminocyclopentane-1,3,4-tricarboxylate (ACPT-I), which showed mildly enhanced potency at mGlu4 and mGlu8 compared to mGlu5 and mGlu6 (Acher et al., 1997; Schann et al., 2006). Similar to the agonists, there are only selective antagonists for Group III mGlu receptors, but not within the group. For example, there are the -methyl analogues of L-AP4 and L-SOP, specifically MAP4 and MSOP, respectively, with affinity in the micromolar range (Wright et al., 2000). In addition to these, there are the hallmark antagonists of mGlu receptors such as DCG-IV and LY341495, which both have reasonable affinity for Group III mGlu receptors, but also have strong affinity at Group I and Group II receptors, respectively; notably, DCG-IV is also a Group II mGlu receptor agonist (Brabet et al., 1998). Allosteric modulators that act in the 7TM domain Group III mGlu receptors have also been characterised, specifically N-Phenyl-7- (hydroxyimino)cyclopropa[b]chromen-1acarboxamide (PHCCC) and cis-2-([(3,5- Dichlorophenyl)amino]carbonyl)cyclohexanecarboxylic acid (VU0155041), which are both PAMs at mGlu4 (Niswender et al., 2008); 6-(4-Methoxyphenyl)-5-methyl-3-(4-pyridinyl) isoxazolo[4,5-c]pyridine-4(5H)-one hydrochloride (MMPIP), a NAM for mGlu7 (Niswender et al., 2010); however there remains a relative paucity of allosteric modulators for mGlu6 and

accounted for.

mGlu8.

**2.4.6 Group III mGlu receptors and their ligands**

One pharmacological avenue that is only beginning to be explored at Class C GPCRs is that of extracellular domain allosteric modulators. For the umami taste receptors, it has been long known that purinergic ribonucleotides, such as inosine- and guanine-monophosphate molecules (IMP and GMP) were potent positive allosteric modulators of the L-glutamate action at the umami receptor (Yamaguchi and Ninomiya, 2000). Interestingly, mutants that altered the effects of glutamate effect were also enhanced by IMP and GMP (Zhang et al., 2008). By employing a chimeric approach along with mutagenesis and molecular modelling, sweet-umami receptors were analysed and the mode of binding and action of IMP was postulated; specifically, the residues lining the IMP binding pocket at the sweet-umami taste receptor, T1R1, were determined (Zhang et al., 2008). It was demonstrated that IMP binds to a novel site that is adjacent to the glutamate binding pocket, the authors thus proposed a model for ligand cooperativity for the mechanism of action of IMP in the T1R1 VFT. The binding of L-glutamate close to the hinge region of the VFT would stabilize the closed conformation of the domain; moreover, binding of 5' ribonucleotides to an adjacent site closer to the putative entrance of the VFT would further stabilize the closed conformation, thereby potentiating the affinity and/or efficacy of L-glutamate. At mGlu receptors, the glutamate-binding pocket is well conserved across the mGlu subtypes, encumbering the discovery selective orthosteric agonists and antagonists (Brauner-Osborne et al., 2007). However, recently, long alkyl chain containing derivatives of (R)-PCEP, a molecule discovered by virtual screening on the VFT of mGlu receptors, revealed a new binding pocket in mGlu4 (Selvam et al., 2010). Indeed, these compounds not only bind in the glutamate-binding pocket itself, but may also interact with a novel, putative binding pocket adjacent to the glutamate-binding site. Given this new interacting region is formed with residues that are less conserved across the eight mGlu subtypes, this mode of targeting mGlu receptors may furnish compounds with greater selectivity. One such compound may already exist in LSP1-2111, with its L-AP4-like moiety and a 4-hydroxy-3-methoxy-5-nitrophenyl moiety, it is possible that this molecule bridges across two distinct binding domains, in a similar fashion to bitopic ligands at muscarinic receptors (Antony et al., 2009; Valant et al., 2008; Valant et al., 2009). Accordingly, this ligand has superior selectivity at mGlu4 and mGlu6 over mGlu7 and mGlu8 (Beurrier et al., 2009).

For an overview of chemical structures of a small range of classical orthosteric mGlu receptor ligands, refer to Figure 3 below.

#### **2.4.7 Group III mGlu receptors and Parkinson's disease**

Parkinson's disease is one of the most common of neurological disorders, which is largely characterised by its effects on motor function, such as bradykinesia and dyskinesia; further to other non-motor symptoms, for example pain and gastrointestinal dysfunction. Parkinson's disease arises mostly due to a progressive degeneration of dopaminergic neurons in the substantia nigra, leading to excessive cholinergic neurotransmission in the striatum (Pisani et al., 2003). Subsequently, the inhibitory effect that dopamine provides in these circuits augments GABAergic firing in the striatopallidal pathway leading to excessive inhibition of GABAergic neurons in the subthalamic nucleus, in turn leading to the abnormal enhancement of glutamatergic neurons (Hirsch, 2000). Currently, the frontline treatment is levo-dopa, which compensates for the diminished dopaminergic function. However, the activation of presynaptic mGlu4 specifically, may result in the diminution of

Metabotropic Receptors for Glutamate and GABA 19

Despite receiving much of the attention within Group III mGlu receptors, mGlu4 is not alone in its involvement in Parkinson's disease. There remains the possibility that post-synaptic mGlu7 and mGlu8 have some effect on the neuronal circuitry in question. The mGlu7 allosteric agonist, *N,N*'-dibenzhydryl-ethane-1,2-diamine dihydrochloride (AMN082) may inhibit the release of [3H]-D-aspartate in substantia nigral slices, suggesting that selective targeting of mGlu7 may yield similar results to those at mGlu4 (unpublished data; Duty, 2010). Despite there being a large amount of doubt surrounding the therapeutic potential of mGlu8 for the treatment of Parkinson's disease, where the semi-selective mGlu8 agonist was failed to reverse haloperidol-induced catalepsy (Lopez et al., 2007); administration of the mixed AMPA antagonist/mGlu8 agonist, (*R,S*)-3-4-DCPG, decreased amphetamine- but not phencyclidine-induced hyperactivity (Ossowska et al., 2004). Concomitantly, (*R,S*)-3-4- DCPG actually enhanced haloperidol-induced catalepsy and induced catalepsy when administered alone. Taken together, and despite similar expression and function compared to mGlu4, does not appear to be a good candidate target for the treatment of Parkinson's disease. Indeed, this scenario highlights the inherent difficulties that are encountered in the

Taken together, it seems that the most appropriate and effective methods for targeting mGlu receptors is via their allosteric ligand-binding site, which increases subtype selectivity and does not impede normal neurotransmission. Refer to Figure 4 for the chemical structures of

It is now well established that mGlu receptors are major targets for numerous central disorders and even for some in the periphery. Accordingly, there are a large number of

Gastro-(o)esophageal reflux disease (GERD) is a chronic condition, in which the major symptom is the abnormal reflux of stomach contents into the oesophagus. The inhibition of mGlu5 is predicted to improve the tone of the cardiac sphincter, thus reducing reflux (Lehmann, 2008). In a recent phase II clinical study performed by Addex pharmaceuticals, reflux and other GERD symptoms are efficiently reduced by a NAM of mGlu5. The same molecule has also entered into a different phase II study targeting migraine, which also

search for mGlu receptor subtype-selective therapeutics.

Fig. 4. Chemical structures of mGlu receptor allosteric ligands.

clinical programs that are running at any one time (Table 1).

**2.4.8 Clinical trials for mGlu receptor ligands**

some allosteric ligands for mGlu receptors.

Fig. 3. Highlighting the structural diversity of agonist and antagonists of mGlu receptors.

increased GABAergic firing in striatopallidal projections. Indeed, compounds that have relatively good selectivity for mGlu4 have been demonstrated to depress the GABAmediated inhibitory synaptic transmission and relive motor symptoms in animal models of Parkinson's disease (Beurrier et al., 2009; Valenti et al., 2003). Given that the dopaminergic dysfunction in the substantia nigra and inhibition of GABA signalling by mGlu4 in the globus pallidus are not inextricably linked there is potential that prolonged mGlu4 receptor activation will result in less compensatory over-activation of the dopaminergic system, therefore maintaining the therapeutic activity of mGlu4 targeting ligands (Nicoletti et al., 2011). Indeed, it has been shown that the *in vivo* treatment with the mGlu4 PAM, PHCCC, reduced dopaminergic neurodegeneration in substantia nigral projections in an MPTPinduced Parkinsonism model (Battaglia et al., 2004; Maj et al., 2003). Along with PHCCC, a more recent PAM of mGlu4 has been characterised and has demonstrated anti-parkinsonian effects (Niswender et al., 2008). VU0155041 is an allosteric agonist and positive allosteric modulator with potency nearly 10-fold of that of PHCCC, moreover, VU0155041 concentration-dependently diminished haloperidol-induced catalepsy and reversed reserpine-mediated akinesia in mice, with an effect that persisted longer than that of the reference Group III orthosteric agonist, L-AP4 (Niswender et al., 2008).

Fig. 3. Highlighting the structural diversity of agonist and antagonists of mGlu receptors.

reference Group III orthosteric agonist, L-AP4 (Niswender et al., 2008).

increased GABAergic firing in striatopallidal projections. Indeed, compounds that have relatively good selectivity for mGlu4 have been demonstrated to depress the GABAmediated inhibitory synaptic transmission and relive motor symptoms in animal models of Parkinson's disease (Beurrier et al., 2009; Valenti et al., 2003). Given that the dopaminergic dysfunction in the substantia nigra and inhibition of GABA signalling by mGlu4 in the globus pallidus are not inextricably linked there is potential that prolonged mGlu4 receptor activation will result in less compensatory over-activation of the dopaminergic system, therefore maintaining the therapeutic activity of mGlu4 targeting ligands (Nicoletti et al., 2011). Indeed, it has been shown that the *in vivo* treatment with the mGlu4 PAM, PHCCC, reduced dopaminergic neurodegeneration in substantia nigral projections in an MPTPinduced Parkinsonism model (Battaglia et al., 2004; Maj et al., 2003). Along with PHCCC, a more recent PAM of mGlu4 has been characterised and has demonstrated anti-parkinsonian effects (Niswender et al., 2008). VU0155041 is an allosteric agonist and positive allosteric modulator with potency nearly 10-fold of that of PHCCC, moreover, VU0155041 concentration-dependently diminished haloperidol-induced catalepsy and reversed reserpine-mediated akinesia in mice, with an effect that persisted longer than that of the Despite receiving much of the attention within Group III mGlu receptors, mGlu4 is not alone in its involvement in Parkinson's disease. There remains the possibility that post-synaptic mGlu7 and mGlu8 have some effect on the neuronal circuitry in question. The mGlu7 allosteric agonist, *N,N*'-dibenzhydryl-ethane-1,2-diamine dihydrochloride (AMN082) may inhibit the release of [3H]-D-aspartate in substantia nigral slices, suggesting that selective targeting of mGlu7 may yield similar results to those at mGlu4 (unpublished data; Duty, 2010). Despite there being a large amount of doubt surrounding the therapeutic potential of mGlu8 for the treatment of Parkinson's disease, where the semi-selective mGlu8 agonist was failed to reverse haloperidol-induced catalepsy (Lopez et al., 2007); administration of the mixed AMPA antagonist/mGlu8 agonist, (*R,S*)-3-4-DCPG, decreased amphetamine- but not phencyclidine-induced hyperactivity (Ossowska et al., 2004). Concomitantly, (*R,S*)-3-4- DCPG actually enhanced haloperidol-induced catalepsy and induced catalepsy when administered alone. Taken together, and despite similar expression and function compared to mGlu4, does not appear to be a good candidate target for the treatment of Parkinson's disease. Indeed, this scenario highlights the inherent difficulties that are encountered in the search for mGlu receptor subtype-selective therapeutics.

Taken together, it seems that the most appropriate and effective methods for targeting mGlu receptors is via their allosteric ligand-binding site, which increases subtype selectivity and does not impede normal neurotransmission. Refer to Figure 4 for the chemical structures of some allosteric ligands for mGlu receptors.

Fig. 4. Chemical structures of mGlu receptor allosteric ligands.

#### **2.4.8 Clinical trials for mGlu receptor ligands**

It is now well established that mGlu receptors are major targets for numerous central disorders and even for some in the periphery. Accordingly, there are a large number of clinical programs that are running at any one time (Table 1).

Gastro-(o)esophageal reflux disease (GERD) is a chronic condition, in which the major symptom is the abnormal reflux of stomach contents into the oesophagus. The inhibition of mGlu5 is predicted to improve the tone of the cardiac sphincter, thus reducing reflux (Lehmann, 2008). In a recent phase II clinical study performed by Addex pharmaceuticals, reflux and other GERD symptoms are efficiently reduced by a NAM of mGlu5. The same molecule has also entered into a different phase II study targeting migraine, which also

Metabotropic Receptors for Glutamate and GABA 21

symptoms of schizophrenia with an mGlu2/mGlu3 agonist was similar to that demonstrated with olanzapine, a common antipsychotic drug; this drug was also tolerated by patients

Preclinical studies strongly suggest that Group III mGlu receptors may play a vital role in the symptomatic control of Parkinson's disease. In particular, increasing mGlu4 activity within the basal ganglia appears to be an interesting approach to reduce akinetic symptoms associated with Parkinson's disease (Beurrier et al., 2009; Lopez et al., 2007). However, to our

The metabotropic GABA (GABAB) receptor is the only known GPCR that is responsive to GABA. Architecturally, it is not composed in the same manner as many other Class C GPCRs. Specifically, it consists of a ligand binding GB1 subunit and a G protein coupling GB2 subunit (Galvez et al., 2001; Kaupmann et al., 1998; Margeta-Mitrovic et al., 2001; White et al., 1998); each subunit consisting of a VFT and 7TM domains, but converse to mGlu receptors they lack a CRD (refer to Figure 2 for schematic overview). The two subunits are not covalently associated, but do interact via a coiled-coil domain in their C-terminal tails, which provides a solid hydrophobic interaction to maintain the integrity of the dimer (Kammerer et al., 1999). Through the use of circular dichroism spectroscopy the authors proposed a region in the C-terminal domains of GB1 and GB2 of approximately 30 amino

Discerning the number of ligands that bind to any one dimer at any one moment is often difficult, especially if there is the possibility for receptors to form higher-order oligomers. It has been shown that in class C heterodimers a single subunit was responsible for the binding of the endogenous ligand, in this case GB1 in the GABAB receptor (Kniazeff et al., 2002). This suggests that a single agonist molecule is sufficient to fully activate heterodimeric receptors, but does not discount multiple binding sites on the same protomer. However, nearly nothing is known of the conformational movement of the GB2 subunit, making it nearly impossible to distinguish between the conformational rearrangement and functional responses of Aco and Acc combinations. The only insights come from the GABAB receptor, whereby the introduction of several large residues, such as tryptophan in the crevice of GB2 VFT leads to a decrease in G protein-mediated functional responses (Kniazeff

It has always been questioned whether GPCRs remain in simple monomeric and dimeric forms or whether they self-associate into higher-order oligomers and, if so, what are the molecular determinants of these interactions. Recently, it has been demonstrated that GABAB are indeed capable of forming tetrameric complexes, which interact via their GB1 subunits (Comps-Agrar et al., 2011; Maurel et al., 2008). By employing the use of a bindingnull GB1 subunit Comps-Agrar et al., (2011) demonstrated that GABAB receptor tetramers could be disrupted and that the resultant complexes are capable of binding approximately twice as much radioligand compared to the wild-type; in addition to increasing the apparent Emax in functional tests. The synthesis of this study was that GABAB receptors that are

knowledge, none of these compounds have reached phase I clinical trials.

(Patil et al., 2007).

**3. Metabotropic GABA receptors**

acids, composed of roughly 5-7 heptads.

et al., 2002).

**3.1 Structure/function of GABAB receptors**

yielded beneficial results. Since glutamate is the main neurotransmitter of the migraine circuit, then inhibition of postsynaptic mGlu5 receptors that are present in this circuit would decrease glutamatergic neurotransmission and hence may pose a useful approach in migraine therapy. However, due to liver toxicity after long-term treatment with this particular molecule, the study was discontinued. Fragile X syndrome is the most common form of inherited mental retardation. Preclinical studies indicate that fragile X phenotypes are linked to an overactivity of mGlu5 (Dolen et al., 2010), suggesting that antagonism of this receptor could be of therapeutic interest. Recently, fenobam, an mGlu5 NAM also known for its anxiolytic properties, entered phase II clinical studies, which so far have demonstrated potential therapeutic benefits on Fragile X symptoms (Berry-Kravis et al., 2009).

Table 1. mGlu receptor ligands currently undergoing clinical trials. Sources: ClinicalTrials.gov and EvaluatePharma.com. \* - Trial discontinued.

mGlu2 and mGlu3 receptors are a major target for the treatment of anxiety and schizophrenia (Conn and Jones, 2009; Conn et al., 2009b). As a result, the activation of these receptors has been exploited for the treatment of said diseases in several clinical studies. Non-selective mGlu2/mGlu3 agonists have reached phase II clinical studies for the treatment of generalised anxiety disorders, but the trial was terminated due to risks of seizure observed in animals (Dunayevich et al., 2008). Allosteric ligands represent an alternative to the use of orthosteric ligands, since they do not interfere with the spatiotemporal profile of the endogenous ligand; therefore they are more targeted and usually produce less deleterious side effects. Recently, a phase I study on anxiety was started by Ortho-McNeil-Janssen Pharmaceuticals Inc. and Addex pharmaceuticals using ADX71149, an mGlu2 PAM, but the conclusions remain known. Altered glutamatergic neurotransmission is also linked in part to schizophrenia and through a phase II study by Eli Lilly, the improvement of

yielded beneficial results. Since glutamate is the main neurotransmitter of the migraine circuit, then inhibition of postsynaptic mGlu5 receptors that are present in this circuit would decrease glutamatergic neurotransmission and hence may pose a useful approach in migraine therapy. However, due to liver toxicity after long-term treatment with this particular molecule, the study was discontinued. Fragile X syndrome is the most common form of inherited mental retardation. Preclinical studies indicate that fragile X phenotypes are linked to an overactivity of mGlu5 (Dolen et al., 2010), suggesting that antagonism of this receptor could be of therapeutic interest. Recently, fenobam, an mGlu5 NAM also known for its anxiolytic properties, entered phase II clinical studies, which so far have demonstrated

potential therapeutic benefits on Fragile X symptoms (Berry-Kravis et al., 2009).

Table 1. mGlu receptor ligands currently undergoing clinical trials. Sources:

mGlu2 and mGlu3 receptors are a major target for the treatment of anxiety and schizophrenia (Conn and Jones, 2009; Conn et al., 2009b). As a result, the activation of these receptors has been exploited for the treatment of said diseases in several clinical studies. Non-selective mGlu2/mGlu3 agonists have reached phase II clinical studies for the treatment of generalised anxiety disorders, but the trial was terminated due to risks of seizure observed in animals (Dunayevich et al., 2008). Allosteric ligands represent an alternative to the use of orthosteric ligands, since they do not interfere with the spatiotemporal profile of the endogenous ligand; therefore they are more targeted and usually produce less deleterious side effects. Recently, a phase I study on anxiety was started by Ortho-McNeil-Janssen Pharmaceuticals Inc. and Addex pharmaceuticals using ADX71149, an mGlu2 PAM, but the conclusions remain known. Altered glutamatergic neurotransmission is also linked in part to schizophrenia and through a phase II study by Eli Lilly, the improvement of

ClinicalTrials.gov and EvaluatePharma.com. \* - Trial discontinued.

symptoms of schizophrenia with an mGlu2/mGlu3 agonist was similar to that demonstrated with olanzapine, a common antipsychotic drug; this drug was also tolerated by patients (Patil et al., 2007).

Preclinical studies strongly suggest that Group III mGlu receptors may play a vital role in the symptomatic control of Parkinson's disease. In particular, increasing mGlu4 activity within the basal ganglia appears to be an interesting approach to reduce akinetic symptoms associated with Parkinson's disease (Beurrier et al., 2009; Lopez et al., 2007). However, to our knowledge, none of these compounds have reached phase I clinical trials.

## **3. Metabotropic GABA receptors**

#### **3.1 Structure/function of GABAB receptors**

The metabotropic GABA (GABAB) receptor is the only known GPCR that is responsive to GABA. Architecturally, it is not composed in the same manner as many other Class C GPCRs. Specifically, it consists of a ligand binding GB1 subunit and a G protein coupling GB2 subunit (Galvez et al., 2001; Kaupmann et al., 1998; Margeta-Mitrovic et al., 2001; White et al., 1998); each subunit consisting of a VFT and 7TM domains, but converse to mGlu receptors they lack a CRD (refer to Figure 2 for schematic overview). The two subunits are not covalently associated, but do interact via a coiled-coil domain in their C-terminal tails, which provides a solid hydrophobic interaction to maintain the integrity of the dimer (Kammerer et al., 1999). Through the use of circular dichroism spectroscopy the authors proposed a region in the C-terminal domains of GB1 and GB2 of approximately 30 amino acids, composed of roughly 5-7 heptads.

Discerning the number of ligands that bind to any one dimer at any one moment is often difficult, especially if there is the possibility for receptors to form higher-order oligomers. It has been shown that in class C heterodimers a single subunit was responsible for the binding of the endogenous ligand, in this case GB1 in the GABAB receptor (Kniazeff et al., 2002). This suggests that a single agonist molecule is sufficient to fully activate heterodimeric receptors, but does not discount multiple binding sites on the same protomer. However, nearly nothing is known of the conformational movement of the GB2 subunit, making it nearly impossible to distinguish between the conformational rearrangement and functional responses of Aco and Acc combinations. The only insights come from the GABAB receptor, whereby the introduction of several large residues, such as tryptophan in the crevice of GB2 VFT leads to a decrease in G protein-mediated functional responses (Kniazeff et al., 2002).

It has always been questioned whether GPCRs remain in simple monomeric and dimeric forms or whether they self-associate into higher-order oligomers and, if so, what are the molecular determinants of these interactions. Recently, it has been demonstrated that GABAB are indeed capable of forming tetrameric complexes, which interact via their GB1 subunits (Comps-Agrar et al., 2011; Maurel et al., 2008). By employing the use of a bindingnull GB1 subunit Comps-Agrar et al., (2011) demonstrated that GABAB receptor tetramers could be disrupted and that the resultant complexes are capable of binding approximately twice as much radioligand compared to the wild-type; in addition to increasing the apparent Emax in functional tests. The synthesis of this study was that GABAB receptors that are

Metabotropic Receptors for Glutamate and GABA 23

internalisation from dendritic spines and shafts in the hippocampus (Guetg et al., 2010). Similarly, prolonged NMDA receptor activation results in the rapid phosphorylation of Ser783 on GB2 in an 5' adenosine-monophosphate-dependent protein kinase- (AMPK) dependent manner (Terunuma et al., 2010). The rapid phosphorylation by AMPK altered the endocytic sorting pathway from receptor recycling to endosomal degradation, Ser783 was then slowly dephosphorylated by protein phosphatase 2A, returning the system back to its receptor recycling processes. Although the modes of which GABAB receptors are phosphorylated and there consequences are not entirely clear, recently there has been a great deal of progress made in understanding how GABAB receptor phosphorylation is

affected by distinct signalling systems and their consequences on receptor function.

The GABAB receptor is extensively expressed throughout the central nervous system, specifically, hippocampus, cortex, thalamus and cerebellum (Bettler and Tiao, 2006; Billinton et al., 1999); and in parts of the peripheral nervous system. They are located both pre- and post-synaptically where they mediate activity of Cav and Kir3 channels, respectively (Dutar and Nicoll, 1988; Lopez-Bendito et al., 2004; Luscher et al., 1997). Presynaptic GABAB receptors can be found at both homo- and hetero-autoreceptors on GABA and, for example, glutamate nerve terminals, respectively (Thompson et al., 1993). Activation of these receptors leads to a hyperpolarisation of the nerve terminal thereby inhibiting further neurotransmitter release. Postsynaptically, GABAB receptors have been demonstrated to mediate slow inhibitory postsynaptic potentials (IPSPs) through the operation of Kir3 channels. It is noteworthy that in the human brain, there are two major isoforms of the GABAB receptors, those that contain a GB1A subunits, and those that possess GB1B subunits, notwithstanding there is no apparent difference in pharmacology or physiology between the two receptors in heterologous cell systems (Ulrich and Bettler, 2007). Despite a lack of obvious differences in function and pharmacology, there is indeed a differential expression pattern, such that GB1A and GB1B are both expressed on GABAergic nerve terminals, yet only GB1A subunits are expressed on glutamatergic synaptic terminals (Kulik et al., 2003). By using different sets of complementary approaches, the authors showed that GB1A-containing heterodimers mainly control presynaptic release of glutamate, whereas receptors possessing

Similar to mGlu receptors, GABAB receptors have two main ligand-binding domains, the orthosteric ligand-binding pocket located within the VFT of GB1; and the allosteric ligandbinding domain, which is within the 7TM region, most likely within the 7TM bundle. There are surprisingly few GABAB receptor full agonists aside from GABA itself and the wellknown baclofen (refer to Figure 5). There are some other agonists such as CGP27492, the tritiated form of which replaced [3H]-baclofen as the radioligand agonist of choice, but was surrounded by controversy when it failed to reproduce the same physiological effects in some key assays (Froestl et al., 1995). A number of GABAB receptor partial agonists have been identified, the most famous of which is the endogenous metabolite of GABA, hydroxybutyric acid (GHB), synthesised from GABA transaminase and semialdehyde reductase. Other partial agonists include CGP44532 and CGP35024, the latter is also a

**3.2 Localisation and physiology of GABAB receptors**

GB1B subunits predominantly mediate post-synaptic inhibition.

**3.3 GABAB receptor pharmacology and clinical relevance**

associated into a tetrameric assembly have reduced binding capacity and functional capability compared to GABAB receptors in dimeric form. Comps-Agrar et al., (2011) attempted to more precisely examine the structural determinants of the molecular construction of the GABAB receptor tetramer. They resolved that an important interaction between the VFTs of the GB1 subunits occurs, and then experimentally demonstrated the disruption of this interaction through mutation and insertion of an *N*-glycosylation site (G380N) increases the apparent *B*max of fluorescent ligand binding and maximal function effect in intracellular calcium mobilisation assays. It is noteworthy that this study demonstrated that there is tetramerisation of GB1A subunit-containing GABAB receptors, but not GB1B subunit-containing receptors.

Stimulation of GABAB receptors results in the activation and dissociation of Gi/o family G proteins, which in turn inhibit the function adeylyl cyclase thereby decreasing intracellular cAMP levels; activate Kir3 channels and inhibit Cav2 channels (Dunlap and Fischbach, 1981; Leaney and Tinker, 2000; Nishikawa et al., 1997). One of the major actions of GABAB receptor activation is the opening of Kir3 channels, where the increase in K+ permeability through these channels hyperpolarises the cell thereby inhibiting the propagation of action potentials (Dascal, 1997; Misgeld et al., 1995).

Many GPCRs undergo rapid receptor phosphorylation and subsequent sequestration from the cell surface, commonly in an arrestin-dependent manner, followed by the recruitment of scaffolding proteins and by clathrin-mediated endocytosis (Shenoy and Lefkowitz, 2005). One interesting feature that is dissimilar to many GPCRs and is the subject of much debate is that GABAB receptors do not appear to undergo activation-dependent phosphorylation and internalisation. Indeed, it has been reported that these receptors are not phosphorylated by the canonical G protein-coupled receptor kinases (GRKs), yet are desensitised by GRK4 in the absence of any apparent phosphorylation (Perroy et al., 2003). It has been demonstrated in chick neurons that upon activation, GABAB receptors form a complex with Cav channels and arrestins, then are consequently internalised as a mechanism of rapid desensitisation of GABAB receptor signalling (Puckerin et al., 2006). This however, is conflicting with evidence provided by Fairfax et al., (2004) whereby GABAB receptors did not associate with arrestins and, indeed, the cAMP-dependent kinase- (PKA) mediated phosphorylation of the GABAB receptor at position Ser892 on the GB2 subunit increases its cell-surface stability; rather than impeding its cellular function. It would appear in these cases that the phosphorylation state and the subsequent events may very well be cell type specific, which may be yet another degree of complexity for texturing GABAB receptormediated signalling. Interestingly, despite the lack of consistent evidence that GABAB receptors are phosphorylated as a consequence of receptor activation, there is an accumulating body of evidence that these receptors are phosphorylated mostly by secondmessenger kinases. For example, protein kinase C (PKC) has been described to phosphorylate the GB1 subunit GABAB receptors after the dissociation of the chaprone protein, *N*-ethylmaleimide-sensitive fusion (NSF) protein, in Chinese hamster ovary (CHO) cells (Pontier et al., 2006). More recently, there have been new developments on how GABAB receptors are phosphorylated and dephosphorylated in neurons. Recent evidence suggests that NMDA receptors can also act as regulators of GABAB receptor function, such that NMDA receptor activation, via calcium/calmodulin-dependent protein kinase, phosphorylates the GB1 subunit at position Ser867, resulting in rapid receptor

associated into a tetrameric assembly have reduced binding capacity and functional capability compared to GABAB receptors in dimeric form. Comps-Agrar et al., (2011) attempted to more precisely examine the structural determinants of the molecular construction of the GABAB receptor tetramer. They resolved that an important interaction between the VFTs of the GB1 subunits occurs, and then experimentally demonstrated the disruption of this interaction through mutation and insertion of an *N*-glycosylation site (G380N) increases the apparent *B*max of fluorescent ligand binding and maximal function effect in intracellular calcium mobilisation assays. It is noteworthy that this study demonstrated that there is tetramerisation of GB1A subunit-containing GABAB receptors, but

Stimulation of GABAB receptors results in the activation and dissociation of Gi/o family G proteins, which in turn inhibit the function adeylyl cyclase thereby decreasing intracellular cAMP levels; activate Kir3 channels and inhibit Cav2 channels (Dunlap and Fischbach, 1981; Leaney and Tinker, 2000; Nishikawa et al., 1997). One of the major actions of GABAB receptor activation is the opening of Kir3 channels, where the increase in K+ permeability through these channels hyperpolarises the cell thereby inhibiting the propagation of action