David E. Jane

Pharmacological agents acting at subtypes of metabotropic glutamate receptors

Metabotropic (G-protein-coupled) glutamate (mGlu) receptors have now emerged as a recognized, but still relatively new area of excitatory amino acid research. Current understanding of the roles and involvement of mGlu receptor subtypes in physiological/pathophysiological functions of the central nervous system has been recently propelled by the emergence of various structurally novel, potent, and mGlu receptor selective pharmacological agents. This article reviews the evolution of pharmacological agents that have been reported to target mGlu receptors, with a focus on the known receptor subtype selectivities of current agents.

(RS)-2-Chloro-5-Hydroxyphenylglycine (CHPG) Activates mGlu5, but not mGlu1, Receptors Expressed in CHO Cells and Potentiates NMDA Responses in the Hippocampus

A new phenylglycine derivative, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), has been synthesized and shown to selectively activate mGlu5a receptors, compared to mGlu1 alpha receptors, when expressed in CHO cells. This selective mGlu5 receptor agonist also potentiates NMDA-induced depolarizations in rat hippocampal slices. CHPG may be a useful tool for studying the role of mGlu5 receptors in the central nervous system.

Competitive antagonism at metabotropic glutamate receptors by (S) -4-carboxyphenylglycine and (RS) -α-methyl-4-carboxyphenylglycine

Two phenylglycine derivates, (S)-4-carboxyphenylglycine and (RS)-alpha-methyl-4-carboxyphenylglycine, competitively antagonised (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD)-stimulated phosphoinositide hydrolysis in rat cerebral cortical slices. The same phenylglycine derivatives selectively antagonized ACPD-induced depolarization in neonatal rat spinal motoneurones and rate thalamic neurones relative to depolarization or excitation induced by N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). Both phenylglycine derivatives also selectively depressed synaptic excitation in thalamic neurones evoked by noxious thermal stimuli, without affecting the synaptic stimulation of the same cells by non-noxious stimuli.

Kainate receptors: Pharmacology, function and therapeutic potential

Compared to the other glutamate receptors, progress in the understanding of the functions of kainate receptors (KARs) has lagged behind, due mainly to the relative lack of specific pharmacological tools. Over the last decade subunit selective agonists (e.g. ATPA and 5-iodowillardiine) and orthosteric (e.g. LY382884 and ACET) and allosteric antagonists for KARs that contain GluK1 (GluR5) subunits have been developed. However, no selective ligands for the other KAR subunits have been identified. The use of GluK1 antagonists has enabled several functions of KARs, that contain this subunit, to be identified. Thus, KARs have been shown to regulate excitatory and inhibitory synaptic transmission. In the case of the regulation of L-glutamate release, they can function as facilitatory autoreceptors or inhibitory autoreceptors during repetitive synaptic activation and can respond to ambient levels of L-glutamate to provide a tonic regulation of L-glutamate release. KARs also contribute a component of excitatory synaptic transmission at certain synapses. They can also act as triggers for both long-term potentiation (LTP) and long-term depression (LTD) and rapid alterations in their trafficking can result in altered synaptic transmission during both synaptic plasticity and neuronal development. KARs also contribute to synchronised rhythmic activity in the brain and are involved in forms of learning and memory. With respect to therapeutic indications, antagonists for GluK1 have shown positive activity in animal models of pain, migraine, epilepsy, stroke and anxiety. This potential has now been confirmed in dental pain and migraine in initial studies in man.

The glutamate story

Glutamatergic synaptic transmission in the mammalian central nervous system was slowly established over a period of some 20 years, dating from the 1950s. Realisation that glutamate and like amino acids (collectively known as excitatory amino acids (EAA)) mediated their excitatory actions via multiple receptors preceded establishment of these receptors as synaptic transmitter receptors. EAA receptors were initially classified as N‐methyl‐D‐aspartate (NMDA) and non‐NMDA receptors, the latter subdivided into quisqualate (later AMPA) and kainate receptors after agonists that appeared to activate these receptors preferentially, and by their sensitivity to a range of differentially acting antagonists developed progressively during the 1970s. NMDA receptors were definitively shown to be synaptic receptors on spinal neurones by the sensitivity of certain excitatory pathways in the spinal cord to a range of specific NMDA receptor antagonists. Importantly, specific NMDA receptor antagonists appeared to be less effective at synapses in higher centres. In contrast, antagonists that also blocked non‐NMDA as well as NMDA receptors were almost universally effective at blocking synaptic excitation within the brain and spinal cord, establishing both the existence and ubiquity of non‐NMDA synaptic receptor systems throughout the CNS. In the early 1980s, NMDA receptors were shown to be involved in several central synaptic pathways, acting in concert with non‐NMDA receptors under conditions where a protracted excitatory postsynaptic potential was effected in response to intense stimulation of presynaptic fibres. Such activation of NMDA receptors together with non‐NMDA receptors led to the phenomenon of long‐term potentiation (LTP), associated with lasting changes in synaptic efficacy (synaptic plasticity) and considered to be an important process in memory and learning. During the 1980s, it was shown that certain glutamate receptors in the brain mediated biochemical changes that were not susceptible to NMDA or non‐NMDA receptor antagonists. This dichotomy was resolved in the early 1990s by the techniques of molecular biology, which identified two families of glutamate‐binding receptor proteins (ionotropic (iGlu) and metabotropic (mGlu) receptors). Development of antagonists binding to specific protein subunits is currently enabling precise identification of discrete iGlu or mGlu receptor subtypes that participate in a range of central synaptic processes, including synaptic plasticity.

Phenylglycine derivatives as new pharmacological tools for investigating the role of metabotropic glutamate receptors in the central nervous system.

The possible roles of G-protein coupled metabotropic glutamate receptors in central nervous function are currently the focus of intensive investigation. The complexity of effects produced by agonists at these receptors probably reflects the activity of a range of sub-types. The metabotropic glutamate receptors first described are linked to phospholipase C, mediating phosphoinositide hydrolysis and release of Ca2+ from intracellular stores. A substance generally considered to be a selective agonist for these receptors is (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD). This substance not only stimulates phosphoinositide hydrolysis, but also inhibits cyclic AMP formation. A family of metabotropic glutamate receptors, incorporating both phospholipase C- and adenylcyclase-linked sub-types has been cloned. Various effects of metabotropic glutamate receptor agonists on membrane ion fluxes and synaptic events have been reported, including neuronal depolarization and/or excitation, hyperpolarization, inhibition of Ca(2+)-dependent and voltage-gated K+ currents, potentiation of N-methyl-D-aspartate-induced responses, depression of synaptic excitation and either induction or augmentation of long-term potentiation. To clarify the role of metabotropic glutamate receptors in central nervous activity and to aid the characterization of the various receptor types that may be involved, a range of highly selective agonists and antagonists is required. To date, currently available antagonists such as L-2-amino-3-phosphonopropionate and L-aspartic acid-beta-hydroxamate appear to be unselective and insufficiently potent. We report here the actions of three phenylglycine derivatives, the particular agonist and/or antagonist properties of which may help to elucidate the roles of metabotropic glutamate receptors in central nervous activity.(ABSTRACT TRUNCATED AT 250 WORDS)

(S)-3,4-DCPG, a potent and selective mGlu8a receptor agonist, activates metabotropic glutamate receptors on primary afferent terminals in the neonatal rat spinal cord

(S)-3,4-Dicarboxyphenylglycine (DCPG) has been tested on cloned human mGlu1-8 receptors individually expressed in AV12-664 cells co-expressing a rat glutamate/aspartate transporter and shown to be a potent and selective mGlu8a receptor agonist (EC(50) value 31+/-2 nM, n=3) with weaker effects on the other cloned mGlu receptors (EC(50) or IC(50) values >3.5 microM on mGlu1-7). Electrophysiological characterisation on the neonatal rat spinal cord preparation revealed that (S)-3,4-DCPG depressed the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) giving a biphasic concentration-response curve showing EC(50) values of 1.3+/-0.2 microM (n=17) and 391+/-81 microM (n=17) for the higher and lower affinity components, respectively. The receptor mediating the high-affinity component was antagonised by 200 microM (S)-alpha-methyl-2-amino-4-phosphonobutyrate (MAP4, K(D) value 5.4+/-1.5 microM (n=3)), a group III metabotropic glutamate (mGlu) receptor antagonist. The alpha-methyl substituted analogue of (S)-3,4-DCPG, (RS)-3,4-MDCPG (100 microM), antagonised the effects of (S)-3,4-DCPG (K(D) value 5.0+/-0.4 microM, n=3) in a similar manner to MAP4. (S)-3,4-DCPG-induced depressions of the fDR-VRP in the low-affinity range of the concentration-response curve were potentiated by 200 microM (S)-alpha-ethylglutamate (EGLU), a group II mGlu receptor antagonist, and were relatively unaffected by MAP4 (200 microM). However, depressions of the fDR-VRP mediated by the AMPA selective antagonist (R)-3,4-DCPG were not potentiated by EGLU, suggesting that the low-affinity component of the concentration-response curve for (S)-3,4-DCPG is not due to antagonism of postsynaptic AMPA receptors. It is suggested that the receptor responsible for mediating the high-affinity component is mGlu8. The receptor responsible for mediating the low-affinity effect of (S)-3,4-DCPG has yet to be identified but it is unlikely to be one of the known mGlu receptors present on primary afferent terminals or an ionotropic glutamate receptor of the AMPA or NMDA subtype.

Metabotropic glutamate receptors contribute to the induction of long-term depression in the CA1 region of the hippocampus.

Long-term depression of synaptic transmission was induced following the prior induction of long-term potentiation in the CA1 region of rat hippocampal slices. We show that the induction of this form of synaptic depression can be prevented by (+)-alpha-methyl-4-carboxyphenylglycine, a selective antagonist of metabotropic glutamate receptors.

Structure–activity analysis of a novel NR2C/NR2D-preferring NMDA receptor antagonist: 1-(phenanthrene-2-carbonyl) piperazine-2,3-dicarboxylic acid

(2S*,3R*)‐1‐(biphenyl‐4‐carbonyl)piperazine‐2,3‐dicarboxylic acid (PBPD) is a moderate affinity, competitive N‐methyl‐D‐aspartate (NMDA) receptor antagonist with an atypical pattern of selectivity among NMDA receptor 2 subunit (NR2) subunits. We now describe the activity of several derivatives of PBPD tested at both rat brain NMDA receptors using L‐[3H]‐glutamate binding assays and at recombinant receptors expressed in Xenopus oocytes. Substituting various branched ring structures for the biphenyl group of PBPD reduced NMDA receptor activity. However, substituting linearly arranged ring structures – fluorenone or phenanthrene groups – retained or enhanced activity. Relative to PBPD, the phenanthrene derivative (2S*,3R*)‐1‐(phenanthrene‐2‐carbonyl)piperazine‐2,3‐dicarboxylic acid (PPDA) displayed a 30‐ to 78‐fold increase in affinity for native NMDA receptors. At recombinant receptors, PPDA displayed a 16‐fold (NR2B) to 94‐fold (NR2C) increase in affinity over PBPD. Replacement of the biphenyl group of PBPD with a 9‐oxofluorene ring system resulted in small changes in receptor affinity and subtype selectivity. 2′‐Bromo substitution on the biphenyl group of PBPD reduced antagonist affinity 3‐ to 5‐fold at NR2A‐, NR2B‐ and NR2D‐containing receptors, but had little effect on NR2C‐containing receptors. In contrast, 4′‐fluoro substitution of the biphenyl ring of PBPD selectively increased NR2A affinity. The aromatic rings of PBPD and PPDA increase antagonist affinity and appear to interact with a region of the NMDA receptor displaying subunit heterogeneity. PPDA is the most potent and selective NR2C/NR2D‐preferring antagonist yet reported and thus may be useful in defining NR2C/NR2D function and developing related antagonists with improved NMDA receptor subtype selectivity.

Pharmacological antagonism of the actions of group II and III mGluR agonists in the lateral perforant path of rat hippocampal slices

1 An understanding of the physiological and pathological roles of metabotropic glutamate receptors (mGluRs) is currently hampered by the lack of selective antagonists. Standard extracellular recording techniques were used to investigate the activity of recently reported mGluR antagonists on agonist‐induced depressions of synaptic transmission in the lateral perforant path of hippocampal slices obtained from 12–16 day‐old rats. 2 The group III specific mGluR agonist, (S)‐2‐amino‐4‐phosphonobutanoate (L‐AP4) depressed basal synaptic transmission in a reversible and dose‐dependent manner. The mean (±s.e.mean) depression obtained with 100 μm L‐AP4 (the maximum concentration tested) was 74 ± 3% and the IC50 value was 3 ± 1 μm (n = 5). 3 The selective group II mGluR agonists, (1S,3S)‐1‐aminocyclopentane‐1,3‐dicarboxylate ((1S,3S)‐ACPD) and (2S,1′R,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine (DCG‐IV) also depressed basal synaptic transmission in a reversible and dose‐dependent manner. The mean depression obtained with 200 μm (1S,3S)‐ACPD was 83 ± 8% and the IC50 value was 12 ± 3 μm (n = 5). The mean depression obtained with 1 μm DCG‐IV was 73 ± 7% and the IC50 value was 88 ± 15 nM (n = 4). 4 Synaptic depressions induced by the actions of 20 μm (1S,3S)‐ACPD and 10 μm L‐AP4 were antagonized by the mGluR antagonists, (+)‐α‐methyl‐4‐carboxyphenylglycine ((+)‐MCPG), (S)‐2‐methyl‐2‐amino‐4‐phosphonobutanoate (MAP4), (2S, 1′S,2′S)‐2‐methyl‐2‐(2′‐carboxycyclopropyl)glycine (MCCG), (RS)‐α‐methyl‐4‐tetrazolylphenylglycine (MTPG), (RS)‐α‐methyl‐4‐sulphonophenylglycine (MSPG) and (RS)‐α‐methyl‐4‐phosphonophenylglycine (MPPG) (all tested at 500 μm). 5 (+)‐MCPG was a weak antagonist of both L‐AP4 and (1S,3S)‐ACPD‐induced depressions. MCCG was selective towards (1S,3S)‐ACPD, but analysis of its effects were complicated by apparent partial agonist activity. MAP4 showed good selectivity for L‐AP4‐induced effects. 6 The most effective antagonist tested against 10 μm L‐AP4 was MPPG (mean reversal 90 ± 3%; n = 4). In contrast, the most effective antagonist tested against 20 μm (1S,3S)‐ACPD induced depressions was MTPG (mean reversal 64 ± 4%; n = 4). Both antagonists produced parallel shifts in agonist dose‐response curves. Schild analysis yielded estimated KD values of 11.7 μm and 27.5 μm, respectively. Neither antagonist had any effect on basal transmission or on depressions induced by the adenosine receptor agonist, 2‐chloroadenosine (500 nM; n = 3). 7 We conclude that both group II and group III mGluRs can mediate synaptic depressions induced by mGluR agonists in the lateral perforant path. The mGluR antagonists MTPG, MPPG and MAP4 should be useful in determining the roles of group II and III mGluRs in the central nervous system.