A.E. Kingston

LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate receptors

The in vitro pharmacology of a structurally novel compound, LY341495, was investigated at human recombinant metabotropic glutamate (mGlu) receptor subtypes expressed in non-neuronal (RGT, rat glutamate transporter) cells. LY341495 was a nanomolar potent antagonist of 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD)-induced inhibition of forskolin-stimulated cAMP formation at mGlu2 and mGlu3 receptors (respective IC50S of 0.021 and 0.014 microM). At group I mGlu receptor expressing cells, LY341495 was micromolar potent in antagonizing quisqualate-induced phosphoinositide (PI) hydrolysis, with IC50 values of 7.8 and 8.2 microM for mGlu1a and mGlu5a receptors, respectively. Among the human group III mGlu receptors, the most potent inhibition of L-2-amino-4-phosphonobutyric acid (L-AP4) responses was seen for LY341495 at mGlu8, with an IC50 of 0.17 microM. LY341495 was less potent at mGlu7 (IC50 = 0.99 microM) and least potent at mGlu4 (IC50 = 22 microM). Binding studies in rat brain membranes also demonstrated nanomolar potent group II mGlu receptor affinity for LY341495, with no appreciable displacement of ionotropic glutamate receptor ligand binding. Thus, LY341495 has a unique range of selectivity across the mGlu receptor subtypes with a potency order of mGlu3 > or = mGlu2 > mGlu8 > mGlu7 >> mGlu1a = mGlu5a > mGlu4. In particular, LY341495 is the most potent antagonist yet reported at mGlu2, 3 and 8 receptors. Thus, it represents a novel pharmacological agent for elucidating the function of mGlu receptors in experimental systems.

The potent mGlu receptor antagonist LY341495 identifies roles for both cloned and novel mGlu receptors in hippocampal synaptic plasticity

Understanding the roles of metabotropic glutamate (mGlu) receptors has been severely hampered by the lack of potent antagonists. LY341495 (2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-y l)propanoic acid) has been shown to block group II mGlu receptors in low nanomolar concentrations (Kingston, A.E., Ornstein, P.L., Wright, R.A., Johnson, B.G., Mayne, N.G., Burnett, J.P., Belagaje, R., Wu, S., Schoepp, D.D., 1998. LY341495 is a nanomolar potent and selective antagonist at group II metabotropic glutamate receptors. Neuropharmacology 37, 1-12) but can be used in higher concentrations to block all hippocampal mGlu receptors, identified so far by molecular cloning (mGlu1-5,7,8). Here we have further characterised the mGlu receptor antagonist activity of LY341495 and have used this compound to investigate roles of mGlu receptors in hippocampal long-term potentiation (LTP) and long-term depression (LTD). LY341495 competitively antagonised DHPG-stimulated PI hydrolysis in AV12-664 cells expressing either human mGlu1 or mGlu5 receptors with Ki-values of 7.0 and 7.6 microM, respectively. When tested against 10 microM L-glutamate-stimulated Ca2+ mobilisation in rat mGlu5 expressing CHO cells, it produced substantial or complete block at a concentration of 100 microM. In rat hippocampal slices, LY341495 eliminated 30 microM DHPG-stimulated PI hydrolysis and 100 microM (1S,3R)-ACPD-inhibition of forskolin-stimulated cAMP formation at concentrations of 100 and 0.03 microM, respectively. In area CA1, it antagonised DHPG-mediated potentiation of NMDA-induced depolarisations and DHPG-induced long-lasting depression of AMPA receptor-mediated synaptic transmission. LY341495 also blocked NMDA receptor-independent depotentiation and setting of a molecular switch involved in the induction of LTP; effects which have previously been shown to be blocked by the mGlu receptor antagonist (S)-MCPG. These effects may therefore be due to activation of cloned mGlu receptors. In contrast, LY341495 did not affect NMDA receptor-dependent homosynaptic LTD; an effect which may therefore be independent of cloned mGlu receptors. Finally, LY341495 failed to antagonise NMDA receptor-dependent LTP and, in area CA3, NMDA receptor-independent, mossy fibre LTP. Since in the same inputs these forms of LTP were blocked by (S)-MCPG, a novel type of mGlu receptor may be involved in their induction.

Neuroprotective activity of the potent and selective mGlu1a metabotropic glutamate receptor antagonist, (+)-2-methyl-4 carboxyphenylglycine (LY367385) : comparison with LY357366, a broader spectrum antagonist with equal affinity for mGlu1a and mGlu5 receptors

(+)-2-Methyl-4-carboxyphenylglycine (LY367385), a potent and selective antagonist of mGlu1a metabotropic glutamate receptors, was neuroprotective in the following in vitro and in vivo models of excitotoxic death: (i) mixed cultures of murine cortical cells transiently exposed to N-methyl-D-aspartate (NMDA); (ii) rats monolaterally infused with NMDA into the caudate nucleus; and (iii) gerbils subjected to transient global ischemia. We have compared the activity of LY367385 with that of the novel compound (+/-)-alpha-thioxantylmethyl-4-carboxyphenylglycine (LY367366), which antagonizes both mGlu1a and -5 receptors at low micromolar concentrations, but also recruits other subtypes at higher concentrations. Although LY367366 was neuroprotective, it was in general less efficacious than LY357385, suggesting that inhibition of mGlu1 receptors is sufficient to confer significant neuroprotection. We conclude that endogenous activation of mGlu1a receptors (or perhaps other mGlu1 receptor splice variants) contributes to the development of neuronal degeneration of excitotoxic origin.

Pharmacological analysis of 4-carboxyphenylglycine derivatives: Comparison of effects on mGluR1α and mGluR5a subtypes

The antagonist effects of the 4-carboxyphenylglycines: (S)-4-carboxy-3hydroxyphenylglycine (4C3HPG), (S)-4-carboxyphenylglycine (4CPG) and (+)-alpha-methyl-4-carboxyphenylglycine (M4CPG) were compared on functional responses of human metabotropic glutamate receptor (mGluR) subtypes mGluR1 alpha and mGluR5a. These receptors both belong to group 1 type mGluRs which couple to the phosphoinositide (PI) hydrolysis/[Ca2+]i mobilization signal transduction pathway and are closely related in both structure and agonist pharmacology. In this study, the IC50 values obtained for quisqualate induced PI hydrolysis responses show that although all the phenylglycines are antagonists for both mGluR1 alpha and mGluR5a, the compounds exhibit differential potencies at these receptor subtypes. The 4C3HPG derivative was the most potent antagonist for both mGluR1 alpha (IC50 range: 19-50 microM) and mGluR5a (IC50 range: 53-280 microM). 4CPG produced an IC50 range of 4r-72 microM for mGluR1 alpha and 150-156 microM for mGluR5a cells. The potency of the M4CPG could not be distinguished from that of 4CPG with IC50 ranges of 29-100 microM and 115-210 microM for mGluR1 alpha and mGluR5a respectively. Further characterization of the dose-response effects of the compounds on quisqualate induced [Ca2+]i mobilization showed that although the magnitude of phenylglycine inhibition was reduced for both mGluR subtypes compared to those observed for stimulation of PI hydrolysis (except for 4C3HPG on mGluR1 alpha), similar differences in the relative potencies of the phenylglycines between mGluR1 alpha (IC50s: 40 +/- 10 microM for 4C3HPG: 300-1000 microM for 4CPG and M4CPG) and mGluR5a (IC50s: > 1000 microM) were evident.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel, competitive mGlu5 receptor antagonist (LY344545) blocks DHPG-induced potentiation of NMDA responses but not the induction of LTP in rat hippocampal slices

We have investigated the pharmacological properties of LY344545, a structurally related epimer of the broad spectrum competitive metabotropic glutamate receptor antagonist, LY341495. We have found that LY344545 also antagonizes competitively nearly all mGlu receptor subtypes, but with a wide spectrum of activity. The order of potency for the human receptor isoforms was mGlu5a (IC50 of 5.5±0.6 μM)>mGlu2=mGlu3>mGlu1α=mGlu7>mGlu6=mGlu8. No significant mGlu4 receptor antagonist activity was detected at the highest concentration used (100 μM). 100 μM LY344545 displaced 50±5% of [3H]‐CGP39653 binding, but less than 30% of [3H]‐kainate or [3H]‐AMPA in radioligand binding assays. LY344545 antagonized L‐glutamate stimulated Ca2+ release in CHO cells transfected with mGlu receptors in a concentration dependent manner with a 10 fold higher affinity for the rat mGlu5a receptor (Ki=2.1±0.6 μM) compared to the rat mGlu1α receptor (Ki=20.5±2.1 μM). 50 μM (1S, 3R)‐ACPD‐induced Ca2+ rises in hippocampal CA1 neurones were also antagonized (IC50=6.8±0.7 μM). LY344545 antagonized 10 μM (S)‐3,5‐DHPG‐induced potentiation of NMDA depolarizations in CA1 neurones (EC50=10.6±1.0 μM). At higher concentrations (100 μM), LY344545 was an NMDA receptor antagonist. LY344545 also blocked the induction, but not the expression, of LTP at CA3 to CA1 synapses with an IC50>300 μM. This effect is consistent with its weak activity at NMDA receptors. These results demonstrate that the binding of ligands to mGlu receptor subtypes is critically dependent on the spatial orientation of the same molecular substituents within a given chemical pharmacophore. The identification of LY344545 as the first competitive antagonist to show selectivity towards mGlu5 receptors supports the potential to design more selective and potent competitive antagonists of this receptor. These results further indicate that mGlu receptor‐mediated potentiation of NMDA responses is not essential for the induction of LTP.

Systemic pre-treatment with a group II mGlu agonist, LY379268, reduces hyperalgesia in vivo

Previous studies investigating the role of metabotropic glutamate (mGlu) receptors in nociceptive processing have been hampered by the lack of systemically active, selective, ligands. This study investigates the possible analgesic and/or anti‐hyperalgesic properties of the most potent compound to date that has systemic agonist activity at group II mGlu receptors, LY379268. In testing the drug in rats as an analgesic to acute noxious stimuli, LY379268 (in doses up to 3 mg kg−1 i.p.) did not affect withdrawal latencies to either mechanical or thermal stimulation. However, when a 3 mg kg−1 dose was given prior to an intraplantar injection of carrageenan, the inflammatory hyperalgesia that developed was significantly delayed compared to saline pre‐treated controls, without affecting the inflammation of the paw. A similar dose of the mGlu‐inactive enantiomer, LY379267, was not anti‐hyperalgesic. In a model of mouse tail withdrawal to warm water, LY379268 (12 mg kg−1 i.p.), given before a subcutaneous tail injection of capsaicin, reduced the subsequent neurogenic hyperalgesia. Rota‐rod testing showed that the drug did not produce a motor impairment in rats at antihyperalgesic doses. The results indicate that systemic activation of this group of mGlu receptors reduces both inflammatory and neurogenic thermal hyperalgesia.

Sulphur-containing amino acids are agonists for group 1 metabotropic receptors expressed in clonal RGT cell lines

Comparison of the pharmacological effects of a range of sulphur-containing amino acids on human mGluR1alpha and mGluR5a has been undertaken. cDNAs of each mGluR were transfected into a Syrian hamster tumour cell line AV12-664 that was previously transfected with the rat glutamate-aspartate transporter protein (GLAST). The L-isomers of cysteine sulphinic acid (CSA), homocysteine sulphinic acid (HCSA), cysteic acid (CA) and serine-O-sulphate (SOS) stimulated PI hydrolysis in human mGluR1alpha and mGluR5a cells with full agonist effects. D-CSA, the only active D-isomer, was a partial agonist for mGluR5a whereas L-sulphocysteine (S-CYS) showed weak agonist-like effects at high concentrations on both mGluR1alpha and mGluR5a. L-Homocysteic acid was inactive on both mGluR1alpha and mGluR5a cells. Treatment of mGluR cultures with glutamate pyruvate transaminase did not alter the potencies of the S-amino acids on PI hydrolysis responses. Inhibitor constants (Ki) obtained for L-HCSA, L-CSA, L-CA and L-SOS in [3H]glutamate receptor binding studies with mGluR1alpha cells indicated that L-HCSA, L-CSA, L-CA and L-SOS can bind specifically to mGluR1 with L-HCSA showing the highest affinity. These results confirm that certain endogenously produced S-amino acids may interact directly with group 1 mGluRs.

Synthesis and structure-activity relationship studies of novel 2-diarylethyl substituted (2-carboxycycloprop-1-yl)glycines as high-affinity group II metabotropic glutamate receptor ligands.

The major excitatory neurotransmitter in the central nervous system, (S)-glutamic acid , activates both ionotropic and metabotropic excitatory amino acid receptors. Its importance in connection to neurological and psychiatric disorders has directed great attention to the development of compounds that modulate the effects of this endogenous ligand. Whereas L-carboxycyclopropylglycine (L-CCG-1) is a potent agonist at, primarily, group II metabotropic glutamate receptors, alkylation of at the alpha-carbon notoriously result in group II mGluR antagonists, of which the most potent compound described so far, LY341495, displays IC(50) values of 23 and 10 nM at the group II receptor subtypes mGlu2 and mGlu3, respectively. In this study we synthesized a series of structural analogues of in which the xanthyl moiety is replaced by two substituted-phenyl groups. The pharmacological characterization shows that these novel compounds have very high affinity for group II mGluRs when tested as their racemates. The most potent analogues demonstrate K(i) values in the range of 5-12 nM, being thus comparable to LY341495.

3-Phenyl-5-isothiazole carboxamides with potent mGluR1 antagonist activity

The disclosed 3-phenyl-5-isothiazole carboxamides are potent allosteric antagonists of mGluR1 with generally good selectivity relative to the related group 1 receptor mGluR5. Pharmacokinetic properties of a member of this series (1R,2R)-N-(3-(4-methoxyphenyl)-4-methylisothiazol-5-yl)-2-methylcyclopropanecarboxamide (14) are good, showing acceptable plasma and brain exposure after oral dosing. Oral administration of isothiazole 14 gave robust activity in the formalin model of persistent pain which correlated with CNS receptor occupancy.