Synthesis and pharmacology of glutamate receptor ligands: new isothiazole analogues of ibotenic acid.

The naturally occurring heterocyclic amino acid ibotenic acid (Ibo) and the synthetic analogue thioibotenic acid (Thio-Ibo) possess interesting but dissimilar pharmacological activity at ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs). Therefore, a series of Thio-Ibo analogues was synthesized. The synthesis included introduction of substituents by Suzuki and Grignard reactions on 4-halogenated 3-benzyloxyisothiazolols, reduction of the obtained alcohols, followed by introduction of the amino acid moiety by use of 2-(N-tert-butoxycarbonylimino)malonic acid diethyl ester. The obtained Thio-Ibo analogues (1, 2a-g) were characterized in functional assays on recombinant mGluRs and in receptor binding assays on native iGluRs. At mGluRs, the activity at Group II was retained for compounds with small substituents (2a-2d), whereas the Group I and Group III receptor activities for all new compounds were lost. Detection of NMDA receptor affinity prompted further characterization, and two-electrode voltage-clamp recordings at recombinant NMDA receptor subtypes NR1/NR2A-D expressed in Xenopus oocytes were carried out for compounds with small substituents (chloro, bromo, methyl or ethyl, compounds 2a-d). This series of Thio-Ibo analogues defines a structural threshold for NMDA receptor activation and reveals that the individual subtypes have different steric requirements for receptor activation. The compounds 2a and 2c are the first examples of agonists discriminating individual NMDA subtypes.

Novel 5-substituted 1-pyrazolol analogues of ibotenic acid: synthesis and pharmacology at glutamate receptors.

5-Substituted 1-pyrazolol analogues of ibotenic acid have been synthesized and pharmacologically characterized on ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs). The syntheses involved introduction of bromide, alkyls, phenyl and arylalkyls in the 5-position of 1-benzyloxypyrazole leading to 5-substituted (RS)-2-amino-(1-hydroxy-4-pyrazolyl)acetic acids (5a-l). The pharmacological activities of the synthesized analogues ranged from the 5-cyclopropylmethyl analogue (5f) with weak but selective affinity for NMDA receptors (IC(50)=35 microM), over the 5-n-propyl analogue (5c), which was a selective mGluR2 agonist (EC(50)=72 microM), to the 5-cyclohexylmethyl analogue (5g), which was a selective mGluR2 antagonist (K(i)=32 microM), and the 5-phenylethyl analogue (5j), which was a weak but apparently selective mGluR1 antagonist (K(i)=230 microM). This series of compounds afforded GluR ligands with a broad spectrum of pharmacological profiles, and showing potential for development of new compounds with subtype-selective activities at various GluRs.

Investigation of a route to ibotenic acid analogues via a reduced pyroglutamate template.

Two alternative "ring switch" based syntheses have been shown to give access to the reduced protected homochiral analogues, 27, 28 and 36, of the CNS active compound ibotenic acid.

Structural determinants of agonist-specific kinetics at the ionotropic glutamate receptor 2.

Glutamate receptors (GluRs) are the most abundant mediators of the fast excitatory neurotransmission in the human brain. Agonists will, after activation of the receptors, induce different degrees of desensitization. The efficacy of agonists strongly correlates with the agonist-induced closure of the ligand-binding domain. However, the differences in desensitization properties are less well understood. By using high-resolution x-ray structure of the GluR2 flop (GluR2o) ligand-binding core protein in complex with the partial glutamate receptor agonist (S)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isothiazolyl)propionic acid [(S)-thio-ATPA], we show that (S)-thio-ATPA induces an 18 degrees closure of the binding core similar to another partial agonist, (S)-2-amino-3-(4-bromo-3-hydroxy-5-isoxazolyl)propionic acid [(S)-Br-HIBO]. Despite the similar closure of the ligand-binding domain, we find in electrophysiological studies that (S)-thio-ATPA induced a 6.4-fold larger steady-state current than (RS)-Br-HIBO, and rapid agonist applications show that (S)-thio-ATPA induces a 3.6-fold higher steady-state/peak ratio and a 2.2-fold slower desensitization time constant than (RS)-Br-HIBO. Structural comparisons reveal that (S)-Br-HIBO, but not (S)-thio-ATPA, induces a twist of the ligand-binding core compared with the apostructure, and the agonist-specific conformation of Leu-650 correlates with the different kinetic profiles pointing at a key role in defining the desensitization kinetics. We conclude that, especially for intermediate efficacious agonists, the desensitization properties are influenced by additional ligand-induced factors beyond domain closure.

4-Alkylated homoibotenic acid (HIBO) analogues: versatile pharmacological agents with diverse selectivity profiles towards metabotropic and ionotropic glutamate receptor subtypes.

4-Alkylated analogues of homoibotenic acid (HIBO) have previously shown high potency and selectivity at ionotropic and metabotropic glutamic acid receptor (iGluR and mGluR) subtypes. Compounds with different selectivity profiles are valuable pharmacological tools for neuropharmacological studies, and the series of 4-alkyl-HIBO analogues have been extended in this paper in the search for versatile agents. Pharmacological characterization of five new analogues, branched and unbranched 4-alkyl-HIBO analogues, have been carried out. The present compounds are all weak antagonists at Group I mGluRs (mGluR1 and 5) presenting only small differences in potencies (Ki values ranging from 89 to 670 microM). Affinities were studied at native and cloned iGluRs, and the compounds described show preference for the AMPA receptor subtypes GluR1 and 2 over GluR3 and 4. However, compared to previous 4-alkyl-HIBO analogues, these compounds show a remarkably high affinity for the Kain preferring subtype GluR5. The observed GluR5 affinities were either similar or higher compared to their GluR1 and 2 affinity. Isopropyl-HIBO showed the highest affinity for GluR5 (Ki=0.16 microM), and represents a unique compound with high affinity towards the three subtypes GluR1, 2 and 5. In general, these compounds represent new selectivity profiles compared to previously reported Glu receptor analogues.

Analogues of homoibotenic acid show potent and selective activity following sensitisation by quisqualic acid.

Quisqualic acid induces sensitisation of neurones to depolarisation by analogues of 2-amino-4-phosphonobutyric acid (AP4), phenylglycine, and homoibotenic acid (HIBO). Thus, after administration of quisqualate these analogues become active at concentrations at which they are otherwise inactive. The mechanisms behind quisqualate-induced sensitisation are poorly understood and have not previously been quantified properly. In this study, we have tested the activity of a number of 4-alkyl- and 4-aryl-substituted analogues of HIBO as regards quisqualate-sensitisation, and present a method for quantifying the sensitisation induced by quisqualate at cortical neurones. These analogues are generally more potent and selective than (S)-AP4 or its homologue (S)-AP5 following quisqualate-sensitisation. Furthermore, we found a statistically significant correlation between the ligands' ability to inhibit CaCl(2)-dependent (S)-[(3)H]glutamate uptake into rat cortical synaptosomes, and their potency following quisqualate-induced depolarisation. This demonstrates the involvement of a transport system in the mechanism underlying the quisqualate-effect.

Beta-lactams as versatile synthons for homochiral ibotenate analogues with potential for activity at glutamate receptors.

The activated beta-lactam aldehydes 37, 41 and 57 were synthesised. Aldehydes 37 and 57 proved to be more versatile substrates for our "ring switching" strategy to homochiral glutamate antagonists than the corresponding compounds in the pyroglutamate or 6-oxopipecolinate series had been. Substantial libraries of homochiral heteroaromatic glycine derivatives with potential for activity at specific glutamate receptor sub-types were prepared from these aldehydes. The aldehyde 41, containing an additional anion stabilising group, underwent a retro-aldol process under "ring switching" conditions.

Identification of amino acid residues in GluR1 responsible for ligand binding and desensitization.

Although GluR1(o) and GluR3(o) are homologous at the amino acid level, GluR3(o) desensitizes approximately threefold faster than GluR1(o). By creating chimeras of GluR1(o) and GluR3(o) and point amino acid exchanges in their S2 regions, two residues were identified to be critical for GluR1(o) desensitization: Y716 and the R/G RNA-edited site, R757. With creation of the double-point mutant (Y716F, R757G)GluR1(o), complete exchange of the desensitization rate of GluR1(o) to that of GluR3(o) was obtained. In addition, both the potency and affinity of the subtype-selective agonist bromohomoibotenic acid were exchanged by the Y716F mutation. A model is proposed of the AMPA receptor binding site whereby a hydrogen-bonding matrix of water molecules plays an important role in determining both ligand affinity and receptor desensitization properties. Residues Y716 in GluR1 and F728 in GluR3 differentially interact with this matrix to affect the binding affinity of some ligands, providing the possibility of developing subtype-selective compounds.

Agonist discrimination between AMPA receptor subtypes.

The lack of subtype-selective compounds for AMPA receptors (AMPA-R) led us to search for compounds with such selectivity. Homoibotenic acid analogues were investigated at recombinant GluR1o, GluR2o(R), GluR3o and GluR1o + 3o receptors expressed in Sf9 insect cells and affinities determined in [3H]AMPA radioligand binding experiments. (S)-4-bromohomoibotenic acid (BrHIBO) exhibited a 126-fold selectivity for GluR1o compared to GluR3o. Xenopus laevis oocytes were used to express functional homomeric and heteromeric recombinant AMPA-R and to determine BrHIBO potency (EC50) at these channels. (R,S)-BrHIBO exhibited a 37-fold selectivity range amongst the AMPA-R. It is hoped that BrHIBO can be used as a lead structure for the development of other subtype-selective compounds.

Glutamate regulates IP3-type and CICR stores in the avian cochlear nucleus.

Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are activated by glutamate released from auditory nerve terminals. If this stimulation is removed, the intracellular calcium ion concentration ([Ca2+]i) of NM neurons rises and rapid atrophic changes ensue. We have been investigating mechanisms that regulate [Ca2+]i in these neurons based on the hypothesis that loss of Ca2+ homeostasis causes the cascade of cellular changes that results in neuronal atrophy and death. In the present study, video-enhanced fluorometry was used to monitor changes in [Ca2+]i stimulated by agents that mobilize Ca2+ from intracellular stores and to study the modulation of these responses by glutamate. Homobromoibotenic acid (HBI) was used to stimulate inositol trisphosphate (IP3)-sensitive stores, and caffeine was used to mobilize Ca2+ from Ca2+-induced Ca2+ release (CICR) stores. We provide data indicating that Ca2+ responses attributable to IP3- and CICR-sensitive stores are inhibited by glutamate, acting via a metabotropic glutamate receptor (mGluR). We also show that activation of C-kinase by a phorbol ester will reduce HBI-stimulated calcium responses. Although the protein kinase A accumulator, Sp-cAMPs, did not have an effect on HBI-induced responses. CICR-stimulated responses were not consistently attenuated by either the phorbol ester or the Sp-cAMPs. We have previously shown that glutamate attenuates voltage-dependent changes in [Ca2+]i. Coupled with the present findings, this suggests that in these neurons mGluRs serve to limit fluctuations in intracellular Ca2+ rather than increase [Ca2+]i. This system may play a role in protecting highly active neurons from calcium toxicity resulting in apoptosis.

Molecular pharmacology of homologues of ibotenic acid at cloned metabotropic glutamic acid receptors.

We have studied the effects of the enantiomers of 2-amino-3-(3-hydroxyisoxazol-5-yl)propionic acid (homoibotenic acid, HIBO) and analogues substituted with a methyl, bromo or butyl group in the four position of the ring at cloned metabotropic glutamate (mGlu) receptors expressed in Chinese hamster ovary (CHO) cells. In contrast to the parent compound ibotenic acid, which is a potent group I and II agonist, the (S)-forms of homoibotenic acid and its analogues are selective and potent group I antagonists whereas the (R)-forms are inactive both as agonists and antagonists at group I, II, and III mGlu receptors. Interestingly, (S)-homoibotenic acid and the analogues display equal potency at both mGlu1alpha and mGlu5a with Ki values in the range of 97 to 490 microM, (S)-homoibotenic acid and (S)-2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid [(S)-4-butylhomoibotenic acid] displaying the lowest and highest potency, respectively. The homoibotenic acid analogues thereby differ from mGlu receptor antagonists derived from phenylglycine such as (S)-4-carboxyphenylglycine which only antagonizes mGlu1alpha (Ki = 18 microM) showing no effect at mGlu5a (Ki > 300 microM).

Synthesis and pharmacology of N-alkylated derivatives of the excitotoxin ibotenic acid.

Three amino-alkylated derivatives of the naturally occurring excitatory amino acid (EAA) receptor agonist ibotenic acid (Ibo) have been synthesized and tested pharmacologically. N-Methyl-Ibo (1a) and N-ethyl-Ibo (1b) were shown to be agonists at NMDA receptors (EC50 = 140 and 320 microM, respectively), though with activities considerably lower than Ibo (EC50 = 9.6 microM). N-Benzyl-Ibo (1c) was inactive at ionotropic EAA receptors and all three compounds were, in contrast to Ibo, inactive at metabotropic EAA receptors. Molecular mechanics calculations have been performed on Ibo, 1a-c and the potent NMDA agonist 2-amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid (AMAA) in order to elucidate the observed structure-activity data.

4-Methylhomoibotenic acid activates a novel metabotropic glutamate receptor coupled to phosphoinositide hydrolysis.

Metabotropic glutamate receptors (mGluRs) are a family of glutamate receptors that are coupled to a variety of second messenger systems through GTP-binding proteins. Of the eight subtypes cloned to date, mGluR1 and mGluR5 are coupled to phosphoinositide hydrolysis in expression systems, and both are activated by the glutamate analogue 1-aminocyclopentane-1S,3R-dicarboxylic acid. Previously, we provided evidence that in rat cortical slices, 4-bromohomoibotenic acid (BrHI) and 4-methylhomoibotenic acid (MHI) activate a 1-aminocyclopentane-1S,3R-dicarboxylic acid-insensitive phosphoinositide hydrolysis-coupled mGluR. We further examine these compounds in expression systems. In a stable cell line expressing mGluR1a, BrHI is a weak partial agonist whereas MHI has no agonist activity. In Xenopus oocytes expressing mGluR1a or mGluR5a, BrHI is a weak agonist at mGluR5a whereas MHI is without effect on either receptor. Both BrHI and MHI have weak agonist activity at mGluRs 4a and 7a expressed in stable BHK cell lines whereas neither compound had any activity on BHK cells expressing mGluR2. Finally, we found that the novel mGluR antagonist LY341495 completely blocked the activation of mGluR1 and mGluR5 and blocked the phosphoinositide hydrolysis response to DHPG in rat cortical slices. In contrast, LY341495 did not block the phosphoinositide hydrolysis response to MHI in rat cortical slices. This provides further evidence that the phosphoinositide hydrolysis response to MHI in rat cortical slices is due to activation of a novel receptor that is distinct from the previously cloned mGluRs.

NMDA receptors mediate heat shock protein induction in the mouse brain following administration of the ibotenic acid analogue AMAA.

Expression of inducible heat shock protein-70 (HSP-70) and hsp-70 mRNA were studied in the adult mouse brain following systemic administration of the ibotenic acid analogue (+/-)-2-amino-3-hydroxy-5-methyl-4-isoxazoleacetic acid (AMAA), which is a potent N-methyl-D-aspartate (NMDA) agonist. At the dose of 20 mg/kg, AMAA produced excitatory behaviours in adult mice but overt convulsions were not seen. This treatment did not result in any detectable morphological brain damage at 4 days following administration. At 2.5 h and 5 h following treatment induction of hsp-70 mRNA expression was found in the pyramidal cell layers of CA1 and, to a lesser extent, CA3 fields of hippocampal Ammon's horn, amygdala, olfactory lobes, tenia tecta, hypothalamic nuclei and a superficial layer of cingulate, frontal and retrosplenial cortices. The presence of HSP-70 was detected by immunochemistry at 24 h following drug administration in those regions previously showing hsp-70 mRNA induction. AMAA-induced hsp-70 mRNA expression was prevented by pre-treatment with the non-competitive NMDA antagonist MK-801. These results suggest that NMDA receptors are involved in the stress response induced by AMAA.

Excitatory amino acid receptor ligands: asymmetric synthesis, absolute stereochemistry and pharmacology of (R)- and (S)-homoibotenic acid.

The (R)- and (S)-forms of 2-amino-3-(3-hydroxyisoxazol-5-yl)propionic acid (homoibotenic acid, HIBO) were synthesized, using (S)-BOC-phenylalanine as a chiral auxiliary and their absolute stereochemistry correlated with that of (R)-Br-HIBO. The enantiomeric excesses for (R)-HIBO (1) (> 99.5%) and (S)-HIBO (2) (99.5%) were determined using chiral HPLC. Whereas compounds 1 and 2 were equipotent inhibitors of the binding of [3H]glutamic acid in the presence of calcium chloride, 2 showed AMPA agonist activity and 1 very weak NMDA agonist activity.

Effects of bromohomoibotenate on metabotropic glutamate receptors.

(S)-Bromohomoibotenic acid [(S)-BrHIbo] stereoselectively antagonized glutamate-stimulated phosphoinositide (PI) hydrolysis in baby hamster kidney (BHK) cells expressing mGluR1a in a competitive manner with an IC50 of 250 microM. However, (S)-BrHIbo did not inhibit (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD]-induced PI hydrolysis in rat hippocampal slices (S)- or (R)-BrHIbo did not show any effects on forskolin-stimulated cAMP-formation in BHK cells expressing mGluR2 or mGluR4 but did displace [3H]2-amino-4-phosphonobutyrate ([3H]AP4) binding from rat corticalmembranes with high affinities (IC50 = 1.0 microM and 1.1 microM, respectively). These data suggest that (S)-BrHIbo may interest with multiple PI-coupled glutamate receptors, however, at concentrations that are several fold higher than for displacement of [3H]AP4 binding from rat cortical membranes.

4-Bromohomoibotenic acid selectively activates a 1-aminocyclopentane-1S,3R-dicarboxylic acid-insensitive metabotropic glutamate receptor coupled to phosphoinositide hydrolysis in rat cortical slices.

Glutamate activates a family of receptors, known as metabotropic glutamate receptors (mGluRs), that are coupled to various second messenger systems through G proteins. All mGluR subtypes characterized to date in rat brain slices are activated by the glutamate analogue 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD). However, few agonists are available that selectively activate specific mGluR subtypes. We report that the glutamate analogue (R,S)-4-bromohomoibotenate (BrHI) stimulates phosphoinositide hydrolysis in rat cerebral cortical slices in a concentration-dependent manner (EC50 = 190 microM). The response to BrHI is stereoselective and is not blocked by ionotropic glutamate receptor antagonists. It is interesting that the responses to BrHI and 1S,3R-ACPD are completely additive, suggesting that these responses are mediated by different receptor subtypes. Consistent with this, the response to BrHI is insensitive to L-2-amino-3-phosphonopropionic acid (L-AP3), whereas the response to 1S,3R-ACPD is partially blocked by L-AP3. BrHI does not activate metabotropic receptors coupled to changes in cyclic AMP accumulation or activation of phospholipase D. Thus, BrHI seems to activate specifically a phosphoinositide hydrolysis-linked mGluR that is insensitive to 1S,3R-ACPD. This compound may prove useful as a tool for elucidating the roles of different mGluR subtypes in mammalian brain.

Excitatory amino acid-induced slow biphasic responses of free intracellular calcium in human neuroblastoma cells.

Effects of an excitatory amino acid, glutamate, and of ionotropic and metabotropic glutamate receptor agonists on the levels of free intracellular calcium, and their specific receptor binding in human SH-SY5Y neuroblastoma cells were studied. The calcium response was always biphasic, except for AMPA, suggesting both stimulatory and inhibitory effects on free intracellular calcium upon glutamate receptor stimulation, both with ionotropic and metabotropic glutamate receptor agonists. Specific binding of glutamate and other glutamate receptor agonists, together with the biphasic calcium response, suggests that human SH-SY5Y neuroblastoma cells express both ionotropic and metabotropic glutamate receptors. These findings shed new light on the use of human SH-SY5Y neuroblastoma cells as a human neuronal tumor cell model.

Aspartate and glutamate mediate excitatory synaptic transmission in area CA1 of the hippocampus.

We examined whether L-aspartate (ASP) and L-glutamate (GLU) both function as endogenous neurotransmitters in area CA1 of the rat hippocampus. Radioligand displacement experiments using 3H-DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (3H-AMPA) to label AMPA/kainate receptors and 3H-cis-4-phosphonomethyl-2-piperidine carboxylic acid (3H-CGS-19755) to label NMDA receptors confirmed that GLU (Ki approximately 500 nM) but not ASP (Ki > 1 mM) has high affinity for AMPA/kainate receptors whereas GLU (Ki approximately 250 nM) and ASP (Ki approximately 1.3 microM) both have high affinity for NMDA receptors. Elevating extracellular potassium concentration (50 mM, 1 min) evoked the calcium-dependent release of both ASP (approximately 50% increase) and GLU (approximately 200% increase) from hippocampal slices and from minislices of area CA1. Reducing extracellular glucose concentration (0.2 mM) reduced GLU release, enhanced ASP release, and reduced AMPA/kainate receptor-mediated responses more than NMDA receptor-mediated responses (to 7% and 34% of control, respectively). Fiber volleys, antidromic population spikes, membrane potential, input resistance, and ATP content all were not affected by glucose reduction. Unlike low glucose, the inhibitory neuromodulator adenosine (5 microM), which reduces ASP and GLU release to a similar extent, reduced AMPA/kainate and NMDA receptor-mediated population EPSPs similarly (to 11% and 12% of control, respectively). AMPA/kainate and NMDA receptor-mediated population EPSPs were also similarly reduced by 0.4 microM TTX (to 32% and 22% of control, respectively) and similarly enhanced by 10 microM 4-aminopyridine (to 206% and 248% of control, respectively). Finally, NMDA receptor-mediated EPSCs measured by whole-cell recording decayed faster in low glucose (73 msec vs 54 msec) but not in adenosine (73 msec vs 78 msec). Together, these results confirm that ASP and GLU are both involved in excitatory synaptic transmission at the Schaffer collateral-commissural terminals in area CA1 of the rat hippocampus.

Differential modulation by cyclothiazide and concanavalin A of desensitization at native alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- and kainate-preferring glutamate receptors.

Concanavalin A, cyclothiazide, and aniracetam, ligands that modulate desensitization at glutamate receptors, were tested for their actions on responses at kainate-preferring receptors in dorsal root ganglion (DRG) neurons and at alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring receptors in hippocampal neurons. In DRG neurons concanavalin A blocked desensitization produced by either kainate or 5-chlorowillardiine and strongly potentiated the peak amplitude of responses to both agonists. However, in hippocampal neurons concanavalin A produced only weak potentiation of responses to kainate and 5-chlorowillardiine, and after treatment with lectin responses to 5-chlorowillardiine remained strongly desensitizing. In contrast, cyclothiazide completely blocked desensitization produced by 5-chlorowillardiine in hippocampal neurons and strongly potentiated responses to kainate; the action of aniracetam was similar but much weaker. In DRG neurons cyclothiazide and aniracetam had no effect on desensitization and instead produced weak inhibition of responses to kainate. The different sensitivities of native AMPA- and kainate-preferring glutamate receptors to cyclothiazide and concanavalin A should prove useful for the differentiation of glutamate receptor subtypes in other areas of the central nervous system.