Metabotropic glutamate 7 receptor subtype modulates motor symptoms in rodent models of Parkinson's disease.

Metabotropic glutamate (mGlu) receptors modulate synaptic transmission in the central nervous system and represent promising therapeutic targets for symptomatic treatment of Parkinson's disease (PD). Among the eight mGlu receptor subtypes, mGlu7 receptor is prominently expressed in the basal ganglia, but its role in restoring motor function in animal models of PD is not known. The effects of N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), the first selective allosteric activator of mGlu7 receptors, were thus tested in different rodent models of PD. Here, we show that oral (5 mg/kg) or intrastriatal administration (0.1 and 0.5 nmol) of AMN082 reverses haloperidol-induced catalepsy in rats. AMN082 (2.5 and 5 mg/kg) reduces apomorphine-induced rotations in unilateral 6-hydroxydopamine (6-OHDA)-lesioned rats. In a more complex task commonly used to evaluate major akinetic symptoms of PD patients, 5 mg/kg AMN082 reverses the increased reaction time to respond to a cue of bilateral 6-OHDA-lesioned rats. In addition, AMN082 reduces the duration of haloperidol-induced catalepsy in a mGlu7 receptor-dependent manner in wild-type but not mGlu7 receptor knockout mice. Higher doses of AMN082 (10 and 20 mg/kg p.o.) have no effect on the same models of PD. Overall these findings suggest that mGlu7 receptor activation can reverse motor dysfunction associated with reduced dopamine activity. Selective ligands of mGlu7 receptor subtypes may thus be considered as promising compounds for the development of antiparkinsonian therapeutic strategies.

mGluR7 facilitates extinction of aversive memories and controls amygdala plasticity.

Formation and extinction of aversive memories in the mammalian brain are insufficiently understood at the cellular and molecular levels. Using the novel metabotropic glutamate receptor 7 (mGluR7) agonist AMN082, we demonstrate that mGluR7 activation facilitates the extinction of aversive memories in two different amygdala-dependent tasks. Conversely, mGluR7 knockdown using short interfering RNA attenuated the extinction of learned aversion. mGluR7 activation also blocked the acquisition of Pavlovian fear learning and its electrophysiological correlate long-term potentiation in the amygdala. The finding that mGluR7 critically regulates extinction, in addition to acquisition of aversive memories, demonstrates that this receptor may be relevant for the manifestation and treatment of anxiety disorders.

Increased seizure susceptibility in mice lacking metabotropic glutamate receptor 7.

To study the role of mGlu7 receptors (mGluR7), we used homologous recombination to generate mice lacking this metabotropic receptor subtype (mGluR7(-/-)). After the serendipitous discovery of a sensory stimulus-evoked epileptic phenotype, we tested two convulsant drugs, pentylenetetrazole (PTZ) and bicuculline. In animals aged 12 weeks and older, subthreshold doses of these drugs induced seizures in mGluR7(-/-), but not in mGluR7(+/-), mice. PTZ-induced seizures were inhibited by three standard anticonvulsant drugs, but not by the group III selective mGluR agonist (R,S)-4-phosphonophenylglycine (PPG). Consistent with the lack of signs of epileptic activity in the absence of specific stimuli, mGluR7(-/-) mice showed no major changes in synaptic properties in two slice preparations. However, slightly increased excitability was evident in hippocampal slices. In addition, there was slower recovery from frequency facilitation in cortical slices, suggesting a role for mGluR7 as a frequency-dependent regulator in presynaptic terminals. Our findings suggest that mGluR7 receptors have a unique role in regulating neuronal excitability and that these receptors may be a novel target for the development of anticonvulsant drugs.

Metabotropic glutamate receptor subtypes as targets for neuroprotective drugs.

Metabotropic glutamate (mGlu) receptors have been considered as potential targets for neuroprotective drugs, but the lack of specific drugs has limited the development of neuroprotective strategies in experimental models of acute or chronic central nervous system (CNS) disorders. The advent of potent and centrally available subtype-selective ligands has overcome this limitation, leading to an extensive investigation of the role of mGlu receptor subtypes in neurodegeneration during the last 2 years. Examples of these drugs are the noncompetitive mGlu1 receptor antagonists, CPCCOEt and BAY-36-7620; the noncompetitive mGlu5 receptor antagonists, 2-methyl-6-(phenylethynyl)pyridine, SIB-1893, and SIB-1757; and the potent mGlu2/3 receptor agonists, LY354740 and LY379268. Pharmacologic blockade of mGlu1 or mGlu5 receptors or pharmacologic activation of mGlu2/3 or mGlu4/7/8 receptors produces neuroprotection in a variety of in vitro or in vivo models. MGlu1 receptor antagonists are promising drugs for the treatment of brain ischemia or for the prophylaxis of neuronal damage induced by synaptic hyperactivity. MGlu5 receptor antagonists may limit neuronal damage induced by a hyperactivity of N-methyl-d-aspartate (NMDA) receptors, because mGlu5 and NMDA receptors are physically and functionally connected in neuronal membranes. A series of observations suggest a potential application of mGlu5 receptor antagonists in chronic neurodegenerative disorders, such as amyotrophic lateral sclerosis and Alzheimer disease. MGlu2/3 receptor agonists inhibit glutamate release, but also promote the synthesis and release of neurotrophic factors in astrocytes. These drugs may therefore have a broad application as neuroprotective agents in a variety of CNS disorders. Finally, mGlu4/7/8 receptor agonists potently inhibit glutamate release and have a potential application in seizure disorders. The advantage of all these drugs with respect to NMDA or AMPA receptor agonists derives from the evidence that mGlu receptors do not "mediate," but rather "modulate" excitatory synaptic transmission. Therefore, it can be expected that mGlu receptor ligands are devoid of the undesirable effects resulting from the inhibition of excitatory synaptic transmission, such as sedation or an impairment of learning and memory.

Molecular cloning, functional expression and pharmacological characterization of the human metabotropic glutamate receptor type 4.

A cDNA encoding the human metabotropic glutamate receptor type 4 (hmGluR4) was isolated from human brain cDNA libraries by cross-hybridization with rat mGluR4 probes. The deduced amino acid sequence of human mGluR4 consists of 912 residues and shows a sequence identity of 96% to the amino acid sequence of rat mGluR4. Northern blot analyses indicate that hmGluR4 is strongly expressed in the cerebellum of the adult human brain but also at low levels in hippocampus, hypothalamus and thalamus. Stimulation of hmGluR4 with L-2-amino-4-phosphonobutyrate (L-AP4), L-serine-O-phosphate (L-SOP), L-glutamate or (1S,3R)-1-aminocyclo-pentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) in stably transfected Chinese hamster ovary (CHO) cells depressed forskolin-induced cAMP accumulation, whereas quisqualate (0.5 mM) was ineffective. The rank order of agonist potencies is: L-AP4 > L-SOP > L-glutamate > (1S,3R)-ACPD >> quisqualate. (R,S)-alpha-methyl-4-carboxyphenylglycine (1 mM), a reported antagonist at some mGluR subtypes, did not reduce the depression of forskolin-induced cAMP accumulation by L-AP4.

Pharmacological characterization of MCCG and MAP4 at the mGluR1b, mGluR2 and mGluR4a human metabotropic glutamate receptor subtypes.

The two reported metabotropic glutamate receptor (mGluR) antagonists, alpha-methyl-cyclopropyl glycine (MCCG) and alpha-methyl-aminophosphonobutyrate (MAP4) were tested on the mGluR1b, mGluR2 and mGluR4a subtypes of human mGluRs. Neither MCCG (500 microM) nor MAP4 (500 microM) antagonized the activation of mGluR1b by 10 microM quisqualate. MCCG was found to potently antagonize the action of 30 microM (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] at mGluR2 (IC50 = 87.5 microM; apparent Kd = 25 microM) but did not block the action of 1 microM S-2-amino-4-phosphonobutyric acid at mGluR4a (IC50 >> 1 mM). MAP4 was found to be a weak antagonist or partial agonist at mGluR4a (IC50 > 500 microM) and, less potently, also antagonized the action of 30 microM (1S,3R)-ACPD) at mGluR2 (IC50 approximately 2 mM).

A novel splice variant of a metabotropic glutamate receptor, human mGluR7b.

Two splice variants of the human metabotropic glutamate receptor 7, named hmGluR7a and hmGluR7b, were isolated from a human brain cDNA library. The isoforms differ by an out-of-frame insertion of 92 nucleotides close to the C-terminus of the hmGluR7 coding region, hmGluR7a has a length of 915 amino acids and represents the human homolog of the recently cloned rat mGluR7. hmGluR7b is seven amino acids longer and exhibits a novel C-terminus of 23 amino acids in length. RT-PCR analysis demonstrated the existence of mGluR7b transcripts in wild-type mouse brain and its absence in mGluR7 knockout mice. Northern blot analysis indicate that mGluR7 expression is developmentally regulated. It is expressed at high levels in human fetal brain and at a lower level in many regions of adult human brain. Stimulation of hmGluR7b with L-2-amino-4-phosphonobutyrate (L-AP4), L-serine-O-phosphate (L-SOP) or L-glutamate in stably transfected Chinese hamster ovary (CHO) cells depressed forskolin-induced cAMP accumulation, whereas (1S,3R)-1-aminocyclopentane-1,3,-dicarboxylic acid ((1S,3R)-ACPD) and quisqualate (both at 1mM) had no significant effects. As described for rat mGluR7, the rank order of agonist potencies is: L-SOP, L-AP4 > L-glutamate > (1S,3R)-ACPD, quisqualate.

Distinct influence of the group III metabotropic glutamate receptor agonist (R,S)-4-phosphonophenylglycine [(R,S)-PPG] on different forms of neuronal damage.

With this study we evaluated the influence of (R, S)-4-phosphonophenylglycine [(R,S)-PPG], a selective group III metabotropic glutamate receptor agonist, on excitotoxic, hypoxic/hypoglycaemic and ischaemic cerebral damage in rodents. Consistent with previous data showing neuroprotective and anticonvulsive effects (Gasparini, F., Bruno, V., Battaglia, G., Lukic, S., Leonhardt, T., Inderbitzin, W., et al., 1999. (R, S)-4-Phosphonophenylglycine, a potent and selective group III metabotropic glutamate receptor agonist, is anticonvulsive and neuroprotective in vivo. Journal of Pharmacology and Experimental Therapeutics 290, 1678-1687), we found pronounced neuroprotective effects with (R,S)-PPG (300 nmol) in a model of excitotoxicity, i.e. quinolinic acid-induced striatal lesions in rats. However, neither in focal cerebral ischaemia in mice nor in global cerebral ischaemia in gerbils or rats did (R,S)-PPG have any significant influence on the extent of neuronal damage. In a model of hypoxia/hypoglycaemia in acutely isolated hippocampal slices, however, (R,S)-PPG led to an improved recovery of population spike amplitude. As acutely isolated hippocampal slices are only viable for a few hours, these electrophysiological recordings can only be performed in a limited time window after the challenge-when most probably excitotoxicity is still the predominant influence in hypoxic pathophysiology. From this we conclude that group III mGluR agonists might be promising drugs against damage mediated mainly by excitotoxicity, but less likely against development of neuronal death due to ischaemia.

Selective blockade of metabotropic glutamate receptor subtype 5 is neuroprotective.

We have used potent and selective non-competitive antagonists of metabotropic glutamate receptor subtype 5 (mGlu5) -- 2-methyl-6-phenylethynylpyridine (MPEP), [6-methyl-2-(phenylazo)-3-pyridinol] (SIB-1757) and [(E)-2-methyl-6-(2-phenylethenyl)pyridine] (SIB-1893) - to examine whether endogenous activation of this particular metabotropic glutamate receptor subtype contributes to neuronal degeneration. In cortical cultures challenged with N-methyl-D-aspartate (NMDA), all three mGlu5 receptor antagonists were neuroprotective. The effect of MPEP was highly specific because the close analogue, 3-methyl-6-phenylethynylpyridine (iso-MPEP), which did not antagonize heterologously expressed mGlu5 receptors, was devoid of activity on NMDA toxicity. Neuroprotection by mGlu5 receptor antagonists was also observed in cortical cultures challenged with a toxic concentration of beta-amyloid peptide. We have also examined the effect of mGlu5 receptor antagonists in in vivo models of excitotoxic degeneration. MPEP and SIB-1893 were neuroprotective against neuronal damage induced by intrastriatal injection of NMDA or quinolinic acid. These results indicate that mGlu5 receptors represent a suitable target for novel neuroprotective agents of potential application in neurodegenerative disorders.

(-)-PHCCC, a positive allosteric modulator of mGluR4: characterization, mechanism of action, and neuroprotection.

Group-III metabotropic glutamate receptors (mGluR4, -6, -7, and -8) modulate neurotoxicity of excitatory amino acids and beta-amyloid-peptide (betaAP), as well as epileptic convulsions, most likely via presynaptic inhibition of glutamatergic neurotransmission. Due to the lack of subtype-selective ligands for group-III receptors, we previously utilized knock-out mice to identify mGluR4 as the primary receptor mediating neuroprotection of unselective group-III agonists such as L-AP(4) or (+)-PPG, whereas mGluR7 is critical for anticonvulsive effects. In a recent effort to find group-III subtype-selective drugs we identified (+/-)-PHCCC as a positive allosteric modulator for mGluR4. This compound increases agonist potency and markedly enhances maximum efficacy and, at higher concentrations, directly activates mGluR4 with low efficacy. All the activity of (+/-)-PHCCC resides in the (-)-enantiomer, which is inactive at mGluR2, -3, -5a, -6, -7b and -8a, but shows partial antagonist activity at mGluR1b (30% maximum antagonist efficacy). Chimeric receptor studies showed that the binding site of (-)-PHCCC is localized in the transmembrane region.Finally, (-)-PHCCC showed neuroprotection against betaAP- and NMDA-toxicity in mixed cultures of mouse cortical neurons. This neuroprotection was additive to that induced by the highly efficacious mGluR1 antagonist CPCCOEt and was blocked by MSOP, a group-III mGluR antagonist. Our data provide evidence for a novel pharmacological site on mGluR4, which may be used as a target-site for therapeutics.

Metabotropic glutamate receptor subtype 5 (mGlu5) and nociceptive function. I. Selective blockade of mGlu5 receptors in models of acute, persistent and chronic pain.

The excitatory neurotransmitter, glutamate, is particularly important in the transmission of pain information in the nervous system through the activation of ionotropic and metabotropic glutamate receptors. A potent, subtype-selective antagonist of the metabotropic glutamate-5 (mGlu5) receptor, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), has now been discovered that has effective anti-hyperalgesic effects in models of inflammatory pain. MPEP did not affect rotarod locomotor performance, or normal responses to noxious mechanical or thermal stimulation in naïve rats. However, in models of inflammatory pain, systemic administration of MPEP produced effective reversal of mechanical hyperalgesia without affecting inflammatory oedema. In contrast to the non-steroidal anti-inflammatory drugs, indomethacin and diclofenac, the maximal anti-hyperalgesic effects of orally administered MPEP were observed without acute erosion of the gastric mucosa. In contrast to its effects in models of inflammatory pain, MPEP did not produce significant reversal of mechanical hyperalgesia in a rat model of neuropathic pain.

2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist.

In the present paper we describe 2-methyl-6-(phenylethynyl)-pyridine (MPEP) as a potent, selective and systemically active antagonist for the metabotropic glutamate receptor subtype 5 (mGlu5). At the human mGlu5a receptor expressed in recombinant cells, MPEP completely inhibited quisqualate-stimulated phosphoinositide (PI) hydrolysis with an IC50 value of 36 nM while having no agonist or antagonist activities at cells expressing the human mGlu1b receptor at concentrations up to 30 microM. When tested at group II and III receptors, MPEP did not show agonist or antagonist activity at 100 microM on human mGlu2, -3, -4a, -7b, and -8a receptors nor at 10 microM on the human mGlu6 receptor. Electrophysiological recordings in Xenopus laevis oocytes demonstrated no significant effect at 100 microM on human NMDA (NMDA1A/2A), rat AMPA (Glu3-(flop)) and human kainate (Glu6-(IYQ)) receptor subtypes nor at 10 microM on the human NMDA1A/2B receptor. In rat neonatal brain slices, MPEP inhibited DHPG-stimulated PI hydrolysis with a potency and selectivity similar to that observed on human mGlu receptors. Furthermore, in extracellular recordings in the CA1 area of the hippocampus in anesthetized rats, the microiontophoretic application of DHPG induced neuronal firing that was blocked when MPEP was administered by iontophoretic or intravenous routes. Excitations induced by microiontophoretic application of AMPA were not affected.

Chiral resolution, pharmacological characterization, and receptor docking of the noncompetitive mGlu1 receptor antagonist (+/-)-2-hydroxyimino- 1a, 2-dihydro-1H-7-oxacyclopropa[b]naphthalene-7a-carboxylic acid ethyl ester.

Racemic CPCCOEt ((1aRS,7aRS)-2-hydroxyimino-1a, 2-dihydro-1H-7-oxacyclopropa[b]naphthalene-7a-carboxylic acid ethyl ester, (+/-)-1) derivatives have been shown to be subtype-selective metabotropic glutamate (mGlu) 1 receptor antagonists (Annoura et al. Bioorg. Med. Chem. Lett. 1996, 6, 763-766). The optical isomers of (+/-)-1 have been separated by chromatography on a chiral stationary phase. The absolute configuration at the C-1a and C-7a positions was determined using X-ray crystallography of an amide derivative with the methyl ester of L-phenylalanine (L-PheOMe) ((+)-6). In a phosphoinositol (PI) turnover assay at the cloned human mGlu1b receptor, (-)-1 and the new amide derivatives (-)-5 and (-)-6, all of which have (1aS,7aS)-stereochemistry on the chromane ring system, showed IC(50) values of 1.5, 0.43, and 0.93 microM, respectively. In contrast, (+)-1 and the new amide derivatives (+)-5 and (+)-6were found to be inactive up to a concentration of 30 microM indicating a selectivity for the (-)-enantiomers of at least 70-fold. In a previous study (Litschig et al. Mol. Pharmacol. 1999, 55, 453-461) we demonstrated using site-directed mutagenesis that the interaction site of (+/-)-1 is located in the transmembrane (TM) domain of hmGlu1b. To suggest a plausible binding mode of (-)-1, we have built a molecular mechanics model of the putative seven TM domain of hmGlu1 based on the alpha-carbon template of the TM helices of rhodopsin. A receptor docking hypothesis suggests that the OH of T815 (TMVII) comes in close contact with the oxime OH of (-)-1 and (-)-5, whereas no such close interactions could be demonstrated by docking of (+)-1.

The agonist selectivity of a class III metabotropic glutamate receptor, human mGluR4a, is determined by the N-terminal extracellular domain.

To test the hypothesis that the determinants for agonist selectivity of class III metabotropic glutamate receptors (mGluRs) are localized in the N-terminal extracellular domain, a chimaeric cDNA was constructed where 519 amino acids of the N-terminal extracellular domain of human mGluR1b were exchanged with the corresponding region of human mGluR4. The pharmacological profile of the chimaera, designated hmGlu(R4)1-519/1b, was analysed by recordings of intracellular calcium concentration ([Ca2+]i) in transiently transfected HEK 293 cells and compared with that of human mGluR1b and human mGluR4a stably expressed in Chinese hamster ovary cells. Application of 100 microM L-2-amino-4-phosphonobutyrate (L-AP4), a class III mGluR-specific agonist, induced a rise in [Ca2+]i in hmGlu(R4)1-519/1b but not in hmGluR1b expressing cells. In contrast, application of quisqualate (100 microM) induced a rise in [Ca2+]i at hmGluR1b but not at hmGlu(R4)1-519/1b. Dose-response analysis with L-AP4 and L-glutamate at hmGlu(R4)1-519/1b revealed a half-maximal effect (EC50) of 16.0 microM and 196 microM, respectively. The EC50 values for quisqualate, glutamate and (1S,3R)-ACPD at hmGluR1b were 10.25 microM, 225 microM and 3060 microM, respectively. The rank order of agonist potency of hmGlu(R4)1-519/1b corresponds to that of hmGluR4 (L-AP4 > L-glutamate > (1S,3R)-ACPD > quisqualate) but is different from that of hmGluR1b (quisqualate > glutamate >> (1S,3R)-ACPD).

Profiling of trans-azetidine-2,4-dicarboxylic acid at the human metabotropic glutamate receptors mGlu1b, -2, -4a and -5a.

We have tested the two enantiomers of trans-azetidine-2,4-dicarboxylic acid, (2S,4S)-azetidine-2,4-dicarboxylic acid ((2S,4S)-ADA) and (2R,4R)-azetidine-2,4-dicarboxylic acid ((2R,4R)-ADA) for activity at the human metabotropic glutamate receptors mGlu1b, mGlu2, mGlu4a and mGlu5a expressed in mammalian cells. In Chinese hamster ovary (CHO) cells expressing human mGlu2 receptors, 500 microM (2S,4S)-ADA inhibited forskolin-stimulated cAMP accumulation by 33 +/- 3% while 100 microM (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid induced an inhibition by 66 +/- 5%. The (2R,4R)-ADA enantiomer was inactive at human mGlu2 receptors. In CHO cells expressing human mGlu4a receptors, 10 microM L-AP4 inhibited forskolin-stimulated cAMP levels by 37 +/- 4% whereas both ADA enantiomers of trans-azetidine-2,4-dicarboxylic acid (500 microM) had no such effect. In CHO cells expressing human mGlu1b receptors and L cells expressing human mGlu5a receptors, both enantiomers, applied at 500 microM or 1 mM, were ineffective in stimulating inositolmonophosphate accumulation and did not affect quisqualate-stimulated inositolmonophosphate accumulation. We conclude that (2S,4S)-azetidine-2,4-dicarboxylic acid is a weak human mGlu2 receptor agonist and that (2R,4R)-azetidine-2,4-dicarboxylic acid is inactive at human mGlu2 receptors. Trans-azetidine-2,4-dicarboxylic acid has no significant agonistic effect on human mGlu4a receptors and neither agonistic nor antagonistic effects on human mGlu1b and mGlu5a receptors.

Discovery and characterization of non-competitive antagonists of group I metabotropic glutamate receptors.

We have investigated the mechanism of inhibition of the new group I mGluR antagonists CPCCOEt and MPEP and determined that both compounds have a non-competitive mode of inhibition. Furthermore using chimeric/mutated receptors constructs we have found that these antagonists act at a novel pharmacological site located in the trans-membrane (TM). Specific non-conserved amino acid residues in the TM domain have been identified which are necessary for the inhibition by CPCCOEt and MPEP of the mGlul and mGlu5 receptors, respectively. Using molecular modeling a model of the TM domain was built for both mGlu1 and mGlu5 receptor subtypes. Docking of CPCCOEt and MPEP into their respective model allowed the modelisation of the novel binding site.

Functional role of mGluR1 and mGluR4 in pilocarpine-induced temporal lobe epilepsy.

Altered expression and distribution of neurotransmitter receptors, including metabotropic glutamate receptors (mGluRs), constitute key aspects in epileptogenesis, impaired hippocampal excitability and neuronal degeneration. mGluR1 mediates predominantly excitatory effects, whereas mGluR4 acts as inhibitory presynaptic receptor. Increased hippocampal expression of mGluR1 and mGluR4 has been observed in human temporal lobe epilepsy (TLE). In this study, we address whether genetic mGluR1 upregulation and mGluR4 knock-down influence seizure susceptibility and/or vulnerability of hippocampal neurons by analyzing transgenic animals in the pilocarpine TLE model. Therefore, we generated transgenic mice expressing mGluR1-enhanced green fluorescent protein (EGFP) fusion protein under control of the human cytomegalovirus (CMV) immediate early promoter. Status epilepticus (SE) was induced in (a) mice overexpressing mGluR1-EGFP and (b) mice deficient for mGluR4 (mGluR4 KO) as well as littermate controls. In the acute epileptic stage after pilocarpine application, mGluR4 KO mice showed a significant increase of severe seizure activity, in contrast to mGluR1 transgenics. Analysis of both transgenic mouse lines in the chronic epileptic phase, using a telemetric EEG-/video-monitoring system, revealed a significant increase in seizure frequency only in mGluR1-EGFP mice. In contrast, enhanced neuronal cell loss was only present in the hippocampus of epileptic mGluR4 KO mice. Our results suggest a role for mGluR1 in promoting seizure susceptibility as well as for mGluR4 to counteract excitatory activity and seizure-associated vulnerability of hippocampal neurons. Therefore, our data strongly recommend both mGluRs as potential drug targets to interfere with the development of hippocampal damage and seizure activity in TLE.

A conserved base pair within helix P4 of the Tetrahymena ribozyme helps to form the tertiary structure required for self-splicing.

Site-specific mutagenesis of the self-splicing Tetrahymena intron has been used to investigate the function of C109-G212, a conserved base pair in the P4 stem of group I introns. Mutation of C109 to G affects splicing only slightly, whereas mutation of G212 to A or C reduces the rate of splicing substantially (500-fold reduction in kcat/Km under standard in vitro splicing conditions for the G212C mutant). Splicing activity of the compensatory double mutant (C109G:G212C) is intermediate between those of the two single mutants. Thus, the stability of the P4 stem as well as the identity of the base at position 212 are important for self-splicing. Single and double mutants containing the G212C substitution have a decreased temperature optimum for self-splicing and are partially Mg2+ suppressible, both indicative of structural destabilization. Chemical structure mapping indicates that the mutations do not redirect the global folding of the RNA, but affect the structure locally and at one other site (A183) that is distant in the secondary structure. We propose that, in addition to its pairing in P4, G212 is involved in a base triplet or an alternate base pair that contributes to the catalytically active tertiary structure of the ribozyme.

(+)-4-phosphonophenylglycine (PPG) a new group III selective metabotropic glutamate receptor agonist.

A new synthesis of (R,S)-PPG (4-phosphonophenylglycine) and the separation of the protected enantiomers leading after deprotection to (+)- and (-)-PPG are described. Pharmacological characterization at the group III metabotropic glutamate receptors hmGluR4a and hmGluR7b revealed (+)-PPG as the active enantiomer.

Synthesis, molecular modeling and preliminary biological evaluation of 1-amino-3-phosphono-3-cyclopentene-1-carboxylic acid and 1-amino-3-phosphono-2-cyclopentene-1-carboxylic acid, two novel agonists of metabotropic glutamate receptors of group III.

On the basis of a pharmacophore definition of mGlu4 agonists, the two novel semi-rigid derivatives 12 and 13 were designed and synthesized. The preliminary biological evaluation demonstrated that both compounds interact with hmGlu4a, while ineffective at group II receptor subtypes. In particular, derivative 13 is a full hmGlu4a agonist with an EC50 = 17 microM.