Inhibition of follicle-stimulating hormone-induced preovulatory follicles in rats treated with a nonsteroidal negative allosteric modulator of follicle-stimulating hormone receptor.

We previously described a negative allosteric modulator (NAM) of FSHR (ADX61623) that blocked FSH-induced cAMP and progesterone production but did not block estradiol production. That FSHR NAM did not affect FSH-induced preovulatory follicle development as evidenced by the lack of an effect on the number of FSH-dependent oocytes found in the ampullae following ovulation with hCG. A goal is the development of a nonsteroidal contraceptive. Toward this end, a high-throughput screen using human FSHR identified an additional nonsteroidal small molecule (ADX68692). Although ADX68692 behaved like ADX61623 in inhibiting production of cAMP and progesterone, it also inhibited FSH-induced estradiol in an in vitro rat granulosa primary cell culture bioassay. When immature, noncycling female rats were injected subcutaneously or by oral dosing prior to exogenous FSH administration, it was found that ADX68692 decreased the number of oocytes recovered from the ampullae. The estrous cycles of mature female rats were disrupted by administration by oral gavage of 25 mg/kg and 10 mg/kg ADX68692. In the highest dose tested (25 mg/kg), 55% of animals cohabited with mature males had implantation sites compared to 33% in the 10 mg/kg group and 77% in the control group. A surprising finding was that a structural analog ADX68693, while effectively blocking progesterone production with similar efficacy as ADX68692, did not block estrogen production and despite better oral availability did not decrease the number of oocytes found in the ampullae even when used at 100 mg/kg. These data demonstrate that because of biased antagonism of the FSHR, nonsteroidal contraception requires that both arms of the FSHR steroidogenic pathway must be effectively blocked, particularly estrogen biosynthesis. Thus, a corollary to these findings is that it seems reasonable to propose that the estrogen-dependent diseases such as endometriosis may benefit from inhibition of FSH action at the ovary using the FSHR NAM approach.

The mGluR5 negative allosteric modulator dipraglurant reduces dyskinesia in the MPTP macaque model.

BACKGROUND: Blocking metabotropic glutamate receptor type 5 (mGluR5) has been proposed as a target for levodopa-induced dyskinesias (LID) in Parkinson's disease (PD). We assessed the effect on LID of dipraglurant, a potent selective mGluR5 receptor negative allosteric modulator in the gold-standard LID macaque model. METHODS: Dipraglurant (3, 10, and 30 mg/kg, by mouth) was tested in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) macaque model of LID in a four-way crossover, single-dose, controlled study (n = 8). RESULTS: Dipraglurant inhibited dyskinesias in the LID macaque model, with best effect reached at 30 mg/kg dose with no alteration of levodopa efficacy. CONCLUSION: Acute challenges of dipraglurant were efficacious on choreic and dystonic LID in the MPTP-macaque model. Dipraglurant pharmacokinetic variables were similar to those of levodopa, suggesting that both drugs can be co-administered simultaneously in further studies.

ADX71743, a potent and selective negative allosteric modulator of metabotropic glutamate receptor 7: in vitro and in vivo characterization.

Metabotropic glutamate receptor 7 (mGlu(7)) has been suggested to be a promising novel target for treatment of a range of disorders, including anxiety, post-traumatic stress disorder, depression, drug abuse, and schizophrenia. Here we characterized a potent and selective mGlu(7) negative allosteric modulator (NAM) (+)-6-(2,4-dimethylphenyl)-2-ethyl-6,7-dihydrobenzo[d]oxazol-4(5H)-one (ADX71743). In vitro, Schild plot analysis and reversibility tests at the target confirmed the NAM properties of the compound and attenuation of L-(+)-2-amino-4-phosphonobutyric acid-induced synaptic depression confirmed activity at the native receptor. The pharmacokinetic analysis of ADX71743 in mice and rats revealed that it is bioavailable after s.c. administration and is brain penetrant (cerebrospinal fluid concentration/total plasma concentration ratio at C(max) = 5.3%). In vivo, ADX71743 (50, 100, 150 mg/kg, s.c.) caused no impairment of locomotor activity in rats and mice or activity on rotarod in mice. ADX71743 had an anxiolytic-like profile in the marble burying and elevated plus maze tests, dose-dependently reducing the number of buried marbles and increasing open arm exploration, respectively. Whereas ADX71743 caused a small reduction in amphetamine-induced hyperactivity in mice, it was inactive in the mouse 2,5-dimethoxy-4-iodoamphetamine-induced head twitch and the rat conditioned avoidance response tests. In addition, the compound was inactive in the mouse forced swim test. These data suggest that ADX71743 is a suitable compound to help unravel the physiologic role of mGlu(7) and to better understand its implication in central nervous system diseases. Our in vivo tests using ADX71743, reported here, suggest that pharmacological inhibition of mGlu(7) is a valid approach for developing novel pharmacotherapies to treat anxiety disorders, but may not be suitable for treatment of depression or psychosis.

Effect of the Metabotropic Glutamate Receptor Type 5 Negative Allosteric Modulator Dipraglurant on Motor and Non-Motor Symptoms of Parkinson's Disease.

Parkinson's disease (PD) patients suffer not only from the primary motor symptoms of the disease but also from a range of non-motor symptoms (NMS) that cause disability and low quality of life. Excessive glutamate activity in the basal ganglia resulting from degeneration of the nigrostriatal dopamine pathway has been implicated in the motor symptoms, NMS and dyskinesias in PD patients. In this study, we investigated the effects of a selective mGlu5 negative allosteric modulator (NAM), dipraglurant, in a rodent motor symptoms model of PD, but also in models of anxiety, depression and obsessive-compulsive disorder, all of which are among the most prevalent NMS symptoms. Dipraglurant is rapidly absorbed after oral administration, readily crosses the blood-brain barrier, and exhibits a high correlation between plasma concentration and efficacy in behavioral models. In vivo, dipraglurant dose-dependently reduced haloperidol-induced catalepsy, increased punished licks in the Vogel conflict-drinking model, decreased immobility time in the forced swim test, decreased the number of buried marbles in the marble-burying test, but had no effect on rotarod performance or locomotor activity. These findings suggest that dipraglurant may have benefits to address some of the highly problematic comorbid non-motor symptoms of PD, in addition to its antidyskinetic effect demonstrated in PD-LID patients.

Lateral allosterism in the glucagon receptor family: glucagon-like peptide 1 induces G-protein-coupled receptor heteromer formation.

Activation of G-protein-coupled receptors (GPCRs) results in a variety of cellular responses, such as binding to the same receptor of different ligands that activate distinct downstream cascades. Additional signaling complexity is achieved when two or more receptors are integrated into one signaling unit. Lateral receptor interactions can allosterically modulate the receptor response to a ligand, which creates a mechanism for tissue-specific fine tuning, depending on the cellular receptor coexpression pattern. GPCR homomers or heteromers have been explored widely for GPCR classes A and C but to lesser extent for class B. In the present study, we used bioluminescence resonance energy transfer (BRET) techniques, calcium flux measurements, and microscopy to study receptor interactions within the glucagon receptor family. We found basal BRET interactions for some of the receptor combinations tested that decreased upon ligand binding. A BRET increase was observed exclusively for the gastric inhibitory peptide (GIP) receptor and the glucagon-like peptide 1 (GLP-1) receptor upon binding of GLP-1 that could be reversed with GIP addition. The interactions of GLP-1 receptor and GIP receptor were characterized with BRET donor saturation studies, shift experiments, and tests of glucagon-like ligands. The heteromer displayed specific pharmacological characteristics with respect to GLP-1-induced ß-arrestin recruitment and calcium flux, which suggests a form of allosteric regulation between the receptors. This study provides the first example of ligand-induced heteromer formation in GPCR class B. In the body, the receptors are functionally related and coexpressed in the same cells. The physiological evidence for this heteromerization remains to be determined.

Fluorinated 9H-xanthene-9-carboxylic acid oxazol-2-yl-amides as potent, orally available mGlu1 receptor enhancers.

Small molecule mGluR1 enhancers, which are 9H-xanthene-9-carboxylic acid [1,2,4]oxadiazol-3-yl- and (2H-tetrazol-5-yl)-amides, have been previously reported. Fluorinated 9H-xanthene-9-carboxylic acid oxazol-2-yl-amides with improved pharmacokinetic properties have been designed and synthesized as useful pharmacological tools for the study of the physiological roles mediated by mGlu1 receptors. The synthesis and the structure-activity relationship of this class of positive allosteric modulators of mGlu1 receptors will be discussed in detail.

Synthesis and characterization of 8-ethynyl-1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives: part 2. New potent non-competitive metabotropic glutamate receptor 2/3 antagonists.

A series of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives was evaluated as non-competitive mGluR2/3 antagonists. Replacement of a cyano group by a five-membered heterocycle produced compounds inhibiting the binding of [(3)H]-LY354740 to rat mGluR2 with low nanomolar affinity and consistent functional effect at both mGluR2 and mGluR3. Further modification to improve the physicochemical properties led eventually to compounds with the ability to reverse LY354740-mediated inhibition of field excitatory postsynaptic potentials in the rat dentate gyrus.

Synthesis and characterization of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives: Part 3. New potent non-competitive metabotropic glutamate receptor 2/3 antagonists.

A series of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives was evaluated as non-competitive mGluR2/3 antagonists. Replacement of the (2-aryl)-ethynyl-moiety in 8-position with smaller less lipophilic substituents produced compounds inhibiting the binding of [3H]-LY354740 to rat mGluR2 with low nanomolar affinity and consistent functional effect at both mGluR2 and mGluR3. These compounds were able to reverse LY354740-mediated inhibition of field excitatory postsynaptic potentials in the rat dentate gyrus and in vivo activity could be demonstrated by reversal of the LY354740-induced hypoactivity in mice after oral administration.

Cellular prion protein signaling in serotonergic neuronal cells.

The cellular prion protein PrP(C) is the normal counterpart of the scrapie prion protein PrP(Sc), the main component of the infectious agent of transmissible spongiform encephalopathies (TSEs). It is a ubiquitous cell-surface glycoprotein, abundantly expressed in neurons, which constitute the targets of TSE pathogenesis. Taking advantage of the 1C11 neuroectodermal cell line, endowed with the capacity to convert into 1C11(5-HT) serotonergic or 1C11(NE) noradrenergic neuronal cells, allowed us to ascribe a signaling function to PrP(C). Antibody-mediated ligation of PrP(C) recruits transduction pathways, which involve nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent reactive oxygen species production and target the extracellular-regulated kinases ERK1/2. In fully differentiated cells only, these effectors are under the control of a PrP(C)-caveolin-Fyn platform, located on neuritic extensions. In addition to its proper signaling activity, PrP(C) modulates the agonist-induced response of the three serotonergic G protein-coupled receptors present on the 1C11(5-HT) differentiated cells. The impact of PrP(C) ligation on the receptor couplings depends on the receptor subtype and the pathway considered. The implementation of the PrP(C)-caveolin complex again is mandatory for PrP(C) to exert its action on 5-HT receptor signaling. Our current data argue that PrP(C) interferes with the intensities and/or dynamics of G protein activation by agonist-bound 5-HT receptors. By mobilizing transduction cascades controlling the cellular redox state and the ERK1/2 kinases and by altering 5-HT receptor-mediated intracellular response, PrP(C) takes part in the homeostasis of serotonergic neuronal cells. These findings may have implications for future research aiming at understanding the fate of serotonergic neurons in prion diseases.

Synthesis and characterization of 8-ethynyl-1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives: new potent non-competitive metabotropic glutamate receptor 2/3 antagonists. Part 1.

A series of 1,3-dihydrobenzo[b][1,4]diazepin-2-one derivatives was evaluated as non-competitive mGluR2/3 antagonists. Attachment of an 8-(2-aryl)-ethynyl-moiety produced compounds inhibiting the binding of [(3)H]-LY354740 to rat mGluR2 with low nanomolar affinity and consistent functional effect at both mGluR2 and mGluR3.

The drug candidate, ADX71441, is a novel, potent and selective positive allosteric modulator of the GABAB receptor with a potential for treatment of anxiety, pain and spasticity.

Positive allosteric modulation of the GABAB receptor is a promising alternative to direct activation of the receptor as a therapeutic approach for treatment of addiction, chronic pain, anxiety, epilepsy, autism, Fragile X syndrome, and psychosis. Here we describe in vitro and in vivo characterization of a novel, potent and selective GABAB positive allosteric modulator (PAM) N-(5-(4-(4-chloro-3-fluorobenzyl)-6-methoxy-3,5-dioxo-4,5-dihydro-1,2,4-triazin-2(3H)-yl)-2-fluorophenyl)acetamide (ADX71441). In vitro, Schild plot and reversibility tests at the target confirmed PAM properties of the compound. In mice and rats ADX71441 is bioavailable after oral administration and is brain penetrant. A single dose of ADX71441 had an anxiolytic-like profile in the mouse marble burying test (minimum effective dose; MED 3 mg/kg) as well as in the elevated plus maze test in mice and rats (both MED 3 mg/kg). Also, in mice, acute administration of ADX71441 reduced visceral pain-associated behaviors in the acetic acid-induced writhing test. ADX71441 dose-dependently reduced time on rotarod in rats (MED 10 mg/kg) indicative of muscle-relaxant qualities. ADX71441 reduced locomotor activity in mice (10 mg/kg) and rats (3 mg/kg) after single dose; however, following sub-chronic administration in mice, 30 mg/kg ADX71441 was associated with normal locomotor activity. While acute administration of ADX71441 reduced body temperature in rats and mice (both MED 10 mg/kg), the effect in the former was transient, rapidly returning to normal levels despite high concentrations of the compound remaining in plasma. Thus, the GABAB PAM ADX71441 represents a valid therapeutic approach for development of novel treatment of anxiety, pain and spasticity.

Control of bioamine metabolism by 5-HT2B and alpha 1D autoreceptors through reactive oxygen species and tumor necrosis factor-alpha signaling in neuronal cells.

Homeostasis of the central nervous system relies on the proper integration of cell-signaling pathways recruited by a variety of neuronal and non-neuronal factors, with the aim of tightly controlling neurotransmitter metabolism, storage, and transport. We took advantage of the 1C11 neuroectodermal cell line, endowed with the capacity to selectively differentiate into serotonergic (1C11(5-HT)) or noradrenergic (1C11(NE)) neurons, to identify functional targets of serotonin (5-hydroxytryptamine [5-HT]) and norepinephrine (NE) autoreceptors possibly involved in the control of neuronal functions. We demonstrate that 5-HT(2B) and adreno alpha(1D) receptors are coupled to reactive oxygen species (ROS) production through NADPH oxidase activation in 1C11(5-HT) and 1C11(NE) neuronal cells, respectively. In the signaling cascade linking 5-HT(2B) receptors to NADPH oxidase, phospholipase A2-mediated arachidonic acid production is required for ROS synthesis. ROS, in turn, act as second message signals and control the activation of TACE (TNF-alpha converting enzyme), a member of a disintegrin and metalloproteinase family. 5-HT(2B) and alpha(1D) receptor stimulation triggers TACE-dependent TNF-alpha shedding in the surrounding milieu of 1C11(5-HT) and 1C11(NE) cells. In these cells, shed TNF-alpha triggers degradation of 5-HT and NE into 5-HIAA and MHPG, respectively. Finally, we observe that 5-HT(2B) and alpha(1D) receptor couplings to the NADPH oxidase-TACE cascade are strictly restricted to 1C11-derived progenies that have implemented a complete neuronal phenotype. Altogether, our data indicate that couplings of 5-HT(2B) and alpha(1D) autoreceptors to ROS and TNF-alpha signaling control neurotransmitter metabolism in 1C11-derived neuronal cells. Eventually, we might explain the origin of oxidative stress and high level of TNF-alpha in neurodegenerative diseases as a consequence of deviation of normal signaling pathways coupled to neurotransmitters.

Reactive oxygen species-dependent TNF-alpha converting enzyme activation through stimulation of 5-HT2B and alpha1D autoreceptors in neuronal cells.

A major determinant of neuronal homeostasis is the proper integration of cell signaling pathways recruited by a variety of neuronal and non-neuronal factors. By taking advantage of a neuroectodermal cell line (1C11) endowed with the capacity to differentiate into serotonergic (1C115-HT) or noradrenergic (1C11NE) neurons, we identified serotonin (5-hydroxytryptamine, 5-HT)- and norepinephrine (NE)-dependent signaling cascades possibly involved in neuronal functions. First, we establish that 5-HT2B receptors and 1D adrenoceptors are functionally coupled to reactive oxygen species (ROS) synthesis through NADPH oxidase activation in 1C115-HT and 1C11NE cells. This observation constitutes the prime evidence that bioaminergic autoreceptors take part in the control of the cellular redox equilibrium in a neuronal context. Second, our data identify TACE (TNF- Converting Enzyme), a member of a disintegrin and metalloproteinase (ADAM) family, as a downstream target of the 5-HT2B and 1D receptor-NADPH oxidase signaling pathways. Upon 5-HT2B or 1D receptor stimulation, ROS fully govern TNF- shedding in the surrounding milieu of 1C115-HT or 1C11NE cells. Third, 5-HT2B and 1Dreceptor couplings to the NADPH oxidase-TACE cascade are strictly restricted to 1C11-derived progenies that have implemented a complete serotonergic or noradrenergic phenotype. Overall, these observations suggest that 5-HT2B and 1D autoreceptors may play a role in the maintenance of neuron- and neurotransmitter-associated functions. Eventually, our study may have implications regarding the origin of oxidative stress as well as up-regulated expression of proinflammatory cytokines in neurodegenerative disorders, which may relate to the deviation of normal signaling pathways.

Severe impairment of NMDA receptor function in mice carrying targeted point mutations in the glycine binding site results in drug-resistant nonhabituating hyperactivity.

NMDA receptor hypofunction has been implicated in the pathophysiology of schizophrenia, and pharmacological and genetic approaches have been used to model such dysfunction. We previously have described two mouse lines carrying point mutations in the NMDA receptor glycine binding site, Grin1(D481N) and Grin1(K483Q), which exhibit 5- and 86-fold reductions in receptor glycine affinity, respectively. Grin1(D481N) animals exhibit a relatively mild phenotype compatible with a moderate reduction in NMDA receptor function, whereas Grin1(K483Q) animals die shortly after birth. In this study we have characterized compound heterozygote Grin1(D481N/K483Q) mice, which are viable and exhibited biphasic NMDA receptor glycine affinities compatible with the presence of each of the two mutated alleles. Grin1(D481N/K483Q) mice exhibited a marked NMDA receptor hypofunction revealed by deficits in hippocampal long-term potentiation, which were rescued by the glycine site agonist d-serine, which also facilitated NMDA synaptic currents in mutant, but not in wild-type, mice. Analysis of striatal monoamine levels revealed an apparent dopaminergic and serotonergic hyperfunction. Behaviorally, Grin1(D481N/K483Q) mice were insensitive to acute dizocilpine pretreatment and exhibited increased startle response but normal prepulse inhibition. Most strikingly, mutant mice exhibited a sustained, nonhabituating hyperactivity and increased stereotyped behavior that were resistant to suppression by antipsychotics and the benzodiazepine site agonist Zolpidem. They also displayed a disruption of nest building behavior and were unable to perform a cued learning paradigm in the Morris water maze. We speculate that the severity of NMDA receptor hypofunction in these mice may account for their profound behavioral phenotype and insensitivity to antipsychotics.

Distribution and abundance of metabotropic glutamate receptor subtype 2 in rat brain revealed by [3H]LY354740 binding in vitro and quantitative radioautography: correlation with the sites of synthesis, expression, and agonist stimulation of [35S]GTPgammas binding.

Until recently, there was a lack of selective radioligands for the subtypes of metabotropic glutamate (mGlu) receptors. [(3)H]LY354740 ((+)-2-aminobicyclo[3,1,0]hexane-2,6-dicarboxylic acid), a selective agonist for group II receptors (mGlu2 and -3, which are negatively coupled to cAMP production), has now been used to map their brain distribution and abundance by in vitro binding and quantitative radioautography. The selective cation dependence of its binding allowed the discrimination between mGlu2 and mGlu3 receptor labeling. Thus, in the presence of Ca(2+) and Mg(2+) ions, the agonist bound selectively to mGlu2 receptors as evidenced by: 1) the correlative distribution and abundance of binding sites (highest in the lacunosum moleculare of the hippocampus and lowest in white matter) with mGlu2 receptor mRNA and protein revealed by in situ hybridization histochemistry and immunohistochemistry, respectively; 2) its selective pharmacology; and 3) the distribution of LY354740-stimulated [(35)S]GTPgammaS binding (25-97% above basal, according to the brain region), revealing G protein-coupled receptor coupling to G(i) proteins. Nonspecific binding (in the presence of 10 muM DCG-IV, a group II-selective, mGlu2-preferring, receptor agonist) was <10% of total. In adjacent sections, the distribution of binding sites for [(3)H]DCG-IV was very similar. This extensive study paves the way for investigations of the regional expression and regulation of mGlu2 receptors in human CNS diseases, such as Alzheimer's disease, which may reveal their functional roles and identify potential therapeutic drug targets. Indeed, it has recently been demonstrated (Higgins et al. [2004] Neuropharmacology 46:907-917) that pharmacological manipulation of mGlu2 receptors influences cognitive performance in the rodent.

Differential regulation of synaptic transmission by mGlu2 and mGlu3 at the perforant path inputs to the dentate gyrus and CA1 revealed in mGlu2 -/- mice.

Group II metabotropic glutamate (mGlu) receptors can act as presynaptic autoinhibitory receptors at perforant path inputs to the hippocampus under conditions of high frequency synaptic activation. We have used mGlu2 -/- mice to examine the relative roles of mGlu2 and mGlu3 in the regulation of perforant path synaptic transmission mediated by both the selective group II receptor agonist, DCG-IV, and by synaptically released glutamate. Field excitatory postsynaptic potentials evoked by stimulation of either the perforant path inputs to the dentate gyrus mid-moleculare or the CA1 stratum lacunosum moleculare were inhibited by DCG-IV with IC(50) values and maximum percentage inhibition of: 169 nM (60%) and 41 nM (72%) in wild-type mice and 273 nM (19%) and 116 nM (49%) in mGlu2 -/- mice, respectively. Activation of presynaptic group II mGlu autoreceptors by synaptically released glutamate, as revealed by a LY341495-mediated increase in the relative amplitude of a test fEPSP evoked after a conditioning burst, was observed in both the dentate gyrus and the stratum lacunosum of wild-type, but not mGlu2 -/- mice. These observations demonstrate that activation of mGlu3 receptors can regulate synaptic transmission at perforant path synapses but suggest that mGlu2 is the major presynaptic group II autoreceptor activated by synaptically released glutamate.

Pharmacological manipulation of mGlu2 receptors influences cognitive performance in the rodent.

Atrophy of the medial temporal lobes, including the glutamatergic cortical-hippocampal circuitry, is an early event in Alzheimer's disease (AD) and probably contributes to the characteristic short-term mnemonic decline. Pharmacological strategies directly targeted to ameliorating this functional decline may represent a novel approach for the symptomatic treatment of AD. Presynaptic group II metabotropic glutamate receptors (i.e. mGlu2 and mGlu3) exert a powerful modulatory influence on the function of these pathways, in particular the perforant pathway. Using a combination of mGlu2 receptor knockout mice and the group II agonist LY354740, we show that activation of mGlu2 receptors produces a cognitive impairment, i.e. a delay-dependent deficit in delayed matching and non-matching to position, and impaired spatial learning in a Morris water maze. Conversely, a group II antagonist, LY341495, improved acquisition of spatial learning. LY354740 potently reduced field excitatory postsynaptic potentials in hippocampal slices from wild type but not mGlu2 receptor knockout mice. Taken together, these results suggest that activation of mGlu2 receptors evokes a powerful inhibitory effect on hippocampal synaptic transmission and mGlu2 agonists produce a cognitive deficit consistent with this change. Conversely, mGlu2 receptor antagonists may improve certain aspects of cognition and thus represent a novel approach for the symptomatic treatment of AD.

Identification and characterization of a novel splice variant of the metabotropic glutamate receptor 5 gene in human hippocampus and cerebellum.

The G-protein coupled metabotropic glutamate receptor mGlu5 plays a pivotal role as a modulator of synaptic plasticity, ion channel activity and excitotoxicity. Two splice variants, hmGlu5a and -5b have been reported previously. During screening of a human brain cDNA library for hmGlu5a, we identified a novel variant (hmGlu5d) generated by alternative splicing at the C-terminal domain. The predicted hmGlu5d protein has a C-terminal 267 amino acid shorter than that of hmGlu5a. The pattern of mRNA expression of mGluR5 variants in human brain were analyzed by RT-PCR and in situ hybridization histochemistry. RT-PCR analysis demonstrated the presence of the hmGlu5d transcript, although at low level, in human whole brain, cerebellum, cerebral cortex and hippocampus. [3H]Quisqualate displayed similar affinity at the hmGlu5 splice variants (K(D) values of 80+/-8 and 54+/-17 nM for hmGlu5a and -5d receptors, respectively). For the five mGlu agonists studied, a similar rank order of potency was observed on both hmGlu5a and -5d receptors: quisqualate>glutamate>DHPG>L-CCGI approximately ACPD. MPEP inhibited the glutamate (2 microM)-induced [Ca(2+)](i) response in hmGlu5a and -5d-HEK293 cells also with similar potency (IC(50) values 25+/-1.5 and 20+/-1.4 nM, respectively). Therefore, the large truncation of the C-terminal tail of mGlu5 does not have any apparent major effect on the potency and efficacy of agonists as measured by the [Ca(2+)](i) responses or by activation of recombinant G-protein coupled inwardly rectifying K(+) (GIRK) channel currents. The only major functional difference is the increased sensitivity of hmGlu5d to protein kinase C (PKC)-mediated desensitization, relative to hmGlu5a.

Alkyl diphenylacetyl, 9H-xanthene- and 9H-thioxanthene-carbonyl carbamates as positive allosteric modulators of mGlu1 receptors.

Starting from the random-screening hit 1a, a series of alkyl diphenylacetyl, 9H-xanthene- and 9H-thioxanthene-carbonyl carbamates 1 has been prepared. These derivatives turned out to be selective positive allosteric modulators of mGlu1 receptors. These compounds do not directly activate mGlu1 receptors but markedly potentiate agonist stimulated responses, increasing potency and maximum efficacy.