Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation.

The forebrain cholinergic system promotes higher brain function in part by signaling through the M(1) muscarinic acetylcholine receptor (mAChR). During Alzheimer's disease (AD), these cholinergic neurons degenerate, therefore selectively activating M(1) receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the M(1) mAChR. BQCA reduces the concentration of ACh required to activate M(1) up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 microM. Furthermore studies in M(1)(-/-) mice demonstrates that BQCA requires M(1) to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and site-directed mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to M(1) allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces beta-arrestin recruitment to M(1), suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the M(1) receptor and represents a promising therapeutic strategy for cognitive disorders.

A novel selective positive allosteric modulator of metabotropic glutamate receptor subtype 5 has in vivo activity and antipsychotic-like effects in rat behavioral models.

We found that 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) is a potent and selective positive allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). In Chinese hamster ovary cells expressing human mGluR5, CDPPB potentiated threshold responses to glutamate in fluorometric Ca2+ assays more than 7-fold with an EC50 value of approximately 27 nM. At 1 microM, CDPPB shifted mGluR5 agonist concentration response curves to glutamate, quisqualate, and (R,S)-3,5-dihydroxyphenylglycine 3- to 9-fold to the left. At higher concentrations, CDPPB exhibited agonist-like activity on cells expressing mGluR5. No other activity was observed on any other mGluR or cell type at concentrations up to 10 microM. CDPPB had no effect on [3H]quisqualate binding to mGluR5 but did compete for binding of [3H]methoxyPEPy, an analog of the selective mGluR5 negative allosteric modulator MPEP. CDPPB was found to be brain penetrant and reversed amphetamine-induced locomotor activity and amphetamine-induced deficits in prepulse inhibition in rats, two models sensitive to antipsychotic drug treatment. These results demonstrate that positive allosteric modulation of mGluR5 produces behavioral effects, suggesting that such modulation serves as a viable approach to increasing mGluR5 activity in vivo. These effects are consistent with the hypothesis that allosteric potentiation of mGluR5 may provide a novel approach for development of antipsychotic agents.

Discovery of positive allosteric modulators for the metabotropic glutamate receptor subtype 5 from a series of N-(1,3-diphenyl-1H- pyrazol-5-yl)benzamides that potentiate receptor function in vivo.

This report describes the discovery of the first centrally active allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGluR5). Appropriately substituted N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides (e.g., 8) have been identified as a novel class of potent positive allosteric modulators of mGluR5 that potentiate the response to glutamate. An iterative analogue library synthesis approach provided potentiators with excellent potency and selectivity for mGluR5 (vs mGluRs 1-4, 7, 8). Compound 8q demonstrated in vivo proof of concept in an animal behavior model where known antipsychotics are active, supporting the development of new antipsychotics based on the NMDA hypofunction model for schizophrenia.

The glycine transporter type 1 inhibitor N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine potentiates NMDA receptor-mediated responses in vivo and produces an antipsychotic profile in rodent behavior.

Glycine acts as a necessary coagonist for glutamate at the NMDA receptor (NMDAR) complex by binding to the strychnine-insensitive glycine-B binding site on the NR1 subunit. The fact that glycine is normally found in the brain and spinal cord at concentrations that exceed those required to saturate this site has led to the speculation that glycine normally saturates NMDAR-containing synapses in vivo. However, additional lines of evidence suggest that synaptic glycine may be efficiently regulated in synaptic areas by the glycine transporter type 1 (GlyT1). The recent description of a potent and selective GlyT1 inhibitor (N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine [NFPS]) provides a tool for evaluation of the hypothesis that inhibition of GlyT1 may increase synaptic glycine and thereby potentiate NMDAR function in vivo. In the present study, we found that (+)-NFPS demonstrated >10-fold greater activity in an in vitro functional glycine reuptake assay relative to the racemic compound. In vivo, (+/-)-NFPS significantly enhanced long-term potentiation in the hippocampal dentate gyrus induced by high-frequency electrical stimulation of the afferent perforant pathway. Furthermore, (+)-NFPS induced a pattern of c-Fos immunoreactivity comparable with the atypical antipsychotic clozapine and enhanced prepulse inhibition of the acoustic startle response in DBA/2J mice, a strain with low basal levels of prepulse inhibition. Collectively, these data suggest that selective inhibition of GlyT1 can enhance NMDAR-sensitive activity in vivo and also support the idea that GlyT1 may represent a novel target for developing therapeutics to treat disorders associated with NMDAR hypofunction.

Metabotropic glutamate subtype 5 receptors modulate locomotor activity and sensorimotor gating in rodents.

Use-dependent N-methyl-d-aspartate receptor (NMDAR) antagonists produce behaviors in human volunteers that resemble schizophrenia and exacerbate those behaviors in schizophrenic patients, suggesting that hypofunction of NMDAR-mediated neuronal circuitry may be involved in the etiology of clinical schizophrenia. Activation of the metabotropic glutamate receptor subtype 5 (mGluR5) enhances NMDAR-mediated currents in vitro. Thus, activation of mGluR5 could potentiate hypofunctional NMDARs in neuronal circuitry relevant to schizophrenia. To further elucidate the role of mGluR5, the present study examined the effects of mGluR5 antagonist administration, with and without coadministration of the use-dependent NMDAR antagonist phencyclidine (PCP), on locomotor activity and prepulse inhibition (PPI) of the acoustic startle response in rodents. We further examined PPI in mGluR5 knockout mice. Finally, we examined PPI after administration of the mGluR5 agonist 2-chloro-5-hydroxyphenylglycine (CHPG) alone and in combination with amphetamine. The data indicate that the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine has no effect on locomotor activity or PPI by itself but does potentiate both PCP-induced locomotor activity and disruption of PPI. We further found that mGluR5 knockout mice display consistent deficits in PPI relative to their wild-type controls. Finally, the data indicate that CHPG has no effect on PPI by itself, but ameliorates amphetamine-induced disruption of PPI. Collectively, these data suggest that mGlu5 receptors play a modulatory role on rodent PPI and locomotor behaviors and are consistent with the hypothesis that mGlu5 agonist/potentiators may represent a novel approach for antipsychotic drug development.

A nonpeptide oxytocin receptor antagonist radioligand highly selective for human receptors.

A novel, potent nonpeptide oxytocin receptor antagonist (1-(1-(2-(2,2,2-trifluoroethoxy)-4-(1-methylsulfonyl-4-piperidinyloxy) phenylacetyl)-4-piperidinyl)-3,4-dihydro-2(1H)-quinolinone) has been identified that can be labeled to high specific activity with [35S]. In binding studies, this compound exhibits sub-nanomolar affinity and a high degree of selectivity (900-1800-fold) for human oxytocin receptors compared to human vasopressin receptors. This compound appears suitable for studying the pharmacology of oxytocin receptors in human and nonhuman primate tissues, for which there is currently a paucity of highly selective tools. It may also be useful as a nonlabeled competitor or as a radioligand in autoradiographic studies of oxytocin receptor localization in these tissues.