Marion Wittmann

N-desmethylclozapine, an allosteric agonist at muscarinic 1 receptor, potentiates N-methyl-d-aspartate receptor activity

The molecular and neuronal substrates conferring on clozapine its unique and superior efficacy in the treatment of schizophrenia remain elusive. The interaction of clozapine with many G protein-coupled receptors is well documented but less is known about its biologically active metabolite, N-desmethylclozapine. Recent clinical and preclinical evidences of the antipsychotic activity of the muscarinic agonist xanomeline prompted us to investigate the effects of N-desmethylclozapine on cloned human M1-M5 muscarinic receptors. N-desmethylclozapine preferentially bound to M1 muscarinic receptors with an IC50 of 55 nM and was a more potent partial agonist (EC50, 115 nM and 50% of acetylcholine response) at this receptor than clozapine. Furthermore, pharmacological and site-directed mutagenesis studies suggested that N-desmethylclozapine preferentially activated M1 receptors by interacting with a site that does not fully overlap with the acetylcholine orthosteric site. As hypofunction of N-methyl-d-aspartate (NMDA) receptor-driven neuronal ensembles has been implicated in psychotic disorders, the neuronal activity of N-desmethylclozapine was electrophysiologically investigated in hippocampal rat brain slices. N-desmethylclozapine was shown to dose-dependently potentiate NMDA receptor currents in CA1 pyramidal cells by 53% at 100 nM, an effect largely mediated by activation of muscarinic receptors. Altogether, our observations provide direct evidence that the brain penetrant metabolite N-desmethylclozapine is a potent, allosteric agonist at human M1 receptors and is able to potentiate hippocampal NMDA receptor currents through M1 receptor activation. These observations raise the possibility that N-desmethylclozapine contributes to clozapines clinical activity in schizophrenics through modulation of both muscarinic and glutamatergic neurotransmission.

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 M1 muscarinic acetylcholine receptor (mAChR). During Alzheimers disease (AD), these cholinergic neurons degenerate, therefore selectively activating M1 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 M1 mAChR. BQCA reduces the concentration of ACh required to activate M1 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 μM. Furthermore studies in M1−/− mice demonstrates that BQCA requires M1 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 M1 allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces β-arrestin recruitment to M1, 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 M1 receptor and represents a promising therapeutic strategy for cognitive disorders.

Distribution and roles of metabotropic glutamate receptors in the basal ganglia motor circuit: implications for treatment of Parkinson's disease and related disorders.

The basal ganglia (BG) are a set of interconnected subcortical structures that play a critical role in motor control. The BG are thought to control movements by a delicate balance of transmission through two BG circuits that connect the input and output nuclei: the direct and the indirect pathways. The BG are also involved in a number of movement disorders. Most notably, the primary pathophysiological change that gives rise to the motor symptoms of Parkinsons Disease (PD) is the loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) that are involved in modulating function of the striatum and other BG structures. This ultimately results in an increase in activity of the indirect pathway relative to the direct pathway and the hallmark PD symptoms of rigidity, bradykinesia, and akinesia. A great deal of effort has been dedicated to finding treatments for this disease. The current pharmacotherapies are aimed at replacing the missing dopamine, while the current surgical treatments are aimed at reducing transmission through the indirect pathway. Dopamine replacement therapy has proven to be helpful, but is associated with severe side effects that limit treatment and a loss of efficacy with progression of the disease. Recently developed surgical therapies have been highly effective, but are highly invasive, expensive, and assessable to a small minority of patients. For these reasons, new effort has been dedicated to finding pharmacological treatment options that will be effective in reducing transmission through the indirect pathway. Members of the metabotropic glutamate receptor (mGluR) family have emerged as interesting and promising targets for such a treatment. This review will explore the most recent advances in the understanding of mGluR localization and function in the BG motor circuit and the implications of those findings for the potential therapeutic role of mGluR-targeted compounds for PD.

Discovery of a selective allosteric M1 receptor modulator with suitable development properties based on a quinolizidinone carboxylic acid scaffold.

One approach to ameliorate the cognitive decline in Alzheimers disease (AD) has been to restore neuronal signaling from the basal forebrain cholinergic system via the activation of the M(1) muscarinic receptor. A number of nonselective M(1) muscarinic agonists have previously shown positive effects on cognitive behaviors in AD patients, but were limited due to cholinergic adverse events thought to be mediated by the activation of the M(2) to M(5) subtypes. One strategy to confer selectivity for M(1) is the identification of positive allosteric modulators, which would target an allosteric site on the M(1) receptor rather than the highly conserved orthosteric acetylcholine binding site. Quinoline carboxylic acids have been previously identified as highly selective M(1) positive allosteric modulators with good pharmacokinetic and in vivo properties. Herein is described the optimization of a novel quinolizidinone carboxylic acid scaffold with 4-cyanopiperidines being a key discovery in terms of enhanced activity. In particular, modulator 4i gave high plasma free fractions, enhanced central nervous system (CNS) exposure, was efficacious in a rodent in vivo model of cognition, and afforded good physicochemical properties suitable for further preclinical evaluation.

Quinolizidinone Carboxylic Acids as CNS Penetrant, Selective M1 Allosteric Muscarinic Receptor Modulators

Positive allosteric modulation of the M1 muscarinic receptor represents an approach to treat the cognitive decline in patients with Alzheimers disease. Replacement of a quinolone ring system in a quinolone carboxylic acid series of M1 modulators with a quinolizidinone bearing a basic amine linkage led to a series of compounds with higher free fraction, enhanced CNS exposure, and improved efficacy in rodent in vivo models of cognition.

Localization and physiological roles of metabotropic glutamate receptors in the direct and indirect pathways of the basal ganglia

Summary. Our current understanding of the circuitry of the basal ganglia, and the pathophysiology of Parkinsons disease has led to major breakthroughs in the treatment of this debilitating movement disorder. Unfortunately, there are significant problems with the currently available pharmacological therapies that focus on dopamine replacement or dopaminergic agonists. Because of this, much effort has been focused on developing novel targets for the treatment of Parkinsons disease. The metabotropic glutamate receptors are a family of G-protein coupled receptors activated by glutamate. These receptors are differentially distributed throughout the basal ganglia in a manner suggesting that they may provide novel targets for the treatment of movement disorders. In this review we summarize anatomical and physiological data from our work and the work of other laboratories describing the distribution and physiological roles of metabotropic glutamate receptors in the basal ganglia with emphasis on possible therapeutic targets.

Parallel synthesis of N-biaryl quinolone carboxylic acids as selective M1 positive allosteric modulators

An iterative analog library synthesis approach was employed in the exploration of a quinolone carboxylic acid series of selective M(1) positive allosteric modulators, and strategies for improving potency and plasma free fraction were identified.

Heterocyclic fused pyridone carboxylic acid M1 positive allosteric modulators

The phenyl ring in a series of quinolone carboxylic acid M(1) positive allosteric modulators was replaced with a variety of heterocycles in order to reduce protein plasma binding and enhance CNS exposure.

Activation of metabotropic glutamate receptor 1 inhibits glutamatergic transmission in the substantia nigra pars reticulata

The substantia nigra pars reticulata is a primary output nucleus of the basal ganglia motor circuit and is controlled by a fine balance between excitatory and inhibitory inputs. The major excitatory input to GABAergic neurons in the substantia nigra arises from glutamatergic neurons in the subthalamic nucleus, whereas inhibitory inputs arise mainly from the striatum and the globus pallidus. Anatomical studies revealed that metabotropic glutamate receptors (mGluRs) are highly expressed throughout the basal ganglia. Interestingly, mRNA for group I mGluRs are abundant in neurons of the subthalamic nucleus and the substantia nigra pars reticulata. Thus, it is possible that group I mGluRs play a role in the modulation of glutamatergic synaptic transmission at excitatory subthalamonigral synapses. To test this hypothesis, we investigated the effects of group I mGluR activation on excitatory synaptic transmission in putative GABAergic neurons in the substantia nigra pars reticulata using the whole cell patch clamp recording approach in slices of rat midbrain. We report that activation of group I mGluRs by the selective agonist (R,S)-3,5-dihydroxyphenylglycine (100 microM) decreases synaptic transmission at excitatory synapses in the substantia nigra pars reticulata. This effect is selectively mediated by presynaptic activation of the group I mGluR subtype, mGluR1. Consistent with these data, electron microscopic immunocytochemical studies demonstrate the localization of mGluR1a at presynaptic sites in the rat substantia nigra pars reticulata. From this finding that group I mGluRs modulate the major excitatory inputs to GABAergic neurons in the substantia nigra pars reticulata we suggest that these receptors may play an important role in basal ganglia functions. Studying this effect, therefore, provides new insights into the modulatory role of glutamate in basal ganglia output nuclei in physiological and pathophysiological conditions.

N-Heterocyclic derived M1 positive allosteric modulators

Replacement of a phenyl ring with N-linked heterocycles in a series of quinolone carboxylic acid M1 positive allosteric modulators was investigated. In particular, a pyrazole derivative exhibited improvements in potency, free fraction, and CNS exposure.