ABSTRACT
Loss of synapses or alteration of synaptic activity is associated with cognitive impairment observed in a number of psychiatric and neurological disorders, such as schizophrenia and Alzheimer's disease. Therefore successful development of in vitro methods that can investigate synaptic function in a high-throughput format could be highly impactful for neuroscience drug discovery. We present here the development, characterisation and validation of a novel high-throughput in vitro model for assessing neuronal function and synaptic transmission in primary rodent neurons. The novelty of our approach resides in the combination of the electrical field stimulation (EFS) with data acquisition in spatially separated areas of an interconnected neuronal network. We integrated our methodology with state of the art drug discovery instrumentation (FLIPR Tetra) and used selective tool compounds to perform a systematic pharmacological validation of the model. We investigated pharmacological modulators targeting pre- and post-synaptic receptors (AMPA, NMDA, GABA-A, mGluR2/3 receptors and Nav, Cav voltage-gated ion channels) and demonstrated the ability of our model to discriminate and measure synaptic transmission in cultured neuronal networks. Application of the model described here as an unbiased phenotypic screening approach will help with our long term goals of discovering novel therapeutic strategies for treating neurological disorders.
Subject(s)
Drug Discovery/instrumentation , Neurons/physiology , Synapses/physiology , Synaptic Transmission/physiology , Voltage-Sensitive Dye Imaging , Animals , Calcium/metabolism , Cations, Divalent/metabolism , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Cerebral Cortex/physiology , Electric Stimulation , Neurons/cytology , Neurons/drug effects , Neurotransmitter Agents/pharmacology , Primary Cell Culture , Rats, Sprague-Dawley , Synapses/drug effects , Synaptic Transmission/drug effects , Voltage-Sensitive Dye Imaging/instrumentation , Voltage-Sensitive Dye Imaging/methodsABSTRACT
N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an important role in synaptic plasticity and learning and memory formation. Malfunctioning of NMDARs, in particular the reduction in NMDAR activity, is thought to be implicated in major neurological disorders. NMDAR positive allosteric modulators (PAMs) represent potential therapeutic interventions for restoring normal NMDAR function. We report a novel screening approach for identification and characterization of NMDAR-PAMs. The approach combines high-throughput fluorescence imaging with automated electrophysiological recording of glutamate-evoked responses in HEK-293 cells expressing NR1/NR2A NMDAR subunits. Initial high-throughput screening (HTS) of a chemical library containing >810,000 compounds using a calcium flux assay in 1536-well plate format identified a total of 864 NMDAR-PAMs. Concentration response determination in both calcium flux and automated electrophysiological assays found several novel chemical series with EC50 values between 0.49 and 10 µM. A small subset (six series) was selected and analyzed for pharmacological properties, subtype selectivity, mode of action, and activity at native NMDARs. Our approach demonstrates the successful application of HTS functional assays that led to identification of NMDAR-PAMs providing the foundation for further medicinal chemistry work that may lead to novel therapies for treatment of cognitive impairment associated with Alzheimer's disease and schizophrenia.
Subject(s)
Drug Discovery/methods , High-Throughput Screening Assays/methods , Receptors, N-Methyl-D-Aspartate/metabolism , Small Molecule Libraries/isolation & purification , Allosteric Regulation/drug effects , Alzheimer Disease/drug therapy , Calcium/chemistry , Glutamic Acid/chemistry , Glutamic Acid/metabolism , HEK293 Cells , Humans , Neuronal Plasticity/drug effects , Receptors, N-Methyl-D-Aspartate/drug effects , Schizophrenia/drug therapy , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacologyABSTRACT
The group II metabotropic glutamate (mGlu) receptors comprised of the mGlu2 and mGlu3 receptor subtypes have gained recognition in recent years as potential targets for psychiatric disorders, including anxiety and schizophrenia. In addition to studies already indicating which subtype mediates the anxiolytic and anti-psychotic effects observed in disease models, studies to help further define the preferred properties of selective group II mGlu receptor ligands will be essential. Comparison of the in vitro properties of these ligands to their in vivo efficacy and tolerance profiles may help provide these additional insights. We have developed a relatively high-throughput native group II mGlu receptor functional assay to aid this characterisation. We have utilised dissociated primary cortical neuronal cultures, which after 7 days in vitro have formed functional synaptic connections and display periodic and spontaneous synchronised calcium (Ca(2+)) oscillations in response to intrinsic action potential bursts. We herein demonstrate that in addition to non-selective group II mGlu receptor agonists, (2R,4R)-APDC, LY379268 and DCG-IV, a selective mGlu2 agonist, LY541850, and mGlu2 positive allosteric modulators, BINA and CBiPES, inhibit the frequency of synchronised Ca(2+) oscillations in primary cultures of rat and mouse cortical neurons. Use of cultures from wild-type, mGlu2(-/-), mGlu3(-/-) and mGlu2/3(-/-) mice allowed us to further probe the contribution of mGlu2 and mGlu3, and revealed LY541850 to be a partial mGlu2 agonist and a full mGlu3 antagonist. Overnight pre-treatment of cultures with these ligands revealed a preferred desensitisation profile after treatment with a positive allosteric modulator. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.
Subject(s)
Calcium Signaling/drug effects , Cerebral Cortex/drug effects , Excitatory Amino Acid Agonists/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Optical Imaging/methods , Receptors, Metabotropic Glutamate , Allosteric Regulation , Amino Acids/pharmacology , Amino Acids, Dicarboxylic/pharmacology , Animals , Biphenyl Compounds/pharmacology , Bridged Bicyclo Compounds/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cerebral Cortex/metabolism , Cyclic AMP/metabolism , Cyclopropanes/pharmacology , Glycine/analogs & derivatives , Glycine/pharmacology , Indans/pharmacology , Ligands , Mice , Mice, Inbred ICR , Mice, Knockout , Mice, Transgenic , Neurons/drug effects , Neurons/metabolism , Optical Imaging/instrumentation , Primary Cell Culture/methods , Proline/analogs & derivatives , Proline/pharmacology , Pyridines/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Metabotropic Glutamate/agonists , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Receptors, Metabotropic Glutamate/biosynthesis , Receptors, Metabotropic Glutamate/genetics , Sulfonamides/pharmacologyABSTRACT
SAR around a known molecule with dual 5-HT(1D) antagonist and 5-HT(transporter) inhibitory activity has led to the discovery of molecules with improved dual activity and reduced cross-reactivity toward other aminergic receptors (5-HT(1B), alpha(1), and D(2)).
Subject(s)
Antidepressive Agents/pharmacology , Naphthalenes/chemistry , Piperazines/pharmacology , Receptor, Serotonin, 5-HT1D/drug effects , Selective Serotonin Reuptake Inhibitors/pharmacology , Serotonin Antagonists/pharmacology , Animals , Antidepressive Agents/chemical synthesis , Dose-Response Relationship, Drug , Guinea Pigs , Kinetics , Models, Chemical , Piperazines/chemical synthesis , Receptor, Serotonin, 5-HT1D/metabolism , Serotonin Antagonists/chemical synthesis , Selective Serotonin Reuptake Inhibitors/chemical synthesis , Stereoisomerism , Structure-Activity RelationshipABSTRACT
A novel series of tertiary alcohol containing 2-substituted benzyl morpholines have been discovered as potent and selective inhibitors of the norepinephrine transporter. Efficient synthetic routes were developed featuring a highly diastereoselective nucleophilic addition of benzyl Grignard reagents to enantiopure (4-benzylmorpholin-2-yl)phenylmethanone (11) as the key synthetic step. In vitro binding affinity for the norepinephrine, dopamine and serotonin transporters and in vivo examination of a select compound (16) in a pharmacodynamic animal model for norepinephrine reuptake inhibition are presented.
Subject(s)
Alcohols/chemistry , Morpholines/pharmacology , Norepinephrine Plasma Membrane Transport Proteins/antagonists & inhibitors , Crystallography, X-Ray , Dopamine Plasma Membrane Transport Proteins/antagonists & inhibitors , Morpholines/chemistryABSTRACT
A novel series of 1-aryl-3,4-dihydro-1H-quinolin-2-ones have been discovered as potent and selective norepinephrine reuptake inhibitors. Efficient synthetic routes have been developed which allow for the multi-gram preparation of both final targets and advanced intermediates for SAR expansion.
Subject(s)
Neurotransmitter Uptake Inhibitors/pharmacology , Norepinephrine/physiology , Quinolines/chemistry , Quinolines/pharmacology , Chromatography, High Pressure Liquid , Neurotransmitter Uptake Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
Novel arylthiomethyl morpholines are potent selective norepinephrine reuptake inhibitors (NERIs) and dual serotonin/norepinephrine reuptake inhibitors (SRI/NERIs). The target compounds were prepared using a stereochemically versatile synthesis featuring an aldol condensation as the key step. One enantiomer of the 2-methoxy-substituted analogue was found to be a potent and selective norepinephrine reuptake inhibitor, whereas the opposite enantiomer was a potent dual serotonin/norepinephrine reuptake inhibitor.
Subject(s)
Adrenergic Uptake Inhibitors/chemical synthesis , Morpholines/chemical synthesis , Norepinephrine/antagonists & inhibitors , Selective Serotonin Reuptake Inhibitors/chemical synthesis , Adrenergic Uptake Inhibitors/pharmacology , Biogenic Amines/antagonists & inhibitors , Biogenic Amines/metabolism , Humans , Morpholines/pharmacology , Selective Serotonin Reuptake Inhibitors/pharmacology , Stereoisomerism , Structure-Activity RelationshipABSTRACT
A series of compounds combining the naphthylpiperazine and thienopyran scaffolds has been prepared and evaluated for 5-HT reuptake inhibition with 5-HT1D antagonist activity. The design of these compounds has been based on the 'overlapping type' strategy where two pharmacophores are linked in a single molecule. The resultant dual pharmacological profile has the potential to deliver a more efficient treatment for depression.
Subject(s)
Pyrans/chemical synthesis , Pyrans/pharmacology , Selective Serotonin Reuptake Inhibitors/chemical synthesis , Selective Serotonin Reuptake Inhibitors/pharmacology , Serotonin 5-HT1 Receptor Antagonists , Serotonin Antagonists/chemical synthesis , Serotonin Antagonists/pharmacology , Animals , Humans , Pyrans/chemistry , Rats , Serotonin Antagonists/chemistry , Selective Serotonin Reuptake Inhibitors/chemistryABSTRACT
Incorporation of an SRI (serotonin reuptake inhibitor) pharmacophore into a selective 5-HT(1D) agonist has led to the discovery of a molecule having both 5-HT(1D) antagonist and SRI activity. RPS methodology was used to develop the SAR and identify potential approaches to reduce unwanted adrenergic alpha 1 and dopamine D(2) cross-reactivities.