ABSTRACT
1. Voltage-sensitive Ca(2+) channels (VSCCs) are often heteromultimeric complexes. The VSCC subtype specifically expressed by skeletal muscle has long been known to contain a gamma subunit, gamma(1), that is only expressed in this tissue. Recent work, initiated by the identification of the mutation present in the stargazer mouse, has led to the identification of a series of novel potential Ca(2+) channel gamma subunits expressed in the CNS. 2. Based on bioinformatic techniques we identified and cloned the human gamma(2), gamma(3) and gamma(4) subunits. 3. TaqMan analysis was used to quantitatively characterise the mRNA expression patterns of all the gamma subunits. All three subunits were extensively expressed in adult brain with overlapping but subunit-specific distributions. gamma(2) and gamma(3) were almost entirely restricted to the brain, but gamma(4) expression was seen in a broad range of peripheral tissues. 4. Using a myc epitope the gamma(2) subunit was tagged both intracellularly at the C-terminus and on a predicted extracellular site between the first and second transmembrane domains. The cellular distribution was then examined immunocytochemically, which indicated that a substantial proportion of the cellular pool of the gamma(2) subunit was present on the plasma membrane and provided initial evidence for the predicted transmembrane topology of the gamma subunits. 5. Using co-transfection techniques we investigated the functional effects of each of the gamma subunits on the biophysics of the T-type VSCC encoded by the alpha(1I) subunit. This revealed a substantially slowed rate of deactivation in the presence of gamma(2). In contrast, there was no significant corresponding effect of either gamma(3) or gamma(4) on alpha(1I) subunit-mediated currents.
Subject(s)
Calcium Channels, T-Type/genetics , Calcium Channels, T-Type/metabolism , Calcium Channels/genetics , Calcium Channels/metabolism , Neurons/chemistry , Animals , Calcium Channels/chemistry , Calcium Channels, T-Type/chemistry , Cell Line , Cloning, Molecular , DNA, Complementary , Electrophysiology , Gene Expression/physiology , Humans , Kidney/cytology , Kinetics , Membrane Potentials/physiology , Mice , Mice, Neurologic Mutants , Molecular Sequence Data , RNA, Messenger/analysis , Sequence Homology, Amino Acid , Taq Polymerase , TransfectionABSTRACT
We have cloned and functionally expressed the human orthologue of the mouse TRAAK gene. When cDNA for hTRAAK is expressed in either Xenopus oocytes or HEK293 cells it forms a K(+)-selective conductance and hyperpolarises the resting membrane potential. Quantitative mRNA expression analysis using Taqman revealed that hTRAAK mRNA is predominantly present in the central nervous system where it exhibits a regionally diverse pattern of expression. Like the related channel TREK-1, the activity of TRAAK was potentiated by arachidonic acid. The neuroprotective agent sipatrigine (10 microM) inhibited both hTREK-1 (73.3+/-4.4%) and hTRAAK (45.1+/-11.2%) in a reversible, voltage-independent manner. Inhibition of both channels was dose-dependent and for TREK-1 occurred with an IC(50) of 4 microM. The related compound lamotrigine, which is a better anticonvulsant but weaker neuroprotective agent than sipatrigine, was a far less effective antagonist of both channels, producing <10% inhibition at a concentration of 10 microM.
Subject(s)
Brain/physiology , Neuroprotective Agents/pharmacology , Piperazines/pharmacology , Potassium Channels, Tandem Pore Domain , Potassium Channels/physiology , Pyrimidines/pharmacology , Amino Acid Sequence , Animals , Cell Line , Female , Humans , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Molecular Sequence Data , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/physiology , Oocytes/drug effects , Oocytes/physiology , Potassium Channel Blockers , Potassium Channels/chemistry , Potassium Channels/genetics , RNA, Messenger/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Transfection , Xenopus laevisABSTRACT
Bisaryl ethers have been identified with excellent 5-HT2C affinity and selectivity over both 5-HT2A and 5-HT2B receptors. Compounds such as 11, 27 and 38 have potent oral activity in a centrally mediated pharmacodynamic model of 5-HT2C function and their potential as novel non-sedating anxiolytic and antidepressants is under investigation.
Subject(s)
Aminopyridines/chemical synthesis , Aminopyridines/pharmacology , Indoles/chemical synthesis , Indoles/pharmacology , Receptors, Serotonin/metabolism , Serotonin Antagonists/chemical synthesis , Administration, Oral , Aminopyridines/administration & dosage , Aminopyridines/chemistry , Animals , Anti-Anxiety Agents/administration & dosage , Anti-Anxiety Agents/chemical synthesis , Anti-Anxiety Agents/chemistry , Anti-Anxiety Agents/pharmacology , Antidepressive Agents/administration & dosage , Antidepressive Agents/chemical synthesis , Antidepressive Agents/chemistry , Antidepressive Agents/pharmacology , Drug Design , Humans , Indoles/administration & dosage , Indoles/chemistry , Kinetics , Molecular Structure , Motor Activity/drug effects , Rats , Receptor, Serotonin, 5-HT2C , Receptors, Serotonin/chemistry , Serotonin Antagonists/administration & dosage , Serotonin Antagonists/chemistry , Serotonin Antagonists/pharmacology , Structure-Activity RelationshipABSTRACT
Bisarylmethoxyethers have been identified with nanomolar 5-HT2C affinity and selectivity over both 5-HT2A and 5-HT2B receptors. Compounds such as 1, 2, 8, 12, 14 and 18 have potent oral activity in a centrally mediated pharmacodynamic model of 5-HT2C function and their therapeutic potential is currently under further investigation.
Subject(s)
Aminopyridines/chemical synthesis , Aminopyridines/pharmacology , Indoles/chemical synthesis , Indoles/pharmacology , Receptors, Serotonin/metabolism , Serotonin Antagonists/chemical synthesis , Serotonin Antagonists/pharmacology , Aminopyridines/administration & dosage , Aminopyridines/chemistry , Animals , Drug Design , Indoles/administration & dosage , Indoles/chemistry , Kinetics , Molecular Structure , Motor Activity/drug effects , Rats , Receptor, Serotonin, 5-HT2C , Serotonin Antagonists/administration & dosage , Serotonin Antagonists/chemistry , Structure-Activity RelationshipSubject(s)
Drug Design , Genome, Human , Combinatorial Chemistry Techniques , Drug Evaluation, Preclinical , HumansABSTRACT
The evolution, synthesis, and biological activity of a novel series of 5-HT(2C) receptor inverse agonists are reported. Biarylcarbamoylindolines have been identified with excellent 5-HT(2C) affinity and selectivity over 5-HT(2A) receptors. In addition, (pyridyloxypyridyl)carbamoylindolines have been discovered with additional selectivity over the closely related 5-HT(2B) receptor. Compounds from this series are inverse agonists at the human cloned 5-HT(2C) receptor, completely abolishing basal activity in a functional assay. The new series have reduced P450 inhibitory liability compared to a previously described series of 1-(3-pyridylcarbamoyl)indolines (Bromidge et al. J. Med. Chem. 1998, 41, 1598) from which they evolved. Compounds from this series showed excellent oral activity in a rat mCPP hypolocomotion model and in animal models of anxiety. On the basis of their favorable biological profile, 32 (SB-228357) and 40 (SB-243213) have been selected for further evaluation to determine their therapeutic potential for the treatment of CNS disorders such as depression and anxiety.
Subject(s)
Anti-Anxiety Agents/chemical synthesis , Antidepressive Agents/chemical synthesis , Indoles/chemical synthesis , Pyridines/chemical synthesis , Receptors, Serotonin/metabolism , Serotonin Receptor Agonists/chemical synthesis , Administration, Oral , Animals , Anti-Anxiety Agents/chemistry , Anti-Anxiety Agents/metabolism , Anti-Anxiety Agents/pharmacology , Antidepressive Agents/chemistry , Antidepressive Agents/metabolism , Antidepressive Agents/pharmacology , Cell Line , Humans , Indoles/chemistry , Indoles/metabolism , Indoles/pharmacology , Models, Molecular , Motor Activity/drug effects , Pyridines/chemistry , Pyridines/metabolism , Pyridines/pharmacology , Radioligand Assay , Rats , Receptor, Serotonin, 5-HT2A , Receptor, Serotonin, 5-HT2B , Receptor, Serotonin, 5-HT2C , Serotonin Receptor Agonists/chemistry , Serotonin Receptor Agonists/metabolism , Serotonin Receptor Agonists/pharmacology , Structure-Activity RelationshipSubject(s)
Phenols/chemical synthesis , Receptors, Serotonin/drug effects , Serotonin Antagonists/chemical synthesis , Sulfonamides/chemical synthesis , Cell Line , Humans , Models, Molecular , Phenols/chemistry , Phenols/metabolism , Radioligand Assay , Receptors, Serotonin/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Serotonin Antagonists/chemistry , Serotonin Antagonists/metabolism , Stereoisomerism , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/metabolismABSTRACT
A model series of 5-HT2C antagonists have been prepared by rapid parallel synthesis. These N-substituted phenyl-N'-pyridin-3-yl ureas were found to have a range of 5-HT2C receptor affinities and selectivities over the closely related 5-HT2A receptor. Extrapolation of simple SAR, derived from this set of compounds, to the more active but synthetically more complex 1-(3-pyridylcarbamoyl)indoline series allowed us to target optimal substitution patterns and identify potent and selective 5-HT(2C/2B) antagonists.
Subject(s)
Phenylurea Compounds/chemistry , Phenylurea Compounds/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Receptors, Serotonin/drug effects , Serotonin Antagonists/chemistry , Serotonin Antagonists/pharmacology , Animals , Humans , In Vitro Techniques , Kinetics , Magnetic Resonance Spectroscopy , Models, Chemical , Phenylurea Compounds/chemical synthesis , Pyridines/chemical synthesis , Receptor, Serotonin, 5-HT2B , Receptor, Serotonin, 5-HT2C , Receptors, Serotonin/metabolism , Serotonin Antagonists/chemical synthesis , Structure-Activity RelationshipSubject(s)
Drosophila Proteins , GTP-Binding Proteins/physiology , Membrane Proteins/classification , Receptors, Cell Surface/physiology , Receptors, Gastrointestinal Hormone/classification , Amino Acid Sequence , Animals , Frizzled Receptors , Humans , Membrane Proteins/chemistry , Membrane Proteins/physiology , Molecular Sequence Data , Receptors, G-Protein-Coupled , Receptors, Gastrointestinal Hormone/chemistry , Rhodopsin/classification , Signal TransductionABSTRACT
The synthesis, biological activity, and molecular modeling of a novel series of substituted 1-(3-pyridylcarbamoyl)indolines are reported. These compounds are isosteres of the previously published indole urea 1 (SB-206553) and illustrate the use of aromatic disubstitution as a replacement for fused five-membered rings in the context of 5-HT2C/2B receptor antagonists. By targeting a region of space previously identified as sterically allowed at the 5-HT2C receptor but disallowed at the 5-HT2A receptor, we have identified a number of compounds which are the most potent and selective 5-HT2C/2B receptor antagonists yet reported. 46 (SB-221284) was selected on the basis of its overall biological profile for further evaluation as a novel, potential nonsedating anxiolytic agent. A CoMFA analysis of these compounds produced a model with good predictive value and in addition good qualitative agreement with both our 5-HT2C receptor model and our proposed binding mode for this class of ligands within that model.
Subject(s)
Anti-Anxiety Agents , Indoles , Models, Molecular , Pyridines , Receptors, Serotonin/drug effects , Serotonin Antagonists , Animals , Anti-Anxiety Agents/chemical synthesis , Anti-Anxiety Agents/chemistry , Anti-Anxiety Agents/metabolism , Anti-Anxiety Agents/pharmacology , Conditioning, Operant/drug effects , Conflict, Psychological , Indoles/chemical synthesis , Indoles/chemistry , Indoles/metabolism , Indoles/pharmacology , Male , Motor Activity/drug effects , Pyridines/chemical synthesis , Pyridines/chemistry , Pyridines/metabolism , Pyridines/pharmacology , Rats , Rats, Sprague-Dawley , Receptor, Serotonin, 5-HT2B , Receptor, Serotonin, 5-HT2C , Receptors, Serotonin/metabolism , Serotonin Antagonists/chemical synthesis , Serotonin Antagonists/chemistry , Serotonin Antagonists/metabolism , Serotonin Antagonists/pharmacology , Social Behavior , Structure-Activity RelationshipABSTRACT
5-HT1 receptors are members of the G-protein-coupled receptor superfamily and are negatively linked to adenylyl cyclase activity. The human 5-HT1B and 5-HT1D receptors (previously known as 5-HT1Dbeta and 5-HT1Dalpha, respectively), although encoded by two distinct genes, are structurally very similar. Pharmacologically, these two receptors have been differentiated using nonselective chemical tools such as ketanserin and ritanserin, but the absence of truly selective agents has meant that the precise function of the 5-HT1B and 5-HT1D receptors has not been defined. In this paper we describe how, using computational chemistry models as a guide, the nonselective 5-HT1B/5-HT1D receptor antagonist 4 was structurally modified to produce the selective 5-HT1B receptor inverse agonist 5, 1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2, 4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6, 7-tetrahydrospiro[furo[2,3-f]indole-3,4'-piperidine] (SB-224289). This compound is a potent antagonist of terminal 5-HT autoreceptor function both in vitro and in vivo.
Subject(s)
Autoreceptors/antagonists & inhibitors , Piperidones/pharmacology , Receptors, Serotonin/drug effects , Serotonin Antagonists/pharmacology , Serotonin Receptor Agonists/pharmacology , Spiro Compounds/pharmacology , Animals , Aspartic Acid/metabolism , Autoreceptors/metabolism , CHO Cells , Cricetinae , Frontal Lobe/drug effects , Frontal Lobe/metabolism , Guinea Pigs , Humans , Hypothermia/chemically induced , Hypothermia/metabolism , In Vitro Techniques , Indoles/toxicity , Male , Models, Molecular , Oxadiazoles/chemistry , Oxadiazoles/metabolism , Oxadiazoles/pharmacology , Piperazines/chemistry , Piperazines/metabolism , Piperazines/pharmacology , Piperidones/chemical synthesis , Piperidones/chemistry , Piperidones/metabolism , Radioligand Assay , Rats , Receptor, Serotonin, 5-HT1B , Receptor, Serotonin, 5-HT1D , Receptors, Serotonin/metabolism , Serotonin Antagonists/chemical synthesis , Serotonin Antagonists/chemistry , Serotonin Antagonists/metabolism , Serotonin Receptor Agonists/chemical synthesis , Serotonin Receptor Agonists/chemistry , Serotonin Receptor Agonists/metabolism , Spiro Compounds/chemical synthesis , Spiro Compounds/chemistry , Spiro Compounds/metabolism , Structure-Activity Relationship , SwineSubject(s)
Piperidines/chemical synthesis , Receptors, Serotonin/drug effects , Serotonin Antagonists/chemical synthesis , Sulfonamides/chemical synthesis , Adenylyl Cyclases/metabolism , Binding, Competitive , Cell Line , Gene Expression , Humans , Magnetic Resonance Spectroscopy , Molecular Structure , Piperidines/pharmacology , Receptors, Serotonin/genetics , Receptors, Serotonin/physiology , Serotonin Antagonists/pharmacology , Stereoisomerism , Structure-Activity Relationship , Sulfonamides/pharmacologyABSTRACT
The synthesis and biological activity are reported for a series of analogues of the previously published indole urea 2 (SB-206553), designed to probe the 5-HT(2C) receptor binding site. Small molecule modeling studies have been used to define a region in space which is allowed at the 5-HT(2C) receptor but disallowed at the 5-HT(2A) receptor. In a complementary approach, docking of 2 into our model of the 5-HT(2C) receptor has allowed us to propose a novel primary binding interaction for this series of diaryl ureas, involving a potential double hydrogen-bonding interaction between the urea carbonyl oxygen of the ligand and two serine residues in the receptor. The difference of two valine residues in the 5-HT(2C) receptor for leucine residues in the 5-HT(2A) receptor is believed to account for the observed 5-HT(2C)/5-HT(2A) selectivity with 2.