Characterisation of a putative antagonist binding domain in the glycine receptor chloride channel
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Chloride channel
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Potentiator
Allodynia
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Competitive antagonist
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Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of ligand-gated ion channels. Typically, channel activation follows the binding of agonists to the orthosteric binding sites of the receptor. α7 nAChRs have a very low probability of channel activation, which can be reversed by the binding of α7 selective positive allosteric modulators (PAMs) to putative sites within the transmembrane domains. Although typical PAMs, like PNU-120596, require coapplication of an orthosteric agonist to produce large channel activations, some, like GAT107 and B-973B [(S)-3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propanamide], are characterized as allosteric activating PAMs, which also bind to an allosteric activation (AA) site in the extracellular domain and activate the α7 ion channel by themselves. We had previously characterized N,N-diethyl-N′-phenylpiperazine analogs with various functions. In this work, we docked members of this family to a homology model of the α7 receptor extracellular domain. The compound 1,1-diethyl-4(naphthalene-2-yl)piperazin-1-ium (2NDEP) a weak partial agonist, showed particularly favorable docking and binding energies at the putative AA site of the receptor. We hypothesized that 2NDEP could couple with PAMs through the AA site. This hypothesis was tested with the α7 mutant C190A, which is not activated by orthosteric agonists but is effectively activated by GAT107. The results showed that 2NDEP acts as an allosteric agonist of α7C190A when coapplied with the PAM PNU-120596. Also, the allosteric activity was nearly abolished upon coapplication with the AA site–selective antagonist 2,3,5,6MP-TQS (cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), consistent with AA site involvement. Overall, our findings show a novel mode of agonism through an allosteric site in the extracellular domain of α7 nAChR.
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Allosteric modulator
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The ligand-gated ion channel superfamily of neurotransmitter receptors are proteins responsible for rapid transmission of nerve impulses at the synapse and have, therefore, been the subject of intensive research for many years. The cys-loop family, of which the 5-HT3 receptor is a member, includes the nicotinic acetylcholine receptor, the GABAA receptor and the glycine receptor. A diverse range of endogenous and artificial ligands activate these receptors, but, nevertheless, the family shares many similarities of structure and function. Several important questions, however, still remain to be determined, including the mechanism of agonist recognition at the binding site, the nature of the connection between the agonist binding and channel domains, the structure of the transmembrane regions and the mechanism of ion permeation and s electivity. This article reviews recent advances in the characterization of the molecular properties ofthe 5-HT3 receptor and their role in its function, and assesses its suitability as a model system for the study of the above questions.
Neurotransmitter receptor
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Inverse agonist
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The Cannabinoid 1 receptor (CB1) is a type A G-protein coupled receptor (GPCR) expressed most widely in the human brain and has been identified as a key receptor in modulating a number of physiological functions of the human body. Therefore, drugs acting on the CB1 have an enormous pharmacotherapeutic potential in disease conditions such as chronic neuropathic pain, cardio-metabolic diseases, obesity, and even in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In order to develop CB1-specific, high-affinity ligands, a better understanding of the structural aspects of the receptor in its native state is essential. Using an E. coli-based cell-free (in vitro) expression system in addition with nanolipid bilayers (Nanodiscs), we developed a convenient method for efficient production of the re-engineered human cannabinoid receptor and showed proof of functionality of the expressed receptor using a radioligand-binding assay. The saturation binding curve with CP55,940, a non-classical bicyclic analog of Δ9-tetrahydrocannabinol (Δ9-THC), showed Bmax of 1358 pmoles/g and a Kd of 8.57 nM. The total amount of functional receptor was calculated to be 500 ng/ml of expression reaction.
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GPR55 is a G protein-coupled receptor activated by l-α-lysophosphatidylinositol and suggested to have roles in pain signaling, bone morphogenesis, and possibly in vascular endothelial cells. It has affinity for certain cannabinoids (molecules that interact with the cannabinoid CB1 and CB2 receptors), but investigation of its functional role in cell-based systems and in tissue has been limited by a lack of selective pharmacological tools. Here, we present our characterization of GPR55 in the yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK293) cells. We describe GSK494581A (1-{2-fluoro-4-[1-(methyloxy)ethyl]phenyl}-4-{[4′-fluoro-4-(methylsulfonyl)-2-biphenylyl]carbonyl}piperazine), a selective small-molecule ligand of GPR55 identified through diversity screening. GSK494581A is one of a series of benzoylpiperazines originally identified and patented as inhibitors of the glycine transporter subtype 1 (GlyT1). The structure–activity relationship between GPR55 and GlyT1 is divergent across this series. The most GPR55-selective example is GSK575594A (3-fluoro-4-(4-{[4′-fluoro-4-(methylsulfonyl)-2-biphenylyl]carbonyl}-1-piperazinyl)aniline), which is approximately 60-fold selective for GPR55 (pEC50 = 6.8) over GlyT1 (pIC50 = 5.0). Several exemplars with activity at GPR55 and GlyT1 have been profiled at a broad range of other molecular targets and are inactive at cannabinoid receptors and all other targets tested. The benzoylpiperazine agonists activate human GPR55 but not rodent GPR55, suggesting that the relatively low level of sequence identity between these orthologs (75%) translates to important functional differences in the ligand-binding site.
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