Fenobam [N-(3-chlorophenyl)-NЈ-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea] is an atypical anxiolytic agent with unknown molecular target that has previously been demonstrated both in rodents and human to exert anxiolytic activity. Here, we report that fenobam is a selective and potent metabotropic glutamate (mGlu)5 receptor antagonist acting at an allosteric modulatory site shared with 2-methyl-6-phenylethynyl-pyridine (MPEP), the protypical selective mGlu5 receptor antagonist. Fenobam inhibited quisqualate-evoked intracellular calcium response mediated by human mGlu5 receptor with IC 50 ϭ 58 Ϯ 2 nM. It acted in a noncompetitive manner, similar to MPEP and demonstrated inverse agonist properties, blocking 66% of the mGlu5 receptor basal activity (in an over expressed cell line) with an IC 50 ϭ 84 Ϯ
Fenobam [N-(3-chlorophenyl)-N¢-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea], a clinically validated non-benzodiazepine anxiolytic, has been shown to be a potent and non-competitive metabotropic glutamate (mGlu)-5 receptor antagonist. In the present study, we have used the site-directed mutagenesis coupled with three-dimensional receptor-based pharmacophore modelling to elucidate the interacting mode of fenobam within the seven-transmembrane domain (7TMD) of mGlu5 receptor and its comparison with that of 2-methyl-6-(phenylethynyl)pyridine (MPEP), the prototype antagonist. The common residues involved in the recognition of MPEP and fenobam include Pro654 3.36 , Tyr658 3.40 , Thr780 6.44 , Trp784 6.48 , Phe787 6.51 , Tyr791 6.55 and Ala809 7.47 . The differentiating residues between both modulators' interacting modes are Arg647 3.29 , Ser657 3.39 and Leu743 5.47 . Our data suggest that these chemically unrelated mGlu5 antagonists act similarly, probing a functionally unique region of the 7TMD. Using [ 3 H]inositol phosphates accumulation assay, we have also identified the critical residues involved in the inverse agonist effect of MPEP. The mutation W784 6.48 A completely blocked the inverse agonist activity of MPEP; two mutations F787 6.51 A and Y791 6.55 A, caused a drastic decrease in the MPEP inverse agonism. Furthermore, these three mutations led to an increased efficacy of quisqualate without having any effect on its potency. The fact that the residues Trp784 6.48 and Phe787 6.51 are essential equally in antagonism and inverse agonism effects emphasizes again the key role of these residues and the involvement of a common transmembrane network in receptor inactivation by MPEP. Keywords: fenobam, inverse agonist, metabotropic glutamate receptor, MPEP, pharmacophore modelling, phosphoinositide hydrolysis. The major excitatory neurotransmitter in the brain, glutamate, acts at two distinct classes of receptors, ionotropic (NMDA, AMPA and kainate) and metabotropic (mGlu; Pin and Acher 2002). On the basis of sequence homology, second messenger coupling and pharmacology, eight mGlu receptors are divided into three groups: group I (mGlu1 and -5) receptors couple to the activation of phospholipase C leading to phosphoinositide hydrolysis and elevation of intracellular Ca 2+ levels ([Ca 2+ ] i ), group II (mGlu2 and -3) and group III (mGlu4, are negatively coupled to cAMP production (Pin and Acher 2002).
G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Here, a comprehensive and automated method allowing fast analysis and comparison of these putative binding pockets across the entire GPCR family is presented. The method relies on a robust alignment algorithm based on conservation indices, focusing on pharmacophore-like relationships between amino acids. Analysis of conservation patterns across the GPCR family and alignment to the rhodopsin X-ray structure allows the extraction of the amino acids lining the TM binding pocket in a so-called ligand binding pocket vector (LPV). In a second step, LPVs are translated to simple 3D receptor pharmacophore models, where each amino acid is represented by a single spherical pharmacophore feature and all atomic detail is omitted. Applications of the method include the assessment of selectivity issues, support of mutagenesis studies, and the derivation of rules for focused screening to identify chemical starting points in early drug discovery projects. Because of the coarseness of this 3D receptor pharmacophore model, however, meaningful scoring and ranking procedures of large sets of molecules are not justified. The LPV analysis of the trace amine-associated receptor family and its experimental validation is discussed as an example. The value of the 3D receptor model is demonstrated for a class C GPCR family, the metabotropic glutamate receptors.
A Sendai virus expression vector in the form of a transcribing copy-back defective interfering RNA was constructed and shown to efficiently express a tagged matrix protein in the only context of a Sendai virus infection. In an attempt to identify relevant M protein domains involved in viral assembly and budding, a series of deletion mutants were tested for their ability to bind to cellular membrane fractions. The deletion of a region spanning amino acids 105-137 significantly decreased this binding when the protein was expressed in a system driven by the T7 RNA polymerase away from any other viral proteins. Plus or minus charges were introduced in the hydrophobic portion of a predicted amphiphilic helix in this region, and M proteins with altered membrane binding properties were produced. The genes encoding these mutant M proteins were then inserted in the Sendai virus vector and shown to be expressed at levels similar to that of the endogenous wild-type M protein. The presence of a negative charge in the hydrophobic region of the putative amphiphilic helix prevented the incorporation of the mutant protein into virus particles and appeared to decrease the efficiency of virus particle budding. In contrast, the introduction of a positive charge appeared to increase the M mutant uptake into virions. The use a Sendai virus vector has therefore been shown instrumental in the identification of mutant M proteins interfering with the viral assembly-budding process.
Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene coding for α-galactosidase A (α-GalA). The deleterious mutations lead to accumulation of α-GalA substrates, including globotriaosylceramide (Gb3) and globotriaosylsphingosine. Progressive glycolipid storage results in cellular dysfunction, leading to organ damage and clinical disease, i.e. neuropathic pain, impaired renal function and cardiomyopathy. Many Fabry patients are treated by bi-weekly intravenous infusions of replacement enzyme. While the only available oral therapy is an α-GalA chaperone, which is indicated for a limited number of patients with specific ‘amenable’ mutations. Lucerastat is an orally bioavailable inhibitor of glucosylceramide synthase (GCS) that is in late stage clinical development for Fabry disease. Here we investigated the ability of lucerastat to lower Gb3, globotriaosylsphingosine and lysosomal staining in cultured fibroblasts from 15 different Fabry patients. Patients’ cells included 13 different pathogenic variants, with 13 cell lines harboring GLA mutations associated with the classic disease phenotype. Lucerastat dose dependently reduced Gb3 in all cell lines. For 13 cell lines the Gb3 data could be fit to an IC50 curve, giving a median IC50 [interquartile range (IQR)] = 11 μM (8.2–18); the median percent reduction (IQR) in Gb3 was 77% (70–83). Lucerastat treatment also dose dependently reduced LysoTracker Red staining of acidic compartments. Lucerastat’s effects in the cell lines were compared to those with current treatments—agalsidase alfa and migalastat. Consequently, the GCS inhibitor lucerastat provides a viable mechanism to reduce Gb3 accumulation and lysosome volume, suitable for all Fabry patients regardless of genotype.
3 Inverse agonism of MPEP at hmGlu5a receptors was partially reduced by mutation C57S, significantly reduced by C99S and C57/99S mutations and totally abolished in the tetramutant. 4 We confirmed the surface expression of all the mutated receptors using [ 3 H]MPEP binding analysis on whole cells. However, B max values were increased for mutant C57S, C99S, and C57/99S but decreased in the C57/93/99/129S receptor. 5 The 24 h preincubation of cells expressing hmGlu5a receptors with 1 mM MPEP followed by extensive washing dramatically increased the wild-type receptor efficacy to quisqualate, to the same levels seen with C57/99S receptors. MPEP preincubation did not affect C57/99S function. 6 We conclude that cysteines 57 and 99 are key residues necessary for modulating hmGlu5a receptor function.
Background: Fabry disease (FD) is a lysosomal storage disorder caused by mutations in the GLA gene coding for α-galactosidase A (α-GalA). These mutations lead to the accumulation of α-GalA substrates, including globotriaosylceramide (Gb3). As a consequence of lipid storage, Fabry patients can suffer from neuropathic pain, impaired kidney function and cardiomyopathy. Existing treatments for FD either require biweekly intravenous infusions of replacement enzyme, or are effective in a limited number of patients with specific "amenable" mutations. Substrate reduction therapy with lucerastat, an orally-available small molecule inhibitor of glucosylceramide synthase (GCS) 1 is an alternative mechanism to reduce Gb3 accumulation, that would be suitable for all FD patients. Methods: Fabry patient-derived fibroblasts with the genotypes R301G (residual -GalA activity; 20%) R220X (<3%) and W162X (<1%) were obtained from the Coriell Institute and cultured for 9 days in the presence of 9 concentrations in duplicate of either lucerastat, migalastat or agalsidase alfa. Lysosomes were stained using LysoTracker® Red DND-99 and area was quantified. Sphingolipids were extracted with methanol and quantified with LC-MS/MS. Fabry mice (Gla-/0 and Gla-/-, n = 5 or 6 for each gender) were treated from 5 weeks of age with lucerastat (1200 mg/kg/day food admix) or normal food for 20 weeks. Mice were sacrificed and sphingolipids were quantified in various organs. Results: In Fabry patient-derived fibroblasts, lucerastat dose-dependently inhibited GCS, reducing glucosylceramide and increasing sphingomyelin, while ceramide remained unchanged. The downstream consequence of GCS inhibition was reduction of Gb3 and lysosome staining, including in cells from patients with no residual α-GalA activity. In Fabry mice, lucerastat treatment reduced lipid storage in two major organs affected by FD: mean Gb3 in the kidneys (-33%, p <0.001) and α-galactoseterminated glycosphingolipids in the dorsal root ganglia (-48%, p <0.05). In the liver of the Fabry mice, mean glucosylceramide (GlcCer (24:0)) was reduced (-59%, p <0.001) in addition to Gb3 (24:1) (-37%, p <0.05), demonstrating substrate reduction through GCS inhibition. Conclusion: Lucerastat, a GCS inhibitor, reduces Gb3 in the absence of residual -GalA activity both in vitro and in vivo. Lucerastat has potential to provide an oral substrate reduction therapy for all Fabry patients independent of genotype. A 12-week exploratory clinical study with lucerastat in Fabry patients has been completed, and a pivotal clinical efficacy study in Fabry patients is being designed. Support: References: 1. Guérard (2017) Orphanet J. Rare Dis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.