title: Ligand modulation of the Epstein-Barr virus-induced seven-transmembrane receptor EBI2: identification of a potent and efficacious inverse agonist.
abstract: The Epstein-Barr virus-induced receptor 2 (EBI2) is a constitutively active seven-transmembrane receptor, which was recently shown to orchestrate the positioning of B cells in the follicle. To date, no ligands, endogenously or synthetic, have been identified that modulate EBI2 activity. Here we describe an inverse agonist, GSK682753A, which selectively inhibited the constitutive activity of EBI2 with high potency and efficacy. In cAMP-response element-binding protein-based reporter and guanosine 5'-3-O-(thio)triphosphate (GTPγS) binding assays, the potency of this compound was 2.6-53.6 nm, and its inhibitory efficacy was 75%. In addition, we show that EBI2 constitutively activated extracellular signal-regulated kinase (ERK) in a pertussis toxin-insensitive manner. Intriguingly, GSK682753A inhibited ERK phosphorylation, GTPγS binding, and cAMP-response element-binding protein activation with similar potency. Overexpression of EBI2 profoundly potentiated antibody-stimulated ex vivo proliferation of murine B cells compared with WT cells, whereas this was equivalently reduced for EBI2-deficient B cells. Inhibition of EBI2 constitutive activity suppressed the proliferation in all cases. Importantly, the suppression was of much higher potency (32-fold) in WT or EBI2-overexpressing B cells compared with EBI2-deficient counterparts. Finally, we screened GSK682753A against an EBI2 mutant library to determine putative molecular binding determinants in EBI2. We identified Phe(111) at position III:08/3.32 as being crucial for GSK682753A inverse agonism because Ala substitution resulted in a >500-fold decrease in IC(50). In conclusion, we present the first ligand targeting EBI2. In turn, this molecule provides a useful tool for further characterization of EBI2 as well as serving as a potent lead compound.
authors.affiliation: Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Tau
authors.initials: T
authors.lastname: Benned-Jensen
authors.affiliation: GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom.
authors.forename: Christopher
authors.initials: C
authors.lastname: Smethurst
authors.affiliation: Department of International Health, Immunology, and Microbiology, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark, and.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Holst
authors.affiliation: GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom.
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Page
authors.affiliation: GlaxoSmithKline, Research Triangle Park, North Carolina 27709.
authors.forename: Howard
authors.initials: H
authors.lastname: Sauls
authors.affiliation: Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Bjørn
authors.initials: B
authors.lastname: Sivertsen
authors.affiliation: Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Thue W
authors.initials: TW
authors.lastname: Schwartz
authors.affiliation: GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom.
authors.forename: Andy
authors.initials: A
authors.lastname: Blanchard
authors.affiliation: GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom.
authors.forename: Robert
authors.initials: R
authors.lastname: Jepras
authors.affiliation: Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark,. Electronic address: rosenkilde@sund.ku.dk.
authors.forename: Mette M
authors.initials: MM
authors.lastname: Rosenkilde
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006576:Heterocyclic Compounds, 4 or More Rings / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D008817:Mice, Mutant Strains; D010080:Oxazoles / Q000737:chemistry / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000502:physiology; D013141:Spiro Compounds / Q000737:chemistry / Q000494:pharmacology*
chemical_list: C561772:8-(3-(4-chlorophenyl)prop-2-enoyl)-3-((3,4-dichlorophenyl)methyl)-1-oxa-3,8-diazaspiro(4.5)decan-2-one; C543809:Gpr183 protein, mouse; D006576:Heterocyclic Compounds, 4 or More Rings; D010080:Oxazoles; D043562:Receptors, G-Protein-Coupled; D013141:Spiro Compounds; D048049:Extracellular Signal-Regulated MAP Kinases