Ligand Modulation of the Epstein-Barr Virus-induced Seven-transmembrane Receptor EBI2

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 (GTP␥S) 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, GTP␥S 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.The Epstein-Barr virus (EBV) induced receptor 2 (EBI2; also known as GPR183) is an orphan member of the 7TM 2 receptor family A. EBI2 is up-regulated up to 200-fold in B cells following EBV infection (1). In agreement with an expression pattern primarily restricted to immunological cell types (1, 2), it was recently demonstrated that EBI2 orchestrates the positioning of B cells in the follicle (3, 4). Specifically, expression of EBI2 allowed activated B cells to translocate to the outer follicular regions; in contrast, the absence of EBI2 permitted the cells to enter the germinal centers. Thus, EBI2 mediates the correct localization of B cells during humoral immune responses. To date, no endogenous ligand for EBI2 has been identified. Given that this receptor does not have a close homolog, the chemical nature of such a ligand is difficult to infer. Accordingly, EBI2 has been placed in varying 7TM receptor subgroups by different phylogenetic analyses as being a target of peptide (1, 5) or lipid ligands (6). Nevertheless, many aspects of EBI2 signaling have been revealed. Thus, we have previously demonstrated that EBI2 is constitutively active through G␣ i (but not G␣ q or G␣ s ) and serum response element (but not NFAT or NFB...

Oxysterol Gradient Generation by Lymphoid Stromal Cells Guides Activated B Cell Movement during Humoral Responses

“…As a result, these cells are less strongly attracted to the B-T zone interface and relocate to the outer follicle in an EBI2-dependent manner. A recent study suggested EBI2 transmits pro-proliferative signals to B cells (Benned-Jensen et al, 2011). We have not found 7α,25-OHC to have mitogenic effects on B cells (T.Y., L.M.…”

Identification of Structural Motifs Critical for Epstein-Barr Virus-Induced Molecule 2 Function and Homology Modeling of the Ligand Docking Site

“…Activation of EBI2 leads to adenylate cyclase inhibition and subsequent reduction of cAMP production. EBI2 also was reported to activate extracellular signal-regulated kinase (Benned-Jensen et al, 2011).…”

“…Previous structure-function studies with EBI2 were focused largely on investigating its constitutive activity. Those studies revealed important structural motifs for the ligand-independent activity of the receptor and suggested potential molecular constraints during receptor transition from the inactive conformation to the active conformation (Benned-Jensen and Rosenkilde, 2008;Benned-Jensen et al, 2011). We decided to explore the molecular mechanisms of ligand-dependent activation of EBI2 by using 7␣,25-OHC as a tool.…”

Patent Highlights April–May 2015

“…B cells with EBI2 knocked out exhibit defective migration, resulting in strongly impaired T-cell-dependent antibody responses. Selective EBI2 antagonists, such as ML401 which has a central piperazine core, have been reported [10], and also a spirocyclic inverse agonist (GSK682753 from GlaxoSmithKline, Brentford, UK) [11] on which the compounds of this invention have been modeled, probably aiming at increasing the metabolic stability beyond that of GSK682753. Biological data are all but absent, although a cell-based luminescent assay is stated to have shown IC 50 values 'below 100 nM' for many of the compounds that were chemically identified in detail.…”