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  • br Introduction The Epstein Barr virus


    Introduction The Epstein–Barr virus induced gene 2 (EBI2 also known as GPR183) is a G protein-coupled seven-transmembrane (7TM) receptor that is predominantly expressed in B and T cells [1,2]. It regulates the trafficking of D609 within lymphoid tissues and is highly important for the generation of humoral immune responses [3,4]. EBI2 remained orphan for years; however, two independent studies recently showed that this receptor is activated by oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC) [5,6]. Binding of oxysterols to EBI2 induce Gαi activation, β-arrestin recruitment and ultimately migration of EBI2-expressing B and T cells. Thus, EBI2 functions as a chemo-attractant receptor. Interestingly, the main oxysterol generating cells within the lymphoid tissue were recently shown to be of stromal origin and these are required for efficient T cell-dependent plasma cell responses [7]. Moreover, we [8,9] and others [10] identified residues critical for oxysterol binding to EBI2 showing that the main anchor points are found in TM-II, -III, -VI and ECL2 of which several are located in the minor binding pocket [11]. The expression of EBI2 has been found to be dysregulated in several types of B cell malignancies and is thus reduced in e.g. diffuse large B-cell lymphomas [12] and chronic lymphocytic leukemia [13] and increased in post-transplantation lymphoproliferative disorders (PTLDs) [14]. EBI2 is also highly expressed in EBV-transformed lymphoblastoid B cells which phenotypically resemble PTLDs [15]. We recently showed that increased expression of EBI2 potentiates antibody-induced proliferation in B cells [16]. Thus, in malignancies where EBI2 expression is increased, this receptor may contribute to pathogenesis possibly by potentiating B cell proliferation. In such cases, blocking EBI2 activity could serve as a target for pharmacotherapy. Furthermore, this could also be envisioned to apply in autoantibody-mediated diseases such as lupus and rheumatoid arthritis. Finally, the up-regulation of EBI2 upon EBV infection may function to position B cells in specific lymphoid zones in order to increase overall viral survival. Blocking EBI2 activity may therefore serve as a novel route to treat EBV infection as no EBV-specific drugs are currently available. Of note, EBI2 is expressed both in the latent and lytic infection stages as opposed to e.g. the EBV-encoded 7TM receptor BILF1 or other EBV genes [1,17]. The desire to develop tool compounds for modulating EBI2 activity is exemplified well by an ongoing uHTS screen at the Sanford-Burnham Center for Chemical Genomics where a range of compounds able to antagonize 7α,25-OHC-mediated β-arrestin recruitment has been identified in a primary screen (PubChem BioAssay ID: 651636). Simultaneous to the deorphanization of EBI2, we provided a characterization of a non-peptide inverse agonist (coined GSK682753A) that suppressed the apparent constitutive activity of the receptor [16]. Here we investigate the antagonistic properties of this compound and find that it potently suppresses 7α,25-OHC-mediated Gαi activation, β-arrestin recruitment and chemotaxis of primary B cells ex vivo. Furthermore, for the first time we demonstrate that 7α,25-OHC-induced activation of EBI2 triggers pertussis toxin (ptx)-sensitive MAP kinase phosphorylation which also is suppressed by GSK682753A.
    Materials and methods
    Discussion Much has been learned about the biology and pharmacology of EBI2 in the past 2 years. Thus, both the endogenous agonist [5,6], the cellular producers of this agonist [7] and the molecular pharmacology of 7α,25-OHC, the most potent agonist, [9,10] have all been characterized within this period. In addition, just prior to the deorphanization, we presented a non-peptide compound, GSK682753A, which inhibited the apparent constitutive activity of EBI2 [16]. Here, we have investigated the antagonistic properties of this molecule showing that it blocks oxysterol-induced G protein activation (Figs. 1 and 2), β-arrestin recruitment (Fig. 3A and B), B cell chemotaxis (Fig. 3C and D) and ERK activation (Fig. 4). Compared to the in vitro assays, the potency measured in the ex vivo chemotaxis assay was much higher. At present we cannot explain this difference. However, we have recently characterized a series of CCR8 antagonists and also observed higher potencies in chemotaxis assays compared to IP3 accumulation [18], and in line with the concomitant higher potency of 7α,25-OHC it is possible that an increase in assay sensitivity could be a contributing factor.