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  • The expression of EBI has been

    2020-02-18

    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 Oseltamivir 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
    Results
    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. Collectively, our results indicate that GSK682753A functions as a competitive antagonist and binds to the receptor in the same region as 7α,25-OHC. First, we observe linearity in the Schild plot analysis (Fig. 2B). Second, GSK682753A is highly dependent on F111 at position III:08/3.32 in TM-III (Fig. 3B). Although this is not the case for 7α,25-OHC (Fig. 3A), it has recently been shown that the TM-III residues Y112 at position III:09/3.33 (just next to F111) and Y116 at III:13/3.37 are crucial for agonist binding [9,10] indicating that the two ligands bind in the same region. Interestingly, the primary uHTS screen at Sanford-Burnham Center for Chemical Genomics identified 2946 compounds out of 364,168 tested that were able to suppress 7α,25-OHC-induced β-arrestin recruitment at 5 μM by more than 50% (PubChem BioAssay ID: 651636). Of note, the most efficacious of the active compounds are very similar in structure to GSK682753A. For instance, the best compound (CID: 5804570) also has two chlorine-substituted benzene-rings at each extremity, an enone moiety and a centrally-located nitrogen-containing ring (in this case a piperazine). Thus, it seems that these structural traits are important for efficacious inhibition of EBI2 activity. It should be noted that we first characterized GSK682753A as an inverse agonist [16]. However, new data indicate that a part of the constitutive activity of EBI2 may be a result of oxysterol contamination in the medium [9]. Thus, the inverse agonism of GSK682753A might have been antagonistic. It is possible that EBI2 is constitutively active; however, presently we cannot assess the magnitude of this (if any) with the tools available.