A

A. being crucial for GSK682753A inverse agonism because Ala substitution resulted in a >500-fold decrease in IC50. In conclusion, we present the first ligand targeting EBI2. In turn, this molecule provides a useful tool for further characterization of EBI2 MAP2K2 as well as serving as a potent lead compound. phenomenon was first exhibited by Costa and Herz (8) and has since been shown to be a physiologically relevant aspect of 7TM receptor signaling. For example, several naturally occurring activating mutations found in endogenous 7TM receptors have been linked to changes in phenotype or induction Tucidinostat (Chidamide) of pathogenesis. In addition to the many endogenous constitutively active 7TM receptors, several virus-encoded 7TM receptors are highly constitutively active, including the human cytomegalovirus (CMV)-encoded receptor US28 (9, 10), the human herpesvirus 8 (HHV8)-encoded ORF74 (11, 12), and the EBV-encoded BILF1 (13, Tucidinostat (Chidamide) 14). Although the precise role of the constitutive activity has to be fully elucidated, it has been speculated that it manipulates signaling pathways of the host cell in order to circumvent immune surveillance, thus increasing overall virus survival (15). Furthermore, in immune-compromised hosts, reactivation of a latent infection of these viruses can lead to severe disease even with fatal outcome like in CMV-encephalitis, HHV8-mediated Kaposi’s sarcomas, and EBV-mediated lymphoproliferative disorders (15, 16). In the case of Kaposi’s sarcoma, ORF74 is usually believed to be one of the primary oncogenes because transgenic mice expressing this receptor develop lesions closely resembling those of the human disease (17, 18) in agreement with its proliferative capability (11). Importantly, the constitutive activity of ORF74 was shown to be essential for this process because Tucidinostat (Chidamide) mice expressing non-constitutively active ORF74 mutants had a marked lower index of tumorigenesis (19). In support of this observation, cells expressing non-constitutively active US28 mutants were less efficient in promoting tumor development compared with WT counterparts when injected into nude mice (20). In turn, this indicates that US28 could function as an oncogene in CMV-related proliferative diseases. Finally, a recent study of BILF1 showed that also this receptor processes proliferative and cell transforming properties directly linked to its constitutive signaling (21). Like HHV8 and CMV, EBV is usually associated with proliferative diseases, including infectious mononucleosis, nasopharyngeal carcinoma, Burkitt’s lymphoma, and posttransplant lymphoproliferative disease (PTLD) (15, 22). The molecular key players in these diseases still have to be elucidated. However, given the association between 7TM receptor constitutive activity and tumorigenesis, combined with the marked increase in EBI2 expression levels upon EBV contamination, we speculate that EBI2 might be one such player. In agreement with this, expression of EBI2 has been found to be elevated in EBV-positive compared with EBV-negative PTLD samples (23). In this study, we present GSK682753A, the first ligand to target EBI2. GSK682753 served as a selective and highly potent inverse agonist for murine as well as human EBI2 with inhibition of G protein-dependent signals as well as signals that are probably G protein-independent. In addition, GSK682753A and two structurally related compounds (GSK682756A and SB477865) inhibited antibody-induced proliferation of murine WT Tucidinostat (Chidamide) B cells more potently than EBI2-deficient counterparts. Finally, by mutational analysis and docking simulation, we propose a binding mode for GSK682753. EXPERIMENTAL PROCEDURES Materials Murine EBI2 was cloned in-house using genomic DNA from murine spleen and corresponded to GenBankTM accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_183031″,”term_id”:”88853592″,”term_text”:”NM_183031″NM_183031. Human EBI2 was kindly provided by H. R. Luttichau (University of Copenhagen) and corresponded to GenBankTM accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_004951″,”term_id”:”1519241875″,”term_text”:”NM_004951″NM_004951. The promiscuous chimeric G protein G6qi4myr (Gqi4myr) was kindly provided by Evi Kostenis (Rheinische Friedrich-Wilhelm University, Bonn, Germany). LipofectamineTM 2000 reagent and Opti-MEM were purchased from Invitrogen. SteadyLite (Lyophilized Substrate Answer) was from Packard (Boston, MA). Goat anti-mouse horseradish peroxidase-conjugated antibody was from Pierce, whereas mouse anti-FLAG (M1) antibody, forskolin, and pertussis toxin were from Sigma. Both SlowFade antifade reagent and goat anti-mouse Alexa Fluor 488- and 568-conjugated antibodies were from Molecular Probes (Carlsbad, CA). The 3,3,5,5-tetramethylbenzidine.