GSK682753A is a novel, potent, and selective inverse agonist of the epstein-barr virus-induced receptor 2 (EBI2) with an IC50 of 53.6 nM. It has recently been demonstrated that the constitutively active seven-transmembrane Epstein-Barr virus-induced receptor 2 (EBI2) coordinates the location of B cells within the follicle. No endogenous or artificial ligand has been found to date that modifies EBI2 activity. Here, we report on the high potency and effectiveness of an inverse agonist, GSK682753A, which specifically inhibited the constitutive activity of EBI2. This compound demonstrated a potency of 2.6-53.6 nm and an inhibitory efficacy of 75% in guanosine 5'-3-O-(thio)triphosphate (GTPγS) binding as well as cAMP-response element-binding protein-based reporter assays. Furthermore, we demonstrate the insensitive activation of extracellular signal-regulated kinase (ERK) by EBI2. The inhibition of ERK phosphorylation, GTPγS binding, and cAMP-response element-binding protein activation by GSK682753A was intriguing because it was equally potent. The proliferation of murine B cells stimulated by antibodies in vivo was significantly enhanced when EBI2 was overexpressed in comparison to WT cells; however, this was likewise diminished in B cells lacking EBI2. The proliferation was always inhibited by blocking EBI2 constitutive activity. Interestingly, compared to EBI2-deficient B cells, WT or EBI2-overexpressing B cells showed a much stronger (32-fold) suppression. To find potential molecular binding determinants in EBI2, GSK682753A was lastly screened against a library of EBI2 mutants. Since Ala substitution caused IC(50) to decrease by more than 500 times, Phe(111) at position III:08/3.32 was found to be essential for GSK682753A inverse agonism. We have now presented the first ligand that targets EBI2. This molecule therefore functions as a powerful lead compound and a helpful tool for additional EBI2 characterization.

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