Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to selfantigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs). Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 [N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of immunoglobulin E (IgE)-and IgG-mediated activation of Fc receptor signaling (EC 50 for degranulation ϭ 56 -64 nM). Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR). R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor (K i ϭ 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Sykindependent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.

GTPases regulate a myriad of cellular functions including signal transduction, cytoskeletal organization and membrane trafficking. Rab GTPases act to coordinate the membrane dynamics of cells by organizing and regulating the activity of effector proteins important in vesicle trafficking. Rab37 is a novel Rab GTPase specifically expressed in the MC-9 mast cell line and bone marrow mast cells. Rab37 is 74% identical to Rab26 and 47% identical to Rab8, a GTPase important in Golgi to plasma membrane vesicle trafficking in mammalian cells. When green fluorescent protein tagged Rab37 is expressed in bone marrow mast cells, the secretory granules are labeled. These data suggest that Rab37 may play an important role in mast cell degranulation making this protein a potentially important target for therapeutic intervention in the treatment of allergy.z 2000 Federation of European Biochemical Societies.