Identification of the Syk kinase inhibitor R112 by a human mast cell screen

Background: Activation of the IgE receptor, FcvarepsilonRI, in mast cells is the key mechanism initiating and propagating pathophysiological responses in allergic rhinitis.

Objective: Identify and characterize a small molecule inhibitor of IgE-dependent mast cell activation for the treatment of allergic diseases.

Methods: A cell-based high-throughput screen for small molecules that block IgE signaling was performed in cultured human mast cells. A potent inhibitor, referred to as R112, was selected and characterized by using biochemical and cell-based assays. R112 effects on IgE-dependent degranulation and cytokine production was measured in mast cells and basophils and compared with other mast cell inhibitors.

Results: R112 inhibited degranulation induced by anti-IgE cross-linking in mast cells (tryptase release, effective concentration for 50% inhibition [EC(50)] = 353 nmol/L) or basophils (histamine release, EC(50) = 280 nmol/L), and by allergen (dust Mite) in basophils (histamine release, EC(50) = 490 nmol/L). R112 also blocked leukotriene C4 production and all proinflammatory cytokines tested. Subsequent molecular characterization indicated that R112 is an ATP-competitive spleen tyrosine kinase (Syk) inhibitor (inhibitory constant [K(i)] = 96 nmol/L). Its onset of action was immediate, and the inhibition was reversible. Incubation of mast cells with R112 showed that cytokine production in mast cells was dependent on sustained activation of the FcvarepsilonRI-Lyn-spleen tyrosine kinase pathway. Unlike other mast cell inhibitors, R112 was able to completely inhibit all three IgE-induced mast cell functions: degranulation, lipid mediator production, and cytokine production.

Conclusion: R112 potently, completely, and rapidly abrogated all mast cell activation cascades triggered by IgE receptor cross-linking.

Clinical implications: R112 and its analogues offer a new modality in the treatment of allergic rhinitis.