The 5,6 dimethylpyridine group of RO9021 projected out over to Gly454 and Pro455, making hydrophobic contacts. A proline at this position in the ATP binding site is rare in kinases, present in only nine out of a total of 433 kinases, so these interactions probably contribute to the high selectivity of this compound for SYK. RO9021 selectively suppresses B cell receptor signaling Since SYK is best studied as a key mediator of BCR acti vating signals within B cells, we first evaluated the effect of RO9021 in blocking BCR dependent responses. The human B cell line, Ramos, was pretreated with 1 uM RO9021 prior to anti IgM antibody induced cross linking of the BCR. The activation of various BCR signaling com ponents was assessed by western blot using phospho specific antibodies.

As shown in Figure 2A, treatment with RO9021 inhibited anti IgM induced phosphorylation of BTK, PLC��2, AKT and ERK, indicating that blockade of SYK kinase activity by RO9021 resulted in attenuation of BCR downstream signaling cascade. Next, we examined the effect of RO9021 in several functional outcomes of BCR signaling using both human B cell lines and primary cells. Consistent with the known biology of SYK, RO9021 blocked anti IgM activated calcium flux in Ramos B cell line with an IC50 value of 78 21 nM. This effect is specific to BCR signaling because RO9021 showed about 12 fold potency shift in blocking T cell receptor induced calcium flux in Jurkat, a human T cell line. Finally, when tested in human PBMCs or whole blood, RO9021 inhibited BCR dependent cell sur face CD69 expression in CD20 B cells with IC50 values of 83 nM and 87 nM, respectively.

Selective inhibition of Fc receptor signaling Drug_discovery in monocytes and mast cells by RO9021 SYK is also recruited into activated Fc receptor through an interaction with the phosphorylated ITAM motifs of the receptor and mediates Fc receptor downstream signal ing. We therefore examined the effects of inhibiting SYK kinase activity with RO9021 on Fc��R signaling in human monocytes and Fc��R signaling in human mast cells. As shown in Figure 2E, the production of the proin flammatory cytokine TNF induced by crosslinking of Fc��R on human monocytes was inhibited by RO9021 with an IC50 value of 63 19 nM. In contrast, RO9021 had very weak effect on Toll like receptor 4 dependent TNF production in monocytes stimulated by lipopolysaccharide, indicating that RO9021 blocks the Fc��R pathway in a specific manner. Further more, RO9021 also displayed a similar inhibitory potency in a Fc��R mediated mast cell acti vation and degranulation assay, as judged by inhibition of IgE/antigen induced histamine release.