Of particu lar interest are JAK1 and JAK3 because pharmaceutical inhibitors of these family sellckchem members have demonstrated clin ical efficacy in RA trials. We therefore examined whether RO9021 had any cellular JAK activity. To that end, PBMCs were pretreated with RO9021 prior to stimulation with ei ther IL 2 to activate the JAK1 3 STAT5 pathway in T cells or IFN to activate the JAK1 2 STAT1 pathway. As a posi tive control, the JAK inhibitor tofacitinib was included in the analysis. As shown in Figure 3A, phospho STAT5 staining in the CD3 T cell population was in duced by IL 2 stimulation. At 1 uM, tofacitinib completely blocked the phosphorylation of STAT5 whereas RO9021 had no significant effect. Concentration dependent inhibitory curves also showed significant potency shift between tofacitinib and RO9021, with average IC50 values of 31 13 nM and 1.

32 0. 66 uM, respectively. Similarly, in IFN treated CD14 monocytes, RO9021 had no effect on the phosphorylation Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries of STAT1, except at the highest concentration tested. In contrast, tofacitinib inhibited STAT1 phosphorylation in a concentration dependent manner with an average IC50 value of 81 50 nM. RO9021 thus did not appear to ap preciably inhibit the JAK STAT pathway in the cell, fur ther supporting the selectivity of the compound. SYK kinase activity is essential for osteoclastogenesis Bone destruction is one of the hallmarks of RA and is mainly attributed to an abnormal activation and differen tiation of macrophages into osteoclasts that mediate bone erosion. We therefore assessed the Inhibitors,Modulators,Libraries effects of RO9021 on osteoclastogenesis using mouse bone marrow derived macrophages.

Mouse bone marrow macrophages were differentiated by treatment with soluble receptor activator Inhibitors,Modulators,Libraries of nuclear factor kappa B ligand and monocyte colony stimulating factor in the presence of RO9021 for about 3 days. As shown in Figure 4, exposure to RO9021 abrogated the formation of multinuclear TRAP osteoclasts in a concentration dependent manner, TRAP cells were barely detectable Inhibitors,Modulators,Libraries in the presence of more than 0. 4 uM RO9021. The results suggest inhibition of SYK kinase activ ity may prevent bone erosion in arthritis, which is consist ent with previous SYK knockout mice studies. RO9021 reveals a novel role for SYK in Toll like receptor 9 dependent signaling As BTK has been implicated in TLR signaling, we next sought to explore the kinase function of SYK in TLR9 mediated responses in human B cells and pDCs with selective SYK inhibitor.

Interestingly, we found that the kinase function of SYK was important for mediating TLR9 responses. As demon strated previously, IFN when used in combination with TLR9 ligand, ODN2006, synergistically Abiraterone molecular weight activated B cells as measured by the production of IL 6, which was inhibited by RO9021. In addition, TLR9 dependent B cell proliferation was also dose dependently inhibited by RO9021.