| In Vitro | In vitro activity: R788 is a prodrug of the spleen tyrosine kinase (Syk) inhibitor R406. R788 is a competitive inhibitor for ATP binding with a Ki of 30 nM. R788 dose-dependently inhibits anti-IgE-mediated CHMC degranulation with an EC50 of 56 nM. R788 also inhibits the anti-IgE-induced production and release of LTC4 and cytokines and chemokines, including TNFα, IL-8, and GM-CSF. Inhibition of Syk by R788 results in inhibition of all phosphorylation events downstream of Syk signaling. Next to FcεRI signaling in CHMC, R788 most potently inhibits the signaling of IL-4 and IL-2 receptors. R788 specifically inhibits FcγR signaling in human mast cells, macrophages, and neutrophils. R788 can inhibit local inflammatory injury mediated by immune complexes. R788 induces apoptosis of the majority of examined DLBCL cell lines. In R788-sensitive DLBCL cell lines, R788 specifically inhibits both tonic- and ligand-induced BCR signaling (autophosphorylation of SYK525/526 and SYK-dependent phosphorylation of the B-cell linker protein [BLNK]).
Kinase Assay: The fluorescence polarization reactions are performed. For Ki determination, duplicate 200-μL reactions are set up at eight different ATP concentrations from 200 μM (2-fold serial dilutions) in the presence of either DMSO or R788 at 125, 62.5, 31.25, 15.5, or 7.8 nM. At different time points, 20 μL of each reaction is removed and quenched to stop the reaction. For each concentration of R788, the rate of reaction at each concentration of ATP is determined and plotted against the ATP concentration to determine the apparent Km and Vmax (maximal rate). Finally the apparent Km (or apparent Ki/Vmax) is plotted against the inhibitor concentration to determine the Ki.
Cell Assay: Cultured human mast cells (CHMC) are derived from cord blood CD34+ progenitor cells and grown, primed, and stimulated and shown in supplemental data. Before stimulation, cells are incubated with R788 or DMSO for 30 minutes. Cells are then stimulated with either 0.25 to 2 mg/mL anti-IgE or anti-IgG or 2 μM ionomycin. For tryptase measurement, ~1500 cells per well are stimulated for 30 min in modified Tyrode's buffer. For LTC4 and cytokine production, 100,000 cells per well are stimulated for 1 or 7 hours, respectively. Tryptase activity is measured by luminescence readout of a peptide substrate, and LTC4 and cytokines are measured using Luminex multiplex technology. |
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| In Vivo | Oral administration of R788 to mice reduces immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. In another study, R788 effectively inhibits BCR signaling in vivo, resulting in reduced proliferation and survival of the malignant B cells and significantly prolongs survival of the treated animals. R788 demonstrates a significant reduction in major inflammatory mediators such as TNFalpha, IL-1, IL-6 and IL-18, leading to reduced inflammation and bone degradation in models of rheumatoid arthritis. |
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