Fostamatinib (R788) is a small-molecule inhibitor and an oral prodrug of R406, a Syk/FLT3-targeted inhibitor (IC50 = 41 nM) that also inhibits Lyn (IC50 = 63 nM) and Lck (IC50 = 37 nM), featuring oral activity and cell permeability, used for the treatment of immune thrombocytopenia.

Lck:37 nM, Syk/FLT3:30 nM (Ki), Syk:41 nM, Lyn:63 nM

Fluorescence polarization kinase assay and Ki determination: 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.

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. (Only for Reference)

福他替尼, R788

H2O: < 1 mg/mL (insoluble or slightly soluble)

Ethanol: < 1 mg/mL (insoluble or slightly soluble)

DMSO: 127.5 mg/mL (219.65 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 4 mg/mL (6.89 mM), Sonication is recommended.