In vitro activity: BMS-935177 demonstrates> 50-fold selectivity over the SRC family of kinases, including 1100-fold selectivity over SRC itself. It inhibited other kinases with a potency less than 150 nM (50-fold selectivity) included TRKA, HER4, TRKB, and RET. It inhibits calcium flux in human Ramos B cells (IC50 = 27 nM) and inhibits CD69 surface expression in peripheral B cells stimulated with anti-IgM and anti-IgG. However, BMS-935177 has no effect on CD69 surface expression in B cells stimulated through the CD40 receptor with CD40 ligand. Against IgG-containing immune complex-driven low affinity activating Fcγ receptor (FcγRIIa and FcγRIII) end points in peripheral blood mononuclear cells (PBMCs), BMS-935177 effectively inhibited TNFα production with an IC50 value of 14 nM.

Kinase Assay: To V-bottom 384- well plates are added BMS-935177 (10 mM in DMSO), human recombinant BTK (1 nM), fluoresceinated peptide (1.5 μM), ATP (20 μM (Km app)), and assay buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, 0.015% Brij 35 surfactant, and 4 mM DTT in 1.6% DMSO), with a final volume of 30 μL. After incubation at room temperature for 60 min, the reaction is terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture is analyzed on the by electrophoretic separation of the fluorescent substrate and phosphorylated product (excitation, 488 nm; emission, 530 nm).

Cell Assay:

Efficacy studies: BMS-935177 (10 and 30 mg/kg) vs vehicle and dexamethasone (Dex) is studied in a mouse anti-collagen antibody-induced arthritis (CAIA) inflammation model. Mice are injected intraperitoneally (ip) with a mixture of four monoclonal antimouse type II collagen antibodies (1 mg of each). Daily oral dosing is immediately started with vehicle (EtOH:TPGS:PEG300, 5:5:90), BMS-935177 (10 or 30 mg/kg), or dexamethasone (dex, 1 mg/kg). Three days later, the mice are injected ip with 1.25 mg/kg LPS. Thereafter, mice are monitored 3×/ week for the development and severity of paw inflammation