In vitro activity: BI-78D3 Inhibits JNK interacting protein 1 (JIP1)-JNK binding and prevents JNK substrate phosphorylation

Kinase Assay: 1) BI 78D3 Inhibits JNK interacting protein 1 (JIP1)-JNK binding (IC50= 500 nM) and prevents JNK substrate phosphorylation; 2) I-78D3 not only blocks JNK dependent Con A-induced liver damage but also restores insulin sensitivity in mouse models of type 2 diabetes. The cell based kinase assays for c-Jun and ATF2 phosphorylation carry out by using the LanthaScreen c-Jun (1-79) HeLa and LanthaScreen ATF2 (19-106) A549 cell lines which stably express GFP-c-Jun 1-79 and GFP-ATF2 19-106, respectively. Phosphorylation is determined by measuring the time resolved FRET (TR-FRET) between a terbium labeled phospho-specific antibody and the GFP-fusion protein. The cells are plated in white tissue culture treated 384 well plates at a density of 10,000 cell per well in 32 μl assay medium (supplemented with 1% charcoal/dextran-treated FBS, 100 U/mL Penicillin and 100 μg/mL Streptomycin, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 25 mM Hepes, pH 7.3, and lacking phenol red). After overnight incubation, cells are pretreated for 60 min with BI-78D3 (0.001, 0.01, 0.1, 1, 10, and 100 μM) followed by 30 min of stimulation with 2 ng/mL of TNF-α that stimulates both JNK and p38. The medium is then removed by aspiration and the cells are lysed by adding 20 μL of lysis buffer (20 mM TrisoHCl, pH 7.6, 5 mM EDTA, 1% Nonidet P-40 substitute, 5 mM NaF, 150 mM NaCl, and 1:100 protease and phosphatase inhibitor mix, SIGMA P8340 and P2850, respectively). The lysis buffer includs 2 nM of the terbium-labeled anti-pc-Jun (pSer73) or anti-pATF2 (pThr71) detection antibodies. After allowing the assay to equilibrate for 1 h at room temperature, TR-FRET emission ratios are determined on a BMG Pherastar fluorescence plate reader (excitation at 340 nm, emission 520 nm and 490 nm; 100 μs lag time, 200 μs integration time, emission ratio=Em520/Em 490).

Cell Assay: Cells [Osteosarcoma cells (SaOS-2, MG-63, U2OS and LM7), primary (non-malignant) human osteoblasts(Hum63, Hum65 and Hum71)] are subjected to a dose range of doxorubicin concentrations in the presence or absence of a non-toxic dose (10 nM) of the small molecule JIP1-inhibitor BI-78D3.