In vitro activity: AS601245 is an inhibitor of the JNK signaling pathway, it showed high JNK1/2/3 inhibition with IC50 of 150, 220, and 70 nM respectively. AS601245 is selective against p38 (IC50 of 4800 nM). The compound showed efficacy in the in vivo rheumatoid arthritis (RA) model by reducing the in vivo production of TNFα and arthritis severity. Extensive biological studies have been conducted on AS601245 to explore the anti-cancer and anti-neurodegenerative potential of this novel JNK inhibitor. AS601245 promotes cell survival after cerebral ischemia.

Kinase Assay: Rat JNK3 assays are performed in 96-well low binding Corning MTT plates: 0.5 μg of recombinant, preactivated GST-JNK3 is incubated with 1 μg of recombinant, biotinylated GST-c-Jun and 2 μM [33Pγ]ATP (2 nCi/μl), in the presence or absence of compounds according to formula I and in a reaction volume of 50 μL containing 50 mM Tris-HCl, pH 8.0; 10 mM MgCl2, 1 mM Dithiothreitol, and 100 μM NaVO4, for 120 min and at room temperature. The reaction is stopped by the addition of 200 μL of a solution containing 250 μg of Streptavidin-coated SPA beads, 5 mM EDTA, 0.1% Triton X-100, and 50 μM ATP, in phosphate saline buffer and further incubation at room temperature for 60 min. After incubation, beads are sedimented by centrifugation at 1500g for 5 min, resuspended in 200 μL of phosphate-buffered saline (PBS) containing 5 mM EDTA, 0.1% Triton X-100, and 50 μM ATP and the radioactivity is measured in a scintillation beta counter, following further sedimentation of the beads by settling down for 60 min at room temperature. Similar method is used to demonstrate inhibition of JNK1 and JNK2.

Cell Assay: CaCo-2 cell proliferation is evaluated by using the kit “CellTiter-Glo Luminescent Cell Viability Assay”. This highly sensitive assay detects the luminescence released by the metabolically active cells. Quantification of luminescence is expressed as RLU (relative light unit). For the proliferation experiments, treatments are performed by adding the drugs (e.g., 0.1 μM AS601245) to the CaCo-2 cells seeded at about 4,000 cells/well in a 96-well plate. HepG2 cell proliferation is analysed through the MTT method. Briefly, 1500 cells/well are seeded in 200 μL of serum-supplemented media and the following day treated with the drugs (e.g, 0.1 μM AS601245). 20 μL of 5 mg/mL thiazolyl blue tetrazolium bromide is subsequently added to the cells and removed 2 hours later. 100 μL of DMSO is added to the cells, and the absorbance is recorded at 570 nm through a 96 well plate ELISA reader. Viability is evaluated through Trypan blue exclusion test.