In vitro activity: 2-D08, a synthetic flavone, is a cell permeable inhibitor of protein sumoylation that has a unique mechanism of action. It is also inhibits Axl, IRAK4, ROS1, MLK4, GSK3β, RET, KDR and PI3Kα with IC50 values of 0.49, 3.9, 5.3, 9.8, 11, 11, 17 and 35 nM respectively in biochemical assays. Protein sumoylation is a dynamic posttranslational modification involved in diverse biological processes during cellular homeostasis and development. Sumoylation has been shown to play a critical role in cancer. 2-D08 showed anti-aggregatory and neuroprotective effect and may be used for the treatment of related diseases.

Kinase Assay: 2-D08 is a cell permeable, mechanistically unique inhibitor of protein sumoylation. It is also inhibits Axl, IRAK4, ROS1, MLK4, GSK3β, RET, KDR and PI3Kα with IC50 values of 0.49, 3.9, 5.3, 9.8, 11, 11, 17 and 35 nM respectively in biochemical assays. IC50 values of the Axl kinase inhibitor (2D08) are determined using kinase-mediated phosphorylation of poly-GAT by AlphaScreen luminescence detection technology. The inhibitor is tested at eight points of dilution in duplicate.

Cell Assay: Human lung multi-potent cells at passage 5 are plated at a density of 250 000 cells per well in six-well plates with growth medium. After 24 h, multi-potent cells are incubated with DMEM+0.5% BSA+penicillin/streptomycin containing 0.1% DMSO (vehicle) or 2D08 (0.1, 1, 10 μM) for 3 h in a humidified 5% CO2 incubator at 37℃. These cells are trypsinized and seeded at 20 000 cells per well in three replicates on 12-well cell culture transwell inserts with 8 μm pore size with DMEM+0.5% BSA+penicillin/streptomycin. Lower transwell chambers contained DMEM+10% FBS+penicillin/streptomycin are used to allow cells to migrate. 0.1% DMSO or 2D08 is added to corresponding upper and lower transwell chambers. After 16 h, non-migrated cells are removed by cotton swabs. Migrated cells are fixed with 4% PFA, permeabilized with methanol and stained with crystal violet. The field-images per transwell are taken by an inverted light microscope.