In vitro activity: At high dose, GNE-3511 resulted in a completesuppression of c-Jun phosphorylation, while the low dose reduced the number of p-c-Jun positive cells to an intermediate level. GNE-3511 protected primary neurons in an in vitro axon degeneration assay as well as reduced phosphorylation of the downstream transcription factor c-Jun.

Kinase Assay: GNE-3511 is a novel, highly potent and selective zipper kinase (e.g. DLK, MAP3K12) inhibitor with IC50 of 0.107 uM for DLK. Dual leucine zipper kinase (DLK, MAP3K12) was recently identified as an essential regulator of neuronal degeneration in multiple contexts. At high dose, GNE-3511 resulted in a completesuppression of c-Jun phosphorylation, while the low dose reduced the number of p-c-Jun positive cells to an intermediate level.

Cell Assay: 7500 HEK293 cells stably transfected with Dox-inducible human DLK in 40 μL of DMEM with 10% serum were seeded into each well of 384-well poly-d-lysine coated plates. The plates were incubated at 37 °C for 20–24 h prior to the addition of 5 μL of 60 μM doxycycline in DMEM. After incubation with doxycycline at 37 °C for approximately 20 h, 5 μL of DLK inhibitors in DMEM was added, and cells were incubated at 37 °C for an additional 5.5 h. The cells were then washed with PBS, permeabilized with 0.1% Triton X-100, blocked for 1 h with SuperBlock before the overnight incubation with the primary antibodies at 4 °C. The secondary antibodies were incubated for 2 h, washed with PBS, and then stained with Hoechst 33342 dye. The cell plates were imaged on Opera Imaging Platform.