Fig. 6: Cellular mechanism of action of Kenpaullone in central neurons.
a Left diagram: DARTS methodology to identify proteins that bind to KP in rat primary cortical neurons, KP applied for 30 h at DIV5 (see the “Methods” section/Supplementary Methods. KP binding to GSK3β is independent of detergent treatment of the protein sample preparation. Of note, KP binding to GSK3ß was documented whereas binding to CDKs was not (see Supplementary Data File S2 DARTS). Right-hand: upper structure is the crystal structure of human GSK3ß (pdb 6V6L), the boxed area shows its ATP-binding pocket, note detail below with KP binding within the ATP pocket, with Val135 forming a hydrogen bond to the KP pyrroline-N, as determined by molecular dynamics simulation (see Supplementary Fig. 6a and Supplementary Information/Supplementary Methods). b Upper diagram: Phosphoproteomics assays reveal S259 phosphorylation target in δ-cat protein after KP treatment (1 µM for 1 h/24 h) of rat primary cortical neurons (DIV5), significant de-phosphorylation resulted after 1 h treatment and was sustained at 24 h (see Supplementary Data File S3 phosphoproteomics). Lower panel: schematic representation of the structure of human δ-cat (CTNND2) showing functional domains. Human residue S276 matches rat S259. The Armadillo domain region plays a key role in transcription factor Kaiso binding to δ-cat. c Top micrograph panel: Representative immuno-labeling of neuronal ßIII-tubulin (green) and δ-cat (red) before and after KP treatment (100 nM, DIV5–8) in rat primary cortical neurons, DAPI counterstain for nuclei (blue). Bottom bar diagram: KP significantly increases δ-cat nuclear abundance (relative abundance normalized for cytoplasmic abundance; see also Supplementary Fig. 6b and Supplementary Methods). Data are represented as mean values ± SEM. n = 33 (vehicle control), n = 34 (KP) neurons/group,****p < 0.001 KP vs. vehicle, two-sided t-test. d Top panel: Representative immuno-labeling of ßIII-tubulin and δ-cat before and after KP treatment (100 nM for 3 days) in differentiated N2a mouse neural cells (see Supplementary Fig. 6e), which were transfected with either hu-δ-cat(WT) or hu-δ-cat(S276A). Bottom Panel: KP significantly enhances the nuclear abundance of δ-cat when transfected with δ-cat(WT); same measurement method as in (c), mean of hu-δ-cat(WT)/vehicle-treated subgroup set as “1” with a subsequent adaptation of all other groups. Mutation hu-δ-cat(S276A) increases nuclear abundance, but treatment with KP has no significant effect on cells expressing it. Data are represented as mean values ± SEM. n = 25–30 neurons/group (n = 30 WT/vehicle control, n = 27 WT/KP; n = 28 S276A/vehicle control, n = 25 S276A/KP) ****p < 0.0001 vs. vehicle, mixed effects statistics. Source data are provided as source data file.
