KenPaullone (9-Bromopaullone), a potent CDK1, CDK2 and CDK5 inhibitor, as new enhancer for iTreg cell differentiation. Kenpaullone promotes iTreg cell differentiation through increased and prolonged transcription of foxp3 gene by enhancing TGFβ-Smad3 signaling pathway.

CDK5-p35:0.85μM, CDK2-CyclinA:0.68μM, CDK2-CyclinE:7.5μM, CDK1-CyclinB:0.4μM, GSK-3β:0.23μM

在HEK-293细胞中，长时间孵育与Kenpaullone后，内源性GSK3α在Tyr279位点的磷酸化显著降低。同时，内源性GSK3β的磷酸化也有所下降，但程度较轻。Kenpaullone还引起SH-SY5Y细胞和PC12细胞中两种GSK3亚型的去磷酸化。不论GSK3在Sf21细胞还是在E. coli中表达，Kenpaullone（20 μM）强烈抑制了GSK3β在Tyr216位点的自磷酸化[2][3]。

IC50 determination: Active GST-LRRK2 (1326-2527), GST-LRRK2 [G2019S] (1326-2527), GST-LRRK2 [A2016T] (1326-2527) and GST-LRRK2 [A2016T+G2019S] (1326-2527) enzyme is purified with glutathione sepharose from HEK293 cell lysate 36 h following transient transfection of the appropriate cDNA constructs. Peptide kinase assays, performed in duplicate, are set up in a total volume of 40 μL containing 0.5 μg LRRK2 kinase (which at approximately 10% purity gives a final concentration of 8 nM) in 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 10 mM MgCl2, 20 μM Nictide, 0.1 μM [γ-32P]ATP (~500 cpm/pmol) and the indicated concentrations of inhibitor dissolved in DMSO. After incubation for 15 min at 30 °C, reactions are terminated by spotting 35 μL of the reaction mix onto P81 phosphocellulose paper and immersion in 50 mM phosphoric acid. Samples are washed extensively and the incorporation of [γ-32P]ATP into Nictide is quantified by Cerenkov counting. IC50 values are calculated with GraphPad Prism using non-linear regression analysis.