D4476 (Casein Kinase I Inhibitor) is an effective, selective, and cell-permeant CK1 (casein kinase 1) inhibitor( IC50=300 nM in a cell-free assay).

CK1:300 nM

D4476（10 μM）对CK1和ALK5的抑制作用分别让其活性降至对照组的约7%和22%。在H4IIE肝癌细胞中，D4476特异性抑制内源性叉头框转录因子O1a（FOXO1a）在Ser322和Ser325上的磷酸化，而对其他位点的磷酸化无影响。[1] D4476在ANBL6、INA6和RPMI8226细胞系中诱导细胞毒性；MM1S和U266细胞系较不敏感；OPM1细胞系完全耐受。高浓度（50 μM）的D4476对所有多发性骨髓瘤（MM）细胞系均产生毒性。在MM细胞中，D4476提高了TP53、P27和FADD的蛋白水平，以及细胞周期的进展和凋亡的诱导。[2] 对白血病干细胞（LSCs）进行D4476处理，显示出对LSCs的高度选择性杀伤作用，而对正常HSPCs的影响较小。[3]

CK1 kinase assay: All protein kinase assays (25 μL) are carried out at room temperature (21℃). Assays are performed for 40 min using a Biomek 2000 Laboratory Automation Workstation in a 96-well format. The concentrations of magnesium acetate and [γ-33P]ATP (800 cpm/pmol) in the assays are 10 mM and 0.1 mM, respectively. Assays are initiated with MgATP and stopped by the addition of 5 μL of 0.5 M orthophosphoric acid. Aliquots are then spotted on to P30 ?ltermats, washed four times in 75 mM phosphoric acid to remove ATP, once in methanol, then dried and counted for radioactivity. CK1 δ (5-20 m-units), diluted in 20 mM Hepes, pH 7.5, 0.15 M NaCl, 0.1 mM EGTA, 0.1% (v/v) Triton X-100, 5 mM dithiothreitol, 50% (v/v) glycerol, is assayed against the peptide RRKDLHDDEEDEAMSITA in an incubation containing 20 mM Hepes, pH 7.5, 0.15 M NaCl, 0.1 mM EDTA, 5 mM DTT, 0.1% (v/v) Triton X-100 and 0.5 mM substrate peptide.

MM cells are seeded in triplicate into 96-well plates in 100 μL culture media. D4476 is added to each well at concentrations of 0, 5, 10, 20, 30, 40, and 50 μM in another 100 μL culture media. Cell viability is measured with MTT at the 72 h drug exposure. Absorbance is measured at 570 nm with spectrophotometer.(Only for Reference)