D 4476

Catalog No.S7642

D 4476 Chemical Structure

Molecular Weight(MW): 398.41

D 4476 is a potent, selective, and cell-permeant CK1 (casein kinase 1) inhibitor with IC50 of 200 nM and 300 nM in a cell-free assay for CK1 from Schizosaccharomyces pombe and CK1δ, respectively. Also acts as an ALK5 inhibitor with IC50 of 500 nM.

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Cited by 2 Publications

1 Customer Review

  • Western blot of signaling effects. 48 hrs after transfection cells were serum‐starved for 12 h before stimulation with Wnt5a for 30 min or 120 min (200 ng/ml) in the presence or absence of casein kinase inhibitor D4476 (100 μM).

    Mol Oncol, 2016, 10(5):677-92. D 4476 purchased from Selleck.

Purity & Quality Control

Choose Selective Casein Kinase Inhibitors

Biological Activity

Description D 4476 is a potent, selective, and cell-permeant CK1 (casein kinase 1) inhibitor with IC50 of 200 nM and 300 nM in a cell-free assay for CK1 from Schizosaccharomyces pombe and CK1δ, respectively. Also acts as an ALK5 inhibitor with IC50 of 500 nM.
Targets
CK1 from Schizosaccharomyces pombe [1] CK1δ [1] ALK5 [1]
200 nM 300 nM 500 nM
In vitro

D4476 (10 μM) effectively inhibits CK1 and ALK5 with the activities of about 7% and 22% versus control group, respectively. In H4IIE hepatoma cells, D4476 specifically inhibits the phosphorylation of endogenous forkhead box transcription factor O1a (FOXO1a) on Ser322 and Ser325, without affecting the phosphorylation of other sites.[1] D4476 induces cytotoxicity in ANBL6, INA6 and RPMI8226 lines; MM1S and U266 lines are less sensitive; and OPM1 line is totally resistant. High concentrations (50 μM) D4476 induced toxicity in all multiple myeloma (MM) lines. In MM cells, D4476 increases the protein levels of TP53, P27, and FADD, as well as the cell cycle progression and induction of apoptosis.[2] Treatment of leukemia stem cells (LSCs) with D4476 shows highly selective killing of LSCs over normal HSPCs.[3]

Cell Data
Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
A375 cells NVP6eI9WTnWwY4Tpc44h[XO|YYm= NVHINlRvOTBvOECg{txO MV20PEBp M{DDTWlvcGmkaYTpc44hd2ZiQ1uxMW1FVTJiY3;tdIxmgCCrbjDoeY1idiCDM{e1JINmdGy|IHHzd4V{e2WmIHHzJIlv[3KnYYPlJIlvKHB{MTDwdo91\WmwIHzleoVtKGG2IEGwJJRwKDhyIIXNJIFnfGW{IES4JIhzeyCkeTDpcY12dm:kbH;0eIlv\yCjbnHsfZNqew>? NITBVGkzPDByN{mxPC=>

... Click to View More Cell Line Experimental Data

Protocol

Kinase Assay:[4]
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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 filtermats, 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.
Cell Research:[2]
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  • Cell lines: MM cell lines including MM1S, RPMI8226, U266, MM1R, OPM1, OPM2, INA6, ANBL6, ANBL-6VR, LR5 and RPMI-DOX40 (DOX40) cells
  • Concentrations: 50 μM
  • Incubation Time: 72 h
  • Method: 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)

Solubility (25°C)

In vitro DMSO 79 mg/mL (198.28 mM)
Water Insoluble
Ethanol Insoluble

* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Information

Molecular Weight 398.41
Formula

C23H18N4O3

CAS No. 301836-43-1
Storage powder
in solvent
Synonyms N/A

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Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID