D 4476

Catalog No.S7642 Batch:S764201

Technical Data

Formula	C₂₃H₁₈N₄O₃
Molecular Weight	398.41	CAS No.	301836-43-1
Solubility (25°C)*	In vitro	DMSO	79 mg/mL (198.28 mM)
		Water	Insoluble
		Ethanol	Insoluble
* <1 mg/ml means slightly soluble or 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. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

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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]

Protocol (from reference)

Kinase Assay:^{^[4]}	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 Assay:^{^[2]}	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.

Kinase Assay:^{^[4]}

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 Assay:^{^[2]}

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.

References

[4] Bain J, et al. Biochem J. 2003, 371(Pt 1), 199-204.

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Customer Product Validation

: Data from [Data independently produced by , , Mol Oncol, 2016, 10(5):677-92]

Selleck's D 4476 has been cited by 21 publications

A kinome-wide CRISPR screen identifies CK1α as a target to overcome enzalutamide resistance of prostate cancer [ Cell Rep Med, 2023, 4(4):101015]	PubMed: 37075701
Golgi retention and oncogenic KIT signaling via PLCγ2-PKD2-PI4KIIIβ activation in gastrointestinal stromal tumor cells [ Cell Rep, 2023, 42(9):113035]	PubMed: 37616163
Neddylation inhibition induces glutamine uptake and metabolism by targeting CRL3SPOP E3 ligase in cancer cells [ Nat Commun, 2022, 13(1):3034]	PubMed: 35641493
CHMP2B regulates TDP-43 phosphorylation and cytotoxicity independent of autophagy via CK1 [ J Cell Biol, 2022, 221(1)e202103033]	PubMed: 34726688
CK1α upregulates the IFNAR1 expression to prompt the anti-HBV effect of type I IFN in hepatoma carcinoma cells [ Virol Sin, 2022, S1995-820X(22)00143-2]	PubMed: 35985475
SPOP and OTUD7A Control EWS-FLI1 Protein Stability to Govern Ewing Sarcoma Growth [ Adv Sci (Weinh), 2021, e2004846]	PubMed: 34060252
SPOPandOTUD7AControlEWS–FLI1Protein Stability to Govern Ewing Sarcoma Growth [ ADVANCED SCIENCE, 2021, 8(14)]	PubMed: None
CK1δ stimulates ubiquitination-dependent proteasomal degradation of ATF4 to promote chemoresistance in gastric Cancer [ Clin Transl Med, 2021, 11(10):e587]	PubMed: 34709767
SKI activates the Hippo pathway via LIMD1 to inhibit cardiac fibroblast activation [ Basic Res Cardiol, 2021, 116(1):25]	PubMed: 33847835
Nicotinamide promotes cardiomyocyte derivation and survival through kinase inhibition in human pluripotent stem cells [ Cell Death Dis, 2021, 12(12):1119]	PubMed: 34845199

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