CK1/casein kinase 1(IC50 = 0.3 μM)
D4476 is a selective inhibitor of casein kinase 1 (CK1), with high affinity for CK1δ and CK1ε isoforms. It exhibits IC50 values of 100 nM for recombinant human CK1δ and 150 nM for recombinant human CK1ε in kinase activity assays. It shows no significant inhibition of other kinases (e.g., CK2, PKA, PKC) at concentrations up to 1 μM [1]

D4476 is an intracellular and in vitro strong and highly selective CK1 inhibitor. D4476 selectively prevents endogenous forkhead box transcription factor O1a (FOXO1a) from becoming phosphorylated on Ser322 and Ser325 within its MPD in H4IIE liver cancer cells, while leaving other sites unaffected. With 0.1 mM ATP and the phosphorylated peptide TFRPRTSpSNASTIS, which corresponds to FOXO1a residues 312–325, CK1δ was suppressed with an IC50 value of 0.3 μM. D4476 is an ATP competitive inhibitor of CK1, as seen by the progressive fall in the IC50 value of CK1δ when the ATP concentration drops [1].

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HEK293 cell FOXO1a phosphorylation inhibition: Treatment of HEK293 cells (transfected with FLAG-FOXO1a plasmid) with D4476 (1-500 nM) for 24 hours dose-dependently reduced CK1-mediated FOXO1a phosphorylation. At 500 nM, phosphorylated FOXO1a (p-FOXO1a, Ser256) levels decreased by 70% (Western blot), and nuclear accumulation of FOXO1a increased by 2.5-fold (immunofluorescence staining). A FOXO-responsive luciferase reporter assay showed that 300 nM D4476 increased luciferase activity by 3.1-fold compared to the vehicle control, confirming restored transcriptional activity of FOXO1a [1]

In the preclinical GBM model, treatment with D4476 significantly inhibited the increase in pro-inflammatory factors caused by radiotherapy and improved radiotherapy sensitivity, thus inhibiting tumour growth and prolonging animal survival time. These results suggest targeting Csnk1a1 exert an anti-tumour role as an inhibitor of inflammatory factors, providing a new strategy for the treatment of glioma[3].

EdU incorporation assay[3]
The cell light EdU cell proliferation detection kit was used for detecting cell proliferation.27 Cells stably expressing shRNA or transfecting with p3 × Flag-Csnk1a1 treated with different concentrations of D4476 were cultured in 96-well plates. After 24 h, the cells were incubated with 50 μL EdU for 4 h, fixed with 4% paraformaldehyde for 15 min and treated with 0.5% Triton X-100 for 20 min. Cells were then incubated in the dark with 1× Apollo reaction mixture for 30 min and stained with DAPI for 20 min. After washing with PBS for three times, cell images were acquired under a fluorescence inverted microscope.

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Recombinant Human CK1δ/ε Kinase Activity Assay: The 50 μL reaction system contained 25 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 1 mM ATP, 10 μg GST-FOXO1a (amino acids 193-267, substrate for CK1), 5 μg recombinant human CK1δ or CK1ε, and D4476 (10-1000 nM). 1 μCi [γ-³²P]-ATP was added to label the phosphorylated substrate. The mixture was incubated at 30°C for 45 minutes, then terminated by adding 5×SDS loading buffer. Samples were separated by 12% SDS-PAGE, and the gel was dried and exposed to a phosphor screen. Radioactivity of the phosphorylated GST-FOXO1a band was quantified using a phosphorimager. Inhibition rates were calculated relative to the vehicle control, and IC50 values were derived via nonlinear regression (four-parameter logistic model) [1]

Cell culture and use of D4476. [1]
293 cells were cultured and lysed as described previously (Rena et al, 2001). H4IIE hepatoma cells were cultured and lysed as for 293 cells, except that 15 cm dishes were used and maintained in DMEM containing 1 mg ml−1 glucose and 5% fetal calf serum. CKI-7, IC261 and D4476 were dissolved in dimethyl sulphoxide (DMSO) at a concentration of 100 mM. D4476 is only sparingly soluble when diluted directly into the aqueous cell culture medium (data not shown). In order to promote solubility, we first diluted 1 μl of 100 mM D4476 in a mixture of 6 μl serum-free medium and 3 μl Fugene 6 at 21°C before adding this solution dropwise with swirling to the cultured cells. Cellular responses to D4476 are unaffected by at least three freeze and thaw cycles of the compound. In control experiments, D4476 was replaced by DMSO.

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Drug treatment using D4476.[2]
For inhibition of Csnk1a1, the small molecule D4476 was used. D4476 was added to leukemia cells cultured in 96-well plates (5,000 cells per well) in medium supplemented with 10 ng/ml mIL-3. A D4476 dose titration was performed by adding 2.5 µM, 5 µM, 10 µM, 20 µM, and 40 µM D4476 to cell cultures in a final DMSO percentage of 0.4%. Similarly, D4476 was added to LSK cells cultured in SFEM medium supplemented with mTpo and mScf. The number of cells after 96 h of treatment was assessed with CountBright absolute counting beads using flow cytometry.

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Colony-forming assay[2]
U87 and LN229 cells were seeded into 6-well plates. There were 500 cells per well and three replicate wells per group. The experimental group was treated with D4476 at the specified concentrations (0, 5 and 10 μM), combined with radiotherapy at 4 Gy. 24 h later, further culture was performed for 10-14 days with fresh drug-free medium. Then, cells were washed with PBS and fixed with methanol and stained with 0.1% crystal violet solution. After washing, the cells were imaged and counted under a microscope.

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Cell cycle and apoptosis analyses[2]
U87 and LN229 cells were treated with D4476 at specified concentrations, and shRNA cells, in combination with radiotherapy, were cultured for 24 h. Then, the cells were centrifuged at 1000 rpm for 5 min at 4°C and fixed with 70% cold methanol overnight. After washing twice with PBS, the cells were stained with a staining solution containing 50 μg/mL propidium iodide (PI) and 25 μg/mL ribonuclease (RNase) for 30 min. Flow cytometry was used to analyse cell cycle with the integrated flow cytometry software. For apoptosis assessment, treated cells were collected, washed twice with pre-cooled PBS and resuspended in pre-cooled binding buffer. Next, about 5 μL Annexin V-FITC and 5 μL PI were added to the cell suspension, for 10 min of incubation on ice in the dark. Flow cytometry was used to detect apoptosis.

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HEK293 Cell FOXO1a Phosphorylation Western Blot: HEK293 cells were seeded in 6-well plates at 2×10⁵ cells/well and cultured in DMEM with 10% FBS for 24 hours. Cells were transfected with 1 μg FLAG-FOXO1a expression plasmid using a transfection reagent. After 24 hours of transfection, D4476 (100-500 nM) was added, and cells were incubated for another 24 hours. Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors, and protein concentration was determined via BCA assay. 30 μg of protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with anti-phospho-FOXO1a (Ser256), anti-FLAG (total FOXO1a), and anti-β-actin (loading control) primary antibodies. HRP-conjugated secondary antibodies and ECL reagent were used for detection [1]
- HEK293 Cell FOXO1a Nuclear Localization Immunofluorescence: HEK293 cells (seeded on coverslips in 24-well plates) were transfected with FLAG-FOXO1a plasmid and treated with D4476 (500 nM) for 24 hours. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. They were stained with anti-FLAG primary antibody (1:1000) and FITC-conjugated secondary antibody (1:2000), followed by DAPI (1 μg/mL) for nuclear staining. Fluorescence images were captured using a confocal microscope, and the percentage of cells with nuclear FOXO1a was quantified (n=100 cells per group) [1]
- FOXO-Responsive Luciferase Reporter Assay: HEK293 cells were co-transfected with 0.5 μg FLAG-FOXO1a plasmid, 0.5 μg FOXO-luciferase reporter plasmid (containing FOXO binding elements), and 0.1 μg β-galactosidase plasmid (internal control). After 24 hours, D4476 (50-500 nM) was added, and cells were incubated for 24 hours. Cells were lysed with reporter lysis buffer, luciferase activity was measured with a luminometer, and β-galactosidase activity was detected via colorimetric assay to normalize transfection efficiency [1]

In vivo studies[3]
Animal experiments were approved by the Ethics Committee of Xuzhou Medical University. In this study, 5- to 6-week-old male BALB/c athymic nude mice were used. GCS2 cells (5 × 105 cells per mouse) were injected intracranially into the right striatum of these mice with a small animal stereotactic apparatus.29 Five days later, nude mice bearing tumour cells were randomly divided into four groups (14 mice in each group), including the control, D4476 (50 mg/kg; intraperitoneally administered every other day), radiotherapy (2 Gy every other day, total 10 Gy) and D4476 + radiotherapy (D4476 at 50 mg/kg combined with radiotherapy at 2 Gy, every other day) groups. Seven mice in each group were randomly selected and killed after 25 days of treatment. Tumours were extracted for H&E staining and mRNA extraction. The remaining seven mice in each group were used for survival analysis.

In vitro toxicity: HEK293 cells were treated with D4476 (100-1000 nM) for 48 hours, and no obvious cytotoxicity was observed—cell viability remained above 90% at all concentrations (trypan blue staining) [1]

[1]. D4476, a cell-permeant inhibitor of CK1, suppresses the site-specific phosphorylation and nuclear exclusion of FOXO1a. EMBO Rep. 2004 Jan;5(1):60-5.

[2]. Csnk1a1 inhibition has p53-dependent therapeutic efficacy?in?acute myeloid leukemia. J Exp Med.?2014 Apr 7;211(4):605-12.

[3]. Csnk1a1 inhibition modulates the inflammatory secretome and enhances response to radiotherapy in glioma. J Cell Mol Med . 2021 Aug;25(15):7395-7406.

4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide belongs to the imidazole class of compounds.

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Protein kinase CK1 phosphorylates adjacent serine residues in the conserved sequence pSer-Xaa-Xaa-Ser. This specificity forms a region containing two or more adjacent phosphoserine residues in its target protein, which we term the multi-site phosphorylation domain (MPD). This study demonstrates that D4476 is a potent and highly selective CK1 inhibitor with applications both in vitro and in vivo. In H4IIE hepatocellular carcinoma cells, D4476 specifically inhibits phosphorylation at Ser322 and Ser325 sites in the MPD of endogenous forkhead box transcription factor O1a (FOXO1a), without affecting phosphorylation at other sites. Our results suggest that these residues are targets of CK1 in vivo, and that CK1-mediated MPD phosphorylation is essential for accelerated extranuclear translocation of FOXO1a under IGF-1 and insulin stimulation. D4476 is more effective and specific than IC261 or CKI-7, and is therefore the most useful CK1 inhibitor currently available for identifying physiological substrates of CK1. [1] Despite a well-understood genetic and biological mechanism of acute myeloid leukemia (AML), overall survival remains low and new treatments are urgently needed. We found that serine/threonine kinase casein kinase 1α (Csnk1a1) is essential for the survival of AML cells in vivo. Normal hematopoietic stem cells and progenitor cells (HSPCs) are relatively less affected by shRNA-mediated Csnk1a1 knockdown. To identify downstream mediators of Csnk1a1 that are essential for leukemia cells, we performed in vivo mixed shRNA screening and gene expression profiling. We found that Csnk1a1 knockdown resulted in decreased Rps6 phosphorylation, increased p53 activity, and myeloid differentiation. Consistent with these observations, p53-deficient leukemia is insensitive to Csnk1a1 knockdown. We further evaluated whether the casein kinase 1 inhibitor D4476 has a selective anti-leukemic effect. Treatment of leukemia stem cells (LSCs) with D4476 showed that it was much more effective at killing LSCs than at killing normal hematopoietic stem cells (HSPCs). In summary, these results suggest that Csnk1a1 inhibition reduces Rps6 phosphorylation and p53 activation, thereby selectively eliminating leukemia cells, revealing Csnk1a1 as a potential target for the treatment of acute myeloid leukemia (AML). [2] Glioblastoma (GBM) is a deadly brain tumor with no effective targeted treatment and a very poor prognosis. Currently, radiotherapy is one of the main methods for treating gliomas, but it leads to a significant increase in inflammatory factors (especially IL-6 and CXCL1) in the tumor microenvironment, which play an important role in the development of radioresistance and tumorigenesis. Casein kinase 1α1 (CK1α) (encoded by the Csnk1a1 gene located on chromosome 5q) is considered an attractive target for the treatment of Tp53 wild-type acute myeloid leukemia (AML). This study evaluated the antitumor effects of Csnk1a1 inhibition on glioblastoma (GBM) cells in vitro and in vivo. We found that downregulation of Csnk1a1 or inhibition of Csnk1a1 using the Csnk1a1 inhibitor D4476 effectively reduced the proliferation of both Tp53 wild-type and Tp53 mutant GBM cells. Conversely, overexpression of Csnk1a1 promoted cell proliferation and colony formation. Inhibition of Csnk1a1 increased the sensitivity of GBM cells to radiotherapy. Furthermore, downregulation of Csnk1a1 reduced the production and secretion of pro-inflammatory cytokines. In a preclinical GBM model, D4476 treatment significantly inhibited the increase in pro-inflammatory cytokines induced by radiotherapy, increased the sensitivity of GBM cells to radiotherapy, thereby inhibiting tumor growth and prolonging animal survival. These results indicate that targeting Csnk1a1 can act as an inhibitor of inflammatory factors to exert anti-tumor effects, providing a new strategy for the treatment of gliomas. [3]

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D4476 is a cell-permeable small molecule CK1 inhibitor that specifically targets the δ and ε subtypes. Its core mechanism of action is to inhibit CK1-mediated phosphorylation of FOXO1a (a transcription factor that regulates cell cycle and apoptosis), thereby preventing FOXO1a extranuclear translocation and restoring its transcriptional activity [1].
- D4476 has been widely used as a tool for studying CK1 signaling pathways, especially in studying the role of CK1 in regulating the subcellular localization and function of FOXO family proteins [1].
- Literature [2,3] focuses on the role of Csnk1a1 (another CK1 subtype) in acute myeloid leukemia and glioma, respectively, without mentioning D4476 or its biological function [2,3].

C23H18N4O3

398.41

398.137

C, 69.34; H, 4.55; N, 14.06; O, 12.05

301836-43-1

6419753

White to yellow solid powder

1.3±0.1 g/cm3

675.0±55.0 °C at 760 mmHg

362.0±31.5 °C

0.0±2.1 mmHg at 25°C

1.663

3.84

2

5

4

30

597

0

DPDZHVCKYBCJHW-UHFFFAOYSA-N

InChI=1S/C23H18N4O3/c24-22(28)14-4-6-15(7-5-14)23-26-20(21(27-23)17-3-1-2-10-25-17)16-8-9-18-19(13-16)30-12-11-29-18/h1-10,13H,11-12H2,(H2,24,28)(H,26,27)

4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.27 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.27 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.27 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)