CK1/casein kinase 1(IC50 = 0.3 μM)

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].

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.

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.

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.

[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-pyridinyl)-1H-imidazol-2-yl]benzamide is a member of imidazoles.

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The protein kinase CK1 phosphorylates serine residues that are located close to another phosphoserine in the consensus pSer-Xaa-Xaa-Ser. This specificity generates regions in its target proteins containing two or more neighbouring phosphoserine residues, termed here multisite phosphorylation domains (MPDs). In this paper, we demonstrate that D4476 is a potent and rather selective inhibitor of CK1 in vitro and in cells. In H4IIE hepatoma cells, D4476 specifically inhibits the phosphorylation of endogenous forkhead box transcription factor O1a (FOXO1a) on Ser322 and Ser325 within its MPD, without affecting the phosphorylation of other sites. Our results indicate that these residues are targeted by CK1 in vivo and that the CK1-mediated phosphorylation of the MPD is required for accelerated nuclear exclusion of FOXO1a in response to IGF-1 and insulin. D4476 is much more potent and specific than IC261 or CKI-7, and is therefore the most useful CK1 inhibitor currently available for identifying physiological substrates of CK1.[1]

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Despite extensive insights into the underlying genetics and biology of acute myeloid leukemia (AML), overall survival remains poor and new therapies are needed. We found that casein kinase 1 α (Csnk1a1), a serine-threonine kinase, is essential for AML cell survival in vivo. Normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected by shRNA-mediated knockdown of Csnk1a1. To identify downstream mediators of Csnk1a1 critical for leukemia cells, we performed an in vivo pooled shRNA screen and gene expression profiling. We found that Csnk1a1 knockdown results in decreased Rps6 phosphorylation, increased p53 activity, and myeloid differentiation. Consistent with these observations, p53-null leukemias were insensitive to Csnk1a1 knockdown. We further evaluated whether D4476, a casein kinase 1 inhibitor, would exhibit selective antileukemic effects. Treatment of leukemia stem cells (LSCs) with D4476 showed highly selective killing of LSCs over normal HSPCs. In summary, these findings demonstrate that Csnk1a1 inhibition causes reduced Rps6 phosphorylation and activation of p53, resulting in selective elimination of leukemia cells, revealing Csnk1a1 as a potential therapeutic target for the treatment of AML.[2]

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Glioblastoma multiforme (GBM), a fatal brain tumour with no available targeted therapies, has a poor prognosis. At present, radiotherapy is one of the main methods to treat glioma, but it leads to an obvious increase in inflammatory factors in the tumour microenvironment, especially IL-6 and CXCL1, which plays a role in tumour to resistance radiotherapy and tumorigenesis. Casein kinase 1 alpha 1 (CK1α) (encoded on chromosome 5q by Csnk1a1) is considered an attractive target for Tp53 wild-type acute myeloid leukaemia (AML) treatment. In this study, we evaluated the anti-tumour effect of Csnk1a1 suppression in GBM cells in vitro and in vivo. We found that down-regulation of Csnk1a1 or inhibition by D4476, a Csnk1a1 inhibitor, reduced GBM cell proliferation efficiently in both Tp53 wild-type and Tp53-mutant GBM cells. On the contrary, overexpression of Csnk1a1 promoted cell proliferation and colony formation. Csnk1a1 inhibition improved the sensitivity to radiotherapy. Furthermore, down-regulation of Csnk1a1 reduced the production and secretion of pro-inflammatory factors. 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]

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.)