D 4476   中文名称: D4476

CK1/casein kinase 1(IC50 = 0.3 μM)

D4476 是一种细胞内和体外强效、高选择性 CK1 抑制剂。 D4476 选择性防止 H4IIE 肝癌细胞中 MPD 内的内源性叉头盒转录因子 O1a (FOXO1a) 在 Ser322 和 Ser325 上磷酸化，而其他位点不受影响。使用 0.1 mM ATP 和磷酸化肽 TFRPRTSpSNASTIS（对应于 FOXO1a 残基 312-325），CK1δ 被抑制，IC50 值为 0.3 μM。 D4476 是 CK1 的 ATP 竞争性抑制剂，当 ATP 浓度下降时，CK1δ 的 IC50 值逐渐下降即可看出 [1]。

在临床前GBM模型中，D4476治疗显著抑制了放疗引起的促炎因子的增加，提高了放疗敏感性，从而抑制了肿瘤生长并延长了动物存活时间。这些结果表明，靶向Csnk1a1作为炎性因子的抑制剂发挥抗肿瘤作用，为胶质瘤的治疗提供了一种新策略[3]。

EdU掺入试验[3]
细胞光EdU细胞增殖检测试剂盒用于检测细胞增殖。27将稳定表达shRNA或用不同浓度D4476处理的p3×Flag-Csnk1a1转染的细胞培养在96孔板中。24小时后，将细胞与50μL EdU孵育4小时，用4%多聚甲醛固定15分钟，用0.5%Triton X-100处理20分钟。然后将细胞与1×Apollo反应混合物在黑暗中孵育30分钟，用DAPI染色20分钟。用PBS洗涤三次后，在荧光倒置显微镜下获取细胞图像。

D4476的细胞培养和使用。[1]
如前所述培养并裂解293细胞（Rena等人，2001）。H4IIE肝癌细胞与293细胞一样进行培养和裂解，不同的是使用15cm的培养皿并将其保存在含有1mg ml-1葡萄糖和5%胎牛血清的DMEM中。将CKI-7、IC261和D4476以100mM的浓度溶解在二甲基亚砜（DMSO）中。当直接稀释到水性细胞培养基中时，D4476仅微溶（数据未显示）。为了提高溶解度，我们首先在21°C下将1μl 100 mM D4476稀释在6μl无血清培养基和3μl Fugene 6的混合物中，然后将该溶液逐滴加入培养的细胞中。细胞对D4476的反应不受化合物至少三次冻融循环的影响。在对照实验中，D4476被DMSO替代。

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使用D4476进行药物治疗。[2]
为了抑制Csnk1a1，使用了小分子D4476。将D4476加入到在补充有10ng/ml mIL-3的培养基中的96孔板（每孔5000个细胞）中培养的白血病细胞中。通过向细胞培养物中加入2.5µM、5µM、10µM、20µM和40µM D4476进行D4476剂量滴定，最终DMSO百分比为0.4%。同样，将D4476加入到在补充了mTpo和mScf的SFEM培养基中培养的LSK细胞中。使用流式细胞术用CountBright绝对计数珠评估处理96小时后的细胞数量。

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集落形成试验[2]
将U87和LN229细胞接种到6孔板中。每个孔有500个细胞，每组有三个重复孔。实验组用指定浓度（0、5和10μM）的D4476治疗，联合4 Gy的放疗。24小时后，用新鲜的无药物培养基进一步培养10-14天。然后，用PBS洗涤细胞，用甲醇固定，用0.1%结晶紫溶液染色。洗涤后，在显微镜下对细胞进行成像和计数。

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细胞周期和凋亡分析[2]
U87和LN229细胞用特定浓度的D4476处理，shRNA细胞与放疗联合培养24小时。然后，在4°C下以1000 rpm离心5分钟，用70%冷甲醇固定过夜。用PBS洗涤两次后，用含有50μg/mL碘化丙啶（PI）和25μg/mL核糖核酸酶（RNase）的染色溶液对细胞进行染色30分钟。使用流式细胞术和集成流式细胞仪软件分析细胞周期。 为了评估凋亡，收集处理过的细胞，用预冷的PBS洗涤两次，并重新悬浮在预冷的结合缓冲液中。接下来，将约5μL Annexin V-FITC和5μL PI加入细胞悬浮液中，在黑暗中冰上孵育10分钟。使用流式细胞术检测细胞凋亡。

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

103.12

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.27 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.27 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.27 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。