SU-5607 (IC-261)

Ckiδ(IC50 = 1 μM); Ckiε(IC50 = 1 μM); Ckiα1 (IC50 = 16 μM)

IC261 对 Ckiδ、Ckiε 和 Ckiα1 的 IC50 值分别为 1 μM、1 μM 和 16 μM。它是一种选择性 ATP 竞争性 CK1 抑制剂。 IC261 的 IC50 > 100 μM，对 PKA、p34cdc2 和 p55fyn 的作用极小 [1]。在 AC1-M88 细胞中，IC261 导致中心体扩增、纺锤体异常和有丝分裂停滞。 12 小时后，IC261 (1 μM) 增加 G2/M 细胞，24 小时后，促进 AC1-M88 细胞死亡。绒毛外滋养层杂交细胞也会响应 IC261 (1 μM) 发生凋亡 [2]。许多胰腺肿瘤细胞系，如 ASPC-1、BxPc3、Capan-1、Colo357、MiaPaCa-2、Panc1、Panc89、PancTu-1 和 PancTu-2 细胞，其增殖能力均被 IC261 (1.25 μM) 抑制。 ）。 IC261 (1.25 μM) 可特异性增强胰腺肿瘤细胞的 CD95 介导的细胞凋亡 [3]。

在 SCID 小鼠中，IC261 (20.5 mg/kg) 抑制 PancTu-2 细胞肿瘤的形成，下调多种抗凋亡蛋白，包括 CK1δ/∊、KRAS 和 IL6，并增加 p21、ATM、CHEK1 和 STAT1 的表达 [3]。

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IC261抑制异种移植模型肿瘤生长[3]
以上结果强烈提示，小分子抑制剂对CK1的抑制影响胰腺肿瘤细胞系的生长。为了比较IC261对体内胰腺肿瘤细胞生长的影响与常用的治疗胰腺肿瘤的吉西他滨的影响，我们将pancu -1细胞固定在基质中，并皮下注射到6周龄的SCID小鼠后腹获得异种移植物。肿瘤植入17天后，将小鼠随机分为4个治疗组（n = 5）：对照组（MOCK治疗）、IC261组、吉西他滨组和IC261/吉西他滨治疗组。小鼠每天治疗8天。在治疗期间对小鼠的临床观察和在研究结束时对所有重要器官的大体病理观察均未显示对其体重或临床病理倾向有任何显着影响。此外，这些体内实验的结果表明，IC261和吉西他滨导致肿瘤生长显著减少。联合治疗并没有进一步降低肿瘤生长（图5）。正如Ki-67的免疫组织学染色所证明的那样，肿瘤率的降低与肿瘤细胞增殖的降低相匹配。各处理组Ki-67阳性染色减少(IC261组减少11.3%；吉西他滨组，14.7%；IC261/吉西他滨组，10.5%)，而对照组（21.0%）（图5B）。此外，与未治疗的对照组相比，IC261治疗组和其他治疗组的细胞凋亡率均有所增加。一个代表性的例子如图5C所示。

磷酸转移酶化验[1]
如前所述，在37°C下测定酪蛋白激酶活性。标准反应（40 μl）含有25 mm2-(N-morpholino)乙磺酸，pH 6.5, 50 mm NaCl, 15 mm MgCl2, 2 mg/ml酪蛋白，2 mm EGTA, 100 μm[γ-32P]ATP （100 - 400 cpm/pmol）。初始速度测量一式两份，以ATP作为不同的底物。动力学常数及其标准误差的计算方法见文献26。对于抑制剂效价（IC50）的测定，[γ -32P]ATP保持恒定（10 μm），而IC261浓度变化（0.1、0.3、1、3和10 μm）。为了评估动力学机制，抑制剂保持恒定(IC261, 20 μm；IC3608, 100 μm)，而[γ -32P]ATP的变化情况如上所述。为了筛选小分子文库，除酪蛋白浓度为10 mg/ml， [γ -32P]ATP浓度为2 μm或1 mm外，CK1亚型（Ckiα1， δ和ε）的测定方法与上述相同。

外滋养层杂交细胞[2]
人上皮外滋养细胞与自然细胞外基质分离后，不可逆地离开细胞周期并死亡。因此，我们采用AC1-M88细胞系进行体外实验，AC1-M88是一种不朽的侵袭性外滋养细胞。该细胞系是由绒毛癌细胞细胞系Jeg-3的突变体AC1-1与胞外滋养细胞融合产生的(Funayama et al., 1997；Frank et al., 2000)。细胞在添加10%胎牛血清（FCS）的DMEM （CV-1）或DMEM/F-12 （AC1-M88）培养基中生长；Biochrom)在37°C下加湿5% CO2的气氛中。在指定的时间点，用5 Gy的γ射线照射细胞并收获细胞进行免疫印迹分析，用1 μM IC261或0.4 μM nocodazole处理12小时后固定进行免疫荧光分析，或用1 μM IC261处理12小时后固定进行流式细胞分析或在指定的时间点裂解进行免疫印迹分析。IC261是按照Mashhoon et al.（2000）的描述合成的。IC261和nocodazole分别溶于DMSO（25和10 mM）中，对照细胞用0.004% DMSO处理。对于免疫细胞化学，细胞在盖片上生长，用甲醇（- 20°C）处理5分钟，然后用丙酮（- 20°C）处理20 - 30秒，然后如上所述用于免疫细胞化学检测。

Five million PancTu-1 cells resuspended in 100 µl of a solution containing 50% Matrigel and 50% DMEM/RPMI-1640 (1:1) were injected into the dorsolateral site of 6-week-old C.B-17/IcrHsd-scid-bg mice. After 17 days, mice were randomised to the control group (n = 5), the IC261 treatment group (n = 5), the gemcitabine group (n = 5) and to the IC261/gemcitabine group (n = 5). Injection of dimethylsulfoxide (DMSO; control group), IC261 (20.5 mg/kg), gemcitabine (0.6 mg/kg) alone or in combination (20.5 mg/kg IC261/0.6 mg/kg gemcitabine) (treatment groups) was performed daily for 8 days. Mice were sacrificed by asphyxiation with CO2 the day after the last treatment. Tumours were measured before and during treatment. Finally, the tumours were excised, measured, weighed and fixed in formalin or shock frozen. Tumour volume was calculated according to the formula for a rotational ellipsoid (length × height × width × 0.5236).[3]

[1]. Crystal structure of a conformation-selective casein kinase-1 inhibitor. J Biol Chem. 2000 Jun 30;275(26):20052-60.

[2]. Inhibition of casein kinase I delta alters mitotic spindle formation and induces apoptosis in trophoblast cells. Oncogene. 2005 Dec 1;24(54):7964-75.

[3]. Anti-apoptotic and growth-stimulatory functions of CK1 delta and epsilon in ductal adenocarcinoma of the pancreas are inhibited by IC261 in vitro and in vivo. Gut. 2008 Jun;57(6):799-806.

Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis. Unlike most protein kinases, they appear to function as constitutively active enzymes. As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes. To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening. Here we report the crystal structure of 3-[(2,4,6-trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 A resolution. The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations. We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor.[1]

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The serine/threonine-specific casein kinase I delta (CKIdelta) is ubiquitously expressed in all tissues, is p53 dependently induced in stress situations and plays an important role in various cellular processes. Our immunohistochemical analysis of the human placenta revealed strongest expression of CKIdelta in extravillous trophoblast cells and in choriocarcinomas. Investigation of the functional role of CKIdelta in an extravillous trophoblast hybrid cell line revealed that CKIdelta was constitutively localized at the centrosomes and the mitotic spindle. Inhibition of CKIdelta with the CKI-specific inhibitor IC261 led to structural alterations of the centrosomes, the formation of multipolar spindles, the inhibition of mitosis and, in contrast to other cell lines, the induction of apoptosis. Our findings indicate that CKIdelta plays an important role in the mitotic progression and in the survival of cells of trophoblast origin. Therefore, IC261 could provide a new tool in treating choriocarcinomas.[2]

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Background: Pancreatic ductal adenocarcinomas (PDACs) are highly resistant to treatment due to changes in various signalling pathways. CK1 isoforms play important regulatory roles in these pathways. Aims: We analysed the expression levels of CK1 delta and epsilon (CK1delta/in) in pancreatic tumour cells in order to validate the effects of CK1 inhibition by 3-[2,4,6-(trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) on their proliferation and sensitivity to anti-CD95 and gemcitabine. Methods: CK1delta/in expression levels were investigated by using western blotting and immunohistochemistry. Cell death was analysed by FACS analysis. Gene expression was assessed by real-time PCR and western blotting. The putative anti-tumoral effects of IC261 were tested in vivo in a subcutaneous mouse xenotransplantation model for pancreatic cancer. Results: We found that CK1delta/in are highly expressed in pancreatic tumour cell lines and in higher graded PDACs. Inhibition of CK1delta/in by IC261 reduced pancreatic tumour cell growth in vitro and in vivo. Moreover, IC261 decreased the expression levels of several anti-apoptotic proteins and sensitised cells to CD95-mediated apoptosis. However, IC261 did not enhance gemcitabine-mediated cell death either in vitro or in vivo. Conclusions: Targeting CK1 isoforms by IC261 influences both pancreatic tumour cell growth and apoptosis sensitivity in vitro and the growth of induced tumours in vivo, thus providing a promising new strategy for the treatment of pancreatic tumours.[3]

C18H17NO4

311.3319

311.115

C, 69.44; H, 5.50; N, 4.50; O, 20.56

186611-52-9

5288600

Light yellow to yellow solid powder

2.8

56.8

1

4

4

23

450

0

O(C([H])([H])[H])C1C([H])=C(C([H])=C(C=1/C(/[H])=C1/C(N([H])C2=C([H])C([H])=C([H])C([H])=C/12)=O)OC([H])([H])[H])OC([H])([H])[H]

JBJYTZXCZDNOJW-JLHYYAGUSA-N

InChI=1S/C18H17NO4/c1-21-11-8-16(22-2)14(17(9-11)23-3)10-13-12-6-4-5-7-15(12)19-18(13)20/h4-10H,1-3H3,(H,19,20)/b13-10+

(3E)-3-[(2,4,6-trimethoxyphenyl)methylidene]-1H-indol-2-one

ic261; 186611-52-9; IC 261; 3-[(2,4,6-TRIMETHOXY-PHENYL)-METHYLENE]-INDOLIN-2-ONE; SU-5607; (3E)-3-[(2,4,6-trimethoxyphenyl)methylidene]-1H-indol-2-one; 3-[(2,4,6-Trimethoxyphenyl)methylidenyl]-indolin-2-one; MFCD00118156;

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)

DMSO : ≥ 33 mg/mL (~106.00 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (8.03 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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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;
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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;
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