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

The IC50 values of IC261 for Ckiδ, Ckiε, and Ckiα1 are 1 μM, 1 μM, and 16 μM, respectively. It is a selective ATP-competitive CK1 inhibitor. With an IC50 > 100 μM, IC261 exhibits minimal action against PKA, p34cdc2, and p55fyn [1]. In AC1-M88 cells, IC261 causes centrosome amplification, spindle abnormalities, and mitotic arrest. After 12 hours, IC261 (1 μM) increases G2/M cells, and 24 hours later, it promotes cell death in AC1-M88. Extravillous trophoblast hybrid cells undergo apoptosis in response to IC261 (1 μM) as well [2]. Numerous pancreatic tumor cell lines, such as ASPC-1, BxPc3, Capan-1, Colo357, MiaPaCa-2, Panc1, Panc89, PancTu-1, and PancTu-2 cells, are inhibited in their ability to proliferate by IC261 (1.25 μM). Pancreatic tumor cells' CD95-mediated apoptosis is specifically enhanced by IC261 (1.25 μM) [3].

In SCID mice, IC261 (20.5 mg/kg) suppresses the formation of PancTu-2 cell tumors, downregulates several anti-apoptotic proteins, including CK1δ/∊, KRAS, and IL6, and increases the expression of p21, ATM, CHEK1, and STAT1 [3].

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IC261 inhibits tumour growth in a xenotransplantation model[3]
The above results strongly suggest that inhibition of CK1 by small molecule inhibitors affects the growth of pancreatic tumour cell lines. To compare the effects of IC261 on pancreatic tumour cell growth in vivo with those of gemcitabine, which is commonly used in pancreatic tumour treatment, PancTu-1 cells were immobilised in matrigel and subcutaneously injected into the hind flank of 6-week-old SCID mice to obtain xenografts. Seventeen days after the tumours had been implanted, the mice were randomised into four treatment groups (n = 5): the control group (MOCK treated), the IC261 group, the gemcitabine group, and the IC261/gemcitabine treated group. The mice were treated daily for 8 days. Clinical observations of the mice during the treatment period and by gross pathological observation of all the vital organs at the termination of the study did not reveal any significant effects either on their weights or on their clinico-pathological disposition. Furthermore, the results of these in vivo experiments indicated that IC261 and gemcitabine led to a significant reduction of tumour growth. A combined treatment did not further reduce tumour growth (fig 5). Reduced tumour rates were matched to decreased tumour cell proliferation, as demonstrated by immunohistological staining for Ki-67. In the treated groups the positive Ki-67 staining was reduced (IC261 group, 11.3%; gemcitabine group, 14.7%; IC261/gemcitabine group, 10.5%) compared to that in the control group (21.0%) (fig 5B). In addition, an increased apoptosis rate was observed in the IC261-treated group and the other treated groups compared to the untreated control group. A representative example is shown in fig 5C.

Phosphotransferase Assays[1]
Casein kinase activity was assayed at 37 °C as described previously. The standard reaction (40 μl) contained 25 mm2-(N-morpholino)ethanesulfonic acid, pH 6.5, 50 mm NaCl, 15 mm MgCl2, 2 mg/ml casein, 2 mm EGTA, 100 μm[γ-32P]ATP (100–400 cpm/pmol). Initial velocity measurements were carried out in duplicate with ATP as the varied substrate. Kinetic constants and their standard errors were calculated as described in Ref. 26. For assay of inhibitor potency (IC50), [γ -32P]ATP was held constant (10 μm), whereas IC261 concentration was varied (0.1, 0.3, 1, 3, and 10 μm). To assess kinetic mechanism, inhibitors were held constant (IC261, 20 μm; IC3608, 100 μm), whereas [γ -32P]ATP was varied as above. For screening small molecule libraries, CK1 isoforms (Ckiα1, δ, and ε) were assayed as above except that casein was used at 10 mg/ml, [γ -32P]ATP was held constant at 2 μm or 1 mm.

Extravillous trophoblast hybrid cells[2]
Human extravillous trophoblast cells irreversibly leave the cell cycle and die when isolated from its natural extracellular matrix. Therefore, we have employed the cell line AC1-M88, an immortal, invasive extravillous trophoblast cell line, for in vitro experiments. This cell line was generated by fusion of extravillous trophoblasts with AC1-1, a mutant of the choriocarcinoma cell line Jeg-3 (Funayama et al., 1997; Frank et al., 2000). Cells were grown in DMEM (CV-1) or DMEM/F-12 (AC1-M88) medium (both Gibco) supplemented with 10% fetal calf serum (FCS; Biochrom) at 37°C in a humidified 5% CO2 atmosphere. Where indicated, cells were γ-irradiated with 5 Gy and harvested at the given time points for Western blot analysis, treated with 1 μM IC261 or 0.4 μM nocodazole for 12 h and fixed for immunofluorescence analysis, or treated with 1 μM IC261 and fixed for flow cytometrical analysis or lysed for Western blot analysis at the indicated time points. IC261 was synthesized as described by Mashhoon et al. (2000). IC261 and nocodazole were dissolved in DMSO as stock solutions (25 and 10 mM, respectively) and control cells were treated with 0.004% DMSO. For immunocytochemistry, the cells were grown on coverslips and were treated with methanol (−20°C) for 5 min, followed by acetone (−20°C) for 20–30 s prior to being used for immunocytochemical detection as described above.

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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.03 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)