IC50: 16 μM
IC261 is a novel inhibitor of CK1.
The p53-targeted kinases casein kinase 1delta (CK1delta) and casein kinase 1epsilon (CK1epsilon) have been reported to be involved in regulating DNA repair and chromosomal segregation.
In vitro: Previoius study found that IC261 could trigger the mitotic checkpoint control. At low micromolar concentrations, IC261 inhibited cytokinesis leading to a transient mitotic arrest. Cells with active p53 arrested in the postmitotic G1 phase by blockage of entry into the S phase. Cells containing non-functional p53 had postmitotic replication developing an 8N DNA content. The increase of DNA content was accompanied by a high amount of micronucleated and apoptotic cells. Immunfluorescence images also showed that IC261 could result in centrosome amplification causing multipolar mitosis at low concentrations [1].
In vivo: Animal study indicated that intrathecal injection of IC261 (0.1-1 nmol) could dose-dependently decrease mechanical allodynia and thermal hyperalgesia that were induced by carrageenan or CFA. In addition, bath-application of IC261 (1 μM) showed only marginal effects on spontaneous excitatory postsynaptic currents recorded in the substantia gelatinosa neurons of control mice. However, IC261 could decrease the frequency of sEPSCs in both inflammatory pain models [2].
Clinical trial: Up to now, IC261 is still in the preclinical development stage.
参考文献:
[1].Behrend L, et al. IC261, a specific inhibitor of the protein kinases casein kinase 1-delta and -epsilon, triggers the mitotic checkpoint and induces p53-dependent postmitotic effects. Oncogene. 2000 Nov 9;19(47):5303-5313.
[2] Kurihara T,et al.? Alleviation of behavioral hypersensitivity in mouse models of inflammatory pain with two structurally different casein kinase 1 (CK1) inhibitors. Mol Pain.2014 Mar 10;10:17
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Kinase experiment: | Casein kinase activity is assayed at 37°C. The standard reaction (40 μL) contains 25 mM 2-(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 are carried out in duplicate with ATP as the varied substrate. Kinetic constants and their standard errors are calculated. For assay of inhibitor potency (IC50), [γ -32P]ATP is held constant (10 μM), whereas IC261 concentration is varied (0.1, 0.3, 1, 3, and 10 μM). To assess kinetic mechanism, inhibitors are held constant (IC261, 20 μM; IC3608, 100 μM), whereas [γ -32P]ATP is varied as above. For screening small molecule libraries, CK1 isoforms (Ckiα1, δ, and ε) are assayed that casein is used at 10 mg/mL, [γ -32P]ATP is held constant at 2 μM or 1 mM[1]. |
Cell experiment: | Human extravillous trophoblast cells irreversibly leave the cell cycle and die when isolated from its natural extracellular matrix. The cell line AC1-M88 is employed in vitro experiments. This cell line is generated by fusion of extravillous trophoblasts with AC1-1. Cells are grown in DMEM (CV-1) or DMEM/F-12 (AC1-M88) medium supplemented with 10% fetal calf serum (FCS) at 37°C in a humidified 5% CO2 atmosphere. Where indicated, cells are 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 and nocodazole are dissolved in DMSO as stock solutions (25 and 10?mM, respectively), and control cells are treated with 0.004% DMSO. For immunocytochemistry, the cells are grown on coverslips and are treated with methanol (?20°C) for 5?min, followed by acetone (?20°C) for 20-30?s prior to being used for immunocytochemical detection[2]. |
Animal experiment: | Five million PancTu-1 cells resuspended in 100 μL of a solution containing 50% Matrigel and 50% DMEM/RPMI-1640 (1:1) are injected into the dorsolateral site of 6-week-old C.B-17/IcrHsd-scid-bg mice. After 17 days, mice are 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) is performed daily for 8 days. Mice are sacrificed by asphyxiation with CO2 the day after the last treatment. Tumours are measured before and during treatment. Finally, the tumours are excised, measured, weighed and fixed in formalin or shock frozen. Tumour volume is calculated according to the formula for a rotational ellipsoid (length × height × width × 0.5236)[3]. |
参考文献: [1]. Mashhoon N, et al. Crystal structure of a conformation-selective casein kinase-1 inhibitor. J Biol Chem. 2000 Jun 30;275(26):20052-60. | |
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