CW069
目录号 : GC11252
An inhibitor of KIFC/HSET
Cas No.:1594094-64-0
Sample solution is provided at 25 µL, 10mM.
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CW069 is a selective allosteric inhibitor of HSET with IC50 value of 75 μM [1].
HSET is a minus-end microtubule (MT) motor protein and is encoded by KIFC1 and Kifc5a in humans and mice, respectively. HSET plays an important role in the assembly of a functional mitotic spindle [1].
CW069 is a selective allosteric inhibitor of HSET. CW069 exhibited significant selectivity over KSP. In silico model for HSET binding, the highly favorable hydrogen bond formed between the carboxylate of CW069 and the guanidinium group of Arg521 in HSET. Also, a cation-πinteraction formed between the exposed phenyl group of CW069 and the Arg521 side chain. Arg521 was critical for CW069 selectivity. The enantiomer of CW069 bound more weakly to HSET due to a weak interaction between the carboxylate of the ligand and Arg521 in HSET. In N1E-115 cancer cells, CW069 significantly increased multipolar spindle formation due to a lack of centrosome clustering. In MDA-MB-231 and BT549 breast cancer cells, CW069 significantly increased multipolar spindles. In N1E-115 cells, CW069 (200 μM) induced multipolar anaphase formation and apoptosis [1].
Reference:
[1]. Watts CA, Richards FM, Bender A, et al. Design, synthesis, and biological evaluation of an allosteric inhibitor of HSET that targets cancer cells with supernumerary centrosomes. Chem Biol, 2013, 20(11): 1399-1410.
Kinase experiment: | The protocol is optimized for use with full-length, N-terminal, 6His-tagged HSET and KSP, and measured the MT-stimulated activity of the proteins. Inhibition of the Gsp synthetase activity of HSET/KSP is observed spectrophotometrically by coupling the hydrolysis of ATP to oxidation of NADH via pyruvate kinase/lactate dehydrogenase reactions. The assay is initiated by adding purified Gsp synthetase/amidase (12.8 nM) to an assay mixture containing the following components (final concentration): 6 nM protein, 0.07 mg/mL MTs, 1.56 mM glutathione, 10 mM spermidine, 2 mM ATP, 2.7 mM MgCl2, 1 mM phospho(enol)-pyruvate, 0.2 mM NADH, 50 μg/mL lactate dehydrogenase, 100 μg/mL pyruvate kinase, and various concentrations of inhibitor (CW-069) all in 50 mM Na PIPES (pH 6.8) at 37°C. The ADP-Glo detection assay is performed. All compound additions are performed using a multidrop BioMek Nxp. Plates are read using a Pherastar microplate reader[1]. |
Cell experiment: | NHDF cells, HeLa cells, BT549, MCF-7 and MDA-MB-231 cells are verified by STR genotyping and all tested negative for mycoplasma. Cells are cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) at 37°C and 5% CO2. All compounds (CW-069) used in the Sulforhodamine B colorimetric (SRB) assay are dissolved in DMSO and diluted in culture medium to a final concentration of 0.2% DMSO. For the SRB assay and live-cell imaging, cells are seeded in 96-well plates at a density of 2,500 cells per well. After 24 hr, the cells are treated with compound (CW-069) for 72 hr, with triplicate wells for each concentration. For the SRB assay, the cells are then fixed with trichloroacetic acid (TCA) and stained with SRB. Fluorescence is quantified using an Infinite 200 PRO plate-reader at a wavelength of 545 nm. Compound (CW-069)-treated wells are compared with solvent control wells and the concentration of compound that resulted in 50% of the solvent-control cell growth is designated as the IC50 concentration, calculated using Graphpad PRISM 6. At least three biological replicates are performed for each assay[1]. |
References: [1]. Watts CA, et al. Design, synthesis, and biological evaluation of an allosteric inhibitor of HSET that targets cancer cells with supernumerary centrosomes. Chem Biol. 2013 Nov 21;20(11):1399-410. | |
| Cas No. | 1594094-64-0 | SDF | |
| 化学名 | 2-[[(2S)-2-(benzylamino)-3-phenylpropanoyl]amino]-5-iodobenzoic acid | ||
| Canonical SMILES | C1=CC=C(C=C1)CC(C(=O)NC2=C(C=C(C=C2)I)C(=O)O)NCC3=CC=CC=C3 | ||
| 分子式 | C23H21IN2O3 | 分子量 | 500.33 |
| 溶解度 | ≥ 50mg/mL in DMSO | 储存条件 | Store at -20° C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 1.9987 mL | 9.9934 mL | 19.9868 mL |
| 5 mM | 0.3997 mL | 1.9987 mL | 3.9974 mL |
| 10 mM | 0.1999 mL | 0.9993 mL | 1.9987 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Quality Control & SDS
- View current batch:
- Purity: >99.50%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet