伊斯平斯作用機(jī)制 - Medchemexpress - MCE中國(guó)

1、Product Data SheetIspinesibCat. No.: HY-50759CAS No.: 336113-53-2分式: CHClNO分量: 517.06作靶點(diǎn): Kinesin; Apoptosis作通路: Cell Cycle/DNA Damage; Cytoskeleton; Apoptosis儲(chǔ)存式: 4C, protect from light* In solvent : -80C, 6 months; -20C, 1 month (protect from light)溶解性數(shù)據(jù)體外實(shí)驗(yàn) DMSO : 125 mg/mL (241.75 mM)H2O : 0.1 m

2、g/mL (insoluble)* means soluble, but saturation unknown.SolventMass1 mg 5 mg 10 mgConcentration制備儲(chǔ)備液1 mM 1.9340 mL 9.6701 mL 19.3401 mL5 mM 0.3868 mL 1.9340 mL 3.8680 mL10 mM 0.1934 mL 0.9670 mL 1.9340 mL請(qǐng)根據(jù)產(chǎn)品在不同溶劑中的溶解度選擇合適的溶劑配制儲(chǔ)備液；旦配成溶液，請(qǐng)分裝保存，避免反復(fù)凍融造成的產(chǎn)品失效。儲(chǔ)備液的保存式和期限：-80C, 6 months; -20C, 1 month (

3、protect from light)。-80C 儲(chǔ)存時(shí)，請(qǐng)?jiān)?6 個(gè)內(nèi)使，-20C 儲(chǔ)存時(shí)，請(qǐng)?jiān)?1 個(gè)內(nèi)使。體內(nèi)實(shí)驗(yàn)請(qǐng)根據(jù)您的實(shí)驗(yàn)動(dòng)物和給藥式選擇適當(dāng)?shù)娜芙獍?。以下溶解案都?qǐng)先按照 In Vitro 式配制澄清的儲(chǔ)備液，再依次添加助溶劑：為保證實(shí)驗(yàn)結(jié)果的可靠性，澄 的儲(chǔ)備液可以根據(jù)儲(chǔ)存條件，適當(dāng)保存；體內(nèi)實(shí)驗(yàn)的作液，建議您現(xiàn)現(xiàn)配，當(dāng)天使； 以下溶劑前顯的百分 指該溶劑在您配制終溶液中的體積占；如在配制過(guò)程中出現(xiàn)沉淀、析出現(xiàn)象，可以通過(guò)加熱和/或超聲的式助溶1. 請(qǐng)依序添加每種溶劑： 10% DMSO 40% PEG300 5% Tween-80 45% salineSolubility:

4、2.5 mg/mL (4.84 mM); Clear solution此案可獲得 2.5 mg/mL (4.84 mM，飽和度未知) 的澄清溶液。以 1 mL 作液為例，取 100 L 25.0 mg/mL 的澄 DMSO 儲(chǔ)備液加到 400 L PEG300 中，混合均勻；向上述體系中加50 L Tween-80，混合均勻；然后繼續(xù)加 450 L 理鹽定容 1 mL。2. 請(qǐng)依序添加每種溶劑： 10% DMSO 90% corn oilSolubility: 2.5 mg/mL (4.84 mM); Clear solution此案可獲得 2.5 mg/mL (4.84 mM，飽和度未知)

5、的澄 溶液，此案不適于實(shí)驗(yàn)周 期在半個(gè)以上的實(shí)驗(yàn)。Page 1 of 2 www.MedChemE以 1 mL 作液為例，取 100 L 25.0 mg/mL 的澄 DMSO 儲(chǔ)備液加到 900 L 油中，混合均勻。BIOLOGICAL ACTIVITY物活性 Ispinesib種特異性的紡錘體驅(qū)動(dòng)蛋 KSP 抑制劑，Ki app 值為 1.7 nM。IC & Target KSP1.7 nM (Ki app)體外研究 Ispinesib is a potent, highly specific inhibitor of KSP, with a Ki app of 1.7 nM1. Ispin

6、esib (150 nM) inhibits BT-474 andMDA-MB-468 cell lines, with GI50s of 45 and 19 nM, respectively2. Ispinesib (SB715992, 15 and 30 nM) suppressesthe proliferation of PC-3 prostate cancer cell by 48.65% and 52.16%, and induces apoptosis of prostate cancer cell by1094.88% and 1516.70%, respectively. Is

7、pinesib up regulates genes responsible for apoptosis and cell cycle arrest, anddown regulates genes responsible for cell proliferation and survival. The anti-proliferation and pro-apoptotic activitiesof Ispinesib can be enhanced by genistein3.體內(nèi)研究 Ispinesib (SCID, 8 mg/kg; nude, 10 mg/kg, q4d 3) red

8、uces tumor volume in mice bearing tumor xenografts of ER-positive (MCF7), HER2-positive (KPL4, HCC1954, and BT-474), and triple-negative (MDA-MB-468) breast cancer cellsvia i.p. one dose every 4 days repeated three times2.PROTOCOLKinase Assay 1 Kinesin specificity analysis is carried out using a pyr

9、uvate kinaselactate dehydrogenase detection system thatcouples the production of ADP to oxidation of NADH. Absorbance changes are monitored at 340 nm. Steady-statestudies using nanomolar concentrations of KSP are performed using a sensitive fluorescence-based assay utilizing apyruvate kinase, pyruva

10、te oxidase, and horseradish peroxidase coupled detection system that couples the generationof ADP to oxidation of Amplex Red to fluorescent resorufin. Generation of resorufin is monitored by fluorescence (excitation = 520 nm and emission= 580 nm). Steady-state biochemical experiments are performed i

11、n PEM25 buffer25 mM Pipes-K+ (pH 6.8), 2 mM MgCl2, 1 mM EGTA supplemented with 10 M paclitaxel for experiments involvingmicrotubules. The IC50 for steady-state inhibition is determined at 500 M ATP, 5 M MTs, and 1 nM KSP in PEM25buffer. Ki app (apparent inhibitor dissociation constant) estimates of

12、Ispinesib are extracted from the concentration-response curves, with explicit correction for enzyme concentration1.MCE has not independently confirmed the accuracy of these methods. They are for reference only.Cell Assay 3 PC-3 prostate cancer cells are seeded in 96 well plates at a density of 4 103

13、 cells/well. PC-3 cells are incubated for24 hours to allow attachment to the surface of each well of the tissue culture plate. Then, the cells are treated withvarying concentration of reagents and incubated for 1 to 3 days. First, PC-3 cells are treated with 15 and 30 nM ofIspinesib, respectively. S

14、econd, PC-3 cells are subjected to combinational treatments with 7.5 or 10 nM of Ispinesibplus 30 M of genistein. Finally, PC-3 cells are pre-treated with 30 M of genistein for 24 hours followed bytreatment with 15 nM of Ispinesib. Control cells are treated with 0.3 mM Na2CO3 (vehicle control). Afte

15、r treatment,PC3 cells are incubated at 37C with MTT (0.5 mg/mL) for 2 hours and isopropyl alcohol at room temperature for 1hour. The spectrophotometric absorbance of each sample is then determined by using ULTRA Multifunctional MicroPlate Reader at 595 nm3.MCE has not independently confirmed the acc

16、uracy of these methods. They are for reference only.Animal Mice with a tumor volume of -250 mm3 receive a single dose of Ispinesib (10 mg/kg). Tumors are dissected, fixed inAdministration 2 10% buffered formalin, and embedded in paraffin, and 5-m tissue sections are prepared. Antigen retrieval is do

17、nePage 2 of 3 www.MedChemEby boiling in 50 mM citrate buffer (pH 5.5), and sections are then incubated in 3% hydrogen peroxide, washed inPBS-0.1% Tween, and blocked in 10% goat serum. Phospho-histone H3 (PH3) antibody is detected using Alexa Fluor488 secondary antibody. Images are taken with a micro

18、scope at 10 magnification and captured using MetaMorphsoftware to quantify PH3 expression by computing the area ratio of PH3-positive cells per total cells. Ki67/cleavedcaspase-3 staining is done. Nonfluorescent images are taken on an Olympus BX41 microscope at 20 magnification2.MCE has not independently confirmed the accuracy of these methods. They are for reference only.戶使本產(chǎn)品發(fā)表的科研獻(xiàn) Redox Biol. 2019 Feb;21:101112.See more customer validations on HYPERLINK www.MedChemE www.MedChemEREFERENCES1. Lad L, et al. Mechanism of inhibition of human KSP by ispine