基本信息

产品描述

JIB-04是一种广谱的选择性Jumonji histone demethylase抑制剂，无细胞试验中对JARID1A，JMJD2E，JMJD3，JMJD2A，JMJD2B，JMJD2C和JMJD2D的IC50分别是230 nM，340 nM，855 nM，445 nM，435 nM，1100 nM和290 nM。

靶点（IC50 & Targe）

JARID1A,230nM

JMJD2A,445nM

JMJD2B,435nM

JMJD2D,290nM

JMJD2E,340nM

体外研究

JIB-04 以癌症选择性的方式引起转录改变，包括下调增殖基因，上调抗增殖/前凋亡基因。JIB-04能够以IC50低至10 nM的水平阻碍肺癌、前列腺癌细胞系的增殖，同时在HBECs和PrSCs/PrECs中产生较少的抗增殖活性。[1]

体内研究

在两个小鼠异种移植模型（H358或A549）中，JIB-04减少肿瘤生长，降低肿瘤中Jumonji组蛋白脱甲基酶的活性，延长肿瘤患者生存时间。[1]

激酶实验

Jumonji脱甲基酶/脯氨酰羟化酶/LSD1活性检测:

活性JMJD2E氨基酸1-350从E.coli中纯化，体外环境中与α-酮戊二酸，5-10 μM铁离子，抗坏血酸，组蛋白肽段基质一起使用，偶联反应时加入甲醛脱氢酶和NAD+来定量NADH的生成量，或使用Epigentek kit P-3081。组蛋白脱甲基反应结果通过Western分析来定量，构建His-tagged hJMJ2D氨基酸1-350表达体系，E.coli表达产物通过Qiagen Ni-NTA琼脂糖手册进行纯化。蛋白用50 mM TrisHCl pH 7.8+0.3 M NaCl + 10%甘油+200 mM immidazol洗脱，后用20 mM TrisHCl pH 7.4，0.15 M NaCl，0.2 mM PMSF，0.5 mM DTT，8%甘油透析。蛋白分装，速冻、储存在-80°C。Western blot中，～1.5 μg酶与0.3 μg H3K9me3基质（Active Motif #31213），10 μM (NH4)2Fe(SO4)2，1 mM α-酮戊二酸，2 mM L-抗坏血酸钠在50 mM pH 7.9溶液中混合，与溶媒或药物在37°C孵育30min-2hrs。加入SDS loading buffer后，煮沸，样品在NuPage 4-12% Bis-Tris胶上电泳，转膜，用Upstate #07-523处理硝化纤维素膜以检测H3K9me3。H3总信号是通过Active Motif #39763和连接IRDye 680的驴抗兔IgG（二抗, LI-COR # 926-32223），在Odyssey Infrared成像系统中获取图像。体外IC50检测和竞争性试验，100-200 ng纯化蛋白与溶媒、JIB-04或类似物共孵育，通过ELISA（H3K9me3去甲基作用：Epigentek kit P-3081，H3K4me3去甲基作用：P-3083，H3K27me3去甲基作用：P-3085）检测活性，反应液包含50mM Hepes pH 7.5，0.01% Tween 20，120nM (NH4)2Fe(SO4)2，1 mMα-酮戊二酸，2 mM L-抗坏血酸钠，50ng肽段基质。最终酶浓度分别为：206 nM JMJD2A，12 nM JMJD2B，60 nM JMJD2C，90 nM JMJD2D E.coli，30 nM JMJD2D Sf9，30 nM JMJD2E，30 nM Jarid1a，35 nM JMJD3。E.coli表达的hJMJD2A（氨基酸1-350）纯化后以每孔400 ng的量进行检测。通过GraphPad Prism软件计算IC50和进行曲线拟合。需要注意的是在基质竞争性试验中， 应该使分析处于线性范围并且不超出ELISA板的结合能力。细胞裂解物的H3K9me3脱甲基酶活性的直接测定：超声处理细胞（细胞接种浓度为2x10E+06/10cm板）或肿瘤PBS匀浆（3x4 sec），等量的蛋白与组蛋白H3K9me3基质在反应液中37°C孵育2h后，用Epigentek kit P-3081进行免疫检测。500 ng E.coli表达的PHD2纯化蛋白，溶于40mM Tris pH 7.4，100mM NaCl，20% glycerol，5mM β-mercapto-ethanol，10mM 麦芽糖备用。从HIF-1 ODD分离出的生物素化的肽段固定在亲和素包裹的96孔板中。酶与被试药物在反应液（20 mM Tris-Cl pH 7.5，5 mM KCl，1.5 mM MgCl2，2 mM DTT，0.12 μM硫酸亚铁，0.5 mM戊邻酮二酸盐，1 mM抗坏血酸）中室温孵育45min。α-酮戊二酸的竞争性类似物DMOG作为抑制作用的阳性对照。肽段羟基化是通过对羟基化HIF肽表位使用多克隆兔抗体，再加入连接羊抗兔HPR的二抗。EnVision上读取发光值。LSD1重组蛋白活性通过使用Epigentek kit P-3075检测。

细胞实验

Cell lines: 人肺癌细胞系(LCa)，原代或非致癌细胞系HBECs，前列腺癌(PCa)，原代前列腺基质细胞(PrSC)和前列腺上皮细胞(PrEC)。

Concentrations: ～10 μM

Incubation Time: 96小时

Method:以每孔1500-3000细胞接种至96孔板，次日开始以递增浓度的化合物处理细胞4天后，用MTS标准分析法用Promega’s Cell Titer或Cell Titer-Glo试剂测试细胞活性，使用Spectra Max或FlouroStar Omega酶标仪读取490 nm和650 nm的吸光度。每种细胞系进行2-5次独立测试，每次包含4-8个测试孔。IC50 通过DIVISA软件计算得到。

(Only for Reference)

动物实验

Animal Models: H358或A549小鼠异种移植模型

Formulation: 12.5%聚氧乙烯蓖麻油，12.5% DMSO水性悬液(A549，灌胃)，10% DMSO 90%芝麻油(H358，腹腔注射)

Dosages: 110 mg/kg (H358，腹腔注射)，55 mg/kg (A549, 灌胃)

Administration: 灌胃或腹腔注射

(Only for Reference)

参考文献

[1] Wang L, et al. Nat Commun. 2013, 4, 2035.

安全信息

图形或危害标志
危险说明	H302 H410
防范说明	P264 P270 P273 P301+P312 P330 P391 P501
UN号码	3077
危险分类	9
包装等级	III

产品说明

应用

JIB-04 has been used in sulforhodamine B cell growth assay. It has also been used as a control to determine the inhibitory action of ML324 on KDM4A (histone lysine demethylase 4A).

包装

5, 25 mg in glass bottle

生化/生理作用

JIB-04 is a selective inihibitor of Jumonji demethylases without inhibiting other α-ketoglutarate-dependent hydroxylases or histone-modifying enzymes including amine oxidase LSD1 (also known as KDM1A), which demethylates histone lysines. JIB-04 is a pan-inhibitor of Jumonji demethylases with JARID1A (KDM5A) being the most sensitive (IC₅₀=230 nM) followed by JMJD2D (IC₅₀=290 nM) and JMJD3 (KDM6B) and JMJD2C (KDM4C) more resistant (IC50 855 and 1100 nM, respectively). JIB-04 selectively inhibited viability of several human cancer cell lines with little toxicity towards normal cells, and also diminished tumor growth in two separate xenograft mouse models.

基本信息

产品性质

安全信息

基本信息

安全信息

图形或危害标志
危险性说明	H302: Harmful if swallowed. H315: Causes skin irritation. H319: Causes serious eye irritation. H335: May cause respiratory irritation.
防范说明	P233: Keep container tightly closed. P260: Do not breathe . P261: Avoid breathing P264: Wash hands thoroughly after handling. P270: Do not eat, drink or smoke when using this product. P271: Use only outdoors or in a well-ventilated area. P280: Wear . P301 + P312: IF SWALLOWED: Call a POISON CENTER or doctor/physician if you feel unwell. P302 + P352: IF ON SKIN: Wash with plenty of soap and water. P304: IF INHALED: . P304 + P340: IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing. P305 + P351 + P338: IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. P312: Call a POISON CENTER or doctor/physician if you feel unwell. P321: Specific treatment (see . on this label). P330: Rinse mouth. P332 + P313: If skin irritation occurs: Get medical advice/ attention. P337 + P313: If eye irritation persists: Get medical advice/attention. P340: Remove victim to fresh air and keep at rest in a position comfortable for breathing. P362: Take off contaminated clothing and wash before reuse. P403: Store in a well-ventilated place. P403 + P233: Store in a well-ventilated place. Keep container tightly closed. P405: Store locked up. P501: Dispose of contents/container to local regulations.