规格 | 价格 | 库存 | 数量 |
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10 mM * 1 mL in DMSO |
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1mg |
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2mg |
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5mg |
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10mg |
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25mg |
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50mg |
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100mg |
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250mg |
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Other Sizes |
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靶点 |
PORCN (IC50 = 74 pM)[1]
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体外研究 (In Vitro) |
通过阻断 Wnt 棕榈酰化、Wnt 与载体蛋白 Wntless/WLS 的相互作用、Wnt 分泌以及 Wnt β-连环蛋白报告活性的激活,确定 Wnt-C59 (C59) 在纳摩尔浓度下可在体外降低 PORCN 功能。 Wnt-C59 的 IC50 为 74 pM,可阻断 WNT3A 介导的驱动荧光素酶的多聚化 TCF 结合位点的激活[1]。
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体内研究 (In Vivo) |
在小鼠中,wnt-C59 表现出良好的生物利用度。 Wnt-C59 下调 Wnt/β-catenin 靶基因,同时预防 MMTV-WNT1 转基因小鼠中乳腺癌的生长[1]。在人类癌症中,Wnt-C59 具有消除癌症干细胞的能力。 Wnt-C59 阻止 HNE1 和 SUNE1 细胞中球体的形成,从而以剂量依赖性方式抑制 NPC 细胞的干性特征 [2]。
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酶活实验 |
Wnt分泌、TOPFlash分析、Dvl2移动转移和PORCN救援[1]
为了测定C59 IC50,用稀释到培养基中1/1000的化合物处理STF3a细胞,48小时后测量荧光素酶活性。为了监测Wnt分泌到培养基中,用C59处理STF3a细胞,C59稀释在以一半体积添加的含有1%FBS的新鲜培养基中。24小时后在4%SDS中收集裂解物。对于HT1080细胞中的SuperTOPflash测定,用50 ng Wnt、100 ng mCherry和650 ng SuperTOPflash在24孔板中转染实验。对于PORCN拯救实验,添加了100 ng 3xHA-mPORCN-D。为了测量Dvl2迁移率偏移,用200ng的每个Wnt质粒和600ng的mCherry转染。在100mM磷酸钠、pH 7.5、150mM NaCl、1%IGEPAL-CA630、完全蛋白酶抑制剂混合物中制备裂解物,并进行蛋白质印迹以检测Dvl2 Wnt WLS交互[1] 用C末端V5标记的WNT3A或WNT8A转染6孔培养皿中的HeLa细胞。转染后6小时,用0.1%DMSO作为对照或加入终浓度为10nM的C59代替培养基。孵育过夜后,用缓冲液裂解细胞:50mM HEPES、150mM NaCl、1mM EDTA、0.5%NP-40和完全蛋白酶抑制剂)。用兔抗WLS C端多克隆抗体免疫沉淀共500μg裂解物。对V5和WLS进行蛋白质印迹(使用针对WLS的小鼠抗体) Alk-C16代谢标记和点击化学[1] 用C-末端V5标记的WNT3A转染在10cm板中以3×106细胞密度铺板的HT1080细胞。转染6小时后,用DMEM代替培养基,DMEM含有5%不含脂肪酸的BSA(Sigma),含有或不含有100μMω-炔基棕榈酸(Alk-C16),如先前报道所合成(3,4)。同时用0.1%DMSO作为对照或100nM C59处理细胞。过夜孵育后,用冷PBS洗涤细胞两次,并在缓冲液中制备细胞裂解物:50mM HEPES、150nM NaCl、1mM EDTA、1%NP-40和蛋白酶抑制剂(Roche)。将蛋白裂解物与抗V5抗体孵育过夜,然后加入30μL蛋白A/G加琼脂糖(50%浆液)2小时。将颗粒用裂解缓冲液洗涤四次,并重悬于磷酸钠缓冲液(100mM磷酸钠,pH 7.5,1%Nonidet P-40,150mM NaCl)中。然后将免疫沉淀的蛋白质在50μl体积中进行点击标记反应1小时RT,最终浓度依次加入以下试剂:0.1 mM生物素叠氮化物(Life Technologies)、1 mM Tris(2-羧乙基)膦盐酸盐(TCEP)、0.2 mM Tris[(1-苄基-1H-1,2,3-三唑-4-基)甲基]胺(TBTA)溶于二甲基亚砜/叔丁醇(20%/80%)和1 mM CuSO4(在水中新制备)。标记的免疫沉淀蛋白通过SDS–PAGE解析,转移到PVDF膜上,用抗V5一级抗体印迹,然后用Dylight 680缀合的抗小鼠抗体和Dylight 800缀合的链亲和素印迹,然后在各自的通道中显现。 |
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细胞实验 |
体外C59毒性研究[1]
HCT116、K562、HL60、A549、U-2-OS、U266、HCC1937、HCC38、MCF7、BT-20、MB157、MDA-MB-435S、MDA-MB-231、MDA-MBA-468、DLD1、SW480、T98G、A172、U-87MG、KATO III、SNU1、SNU5、SNU16、SK-MEL-5、SK-MEL-28、HS294T、A375、A2058、MEWO、FU97、IM95、IM95M、NUGC-3、NUGC-4、OCUM-1、SCH、MKN1、MKN7、MKN45 MKN74、AZ521、JHH2、SNU216、YCC-3(韩国细胞系库,韩国)、EOL-1在供应商规定的培养条件下生长。对于测定,将75μl培养基中的5000个细胞接种在黑色96孔板的每个孔中,并在37°C下孵育过夜。C59在培养基中以4倍原液的形式连续稀释,然后将25μl的每种稀释液加入细胞中,得到50μM至1.5 nM的最终浓度。处理2天后,向每个孔中加入100μl CellTiter-Glo®发光细胞活力测定试剂,并在室温下孵育10分钟。使用Tecan Safire仪器测量发光。 |
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动物实验 |
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参考文献 |
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分子式 |
C25H21N3O
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分子量 |
379.45
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精确质量 |
379.168
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元素分析 |
C, 79.13; H, 5.58; N, 11.07; O, 4.22
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CAS号 |
1243243-89-1
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相关CAS号 |
Wnt-C59;1243243-89-1
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PubChem CID |
57519544
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外观&性状 |
White to yellow solid
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密度 |
1.2±0.1 g/cm3
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沸点 |
628.3±55.0 °C at 760 mmHg
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闪点 |
333.8±31.5 °C
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蒸汽压 |
0.0±1.8 mmHg at 25°C
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折射率 |
1.648
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LogP |
4.19
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tPSA |
54.88
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氢键供体(HBD)数目 |
1
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氢键受体(HBA)数目 |
3
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可旋转键数目(RBC) |
5
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重原子数目 |
29
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分子复杂度/Complexity |
508
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定义原子立体中心数目 |
0
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SMILES |
O=C(NC1=CC=C(C2=CC=CN=C2)C=C1)CC3=CC=C(C4=CC(C)=NC=C4)C=C3
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InChi Key |
KHZOJCQBHJUJFY-UHFFFAOYSA-N
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InChi Code |
InChI=1S/C25H21N3O/c1-18-15-22(12-14-27-18)20-6-4-19(5-7-20)16-25(29)28-24-10-8-21(9-11-24)23-3-2-13-26-17-23/h2-15,17H,16H2,1H3,(H,28,29)
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化学名 |
2-(4-(2-methylpyridin-4-yl)phenyl)-N-(4-(pyridin-3-yl)phenyl)acetamide
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别名 |
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HS Tariff Code |
2934.99.9001
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存储方式 |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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运输条件 |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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溶解度 (体外实验) |
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溶解度 (体内实验) |
配方 1 中的溶解度: ≥ 2.5 mg/mL (6.59 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加,逐一添加), 澄清溶液。
例如,若需制备1 mL的工作液,可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中,混匀;然后向上述溶液中加入50 μL Tween-80,混匀;加入450 μL生理盐水定容至1 mL。 *生理盐水的制备:将 0.9 g 氯化钠溶解在 100 mL ddH₂O中,得到澄清溶液。 配方 2 中的溶解度: ≥ 2.5 mg/mL (6.59 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加,逐一添加), 澄清溶液。 例如,若需制备1 mL的工作液,可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中,混匀。 *20% SBE-β-CD 生理盐水溶液的制备(4°C,1 周):将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中,得到澄清溶液。 View More
配方 3 中的溶解度: ≥ 2.5 mg/mL (6.59 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加,逐一添加), 澄清溶液。 配方 4 中的溶解度: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O:5 mg/mL 1、请先配制澄清的储备液(如:用DMSO配置50 或 100 mg/mL母液(储备液)); 2、取适量母液,按从左到右的顺序依次添加助溶剂,澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明,并不一定代表此产品 的实际溶解配方): 10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline); 假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中,混合均匀/澄清;向上述体系中加入50 μL Tween-80,混合均匀/澄清;然后继续加入450 μL ddH2O (或 saline)定容至 1 mL; 3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例; 4、 如产品在配制过程中出现沉淀/析出,可通过加热(≤50℃)或超声的方式助溶; 5、为保证最佳实验结果,工作液请现配现用! 6、如不确定怎么将母液配置成体内动物实验的工作液,请查看说明书或联系我们; 7、 以上所有助溶剂都可在 Invivochem.cn网站购买。 |
制备储备液 | 1 mg | 5 mg | 10 mg | |
1 mM | 2.6354 mL | 13.1770 mL | 26.3539 mL | |
5 mM | 0.5271 mL | 2.6354 mL | 5.2708 mL | |
10 mM | 0.2635 mL | 1.3177 mL | 2.6354 mL |
1、根据实验需要选择合适的溶剂配制储备液 (母液):对于大多数产品,InvivoChem推荐用DMSO配置母液 (比如:5、10、20mM或者10、20、50 mg/mL浓度),个别水溶性高的产品可直接溶于水。产品在DMSO 、水或其他溶剂中的具体溶解度详见上”溶解度 (体外)”部分;
2、如果您找不到您想要的溶解度信息,或者很难将产品溶解在溶液中,请联系我们;
3、建议使用下列计算器进行相关计算(摩尔浓度计算器、稀释计算器、分子量计算器、重组计算器等);
4、母液配好之后,将其分装到常规用量,并储存在-20°C或-80°C,尽量减少反复冻融循环。
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)。如该浓度超过该批次药物DMSO溶解度,请首先与我们联系。
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 ddH2O,混匀澄清。
(1) 请确保溶液澄清之后,再加入下一种溶剂 (助溶剂) 。可利用涡旋、超声或水浴加热等方法助溶;
(2) 一定要按顺序加入溶剂 (助溶剂) 。
NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
NCT03436134 | UNKNOWN STATUS | Other:Sessions of conventional apheresis Other:Sessions of news apheresis with double filtration |
Antibody Mediated Rejection Kidney Transplantation |
University Hospital,Grenoble | 2018-07-01 | Not Applicable |
C59 is a bona fide inhibitor of PORCN activity. A, C59 is a potent inhibitor of Wnt/β-catenin signaling. HEK293 cells constitutively expressing WNT3A and the β-catenin reporter STF were treated with C59 or dimethyl sulfoxide (DMSO). After 48 hours, luciferase activity was measured. Error bars represent SD. Structure of C59 is shown above. Inset, WNT3A secretion into culture medium was blocked by 0.1 nmol/L C59. Uncut immunoblots are shown in Supplementary Fig. S1A. B, PORCN overexpression reverses the effects of C59. HT1080 cells were transfected with empty vector (EV) or mPORCN-D expression plasmids followed by treatment with C59 (1 nmol/L) or IWP1 (1 μmol/L). Luciferase activity was measured after 24 hours. Error bars represent SD. C, C59 blocks the palmitoylation-dependent Wnt–WLS interaction. HeLa cells were transfected with either WNT3A-V5 or WNT8A-V5 plasmids, then treated with DMSO or C59 (10 nmol/L). WLS was immunoprecipitated and precipitates were probed for WLS and V5. Uncut immunoblots are shown in Supplementary Fig. S1B. D, C59 blocks palmitoylation of Wnts. Alk-C16 was added to HeLa cells transfected with WNT3A-V5 and cotreated with either DMSO, C59 (100 nmol/L), or IWP1 (1 μmol/L). Lysates were prepared and Wnt was immunoprecipitated with antibody to V5. Click chemistry was conducted to attach azido-biotin to alkyne-palmitate groups. Finally, samples were separated by SDS-PAGE and probed for biotin and WNT3A-V5. This result was reproduced in HT1080 cells (Supplementary Fig. S1C). Cancer Res . 2013 Jan 15;73(2):502-7. td> |
C59 is bioavailable and prevents MMTV-WNT1 tumor growth. A, C59 is bioavailable. Mice were given a single dose of 2.5 mg/kg C59 intravenously or 5 mg/kg orally. At times indicated after treatment, mice were sacrificed and C59 plasma concentration was measured by liquid chromatography/tandem mass spectrometry. Dotted line indicates calculated IC50. Error bars represent SD. B, C59 prevents growth of MMTV-WNT1 tumors. Female nude mice orthotopically transplanted with independent MMTV-WNT1 tumors were treated with vehicle (line 1, n = 8; line 2, n = 10) or C59 10 mg/kg (line 1, n = 10; line 2, n = 12) once daily for 17 days. Tumor volumes were measured on alternate days. Data is presented as mean ± SD. P < 0.001 (d7-17) using 2-tailed t test. C, C59 significantly decreased tumor weight. Tumor weights at sacrifice from the transplanted mice are shown. Data analyzed using 2-tailed t test. D, C59 prevents growth of primary MMTV-WNT1 tumors. Female virgin MMTV-WNT1 mice with measurable mammary tumors were treated with vehicle (6 mice) or 5 mg/kg C59 (5 mice) for 21 days. Data represents change in tumor volume. Data is presented as mean ± SEM. P < 0.05 from days 7 to 21 using 2-tailed t test. Cancer Res . 2013 Jan 15;73(2):502-7. td> |
C59 decreases Wnt pathway activity in MMTV-WNT1 tumors. A, C59 inhibits β-catenin target gene expression. Total RNA was isolated from orthotopically transplanted tumors, and transcript levels for Axin2, Ccnd2, C-myc, and Tcf7 were measured by qRT-PCR. Expression was normalized to Actb. ***, P < 0.001, 2-tailed t test. B, C59 decreases proliferation. Ki67 immunostaining in sections from the primary tumors (open symbols) and orthotopically transplanted tumors (closed symbols) was digitally quantified. Percentages of Ki67-positive nuclei are shown. Data analyzed using 2-tailed t test. C, C59 decreases cytoplasmic and nuclear β-catenin in tumors. β-catenin staining in MMTV-WNT1 tumor sections. Two representative samples from each treatment arm are shown. Right, outset, are enlargement of areas indicated in middle. Scale bars, 50 μm. D, C59 at therapeutically effective dose does not affect intestinal nuclear β-catenin. Intestinal sections from mice treated with vehicle or C59 for 21 days were stained for β-catenin. Cancer Res . 2013 Jan 15;73(2):502-7. td> |