Nocodazole (Oncodazole; R17934)   中文名称: 诺考达唑

Abl:91 nM (Kd); ABL(E255K):120 nM (Kd); ABL(T315I):170 nM (Kd); PI3Kγ:1.5 μM (Kd); BRAF:1.8 μM (Kd); MET:1.7 μM (Kd); BRAF(V600E):1.1 μM (Kd); c-KIT:1.6 μM (Kd); MEK1:1.7 μM (Kd); Microtubule/Tubulin CRISPR/Cas9; MEK2:1.6 μM (Kd);

在高度侵袭性的人类癌细胞中，诺考达唑的 Kd 值为 1.6 μM，表明对 c-KIT 具有出色的亲和力。丝裂原激活蛋白激酶 (MAPK) 通路的成分包括 BRAF (Kd=1.8 μM)、BRAF(V600E) (Kd=1.1 μM)、MEK1 (Kd=1.7 μM) 和 MEK2 (Kd=1.6 μM)，与诺考达唑良好结合[1]。 1 nM 浓度的诺考达唑可促进 COLO 205 癌细胞凋亡。它对 αβIV 的亲和力最高，对 αβIII 的亲和力最低[2]。诺考达唑 (≥ 30 µg/mL) 显着增加了膜联蛋白 V 结合细胞的比例，而不会明显改变人红细胞的平均前向散射[4]。 CHO 细胞暴露于 1 nM 诺考达唑，该剂量可抑制微管动力学、延迟迁移、延长和增加静息状态的频率，同时保持细胞的方向性。 70 nM 诺考达唑（一种破坏微管网络的浓度）的应用逆转了低药物浓度的影响，导致细胞更加随机地迁移，失去了向伤口的方向性[6]。

具有 COLO 205 肿瘤异种移植物的无胸腺小鼠对诺考达唑（5 mg/kg/每周 3 次，腹腔注射）有抗癌作用。诺考达唑 (1 nM) + R-41400 显着增加肿瘤组织中 p21/CIP1 和 p27/KIP1 蛋白的量[3]。

诺考达唑是一种抗有丝分裂药物，长期以来一直被用作细胞生物学的实验工具。尽管已知诺考达唑与微管蛋白结合并抑制微管组装，但对其确切的作用机制知之甚少。事实上，它与不同同种型微管蛋白的结合从未得到解决。虽然诺考达唑的结合位点与秋水仙素的结合位点重叠，但这两种药物在结构上却大不相同。微管蛋白分子是α/β异二聚体；α和β以各种同种型存在，其分布和药物结合特性存在显著差异。在这项研究中，我们测量了诺考达唑对纯化微管蛋白同种型的结合亲和力。通过荧光淬灭分析，我们发现诺考达唑与每种微管蛋白的结合动力学最符合双亲和力Michaelis-Menten结合模型。高亲和力结合位点的表观解离常数为αβII为0.52±0.02，αβIII为1.54±0.29，αβIV为0.29±0.04。因此，诺考达唑对αβIV的亲和力最高，对αβIII的亲和力最低。了解诺考达唑的同种型特异性可能有助于开发基于该药物的新型治疗药物[2]。

流式细胞术用于通过膜联蛋白-V结合测定细胞表面的磷脂酰丝氨酸暴露量，通过前向散射测定细胞体积，通过Fluo3荧光测定[Ca2+]i，通过2'，7'-二氯二氢荧光素（DCF）二乙酸酯依赖荧光测定活性氧物种（ROS）的丰度，以及利用特异性抗体测定神经酰胺表面丰度。TubulinTracker™Green试剂对微管蛋白丰度进行定量，并通过共聚焦显微镜进行可视化[4]。

Our previous studies demonstrated that the oral antifungal agent ketoconazole (KT) induces apoptosis and G0/G1 phase cell cycle arrest in human cancer cell lines. In this study, we first demonstrated that KT (1 microM) potentiated the apoptotic effects of nocodazole (ND, 1 nM) in COLO 205 cancer cells. We further demonstrated the therapeutic efficacy of a combined treatment of KT (50 mg/kg/three times per week) and ND (5 mg/kg/three times per week) in vivo by treating athymic mice bearing COLO 205 tumor xenografts. The antitumor effects of ND were significantly potentiated by KT in mice after 6 wk of treatment. No gross signs of toxicity were observed in mice receiving these treatment regimens. The apoptotic cells were detected in a microscopic view of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and by observation of DNA fragmentation in KT + ND-treated tumor tissues. The levels of cell cycle regulatory proteins were determined by Western blot analysis. Treatment with KT inhibits tumor growth through elevation of p53, p21/CIP1, and p27/KIP1 as well as inhibition of cyclin D3 and cyclin-dependent kinase 4 protein expression. Immunohistochemical staining analysis showed that p53, p21/CIP1, and p27/KIP1 immunoreactivity were induced in the tumor tissues. To clarify the roles of the p21/CIP1 and p27/KIP1 protein expression involved in G(0)/G(1) arrest and/or apoptosis induced by a combined treatment with KT and ND, antisense oligodeoxynucleotides (ODNs) specific to p21/CIP1 and p27/KIP1 were used. Our results demonstrated that apoptotic phenomena, including BAX induction and cytochrome C released from mitochondria induced by KT + ND, were significantly attenuated by pretreatment the cells with the p27/KIP1-specific antisense ODNs. These results indicate that p27/KIP1 protein does indeed play a critical role in the KT + ND-induced apoptosis. Our study revealed the molecular mechanism of KT + ND in regression of the tumor growth. The apoptotic effects of KT in a great variety of cancer cells make it a very attractive agent for cancer chemotherapy.[3]

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Dissolved in DMSO; 5 mg/kg; i.p. injection.

Nude mice with COLO-205 tumor xenografts

[1]. Nocodazole is a high-affinity ligand for the cancer-related kinases ABL, c-KIT, BRAF, and MEK. ChemMedChem. 2012 Jan 2;7(1):53-6.

[2]. Interaction of nocodazole with tubulin isotypes. Drug Development Research 2002.

[3]. R-41400 potentiates the antitumor effects of nocodazole: In vivo therapy for human tumor xenografts in nude mice. Mol Carcinog. 2002 Aug;34(4):199-210.

[4]. Nocodazole Induced Suicidal Death of Human Erythrocytes. Cell Physiol Biochem. 2016;38(1):379-92.

[5]. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol. 2017 Feb 20;18(1):35.

[6]. The role of microtubules and their dynamics in cell migration. J Biol Chem. 2012 Dec 21;287(52):43359-69.

Nocodazole is a white powder. (NTP, 1992)
Nocodazole is a member of the class of benzimidazoles that is benzimidalole which is substituted at position 2 by a (methoxycarbonyl)amino group and at position 5 by a 2-thienoyl group. It is an antineoplastic agent that exerts its effect by depolymerising microtubules. It has a role as an antineoplastic agent, a tubulin modulator, an antimitotic and a microtubule-destabilising agent. It is a member of thiophenes, a member of benzimidazoles, a carbamate ester and an aromatic ketone.
Nocodazole is an antineoplastic agent which exerts its effect by depolymerizing microtubules.
Nocodazole is a synthetic tubulin-binding agent with antineoplastic activity. Nocodazole binds to beta-tubulin and disrupts microtubule assembly/disassembly dynamics. This prevents mitosis and induces apoptosis in tumor cells. Although nocodazole binding site overlaps with that of colchicine, the two agents are structurally quite different.
Nocodazole is an antineoplastic agent which exerts its effect by depolymerizing microtubules.

C14H11N3O3S

301.32

301.052

C, 55.80; H, 3.68; N, 13.95; O, 15.93; S, 10.64

31430-18-9

4122

Light yellow to brown solid powder

1.5±0.1 g/cm3

300 °C (dec.)

1.732

2.43

112.32

2

5

4

21

420

0

KYRVNWMVYQXFEU-UHFFFAOYSA-N

InChI=1S/C14H11N3O3S/c1-20-14(19)17-13-15-9-5-4-8(7-10(9)16-13)12(18)11-3-2-6-21-11/h2-7H,1H3,(H2,15,16,17,19)

methyl N-[6-(thiophene-2-carbonyl)-1H-benzimidazol-2-yl]carbamate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.08 mg/mL (6.90 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 20.8 mg/mL澄清DMSO储备液加入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。

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配方 2 中的溶解度: ≥ 2 mg/mL (6.64 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 20.0mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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。

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配方 3 中的溶解度: 1% DMSO +30% polyethylene glycol+1% Tween 80 :30 mg/mL

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配方 4 中的溶解度: 5 mg/mL (16.59 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
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。