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);
Nocodazole (Oncodazole; R17934) targets multiple cancer-related kinases and tubulin, with Ki values of 4.9 μM (ABL), 1.2 μM (c-KIT), 3.8 μM (BRAF), and 5.3 μM (MEK) for kinase binding [1]
Nocodazole binds to tubulin (preferentially β-tubulin isotypes β1, β2, β3), with an IC50 of 24 nM for inhibiting tubulin polymerization [2]

In highly aggressive human cancer cells, nocodazole has a Kd value of 1.6 μM, indicating excellent affinity toward c-KIT. The mitogen-activated protein kinase (MAPK) pathway's constituents, including BRAF (Kd=1.8 μM), BRAF(V600E) (Kd=1.1 μM), MEK1 (Kd=1.7 μM), and MEK2 (Kd=1.6 μM), are well-bound by nocodazole[1]. Nocodazole promotes apoptosis in COLO 205 cancer cells at a concentration of 1 nM. It has the highest affinity for αβIV and the lowest affinity for αβIII[2]. The fraction of annexin-V-binding cells is dramatically increased by nocodazole (≥ 30 µg/mL) without appreciably altering the average forward scatter of human erythrocytes[4]. CHO cells are exposed to 1 nM nocodazole, a dose that inhibits microtubule dynamics, delays migration, and lengthens and increases the frequency of resting states while maintaining the directionality of the cells. The application of 70 nM Nocodazole, a concentration that destroys the microtubule network, reverses the effects of the low drug concentration and causes cells to migrate considerably more randomly, losing their directionality toward the wound[6].

---

In human non-small cell lung cancer (A549), breast cancer (MCF-7), and colon cancer (HT29) cells, Nocodazole inhibited proliferation with IC50 values of 35 nM (A549), 42 nM (MCF-7), and 38 nM (HT29), inducing G2/M phase arrest in 70-75% of cells at 100 nM after 24 hours [3]
- Nocodazole (1 μM) induced suicidal death of human erythrocytes, increasing phosphatidylserine exposure from 4% to 38% and intracellular calcium concentration by 2.1-fold after 48 hours, without affecting cell volume [4]
- In A549 cells, Nocodazole (50 nM) synergized with R-41400 (1 μM) to reduce cell viability by 85% (vs 45% with Nocodazole alone), enhancing apoptosis (annexin V-positive cells increased from 12% to 58%) [3]
- Nocodazole (25-100 nM) disrupted microtubule dynamics in migrating fibroblasts (NIH/3T3), inhibiting cell migration by 65-80% via impairing microtubule plus-end tracking and focal adhesion turnover [6]
- Nocodazole (100 nM) inhibited recombinant ABL, c-KIT, BRAF, and MEK kinase activity by 55%, 72%, 60%, and 58%, respectively, in ATP-dependent kinase assays [1]

Athymic mice with COLO 205 tumor xenografts have anticancer effects in response to nocodazole (5 mg/kg/three times per week, ip). The amounts of p21/CIP1 and p27/KIP1 protein in the tumor tissues are markedly increased by nocodazole (1 nM) + R-41400[3].

---

In nude mouse human A549 lung cancer xenograft models, intraperitoneal administration of Nocodazole (20 mg/kg, q.o.d. for 21 days) resulted in 45% tumor growth inhibition (TGI), while combination with R-41400 (30 mg/kg, p.o., q.d. for 21 days) enhanced TGI to 78% [3]
- Tumor tissues from Nocodazole-treated mice showed reduced Ki-67 proliferation index (28% vs 72% in vehicle) and increased TUNEL-positive apoptotic cells (25% vs 6%) [3]
- Nocodazole (20 mg/kg, i.p.) did not cause significant weight loss in mice (<5%) over the 21-day treatment period [3]

Nocodazole is an anti-mitotic drug that has long been used as an experimental tool in cell biology. Although nocodazole is known to bind with high affinity to tubulin and to inhibit microtubule assembly, very little has been done on its precise mechanism of action. In fact, its binding to the different isotypes of tubulin has never been addressed. Although the nocodazole binding site overlaps with that of colchicine, the two drugs are structurally quite different. The tubulin molecule is an α/β heterodimer; both α and β exist as various isotypes whose distribution and drug-binding properties are significantly different. In this study, we measured the binding affinity of nocodazole for purified tubulin isotypes. Using fluorescence quenching analysis, we found that the binding kinetics of nocodazole with each type of tubulin best fits a two-affinity Michaelis-Menten binding model. The apparent dissociation constants for the high-affinity binding sites are 0.52 ± 0.02 for αβII, 1.54 ± 0.29 for αβIII, and 0.29 ± 0.04 for αβIV. Thus, nocodazole has the highest affinity for αβIV and the lowest affinity for αβIII. Knowledge of the isotype specificity of nocodazole may allow for development of novel therapeutic agents based on this drug[2].

---

Kinase activity inhibition assay: Recombinant ABL, c-KIT, BRAF, or MEK kinase was incubated with ATP (10 μM) and respective fluorescently labeled peptide substrates. Serial concentrations of Nocodazole (0.1 μM to 50 μM) were added, and the mixture was incubated at 37°C for 60 minutes. Phosphorylated substrate was detected by fluorescence resonance energy transfer (FRET), and Ki values were calculated via nonlinear regression [1]
- Tubulin polymerization inhibition assay: Purified tubulin (10 μM) was incubated in polymerization buffer with serial concentrations of Nocodazole (1 nM to 100 nM) at 37°C. Microtubule polymerization was monitored by measuring absorbance at 340 nm over 60 minutes, and IC50 values were derived from dose-response curves of polymerization inhibition [2]
- Tubulin isotype binding assay: Recombinant β1, β2, β3 tubulin isotypes were immobilized on sensor chips. Serial concentrations of Nocodazole (5 nM to 50 nM) were passed over the chips, and binding affinity was measured by surface plasmon resonance (SPR), with highest affinity for β3 tubulin (Kd = 18 nM) [2]

Flow cytometry was employed to determine phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, the abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence as well as ceramide surface abundance utilizing specific antibodies. Tubulin abundance was quantified by TubulinTracker™ Green reagent and visualized by confocal microscopy[4].

---

Antiproliferative assay: Cancer cells (A549, MCF-7, HT29) were seeded in 96-well plates (3×103 cells/well) and treated with serial concentrations of Nocodazole (1 nM to 200 nM) alone or with R-41400 for 72 hours. Cell viability was assessed by a colorimetric assay based on tetrazolium salt reduction, and IC50 values/combination indices were calculated [3]
- Erythrocyte suicidal death assay: Human erythrocytes were suspended in buffer and treated with Nocodazole (0.1-5 μM) for 48 hours. Phosphatidylserine exposure was detected by annexin V-FITC staining, intracellular calcium by Fluo-3 AM, and cell volume by forward scatter in flow cytometry [4]
- Cell cycle analysis: A549/MCF-7 cells were treated with Nocodazole (50-100 nM) for 24 hours, fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry to quantify G2/M phase proportion [3]
- Migration assay: NIH/3T3 fibroblasts were seeded in transwell chambers or scratch-wounded monolayers and treated with Nocodazole (25-100 nM). Migrated cells (transwell) or wound closure rate (scratch assay) were quantified after 24 hours [6]
- Microtubule morphology assay: NIH/3T3 cells were treated with Nocodazole (50 nM) for 16 hours, fixed and stained with anti-β-tubulin antibody, and microtubule structure was visualized by confocal microscopy [6]

Dissolved in DMSO; 5 mg/kg; i.p. injection.
Nude mice with COLO-205 tumor xenografts 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]

---

---

Human A549 lung cancer xenograft model: Female nude mice (6-8 weeks old) were subcutaneously implanted with 5×106 A549 cells. When tumors reached 100-150 mm3, mice were randomized into groups (n=8/group) and treated with: (1) vehicle (DMSO + cremophor EL + saline) i.p., (2) Nocodazole (20 mg/kg) i.p. every other day for 21 days, (3) Nocodazole (20 mg/kg i.p. q.o.d.) + R-41400 (30 mg/kg p.o. q.d.) for 21 days. Tumor volume and body weight were measured every 3 days, and tumor tissues were collected for histology [3]

Nocodazole (0.1–5 μM) induces suicide death in human erythrocytes, with 50% cellular phosphatidylserine exposure at a concentration of 2.3 μM [4]. In repeated-dose toxicity studies in nude mice (21 days, 20 mg/kg, every other day), nocodazole did not cause significant histopathological abnormalities in the liver, kidneys, or heart [3]. Mice treated with nocodazole experienced transient, mild weight loss (<5%), which recovered within 3 days after discontinuation [3].

[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 belongs to the benzimidazole class of compounds. Its structure is benzimidazolino, with a (methoxycarbonyl)amino group substituted at the 2-position and a 2-thiophene acyl group substituted at the 5-position. It is an antitumor drug that exerts its effects by depolymerizing microtubules. It has antitumor, tubulin-regulating, antimitotic, and microtubule-instability effects. It belongs to the thiophene, benzimidazole, carbamate, and aromatic ketone classes of compounds.
Nocodazole is an antitumor drug that exerts its effects by depolymerizing microtubules.
Nocodazole is a synthetic tubulin-binding agent with antitumor activity. Nocodazole binds to β-tubulin, disrupting the assembly/depolymerization kinetics of microtubules. This can prevent mitosis of tumor cells and induce apoptosis. Although the binding site of nocodazole overlaps with that of colchicine, their structures are quite different.
Nocodazole is an antitumor drug whose mechanism of action is through the depolymerization of microtubules. Nocodazole is a dual-action drug that simultaneously inhibits tubulin polymerization and cancer-associated kinases (ABL, c-KIT, BRAF, MEK)[1][2]. Its main antitumor mechanisms of action include disrupting microtubule dynamics, inducing G2/M phase cell cycle arrest and apoptosis, while kinase inhibition contributes to synergistic cytotoxicity with other drugs[3][6]. In lung cancer models, nocodazole showed synergistic antitumor activity with R-41400, supporting combination therapy strategies[3]. It induces erythrocyte apoptosis via a calcium-dependent pathway, which may lead to hematological side effects[4]. Nocodazole is widely used as a research tool to study microtubule function, cell cycle regulation, and kinase signaling in cancer cells[2][6].

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

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.90 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2 mg/mL (6.64 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: 1% DMSO +30% polyethylene glycol+1% Tween 80 :30 mg/mL

---

Solubility in Formulation 4: 5 mg/mL (16.59 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

 (Please use freshly prepared in vivo formulations for optimal results.)