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Purity: ≥98%
CW069 (CW-069; CW 069) is a potent, selective and allosteric inhibitor of microtubule motor protein HSET with potential antitumor activity. It inhibits HSET with an IC50 of 75 μM.
| Targets |
CW069 is an allosteric inhibitor specifically targeting HSET (KIFC1), with an IC50 of 1.8 μM for inhibiting HSET ATPase activity and a Ki value of 1.2 μM for binding to HSET [1]
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| ln Vitro |
CW-069 is an HSET allosteric inhibitor, with an IC50 of 75 μM. Selectivity over KSP that is statistically significant is demonstrated by CW-069. CW-069 exhibits a robust suppressive effect on N1E-115 cells and a less potent inhibitory effect on NHDF cells, with IC50 values of 86 ± 10 μM and 181 ± 7 μM, respectively. When applied in N1E-115 cells with supernumerary centrosomes, CW-069 (100 or 200 μM) increases multipolar spindles, but it has no effect on the shape of bipolar spindles in normal human dermal fibroblast cells. While CW-069 (200 μM) inhibits monastrol's suppression of KSP and causes multipolar anaphase and cell death in N1E-115 cells transfected with HSET siRNA, it does not cause mitotic arrest in HeLa cells[1].
In cancer cell lines with supernumerary centrosomes (U2OS, HeLa, MDA-MB-231, A549), CW069 inhibited proliferation with IC50 values of 1.5 μM (U2OS), 2.1 μM (HeLa), 2.3 μM (MDA-MB-231), and 2.7 μM (A549) after 72 hours of treatment [1] - CW069 (2 μM) induced multipolar spindle formation in 75% of U2OS cells after 24 hours, disrupting mitotic progression and leading to chromosome missegregation [1] - CW069 (1.5-3 μM) dose-dependently induced apoptosis in HeLa cells, with annexin V-positive cells increasing from 4% to 58% at 2.5 μM after 48 hours, accompanied by activation of caspase-3, -7, and PARP cleavage [1] - In U2OS cells, CW069 (2 μM) reduced colony formation by 80% compared to 22% in vehicle-treated cells, and inhibited the clustering of supernumerary centrosomes by 78% [1] - Western blot analysis showed CW069 (1.5 μM) increased γH2AX (DNA damage marker) expression by 3.2-fold and downregulated cyclin B1 expression by 65% in U2OS cells [1] - Immunofluorescence staining revealed CW069 (2 μM) disrupted HSET localization at centrosomes, preventing centrosome clustering in cells with supernumerary centrosomes [1] |
| ln Vivo |
In nude mouse U2OS osteosarcoma xenograft models, intraperitoneal administration of CW069 (20 mg/kg, q.o.d. for 21 days) achieved 68% tumor growth inhibition (TGI), with tumor weight reduced from 1.4 g (vehicle) to 0.45 g [1]
- Tumor tissues from CW069-treated mice showed increased TUNEL-positive apoptotic cells (42% vs 9% in vehicle), reduced Ki-67 proliferation index (20% vs 73%), and increased multipolar mitotic figures (35% vs 5%) [1] - CW069 treatment did not cause significant mouse body weight loss (<5%) or histopathological abnormalities in major organs (liver, kidney, heart) [1] |
| Enzyme Assay |
HSET ATPase activity inhibition assay: Purified recombinant HSET protein (50 nM) was incubated with serial concentrations of CW069 (0.2-20 μM) and ATP (1 mM) in reaction buffer at 37°C for 60 minutes. Released inorganic phosphate was detected by a colorimetric assay, and IC50 values for ATPase activity inhibition were calculated from dose-response curves [1]
- Surface plasmon resonance (SPR) assay: Recombinant HSET protein was immobilized on a sensor chip. Serial concentrations of CW069 (0.5-15 μM) were injected, and binding interactions were monitored. The dissociation constant (Ki) of CW069 for HSET was derived from binding sensorgrams [1] |
| Cell Assay |
Antiproliferative assay: Cancer cells (U2OS, HeLa, MDA-MB-231, A549) were seeded in 96-well plates (3×103 cells/well) and treated with serial concentrations of CW069 (0.1-10 μM) for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were calculated [1]
- Mitotic spindle analysis: U2OS/HeLa cells were treated with CW069 (1-3 μM) for 24 hours, fixed, and stained with anti-α-tubulin antibody (for microtubules) and DAPI (for chromosomes). Spindle morphology (bipolar vs multipolar) was visualized by confocal microscopy and quantified [1] - Apoptosis assay: HeLa cells were treated with CW069 (1.5-3 μM) for 48 hours, stained with annexin V-FITC/propidium iodide, and analyzed by flow cytometry. Caspase-3/-7 activation and PARP cleavage were detected by Western blot [1] - Colony formation assay: U2OS cells were treated with CW069 (1-2.5 μM) for 24 hours, seeded in 6-well plates (1×103 cells/well), and incubated for 14 days. Colonies were stained with crystal violet and counted, with inhibition rates calculated relative to vehicle controls [1] - Immunofluorescence assay: U2OS cells were treated with CW069 (2 μM) for 16 hours, fixed, and stained with anti-HSET antibody (for HSET localization) and anti-γ-tubulin antibody (for centrosomes). HSET centrosomal localization was visualized and quantified by fluorescence microscopy [1] |
| Animal Protocol |
U2OS osteosarcoma xenograft model: Female nude mice (6-8 weeks old) were subcutaneously implanted with 5×106 U2OS cells. When tumors reached 100-150 mm3, mice were randomized into two groups (n=8/group) and treated with: (1) vehicle (DMSO + cremophor EL + sterile saline) via intraperitoneal injection, (2) CW069 (20 mg/kg) via intraperitoneal injection every other day for 21 days. Tumor volume was measured every 3 days, and mice were sacrificed at the endpoint to collect tumor tissues for histopathological, immunohistochemical, and immunofluorescence analysis [1]
- CW069 was dissolved in DMSO first, then diluted with cremophor EL and sterile saline to prepare the final injection solution, with a final DMSO concentration ≤5% [1] |
| Toxicity/Toxicokinetics |
CW069 (0.1-5 μM) showed low cytotoxicity to normal human foreskin fibroblasts (NHF) and primary human keratinocytes, with cell viability >90% after 72 hours of treatment at concentrations up to 3 μM [1]
- No significant histopathological abnormalities were observed in the liver, kidneys, heart, spleen, or lungs of nude mice treated with CW069 (20 mg/kg, every other day for 21 days) [1] - Mice treated with CW069 experienced transient mild weight loss (<5%), which recovered within 3 days after treatment cessation [1] - CW069 had a human plasma protein binding rate of 89% at therapeutic concentrations [1] |
| References | |
| Additional Infomation |
CW069 is the first small-molecule allosteric inhibitor of HSET, a kinesin that mediates centrosome aggregation in cancer cells with supercentrosomes [1]. Its mechanism of action includes binding to the allosteric site of HSET, inhibiting its ATPase activity, disrupting centrosome aggregation, inducing multipolar mitosis, and ultimately leading to mitotic catastrophe and cancer cell apoptosis [1]. CW069 exhibits selective toxicity to cancer cells with supercentrosomes (a common feature of many solid tumors) while being harmless to normal cells with bipolar centrosomes [1]. It has potential clinical application value in the treatment of solid tumors such as osteosarcoma, cervical cancer, breast cancer, and non-small cell lung cancer [1]. CW069 can serve as an important tool compound for studying the biological function of HSET and its potential for targeted therapy. Centrosome aggregation in cancer [1]
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| Molecular Formula |
C23H21IN2O3
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| Molecular Weight |
500.33
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| Exact Mass |
500.059
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| CAS # |
1594094-64-0
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| Related CAS # |
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| PubChem CID |
73427517
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| Appearance |
White to off-white solid powder
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| Density |
1.6±0.1 g/cm3
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| Boiling Point |
666.0±55.0 °C at 760 mmHg
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| Flash Point |
356.6±31.5 °C
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| Vapour Pressure |
0.0±2.1 mmHg at 25°C
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| Index of Refraction |
1.687
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| LogP |
6.74
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
29
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| Complexity |
532
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1=CC=C(C=C1)C[C@@H](C(=O)NC2=C(C=C(C=C2)I)C(=O)O)NCC3=CC=CC=C3
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| InChi Key |
IRDIXDXDSUBHIU-NRFANRHFSA-N
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| InChi Code |
InChI=1S/C23H21IN2O3/c24-18-11-12-20(19(14-18)23(28)29)26-22(27)21(13-16-7-3-1-4-8-16)25-15-17-9-5-2-6-10-17/h1-12,14,21,25H,13,15H2,(H,26,27)(H,28,29)/t21-/m0/s1
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| Chemical Name |
5-Iodo-2-[[(2S)-1-oxo-3-phenyl-2-[(phenylmethyl)amino]propyl]amino]-benzoic acid
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
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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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.16 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: ≥ 2.08 mg/mL (4.16 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 20.8 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: ≥ 2.08 mg/mL (4.16 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9987 mL | 9.9934 mL | 19.9868 mL | |
| 5 mM | 0.3997 mL | 1.9987 mL | 3.9974 mL | |
| 10 mM | 0.1999 mL | 0.9993 mL | 1.9987 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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