CW069

Catalog No.S7336 Batch:S733601

Technical Data

Formula

C₂₃H₂₁IN₂O₃

Molecular Weight

500.33

CAS No.

1594094-64-0

Solubility (25°C)*

In vitro

DMSO

100 mg/mL (199.86 mM)

Ethanol

4 mg/mL (7.99 mM)

Water

Insoluble

In vivo (Add solvents to the product individually and in order)

Clear solution	5%DMSO 1 40%PEG300 2 5%Tween80 3 50%ddH2O	5.0mg/ml	Taking the 1 mL working solution as an example, add 50 μL of 100 mg/ml clarified DMSO stock solution to 400 μL of PEG300, mix evenly to clarify it; add 50 μL of Tween80 to the above system, mix evenly to clarify; then continue to add 500 μL of ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.
Clear solution	5% DMSO 1 95% Corn oil	1.0mg/ml	Taking the 1 mL working solution as an example, add 50 μL of 20 mg/ml clear DMSO stock solution to 950 μL of corn oil and mix evenly. The mixed solution should be used immediately for optimal results.

* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
* Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

Preparing Stock Solutions

Biological Activity

Description

CW069 is an allosteric, and selective inhibitor of microtubule motor protein HSET with IC50 of 75 μM, significant selectivity over KSP.

Targets

HSET ^[1]

75 μM

In vitro

CW069 increases multipolar spindles in N1E-115 cells with supernumerary centrosomes without altering bipolar spindle morphology in normal human dermal fibroblast cells. CW069 inhibits growth in cancer N1E-115 cells with IC50 of 10 μM, but not in NHDF or primary human bone marrow cells. ^[1]

Features

Allosteric, HSET-selective inhibitor.

Protocol (from reference)

Kinase Assay:^{^[1]}	In Vitro Enzymatic ATPase Assay The protocol is optimized for use with full-length, N-terminal, 6His-tagged HSET and KSP, and measured the MT-stimulated activity of the proteins. Inhibition of the Gsp synthetase activity of HSET/KSP is observed spectrophotometrically by coupling the hydrolysis of ATP to oxidation of NADH via pyruvate kinase/lactate dehydrogenase reactions. The assay is initiated by adding purified Gsp synthetase/amidase (12.8 nM) to an assay mixture containing the following components (final concentration): 6 nM protein, 0.07 mg/ml MTs (University Biologicals), 1.56 mM glutathione, 10 mM spermidine, 2 mM ATP, 2.7 mM MgCl₂, 1 mM phospho(enol)-pyruvate, 0.2 mM NADH, 50 μg/ml lactate dehydrogenase, 100 μg/ml pyruvate kinase, and various concentrations of inhibitor all in 50 mM Na PIPES (pH 6.8) at 37°C. The ADP-Glo detection assay (Promega) is performed as described in the manufacturer’s instructions. All compound additions were performed using a multidrop BioMek Nxp. Plates were read using a Pherastar microplate reader.
Cell Assay:^{^[1]}	Cell lines N1E-115 cells, NHDF and primary human bone marrow cells. Concentrations ~400 μM Incubation Time 72 hours Method Cells are cultured in DMEM supplemented with 10% fetal calf serum (FCS) at 37°C and 5% CO2. All compounds used in the Sulforhodamine B colorimetric (SRB) assay are dissolved in DMSO and diluted in culture medium to a final concentration of 0.2% DMSO. For the SRB assay and live-cell imaging, cells are seeded in 96-well plates at a density of 2,500 cells per well. After 24 hr, the cells are treated with compound for 72 hr, with triplicate wells for each concentration. For the SRB assay, the cells are then fixed with trichloroacetic acid (TCA) and stained with SRB. Fluorescence is quantified using an Infinite 200 PRO plate-reader at a wavelength of 545 nm. Compound-treated wells are compared with solvent control wells and the concentration of compound that results in 50% of the solvent-control cell growth is designated as the IC50 concentration, calculated using Graphpad PRISM 6. At least three biological replicates are performed for each assay.

Kinase Assay:^{^[1]}

In Vitro Enzymatic ATPase Assay
The protocol is optimized for use with full-length, N-terminal, 6His-tagged HSET and KSP, and measured the MT-stimulated activity of the proteins. Inhibition of the Gsp synthetase activity of HSET/KSP is observed spectrophotometrically by coupling the hydrolysis of ATP to oxidation of NADH via pyruvate kinase/lactate dehydrogenase reactions. The assay is initiated by adding purified Gsp synthetase/amidase (12.8 nM) to an assay mixture containing the following components (final concentration): 6 nM protein, 0.07 mg/ml MTs (University Biologicals), 1.56 mM glutathione, 10 mM spermidine, 2 mM ATP, 2.7 mM MgCl₂, 1 mM phospho(enol)-pyruvate, 0.2 mM NADH, 50 μg/ml lactate dehydrogenase, 100 μg/ml pyruvate kinase, and various concentrations of inhibitor all in 50 mM Na PIPES (pH 6.8) at 37°C. The ADP-Glo detection assay (Promega) is performed as described in the manufacturer’s instructions. All compound additions were performed using a multidrop BioMek Nxp. Plates were read using a Pherastar microplate reader.

Cell Assay:^{^[1]}

Cell lines
N1E-115 cells, NHDF and primary human bone marrow cells.
Concentrations
~400 μM
Incubation Time
72 hours
Method
Cells are cultured in DMEM supplemented with 10% fetal calf serum (FCS) at 37°C and 5% CO2. All compounds used in the Sulforhodamine B colorimetric (SRB) assay are dissolved in DMSO and diluted in culture medium to a final concentration of 0.2% DMSO. For the SRB assay and live-cell imaging, cells are seeded in 96-well plates at a density of 2,500 cells per well. After 24 hr, the cells are treated with compound for 72 hr, with triplicate wells for each concentration. For the SRB assay, the cells are then fixed with trichloroacetic acid (TCA) and stained with SRB. Fluorescence is quantified using an Infinite 200 PRO plate-reader at a wavelength of 545 nm. Compound-treated wells are compared with solvent control wells and the concentration of compound that results in 50% of the solvent-control cell growth is designated as the IC50 concentration, calculated using Graphpad PRISM 6. At least three biological replicates are performed for each assay.

References

[1] Watts CA, et al. Chem Biol. 2013, 20(11), 1399-1410.

Customer Product Validation

: Data from [Data independently produced by , , J Cell Sci, 2017, 130(21):3676-3684]

Selleck's CW069 has been cited by 7 publications

Molecular landscape and functional characterization of centrosome amplification in ovarian cancer [ Nat Commun, 2023, 14(1):6505]	PubMed: 37845213
KIF24 depletion induces clustering of supernumerary centrosomes in PDAC cells [ Life Sci Alliance, 2022, 5(11)e202201470]	PubMed: 35803737
Terminally differentiated osteoclasts organize centrosomes into large clusters for microtubule nucleation and bone resorption [ Mol Biol Cell, 2022, mbcE22030098]	PubMed: 35511803
IFT proteins interact with HSET to promote supernumerary centrosome clustering in mitosis. [ EMBO Rep, 2020, 9:e49234]	PubMed: 32270908
Unraveling mitotic protein networks by 3D multiplexed epitope drug screening [ Mol Syst Biol, 2018, 14(8):e8238]	PubMed: 30104419
Centrosome Clustering Is a Tumor-selective Target for the Improvement of Radiotherapy in Breast Cancer Cells [Choe MH Anticancer Res, 2018, 38(6):3393-3400]	PubMed: 29848688
Precocious Centriole Disengagement and Centrosome Fragmentation Induced by Mitotic Delay [ Nat Commun, 2017, 13;8:15803]	PubMed: 28607478

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SHIPPING AND STORAGE
Selleck products are transported at room temperature. If you receive the product at room temperature, please rest assured, the Selleck Quality Inspection Department has conducted experiments to verify that the normal temperature placement of one month will not affect the biological activity of powder products. After collecting, please store the product according to the requirements described in the datasheet. Most Selleck products are stable under the recommended conditions.

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