Home Cytoskeletal Signaling Microtubule Associated inhibitor CW069 For research use only.

CW069

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

CW069 Chemical Structure

CW069 Chemical Structure

CAS No. 1594094-64-0

Purity & Quality Control

Batch: S733601 DMSO]100 mg/mL]false]Ethanol]4 mg/mL]false]Water]Insoluble]false Purity: 99.02%
99.02

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Biological Activity

Description CW069 is an allosteric, and selective inhibitor of microtubule motor protein HSET with IC50 of 75 μM, significant selectivity over KSP.
Features Allosteric, HSET-selective inhibitor.
Targets
HSET [1]
75 μM
In vitro
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]
Kinase Assay 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 Research 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.

Chemical Information & Solubility

Molecular Weight 500.33 Formula

C23H21IN2O3
CAS No. 1594094-64-0 SDF Download CW069 SDF
Smiles C1=CC=C(C=C1)CC(C(=O)NC2=C(C=C(C=C2)I)C(=O)O)NCC3=CC=CC=C3
Storage (From the date of receipt)

In vitro
Batch:

DMSO : 100 mg/mL ( (199.86 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Ethanol : 4 mg/mL

Water : Insoluble


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Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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