Kinase experiment:

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, 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 (CW-069) all in 50 mM Na PIPES (pH 6.8) at 37℃. The ADP-Glo detection assay is performed. All compound additions are performed using a multidrop BioMek Nxp. Plates are read using a Pherastar microplate reader[1].

Cell experiment:

NHDF cells, HeLa cells, BT549, MCF-7 and MDA-MB-231 cells are verified by STR genotyping and all tested negative for mycoplasma. Cells are cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) at 37℃ and 5% CO2. All compounds (CW-069) 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 (CW-069) 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 (CW-069)-treated wells are compared with solvent control wells and the concentration of compound that resulted 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[1].

CW069 is a selective allosteric inhibitor of HSET with IC50 value of 75 μM [1].

HSET is a minus-end microtubule (MT) motor protein and is encoded by KIFC1 and Kifc5a in humans and mice, respectively. HSET plays an important role in the assembly of a functional mitotic spindle [1].

CW069 is a selective allosteric inhibitor of HSET. CW069 exhibited significant selectivity over KSP. In silico model for HSET binding, the highly favorable hydrogen bond formed between the carboxylate of CW069 and the guanidinium group of Arg521 in HSET. Also, a cation-πinteraction formed between the exposed phenyl group of CW069 and the Arg521 side chain. Arg521 was critical for CW069 selectivity. The enantiomer of CW069 bound more weakly to HSET due to a weak interaction between the carboxylate of the ligand and Arg521 in HSET. In N1E-115 cancer cells, CW069 significantly increased multipolar spindle formation due to a lack of centrosome clustering. In MDA-MB-231 and BT549 breast cancer cells, CW069 significantly increased multipolar spindles. In N1E-115 cells, CW069 (200 μM) induced multipolar anaphase formation and apoptosis [1].

Reference:
[1]. Watts CA, Richards FM, Bender A, et al. Design, synthesis, and biological evaluation of an allosteric inhibitor of HSET that targets cancer cells with supernumerary centrosomes. Chem Biol, 2013, 20(11): 1399-1410.