In vitro activity: K-858 is a novel, potent and ATP-uncompetitive inhibitor of mitotic kinesin Eg5 that has potential antitumor activity. K858 is a potent inhibitor of replication and induces apoptosis in breast tumor cells, independently from the tumor phenotype. This anti-proliferative response of tumor cells to K858 can be limited by the contemporaneous over-expression of survivin; consequently, the reduction of survivin levels, obtained with AKT inhibitors, can sensitize tumor cells to K858-induced apoptosis. K858 blocked centrosome separation, activated the spindle checkpoint, and induced mitotic arrest in cells accompanied by the formation of monopolar spindles. K858 induces mitotic arrest and growth inhibition through the activation of the Mad2-mediated spindle checkpoint. K858 induces mitotic cell death in cancer cells but not in normal cells. K858 induces mitotic arrest, apoptosis, and cell growth inhibition and has no effect on microtubule polymerization.

Kinase Assay: K858 is an ATP-uncompetitive inhibitor of Eg5 that is at least 150-fold more selective for Eg5 than other members of the kinesin superfamily. K858 induces mitotic arrest and growth inhibition through the activation of the Mad2-mediated spindle checkpoint. K858 induces mitotic cell death in cancer cells but not in normal cells. K858 induces mitotic arrest, apoptosis, and cell growth inhibition and has no effect on microtubule polymerization.

Cell Assay: To determine cytotoxicity, sulforhodamine B colorimetric assay is performed: 1.5 × 104 cells are plated on 96 well plates, grown for 24 h and treated with different concentrations of K858 (1 μM, 10 μM, 100 μM) for 24 and 48 h. Cells are then fixed with 50 % trichloroacetic acid for 1 h at 4°C and stained for 30 min at room temperature (RT) with 0.4 % sulforhodamine B in 1 % acetic acid. Excess dye is removed by washing four times with 1 % acetic acid. Protein bound dye is dissolved in 10 mM TRIS pH 10, and optical density (OD) is determined at 510 nm using a microplate reader.