In vitro activity: OTS964 inhibits the growth of TOPK-positive cells with low IC50 values [A549 (31 nM), LU-99 (7.6 nM), DU4475 (53 nM), MDAMB- 231 (73 nM), T47D (72 nM), Daudi (25 nM), UM-UC-3 (32 nM), HCT-116 (33 nM),MKN1 (38 nM), MKN45 (39 nM), HepG2 (19 nM), MIAPaca-2 (30 nM), and 22Rv1 (50 nM)], whereas its growth inhibitory effect against TOPK-negative HT29 cancer cells is significantly weaker, with IC50 of 290nM. Although OTS964 reveals some suppressive effect on Src family kinases, the response to OTS964 in these cancer cells is not correlated with the expression of Src family kinases c-Src, Fyn, and Lyn, supporting the TOPK-dependent growth inhibitory effects of OTS964. Time lapse imaging in T47D cells shows that treatment with OTS964 induces cytokinesis defects followed by apoptosis.

Kinase Assay: OTS964, a dimethylated derivative of OTS514, is a potent and selective TOPK ((T-lymphokine-activated killer cell-originated protein kinase) inhibitor. It inhibits TOPK with high affinity and selectivity (IC50 value is 28 nM). TOPK is a protein that is found in a wide variety of cancers/tumors and is believed to work as an oncogene that promote tumor growth. Therefore, as an inhibitor of TOPK, OTS964 has potential anticancer activity. It causes a cytokinesis defect and the subsequent apoptosis of cancer cells in vitro as well as in xenograft models of human lung cancer.

Cell Assay: Cells (100 μl, A549 cells, LU-99 cells, DU4475 cells, MDA-MB-231 cells, T47D cells, Daudi cells, UM-UC-3 cells, HCT-116 cells, MKN1 cells, MKN45 cells, HepG2 cells, MIAPaca-2 cells, 22Rv1 cells and HT29 cells) are plated in 96-well plates at a certain density. The cells are allowed to adhere overnight before exposure to compounds for 72 hours at 37°C. Plates are read with a spectrophotometer at a wavelength of 450 nm. All assays are carried out in triplicate. After measuring IC50 values, we calculates the z scores to produce P values. After log transformation (base 10) of IC50 values (nM), the mean and SD are calculated for the log values of the IC50 for the 13 TOPK-positive cell lines.