OTS514

OTS514 is a highly potent TOPK inhibitor, which inhibits TOPK kinase activity with a median inhibitory concentration (IC50) value of 2.6 nM.

Signaling Pathways >> Cell Cycle/DNA Damage >> TOPK

Research Areas >> Cancer

IC50: 2.6 nM (TOPK)[1]

To confirm the specificity of OTS514 against TOPK, a panel of 60 diverse human protein kinases is used. The activity of each kinase is measured after a 2 hour incubation with 0.2 μM OTS514. The highest inhibition is observed for TOPK (83.5% inhibition), whereas the mean and the SD of the inhibitory effects against other kinases are 11.5 and 18.5%, respectively, indicating the specificity of the TOPK inhibitory effect of OTS514. TOPK expression is examined in cancer cell lines derived from various cancer types, such as lung cancer (A549, LU-99), breast cancer (DU4475, MDA-MB-231, and T47D), Burkitt lymphoma (Daudi), bladder cancer (UM-UC-3), colon cancer (HCT-116 and HT29), gastric cancer (MKN1 and MKN45), liver cancer (HepG2), pancreatic cancer (MIAPaca-2), and prostate cancer (22Rv1). High expression of TOPK is observed in all of these cell lines except HT29. The growth inhibitory effects of OTS514 are examined ton each cell line and strong growth inhibitory effects are found with low IC50 values ranging from 1.5 to 14 nM for the TOPK-positive cancer cell lines. On the other hand, the IC50 value for HT29 cells, in which TOPK expression is hardly detectable, is significantly higher at 170 nM (P=5.92×10-11).[1].

The in vivo antitumor effect of OTS514 is first investigated t in a xenograft model of A549 cells (TOPK-positive lung cancer cells). Intravenous administration of free OTS514 at 1, 2.5, and 5 mg/kg once a day for 2 weeks results in tumor growth inhibition (TGI) of 5.7, 43.3, and 65.3% on day 15, respectively, without any body weight loss. The antitumor effect of OTS514 is further investigated in another lung cancer xenograft model of LU-99 cells. Intravenous administration of OTS514 (5 mg/kg) once a day for 2 weeks achieve good growth-suppressive effect with TGI of 104% without any body weight loss. However, although the antitumor effect against LU-99 xenograft is stronger than that against A549 xenograft, the treatment still causes a significant reduction of white blood cells (WBCs) (P<0.01)[1].

CD34+ HSCs are cultured in RPMI supplemented with 20% fetal bovine serum and 1× StemSpan CC100. Cells are treated with OTS514 (20 or 40 nM) or OTS-964 (100 or 200 nM) for 48 hours. Collected cells are washed with phosphate-buffered saline (PBS) and resuspended in 100 μL of PBS followed by staining with CD41a antibody for 20 min at room temperature. Finally, the cells are washed with PBS again and then analyzed for CD41a staining by flow cytometry on the BD FACSCalibur. Expression of STAT5 is examined by Western blot with an anti-STAT5 antibody[1].

Mice[1] A549 (1×107 cells) or LU-99 cells (5×106 or 1×107 cells) are injected subcutaneously in the left flank of female BALB/cSLC-nu/nu mice. When A549 xenografts have reached an average volume of 200 mm3 or when LU-99 xenografts have reached an average volume of 150 or 200 mm3, animals are randomized into groups of six mice. The in vivo antitumor effect of OTS514 is first investigated in a xenograft model of A549 cells (TOPK-positive lung cancer cells). OTS514 is administered to mice bearing A549 cells after the tumor size reaches about 200 mm3. Mice are intravenously treated with OTS514 (1, 2.5, and 5 mg/kg) once a day for 2 weeks. The tumor size is measured as a surrogate marker of drug response, and the percentage of tumor growth inhibition (TGI) is calculated. The antitumor effect of OTS514 is further investigated tin another lung cancer xenograft model of LU-99 cells. OTS514 is administered to mice bearing LU-99 cancer cells after the tumor size reaches about 200 mm3. Mice are intravenously treated with OTS514 (5 mg/kg) once a day for 2 weeks. Tumor volumes are determined using a caliper. The weight of the mice is determined as an indicator of tolerability on the same days[1].

[1]. Matsuo Y, et al. TOPK inhibitor induces complete tumor regression in xenograft models of human cancer through inhibition of cytokinesis. Sci Transl Med. 2014 Oct 22;6(259):259ra145.

OTS-964 | HI TOPK 032