TOPK (IC50 = 2.6 nM)

In a xenograft model of A549 cells (TOPK-positive lung cancer cells), OTS514 (1–5 mg/kg; once daily for 2 weeks; intravenous administration) induces tumor regression[1].

Expression of TOPK and phosphorylation of histone H3 (Ser10) were examined by Western blot, as described previously. Other antibodies used for Western blots are as follows: c-Src (1:1000), Fyn (1:1000), and Lyn (1:1000). In vitro cell viability was measured by the colorimetric assay using Cell Counting Kit-8. Cells (100 μl) were plated in 96-well plates at a density that generated continual linear growth (A549, 1 × 103 cells; LU-99, 2 × 103 cells; DU4475, 4 × 103 cells; MDA-MB-231, 3 × 103 cells; T47D, 3 × 103 cells; Daudi, 5 × 103 cells; UM-UC-3, 1 × 103 cells; HCT-116, 1 × 103 cells; MKN1, 2 × 103 cells; MKN45, 4 × 103 cells; HepG2, 4 × 103 cells; MIAPaca-2, 2 × 103 cells; 22Rv1, 6 × 103 cells; and HT29, 3 × 103 cells). The cells were allowed to adhere overnight before exposure to compounds for 72 hours at 37°C. Plates were read with a spectrophotometer at a wavelength of 450 nm. All assays were carried out in triplicate. After measuring IC50 values, we calculated the z scores to produce P values. After log transformation (base 10) of IC50 values (nM), the mean and SD were calculated for the log values of the IC50 for the 13 TOPK-positive cell lines. The mean and SD were 0.76 and 0.23 for OTS514 and 1.53 and 0.26 for OTS964. Then, the z scores from the HT29 IC50 values of OTS514 and OTS964 were 6.44 and 3.62, respectively[1].

In vitro differentiation of human HSCs[1]
CD34+ HSCs were purified from growth factor–mobilized peripheral blood of healthy donors, and then cells were cultured in RPMI supplemented with 20% fetal bovine serum and 1× StemSpan CC100. Cells were treated with OTS514 (20 or 40 nM) or OTS964 (100 or 200 nM) for 48 hours. Collected cells were 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 were washed with PBS again and then analyzed for CD41a staining by flow cytometry on the BD FACSCalibur. Expression of STAT5 was examined by Western blot with an anti-STAT5 antibody.

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Microarray analysis[2]
5 × 105 H929 cells were treated with 0.015% DMSO, 15 nM OTS514, 15 µM lenalidomide (LEN), or 5 nM carfilzomib (CFZ) for 24 hours. Additionally, each active drug combination was performed (OTS514/LEN, OTS514/CFZ, OTS514/LEN/CFZ, and LEN/CFZ). RNA from three independent experiments (a total of 24 samples) was extracted with the Qiagen RNeasy mini kit and analyzed on two Human HT12v4 bead arrays at the University of Chicago functional genomics core facility. Gene Set Enrichment Analysis (GSEA) was performed on quantile‐normalized, background‐subtracted data using hallmark gene sets from the Molecular Signatures Database v6.1.27, 28 Upstream regulator analysis was generated through the use of Ingenuity Pathway Analysis.

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The cells were cultivated in RPMI supplemented with 1×StemSpan CC100 and 20% fetal bovine serum. For 48 hours, cells were exposed to either OTS964 (100 or 200 nM) or OTS514 (20 or 40 nM). Following a PBS wash and resuspension in 100 milliliters of PBS, the collected cells were stained for 20 minutes at room temperature using CD41a antibody. Ultimately, the cells underwent one more PBS wash before being subjected to flow cytometry analysis for CD41a staining. Using an anti-STAT5 antibody, a Western blot was used to measure STAT5 expression.

Female BALB/cSLC-nu/nu mice bearing a xenograft model of A549 cells[1]
1, 2.5, and 5 mg/kg
Intravenously treated; once every day for 2 weeks

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In vivo xenograft study[1]
A549 (1 × 107 cells) or LU-99 cells (5 × 106 or 1 × 107 cells) were injected subcutaneously in the left flank of female BALB/cSLC-nu/nu mice. When A549 xenografts had reached an average volume of 200 mm3 or when LU-99 xenografts had reached an average volume of 150 or 200 mm3, animals were randomized into groups of six mice. The starting tumor volume of 150 mm3 was used for LU-99 xenografts when tumors were monitored for a longer time period (>14 days), because LU-99 cells grew very rapidly, and thus the starting volume of 200 mm3 prevented longer observation considering animal ethics (for example, 200 mm3 of inoculated LU-99 tumor reached an average tumor volume of about 1100 mm3, whereas A549 tumor reached about 490 mm3 on day 15). For intravenous administration, compounds were formulated in 5% glucose and injected into the tail vein. For oral administration, compounds (e.g. OTS514) were prepared in a vehicle of 0.5% methylcellulose and given by oral gavage at the indicated dose and schedule. An administration volume of 10 ml/kg of body weight was used for both administration routes. Concentrations were indicated in the main text and figures. Tumor volumes were determined using a caliper. The results were converted to tumor volume (mm3) by the formula length × width2 × 1/2. The weight of the mice was determined as an indicator of tolerability on the same days. The animal experiments were conducted at KAC Co. Ltd. for A549 xenograft or at OncoTherapy Science Inc. for LU-99 xenograft, in accordance with the Institutional Guidelines for the Care and Use of Laboratory Animals of each site. TGI was calculated according to the formula [1 − (T − T0)/(C − C0)] × 100, where T and T0 are the mean tumor volumes at day 15 or 22 and day 1, respectively, for the experimental group, and C and C0 are those for the vehicle control group. WBCs were counted with Sysmex XT-1800iV Analyzer (Sysmex Corporation) at KAC Co. Ltd. or with a cell counting chamber. Blood was collected in a blood collection tube with EDTA to prevent coagulation and to perform the blood cell count.

[1]. 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.

[2]. Potent anti-myeloma activity of the TOPK inhibitor OTS514 in pre-clinical models. Cancer Med. 2020 Jan;9(1):324-334.

TOPK (T-lymphokine-activated killer cell-originated protein kinase) is highly and frequently transactivated in various cancer tissues, including lung and triple-negative breast cancers, and plays an indispensable role in the mitosis of cancer cells. We report the development of a potent TOPK inhibitor, OTS964 {(R)-9-(4-(1-(dimethylamino)propan-2-yl)phenyl)-8-hydroxy-6-methylthieno[2,3-c]quinolin-4(5H)-one}, which inhibits TOPK kinase activity with high affinity and selectivity. Similar to the knockdown effect of TOPK small interfering RNAs (siRNAs), this inhibitor causes a cytokinesis defect and the subsequent apoptosis of cancer cells in vitro as well as in xenograft models of human lung cancer. Although administration of the free compound induced hematopoietic adverse reactions (leukocytopenia associated with thrombocytosis), the drug delivered in a liposomal formulation effectively caused complete regression of transplanted tumors without showing any adverse reactions in mice. Our results suggest that the inhibition of TOPK activity may be a viable therapeutic option for the treatment of various human cancers.[1]

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Multiple myeloma (MM) continues to be considered incurable, necessitating new drug discovery. The mitotic kinase T-LAK cell-originated protein kinase/PDZ-binding kinase (TOPK/PBK) is associated with proliferation of tumor cells, maintenance of cancer stem cells, and poor patient prognosis in many cancers. In this report, we demonstrate potent anti-myeloma effects of the TOPK inhibitor OTS514 for the first time. OTS514 induces cell cycle arrest and apoptosis at nanomolar concentrations in a series of human myeloma cell lines (HMCL) and prevents outgrowth of a putative CD138+ stem cell population from MM patient-derived peripheral blood mononuclear cells. In bone marrow cells from MM patients, OTS514 treatment exhibited preferential killing of the malignant CD138+ plasma cells compared with the CD138- compartment. In an aggressive mouse xenograft model, OTS964 given orally at 100 mg/kg 5 days per week was well tolerated and reduced tumor size by 48%-81% compared to control depending on the initial graft size. FOXO3 and its transcriptional targets CDKN1A (p21) and CDKN1B (p27) were elevated and apoptosis was induced with OTS514 treatment of HMCLs. TOPK inhibition also induced loss of FOXM1 and disrupted AKT, p38 MAPK, and NF-κB signaling. The effects of OTS514 were independent of p53 mutation or deletion status. Combination treatment of HMCLs with OTS514 and lenalidomide produced synergistic effects, providing a rationale for the evaluation of TOPK inhibition in existing myeloma treatment regimens.[2]

C21H20N2O2S

364.46

364.124

C, 69.21; H, 5.53; N, 7.69; O, 8.78; S, 8.80

1338540-63-8

OTS514 hydrochloride;2319647-76-0; OTS514;1338540-63-8; 1338544-87-8 (HBr); 1338545-92-8 (S-isomer HCl); 1338541-25-5 (s-isomer);

67448836

Light yellow to yellow solid

1.3±0.1 g/cm3

501.3±50.0 °C at 760 mmHg

256.9±30.1 °C

0.0±1.3 mmHg at 25°C

1.665

3.25

3

4

3

26

522

1

Cl[H].S1C([H])=C([H])C2=C1C(N([H])C1C(C([H])([H])[H])=C([H])C(=C(C3C([H])=C([H])C(=C([H])C=3[H])[C@@]([H])(C([H])([H])[H])C([H])([H])N([H])[H])C=12)O[H])=O

OETLNMOJNONWOY-LBPRGKRZSA-N

InChI=1S/C21H20N2O2S/c1-11-9-16(24)17(14-5-3-13(4-6-14)12(2)10-22)18-15-7-8-26-20(15)21(25)23-19(11)18/h3-9,12,24H,10,22H2,1-2H3,(H,23,25)/t12-/m0/s1

9-[4-[(2R)-1-aminopropan-2-yl]phenyl]-8-hydroxy-6-methyl-5H-thieno[2,3-c]quinolin-4-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.86 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.86 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.86 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)