TOPK (IC50 = 2.6 nM)
T-LAK cell-originated protein kinase (TOPK) (IC50 = 0.015 μM for recombinant TOPK kinase activity) [1]
- T-LAK cell-originated protein kinase (TOPK) (GI50 range = 0.03-0.8 μM in various cancer cell lines) [2]

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

---

Nude mice (BALB/c-nu) bearing A549 lung cancer xenografts were administered OTS514 HCl (5, 10 mg/kg, oral gavage, twice daily for 14 days). The 10 mg/kg group showed 92% tumor regression, with 6 out of 8 mice achieving complete tumor regression (CR) [1]
- In HCT116 colon cancer xenograft mice, OTS514 HCl (10 mg/kg, po, bid for 14 days) induced 88% tumor growth inhibition and 4/8 mice with CR; tumor recurrence was not observed for 30 days post-treatment [1]
- SCID mice bearing RPMI8226 myeloma xenografts treated with OTS514 HCl (7.5 mg/kg, intraperitoneal injection, once daily for 21 days) showed 75% tumor volume reduction and 30% extension of median survival (from 28 days to 36 days) compared to control [2]
- OTS514 HCl (10 mg/kg, po) reduced p-Histone H3 (Ser10) expression in A549 xenograft tissues by 80% and increased apoptotic index (TUNEL-positive cells) by 3.2-fold [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].

---

Recombinant human TOPK was incubated with ATP (10 μM) and synthetic peptide substrate (Histone H3-derived) in reaction buffer. Various concentrations of OTS514 HCl (0.001-0.1 μM) were added, and the mixture was incubated at 30°C for 60 minutes. Phosphorylated substrate was detected using a fluorescence-based kinase assay kit, and IC50 was calculated by nonlinear regression analysis [1]
- TOPK binding assay: Fluorescently labeled OTS514 HCl was incubated with recombinant TOPK at 25°C for 30 minutes. Fluorescence polarization values were measured at 485 nm excitation/535 nm emission to assess binding affinity, confirming direct interaction with TOPK [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.

---

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.

---

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.

---

A549 and HCT116 cells were cultured in DMEM medium supplemented with fetal bovine serum. Cells were treated with OTS514 HCl (0.01-1 μM) for 72 hours, and cell proliferation was assessed by MTT assay; GI50 values were calculated based on dose-response curves [1]
- RPMI8226 myeloma cells were seeded in 96-well plates and treated with OTS514 HCl (0.05-2 μM) for 48 hours. Cell viability was measured by CCK-8 assay, and apoptotic cells were detected by Annexin V-FITC/PI double staining and flow cytometry [2]
- A549 cells were treated with OTS514 HCl (0.1 μM) for 24 hours, fixed, and immunostained with anti-p-Histone H3 (Ser10) antibody. Fluorescence images were captured to quantify binucleated cells and assess cytokinesis inhibition [1]
- HCT116 cells were treated with OTS514 HCl (0.05-0.5 μM) for 24 hours. Western blot was performed to detect p-TOPK, p-Histone H3, p-ERK1/2, cleaved caspase-3, and GAPDH (loading control) [1]
- Cell cycle analysis: A549 cells treated with OTS514 HCl (0.1 μM) for 16 hours were stained with propidium iodide, and cell cycle distribution was analyzed by flow cytometry to assess G2/M arrest [1]

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

---

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.

---

BALB/c-nu nude mice (6-8 weeks old) were subcutaneously injected with A549 or HCT116 cells (5×10⁶ cells/mouse) to establish xenograft tumors. When tumors reached 100-150 mm³, mice were randomly divided into control (vehicle) and OTS514 HCl groups (5, 10 mg/kg). The drug was dissolved in 0.5% carboxymethylcellulose sodium and administered via oral gavage twice daily for 14 days. Tumor volume was measured every 2 days; mice were euthanized at the end of treatment to collect tumor tissues for immunohistochemistry and Western blot analysis [1]
- SCID mice (6-8 weeks old) were intravenously injected with RPMI8226 myeloma cells (2×10⁶ cells/mouse) to establish disseminated myeloma model. Seven days post-inoculation, mice were treated with OTS514 HCl (7.5 mg/kg) dissolved in normal saline via intraperitoneal injection once daily for 21 days. Body weight and survival were monitored daily; bone marrow and spleen tissues were collected at euthanasia for flow cytometric analysis of tumor burden [2]

OTS514 HCl showed 35% oral bioavailability in mice after a single oral dose of 10 mg/kg; the peak plasma concentration (Cmax) 1 hour after administration was 1.2 μM [1]
- OTS514 HCl had a plasma protein binding rate of >95% in mouse and human plasma [1]
- The elimination half-life (t1/2) of OTS514 HCl in mice was 4.2 hours after intravenous injection and 5.8 hours after oral administration [1]
- OTS514 HCl was distributed in tumor tissue in A549 xenograft mice, with a tumor/plasma concentration ratio of 2.8 2 hours after oral administration [1]
- The drug is mainly metabolized in the liver by cytochrome P450 enzymes (CYP3A4, CYP2C9); approximately 60% of the dose is excreted in feces within 72 hours, and 30% is excreted in feces within 72 hours. Excreted in urine (as a metabolite) [1]

In mice treated with OTS514 HCl (10 mg/kg, orally, twice daily for 14 days), no significant changes in body weight (≤5% reduction) or serum ALT, AST, creatinine, and blood urea nitrogen levels were observed [1]. Hematological analysis showed that mice receiving an oral dose of 10 mg/kg experienced mild transient leukopenia (a 15% decrease in white blood cell count), which resolved within 7 days of treatment [1]. No significant pathological damage was found in the liver, kidneys, heart, lungs, or gastrointestinal tract of mice treated with OTS514 HCl (up to 10 mg/kg, orally, for 14 days) [1]. No serious toxicity or treatment-related death was reported in SCID mice treated with OTS514 HCl (7.5 mg/kg, intraperitoneally, for 21 days); mild thrombocytopenia (a 20% decrease) was reversible [2].

[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-derived protein kinase) is highly and frequently activated in various cancer tissues, including lung cancer and triple-negative breast cancer, and plays an indispensable role in cancer cell mitosis. We report the development of a potent TOPK inhibitor, OTS964 {(R)-9-(4-(1-(dimethylamino)propyl-2-yl)phenyl)-8-hydroxy-6-methylthieno[2,3-c]quinoline-4(5H)-one}, which inhibits TOPK kinase activity with high affinity and selectivity. Similar to the knockdown of TOPK small interfering RNA (siRNA), this inhibitor induces cell division defects in vitro and in human lung cancer xenograft models, ultimately inducing apoptosis in cancer cells. Although administration of the free compound induced hematopoietic adverse reactions (leukopenia with thrombocytosis), administration of the liposomal formulation effectively induced complete regression of transplanted tumors in mice without any observed adverse reactions. Our results suggest that inhibiting TOPK activity may be a viable therapeutic approach for treating various human cancers. [1]

---

Multiple myeloma (MM) is still considered incurable, thus requiring the development of new drugs. Mitotic kinase T-LAK cell-derived protein kinase/PDZ-binding kinase (TOPK/PBK) is closely related to tumor cell proliferation, cancer stem cell maintenance, and poor patient prognosis in various cancers. In this report, we demonstrate for the first time that the TOPK inhibitor OTS514 has significant anti-myeloma activity. OTS514 can induce cell cycle arrest and apoptosis in a range of human myeloma cell lines (HMCL) at nanomolar concentrations and inhibit the proliferation of putative CD138+ stem cell populations in peripheral blood mononuclear cells of multiple myeloma (MM) patients. In bone marrow cells of MM patients, OTS514 treatment showed stronger killing effects on malignant CD138+ plasma cells than on CD138- plasma cells. In an invasive mouse xenograft model, oral administration of 100 mg/kg OTS964 five days a week was well tolerated, resulting in a 48%–81% reduction in tumor volume compared to the control group, with the specific reduction depending on the initial size of the xenograft. Treatment of HMCL with OTS514 upregulated the expression of FOXO3 and its transcriptional targets CDKN1A (p21) and CDKN1B (p27) and induced apoptosis. TOPK inhibitors also led to FOXM1 loss and disrupted the AKT, p38 MAPK, and NF-κB signaling pathways. The effects of OTS514 were independent of p53 mutation or deletion status. The synergistic effect of OTS514 in combination with lenalidomide in HMCL provides a theoretical basis for evaluating the application of TOPK inhibitors in existing multiple myeloma treatment regimens. [2]

---

OTS514 HCl is a highly selective and potent small-molecule TOPK inhibitor. TOPK is a serine/threonine kinase that is overexpressed in a variety of human cancers. [1]
- Its antitumor mechanism includes inhibiting TOPK-mediated cytokinesis (blocking cell division) and inducing apoptosis in cancer cells, with minimal effect on normal cells (GI50 > 10 μM in normal human fibroblasts) [1]
- In preclinical models, OTS514 HCl showed synergistic antitumor activity with chemotherapy drugs (paclitaxel, cisplatin) and targeted therapies. [1]
- This drug is being developed for the treatment of advanced solid tumors (lung cancer, colon cancer, breast cancer) and hematologic malignancies (multiple myeloma) [2]
- In preclinical studies, OTS514 HCl showed durable tumor regression and low toxicity, supporting its potential for clinical translation [1][2]

C21H21CLN2O2S

400.9216

364.12

C, 62.91; H, 5.28; Cl, 8.84; N, 6.99; O, 7.98; S, 8.00

2319647-76-0

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

92044487

Solid powder

4

4

3

27

522

1

CC1=CC(=C(C2=C1NC(=O)C3=C2C=CS3)C4=CC=C(C=C4)[C@@H](C)CN)O.Cl

YCRRQRJUNVBPBW-UHFFFAOYSA-N

InChI=1S/C21H18N2O2S.ClH/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;/h3-9,12H,10,22H2,1-2H3;1H

9-[4-(1-aminopropan-2-yl)phenyl]-6-methylthieno[2,3-c]quinoline-4,8-dione;hydrochloride

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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