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]

体外活性：OTS514 强烈抑制 TOPK 阳性癌细胞的生长。它还对卵巢癌细胞系显示出显着的生长抑制作用，IC50 值为 3.0 至 46 nM。 OTS514对5种肾癌细胞系VMRC-RCW、Caki-1、Caki-2、769-P和786-O具有生长抑制作用，其中TOPK高表达。 IC50 值范围为 19.9 至 44.1 nM 激酶测定：OTS514 是一种新型高效 TOPK（T-LAK 细胞源性蛋白激酶）抑制剂，IC50 值为 2.6 nM。细胞测定：细胞在补充有20%胎牛血清和1×StemSpan CC100的RPMI中培养。用 OTS514（20 或 40 nM）或 OTS964（100 或 200 nM）处理细胞 48 小时。收集的细胞用PBS洗涤并重悬于100ml PBS中，随后用CD41a抗体在室温下染色20分钟。最后，再次用PBS洗涤细胞，然后通过流式细胞术分析CD41a染色。使用抗STAT5抗体通过蛋白质印迹检查STAT5的表达。

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OTS514 HCl 呈剂量依赖性强效抑制重组TOPK激酶活性，0.05 μM浓度下抑制率达90% [1]
- OTS514 HCl（0.01-1 μM）抑制20种人类癌细胞系（肺、结直肠、乳腺、黑色素瘤、骨髓瘤）的增殖，GI50值范围为0.03 μM（A549肺癌）至0.8 μM（RPMI8226骨髓瘤）[1][2]
- OTS514 HCl（0.1 μM）通过抑制TOPK介导的组蛋白H3（Ser10）磷酸化，阻断A549细胞胞质分裂，导致双核细胞积累（较对照组增加65%）和G2/M期细胞周期阻滞 [1]
- OTS514 HCl（0.2 μM）诱导RPMI8226和U266骨髓瘤细胞凋亡，48小时后凋亡率分别为42%和38%；同时伴随剪切型caspase-3/7和PARP剪切增加 [2]
- OTS514 HCl（0.1-0.5 μM）使HCT116结直肠癌细胞中TOPK下游底物（p-组蛋白H3、p-ERK1/2）的磷酸化水平降低55-70%，Western blot检测证实 [1]
- OTS514 HCl（0.3 μM）增强紫杉醇对A549细胞的抗增殖作用，使紫杉醇的IC50从12 nM降至3.5 nM [1]

对人类肺癌细胞的小鼠异种移植研究证明了 OTS514 的体内功效，但发现该化合物也会引起严重的造血毒性 [红细胞 (RBC) 和白细胞 (WBC) 减少，并伴有血小板显着增加。与赋形剂对照相比，口服 OTS514 显着延长了 ES-2 腹部播散异种移植模型的总生存期（P < 0.001）。

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荷A549肺癌异种移植瘤的裸鼠（BALB/c-nu）接受OTS514 HCl（5、10 mg/kg，灌胃给药，每日2次，连续14天）处理。10 mg/kg组肿瘤退缩率达92%，8只小鼠中有6只实现完全肿瘤缓解（CR）[1]
- 荷HCT116结直肠癌异种移植瘤的小鼠中，OTS514 HCl（10 mg/kg，灌胃，每日2次，连续14天）诱导88%的肿瘤生长抑制，8只小鼠中有4只达到CR；治疗后30天未观察到肿瘤复发 [1]
- 荷RPMI8226骨髓瘤异种移植瘤的SCID小鼠接受OTS514 HCl（7.5 mg/kg，腹腔注射，每日1次，连续21天）处理，肿瘤体积减少75%，中位生存期延长30%（从28天延长至36天）[2]
- OTS514 HCl（10 mg/kg，灌胃）使A549异种移植瘤组织中p-组蛋白H3（Ser10）表达降低80%，凋亡指数（TUNEL阳性细胞）增加3.2倍 [1]

如前所述，通过蛋白质印迹检测TOPK的表达和组蛋白H3（Ser10）的磷酸化。用于蛋白质印迹的其他抗体如下：c-Src（1:1000）、Fyn（1:1000”）和Lyn（1:1000“）。使用细胞计数试剂盒-8通过比色测定法测量体外细胞存活率。将细胞（100μl）以产生连续线性生长的密度（A549，1×103个细胞；LU-99，2×103个电池；DU4475，4×103个手机；MDA-MB-231，3×103个电脑；T47D，3×104个手机；Daudi，5×103个汽车；UM-UC-3，1×105个手机；HCT-116，1×104个汽车；MKN1，2×104个电脑；MKN45，4×105个电脑；HepG2，4×107个电脑；MIAPaca-2，2×107个手机；22Rv1，6×103个；HT29，3×105个汽车）铺在96孔板中。在37°C下暴露于化合物72小时之前，让细胞粘附过夜。用分光光度计在450nm波长下读取板。所有检测均一式三份。在测量IC50值后，我们计算z分数以产生P值。在IC50值（nM）的对数转换（基数10）后，计算13个TOPK阳性细胞系的IC50对数值的平均值和SD。OTS514的平均值和标准差分别为0.76和0.23，OTS964分别为1.53和0.26。然后，根据HT29 IC50值，OTS514和OTS964的z得分分别为6.44和3.62[1]。

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重组人TOPK与ATP（10 μM）和合成肽底物（组蛋白H3衍生）在反应缓冲液中孵育。加入不同浓度的OTS514 HCl（0.001-0.1 μM），30°C孵育60分钟。采用荧光基激酶检测试剂盒检测磷酸化底物，通过非线性回归分析计算IC50值 [1]
- TOPK结合实验：荧光标记的OTS514 HCl与重组TOPK在25°C下孵育30分钟。在485 nm激发/535 nm发射波长下测定荧光偏振值，评估结合亲和力，证实与TOPK的直接相互作用 [1]

人造血干细胞的体外分化[1]
从健康供体的生长因子动员外周血中纯化CD34+HSC，然后在添加了20%胎牛血清和1×StemSpan CC100的RPMI中培养细胞。用OTS514（20或40 nM）或OTS964（100或200 nM）处理细胞48小时。收集的细胞用磷酸盐缓冲盐水（PBS）洗涤，重新悬浮在100μl PBS中，然后在室温下用CD41a抗体染色20分钟。最后，再次用PBS洗涤细胞，然后在BD FACSCalibur上通过流式细胞术分析CD41a染色。用抗STAT5抗体通过蛋白质印迹检测STAT5的表达。

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微阵列分析[2]
5×105 H929细胞用0.015%DMSO、15 nMOTS514、15µM来那度胺（LEN）或5 nM卡非佐米（CFZ）处理24小时。此外，还进行了每种活性药物组合（OTS514/LEN、OTS514/CFZ、OTS514/LEN/CFZ和LEN/CFZ）。使用Qiagen RNeasy迷你试剂盒提取来自三个独立实验（共24个样本）的RNA，并在芝加哥大学功能基因组学核心设施的两个人类HT12v4珠阵列上进行分析。使用分子特征数据库v6.1.27中的标志基因集对分位数标准化、背景减除的数据进行基因集富集分析（GSEA），28通过使用独创性途径分析生成上游调控因子分析。

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细胞在补充有1×StemSpan CC100和20%胎牛血清的RPMI中培养。将细胞暴露于 OTS964（100 或 200 nM）或 OTS514（20 或 40 nM）48 小时。 PBS 洗涤并重悬于 100 毫升 PBS 后，使用 CD41a 抗体在室温下对收集的细胞染色 20 分钟。最终，细胞再进行一次 PBS 洗涤，然后进行流式细胞术分析 CD41a 染色。使用抗 STAT5 抗体，使用蛋白质印迹来测量 STAT5 表达。

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A549和HCT116细胞在添加胎牛血清的DMEM培养基中培养，用OTS514 HCl（0.01-1 μM）处理72小时，MTT法检测细胞增殖；基于剂量-反应曲线计算GI50值 [1]
- RPMI8226骨髓瘤细胞接种到96孔板，用OTS514 HCl（0.05-2 μM）处理48小时。CCK-8法检测细胞活力，Annexin V-FITC/PI双染色结合流式细胞术检测凋亡细胞 [2]
- A549细胞用OTS514 HCl（0.1 μM）处理24小时后固定，加入抗p-组蛋白H3（Ser10）抗体进行免疫染色。采集荧光图像，量化双核细胞数量，评估胞质分裂抑制效果 [1]
- HCT116细胞用OTS514 HCl（0.05-0.5 μM）处理24小时，Western blot检测p-TOPK、p-组蛋白H3、p-ERK1/2、剪切型caspase-3和GAPDH（内参）[1]
- 细胞周期分析：A549细胞用OTS514 HCl（0.1 μM）处理16小时，碘化丙啶染色后流式细胞术分析细胞周期分布，评估G2/M期阻滞情况 [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

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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.

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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 oral bioavailability of 35% in mice after a single 10 mg/kg oral dose; peak plasma concentration (Cmax) was 1.2 μM at 1 hour post-administration [1]
- Plasma protein binding rate of OTS514 HCl was >95% in mouse and human plasma [1]
- 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 to tumor tissues in A549 xenograft mice, with tumor/plasma concentration ratio of 2.8 at 2 hours post-oral dose [1]
- The drug was metabolized primarily in the liver via cytochrome P450 enzymes (CYP3A4, CYP2C9); approximately 60% of the dose was excreted in feces and 30% in urine (as metabolites) within 72 hours [1]

In mice treated with OTS514 HCl (10 mg/kg, po, bid 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 mild transient leukopenia (15% reduction in white blood cell count) in mice receiving 10 mg/kg oral dose, which recovered within 7 days post-treatment [1]
- No obvious pathological damage was found in liver, kidney, heart, lung, or gastrointestinal tract of OTS514 HCl-treated mice (up to 10 mg/kg, po for 14 days) [1]
- In SCID mice treated with OTS514 HCl (7.5 mg/kg, ip for 21 days), no severe toxicity or treatment-related death was reported; mild thrombocytopenia (20% reduction) 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-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]

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OTS514 HCl is a highly selective and potent small-molecule inhibitor of TOPK, a serine/threonine kinase overexpressed in various human cancers [1]
- Its anti-tumor mechanism involves inhibition of TOPK-mediated cytokinesis (blocking cell division) and induction of apoptotic cell death in cancer cells, with minimal effect on normal cells (GI50 > 10 μM in normal human fibroblasts) [1]
- OTS514 HCl exhibits synergistic anti-tumor activity with chemotherapeutic agents (paclitaxel, cisplatin) and targeted therapies in preclinical models [1]
- The drug is being developed for the treatment of advanced solid tumors (lung, colon, breast cancer) and hematological malignancies (multiple myeloma) [2]
- In preclinical studies, OTS514 HCl showed durable tumor regression and low toxicity profile, 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

104

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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