TOPK (IC50 = 2.6 nM)
Mitogen-Activated Protein Kinase Kinases (TOPK/PBK) [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 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。

---

在一系列人骨髓瘤细胞系（HMCL）中，OTS514 在纳摩尔浓度下即可诱导细胞周期停滞和凋亡。[2]
- 它能抑制多发性骨髓瘤（MM）患者来源的外周血单个核细胞中推定的 CD138⁺ 干细胞群的增殖。[2]
- 在 MM 患者的骨髓细胞中，与 CD138⁻ 细胞亚群相比，OTS514 处理能优先杀伤恶性 CD138⁺ 浆细胞。[2]
- OTS514 处理 HMCL 后，可上调 FOXO3 及其转录靶点 CDKN1A（p21）和 CDKN1B（p27）的表达，诱导细胞凋亡，降低 FOXM1 的表达，并破坏 AKT、p38 MAPK 和 NF-κB 信号通路。[2]
- OTS514 的作用不受 p53 突变或缺失状态的影响。[2]
- OTS514 与来那度胺联合处理 HMCL 时，产生协同的抗骨髓瘤效应。[2]
- 对于 OTS514 耐药细胞系 8226 Dox40，在存在 10 µM 维拉帕米（可阻断 ABCB1 活性）的条件下培养，可恢复其对 OTS514 的敏感性。[2]
- 经血清饥饿诱导 G1 期停滞的 MM1.S 和 U266 细胞，在解除饥饿后用 10 nM OTS514 处理 24 h，通过碘化丙啶染色流式细胞术分析显示细胞 DNA 含量发生改变。[2]
- 用 100 nM OTS514 处理 HMCL 0-24 h，Western blot 检测显示 4 h 后出现半胱天冬酶介导的 PARP 切割（凋亡标志）。[2]
- 用 OTS514 处理 MM1.S 和 U266 细胞 24 h，Western blot 分析显示 TOPK 蛋白水平呈剂量依赖性降低。[2]

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）。

---

在使用 NSG 小鼠（NOD/SCID/IL2Rg）建立的 H929 细胞侵袭性异种移植模型中，每周 5 次口服给予 100 mg/kg OTS514 耐受性良好。根据初始移植细胞量（1×10⁶ 或 2×10⁶ 个细胞）的不同，与对照组相比，OTS514 可使肿瘤体积缩小 48%-81%。[2]

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]。

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。

---

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。

---

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。

---

MTT 法细胞活力检测：将人骨髓瘤细胞系（HMCL）与不同浓度的 OTS514 共同培养 72 h，采用 MTT 法评估细胞活力。对于 OTS514 耐药的 8226 Dox40 细胞系，同时加入 10 µM 维拉帕米以阻断 ABCB1 活性，再进行 MTT 检测。[2]
- 细胞周期分析：将 MM1.S 和 U266 细胞经血清饥饿过夜诱导 G1 期停滞，解除饥饿后，分为未处理组和 10 nM OTS514 处理组，培养 24 h 后，采用碘化丙啶染色流式细胞术分析细胞 DNA 含量，以评估细胞周期分布。[2]
- PARP 切割 Western blot 检测：用 100 nM OTS514 处理 HMCL 0-24 h，通过 Western blot 检测 PARP 切割情况，以评估细胞凋亡程度。[2]
- TOPK 蛋白 Western blot 检测：用不同浓度 OTS514 处理 MM1.S 和 U266 细胞 24 h，通过 Western blot 分析 TOPK 蛋白表达水平，以观察药物对靶点蛋白的剂量依赖性影响。[2]
- 患者来源细胞凋亡流式细胞术检测：将 MM 患者的骨髓单个核细胞与不同浓度的 OTS514 共同培养，18 h 后收集细胞，采用 Annexin V/PI 双染色流式细胞术分析 CD138⁺ 浆细胞亚群和 CD138⁻ 细胞亚群的晚期凋亡情况。[2]
- CD138⁺ 细胞增殖抑制实验：将 MM 患者来源的外周血单个核细胞（PBMC）分为加或不加 5 ng/mL IL-3 和 IL-6、加或不加 10 nM OTS514 的各组，培养 6 天后，通过磁珠分选法分离 CD138⁺ 细胞，评估 OTS514 对 CD138⁺ 细胞增殖的抑制作用。[2]
- 与来那度胺的协同作用实验：采用 MTT 法，分别用 OTS514、来那度胺以及两者的恒定比例组合（OTS514:来那度胺 = 1:6 和 1:12 摩尔当量）处理细胞，评估协同抗骨髓瘤效应。用不同浓度 OTS514 单独或联合来那度胺处理 H929 细胞 24 h，通过 Western blot 检测 IKAROS（IKZF1）、IRF4 和 FOXM1 的表达水平。此外，用 20 µM 来那度胺、100 nM OTS514 或两者组合处理 MM1.S 细胞，以 DMSO 为对照，采用 Amplex 红色过氧化物酶活性测定法检测相关活性。[2]
- 下游信号通路 Western blot 分析：用不同浓度 OTS514 处理 H929、8226 和 KMS11 细胞 24 h，采用 LI-COR 近红外检测系统进行 Western blot 分析，评估 p21/p27 及其他相关蛋白的表达水平。用 15 nM OTS514 处理 H929、U266、MM1.S、8226 和 KMS11 细胞 24 h，采用磷酸化特异性抗体进行 Western blot 检测 IκBα、p38、AKT 和 FOXM1 的磷酸化水平，随后剥离膜并重新杂交以检测这些靶点的总蛋白水平。[2]

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.

---

Xenograft model establishment: NSG mice (NOD/SCID/IL2Rg) are used to establish xenografts of H929 cells, with 6 mice per group. [2]
- Drug administration: When the tumor volume reaches 100 mm³, mice begin receiving OTS514 orally at a dose of 100 mg/kg, 5 days per week. [2]
- Monitoring: Tumor volume is measured regularly to assess the anti-tumor effect, and body weight is monitored throughout the treatment course to evaluate tolerability. [2]

In the mouse xenograft model, oral administration of OTS514 at 100 mg/kg 5 days per week is well tolerated, with no significant adverse effects observed as indicated by stable body weight throughout the treatment period. [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]

---

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]

---

OTS514 is a potent TOPK inhibitor. TOPK/PBK (T-LAK cell-originated protein kinase/PDZ-binding kinase) is associated with the proliferation of tumor cells, maintenance of cancer stem cells, and poor patient prognosis in many cancers. [2]
- Multiple myeloma (MM) is considered incurable, necessitating new drug discovery, and OTS514 shows potent anti-myeloma activity in pre-clinical models, 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

104

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.86 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

配方 2 中的溶解度: ≥ 2.5 mg/mL (6.86 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

---

View More

配方 3 中的溶解度: ≥ 2.5 mg/mL (6.86 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

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：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。