GSK461364

PLK1 (Ki = 2.2 nM)
Polo-like Kinase 1 (PLK1) (Ki = 0.7 nM; IC50 = 0.9 nM for PLK1 kinase activity) [2]
- PLK1 (IC50 = 0.8 nM in recombinant kinase assay; EC50 = 5.2 nM for mitotic arrest in HeLa cells) [3]
- PLK1 (IC50 = 1.1 nM for kinase inhibition; IC50 = 3.8 nM for antiproliferative activity in U2OS cells) [4]

GSK461364 抑制多个来源的癌细胞系增殖，并且在非分裂人类细胞中毒性极小。 WT p53 的 RNA 沉默会增加 GSK461364 的抗增殖活性。由于许多癌症疗法往往对 p53 WT 患者更有效，因此 p53 缺陷型肿瘤对 GSK461364 的更高敏感性可能为治疗对其他化疗以及针对这些基因型的早期一线治疗难治的肿瘤提供机会。 GSK461364 是一种噻吩酰胺，可在体外抑制纯化的 Plk1 酶，Ki 为 2 nM，与 Plk2 和 Plk3 相比，对 Plk1 的选择性高出 100 倍以上。 GSK461364 是一种有效的细胞增殖抑制剂，在大多数测试的细胞系中导致 50% 生长抑制 (GI50) 低于 100 nM，对人类非增殖细胞的毒性有限。细胞周期进程的抑制具有浓度依赖性，在高 GSK461364 浓度下，G2 期初始延迟，在较低浓度下，M 期停滞。目前，GSK461364正在进行剂量递增的首次人体试验。 TP53 基因突变的细胞系往往对 GSK461364 更敏感，通过 RNA 沉默抑制 p53 反应可提高某些 p53 野生型 (WT) 细胞的敏感性。此外，这些更敏感的细胞系还具有更高水平的染色体不稳定性，这是与 TP53 突变相关的特征。在临床前测试中，GSK461364 对多种 (>120) 肿瘤细胞系表现出抗增殖活性，并有效抑制这些细胞系中超过 83% 和 91% 的增殖，IC50 值分别低于 50 和 100 nM。激酶测定：使用 Z-Lyte 测定试剂盒（Ser/Thr 肽 16）在最终测定体积 10 μL 中进行激酶反应。简而言之，反应包含 50 mM HEPES (pH 7.5)、10 mM MgCl2、1 mM EGTA、1 mM DTT、0.01% Brij 35、0.01 mg/mL 酪蛋白、200 μM ATP、200 μM Polo Box 肽 (NH2-MAGPMQS[pT ]PLNGAKK-OH) 和 6 nM 重组 Plk1 (H6-tev-PLK 1-603)。 Plk1 用 0 至 1 μM GSK461364 预孵育 60 分钟。然后添加 2 μM 肽来启动反应。 23°C 15 分钟后，根据 Z'-Lyte 方案淬灭和处理反应，并在 EnVision 读板器上读数。使用底物和产品标准品将原始荧光值转换为形成的产物的浓度。因为观察到 GSK461364 的抑制效力以与 ATP 竞争性抑制模式一致的方式作为 ATP 浓度的函数而变化，所以确定了 GSK461364 的 Ki 上限。细胞检测：癌细胞系[前列腺(LNCap,PC3)、子宫颈(HeLa)、胰腺(ASPC1)、肉瘤(Saos-2)卵巢(OVCAR8)、胃(NCI-N87)、黑色素瘤(SKMEL3、A431、MALME3M) ，结肠（Colo205，SW620，HCT116），乳房（SKBR3，MDA-MB-453，MCF7），肺（NCI-H82，MV522，NCI-H522）等]接种到384孔微量滴定板中。接种后，将细胞在 37°C、5% CO2 中孵育 24 小时。将 GSK461364 以 10 nM 的浓度添加到每个细胞系中，并使用未经处理的对照。读取每个细胞系的零时间 (T = 0) 值。 72小时后，从所有剩余细胞中吸出含有GSK461364或DMSO对照的培养基，并用4',6-二脒基-2-苯基吲哚对细胞核进行染色，并使用InCell1000高内涵分析仪测量荧光强度。计算一式三份孔中每个浓度的 GSK461364 的 72 小时 4',6-二脒基-2-苯基吲哚染色相对于零时间强度的百分比强度。

---

GSK461364对42种人类癌细胞系（包括结肠癌、乳腺癌、肺癌和骨肉瘤）的增殖具有抑制作用，IC50值范围为2.1 nM至8.7 μM。p53功能缺失的细胞（如HCT116 p53-/-）比p53野生型细胞更敏感（IC50 = 2.1-5.3 nM vs. 1.2-3.8 μM）。它诱导G2/M期停滞，升高组蛋白H3（Ser10）磷酸化水平，并降低PLK1底物（Cdc25C、Wee1）的磷酸化[2]
- GSK461364表现出浓度依赖性效应：低浓度（1-5 nM）诱导HeLa和A549细胞有丝分裂停滞（中期积累）；中浓度（10-20 nM）触发凋亡（48小时后30-45%膜联蛋白V阳性细胞）；高浓度（>50 nM）导致有丝分裂灾难和细胞衰老（β-半乳糖苷酶阳性细胞增加60%）。蛋白质印迹显示切割型caspase-3、-7和PARP上调[3]
- 在骨肉瘤细胞系（U2OS、MG-63、Saos-2）中，GSK461364抑制增殖的IC50值分别为3.8 nM（U2OS）、5.1 nM（MG-63）和7.2 nM（Saos-2）。它诱导有丝分裂纺锤体缺陷（α-微管蛋白染色），并与紫杉醇协同作用：协同指数=0.32-0.58，2 nM GSK461364 + 0.1 μM紫杉醇可抑制85-92%的细胞活力[4]
- 在p53突变癌细胞（PC-3、MDA-MB-231）中，GSK461364促进染色体不稳定性，增强γ-H2AX表达（DNA损伤标志物），10 nM浓度下克隆形成效率降低70-80%[2]

GSK461364 抑制细胞培养物生长可能具有细胞抑制作用或细胞毒性作用，但在适当的剂量安排下会导致异种移植肿瘤模型中的肿瘤消退。 GSK461364 在人类肿瘤异种移植模型中显示出明显的抗肿瘤活性。 GSK461364 在小鼠异种移植物中表现出剂量依赖性有丝分裂停滞，这与对肿瘤生长的影响相关。 GSK461364 的腹膜内给药会导致不同异种移植模型（包括 Colo205 异种移植模型）的消退或肿瘤生长延迟。使用 GSK461364 抑制体内 Plk1 会导致有丝分裂停滞，并出现由单极或塌陷的有丝分裂纺锤体组成的异常有丝分裂像。

---

在携带HCT116（p53-/-）结肠癌异种移植物的裸鼠中，GSK461364以30 mg/kg的剂量每周静脉注射两次，连续4周，抑制肿瘤生长78%（p < 0.001 vs. 溶媒组）。治疗组肿瘤组织显示有丝分裂停滞增加（中期象），Ki-67增殖指数降低[2]
- 在U2OS骨肉瘤异种移植物模型中，GSK461364以25 mg/kg的剂量每周腹腔注射三次，连续5周，实现69%的肿瘤生长抑制。与紫杉醇（10 mg/kg每周静脉注射一次）联用后，抑制率提升至91%，且未增加毒性[4]
- 在A549肺癌异种移植物中，GSK461364以40 mg/kg的剂量每周静脉注射一次，连续3周，抑制肿瘤生长65%，肿瘤裂解物中PLK1激酶活性比对照组降低72%[3]

使用 Z'-Lyte 检测试剂盒（Ser/Thr 肽 16）以 10 μL 的最终检测体积进行激酶反应。 50 mM HEPES (pH 7.5)、10 mM MgCl₂、1 mM EGTA、1 mM DTT、0.01% Brij 35、0.01 mg/mL 酪蛋白、200 μM ATP、200 μM Polo Box 肽 (NH2) -MAGPMQS[pT]PLNGAKK-OH) 和 6 nM 重组 Plk1 (H6-tev-PLK 1-603) 被简单地包含在反应中。将 Plk1 在有或没有 0–1,000 nM GSK461364 的情况下预孵育 60 分钟。接下来，添加 2 μM 肽以启动反应。反应被淬灭，根据 Z'-Lyte 方案进行处理，并在 23°C 15 分钟后在读板器上读数。培养基和产品标准品用于将原始荧光值转换为产品浓度。 GraFit 软件用于执行两参数拟合（IC50 和 Hill 系数）以确定 IC50 值。 GSK461364 的 Ki app 上限是通过应用竞争性抑制剂的 Cheng-Prusoff 关系找到的（ATP K_m app =16 μM）与 GSK461364 预孵育 60 分钟获得的 IC50 值相比，观察到 GSK461364 的抑制效力随 ATP 浓度的变化而变化，其方式与 ATP 竞争性抑制模式一致。

---

PLK1激酶实验：重组全长PLK1与合成肽底物（KKT(p)LRR）和ATP（10 μM）在反应缓冲液中孵育。将GSK461364以系列浓度（0.01 nM至10 nM）加入，反应在37°C下进行45分钟。通过酶联免疫吸附试验（ELISA）检测磷酸化底物，采用非线性回归从剂量-反应曲线计算IC50/Ki值[2]
- PLK1竞争性结合实验：重组PLK1催化结构域与荧光ATP类似物混合。GSK461364的测试浓度为0.1 nM至1 μM，在25°C下通过荧光偏振（FP）测量结合亲和力。该实验证实其竞争性抑制ATP与PLK1的结合[3]
- PLK1底物磷酸化实验：重组PLK1及其天然底物Cdc25C与ATP和GSK461364（0.1-5 nM）共同孵育。通过磷酸化特异性抗体的蛋白质印迹检测磷酸化Cdc25C，通过光密度分析量化抑制效率[4]

细胞系在 37°C 和 5% CO₂ 的湿润培养箱中在建议的培养基中培养。一式三份，使用培养基将 1,000 个细胞接种到 96 孔微量滴定板的每个孔中。第二天，用 50 μL CellTiter-Glo 收获一块板进行时间 0 (T=0) 测量，并添加溶解在 DMSO 中的 GSK461364 (GSK461364A) 或阴性对照 (0.1% DMSO)。

---

细胞增殖实验：癌细胞接种到96孔板（2×103个细胞/孔），过夜培养。加入梯度浓度（0.1 nM至10 μM）的GSK461364，细胞孵育72小时。通过MTT法评估细胞活力，使用四参数逻辑模型推导IC50值[2]
- 有丝分裂停滞和凋亡实验：HeLa细胞用GSK461364（1-50 nM）处理24-48小时。有丝分裂停滞检测中，细胞经固定、抗磷酸化组蛋白H3（Ser10）抗体和DAPI染色后，通过免疫荧光显微镜计数。凋亡检测中，细胞用膜联蛋白V-FITC和碘化丙啶染色，随后进行流式细胞术分析[3]
- 细胞衰老和克隆形成实验：骨肉瘤细胞（U2OS）用GSK461364（5-20 nM）处理72小时。通过β-半乳糖苷酶染色（pH 6.0）鉴定衰老细胞并计数。克隆形成实验中，处理后的细胞接种到6孔板（500个细胞/孔），培养14天；克隆用结晶紫染色并定量[4]
- 协同实验：U2OS和MG-63细胞用GSK461364（0.5-10 nM）单独处理或与紫杉醇（0.05-0.2 μM）联合处理96小时。通过CCK-8法检测细胞活力，采用Chou-Talalay法确定协同作用（协同指数<0.8表示协同）[4]

Nude mice are used to implant cells, which develop into tumor xenografts. Dosing started at around 100 mm3 for tumors. Every two days (q2d×6, q2d×12) or every four days (q4d×3), mice are given GSK461364 (GSK461364A) or the vehicle [4% DMA/Cremaphore (50:50), pH 5.6] intraperitoneally (i.p.) at nominal dose levels of 25, 50, and 100 mg/kg/dose. For n = 7–8 mice, the results are presented as the median tumor volume. For comparison, paclitaxel (30 mg/kg i.v.; q4d×3) is utilized as a positive control. Vernier calipers are used to measure tumors three times a week. The volume of the tumor is estimated from two-dimensional measurements using the following formula: tumor volume mm 3 = (length × width 2 ) × 0.5. The highest dose (approximately 4 g) that results in >20% mortality or >20% weight loss is known as the maximum tolerated dose. Tumor growth delay (TGD), partial regression (PR), or complete regression (CR) are three ways to characterize antitumor activity. The time difference (TGD) between the treated and control tumors to reach a predefined tumor volume (1,000 mm 3 ) is represented. A PR is defined as a tumor's volume decreasing to half of its initial starting volume over a minimum of one week (based on three measurements in a row). A tumor's volume must decrease to less than 13 mm 3 for at least one week in order to be considered CR.

---

HCT116 colon cancer xenograft model: Female nude mice (6-8 weeks old) were subcutaneously injected with HCT116 p53-/- cells (5×106 cells) into the right flank. When tumors reached 100-120 mm3, mice were randomized into treatment (n=8) and vehicle control (n=8) groups. GSK461364 was dissolved in DMSO:PEG400:saline (10:40:50 v/v/v) and administered intravenously via tail vein at 30 mg/kg twice weekly for 4 weeks. Tumor volume and body weight were measured twice weekly [2]
- U2OS osteosarcoma xenograft model: Nude mice bearing U2OS xenografts (1×107 cells) were treated when tumors reached 130-150 mm3. GSK461364 was formulated in 5% dextrose solution and given intraperitoneally at 25 mg/kg three times weekly for 5 weeks. The combination group received additional paclitaxel (10 mg/kg) via intravenous injection once weekly. Tumors were harvested at the end of treatment for histopathological analysis [4]
- A549 lung cancer xenograft model: Male nude mice with A549 xenografts (8×106 cells) were treated with GSK461364 dissolved in 10% ethanol:90% saline (v/v) at 40 mg/kg intravenously once weekly for 3 weeks. Tumor growth was monitored by caliper measurement, and PLK1 activity in tumor lysates was assayed post-treatment [3]

In patients with advanced solid malignancies (Phase I study), intravenous administration of GSK461364 at doses ranging from 0.3 to 12 mg/m² resulted in a terminal half-life (t1/2) of 1.8-2.5 hours [1]
- Plasma clearance (CL) was 15-22 L/h/m², and volume of distribution at steady state (Vdss) was 18-25 L/m² in human subjects. No accumulation was observed after repeated dosing (twice weekly for 3 weeks) [1]
- In mice, oral bioavailability of GSK461364 was <5% after a single oral dose of 50 mg/kg, while intravenous bioavailability was 100% [3]
- The drug was metabolized primarily via cytochrome P450 3A4 (CYP3A4) in human liver microsomes, with minimal metabolism by other CYP isoforms [1]

In Phase I clinical trials, the most common treatment-related adverse events (AEs) were myelosuppression (neutropenia: 62%, thrombocytopenia: 45%), gastrointestinal toxicity (nausea: 38%, diarrhea: 27%), and fatigue (32%). Grade 3/4 neutropenia occurred in 28% of patients at doses ≥8 mg/m² [1]
- In mice, the maximum tolerated dose (MTD) of GSK461364 via intravenous injection was 60 mg/kg, with mortality observed at 80 mg/kg. High-dose treatment (50 mg/kg IP) caused mild renal tubular vacuolation, which was reversible after treatment cessation [3]
- Human plasma protein binding rate of GSK461364 was 97-99% (determined by equilibrium dialysis). No significant drug-drug interactions were observed with CYP3A4 substrates in in vitro studies [1]
- In long-term animal studies (8 weeks), GSK461364 at therapeutic doses (25-30 mg/kg) did not induce significant cardiotoxicity or neurotoxicity [4]

[1]. Phase I study of GSK461364, a specific and competitive Polo-like kinase 1 inhibitor, in patients with advanced solid malignancies. Clin Cancer Res. 2011 May 15;17(10):3420-30.

[2]. Sensitivity of cancer cells to Plk1 inhibitor GSK461364A is associated with loss of p53 function and chromosome instability. Mol Cancer Ther. 2010 Jul;9(7):2079-89.

[3]. Distinct concentration-dependent effects of the polo-like kinase 1-specific inhibitor GSK461364A, including differential effect on apoptosis. Cancer Res. 2009 Sep 1;69(17):6969-77.

[4]. Cytotoxic mechanism of PLK1 inhibitor GSK461364 against osteosarcoma: Mitotic arrest, apoptosis, cellular senescence, and synergistic effect with paclitaxel. Int J Oncol. 2016 Mar;48(3):1187-94.

5-[6-[(4-methyl-1-piperazinyl)methyl]-1-benzimidazolyl]-3-[(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy]-2-thiophenecarboxamide is a member of (trifluoromethyl)benzenes.
Polo-like Kinase 1 Inhibitor GSK461364 is a small molecule Polo-like kinase 1 (PLK1) inhibitor with potential antineoplastic activity. Polo-like kinase 1 inhibitor GSK461364 selectively inhibits Plk1, inducing selective G2/M arrest followed by apoptosis in a variety of tumor cells while causing reversible cell arrest at the G1 and G2 stage without apoptosis in normal cells. Plk1, named after the polo gene of Drosophila melanogaster, is a serine/threonine protein kinase involved in regulating mitotic spindle function in a non-ATP competitive manner.

---

GSK461364 is a highly specific, competitive inhibitor of PLK1 that binds to the ATP-binding pocket of the kinase domain, blocking its catalytic activity [2]
- Sensitivity to GSK461364 correlates with p53 functional loss and chromosome instability, making p53-mutant cancers potential therapeutic targets [2]
- The compound exhibits distinct concentration-dependent effects in cancer cells: mitotic arrest at low concentrations, apoptosis at intermediate concentrations, and mitotic catastrophe/senescence at high concentrations [3]
- It has advanced to Phase I clinical trials for advanced solid malignancies, showing manageable toxicity and preliminary antitumor activity [1]
- Synergistic antitumor effects with paclitaxel support its potential use in combination therapy for osteosarcoma and other solid tumors [4]

C27H28F3N5O2S

543.6

543.191

C, 59.66; H, 5.19; F, 10.48; N, 12.88; O, 5.89; S, 5.90

929095-18-1

15983966

white solid powder

1.4±0.1 g/cm3

658.0±65.0 °C at 760 mmHg

351.7±34.3 °C

0.0±2.0 mmHg at 25°C

1.645

3.34

104.86

1

9

7

38

814

1

C(N1CCN(C)CC1)C1C=CC2=C(N(C3SC(C(=O)N)=C(O[C@@H](C4C=CC=CC=4C(F)(F)F)C)C=3)C=N2)C=1

ZHJGWYRLJUCMRT-QGZVFWFLSA-N

InChI=1S/C27H28F3N5O2S/c1-17(19-5-3-4-6-20(19)27(28,29)30)37-23-14-24(38-25(23)26(31)36)35-16-32-21-8-7-18(13-22(21)35)15-34-11-9-33(2)10-12-34/h3-8,13-14,16-17H,9-12,15H2,1-2H3,(H2,31,36)/t17-/m1/s1

5-[6-[(4-methylpiperazin-1-yl)methyl]benzimidazol-1-yl]-3-[(1R)-1-[2-(trifluoromethyl)phenyl]ethoxy]thiophene-2-carboxamide

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 (4.60 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 (4.60 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 (4.60 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 储备液添加到 900 μL 玉米油中并混合均匀。

---

配方 4 中的溶解度: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30 mg/mL

---

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。