FAK (Ki = 0.4 nM)

体外活性：GSK2256098是葛兰素史克开发的一种小分子，作为选择性FAK（粘着斑激酶）激酶抑制剂，通过靶向FAK酪氨酸（Y）397的磷酸化位点来抑制FAK活性。GSK2256098抑制胰腺导管的生长和存活腺癌细胞。与最近的 FAK 家族成员 Pyk2 相比，它对 FAK 的选择性高出一千倍。 GSK2256098 通过靶向 FAK 酪氨酸 (Y) 397 的磷酸化位点来抑制 FAK 活性。GSK2256098 处理导致 PTEN 突变 (Ishikawa) 细胞对 pFAK(Y397) 的抑制作用强于 PTEN 野生型 (Hec1A) 细胞。 Ishikawa 细胞比处理过的 Hec1A 细胞对 GSK2256098 更敏感。用野生型 PTEN 构建体转染 Ishikawa 细胞，用 GSK2256098 处理后，pFAK(Y397) 表达没有变化。与 Hec1a 细胞相比，在 Ishikawa 细胞中观察到细胞活力降低，对化疗（紫杉醇和托泊替康）与 GSK2256098 组合的敏感性增强。激酶检测：与最接近的 FAK 家族成员 Pyk2 相比，GSK2256098 对 FAK 的选择性高出千倍。 GSK2256098 通过靶向 FAK 的磷酸化位点酪氨酸 (Y) 397 抑制 FAK 活性。GSK2256098 抑制癌细胞系 OVCAR8（卵巢）、U87MG（脑）和 A549（肺）中的 FAK 活性或 Y397 磷酸化，IC50 为 15分别为 8.5 和 12 nM。细胞测定：PDAC细胞在6孔板上培养。当细胞在常规培养基中汇合达到约70%时，将细胞在含有0.1-10μM GSK2256098的培养基中孵育48或72小时。处理结束后，重新接种细胞并保存 9 天，然后使用克隆试剂对细胞进行染色，并对蓝色菌落进行计数。 GSK2256098 已开发用于通过靶向 FAK 酪氨酸磷酸化位点来抑制 FAK 活性(Y) 397. 孵育 30 分钟后，GSK2256098 抑制癌细胞系 OVCAR8（卵巢）、U87MG（脑）和 A549（肺）中的 FAK 活性或 Y397 磷酸化，IC50 值为 15、8.5 和 12 nM ， 分别。此外，数据表明，GSK2256098 对 FAK 的细胞抑制最早可在培养细胞中 30 分钟发生，在小鼠肿瘤异种移植物中持续长达 12 小时。 GSK2256098 抑制 FAK 激酶活性可降低 Akt 和 ERK 活性。 PI3K/Akt 和 ERK 信号传导有助于细胞存活，这意味着 GSK2256098 在减弱特定类型 PDAC 细胞的异常存活途径方面具有药理学价值。 GSK2256098可以通过caspase-9/PARP相关途径促进L3.6P1细胞凋亡。它以 FAK 特异性方式减弱 PDAC 细胞的异常生长和异常运动。

在血脑屏障完整的小鼠和大鼠中进行的药代动力学 (PK) 研究表明，GSK2256098 对中枢神经系统的渗透性较差。然而，它在 GBM（胶质母细胞瘤）患者肿瘤中的浓度超过了与临床前活性相关的浓度。 在 Ishikawa 原位小鼠模型中，与接种 Hec1A 细胞的小鼠相比，用 GSK2256098 治疗可导致肿瘤重量降低和转移减少。与 Hec1a 模型相比，Ishikawa 模型中用 GSK2256098 治疗的肿瘤具有较低的微血管密度 (CD31)、较少的细胞增殖 (Ki67) 和较高的细胞凋亡 (TUNEL) 率。 GSK2256098 可能对 PTEN 突变子宫癌患者有治疗益处，PTEN 代表了一种潜在的预测生物标志物。众所周知，FAK 在血管生成、增殖和细胞凋亡中发挥重要作用，因此对从治疗实验中收集的肿瘤样本进行了检查。评估 CD31，观察到用 GSK2256098 和紫杉醇治疗的小鼠肿瘤中的微血管密度显着低于媒介物对照组小鼠的肿瘤中（P<0.05）。这在两种模型中都是一致的，但石川肿瘤的微血管密度最低。使用 GSK2256098 治疗的小鼠中的所有肿瘤模型均表现出比对照小鼠更少的通过 Ki67 的增殖。 Ishikawa 肿瘤对治疗的反应具有最低的 Ki67 表达。 GSK2256098 治疗后，Ishikawa 肿瘤比 Hec1A 肿瘤具有更高的细胞凋亡指数。在使用 GSK2256098 和紫杉醇联合治疗的所有模型中均观察到显着的细胞凋亡率

FAK 家族中最接近的成员 Pyk2 对 FAK 的选择性比 GSK2256098 低一千倍。通过关注 FAK 的磷酸化位点酪氨酸 (Y) 397，GSK2256098 抑制 FAK 活性。对于 OVCAR8（卵巢）、U87MG（脑）和 A549（肺癌）癌细胞系，GSK2256098 抑制 FAK 活性或 Y397 磷酸化，IC50 分别为 15、8.5 和 12 nM。

PDAC 细胞在 96 孔板的孔中生长。孔中装有 10 微升 MTS（总共 100 μL）。将板在 37°C 下孵育 10 至 30 分钟后，在酶标仪上测量反应后的 MTS 在 450 nm 波长处的吸光度。 GSK2256098 对细胞活力的 IC50 使用 Sigma 图程序确定。使用 6 孔板培养 PDAC 细胞。当细胞在常规培养基中汇合达到大约 70% 后，将细胞在含有 0.1–10 μM GSK2256098 的培养基中孵育 48 或 72 小时。处理结束后重新接种细胞并储存九天。用克隆试剂对细胞进行染色后，对蓝色菌落进行计数[1]。

Mice: The mice used are female athymic nude mice aged 8–12 weeks. In experimental therapy, 4x10⁶ Ishikawa or Hec1A cells are injected into the uterus horn. The mice are randomized (n = 10 mice per group) based on the following groups after receiving tumor cell injection: 1) 100 microliters of a vehicle control (orally, daily); 2) 75 milligrams per kilogram of GSK2256098 in 100 microliters of vehicle (orally, daily); 3) 2.5 milligrams per kilogram of Paclitaxel in 200 microliters of PBS (intraperitoneally, weekly); and 4) GSK2256098 and Paclitaxel (doses and frequencies mentioned above). After the tumor injection, therapy is started 10–14 days later. Four to six weeks after treatment initiation, the mice are checked for side effects and cervically dislocated for death. Each mouse's weight, the total weight of the tumor, the location and quantity of tumor nodules are noted for each treatment group at the end of the experiment. In order to process tumor samples for additional analysis, they are either frozen in a medium with the ideal cutting temperature or paraffin-embedded in a section fixed in formalin.

[1]. A small molecule FAK kinase inhibitor, GSK2256098, inhibits growth and survival of pancreatic ductal adenocarcinoma cells. Cell Cycle. 2014;13(19):3143-9.

[2]. PTEN Expression as a Predictor of Response to Focal Adhesion Kinase Inhibition in Uterine Cancer. Mol Cancer Ther. 2015 Jun;14(6):1466-75.

GSK2256098 is under investigation in clinical trial NCT02523014 (Vismodegib and FAK Inhibitor GSK2256098 in Treating Patients With Progressive Meningiomas).
FAK Inhibitor GSK2256098 is a focal adhesion kinase-1 (FAK) inhibitor with potential antiangiogenic and antineoplastic activities. FAK inhibitor GSK2256098 inhibits FAK, which may prevent the integrin-mediated activation of several downstream signal transduction pathways, including ERK, JNK/MAPK and PI3K/Akt, thereby inhibiting tumor cell migration, proliferation and survival, and tumor angiogenesis. The tyrosine kinase FAK is normally activated by binding to integrins in the extracellular matrix (ECM) but may be upregulated and constitutively activated in various tumor cell types.

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Focal adhesion kinase (FAK) hyperactivation is common in pancreatic ductal adenocarcinoma (PDAC). A small molecule, GSK2256098 (GlaxoSmithKline), has been developed to inhibit FAK activity through targeting the phosphorylation site of FAK, tyrosine (Y) 397. We sought to determine whether GSK2256098 inhibition of FAK Y397 phosphorylation attenuates PDAC-associated cell proliferation, motility and survival. Cultured PDAC cells were used as cellular models of GSK2256098-impaired abnormal growth. Western blot analysis, cell viability analysis, clonogenic survival, soft-agar and wound healing assays were performed. The responses of 6 PDAC cell lines in regards to FAK Y397 phosphorylation or activity to GSK2256098 treatments (0.1-10 μM) ranged from low (less than 20% inhibition) to high (more than 90% inhibition). The least and most sensitive cell lines (PANC-1 and L3.6P1) were selected for further analysis. GSK2256098 inhibition of FAK Y397 phosphorylation correlated with decreased levels of phosphorylated Akt and ERK in L3.6P1 cells. GSK2256098 decreased cell viability, anchorage-independent growth, and motility in a dose dependent manner. Current studies demonstrate that small molecule kinase inhibitors targeting FAK Y397 phosphorylation can inhibit PDAC cell growth. Assessments of FAK Y397 phosphorylation in biopsies may be used as a biomarker to select the subgroup of responsive patients and/or monitor the effects of GSK2256098 on FAK-modulated tumor growth during treatment.[1]

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PTEN is known to be frequently mutated in uterine cancer and also dephosphorylates FAK. Here, we examined the impact of PTEN alterations on the response to treatment with a FAK inhibitor (GSK2256098). In vitro and in vivo therapeutic experiments were carried out using PTEN-mutated and PTEN-wild-type models of uterine cancer alone and in combination with chemotherapy. Treatment with GSK2256098 resulted in greater inhibition of pFAK(Y397) in PTEN-mutated (Ishikawa) than in PTEN-wild-type (Hec1A) cells. Ishikawa cells were more sensitive to GSK2256098 than the treated Hec1A cells. Ishikawa cells were transfected with a wild-type PTEN construct and pFAK(Y397) expression was unchanged after treatment with GSK2256098. Decreased cell viability and enhanced sensitivity to chemotherapy (paclitaxel and topotecan) in combination with GSK2256098 was observed in Ishikawa cells as compared with Hec1a cells. In the Ishikawa orthoptopic murine model, treatment with GSK2256098 resulted in lower tumor weights and fewer metastases than mice inoculated with Hec1A cells. Tumors treated with GSK2256098 had lower microvessel density (CD31), less cellular proliferation (Ki67), and higher apoptosis (TUNEL) rates in the Ishikawa model when compared with the Hec1a model. From a large cohort of evaluable patients, increased FAK and pFAK(Y397) expression levels were significantly related to poor overall survival. Moreover, PTEN levels were inversely related to pFAK(Y397) expression. These preclinical data demonstrate that PTEN-mutated uterine cancer responds better to FAK inhibition than does PTEN wild-type cancer. Therefore, PTEN could be a biomarker for predicting response to FAK-targeted therapy during clinical development.[2]

C20H23CLN6O2

414.89

414.157

C, 57.90; H, 5.59; Cl, 8.54; N, 20.26; O, 7.71

1224887-10-8

1416771-10-2 (HCl);1224887-10-8;

46214930

White to off-white solid powder

1.3±0.1 g/cm3

545.7±60.0 °C at 760 mmHg

283.8±32.9 °C

0.0±1.5 mmHg at 25°C

1.638

7.34

93.1

3

6

7

29

539

0

ClC1=C([H])N=C(C([H])=C1N([H])C1=C([H])C([H])=C([H])C([H])=C1C(N([H])OC([H])([H])[H])=O)N([H])C1=C([H])C(C([H])([H])[H])=NN1C([H])(C([H])([H])[H])C([H])([H])[H]

BVAHPPKGOOJSPU-UHFFFAOYSA-N

InChI=1S/C20H23ClN6O2/c1-12(2)27-19(9-13(3)25-27)24-18-10-17(15(21)11-22-18)23-16-8-6-5-7-14(16)20(28)26-29-4/h5-12H,1-4H3,(H,26,28)(H2,22,23,24)

2-[[5-chloro-2-[(5-methyl-2-propan-2-ylpyrazol-3-yl)amino]pyridin-4-yl]amino]-N-methoxybenzamide

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 (6.03 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。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.03 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。

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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：
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；
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