PF-573228

FAK (IC50 = 4 nM)

PF-573228 抑制纯化的 FAK 重组催化部分，IC50 为 4 nM [1]。 PF-573228 抑制 Tyr397 上的 FAK 磷酸化，IC50 为 30-100 nM[1]。 PF-573228 显着降低 FAK Tyr397 磷酸化 [1]。 PF-573228 减少趋化性和趋化性迁移，同时也减少粘着斑周转 [1]。

在几种人类 sc 异种移植模型中，PF-562271 表现出剂量依赖性肿瘤生长抑制作用，并在 25 至 50 mg/剂量时对 PC-3M、BT474、BxPc3 和 LoVo 产生最大肿瘤抑制作用，抑制范围为 78% 至 94%每天两次体重减轻，没有体重减轻、发病或死亡。 PF-562271（25 mg/kg，口服）可显着减少皮下和骨转移 PC3M-luc-C6 异种移植模型中的肿瘤进展。在 Huh7.5 肝细胞癌异种移植模型中，舒尼替尼和 PF-562271 的联合治疗针对血管生成和肿瘤侵袭性，通过阻断肿瘤生长并影响肿瘤停药后恢复的能力，产生比单药更显着的抗肿瘤效果的治疗。

重组激酶试验[1]
纯化活化的FAK激酶结构域（氨基酸410-689）与50 μm ATP和10 μg/孔的随机肽聚合物Glu和Tyr（摩尔比为4:1），poly（Glu/Tyr）在激酶缓冲液（50 mm HEPES, pH 7.5, 125 mm NaCl, 48 mm MgCl2）中反应15分钟。从1 μm的最高浓度开始，用1 / 2 -Log浓度的连续稀释化合物对poly（Glu/Tyr）的磷酸化进行挑战。每个浓度运行三次。用抗磷酸酪氨酸（PY20）抗体检测聚Glu/Tyr的磷酸化，然后用辣根过氧化物酶偶联山羊抗小鼠IgG抗体检测聚Glu/Tyr的磷酸化。加入标准辣根过氧化物酶底物3,3 ',5,5 ' -四甲基联苯胺，加入停止液（2 m H2SO4）后，在450 nm处获得光密度读数。IC50值采用Hill斜率模型确定。广泛的激酶选择性分析使用KinaseProfiler™选择性筛选服务，可通过获得。欲了解更多信息，请访问：www.upstate.com/discovery/services/kp_overview.q.

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细胞激酶测定[1]
利用Invitrogen公司的GeneSwitch诱导系统，在米非司酮的诱导调节下，生成稳定的A431上皮癌克隆，表达野生型v5标记的FAK蛋白或突变型FAK Y397F v5标记的蛋白。稳定克隆在Dulbecco改良Eagle培养基、10%胎牛血清、750 μg/ml Zeocin和50 μg/ml Hygromycin中生长。在进行FAK细胞ELISA检测前一天，将A431·FAKwt细胞以1.2 × 106个/ml的浓度接种于96孔u型板的生长培养基中。37℃、5% CO2作用4 ~ 6 h后，用0.1 nm米非司酮诱导FAK表达。包括非诱导对照组。随后在山羊抗小鼠或抗兔板上涂有抗v5或抗fak （1.0 μg/ml）或在Superblock tris缓冲盐水缓冲液中涂有不相关的抗体对照。抗v5或抗fak包被板在3%牛血清白蛋白，0.5% Tween中阻断，室温下1小时。细胞在37°C， 5% CO2条件下，以1 μm的最高浓度开始½-Log连续稀释30 min。用指定浓度的化合物处理细胞的裂解物在p -裂解缓冲液中制备（50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25%脱氧胆酸钠，150 mm NaCl, 1 mm EDTA, 1 mm Na3VO4, 1 mm NaF和蛋白酶抑制剂），并将其转移到抗v5或抗FAK包被板上以捕获总诱导蛋白或总FAK蛋白。采用抗磷特异性FAK[Y397]检测自磷酸化FAK Tyr397，然后检测二级报告抗体。加入辣根过氧化物酶底物，在450 nm处读板。IC50值采用Hill斜率模型确定。为了进行Western blot分析，在CH缓冲液（50 mm HEPES, 0.15 m NaCl, 2mm EDTA， 1% Nonidet P-40和0.5%脱氧胆酸钠，pH 7.2）裂解前，用指定浓度的抑制剂处理REF52细胞，缓冲液中含有1 mm苯基甲基磺酰氟，100 mm lepeptin和0.05 TIU/ml抑酶蛋白，1 mm Na3VO4, 40 mm NaF和10 mm Na4P2O7。在悬浮/复制实验中，无论是否存在指定浓度的抑制剂，将REF52细胞悬浮在无血清培养基中，在继续存在或不存在抑制剂的情况下，允许重新附着在5 μg/ml FN包被的板上30分钟。用ch缓冲液裂解细胞，用25-50 μg的蛋白对全细胞裂解液进行Western blot分析。

伤口愈合试验[1]
用10 μl移液针尖在35 mm Bioptechs delta-T培养皿上损伤融合的REF52单层，在37℃下用延时显微镜拍摄细胞迁移到创口9 h。划痕时加入PF-573,228，划痕1 h后开始拍摄。延时显微镜采用尼康TE200倒置显微镜，配有20倍差干涉对比物镜和Bioptechs加热台。这些图像是用滨松虎鲸相机拍摄的，并使用Improvision Openlab软件进行编译。在图像捕获之后，随着时间的推移，单个细胞的细胞核被跟踪，因为它们迁移到伤口，细胞速度通过细胞轨迹的长度除以电影的总时间来计算。

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免疫荧光与细胞扩散[1]
在无血清的情况下，对贴壁的REF52细胞进行洗涤，并用浓度逐渐增加的PF-573,228处理1小时。在重复实验中，将REF52细胞在无血清和增加FAK抑制剂量的情况下悬浮30分钟。在抑制剂持续存在的情况下，将细胞镀于fn包被（5 μg/ml）的盖片（用于免疫荧光）或delta-T皿（用于差干涉对比延时电影）上60分钟。用新鲜的4%多聚甲醛固定细胞20分钟，用0.5% Triton X-100渗透2分钟，用20%山羊血清、2%牛血清白蛋白阻断30分钟。然后用paxillin或FAK Tyr(P)397抗体染色。二抗用Alexa Fluor 488或594染料标记。使用尼康Eclipse E600显微镜（60倍油物镜）、滨松数码相机和Openlab成像软件包获取图像。

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TIRF显微镜与粘附周转分析[1]
用FLP-In系统稳定转染NIH-3T3细胞。用PF-573,228处理δ - t培养皿上的融合单层，并按上述方法用移液管尖端损伤。使用配备60 × 1.45 N.A. TIRF物镜的Nikon TE2000-E Eclipse倒置TIRF显微镜、Retiga数码相机和QCapture Pro采集软件，在37°C下观察荧光粘附。每5分钟采集一次图像，持续60-90分钟。使用Openlab软件进行图像分析。随着时间的推移，测量每种粘附的强度，并确定每种粘附的峰值（最大）强度。粘附寿命计算为粘附从其半峰强度达到峰值强度所需的时间和粘附从其峰值强度返回到其半峰强度所需的时间的总和。每个细胞至少分析了7个粘连。

To better understand the role of FAK in leukocyte recruitment, we used a FAK-specific inhibitor (PF-573228) and determined the effect on IL-4 induced eosinophil recruitment in vivo. PF-573228 prevented the expression of VCAM-1 and CCL26 expression in IL-4-stimulated human endothelial cells in vitro. As a result, eosinophil adhesion and transmigration were blocked. PF-572338 also prevented IL-4-induced VCAM-1 expression in vivo. Using brightfield intravital microscopy, we found that PF-573228 decreased leukocyte rolling flux, adhesion, and emigration. We specifically examined eosinophil recruitment in vivo by using an eosinophil-GFP reporter mouse and found PF-573228 attenuated eosinophil emigration. This study reveals that a FAK inhibitor influences inflammation through its action on eosinophil recruitment. [2]

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25 mg/kg; Oral gavage

PC3M-luc-C6 xenograft models

[1]. Cellular characterization of a novel focal adhesion kinase inhibitor. J Biol Chem. 2007 May 18;282(20):14845-52.

[2]. Inhibiting focal adhesion kinase (FAK) blocks IL-4 induced VCAM-1 expression and eosinophil recruitment in vitro and in vivo. J Leukoc Biol . 2018 Jul;104(1):147-158.

6-[[4-[(3-methylsulfonylphenyl)methylamino]-5-(trifluoromethyl)-2-pyrimidinyl]amino]-3,4-dihydro-1H-quinolin-2-one is a member of quinolines.

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Focal adhesion kinase (FAK) is a member of a family of non-receptor protein-tyrosine kinases that regulates integrin and growth factor signaling pathways involved in cell migration, proliferation, and survival. FAK expression is increased in many cancers, including breast and prostate cancer. Here we describe perturbation of adhesion-mediated signaling with a FAK inhibitor, PF-573,228. In vitro, this compound inhibited purified recombinant catalytic fragment of FAK with an IC(50) of 4 nM. In cultured cells, PF-573,228 inhibited FAK phosphorylation on Tyr(397) with an IC(50) of 30-100 nM. Treatment of cells with concentrations of PF-573,228 that significantly decreased FAK Tyr(397) phosphorylation failed to inhibit cell growth or induce apoptosis. In contrast, treatment with PF-573,228 inhibited both chemotactic and haptotactic migration concomitant with the inhibition of focal adhesion turnover. These studies show that PF-573,228 serves as a useful tool to dissect the functions of FAK in integrin-dependent signaling pathways in normal and cancer cells and forms the basis for the generation of compounds amenable for preclinical and patient trials.[1]

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Leukocyte recruitment plays a critical role during both normal inflammation and chronic inflammatory diseases, and ongoing studies endeavor to better understand the complexities of this process. Focal adhesion kinase (FAK) is well known for its role in cancer, yet it also has been shown to regulate aspects of neutrophil and B16 melanoma cell recruitment by rapidly influencing endothelial cell focal adhesion dynamics and junctional opening. Recently, we found that FAK related non-kinase (FRNK), a protein that is often used as a FAK dominant negative, blocked eosinophil transmigration by preventing the transcription of vascular cell adhesion molecule-1 (VCAM-1) and eotaxin-3 (CCL26). Surprisingly, the blocking occurred even in the absence of endogenous FAK. To better understand the role of FAK in leukocyte recruitment, we used a FAK-specific inhibitor (PF-573228) and determined the effect on IL-4 induced eosinophil recruitment in vitro and in vivo. PF-573228 prevented the expression of VCAM-1 and CCL26 expression in IL-4-stimulated human endothelial cells in vitro. As a result, eosinophil adhesion and transmigration were blocked. PF-572338 also prevented IL-4-induced VCAM-1 expression in vivo. Using brightfield intravital microscopy, we found that PF-573228 decreased leukocyte rolling flux, adhesion, and emigration. We specifically examined eosinophil recruitment in vivo by using an eosinophil-GFP reporter mouse and found PF-573228 attenuated eosinophil emigration. This study reveals that a FAK inhibitor influences inflammation through its action on eosinophil recruitment.[2]

C22H20F3N5O3S

491.49

491.123

C, 53.76; H, 4.10; F, 11.60; N, 14.25; O, 9.77; S, 6.52

869288-64-2

11612883

White to off-white solid powder

1.5±0.1 g/cm3

1.616

1.03

121.46

3

10

6

34

822

0

HESLKTSGTIBHJU-UHFFFAOYSA-N

InChI=1S/C22H20F3N5O3S/c1-34(32,33)16-4-2-3-13(9-16)11-26-20-17(22(23,24)25)12-27-21(30-20)28-15-6-7-18-14(10-15)5-8-19(31)29-18/h2-4,6-7,9-10,12H,5,8,11H2,1H3,(H,29,31)(H2,26,27,28,30)

3,4-Dihydro-6-[[4-[[[3-(methylsulfonyl)phenyl]methyl]amino]-5-(trifluoromethyl)-2-pyrimidinyl]amino]-2(1H)-quinolinone

PF573,228; PF 573,228; PF-573,228; PF573228; 869288-64-2; PF-573228; PF 573228; PF573228; PF-228; 3,4-Dihydro-6-[[4-[[[3-(methylsulfonyl)phenyl]methyl]amino]-5-(trifluoromethyl)-2-pyrimidinyl]amino]-2(1H)-quinolinone; 6-((4-((3-(Methylsulfonyl)benzyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-3,4-dihydroquinolin-2(1H)-one; 6-(4-(3-(methylsulfonyl)benzylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-3,4-dihydroquinolin-2(1H)-one; PF 573228; PF-573228;

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 (5.09 mM) 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), 悬浮液；超声助溶。
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。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (4.23 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 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 3 中的溶解度: ≥ 2.08 mg/mL (4.23 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 20.8 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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配方 4 中的溶解度: 30% PEG400+0.5% Tween80+5% propylene glycol:30 mg/mL

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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；
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。