KU-60019

ATM ( IC50 = 6.3 nM ); DNA-PKcs ( IC50 = 1.7 μM )

体外活性：与 KU-55933 相比，KU-60019 是一种改进的水溶性更好的 ATM 激酶抑制剂，同时表现出相似的靶点选择性。 KU-60019 对 DNA-PKcs 和 ATR 以及 229 种其他蛋白激酶（如 PI3K、mTOR 和 mTOR/FKBP12）几乎没有活性，IC50 值分别为 1.7 μM 和 >10 μM。在人胶质瘤 U87 和 U1242 细胞中，KU-60019 在阻断辐射诱导的关键 ATM 蛋白靶标（如 p53、γ-H2AX 和 CHK2）磷酸化方面的效力比 KU-55933 高 3 至 10 倍，浓度为 1 μM KU-60019 显着诱导 p53 (S15) 磷酸化降低 >70%，达到该程度需要约 10 μM 的 KU-55933 才能实现。 KU-60019 在 1 μM 和 10 μM 时有效地使人胶质瘤细胞放射增敏，剂量增强比分别为 1.7 和 4.4，并且还使正常成纤维细胞放射增敏，但不使 AT 成纤维细胞放射增敏。 KU-60019 治疗 (3 μM) 可分别阻断基础和胰岛素诱导的 AKT S473 磷酸化 70% 和约 50%，并将辐射诱导的 AKT 磷酸化完全降低至对照水平以下。 KU-60019 对 AKT S473 磷酸化的影响可以在神经胶质瘤细胞系和正常成纤维细胞中观察到，但在 AT (h-TERT) 细胞中观察不到，并且可以被磷酸酶抑制剂冈田酸显着阻断，这表明 ATM 激酶在通过未知磷酸酶调节 AKT 磷酸化。与抑制促存活 AKT 信号传导相一致，3 μM 的 KU-60019 显着抑制人胶质瘤 U87 细胞的迁移和侵袭，分别 >70% 和 ~60%，以及 U1242 细胞分别 >50% 和 ~60% 。激酶测定：KU-55933 的体外 IC50 为 13 nM，Ki 为 2.2 nM，并且使用一组 60 种蛋白激酶对 ATM 激酶具有高度特异性。 KU-60019 是一种改进的 ATM 激酶抑制剂，IC50 为 6.3 nM，大约是 KU-55933 的一半。 DNA-PKcs 和 ATR 的 IC50 值分别为 1.7 和 >10 μM，几乎比 ATM 高 270 倍和 1600 倍。 KU-60019 在阻断辐射诱导的人胶质瘤细胞中关键 ATM 靶点磷酸化方面的效果比 KU-55933 有效 10 倍。在人 U87 神经胶质瘤细胞中，KU-55933 在 10 μM 浓度下完全抑制 p53 (S15) 的磷酸化，但在 3 μM 浓度下则不然，而在 10 μM 浓度下照射后 1 小时，γ-H2AX 水平仅部分降低。相比之下，3 μM KU-60019 完全抑制 p53 磷酸化，1 μM 则部分抑制。细胞测定：将细胞（U87 和 U1242）暴露于 KU-60019 1、3 和 5 天。细胞生长由 AlamarBlue 测定。将 AlamarBlue 添加到培养基中至推荐的最终浓度。将板在 37°C 下孵育 1 小时，在 Fluoro-Count 读板器上测定荧光（激发波长 530 nm，发射波长 590 nm），并将值作为细胞生长的测量值。通过台盼蓝/荧光激活细胞分选（FACS）测定法测定细胞存活率。

与单独放疗、单独使用KU-60019或不治疗相比，放疗加KU-60019可显著提高小鼠在原位胶质瘤U1242/luc-GFP异种移植模型中的存活率。此外，p53突变胶质瘤对KU-60019放射增敏的反应远比野生型胶质瘤强烈。[2]

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为了研究ATM特异性抑制PTEN缺陷细胞的体内效果，我们在皮下异种移植物环境中使用了PC3 PTEN四环素诱导细胞系模型。首先，我们建立了四环素衍生物多西环素在体外（补充图4A）和体内（补充图4B）有效诱导PTEN表达。卡尺测量肿瘤体积显示，使用强力霉素诱导PTEN，导致肿瘤生长从72小时开始减慢（补充图4C）。接下来，我们研究了ATM特异性激酶抑制剂KU-60019作为单一模态在PC3-PTEN诱导模型中的选择性毒性。之所以选择这种抑制剂，是因为它是一种有效的ATM抑制剂，与KU-55933不同，它在动物系统中具有活性。尽管在pten野生型对照之前，pten缺陷对照肿瘤的大小增加了4倍，但KU-60019治疗的pten缺陷肿瘤的生长速度在统计学上显着减慢（图4A）。在KU-60019给药后（第1-5天），这种生长抑制在实验开始时（第5-12天）尤为明显。各治疗组的平均相对体重无显著变化（图4B）。采用免疫荧光法分析PTEN在切除肿瘤中的体内诱导表达[2]。

用AlamarBlue®检测细胞生长情况。将U1242细胞连续稀释，贴壁6 h后暴露于3 μM KU-60019中。在播种后第1、3和5天，将AlamarBlue®添加到培养基中至推荐的终浓度。在37℃下孵育1小时，在FluoroCount平板阅读器上测定荧光（激发530 nm，发射590 nm），并取其值作为细胞生长的度量[1]

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KU-60019 暴露于细胞 1、3 和 5 天。 AlamarBlue 是细胞生长的衡量标准。培养基中添加 AlamarBlue，直至达到建议的最终浓度。 Fluoro-Count 读板器（激发波长 530 nm，发射波长 590 nm）用于测量 37 °C 孵育一小时后板上的荧光。获得的值代表细胞的生长。台盼蓝/荧光激活细胞分选 (FACS) 测定用于评估细胞存活率。

[1]. Improved ATM kinase inhibitor KU-60019 radiosensitizes glioma cells, compromises insulin, AKT and ERK prosurvival signaling, and inhibits migration and invasion. Mol Cancer Ther. 2009 Oct;8(10):2894-902.

[2]. Mechanistic Rationale to Target PTEN-Deficient Tumor Cells with Inhibitors of the DNA Damage Response Kinase ATM. Cancer Res. 2015 Jun 1;75(11):2159-65.

Ataxia telangiectasia (A-T) mutated (ATM) is critical for cell cycle checkpoints and DNA repair. Thus, specific small molecule inhibitors targeting ATM could perhaps be developed into efficient radiosensitizers. Recently, a specific inhibitor of the ATM kinase, KU-55933, was shown to radiosensitize human cancer cells. Herein, we report on an improved analogue of KU-55933 (KU-60019) with K(i) and IC(50) values half of those of KU-55933. KU-60019 is 10-fold more effective than KU-55933 at blocking radiation-induced phosphorylation of key ATM targets in human glioma cells. As expected, KU-60019 is a highly effective radiosensitizer of human glioma cells. A-T fibroblasts were not radiosensitized by KU-60019, strongly suggesting that the ATM kinase is specifically targeted. Furthermore, KU-60019 reduced basal S473 AKT phosphorylation, suggesting that the ATM kinase might regulate a protein phosphatase acting on AKT. In line with this finding, the effect of KU-60019 on AKT phosphorylation was countered by low levels of okadaic acid, a phosphatase inhibitor, and A-T cells were impaired in S473 AKT phosphorylation in response to radiation and insulin and unresponsive to KU-60019. We also show that KU-60019 inhibits glioma cell migration and invasion in vitro, suggesting that glioma growth and motility might be controlled by ATM via AKT. Inhibitors of MEK and AKT did not further radiosensitize cells treated with KU-60019, supporting the idea that KU-60019 interferes with prosurvival signaling separate from its radiosensitizing properties. Altogether, KU-60019 inhibits the DNA damage response, reduces AKT phosphorylation and prosurvival signaling, inhibits migration and invasion, and effectively radiosensitizes human glioma cells.[1]

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Ataxia telangiectasia mutated (ATM) is an important signaling molecule in the DNA damage response (DDR). ATM loss of function can produce a synthetic lethal phenotype in combination with tumor-associated mutations in FA/BRCA pathway components. In this study, we took an siRNA screening strategy to identify other tumor suppressors that, when inhibited, similarly sensitized cells to ATM inhibition. In this manner, we determined that PTEN and ATM were synthetically lethal when jointly inhibited. PTEN-deficient cells exhibited elevated levels of reactive oxygen species, increased endogenous DNA damage, and constitutive ATM activation. ATM inhibition caused catastrophic DNA damage, mitotic cell cycle arrest, and apoptosis specifically in PTEN-deficient cells in comparison with wild-type cells. Antioxidants abrogated the increase in DNA damage and ATM activation in PTEN-deficient cells, suggesting a requirement for oxidative DNA damage in the mechanism of cell death. Lastly, the ATM inhibitor KU-60019 was specifically toxic to PTEN mutant cancer cells in tumor xenografts and reversible by reintroduction of wild-type PTEN. Together, our results offer a mechanistic rationale for clinical evaluation of ATM inhibitors in PTEN-deficient tumors.[2]

C30H33N3O5S

547.67

547.214

925701-49-1

15953870

Light yellow to khaki solid powder

1.3±0.1 g/cm3

786.6±60.0 °C at 760 mmHg

429.5±32.9 °C

0.0±2.7 mmHg at 25°C

1.645

5.9

109.55

1

8

5

39

972

2

C[C@@H]1CN(C[C@@H](O1)C)CC(=O)NC2=CC3=C(C=C2)SC4=C(C3)C=CC=C4C5=CC(=O)C=C(O5)N6CCOCC6

SCELLOWTHJGVIC-BGYRXZFFSA-N

InChI=1S/C30H33N3O5S/c1-19-16-32(17-20(2)37-19)18-28(35)31-23-6-7-27-22(13-23)12-21-4-3-5-25(30(21)39-27)26-14-24(34)15-29(38-26)33-8-10-36-11-9-33/h3-7,13-15,19-20H,8-12,16-18H2,1-2H3,(H,31,35)/t19-,20+

2-[(2S,6R)-2,6-dimethylmorpholin-4-yl]-N-[5-(6-morpholin-4-yl-4-oxopyran-2-yl)-9H-thioxanthen-2-yl]acetamide

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)