GSK1324726A (I-BET-726)   中文名称: GSK-1324726A

BRD4/3/2 (IC50= 22/31/41 nM)

使用 GSK1324726A (I-BET726) 处理一组神经母细胞瘤细胞系，报告显示，无论 MYCN 拷贝数或表达水平如何，大多数细胞系都具有显着的生长抑制和细胞毒性。研究的所有神经母细胞瘤细胞系均表现出显着的生长抑制，中位生长 IC50 值（gIC50；抑制剂浓度导致 50% 生长抑制）等于 75 nM[1]。

I-BET726 (GSK1324726A) 抑制神经母细胞瘤的生长。由于肿瘤的范围，SK-N-AS模型中载体组的小鼠在第14天被处死。在研究的第14天，GSK1324726A中发现58％的肿瘤生长抑制（TGI） (15 mg/kg) 组，而载体组和 GSK1324726A (5 mg/kg) 组之间的肿瘤生长没有显着差异 (n=9；p=0.006)。 GSK1324726A (15 mg/kg) 组小鼠再接受 7 天的治疗，直到肿瘤体积等于媒介物组的肿瘤体积。那时，研究就停止了。在 CHP-212 模型中，肿瘤生长速度明显减慢。 42 天后，试验结束时（第 14 天），用媒介物治疗的小鼠体内的肿瘤大小几乎只有 SK-N-AS 模型中肿瘤大小的一半。在 CHP-212 模型中用 5 mg/kg GSK1324726A 治疗导致 TGI 等于 50%（n=8；p=0.1816），而在研究结论中，15 mg/kg 组的小鼠显示 TGI 为 82%（n= 5；p=0.0488)[1]。

热位移测定（Tm）[2]
在FluoDiaT70仪器上分析溴结构域的热位移。在存在或不存在100μM配体的情况下，将蛋白质样品的温度从26/30升高到74°C。在10%甘油的PBS缓冲液中使用荧光Sypro橙色染料的1:1000稀释液观察变性。温度以1°C/分钟的速率倾斜，每1°C读取一次荧光读数。测试的溴代胺包括ATAD2、BAZ2B、BRD2（串联溴结构域）、BRD4（N-和C-末端以及串联溴结构区）、CREBBP、PCAF、SMARCA2、SP140和TAF1（串联溴构域）。报告的结果是至少两个实验的平均值。[2]

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与BRD2、3和4结合的表面等离子体共振分析[2]
在25°C的T100 BIAcore仪器上采集并分析His6标记的BRD2（1–473）、BRD3（1–434）和BRD4（1–477）的BIAcore数据。在所有情况下，将固定在右旋糖酐表面的具有~5-12kU胺偶联蛋白的CM5芯片与30 mM Hepes pH 7、150 mM NaCl、1 mM EGTA和NaN3的运行缓冲液一起使用。从10μM开始，以三倍稀释度对化合物进行滴定。使用1:1结合模型通过BIA评估分析传感图和结合曲线。使用响应=浓度×Rmax/（浓度+KD）+偏移量来计算平衡KD。[2]

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等温滴定量热法。[2]
BRD4的ITC滴定是使用Microcal VPITC在25°C、pH 7.5、150mM NaCl的50mM Hepes中进行的。首先，将9μM His标记的BRD4（1-477）串联溴结构域放入细胞中，并将200μM配体滴定到其中，以获得最终配体：蛋白质过量~4:1。然后在Origin（Microcal版本）中拟合数据，得出以下参数：化学计量为1.93，KD为4.4±0.9 nM，ΔH−15.9±0.08 kcal/mol，ΔS−15.2 cal/mol/deg。 CREBBP的ITC滴定是使用Microcal AutoITC200在25°C、pH 7.4、150mM NaCl的50mM Hepes中进行的。首先，将44μM的CREBBP溴结构域放入细胞中，并将500μM配体滴定到其中，以获得最终配体：蛋白质过量~2.2:1。然后在Origin（Microcal版本）中拟合数据，得出以下参数：化学计量为1.1，KD为6.3±0.5μM，ΔH−4.35±0.08 kcal/mol，ΔS 9.2 cal/mol/deg。

poA1萤光素酶测定法[2]
在人ApoA1启动子区（−1162，+232）和人ApoA-1基因的3′-UTR（+1037，+1091）的控制下，用编码萤火虫萤光素酶的质粒稳定转染HepG2细胞。ApoA1效力定义为化合物的浓度，导致荧光素酶发光增加70%（ApoA1-Luc EC170）。请参阅支持信息，补充方法。在HepG2细胞中，ApoA1萤光素酶报告基因活性与ApoA1蛋白分泌之间已显示出高度相关性

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IL-6 PBMC和全血检测[2]
将从全血中纯化的人全血和外周血单核细胞（PBMC）与LPS和不同浓度的测试化合物孵育18-24小时。制备样品，并测量IL-6的抑制作用，如支持信息，补充方法中所述。

In vivo Studies[1]
CHP-212 (1x107) or SK–N-AS (5x106) cells in 100% matrigel were implanted subcutaneously into the right flank of approximately 9 week old female nude (Crl:CD-1-Foxn1 nu) mice. Tumors were measured with calipers and randomized using stratified sampling according to tumor size into treatment groups of 10 mice. GSK1324726A (I-BET726) formulated as a spray dried dispersion was prepared as a suspension in 1% methylcellulose vehicle. GSK1324726A (I-BET726) in vehicle or vehicle alone was administered orally by individual body weight at 10mls/kg. Mice were weighed and tumors were measured with calipers twice weekly, and mice were observed daily for any adverse treatment affects. Mice were euthanized using CO2 inhalation according to AVMA guidelines after two consecutive tumor measurements greater than 2500mm3, or if body weight loss greater than 20% was observed. For mouse pharmacodynamic studies, mice were euthanized as described above. Tumors were harvested from euthanized mice and placed in RNAlater for RNA isolation as described in Methods S1. Blood was collected after euthanasia via cardiac puncture.

Suspended in 1% methylcellulose; 15 mg/kg; p.o.

Xenograft models of non-MYCN-amplified and MYCN-amplified neuroblastoma in immunocompromised mice using the SK–N-AS and CHP-212 cell lines

[1]. BET inhibition silences expression of MYCN and BCL2 and induces cytotoxicity in neuroblastoma tumor models. PLoS One. 2013 Aug 23;8(8):e72967.

[2]. The discovery of I-BET726 (GSK1324726A), a potent tetrahydroquinoline ApoA1 up-regulator and selective BET bromodomain inhibitor. J Med Chem. 2014 Oct 9;57(19):8111-31.

BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.[1]

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hrough their function as epigenetic readers of the histone code, the BET family of bromodomain-containing proteins regulate expression of multiple genes of therapeutic relevance, including those involved in tumor cell growth and inflammation. BET bromodomain inhibitors have profound antiproliferative and anti-inflammatory effects which translate into efficacy in oncology and inflammation models, and the first compounds have now progressed into clinical trials. The exciting biology of the BETs has led to great interest in the discovery of novel inhibitor classes. Here we describe the identification of a novel tetrahydroquinoline series through up-regulation of apolipoprotein A1 and the optimization into potent compounds active in murine models of septic shock and neuroblastoma. At the molecular level, these effects are produced by inhibition of BET bromodomains. X-ray crystallography reveals the interactions explaining the structure-activity relationships of binding. The resulting lead molecule, I-BET726, represents a new, potent, and selective class of tetrahydroquinoline-based BET inhibitors.[2]

C25H23CLN2O3

434.91

434.139

C, 69.04; H, 5.33; Cl, 8.15; N, 6.44; O, 11.04

1300031-52-0

52912222

White to light yellow solid

1.3±0.1 g/cm3

707.3±60.0 °C at 760 mmHg

381.6±32.9 °C

0.0±2.4 mmHg at 25°C

1.650

5.67

69.64

2

4

4

31

643

2

ClC1C([H])=C([H])C(=C([H])C=1[H])N([H])[C@@]1([H])C2C([H])=C(C3C([H])=C([H])C(C(=O)O[H])=C([H])C=3[H])C([H])=C([H])C=2N(C(C([H])([H])[H])=O)[C@@]([H])(C([H])([H])[H])C1([H])[H]

FAWSUKOIROHXAP-NPMXOYFQSA-N

InChI=1S/C25H23ClN2O3/c1-15-13-23(27-21-10-8-20(26)9-11-21)22-14-19(7-12-24(22)28(15)16(2)29)17-3-5-18(6-4-17)25(30)31/h3-12,14-15,23,27H,13H2,1-2H3,(H,30,31)/t15-,23+/m0/s1

4-[(2S,4R)-1-Acetyl-4-[(4-chlorophenyl)amino]-2-methyl-1,2,3,4-tetrahydro-6-quinolinyl]benzoic acid

GSK1324726A, I-BET726, I-BET-726, I-BET 726, GSK1324726A (I-BET726); 4-[(2s,4r)-1-Acetyl-4-[(4-Chlorophenyl)amino]-2-Methyl-1,2,3,4-Tetrahydroquinolin-6-Yl]benzoic Acid; CHEMBL2177300; 4-[(2S,4R)-1-acetyl-4-(4-chloroanilino)-2-methyl-3,4-dihydro-2H-quinolin-6-yl]benzoic acid; GSK-1324726A, GSK 1324726A

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 (5.75 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 (5.75 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；
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。