BRD4 (1/2) (IC50= 77/33 nM)

(+)-JQ-1 是 Bromodomain BET 家族的一种强效、高选择性和 Kac 竞争性抑制剂。 (+)-JQ-1（100 nM，48 小时）可促进鳞状细胞发育，细胞纺锤体形成、变平和角蛋白表达增强即可证明这一点。通过定量免疫组织化学评估，与 (-)-JQ1 (250 nM) 和载体对照相比，(+)-JQ-1 (250 nM) 刺激处理的 NMC 797 细胞中角蛋白的快速表达。 (+)-JQ-1（相对于 (-)-JQ1 (250 nM)）在处理的 NMC 797 细胞中引起时间依赖性强 (3+) 角蛋白染色 [1]。添加 (+)-JQ-1 后几乎立即观察到自噬基因的去抑制[2]。 (+)-JQ-1 是 BET 家族共激活蛋白 BRD4 的强效噻吩二氮卓抑制剂 (Kd=90 nM)，该蛋白通过 MYC 癌基因的转录调节参与癌症的发展。 (+)-JQ-1 的剂量范围实验表明可有效抑制 H4Kac4 结合，小鼠 BRDT (1) 的 IC50 值为 10 nM，人 BRDT (1) 的 IC50 值为 11 nM [3]。

将与已形成肿瘤相匹配的小鼠队列随机每天接受载体或 (+)-JQ1 (50 mg/kg) 腹膜内注射。 FDG-PET 成像用于在治疗后四天和随机分组之前评估小鼠。当施用 (+)-JQ1 时，FDG 摄取显着减少。肿瘤体积的评估表明，JQ1 治疗抑制了肿瘤生长。 CD1 小鼠用于口服和静脉给药后 (+)-JQ1 的药代动力学研究。静脉注射（5 mg/kg）后（+）-JQ1 平均血浆浓度的时间曲线。静脉注射(+)-JQ1的半衰期(T1/2)约为1小时，其药代动力学特征显示出良好的药物暴露(AUC=2090 hr*ng/mL)。口服剂量（10 mg/kg）后，创建了 (+)-JQ1 的平均血浆浓度-时间曲线。口服(+)-JQ1药代动力学参数显示出良好的药物暴露（AUC=2090 hr*ng/mL）、血浆峰浓度（Cmax=1180 ng/mL）和口服生物利用度（F=49%）[1]。

乙酰组蛋白结合测定。[1]
如前所述51进行分析，对制造商的方案（PerkinElmer，USA）稍作修改。将所有试剂在补充有0.05%CHAPS的50mM HEPES、100mM NaCl、0.1%BSA、pH 7.4中稀释，并在加入平板之前使其平衡至室温。在150–0μM的范围内制备配体的24点1:2系列稀释液，并将4μl转移到低体积384孔板中，然后加入4μl His标记蛋白（BRD4（1），250 nM，BRD4（2）和CREBBP，2000 nM）。将板密封并在室温下孵育30分钟，然后将4μl等摩尔浓度的生物素化肽添加到蛋白质中[BRD4（1）和BRD4（2）的肽：H4K5acK8acK12acK16ac，HSGRGK（Ac）GGK（Ac。在弱光条件下加入4μl链霉亲和素包被的供体珠（25μg/ml）和4μl镍螯合受体珠（25µg/ml）之前，将板密封并再孵育30分钟。将板箔密封以避光，在室温下孵育60分钟，并使用AlphaScreen 680激发/570发射滤光片组在PHERAstar FS板读取器上读取。对照相应的DMSO对照标准化后，在Prism 5（GraphPad Software，USA）中计算IC50值，并作为20μl反应体积中化合物的最终浓度给出。

细胞增殖测定。[1]
将细胞接种到白色384孔微量滴定板（Nunc）中，在总体积为50μl的培养基中，每孔500个细胞。797、TT和TE10细胞生长在含有1%青霉素/链霉素和10%FBS的DMEM中。Per403细胞生长在含1%青霉素/链霉菌和20%FBS的DMEM中。患者来源的NMC 11060细胞生长在具有10%FBS和1%青霉素/链球菌素的RPMI中。通过机器人销钉转移（PerkinElmer JANUS配备有V＆P Scientific 100 nl销钉工具）将化合物递送至微量滴定分析板。在37ºC下孵育48小时后，裂解细胞，并使用商业增殖测定法评估孔的总ATP含量。根据剂量分析重复测量，并通过逻辑回归计算IC50的估计值。[1]

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细胞生长测定。[1]
将细胞以每孔1.5×104个细胞的浓度接种在6孔组织培养皿中。细胞在2ml DMEM（797）或RPMI（11060）中生长，所述DMEM或RPMI含有10%胎牛血清、1%青霉素/链霉素和250nM（+）-JQ1或等效体积的DMSO（0.025%）。每天更换每口井中一半的介质。在第0天、第1天、第3天、第7天和第10天，将分配给每个时间点的细胞培养皿进行胰蛋白酶消化，以1:1的比例与0.4%台盼蓝混合，并使用Countess自动细胞计数器计数。

Xenograft Efficacy Studies.[1]
NMC 797 xenografts were established by injecting NMC 797 cells (107) in 30 % Matrigel (BD Biosciences) into the flank of 6 week-old female NCr nude mice. Twelve days after injection, mice with measureable tumors were divided into cohorts to be treated with JQ1 at 50 mg kg-1 IP or vehicle (5 % DMSO in 5 % dextrose). For FDG-PET studies, mice with established tumors measuring approximately 1 cm in the largest linear dimension underwent baseline CT/PET imaging 1 h after injection of 250 μCi of FDG (Pre-treatment). Mice were then treated with four daily doses of 50 mg kg-1 of racemic JQ1 by intraperitoneal injection. Two hours after the fourth dose of JQ1 or vehicle, mice underwent repeat FDG-PET imaging (Post-treatment). The integrated signal encompassed within the entire tumor volume is expressed as the percent of injected dose per gram (% ID/gm). Tumors were fixed in 10 % buffered formalin for histopathological analysis. For tumor caliper studies, the average size of tumors in the JQ1 treatment group (n = 8) and vehicle group (n = 7) were similar (63.8 ± 17.1 and 73.6 ± 14.4 mm3 respectively) at the start of treatment. Animals were followed daily for clinical symptoms. Tumor measurements were assessed by caliper measurements, and volume calculated using the formula Vol = 0.5 x L x W2. After 2 weeks of treatment, all mice were humanely euthanized, and tumors were fixed in 10 % formalin for histopathological examination. Statistical significance of tumor volumes was calculated by two-sided Students t-test.[1]

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Primary NMC Xenograft Studies.[1]
A primary xenograft model of NMC was established by injecting NCr nude mice with primary cells (107 cells in 100 μl of 30 % Matrigel in 70 % PBS) collected from malignant pleural fluid obtained with IRB approval and informed consent from a patient at the Dana-Farber Cancer Institute and Brigham & Women’s Hospital. As above, four mice with established tumors measuring approximately 1 cm in the largest linear dimension underwent baseline CT/PET imaging 1 h after injection of 250 μCi of FDG (Pre-treatment). Mice were then treated with four daily doses of 50 mg kg-1 of (+)-JQ1 by intraperitoneal injection. Animals were followed daily for clinical symptoms. Two hours after the fourth dose of (+)-JQ1 or vehicle, mice underwent repeat FDG-PET imaging (Post-treatment). The integrated signal encompassed within the entire tumor volume is expressed as the percent of injected dose per gram (%ID/gm). Tumors were fixed in 10 % buffered formalin for histopathological analysis. At the conclusion of the study, all mice were humanely euthanized, and tumors were fixed in 10 % formalin for histopathological examination. Survival (30 days) studies performed with NMC Per403 and 11060 xenografts were initiated as above. For these studies, (+)-JQ1 was administered at a dose of 50 mg kg-1 by daily intraperitoneal injection. The average size of tumors in the (+)-JQ1 treatment group (n = 10) and vehicle group (n = 10) were similar at the start of treatment. Animals were followed daily for clinical symptoms. Tumor measurements were assessed by caliper measurements, and volume calculated using the formula Vol = 0.5xLxW2. Statistical significance of tumor volumes was calculated by two-sided Students t-test. Comparative survival analysis was performed using the Log-rank (Mantel-Cox) Test, and data were presented as a Kaplain-Meier plot annotated with a measure of statistical significance (pvalue). All animal studies were approved by the IACUC of the DFCI.

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Pharmacokinetic Studies in Mice.[1]
Male CD1 mice (24 – 29 gm) were treated with a single dose of (+)-JQ1 at 5 mg kg-1 for intravenous tail vein injection studies and 10 mg kg-1 for oral gavage studies. Approximately 150 μl of blood were taken from animals by retro-orbital puncture under anesthesia with Isoflurane into EDTA tubes at pre-specified time intervals: 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 5, 8 and 24 hours. Three animals were analyzed per time point. Blood samples were put on ice and centrifuged to obtain plasma samples (2000 x.g, 5 min under 4 °C) within 15 minutes post-sampling. Plasma samples were stored at approximately -70 °C until analysis was performed. Mice were provided free access to food and water throughout the study. Compound was formulated for intravenous injection in 10 % DMSO and 10 % HP-β-CD. Pharmcokinetic studies and pharmacologic assay development was performed at ChemPartner (Shanghai, CHINA). Data were analyzed by J.E.B. using Microsoft Excel and GraphPad Prism 5.02.

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In vivo formulations used (reported):

1. Dissolved in 5% dextrose; 50 mg/kg; i.p. injection; Nature. 2010 Dec 23;468(7327):1067-73; 2. Dissolved in 10% DMSO and 90% of a 10% 2-hydroxypropyl-β-cyclodextrin solution; Leukemia. 2017 Oct;31(10):2037-2047.; 3. Dissolved in 1% DMSO+5% Glucose+ddH2O; Cell. 2018 Sep 20;175(1):186-199.e19.; 4. Dissolved in 20% hydroxypropyl-β-cyclodextrin, 5% DMSO, 0.2% Tween-80 in saline; Mol Cancer Ther. 2016 Jun;15(6):1217-26.; 5. Dissolved in 1:1 propylene glycol:water; J Biol Chem. 2016 Nov 4;291(45):23756-23768.; 6. Dissolved in 5% DMSO in 10% 2-hydroxypropyl-β-cyclodextrin solution; Cancer Lett. 2017 Aug 28;402:100-109.

[1]. Selective inhibition of BET bromodomains. Nature. 2010 Dec 23;468(7327):1067-73.

[2]. Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and LysosomalFunction. Mol Cell. 2017 May 18;66(4):517-532.e9.

[3]. Small-molecule inhibition of BRDT for male contraception. Cell. 2012 Aug 17;150(4):673-84.

JQ1 is a member of the class of thienotriazolodiazepines that is the tert-butyl ester of [(6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl]acetic acid. An inhibitor of bromodomain-containing protein 4 that exhibits anti-cancer and cardioprotective properties. It has a role as a bromodomain-containing protein 4 inhibitor, a cardioprotective agent, an antineoplastic agent, an anti-inflammatory agent, an angiogenesis inhibitor, an apoptosis inducer and a ferroptosis inducer. It is a thienotriazolodiazepine, an organochlorine compound, a carboxylic ester and a tert-butyl ester.

C23H25CLN4O2S

456.99

456.138

C, 60.45; H, 5.51; Cl, 7.76; N, 12.26; O, 7.00; S, 7.02

1268524-70-4

(R)-(-)-JQ1 Enantiomer;1268524-71-5;JQ-1 (carboxylic acid);202592-23-2

46907787

White to yellow solid

1.3±0.1 g/cm3

610.4±65.0 °C at 760 mmHg

322.9±34.3 °C

0.0±1.7 mmHg at 25°C

1.657

4.49

97.61

0

6

5

31

706

1

ClC1C([H])=C([H])C(=C([H])C=1[H])C1C2C(C([H])([H])[H])=C(C([H])([H])[H])SC=2N2C(C([H])([H])[H])=NN=C2[C@]([H])(C([H])([H])C(=O)OC(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])N=1

DNVXATUJJDPFDM-KRWDZBQOSA-N

InChI=1S/C23H25ClN4O2S/c1-12-13(2)31-22-19(12)20(15-7-9-16(24)10-8-15)25-17(11-18(29)30-23(4,5)6)21-27-26-14(3)28(21)22/h7-10,17H,11H2,1-6H3/t17-/m0/s1

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 (5.47 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 (5.47 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。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.47 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。

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配方 4 中的溶解度: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O:5mg/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。