BET (IC50 = 32.5-42.5 nM)[1]

Molibresib (I-BET 762) 显示出与 BET 最强的亲和力相互作用。 Molibresib 以高亲和力与 BET 的串联溴结构域结合（解离常数 Kd 为 50.5-61.3 nM）。 Molibresib 高效（半数最大抑制浓度 IC50 为 32.5-42.5 nM）取代已预先结合至 BET 串联溴结构域的四乙酰化 H4 肽[1]。 Molibresib 对 BRD2/3/4 蛋白的 BD1/BD2 结构域表现出高亲和力。 Molibresib 治疗会导致所有三种蛋白质向染色质的募集减少[2]。 Molibresib 抑制 OPM-2 细胞生长，IC50 为 60.15 nM[3]。

使用通过将 OPM-2 细胞注射到 NOD-SCID 小鼠中创建的体内系统异种移植模型，口服检查 Molibresib (I-BET 762) 的抗骨髓瘤活性。 Molibresib 口服剂量高达 10 mg/kg 和 30 mg/kg，每隔一天给药一次，耐受性良好，与载体对照相比，不会显着影响体重。当小鼠服用 Molibresib 后，其血浆中 hLC 的浓度显着降低[3]。

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接下来，我们在通过将OPM-2细胞注射到NOD-SCID小鼠体内产生的体内全身异种移植物模型中测试了口服I-BET762的抗骨髓瘤活性。与赋形剂对照组相比，I-BET762的每日口服剂量高达10mg/kg和每隔一天服用30mg/kg，耐受性良好，对体重没有明显影响（图6B）。我们发现，用I-BET762治疗的小鼠血浆hLC浓度显著降低（图6C）。具体来说，随着疾病的进展，携带骨髓瘤的小鼠血液中的hLC浓度急剧增加。正如预期的那样，在载体治疗的动物中，hLC水平持续升高，直至终止，与进展性骨髓瘤一致。尽管在用I-BET762治疗的小鼠中发现hLC水平升高，但用3个最高剂量治疗的小鼠在所有4个研究时间点的hLC浓度均显著降低（P≤0.001）（图6C）。在载体处理的动物中，人CD38+BM细胞为10%，而在用3种最高剂量处理的动物（P≤.001）中，它们<1%（图6D；补充图4A）。同样，安乐死时椎骨的组织病理学分析显示，I-BET762治疗动物的OPM-2细胞浸润明显降低（补充图4B）。最后，本研究中给药后30分钟的药代动力学取样与BALB/c小鼠静脉或口服3和30mg/kg给药的预期浓度一致（补充方法和补充表2）。 这种显著的抗骨髓瘤活性导致在所有4个I-BET762治疗组的小鼠中观察到显著的（P≤0.002）生存优势，用3个最高剂量的I-BET772治疗的动物没有达到中位生存率（图6E），特别是包括每天给药20至30 mg/kg的小鼠组（在研究期间有一个给药假期）和每隔一天给药30 mg/kg的组（图6E）。这些数据代表了口服活性BET抑制剂在体内显著延缓骨髓瘤进展的第一个例子。[3]

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然后，我们研究了Bim调节的细胞死亡对GEM和I-BET762在异种移植物小鼠中的抗癌功能的必要性。在Panc-1荷瘤小鼠中，GEM和I-BET762降低了肿瘤的重量和体积。与单独使用任何一种药物相比，GEM和I-BET762的组合引发了肿瘤重量和体积的显著下降（图6A）。TUNEL和Ki67检测表明，与联合治疗相比，I-BET762和GEM单独使用时诱导的细胞凋亡较少（图6B和C）。相比之下，与亲本肿瘤相比，Bim-KD肿瘤对联合治疗的生长抑制明显较弱（图6A-C）。此外，为了评估I-BET762及其与GEM的联合对小鼠的毒性作用，我们测量了治疗后ALT、AST和BUN水平。我们发现I-BET762不影响血清样本中的ALT或AST或其GEM诱导的升高。BUN不受上述任何治疗的影响（图6D）。[5]

根据文献报道方法[J.Med.Chem.，54（2011），p.3827]，在BRD2、BRD3和BRD4荧光各向异性（FP）测定中评估了化合物的靶点结合活性。异恶唑喹啉类似物与FP配体竞争，以亚微摩尔IC50与溴结构域结合，如表1所示。通过浸泡在BRD2 N-末端溴结构域的晶体中，获得了化合物1的1.8Å分辨率X射线晶体结构，6揭示了其结合模式（图1A）[1]。

对于体外细胞增殖和凋亡测定，骨髓瘤细胞系通过使用补充有10%胎牛血清、2 mM l-谷氨酰胺、青霉素500 IU/mL和链霉素500μg/mL的RPMI 1640培养基进行培养。细胞放置在96孔U形底板中，终浓度为0.2×106个细胞/毫升，在37°C、5%CO2的加湿培养箱中。对于基质与非基质实验，骨髓瘤细胞被放置在平底96孔板中，MS5细胞融合率>90%，或放置在没有基质的孔中。将化合物（即I-BET151、I-BET762、无活性异构体I-BET768和JQ1）连续稀释到培养基中，并从10mM二甲亚砜（DMSO）储备溶液开始以指定浓度加入培养物中。 在MS5基质细胞存在的情况下，使用上述完全培养基在平底96孔板中培养原发性骨髓瘤细胞，并补充5 ng/mL的白细胞介素-6（IL-6）。[3]

Xenotransplantation experiments[3]
The antimyeloma efficacy of orally administered I-BET762 was tested in a systemic xenograft myeloma model. For this purpose, sublethally irradiated (200 cGy) NOD/SCID mice age 9 to 11 weeks were given 107 OPM-2 myeloma cells via tail vein injection. On day 15 following inoculation, animals were started on oral treatment with I-BET762 at escalating doses or vehicle (1% methylcellulose and 0.2% sodium lauryl sulfate), which was continued up to day 83. Specifically, we treated 1 group of mice with vehicle and 4 groups with different dosing schedules of I-BET762: 3 mg/kg per day; 10 mg/kg per day; 30 mg/kg on alternate days; and 30 to 20 mg/kg per day (ie, 30 mg/kg per day for 14 days, followed by 2 weeks [days 15 to 31] off treatment [drug was withheld due to a decline in body weight until animals had regained weight], followed by 20 mg/kg per day until termination of the experiment [days 43 to 82]). Blood samples (∼70 μL) were removed at 0.5 hours after oral administration of I-BET762 on day 15 (treatment initiation); days 27, 45, and 82 (3, 10, and 20 to 30 mg/kg once per day groups only); and day 83 (30 mg/kg once every other day group only). The blood was centrifuged to obtain 20 μL plasma and stored at −20°C prior to analysis for I-BET762 by using a specific liquid chromatography/mass spectrometry/mass spectrometry assay.
Serum human λ light chain (hLC) was measured with enzyme-linked immunosorbent assay, and the frequency of BM CD38+ human myeloma cells was measured by flow cytometry and by histologic examination (in euthanized animals).

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BALB/c nude mice were subcutaneously injected with pancreatic cancer cells in their right flanks. When the tumor volume reached 150–200 mm3, 24 tumor-bearing mice were randomly divided into 4 groups (I-BET762, GEM, both, and control). The mice in the GEM group were injected with GEM (25 mg/kg/day) through the caudal vein every 3 days for 13 days, and those in the I-BET762 group received an intraperitoneal injection of I-BET762 (30 mg/kg/day) daily for 13 days. The mice in the combination group were treated with both I-BET762 (30 mg/kg/day) and GEM (25 mg/kg/day). In the control group, mice were treated with an equivalent amount of vehicle. Changes in body weight were monitored throughout the experiment. Tumor growth was measured every other day according to the following formula: tumor volume = length × width2/2. Mice were sacrificed on day 22 of the treatment. The tumors were excised and weighed, and the tumor volume was measured. Finally, 0.5 ml of blood was drawn from every mouse by cardiac puncture and was sent to clinical laboratories to evaluate the hepatic and renal activities.[5]

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Dissolved in 20% beta-cyclodextrin, 2% DMSO in 0.9% saline; 30mg/kg; i.v. injection

Mouse model

[1]. Nicodeme E, et al. Suppression of inflammation by a synthetic histone mimic. Nature. 2010 Dec 23;468(7327):1119-23.
[2]. Asangani IA, et al. Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer. Nature. 2014 Jun 12;510(7504):278-82.
[3]. Chaidos A, et al. Potent antimyeloma activity of the novel bromodomain inhibitors I-BET151 and I-BET762. Blood. 2014 Jan 30;123(5):697-705.
[4]. Identification of a novel series of BET family bromodomain inhibitors: Binding mode and profile of I-BET151 (GSK1210151A). Bioorg Med Chem Lett. 2012 Apr 15;22(8):2968-72.
[5]. RETRACTED ARTICLE: The BET inhibitor I-BET762 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcitabine. Sci Rep. 2018; 8: 8102.

2-[(4S)-6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]-N-ethylacetamide is a benzodiazepine.
Molibresib is under investigation in clinical trial NCT01943851 (A Dose Escalation Study to Investigate the Safety, Pharmacokinetics (PK), Pharmacodynamics (PD) and Clinical Activity of GSK525762 in Subjects With Relapsed, Refractory Hematologic Malignancies).
Molibresib is a small molecule inhibitor of the BET (Bromodomain and Extra-Terminal) family of bromodomain-containing proteins with potential antineoplastic activity. Upon administration, molibresib binds to the acetylated lysine recognition motifs on the bromodomain of BET proteins, thereby preventing the interaction between the BET proteins and acetylated histone peptides. This disrupts chromatin remodeling and gene expression. Prevention of the expression of certain growth-promoting genes may lead to an inhibition of tumor cell growth. Characterized by a tandem repeat of bromodomain at the N-terminus, BET proteins, comprising of BRD2, BRD3, BRD4 and BRDT, are transcriptional regulators that play an important role during development and cellular growth.

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Drug Indication
Treatment of breast cancer

C22H22CLN5O2

423.895383358002

423.146

C, 62.34; H, 5.23; Cl, 8.36; N, 16.52; O, 7.55

1260907-17-2

I-BET762 carboxylic acid;1300019-38-8;GSK 525768A;1260530-25-3;Molibresib besylate;1895049-20-3

46943432

Typically exists as Off-white to yellow solids at room temperature

1.4±0.1 g/cm3

1.666

1.99

84.89

1

5

5

30

639

1

ClC1=CC=C(C2=N[C@@H](CC(NCC)=O)C3=NN=C(C)N3C4=CC=C(OC)C=C24)C=C1

AAAQFGUYHFJNHI-SFHVURJKSA-N

InChI=1S/C22H22ClN5O2/c1-4-24-20(29)12-18-22-27-26-13(2)28(22)19-10-9-16(30-3)11-17(19)21(25-18)14-5-7-15(23)8-6-14/h5-11,18H,4,12H2,1-3H3,(H,24,29)/t18-/m0/s1

(S)-2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diazepin-4-yl)-N-ethylacetamide

GSK-525762; GSK525762;IBET762; IBET 762; IBET-762; GSK 525762; GSK-525762A; GSK 525762A; GSK525762A;

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)

DMSO : ~200 mg/mL (~471.81 mM)
1M HCl : 100 mg/mL (~235.90 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.90 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 (5.90 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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: ≥ 2.5 mg/mL (5.90 mM) (饱和度未知) in 5% DMSO + 95% (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。
*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 4 中的溶解度: ≥ 0.5 mg/mL (1.18 mM) (饱和度未知) in 1% DMSO 99% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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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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。