FL-411 (BRD4-IN-1) is a potent and selective BRD4 inhibitor with an IC50 of 0.43±0.09 μM for BRD4.

BRD4:0.43 μM

FL-411是一种选择性的BRD4抑制剂。通过TR-FRET测量FL-411对BRD2(1)、BRD4(1)和BRD4(2)的第一和第二bromodomains的结合亲和力，IC50分别为24.60±0.70 μM、0.47±0.02 μM和0.93±0.05 μM。FL-411显示出良好的BRD4(1)抑制活性（IC50=0.43±0.09 μM）、抗增殖活性（MCF-7, IC50=1.62±0.06 μM；MDA-MB-231, IC50=3.27±0.14 μM）和自噬活性（MCF-7细胞中42.29%），同时对MCF10A细胞的毒性低。FL-411通过阻断BRD4-AMPK相互作用，从而激活AMPK-mTOR-ULK1调节的自噬途径，在乳腺癌细胞中诱导ATG5依赖的自噬相关细胞死亡（ACD）。

为了评估FL-411在体内的抗肿瘤活性，本研究采用了两种乳腺肿瘤异种移植模型，即MCF-7和MDA-MB-231细胞系模型。通过三种不同剂量的FL-411（25 mg/kg、50 mg/kg、100 mg/kg）进行体内研究。结果显示，各模型中FL-411均以剂量依赖性方式显著抑制肿瘤生长，MCF-7和MDA-MB-231细胞模型的肿瘤生长抑制比率分别为80%和76%。所有剂量组的肿瘤重量均观察到显著减轻（p<0.001）。FL-411对所有治疗组体重无明显影响。为探究FL-411介导的体内肿瘤生长抑制是否与细胞增殖减少和自噬相关细胞死亡增加有关，对照组和FL-411处理组小鼠的肿瘤组织进行了Ki-67和LC3的免疫组化分析。FL-411处理显著减少了Ki-67阳性细胞的数量（p<0.001），同时增加了自噬水平，后者通过LC3表达增加确定（p<0.001）。

The MCF-7 and MDA-MB-231 cells are dispensed in 96-well flat bottom microtiter plates at a density of 5×104 cells/mL. After 24 h incubation, MCF-7 or MDA-MB-231 cells are treated with 1.5 and 3 μM FL-411, respectively. 3-MA (1 mM) is added 1 h before treated with FL-411. After treatment, cell viability is measured by the MTT assay. 5 mg/mL MTT is added to each well. After 4 h incubation, the medium is removed and 150 μL of DMSO is added to each well to dissolve the crystal formazan dye. Absorbance is measured at 570 nm on an enzyme-linked immunosorbent assay reader

Mice52 female nude mice (BALB/c, 6-8 weeks, 20-22 g) are injected subcutaneously with MCF-7 cells or MDA-MB-231 cells (5×106 cells/mouse), respectively. When the tumors reach 100 mm3 in volume, the mice are divided into four groups for each cell line. Three groups are treated with different doses of FL-411 (low dose, 25 mg/kg; median dose, 50 mg/kg; high dose, 100 mg/kg) once a day by intragastric administration for 24 or 27 days, whereas the control group is treated with vehicle control. During the treatment, the tumor volumes and body weight are measured every 3 days until the end of the study. At the end of treatment, all mice are sacrificed. The tumor tissues are harvested, weighed, and photographed. Then, the tumor tissues are frozen in liquid nitrogen or fixed in formalin immediately