Galunisertib (LY2157299) is a TGF-β receptor type I (TGF-βRI) inhibitor (IC50=56 nM) that is selective. Galunisertib has antitumor activity and can be used in combination with PD-1 inhibitors.

TβRI:56 nM (cell free)

方法: 小鼠胚胎成纤维细胞 NIH3T3 用 Berzosertib (0.0001-10 µM) 处理 2 h，随后用 TGFβ1 孵育过夜，使用 3H-thymidine proliferation assay 检测细胞增殖。
结果: Berzosertib 抑制 TGFβ1 诱导的增殖，IC50 为 0.396 µM。[1]
方法: 七种 HCC 细胞系 JHH6、SK-HEP1、SK-Suni、SK-Sora、HepG2、Hep3B、HuH7 用 Galunisertib (1-10 µM) 和 TGF-β (5 ng/mL) 处理 5-24 h，使用 Western Blot 检测靶点蛋白表达水平。
结果: 添加 Galunisertib 以剂量和时间依赖的方式降低了所有细胞系中 p-Smad2 的表达水平，与 TGF-β 诱导无关。[2]

方法: 为检测体内抗肿瘤活性，用 Galunisertib (75 mg/kg，10% beta-cyclodextrin-HCl) 灌胃给药携带 MX1、Calu6 或 4T1 肿瘤的 athymic nu/nu 小鼠，每天两次，持续 20-40 天。
结果: Galunisterib 单药治疗都导致了明显的肿瘤生长延迟。对于MX1，Galunisertib 治疗导致肿瘤生长延迟 10.3±4.3天。对于 Calu6，Galunisertib 治疗导致肿瘤生长推迟 8.3±2.6天。对于 4T1，Galunisertib 治疗导致肿瘤生长延迟 13±2.4天。[1]

Briefly, the assay was first done at 30°C for 4 h in a 96-well plate containing 2 ng/mL TGF-bR KD, 100 mM Hepes pH 7.5, 4 mM MgCl2, 0.2 mM MnCl2, 0.4 mM sodium orthovanadate, 2 mM DL-dithiothreitol, and 10mM ATP. After incubation, 50mL of Kinase-Glo plus reagent was added and further incubated at 25°C for 30 min. Subsequently, a 100mL aliquot of each reaction mixture was transferred to a black mictotiter plate and the luminescence was measured by a vector counter. The inhibitory activity IC50 was tested in duplicate for each sample [1].

Cell survival was determined using the MTT assay. The conversion of yellow water-soluble tetrazolium MTT into purple insoluble formazan is catalyzed by mitochondrial dehydrogenases and used to estimate the number of viable cells. In brief, cells were seeded in 96-well tissue culture plates at a density of 2 × 103 cells/well. After drug exposure, cells were incubated with 0.4 mg/mL MTT for 4 hours at 37°C. After incubation, the supernatant was discarded, insoluble formazan precipitates were dissolved in 0.1 mL of DMSO, and the absorbance was measured at 560 nm by use of a microplate reader. Wells with untreated cells or with drug-containing medium without cells were used as positive and negative controls respectively. For proliferation assay, MTT assay was done daily to determine the number of viable cells in untreated control and galunisertib-treated group [2].

Transgenic mice expressing a fusion gene (Alb/TGF) consisting of modified porcine TGF-β1 cDNA under the control of the regulatory elements of the mouse albumin gene (26) were used under animal institute approved protocol. Mice were given LY-2157299 at a dose of 100mg/kg/day in NaCMC/SLS/PVP/antifoam solution by gastric lavage using a curved 14 G needle. Blood counts were analyzed by the Advia machine. Mice femurs were flushed and bone marrows cells were used for clonogenic assays [3].