SB-431542 is an inhibitor of ALK5/TGF-β type I Receptor (IC50=94 nM) and is selective. SB 431542 also has inhibitory activity against ALK4 and ALK7 but not other proteins. SB 431542 can be used for induced differentiation of stem cells.

ALK5:94 nM, ALK4:140 nM

方法：人肾细胞 293T、人类结肠癌来源的非致瘤性细胞 FET 和人肺腺癌细胞 A549 用 SB-431542 (2-10 μM) 和 TGF-β1 (12.5 ng/mL) 处理 1 h，使用 Western Blot 方法检测靶点蛋白表达水平。
结果：TGF-β1 诱导了 Smad2/3 和 Smad4 之间的复合物形成。SB-431542 以剂量依赖的方式，通过阻断 TGF-β 诱导的磷酸化和Smad2/3的激活，阻断复合物形成。[1]
方法：人胶质母细胞瘤细胞 D54MG 用 SB-431542 (1-10 μM) 处理 24 h，使用 Transwell 方法检测细胞迁移情况。
结果： SB-431542抑制 TGF-βRI 信号传导以浓度依赖的方式阻断 D54MG 细胞的细胞迁移。[2]
方法：人多能干细胞 hESCs 用含 SB-431542 (10 nM) 和 Noggin (500 ng/mL) 的 KSR 培养基培养 11 天，使用 Immunofluorescence 方法检测细胞分化情况。
结果：神经诱导通过 PAX6 的表达来监测，PAX6 是神经直肠真皮分化的早期标志物。与单独使用 Noggin 或 SB-431542 时不到 10% 的 PAX6+ 细胞相比，用 Noggin 和 SB-431542 联合处理显著提高了神经诱导的效率，使其超过总细胞的 80%。[3]

方法：为检测体内抗肿瘤活性，将 SB-431542 (10 mg/kg in 20% DMSO/80% corn oil) 腹腔注射给携带小鼠乳腺癌肿瘤 4T1 的 Balb/c 小鼠，每周三次，持续四周。
结果：SB-431542 可显著抑制 4T1 乳腺肿瘤的肺转移。[4]
方法：为研究肌腱损伤的治疗方法，将 SB-431542 (10 mg/kg) 腹腔注射给大面积肩袖撕裂的 C57B/6J 小鼠模型，每天一次，持续二或六周。
结果：SB-431542 抑制 TGF-β1 信号传导可减少纤维化、脂肪浸润和肌肉重量减轻。SB-431542 治疗通过促进纤维/脂肪生成祖细胞 (FAP) 的凋亡，减少了损伤肌肉中 FAP 的数量，FAP 是肩袖肌纤维化和脂肪浸润的重要细胞来源。[5]

Kinase assays were performed with 65 nM GSTALK5 and 184 nM GST-Smad3 in 50 mM HEPES, 5 mM MgCl2, 1 mM CaCl2, 1 mM dithiothreitol, and 3 M ATP. Reactions were incubated with 0.5 μCi of [33P]γATP for 3 h at 30°C. Phosphorylated protein was captured on P-81 paper, washed with 0.5% phosphoric acid, and counted by liquid scintillation. Alternatively, Smad3 or Smad1 protein was also coated onto FlashPlate Sterile Basic Microplates. Kinase assays were then performed in FlashPlates with same assay conditions using either the kinase domain of ALK5 with Smad3 as a substrate or the kinase domain of ALK6 (BMP receptor) with Smad1 as substrate. Plates were washed three times with phosphate buffer and counted by TopCount [2].

A498 cells were seeded at 5,000 to 10,000 cells/well in 96-well plates. The cells were serum-deprived for 24 h and then treated with compounds for 48 h to assess the cellular toxicity. Cell viability is determined by incubating cells for 4 h with XTT labeling and electron coupling reagent. Live cells with active mitochondria produce an orange-colored product, formazan, which is detected using a plate reader at between A450 nm and A500 nm with a reference wavelength greater than 600 nm. The absorbance values correlate with the number of viable cells [2].

BALB/c mice received intraperitoneal (i.p.) injections of colon-26 tumor cells. Three days after tumor cell inoculation, SB-431542 (1 μM solution, 100 μl/animal) or vehicle alone was directly injected into the peritoneal cavity. CTL activities were measured by a standard 4 h 51Cr release assay after culturing spleen cells with γ-irradiated tumor cells for five days in the absence of added growth factors. In vitro experiments, cell lysate of HLA-A*2402 positive gastric cancer cell line, OCUM-8, was incubated with human DC cultures for 4 h. After washing extensively, PBMCs obtained from the same volunteer as DCs were incubated for 7 days and measured CTL activity by 51Cr release assay. NK activity was tested using 51Cr release assay against K562 [4].