LDN193189 (DM-3189) is a selective inhibitor of the BMP type I receptor that inhibits ALK2 and ALK3 (IC50=5/30 nM), with less activity against ALK4, ALK5, and ALK7. LDN193189 can be used in studies of progressive ossifying fibrous dysplasia.

ALK3:30 nM (C2C12 cells), ALK2:5 nM (C2C12 cells)

方法：肺动脉平滑肌细胞 PASMCs 用 LDN193189 (2-32000 nM) 预处理 10 min，再用 BMP4 (10 ng/mL) 或 TGF-β (0.5 ng/mL) 处理 30 min，使用 Western Blot 检测靶点蛋白表达水平。
结果：用 LDN193189 处理的 PASMC 的显示对 BMP4 或 TGF-β 信号传导的不同抑制，IC50 分别为 5 nM 和 ≥1 µM。[1]
方法：未分化的 C2C12 细胞用 Smad3/4 反应性 (CAGA)12-荧光素酶或 Smad1/5 反应性 BRE 荧光素酶报道基因构建体连同组成型表达的海肾荧光素酶转染过夜。细胞被血清饥饿，并用 LDN193189 (0.05-5 µM) 和 GDF8、TGF-β 或 BMP2 处理 6 h，检测荧光素酶活性。
结果：LDN193189 从 0.05 µM 抑制 GDF8 诱导的 (CAGA)12-荧光素酶活性。0.05 µM 的 LDN193189 可有效抑制 BMP2 诱导的 BRE 荧光素酶活性。TGF-β诱导的 (CAGA)12-荧光素酶活性被 0.5 µM 的 LDN193189 抑制。[2]

方法：为研究 ALK2 激酶抑制对体内异位钙化的影响，将 LDN193189 (3 mg/kg) 腹腔注射给 caALK2 转基因和野生型小鼠，每 12 h 一次，持续 60 天。
结果：在 P30 时，LDN193189 在大约三分之二的小鼠中预防了异位骨，并减轻了其余小鼠的损伤。而在 P60 时，LDN193189 在三分之一的小鼠中防止了异位骨并减轻了剩余小鼠的损伤。[1]

C2C12 cells were seeded into 96-well plates at 2,000 cells per well in DMEM supplemented with 2% FBS. We treated the wells in quadruplicate with BMP ligands and LDN-193189 or vehicle. We collected the cells after 6 d in culture in 50 μl Tris-buffered saline and 1% Triton X-100. We added the lysates to p-nitro-phenylphosphate reagent in 96-well plates for 1 h and then evaluated alkaline phosphatase activity (absorbance at 405 nm). We measured cell viability and quantity by Cell Titer Aqueous One (absorbance at 490 nm), using replicate wells treated identically to those used for alkaline phosphatase measurements [1].

In the first experiment, SCID mice were implanted with MDA-PCa-118b tumors. After 7 days when tumors reached measurable sizes, mice were injected with LDN-193189 (3 mg/kg) or with vehicle intraperitoneally twice a day. Tumor sizes and body weights were measured weekly. Mice were injected with calcein at three days and one day prior to sacrifice. Blood was collected and tumors were weighed. A portion of the tumors were fixed in formaldehyde for micro-computed tomography, using EVS CT, or further decalcified for bone histomorphometric analysis, using the OsteoMeasure Analysis System, or flash frozen for RNA preparation. Osteocalcin in the mouse serum was determined by ELISA. In the second experiment, PCa-118b tumors were first digested with Accumax, and the isolated cells were plated overnight, digested by Accutase, resuspended in Matrigel in 1:1 ratio, and injected into SCID mice (1 × 10^6 cells/mouse) subcutaneously. Mice were treated with LDN-193189 five days post-injection [3].