DMH-1 is a BMP inhibitor that inhibits ALK1, ALK2, ALK3 and ALK6 (IC50=27/107.9/<5/47.6 nM) selectively. DMH-1 promotes pluripotent stem cell differentiation.

ALK2:107.9 nM

方法: HEK293 细胞用 DMH-1 (1-20 µM) 处理 30 min，然后用 BMP4 (50 ng/mL) 或 Activin A (40 ng/mL) 孵育30 min，使用 Western Blot 检测靶点蛋白表达水平。
结果: DMH-1 阻断了 BMP4 诱导的 HEK293 细胞中 Smad 1/5/8 磷酸化。相反，在 HEK293 细胞中，DMH-1 对 BMP4 诱导的 p38 MAPK 磷酸化和 Activin A 诱导的 Smad2 磷酸化没有影响。[1]
方法: hiPSC 细胞用 SB43154230 与 Noggin (500 ng/mL) 或 DMH-1 (0.5 µM) 处理 7 天，使用 qRT-PCR 检测神经前体标记物的表达。
结果: 所检查的八种细胞系中的六种中，DMH-1 和 Noggin 的神经化在第 7 天产生了相当百分比的 PAX6阳性细胞。在两种细胞系 SM5 和 TSC-12B4 中，用 Noggin 神经化的培养物中 PAX6 阳性细胞的数量更高。[2]

方法: 为检测体内抗肿瘤活性，将 DMH-1 (5 mg/kg，12.5% 2-hydroxypropyl-β-cyclodextrin) 腹腔注射给携带 A549 异种移植物的 SCID 小鼠，每两天一次，持续四周。
结果: DMH-1 治疗显著降低了人肿瘤异种移植物模型中的肿瘤生长。在 4周治疗结束时，与载体对照组相比，DMH-1 治疗导致肿瘤体积显著减少约 50%。[3]

Kinase assay: All kinase assays are conducted by Reaction Biology Corp. In brief, compounds are tested at 10 concentrations by 3-fold serial dilutions starting at 30 μM, using nonspecific kinase inhibitor staurosporine as control. In vitro kinase reactions are carried out in the presence of 10 μM (33P)γATP. Five kinases tested are the human BMP type-I receptor activin receptor-like kinase 2 (ALK-2/ACVR1), the human TGFβ type-I receptor activin receptor-like kinase 5 (Alk5/TGFβR1), the human VEGF type-II receptor (KDR/Flk-1/VEGFR2), the human AMP-activated protein kinase (AMPK/A1/B1/G1) and the human platelet-derived growth factor receptor-β (PDGFRβ).

About 10,000 A549 cells per well are seeded in 96-well plates and incubated for overnight. The culture medium is then changed to fresh medium containing DMSO or DMH1 at various concentrations. The cells are then incubated for 48 hours and 96 hours before treatment termination by replacing the medium with 100 μL of 10% trichloroacetic acid in 1× PBS, followed by incubation at 4°C for at least 1 hour. Subsequently, the plates are washed with water and air dried. The plates are stained with 50 μL 0.4% sulphorhodamine assay in 1% acetic acid for 30 minutes at room temperature. Unbound dye is washed off with 1% acetic acid. After air drying and solubilization of the protein-bound dye in 10 mM Tris solution, absorbance is read in a microplate reader at 565 nm.(Only for Reference)