ACVR1 (IC50 = 1.3 nM); ALK1 (IC50 = 2.4 nM); BMPR1A (IC50 = 85.8 nM); ALK4 (IC50 = 2133 nM); ALK5 (IC50 = 9276 nM)

In BMPR2−/− cells, BMP7-induced phosphorylation of SMAD1/5/8 is blocked by LDN-212854 (0-3.815 μM)[1]. In Huh7 and MT cells, LDN-212854 (2.5 μM, 5 days) suppresses cell proliferation[2]. Huh7 and MT cells' expression of ID1 and EpCAM is suppressed by LDN-212854 (0.5 μM, 48 h)[2].

In an inducible transgenic mutant ALK2 mouse model of fibrodysplasia ossificans progressiva, potently suppresses heterotopic ossification when administered intraperitoneally twice daily for four weeks at a dose of 6 mg/kg [1]. By suppressing ID1, LDN-212854 (intraperitoneal injection, 6 mg/kg, twice daily for 4 weeks) inhibits the growth of HCC tumors in HCC xenograft models[2].

Kinase Assay[1]
Purified recombinant ALK1-5 and other kinase proteins, ATP, ATP [γ-32P], and dephosphorylated casein at final concentrations of 2.5nM, 6μM, 0.05 μCi μL−1, and 0.5 mg mL−1 respectively were aliquoted in kinase buffer containing 0.2% bovine serum albumin supplemented with 10mM MnCL2 into 96-well plates, in combination with inhibitor compounds diluted at varying concentrations in kinase buffer (0.01nM to 10 μM) in triplicate. Positive control samples lacking inhibitor compounds, and negative controls lacking recombinant kinase were also measured in triplicate. The mixture was reacted at RT for 45 min., quenched with a final concentration of 2% phosphoric acid. The reaction mixture was transferred to 96-well P81 phosphocellulose filter plates and bound for 5 min. The plates were washed twenty-times with 150 μL of 1% phosphoric acid solution per well by vacuum manifold. Plates were dried at RT for 1 h, sealed, and assayed with Microscint 20 scintillation fluid using a Spectramax L luminometer using the photon counting setting with an integration time of one second per well. Data was normalized to positive controls at 100% enzyme activity with negative controls being subtracted as background. GraphPad was used for graphing and regression analysis by sigmoidal dose-response with variable Hill coefficient.

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Luciferase Reporter Assay[1]
C2C12 Bre-Luc and 293T CAGA-Luc cells were seeded at 20,000 cells in DMEM supplemented with 2% FBS per well in tissue culture treated 96-well plates. The cells were incubated for 1 h (37°C and 10% CO2) and allowed to settle and attach. Compounds of interest or DMSO were diluted in DMEM and added at final compound concentrations of 1 nM to 10 μM. Cells were then incubated for 30 min. Adenovirus expressing constitutively active BMP and TGF-β type I receptors (Ad.caALK1-5) were added to achieve a multiplicity of infection (MOI) of 100. Plates were incubated overnight at 37°C. Cell viability was assayed with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay per the manufacturer’s instructions. Media was discarded, and firefly luciferase activity was measured according to manufacturer’s protocol. Light output was measured using a Spectramax L luminometer with an integration time of one second per well. Data was normalized to 100% of incremental BRE-Luc activity due to adenoviruses specifying caALK1, 2, or 3, or the incremental CAGA-Luc activity due to adenoviruses specifying caALK4 or 5. Graphing and regression analysis by sigmoidal dose-response with variable Hill coefficient was performed using GraphPad Prism software.

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BMP Induced ALP Activity[1]
C2C12 myofibroblasts cells were seeded in 96-well plates at 2,000 cells per well in DMEM supplemented with 2% FBS as previously described. Compounds diluted in DMEM and were added at final concentrations ranging from 1nM to 10 μM, in quadruplicate. BMP4 and BMP6 ligands diluted in DMEM were added to final concentrations of 20 ng mL−1. Positive controls were generated by omitting compounds and negative controls were generated by omitting both compounds and ligands. Cells were incubated for 6 days at 37°C and 5% CO2 and subsequently harvested using 1% Triton X-100. Extracts from each well were incubated at RT for 30 min with alkaline phosphatase (ALP) yellow (pNPP) liquid substrate for ELISA and ALP activity was measured by absorbance at 405 nM per the manufacturer’s instructions. Absorbance data was analyzed with positive controls as 100% ALP activity and negative controls being subtracted as background. Graphing and regression analysis by sigmoidal dose-response with variable Hill coefficient was performed using GraphPad Prism.

Western Blot Analysis[1]
Cell Types: BMPR2-deficient pulmonary vascular smooth muscle cells
Tested Concentrations: 0, 1, 3, 6, 16, 39, 98, 244, 610, 1530, 3815 nM
Incubation Duration:
Experimental Results: Inhibited the phosphorylation of SMAD1/5/8 induced by BMP7 with an IC50 value of 37 nM.

Animal/Disease Models: Murine inducible transgenic ALK2Q207D model of heterotopic ossification[1]
Doses: 6 mg/kg
Route of Administration: intraperitoneal (ip)injection , twice (two times) daily for 4 weeks
Experimental Results: Prevented the formation of heterotopic bone and preserved limb range of motion with minimal or no impairment in the majority of mice.

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Animal/Disease Models: HCC xenografts (Huh7 or MT cell)[1]
Doses: 6 mg/kg
Route of Administration: intraperitoneal (ip)injection, twice (two times) daily for 10-14 days.
Experimental Results: Inhibited tumor growth and demonstrated less spheroid/colony formation ability than PBS-treated tumor cells.

[1]. Development of an ALK2-biased BMP type I receptor kinase inhibitor. ACS Chem Biol. 2013;8(6):1291-302.

[2]. BMP9-ID1 signaling promotes EpCAM-positive cancer stem cell properties in hepatocellular carcinoma. Mol Oncol. 2021 Aug;15(8):2203-2218.

LDN-212854 is a member of the class of quinolines that is quinoline substituted by a 6-[4-(piperazin-1-yl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl group at position 5. It is a potent ALK inhibitor (IC50 values of 2.4, 1.3, 85.8, 2,133 and 9,276 nM for ALK1, ALK2, ALK3, ALK4 and ALK5, respectively). It has a role as an antineoplastic agent, an angiogenesis inhibitor and an EC 2.7.11.30 (receptor protein serine/threonine kinase) inhibitor. It is a pyrazolopyrimidine, a member of quinolines and a N-arylpiperazine.

C25H22N6

406.48

406.19

C, 73.87; H, 5.46; N, 20.67

1432597-26-6

60182388

Light yellow to yellow solid powder

1.3±0.1 g/cm3

1.740

1.71

1

5

3

31

587

0

BBDGBGOVJPEFBT-UHFFFAOYSA-N

InChI=1S/C25H22N6/c1-3-21(22-4-2-10-27-24(22)5-1)23-16-29-31-17-19(15-28-25(23)31)18-6-8-20(9-7-18)30-13-11-26-12-14-30/h1-10,15-17,26H,11-14H2

5-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.5 mg/mL (6.15 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.15 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)