AMPK (Ki = 109 nM); ACVR1; BMPR1A; ALK6; Autophagy

Dorsomorphin inhibits ACC inactivation by either AICAR or metformin, and also attenuates AICAR and metformin’s effect to increase fatty acid oxidation or suppress lipogenic genes in hepatocytes.[1] In HT-29 cells, Dorsomorphin almost entirely prevents autophagic proteolysis by inhibiting AMPK activity.[2] Additionally, Dorsomorphin blocks the BMP-mediated phosphorylation of SMAD1/5/8, target gene transcription, and osteogenic differentiation by specifically inhibiting the BMP type I receptors ALK2, ALK3, and ALK6.[3]

Dorsomorphin (10 mg/kg) reduces basal levels of hepcidin expression and increases serum iron concentrations in adult mice. [3] Dorsomorphin (0.2 mg/kg, i.v.) significantly reduces VCAM-1 and ICAM-1 expression in the thoracic aorta of LPS-treated rats. [4]

Liver AMPK is partially purified from male SD rats to the blue-Sepharose step. In a buffer of 40 mM HEPES, pH 7.0, 80 mM NaCl, 0.8 mM EDTA, 5 mM MgCl2, 0.025% BSA, and 0.8 mM DTT, 100 μl AMP, 100 μl ATP (0.5 μCi 33P-ATP per reaction), and 50 μM SAMS are present in the 100-l reaction mixture. As soon as the enzyme is added, the reaction starts. After 30-minute incubation at 30°C, the reaction is stopped by addition of 80 μl 1% H3PO4. Aliquots (100 μl) are transferred to 96-well MultiScreen plates. The plate is washed three times with 1% H3PO4 followed by detection in a Top-count. The in vitro AMPK inhibition data obtained with compound C — (6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyyrazolo[1,5-a] pyrimidine — are fit to the following equation for competitive inhibition by nonlinear regression using a least-squares Marquardt algorithm in a computer program written by N. Thornberry of Merck Research Laboratories: Vi/Vo = (Km + S)/[S + Km × (1 + I/Ki)], where Vi is the inhibited velocity, Vo is the initial velocity, S is the substrate (ATP) concentration, Km is the Michaelis constant for ATP, I is the inhibitor (compound C) concentration, and Ki is the dissociation constant for compound C.

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AMPK Kinase Assay (ELISA Assay)[5]
HT1080 cells were seeded in 24-well plates (2×104 cells per well) and treated with compound C (Dorsomorphin) in the presence or absence of glucose or 10 mM 2DG for 2 h. HT1080 cells that overexpressed the wild-type and dominant negative AMPKα1 were prepared by transfecting plasmid DNA (pAMPKα1-wt, pAMPKα1-D168A and pcFlag as a control) in 6-well plates, seeding in 24-well plate and treating with UPR inhibitors. Cells were lysed with cell lysis buffer (20 mM Tris-HCl, pH 7.5, 250 mM NaCl, 10% glycerol, 0.5% NP-40, 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, 1 µg/mL pepstatin, 0.5 µg/mL leupeptin, 5 mM NaF, 2 mM Na3Vo4, 2 mM β-glycerophosphate, 1 mM DTT). Relative AMPK kinase activity (mean ± SD of duplicate determinations) to control sample (vehicle or pcFlag under normal growth conditions) was determined using the CycLex AMPK kinase assay kit according to the manufacturer’s instructions.[5]

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Alkaline phosphatase activity[3]
C2C12 cells were seeded into 96-well plates at 2,000 cells per well in DMEM supplemented with 2% FBS. Wells were treated in quadruplicate with BMP ligands and Dorsomorphin or vehicle. Cells were harvested after 5 d in culture with 50 µl Tris buffered saline, 1% Triton X-100. Lysates were added to p-nitro-phenylphosphate reagent in 96-well plates for 1 h, and alkaline phosphatase activity expressed as absorbance at 405 nM. Cell viability and quantity were measured by Cell-titer Glo and binding of nuclear dye CyQuant, respectively, using replicate wells treated identically to those used for alkaline phosphatase measurements.

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Dorsomorphin dihydrochloride (BML-275 dihydrochloride; Compound C dihydrochloride) is a potent, selective and ATP-competitive AMPKinhibitor, with aKiof 109 nM. Dorsomorphin dihydrochloride inhibits BMP pathway by targeting the type I receptors ALK2, ALK3 and ALK6.

For in vitro study, about 1×106/mL RAW264.7 cells were seeded in 48-well plates. The cells were treated with AICAR or/and Dorsomorphin (compound C) for different time points. Alternatively, the cells were pretreated with AICAR or/and Dorsomorphin (compound C) from 15 min to 1 hour, prior to 100 ng/mL or 1 µg/mL lipopolysaccharide (LPS, from Escherichia coli 0111:B4) challenge.[4]

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Reactive Oxygen Species (ROS) Detection[4]
The cells were pretreated with 2 µM Carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) in the presence or absence of 10 µM diphenyleneiodonium chloride (DPI), AICAR, or Dorsomorphin (compound C) for 15 min, followed by LPS (1 µg/mL) stimulation. After 15 min, ROS generation was determined by flow cytometric analysis. The H2DCFDA-untreated cells were defined as negative control.

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Dorsomorphin (compound C) (0-10 μM, 18 h) suppresses 2DG-induced GRP78 promoter activity in human fibrosarcoma HT1080 cells in a dose-dependent manner but has little effect on tunicamycin-induced GRP78 promoter activity. Dorsomorphin (compound C) also suppresses GRP78 promoter activity induced by glucose withdrawal. Dorsomorphin (compound C) has no effect on 2DG-induced PERK activation and reduces the both basal and 2DG-induced AMPK phosphorylation levels in HT1080 cells.

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Cells were treated with hydrogen peroxide and incubated in complete medium with or without treatment with the AMPK activators metformin (10 μM), AICAR (10 μM), alone or plus AMPK inhibitor Compound C (CC, 10 μM) for 3 days.

Iron-replete mice
~10 mg/kg
i.v.

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12-week-old C57BL/6 mice raised on a standard diet are injected via the tail vein with 0.2 g/kg of Dextran or 0.2 g/kg of iron-dextran USP. Dextran is injected with vehicle only, whereas iron-dextran is injected with either vehicle or Dorsomorphin (10 mg/kg). 1 h after injection, mice are killed and liver segments are collected in 500 µL of SDS-lysis buffer and mechanically homogenized. 20 µL of liver extracts are resolved by SDS-PAGE and immunoblotted. Total RNA is harvested using Trizol from mechanically homogenized mouse livers (6 h after injection with a single intraperitoneal dose of Dorsomorphin (10 mg/kg) or DMSO).[3]

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Zebrafish bone mineralization[3]
WT zebrafish embryos were raised in E3 buffer containing phenylthiourea. At 1 day post fertilization (d.p.f.), embryos were treated with dorsomorphin (1–4 µM) or DMSO vehicle. At 5 d.p.f. and onward, larvae were fed for 1 h every other day. Following each feeding, residual food was washed out and medium was replaced with E3 containing dorsomorphin or vehicle. At 10 d.p.f., larvae were immersed in 0.2% calcein for 30 min. Embryos were washed repeatedly in E3 buffer for 3 h to remove unbound calcein and anesthetized with tricaine. Calcified skeletal structures were visualized by green fluorescence, and the number of vertebral bodies were counted. Iron-dextran injections[3]
Adult fish were anesthetized with tricaine and injected with 10 µl of iron-dextran solution (100 mg ml−1, average dextranMW= 5,000) into the abdominal cavity with dorsomorphin (23 µg/g) or vehicle (DMSO). Control fish were injected with 10 µl of dextran (average MW = 5,000, Sigma). Fish were revived in water. 1 h after injection, fish were anesthetized on ice, and livers were collected into 200 µl SDS-lysis buffer and homogenized mechanically. 15 µl of protein extract was fractionated by SDS-PAGE and immunoblotted, as described above. 3 h after injection, total RNA was extracted from mechanically homogenized zebrafish livers using Trizol reagent.[3]
12-week-old C57BL/6 mice raised on a standard diet are injected via the tail vein with 0.2 g/kg of Dextran or 0.2 g/kg of iron-dextran USP. Dextran is injected with vehicle only, whereas iron-dextran is injected with either vehicle or Dorsomorphin (10 mg/kg). 1 h after injection, mice are killed and liver segments are collected in 500 µL of SDS-lysis buffer and mechanically homogenized. 20 µL of liver extracts are resolved by SDS-PAGE and immunoblotted. Total RNA is harvested using Trizol from mechanically homogenized mouse livers (6 h after injection with a single intraperitoneal dose of Dorsomorphin (10 mg/kg) or DMSO).[3]

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Animal Studies[4]
Male BALB/c mice at 6–7 weeks of age weighing 20–22 g were fed with food and water ad libitum, and housed in a standard animal facility with 12 h light/dark cycle and 50%–70% humidity) for 3 days before the study.
BALB/c mice were randomly divided into five experimental groups: Control (intraperitoneally (i.p.) injection of PBS), LPS (i.p. injection of 2 mg/kg body weight), LPS+AICAR (i.p. injection, 500 mg/kg body weight 60 min before LPS injection), LPS+Compound C (CC or Dorsomorphin) (i.p. injection, 25 mg/kg body weight 60 min before LPS challenge), and LPS+AICAR+Compound C (CC or Dorsomorphin) (i.p. injection of 500 mg of AICAR and 25 mg of Compound C (CC or Dorsomorphin) per kilogram of body weight 60 min before i.p. injection of LPS). Six or twelve hours after injection of LPS, the mice were anesthetized with pentobarbital and euthanized thereafter by cervical dislocation, and blood and tissues were collected for analysis.
For survival experiment, the grouped mice as mentioned above were injected i.p. with LPS (20 mg/kg body weight). To investigate the effect of AICAR or compound C/Dorsomorphin, the mice received injection (i.p.) of 500 mg of AICAR or/and 25 mg of compound C per kilogram of body weight 60 min before administration of LPS. Survival of animals was monitored every 2 hours for up to 24 hours. Severity of sepsis was monitored according to general appearance, breathing frequency, and provoked behavior. The mice were euthanized by cervical dislocation under deep anaesthesia, if the mice exhibited a disease point of no return. After 24 hours, the number of the survival mice of each group was recorded: 8 mice survived in total 8 Control mice (8/8), 0/20, 8/19, 12/19, and 3/19 surviving mice in LPS, LPS+AICAR, LPS+CC, LPS+AICAR+CC respectively. All surviving mice were anesthetized and euthanized with the same protocol described above.

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Sprague-Dawley (SD) of 9–12 weeks old female rats (220–270 g) were used in this research. The mechanistic group was given Dorsomorphin (0.2 mg/kg, i.p dissolved in 1% DMSO solution, for the next 21 days) 30 min prior to fisetin HD (40 mg/kg, p.o.). Dorsomorphin was employed as an inhibitor of the activity of AMPK and SIRT1. [6]

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The rats were randomised into four groups, that is control, leptin-, leptin + dorsomorphin (AMPK inhibitor)- and leptin + LY294002 (PI3K inhibitor)-treated groups, with each group consisting of six rats. Leptin was given once daily for 14 days via the intraperitoneal (i.p.) route at a dose of 60 µg/kg body weight (Recombinant Rat Leptin; Purity >95%, Biovision, USA). In the leptin- and inhibitor-treated groups, the animals were given either Dorsomorphin (5 mg kg−1 day−1) or LY294002 (PI3K inhibitor; 1.2 mg kg−1 day−1) i.p. together with leptin for 14 days. Control rats received 0.1 ml of normal saline i.p. for 14 days. Body weight of both the control and experimental animals was recorded weekly. The doses of leptin and dorsomorphin used in this study were according to Almabhouh et al., 2015, and Pachori et al., 2010, respectively. The dose of LY294002 used was according to Shan et al., 2008. The duration of treatment was based on Haron et al., 2010.[7]

[1]. J Clin Invest. 2001 Oct;108(8):1167-74.

[2]. J Biol Chem. 2006 Nov 17;281(46):34870-9.

[3]. Nat Chem Biol. 2008 Jan;4(1):33-41.

[4]. PLoS One. 2014 Jan 24;9(1):e86881.

[5]. PLoS One. 2012;7(9):e45845.

[6]. Naunyn Schmiedebergs Arch Pharmacol. 2024 Jul 4. doi: 10.1007/s00210-024-03257-7

[7]. Andrologia. 2019 Apr;51(3):e13196.

Dorsomorphin is a pyrazolopyrimidine that is pyrazolo[1,5-a]pyrimidine which is substituted at positions 3 and 6 by pyridin-4-yl and p-[2-(piperidin-1-yl)ethoxy]phenyl groups, respectively. It is a potent, selective, reversible, and ATP-competitive inhibitor of AMPK (AMP-activated protein kinase, EC 2.7.11.31) and a selective inhibitor of bone morphogenetic protein (BMP) signaling. It has a role as an EC 2.7.11.31 {[hydroxymethylglutaryl-CoA reductase (NADPH)] kinase} inhibitor and a bone morphogenetic protein receptor antagonist. It is a pyrazolopyrimidine, a member of piperidines, an aromatic ether and a member of pyridines.
Dorsomorphin has been reported in Trigonella foenum-graecum.

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Activation of the NF-κB and mitogen activated protein (MAP) kinases plays an important role in the expression of inflammatory genes such as adhesion molecules. Although compound C (Dorsomorphin) is known as an AMPK inhibitor, AMPK-independent action of it has been recognized. Effects on the expression of ICAM-1 and VCAM-1 by compound C (Dorsomorphin) were investigated in TNF-α-activated human umbilical vein endothelial cells (HUVECs) in vitro and in thoracic aorta of rats treated with lipopolysaccharide (LPS) in vivo. Compound C inhibited ICAM-1 and VCAM-1 expression at the transcriptional as well as translational level in TNF-α-activated HUVECs. In both DN-AMPK- and AMPKα(1)-siRNA-transfected HUVECs, compound C still inhibited TNF-α-induced VCAM-1 and ICAM-1 expression, indicating that this is AMPK-independent action. Interestingly, compound C significantly inhibited NF-κB activity and translocation of p65 to nucleus in HUVECs when activated with TNF-α. Importantly, administration of compound C (0.2 mg/kg) significantly reduced expression of both ICAM-1 and VCAM-1 in LPS-treated rat thoracic aortas. In addition, compound C significantly inhibited iNOS and production of NO in both TNF-α- and LPS-activated RAW 264.7 cells. Finally, compound C significantly inhibited phosphorylation of Akt and p-38MAPK but not protein kinase c or ERK1/2 in HUVECs. Taken together, we conclude that adhesion molecules (ICAM-1, VCAM-1) are to be the novel targets of compound C in preventing inflammatory insult to endothelial cells independent of AMPK inhibition via inhibition of NF-κB activity along with inhibition of phosphorylation of PI3K and P38 MAPK.[Atherosclerosis. 2011 Nov;219(1):57-64. ]

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Inhibiting the unfolded protein response (UPR) can be a therapeutic approach, especially for targeting the tumor microenvironment. Here, we show that compound C (Dorsomorphin) (also known as dorsomorphin), a small-molecule inhibitor of AMP-activated protein kinase (AMPK) and bone morphogenetic protein (BMP) signaling, inhibit the UPR-induced transcription program depending on the glucose deprivation conditions. We found that compound C prevented UPR marker glucose-regulated protein 78 (GRP78) accumulation and exerted enhanced cytotoxicity during glucose deprivation. Gene expression profiling, together with biochemical analysis, revealed that compound C (Dorsomorphin) had a unique mode of action to suppress the transcriptional activation of UPR-targeted genes, as compared with the classic UPR inhibitors versipelostatin and biguanides. Surprisingly, the UPR-inhibiting activity of compound C was not associated with either AMPK or BMP signaling inhibition. We further found that combination treatments of compound C and the classic UPR inhibitors resulted in synergistic cell death with UPR suppression during glucose deprivation. Our findings demonstrate that compound C could be a unique tool for developing a UPR-targeted antitumor therapy.[PLoS One. 2012;7(9):e45845.]

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Bone morphogenetic protein (BMP) signals coordinate developmental patterning and have essential physiological roles in mature organisms. Here we describe the first known small-molecule inhibitor of BMP signaling-Dorsomorphin, which we identified in a screen for compounds that perturb dorsoventral axis formation in zebrafish. We found that dorsomorphin selectively inhibits the BMP type I receptors ALK2, ALK3 and ALK6 and thus blocks BMP-mediated SMAD1/5/8 phosphorylation, target gene transcription and osteogenic differentiation. Using dorsomorphin, we examined the role of BMP signaling in iron homeostasis. In vitro, dorsomorphin inhibited BMP-, hemojuvelin- and interleukin 6-stimulated expression of the systemic iron regulator hepcidin, which suggests that BMP receptors regulate hepcidin induction by all of these stimuli. In vivo, systemic challenge with iron rapidly induced SMAD1/5/8 phosphorylation and hepcidin expression in the liver, whereas treatment with dorsomorphin blocked SMAD1/5/8 phosphorylation, normalized hepcidin expression and increased serum iron levels. These findings suggest an essential physiological role for hepatic BMP signaling in iron-hepcidin homeostasis.[3]

C24H27CL2N5O

472.41008

471.159

C, 61.02; H, 5.76; Cl, 15.01; N, 14.82; O, 3.39

1219168-18-9

Dorsomorphin;866405-64-3;Dorsomorphin dihydrochloride;1219168-18-9

49761481

Light yellow to yellow solid powder

5.864

2

5

6

32

514

0

[H]Cl.[H]Cl.C12=C(C3=CC=NC=C3)C=NN1C=C(C4=CC=C(OCCN5CCCCC5)C=C4)C=N2

RJDVIJJQKMGPMV-UHFFFAOYSA-N

InChI=1S/C24H25N5O.2ClH/c1-2-12-28(13-3-1)14-15-30-22-6-4-19(5-7-22)21-16-26-24-23(17-27-29(24)18-21)20-8-10-25-11-9-20;;/h4-11,16-18H,1-3,12-15H2;2*1H

6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine;dihydrochloride

Compound C; CpdC; BML275; Dorsomorphin dihydrochloride; 1219168-18-9; Dorsomorphin 2HCl; Dorsomorphin (dihydrochloride); 6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine;dihydrochloride; 6-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidine dihydrochloride; Dorsomorphin (dihydrochloride) (GMP); Pyrazolo[1,5-a]pyrimidine, 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-, hydrochloride (1:2); BML-275; BML 275

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO: ~54 mg/mL (~11 mM)
Water: ~100 mg/mL (212 mM) mg/mL

Solubility in Formulation 1: 20 mg/mL (42.34 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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Solubility in Formulation 2: PBS: 15mg/mL

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