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    • Dorsomorphin (Compound C): Selective ATP-Competitive AMPK...

    2025-12-25

    Dorsomorphin (Compound C): Selective ATP-Competitive AMPK Inhibitor

    Executive Summary: Dorsomorphin (Compound C, B3252) is a cell-permeable, reversible ATP-competitive inhibitor of AMP-activated protein kinase (AMPK) with a Ki of 109 nM, exhibiting high selectivity over related kinases such as PKA and PKC (APExBIO); it robustly inhibits downstream phosphorylation of acetyl-CoA carboxylase (ACC) by up to 80% in mammalian cell systems. Dorsomorphin also blocks bone morphogenetic protein (BMP) signaling via Smad1/5/8 inhibition, reducing hepatic hepcidin expression and modulating iron metabolism (Ren et al., 2025). Its unique dual-pathway activity enables precise dissection of energy and differentiation signals, supporting workflows in autophagy, metabolism, and stem cell biology. The compound is insoluble in water and ethanol but dissolves in DMSO at ≥8.49 mg/mL with warming and ultrasonic treatment, and is recommended at 4–40 μM in cell culture and 10 mg/kg i.p. in animal models. Dorsomorphin is supplied by APExBIO and should be stored at -20°C for optimal stability.

    Biological Rationale

    AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis, activated by increased AMP/ATP ratios. It orchestrates metabolic shifts toward catabolism in response to energetic stress, phosphorylating targets such as ACC to inhibit lipid synthesis and promote fatty acid oxidation (see prior review). Precise inhibition of AMPK allows researchers to dissect energy-driven signaling in cancer, neurobiology, and metabolic diseases. Dorsomorphin (Compound C) was the first small molecule identified to directly inhibit AMPK in an ATP-competitive, reversible manner (APExBIO).

    In addition, Dorsomorphin uniquely inhibits BMP type I receptor signaling, affecting the phosphorylation of Smad1/5/8 and thereby influencing cell fate, stem cell differentiation, and iron homeostasis via hepatic hepcidin regulation (Ren et al., 2025). This dual specificity renders Dorsomorphin a valuable probe for studies in autophagy regulation, muscle atrophy, iron metabolism, and neural induction.

    Mechanism of Action of Dorsomorphin (Compound C)

    • AMPK Inhibition: Dorsomorphin binds the ATP-binding pocket of AMPK, competitively inhibiting kinase activity with a Ki of 109 nM. This results in rapid suppression of AMPK-mediated phosphorylation events in hepatocytes and HeLa cells (APExBIO).
    • Downstream Effects: Inhibition of AMPK reduces phosphorylation of acetyl-CoA carboxylase (ACC) by up to 80%, impairing fatty acid oxidation and increasing lipid synthesis. It also attenuates autophagic proteolysis by disrupting AMPK-driven autophagy signaling (Ren et al., 2025).
    • BMP/Smad Pathway Blockade: Dorsomorphin blocks BMP type I receptors, preventing phosphorylation of Smad1/5/8. This downregulates hepcidin gene transcription, increases serum iron, and modulates stem cell fate decisions (see detailed mechanism).
    • Dual-Pathway Modulation: The capacity to simultaneously inhibit AMPK and BMP/Smad pathways allows for targeted investigation of metabolic, autophagic, and differentiation processes.

    Evidence & Benchmarks

    • Dorsomorphin inhibits AMPK with a Ki of 109 nM, showing high selectivity over PKA, PKC, and JAK3 (APExBIO).
    • Suppresses AMPK-driven ACC phosphorylation by 80% in cultured mammalian cells at ≤40 μM (Ren et al., 2025).
    • Inhibits BMP4-induced Smad1/5/8 phosphorylation with an IC50 of 0.47 μM (APExBIO).
    • Reduces hepatic hepcidin mRNA and elevates serum iron in mouse models at 10 mg/kg i.p. dosing (Ren et al., 2025).
    • Blocks AMPK/PINK1/Parkin-mediated mitophagy, negating muscle-protective effects in high-fat diet mouse models (Ren et al., 2025).

    This article extends prior mechanistic reviews by formally integrating benchmarks from the latest translational studies and clarifying context-specific usage, compared to Dorsomorphin (Compound C): AMPK Inhibition and Mitochondr..., which focused on mechanistic pathways in isolation.

    For an exploration of metabolic rewiring and stem cell differentiation mechanisms not fully addressed here, see Dorsomorphin (Compound C): Unraveling Metabolic Rewiring ....

    Applications, Limits & Misconceptions

    Dorsomorphin (Compound C) is used in workflows targeting:

    • Inhibition of AMPK activity in hepatocytes and HeLa cells for dissecting energy sensing pathways.
    • Autophagy regulation via suppression of AMPK/PINK1/Parkin-mediated mitophagy (Ren et al., 2025).
    • Inhibition of BMP4-induced Smad phosphorylation to study differentiation and iron metabolism.
    • Induction of dorsalization in zebrafish embryos by modulating BMP signaling.
    • Promotion of self-renewal and neural induction in human embryonic stem cells by BMP pathway inhibition.

    Recent studies confirm that Dorsomorphin blocks the mitochondrial quality control benefits of Lycium barbarum polysaccharide in high-fat diet mouse models, illustrating a mechanistic link between AMPK inhibition and diminished mitophagy (Ren et al., 2025).

    Common Pitfalls or Misconceptions

    • Non-selectivity at high concentrations: Dorsomorphin shows off-target effects above 40 μM, including inhibition of unrelated kinases.
    • Irreversible storage degradation: Solutions are unstable; freshly prepare in DMSO, avoid long-term storage.
    • Insolubility in aqueous buffers: The compound is insoluble in water/ethanol, requiring DMSO and warming for dissolution.
    • Not a pure BMP inhibitor: Dorsomorphin affects both AMPK and BMP/Smad pathways—interpret phenotypes accordingly.
    • Does not mimic genetic AMPK knockout: Pharmacological inhibition is reversible and context-dependent; controls are essential.

    Workflow Integration & Parameters

    • Stock Preparation: Dissolve in DMSO at ≥8.49 mg/mL with gentle warming and ultrasonic treatment.
    • Cell Culture: Recommended working concentrations are 4–40 μM; titrate for cell type and endpoint.
    • Animal Models: Intraperitoneal dosing at 10 mg/kg is effective in mice for hepatic and metabolic endpoints.
    • Storage: Store powder at -20°C; do not freeze/thaw repeatedly. Use solutions immediately.
    • Controls: Always include DMSO vehicle and, where possible, genetic AMPK or BMP pathway controls.

    For protocol optimization, troubleshooting, and advanced workflow guidance, see Dorsomorphin (Compound C): Precision AMPK Inhibitor for A.... This guide offers actionable strategies not detailed here.

    Conclusion & Outlook

    Dorsomorphin (Compound C, B3252) is a rigorously benchmarked, dual-pathway inhibitor supplied by APExBIO. Its selectivity for AMPK and BMP/Smad kinases positions it as a gold-standard probe for dissecting metabolic, autophagic, and differentiation processes in cell and animal models. New research confirms its role in modulating mitophagy, iron metabolism, and stem cell fate, but highlights the necessity of context-specific controls and careful dosing. As the field advances, Dorsomorphin will remain a fundamental tool for interrogating the intersection of energy sensing and developmental signaling.