Mechanism and cellular actions of the potent AMPK inhibitor BAY-3827

Inhibition of adenosine 5′-monophosphate (AMP)–activated protein kinase (AMPK) is under increasing investigation for its therapeutic potential in many diseases. Existing AMPK inhibitors are however limited, with poor selectivity and substantial off-target effects. Here, we provide mechanistic insights and describe the cellular selectivity of the recently identified AMPK inhibitor BAY-3827. A 2.5-Å cocrystal structure of the AMPK kinase domain with BAY-3827 revealed distinct features including a disulfide bridge between the αD helix Cys¹⁰⁶ and the activation loop residue Cys¹⁷⁴. This bridge appears to stabilize the activation loop such that Asn¹⁶² repositions the Asp-Phe-Gly (DFG) motif Phe¹⁵⁸ toward the C-terminal lobe, displacing His¹³⁷ and disrupting the regulatory spine, promoting an inactive kinase state. In hepatocytes, BAY-3827 blocked AMPK activator (MK-8722)–mediated phosphorylation of ACC1 and corresponding inhibition of lipogenesis. Transcriptome analysis revealed that BAY-3827 down-regulated ~30% of MK-8722–stimulated AMPK-dependent genes. We establish the molecular and cellular basis of BAY-3827’s selectivity and utility for delineating AMPK functions while highlighting its limitations.