biostudies-literatureBringas CFWellcome Trusteadx2434https://www.ebi.ac.uk/biostudies/studies/S-EPMC12372887biostudies-literatureUnknown11(34)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.Science advancesMechanism and cellular actions of the potent AMPK inhibitor BAY-3827.PMC12372887222531/Z/21/ZLiu HGoransson OAhangar MSScott JWSakamoto KAddinsall ABCuenco JFebbraio MALindahl MForetz MZeqiraj EOvens AJBringas CFfalseMechanism 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.2025-01-01T00:00:00Z2025 Aug2026-05-09T10:41:26.925Z2026-04-08T00:48:45.296ZS-EPMC123728874084509710.1126/sciadv.adx2434