Abstact

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-A cocrystal structure of the AMPK kinase domain with BAY-3827 revealed distinct features including a disulfide bridge between the alphaD helix Cys(106) and the activation loop residue Cys(174). This bridge appears to stabilize the activation loop such that Asn(162) repositions the Asp-Phe-Gly (DFG) motif Phe(158) toward the C-terminal lobe, displacing His(137) 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.