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

Inhibition of AMP-activated protein kinase (AMPK) is under increasing investigation for its therapeutic potential in many diseases, including certain cancers. However, existing AMPK- inhibitors available as tool compounds are largely limited to compound C/dorsomorphin and SBI-0206965, both of which suffer from poor selectivity and off-target effects. Here we describe the structure-based molecular insights and cellular actions of a recently identified potent AMPK inhibitor, BAY-3827. Kinase selectivity profiling and sequence analyses of kinases that are highly or weakly inhibited by BAY-3827 uncovered key conserved residues involved in its inhibitory mechanism. A 2.5 Å co-crystal structure of the AMPK kinase domain (KD)-BAY-3827 complex and comparison with known KD-inhibitor structures, revealed an overlapping site in the ATP-binding pocket and an αC helix-out conformation. A distinct feature of the BAY-3827-bound state is the formation of a disulfide bridge between the αD helix Cys ¹⁰⁶ and the activation loop residue Cys ¹⁷⁴ . This bridge appears to stabilize the activation loop such that Asn ¹⁶² repositions the DFG motif Phe ¹⁵⁸ toward the C-terminal kinase lobe, displacing His ¹³⁷ and disrupting the regulatory spine, thereby promoting an inactive state. In hepatocytes, 2.5-5 μM BAY-3827, but not the structurally resembling inactive BAY-974, fully blocked AMPK activator (MK-8722)-mediated phosphorylation of ACC1 and corresponding inhibition of lipogenesis. Unbiased transcriptome analysis in MK- 8722-treated wild-type and AMPK-null hepatocytes revealed that 5 μM BAY-3827 downregulated >30% of MK-8722-stimulated AMPK-dependent genes. Based on its greater selectivity and potency substantiated by comprehensive structural and cellular investigations, BAY-3827 is a powerful tool to delineate AMPK functions.