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

Fraguas Bringas, C.; Ahangar, M. S.; Cuenco, J.; Liu, H.; Addinsall, A. B.; Lindahl, M.; Foretz, M.; Göransson, O.; Scott, J. W.; Zeqiraj, E.; Sakamoto, K.

doi:10.1101/2025.02.28.640688

bioRxiv · 10.1101/2025.02.28.640688

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

Fraguas Bringas, C.·Ahangar, M. S.·Cuenco, J.·Liu, H.·Addinsall, A. B.·Lindahl, M.·Foretz, M.·Göransson, O.·Scott, J. W.·Zeqiraj, E.·Sakamoto, K.

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Abstract

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 [A] 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 C helix-out conformation. A distinct feature of the BAY-3827-bound state is the formation of a disulfide bridge between the D helix Cys106 and the activation loop residue Cys174. This bridge appears to stabilize the activation loop such that Asn162 repositions the DFG motif Phe158 toward the C-terminal kinase lobe, displacing His137 and disrupting the regulatory spine, thereby promoting an inactive state. In hepatocytes, 2.5-5 M 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 M 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. One-sentence summaryWe provide the mechanism of action of the potent and selective AMPK inhibitor BAY-3827, which blocks AMPK-dependent cellular functions.

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Fraguas Bringas, C., Ahangar, M. S., Cuenco, J., Liu, H., Addinsall, A. B., Lindahl, M., Foretz, M., Göransson, O., Scott, J. W., Zeqiraj, E., Sakamoto, K.. 2025-03-03. Mechanism and cellular actions of the potent AMPK inhibitor BAY-3827. https://doi.org/10.1101/2025.02.28.640688

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