{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T01:28:42Z","timestamp":1792805322016,"version":"4.5.0"},"reference-count":59,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2023,12,29]],"date-time":"2023-12-29T00:00:00Z","timestamp":1703808000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Wellcome Trust (UK)","award":["204766\/Z\/16\/Z"],"award-info":[{"award-number":["204766\/Z\/16\/Z"]}],"id":[{"id":"https:\/\/ror.org\/029chgv08","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>AMP-activated protein kinase (AMPK) is the central component of a signalling pathway that senses energy stress and triggers a metabolic switch away from anabolic processes and towards catabolic processes. There has been a prolonged focus in the pharmaceutical industry on the development of AMPK-activating drugs for the treatment of metabolic disorders such as Type 2 diabetes and non-alcoholic fatty liver disease. However, recent findings suggest that AMPK inhibitors might be efficacious for treating certain cancers, especially lung adenocarcinomas, in which the PRKAA1 gene (encoding the \u03b11 catalytic subunit isoform of AMPK) is often amplified. Here, we study two potent AMPK inhibitors, BAY-3827 and SBI-0206965. Despite not being closely related structurally, the treatment of cells with either drug unexpectedly caused increases in AMPK phosphorylation at the activating site, Thr172, even though the phosphorylation of several downstream targets in different subcellular compartments was completely inhibited. Surprisingly, the two inhibitors appear to promote Thr172 phosphorylation by different mechanisms: BAY-3827 primarily protects against Thr172 dephosphorylation, while SBI-0206965 also promotes phosphorylation by LKB1 at low concentrations, while increasing cellular AMP:ATP ratios at higher concentrations. Due to its greater potency and fewer off-target effects, BAY-3827 is now the inhibitor of choice for cell studies, although its low bioavailability may limit its use in vivo.<\/jats:p>","DOI":"10.3390\/ijms25010453","type":"journal-article","created":{"date-parts":[[2023,12,29]],"date-time":"2023-12-29T06:18:13Z","timestamp":1703830693000},"page":"453","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":27,"title":["BAY-3827 and SBI-0206965: Potent AMPK Inhibitors That Paradoxically Increase Thr172 Phosphorylation"],"prefix":"10.3390","volume":"25","author":[{"given":"Simon A.","family":"Hawley","sequence":"first","affiliation":[{"name":"Division of Cell Signalling & Immunology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4995-2734","authenticated-orcid":false,"given":"Fiona M.","family":"Russell","sequence":"additional","affiliation":[{"name":"Division of Cell Signalling & Immunology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fiona A.","family":"Ross","sequence":"additional","affiliation":[{"name":"Division of Cell Signalling & Immunology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8373-7379","authenticated-orcid":false,"given":"D. Grahame","family":"Hardie","sequence":"additional","affiliation":[{"name":"Division of Cell Signalling & Immunology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,12,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"472","DOI":"10.1016\/j.cmet.2020.01.015","article-title":"AMPK and TOR: The Yin and Yang of cellular nutrient sensing and growth control","volume":"31","author":"Gonzalez","year":"2020","journal-title":"Cell Metab."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"3677","DOI":"10.1016\/j.molcel.2021.08.015","article-title":"AMPK: Restoring metabolic homeostasis over space and time","volume":"81","author":"Trefts","year":"2021","journal-title":"Mol. Cell"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1038\/s41580-022-00547-x","article-title":"New insights into activation and function of the AMPK","volume":"24","author":"Steinberg","year":"2023","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"496","DOI":"10.1038\/nature06161","article-title":"Structural basis for AMP binding to mammalian AMP-activated protein kinase","volume":"449","author":"Xiao","year":"2007","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"12653","DOI":"10.1074\/jbc.M117.793018","article-title":"Deconvoluting AMP-activated protein kinase (AMPK) adenine nucleotide binding and sensing","volume":"292","author":"Gu","year":"2017","journal-title":"J. Biol. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1126\/science.abe7565","article-title":"Structure of an AMPK complex in an inactive, ATP-bound state","volume":"373","author":"Yan","year":"2021","journal-title":"Science"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"18331","DOI":"10.1074\/jbc.M708379200","article-title":"Structural properties of AMP-activated protein kinase. Dimerization, molecular shape, and changes upon ligand binding","volume":"283","author":"Riek","year":"2008","journal-title":"J. Biol. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"556","DOI":"10.1016\/j.cmet.2013.08.019","article-title":"AMP is a true physiological regulator of AMP-activated protein kinase by both allosteric activation and enhancing net phosphorylation","volume":"18","author":"Gowans","year":"2013","journal-title":"Cell Metab."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"19237","DOI":"10.1073\/pnas.1009705107","article-title":"beta-Subunit myristoylation is the gatekeeper for initiating metabolic stress sensing by AMP-activated protein kinase (AMPK)","volume":"107","author":"Oakhill","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"32207","DOI":"10.1074\/jbc.M606357200","article-title":"Dissecting the role of 5\u2032-AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase","volume":"281","author":"Suter","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2308","DOI":"10.1016\/S0021-9258(19)85891-X","article-title":"Regulation of rat liver acetyl-CoA carboxylase. Regulation of phosphorylation and inactivation of acetyl-CoA carboxylase by the adenylate energy charge","volume":"255","author":"Yeh","year":"1980","journal-title":"J. Biol. Chem."},{"key":"ref_12","first-page":"E1","article-title":"The AMP-activated protein kinase, a metabolic master switch: Possible roles in Type 2 diabetes","volume":"277","author":"Winder","year":"1999","journal-title":"Am. J. Physiol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1016\/j.cmet.2006.05.005","article-title":"Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome","volume":"3","author":"Cool","year":"2006","journal-title":"Cell Metab."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"507","DOI":"10.1126\/science.aah5582","article-title":"Systemic pan-AMPK activator MK-8722 improves glucose homeostasis but induces cardiac hypertrophy","volume":"357","author":"Myers","year":"2017","journal-title":"Science"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1147","DOI":"10.1016\/j.cmet.2017.04.010","article-title":"Activation of skeletal muscle AMPK promotes glucose disposal and glucose lowering in non-human primates and mice","volume":"25","author":"Cokorinos","year":"2017","journal-title":"Cell Metab."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"889","DOI":"10.1016\/S2468-1253(21)00300-9","article-title":"Efficacy and safety of PXL770, a direct AMP kinase activator, for the treatment of non-alcoholic fatty liver disease (STAMP-NAFLD): A randomised, double-blind, placebo-controlled, phase 2a study","volume":"6","author":"Cusi","year":"2021","journal-title":"Lancet Gastroenterol. Hepatol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1038\/cr.2014.163","article-title":"Choreography of AMPK activation","volume":"25","author":"Langendorf","year":"2015","journal-title":"Cell Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3017","DOI":"10.1038\/ncomms4017","article-title":"Structural basis of AMPK regulation by small molecule activators","volume":"4","author":"Xiao","year":"2013","journal-title":"Nat. Commun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"873","DOI":"10.1038\/s42255-020-0245-2","article-title":"Long-chain fatty acyl-CoA esters regulate metabolism via allosteric control of AMPK beta1 isoforms","volume":"2","author":"Pinkosky","year":"2020","journal-title":"Nat. Metab."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Vara-Ciruelos, D., Russell, F.M., and Hardie, D.G. (2019). The strange case of AMPK and cancer: Dr Jekyll or Mr Hyde?. Open Biol., 9.","DOI":"10.1098\/rsob.190099"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"690","DOI":"10.1016\/j.celrep.2019.03.067","article-title":"Phenformin, but not metformin, delays development of T cell acute lymphoblastic leukemia\/lymphoma via cell-autonomous AMPK activation","volume":"27","author":"Dandapani","year":"2019","journal-title":"Cell Rep."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"649","DOI":"10.1016\/j.cmet.2016.03.008","article-title":"AMPK is essential to balance glycolysis and mitochondrial metabolism to control T-ALL cell stress and survival","volume":"23","author":"Kishton","year":"2016","journal-title":"Cell Metab."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"4658","DOI":"10.1111\/febs.13534","article-title":"Evidence of cancer promoting roles for AMPK and related kinases","volume":"282","author":"Monteverde","year":"2015","journal-title":"FEBS J."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1167","DOI":"10.1172\/JCI13505","article-title":"Role of AMP-activated protein kinase in mechanism of metformin action","volume":"108","author":"Zhou","year":"2001","journal-title":"J. Clin. Investig."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1038\/nchembio.2007.54","article-title":"Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism","volume":"4","author":"Yu","year":"2008","journal-title":"Nat. Chem. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1042\/BJ20070797","article-title":"The selectivity of protein kinase inhibitors: A further update","volume":"408","author":"Bain","year":"2007","journal-title":"Biochem. J."},{"key":"ref_27","unstructured":"International Centre for Kinase Profiling (2023, November 14). Kinase Profiling Inhibitor Database. Available online: https:\/\/www.kinase-screen.mrc.ac.uk\/kinase-inhibitors."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1016\/j.molcel.2015.05.031","article-title":"Small molecule inhibition of the autophagy kinase ULK1 and identification of ULK1 substrates","volume":"59","author":"Egan","year":"2015","journal-title":"Mol. Cell"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"8874","DOI":"10.1074\/jbc.RA118.003547","article-title":"AMP-activated protein kinase selectively inhibited by the type II inhibitor SBI-0206965","volume":"293","author":"Dite","year":"2018","journal-title":"J. Biol. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2977","DOI":"10.1042\/BCJ20210284","article-title":"Investigation of the specificity and mechanism of action of the ULK1\/AMPK inhibitor SBI-0206965","volume":"478","author":"Ahwazi","year":"2021","journal-title":"Biochem. J."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1007\/s13402-020-00584-8","article-title":"The potent AMPK inhibitor BAY-3827 shows strong efficacy in androgen-dependent prostate cancer models","volume":"44","author":"Lemos","year":"2021","journal-title":"Cell Oncol."},{"key":"ref_32","unstructured":"Lemos, C., and Schulze, A. (2023, November 14). Overview for BAY-3827, an Inhibitor of PRKAA1, RPS6KA1. Available online: https:\/\/www.sgc-ffm.uni-frankfurt.de\/chemProbes#!specificprobeoverview\/BAY-3827."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1042\/bj1590001","article-title":"Concentration of MgATP2- and other ions in solution. Calculation of the true concentrations of species present in mixtures of associating ions","volume":"159","author":"Storer","year":"1976","journal-title":"Biochem. J."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"769","DOI":"10.1146\/annurev-biochem-090308-173656","article-title":"The evolution of protein kinase inhibitors from antagonists to agonists of cellular signaling","volume":"80","author":"Dar","year":"2011","journal-title":"Annu. Rev. Biochem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"619","DOI":"10.1016\/j.chembiol.2014.03.006","article-title":"Small molecule drug A-769662 and AMP synergistically activate naive AMPK independent of upstream kinase signaling","volume":"21","author":"Scott","year":"2014","journal-title":"Chem. Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"4430","DOI":"10.1074\/jbc.M708296200","article-title":"AMP-activated protein kinase phosphorylates Golgi-specific brefeldin A resistance factor 1 at Thr1337 to induce disassembly of Golgi apparatus","volume":"283","author":"Miyamoto","year":"2008","journal-title":"J. Biol. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1016\/j.molcel.2008.03.003","article-title":"AMPK phosphorylation of raptor mediates a metabolic checkpoint","volume":"30","author":"Gwinn","year":"2008","journal-title":"Mol. Cell"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1186\/s13024-016-0141-0","article-title":"ULK1-mediated phosphorylation of ATG14 promotes autophagy and is impaired in Huntington\u2019s disease models","volume":"11","author":"Wold","year":"2016","journal-title":"Mol. Neurodegener."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"547","DOI":"10.1080\/15548627.2016.1140293","article-title":"The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14","volume":"12","author":"Park","year":"2016","journal-title":"Autophagy"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1092","DOI":"10.1124\/dmd.104.000349","article-title":"Effective dosing regimen of 1-aminobenzotriazole for inhibition of antipyrine clearance in guinea pigs and mice using serial sampling","volume":"32","author":"Balani","year":"2004","journal-title":"Drug Metab. Dispos."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1038\/cr.2014.150","article-title":"Structural basis of AMPK regulation by adenine nucleotides and glycogen","volume":"25","author":"Li","year":"2015","journal-title":"Cell Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1220","DOI":"10.1016\/j.chembiol.2008.10.005","article-title":"Thienopyridone drugs are selective activators of AMP-activated protein kinase beta1-containing complexes","volume":"15","author":"Scott","year":"2008","journal-title":"Chem. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"554","DOI":"10.1016\/j.cmet.2010.04.001","article-title":"Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation","volume":"11","author":"Hawley","year":"2010","journal-title":"Cell Metab."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"813","DOI":"10.1016\/j.chembiol.2017.05.021","article-title":"Mechanisms of paradoxical activation of AMPK by the kinase inhibitors SU6656 and sorafenib","volume":"24","author":"Ross","year":"2017","journal-title":"Cell Chem. Biol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1074\/jbc.M112.422659","article-title":"Ligand binding to the AMP-activated protein kinase active site mediates protection of the activation loop from dephosphorylation","volume":"288","author":"Chandrashekarappa","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"e84319","DOI":"10.7554\/eLife.84319","article-title":"Snf1\/AMPK fine-tunes TORC1 signaling in response to glucose starvation","volume":"12","author":"Caligaris","year":"2023","journal-title":"eLife"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"944","DOI":"10.1007\/s10637-022-01278-8","article-title":"Brain pharmacokinetics and metabolism of the AMP-activated protein kinase selective inhibitor SBI-0206965, an investigational agent for the treatment of glioblastoma","volume":"40","author":"Desai","year":"2022","journal-title":"Investig. New Drugs"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"e25555","DOI":"10.7554\/eLife.25555","article-title":"Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy","volume":"6","author":"Grumati","year":"2017","journal-title":"eLife"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1016\/S0014-5793(96)01209-4","article-title":"The alpha1 and alpha2 isoforms of the AMP-activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro","volume":"397","author":"Woods","year":"1996","journal-title":"FEBS Lett."},{"key":"ref_50","unstructured":"Anand, R., Apgar, J.M., Biftu, T., Chen, P., Chu, L., Colandrea, V.J., Dong, G., Dropinski, D.F., Feng, D., and Hicks, J.D. (2012). Novel Cyclic Azabenzimidazole Derivatives Useful as Anti-Diabetic Agents. (WO\/2012\/116145 23)."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1016\/S1046-5928(03)00126-8","article-title":"Mammalian AMP-activated protein kinase: Functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli","volume":"30","author":"Neumann","year":"2003","journal-title":"Protein Expr. Purif."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1707","DOI":"10.1126\/science.1178377","article-title":"Structure of the LKB1-STRAD-MO25 complex reveals an allosteric mechanism of kinase activation","volume":"326","author":"Zeqiraj","year":"2009","journal-title":"Science"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.cmet.2005.05.009","article-title":"Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase","volume":"2","author":"Hawley","year":"2005","journal-title":"Cell Metab."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"421","DOI":"10.1016\/0014-5793(95)01368-7","article-title":"5\u2032-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2C alpha and native bovine protein phosphatase-2AC","volume":"377","author":"Davies","year":"1995","journal-title":"FEBS Lett."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1007\/978-1-4939-7598-3_16","article-title":"Intact cell assays to monitor AMPK and determine the contribution of the AMP-binding or ADaM sites to activation","volume":"1732","author":"Hawley","year":"2018","journal-title":"Methods Mol. Biol."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1007\/978-1-4939-7598-3_5","article-title":"Cell-free assays to measure effects of regulatory ligands on AMPK","volume":"1732","author":"Fyffe","year":"2018","journal-title":"Methods Mol. Biol."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"11929","DOI":"10.1074\/jbc.M110.189779","article-title":"Selective expression in carotid body Type I cells of a single splice variant of the large conductance calcium- and voltage-activated potassium channel confers regulation by AMP-activated protein kinase","volume":"286","author":"Ross","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1016\/0003-2697(76)90527-3","article-title":"A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding","volume":"72","author":"Bradford","year":"1976","journal-title":"Anal. Biochem."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1038\/nature23275","article-title":"Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK","volume":"548","author":"Zhang","year":"2017","journal-title":"Nature"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/1\/453\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T21:43:55Z","timestamp":1760132635000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/1\/453"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,12,29]]},"references-count":59,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2024,1]]}},"alternative-id":["ijms25010453"],"URL":"https:\/\/doi.org\/10.3390\/ijms25010453","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,12,29]]}}}