Investigation of the specificity and mechanism of action of the ULK1/AMPK inhibitor SBI-0206965

Journal article


Ahwazi, Danial, Neopane, Katyayanee, Markby, Greg, Kopietz, Franziska, Ovens, Ashley, Dall, Morten, Hassing, Anna, Graesle, Pamina, Alshuweishi, Yazeed, Treebak, Jonas, Salt, Ian, Goransson, Olga, Zeqiraj, Elton, Scott, John Walter and Sakamoto, Kei. (2021). Investigation of the specificity and mechanism of action of the ULK1/AMPK inhibitor SBI-0206965. Biochemical Journal. 478(15), pp. 2977-2997. https://doi.org/10.1042/BCJ20210284
AuthorsAhwazi, Danial, Neopane, Katyayanee, Markby, Greg, Kopietz, Franziska, Ovens, Ashley, Dall, Morten, Hassing, Anna, Graesle, Pamina, Alshuweishi, Yazeed, Treebak, Jonas, Salt, Ian, Goransson, Olga, Zeqiraj, Elton, Scott, John Walter and Sakamoto, Kei
Abstract

SBI-0206965, originally identified as an inhibitor of the autophagy initiator kinase ULK1, has recently been reported as a more potent and selective AMP-activated protein kinase (AMPK) inhibitor relative to the widely used, but promiscuous inhibitor Compound C/Dorsomorphin. Here, we studied the effects of SBI-0206965 on AMPK signalling and metabolic readouts in multiple cell types, including hepatocytes, skeletal muscle cells and adipocytes. We observed SBI-0206965 dose dependently attenuated AMPK activator (991)-stimulated ACC phosphorylation and inhibition of lipogenesis in hepatocytes. SBI-0206965 (≥25 μM) modestly inhibited AMPK signalling in C2C12 myotubes, but also inhibited insulin signalling, insulin-mediated/AMPK-independent glucose uptake, and AICA-riboside uptake. We performed an extended screen of SBI-0206965 against a panel of 140 human protein kinases in vitro, which showed SBI-0206965 inhibits several kinases, including members of AMPK-related kinases (NUAK1, MARK3/4), equally or more potently than AMPK or ULK1. This screen, together with molecular modelling, revealed that most SBI-0206965-sensitive kinases contain a large gatekeeper residue with a preference for methionine at this position. We observed that mutation of the gatekeeper methionine to a smaller side chain amino acid (threonine) rendered AMPK and ULK1 resistant to SBI-0206965 inhibition. These results demonstrate that although SBI-0206965 has utility for delineating AMPK or ULK1 signalling and cellular functions, the compound potently inhibits several other kinases and critical cellular functions such as glucose and nucleoside uptake. Our study demonstrates a role for the gatekeeper residue as a determinant of the inhibitor sensitivity and inhibitor-resistant mutant forms could be exploited as potential controls to probe specific cellular effects of SBI-0206965.

KeywordsAMPK; AMPK-related kinase; glucose uptake; lipogenesis; SBI-0206965; ULK1
Year2021
JournalBiochemical Journal
Journal citation478 (15), pp. 2977-2997
PublisherPortland Press Ltd.
ISSN0264-6021
Digital Object Identifier (DOI)https://doi.org/10.1042/BCJ20210284
PubMed ID34259310
Scopus EID2-s2.0-85113792702
PubMed Central IDPMC8370752
Open accessPublished as ‘gold’ (paid) open access
Research or scholarlyResearch
Page range2977-2997
FunderAustralian Catholic University
National Health and Medical Research Council
Publisher's version
License
File Access Level
Open
Output statusPublished
Publication dates
Online10 Aug 2021
Publication process dates
Accepted13 Jul 2021
Deposited17 Nov 2021
Grant IDNHMRC/1138102
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