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

Ahwazi, Danial ; Neopane, Katyayanee ; Markby, Greg R. ; Kopietz, Franziska LU orcid ; Ovens, Ashley J. ; Dall, Morten ; Hassing, Anna S. ; Gräsle, Pamina ; Alshuweishi, Yazeed and Treebak, Jonas T. , et al. (2021) In Biochemical Journal 478(15). p.2977-2997
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... (More)

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.

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publishing date
type
Contribution to journal
publication status
published
subject
in
Biochemical Journal
volume
478
issue
15
pages
21 pages
publisher
Portland Press
external identifiers
  • scopus:85113792702
  • pmid:34259310
ISSN
0264-6021
DOI
10.1042/BCJ20210284
language
English
LU publication?
yes
id
38a16ac1-60c3-484b-998f-aadaa39e1cf5
date added to LUP
2021-09-20 15:31:10
date last changed
2024-12-29 13:07:20
@article{38a16ac1-60c3-484b-998f-aadaa39e1cf5,
  abstract     = {{<p>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.</p>}},
  author       = {{Ahwazi, Danial and Neopane, Katyayanee and Markby, Greg R. and Kopietz, Franziska and Ovens, Ashley J. and Dall, Morten and Hassing, Anna S. and Gräsle, Pamina and Alshuweishi, Yazeed and Treebak, Jonas T. and Salt, Ian P. and Göransson, Olga and Zeqiraj, Elton and Scott, John W. and Sakamoto, Kei}},
  issn         = {{0264-6021}},
  language     = {{eng}},
  number       = {{15}},
  pages        = {{2977--2997}},
  publisher    = {{Portland Press}},
  series       = {{Biochemical Journal}},
  title        = {{Investigation of the specificity and mechanism of action of the ULK1/AMPK inhibitor SBI-0206965}},
  url          = {{http://dx.doi.org/10.1042/BCJ20210284}},
  doi          = {{10.1042/BCJ20210284}},
  volume       = {{478}},
  year         = {{2021}},
}