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

Ahwazi, D. et al. (2021) Investigation of the specificity and mechanism of action of the ULK1/AMPK inhibitor SBI-0206965. Biochemical Journal, 478(15), pp. 2977-2997. (doi: 10.1042/BCJ20210284) (PMID:34259310)

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Abstract

SBI-0206965, originally identified as an inhibitor of the autophagy initiator kinase ULK1, has recently been reported as a more potent and selective 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.

Item Type:Articles
Additional Information:This work was financially supported by The Swedish Research Council (project grant Dnr 2017-01295), The Swedish Diabetes Foundation, Royal Physiographic Society of Lund and The Påhlsson Foundation (to O.G.). J.W.S. was supported by National Health and Medical Research Council (NHMRC) project grant (GNT1138102). This project was also supported in part by the Victorian Government’s Operational Infrastructure Support Program. A.J.O. is supported by a PhD scholarship funded by the Australian Catholic University. M.D is supported by a postdoc fellowship and A.S.H by a PhD scholarship from the Danish Diabetes Academy, which is funded by the Novo Nordisk Foundation (NNF17SA0031406). P.G. was supported by Erasmus+ programme. Y.A. was supported by a research scholarship from King Saud University. E.Z. was supported by a Sir Henry Dale Fellowship (Wellcome Trust and the Royal Society; 200523/Z/16/Z). The Novo Nordisk Foundation Center for Basic Metabolic Research is an independent Research Center based at the University of Copenhagen, Denmark, and partially funded by an unconditional donation from the Novo Nordisk Foundation (Grant number NNF18CC0034900).
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Salt, Dr Ian and Alshuweishi, Yazeed Abdullah I
Authors: Ahwazi, D., Neopane, K., Markby, G. R., Kopietz, F., Ovens, A. J., Dall, M., Hassing, A. S., Graesle, P., Alshuweishi, Y. A. I., Treebak, J. T., Salt, I., Göransson, O., Zeqiraj, E., Scott, J. W., and Sakamoto, K.
College/School:College of Medical Veterinary and Life Sciences
College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Journal Name:Biochemical Journal
Publisher:Portland Press
ISSN:0264-6021
ISSN (Online):1470-8728
Published Online:14 July 2021
Copyright Holders:Copyright © 2021 The Authors
First Published:First published in Biochemical Journal 478(15): 2977-2997
Publisher Policy:Reproduced under a Creative Commons License

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