Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases

ARTICLE DOI: 10.1038/s41467-018-06787-w OPEN Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases 1234567890():,; Jacob Lauwring Andersen1, Borbala Gesser2, Erik Daa Funder3, Christine Juul Fælled Nielsen1, Helle Gotfred-Rasmussen1, Mads Kirchheiner Rasmussen2,4, Rachel Toth5,6, Kurt Vesterager Gothelf J. Simon C. Arthur5,6, Lars Iversen2,4 & Poul Nissen 1 3, Dimethyl fumarate (DMF) has been applied for decades in the treatment of psoriasis and now also multiple sclerosis. However, the mechanism of action has remained obscure and involves high dose over long time of this small, reactive compound implicating many potential targets. Based on a 1.9 Å resolution crystal structure of the C-terminal kinase domain of the mouse p90 Ribosomal S6 Kinase 2 (RSK2) inhibited by DMF we describe a central binding site in RSKs and the closely related Mitogen and Stress-activated Kinases (MSKs). DMF reacts covalently as a Michael acceptor to a conserved cysteine residue in the αF-helix of RSK/MSKs. Binding of DMF prevents the activation loop of the kinase from engaging substrate, and stabilizes an auto-inhibitory αL-helix, thus pointing to an effective, allosteric mechanism of kinase inhibition. The biochemical and cell biological characteristics of DMF inhibition of RSK/MSKs are consistent with the clinical protocols of DMF treatment. 1 Danish Research Institute of Translational Neuroscience – DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark. 2 Department of Dermatology, Aarhus University Hospital, P.P. Oerumsgade 11, DK-8000 Aarhus C, Denmark. 3 Department of Chemistry and iNANO, Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark. 4 Department of Clinical Medicine, Aarhus University, P.P. Oerumsgade 11, DK-8000 Aarhus C, Denmark. 5 Division of Cell Signaling and Immunology and University of Dundee, Dow Street, Dundee DD1 5EH, UK. 6 Medical Research Council Protein Phosphorylation Unit, School of Life Sciences, Wellcome Trust Building, University of Dundee, Dow Street, Dundee DD1 5EH, UK. Correspondence and requests for materials should be addressed to L.I. (email: ) or to P.N. (email: ) NATURE COMMUNICATIONS | (2018)9:4344 | DOI: 10.1038/s41467-018-06787-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06787-w S ystemic psoriasis treatment with fumaric acid esters has been known for >50 years, and dimethyl fumarate (DMF) is considered the clinically active agent1 (Fig. 1a). Most recently the European Medicines Agency (EMA) has approved a new oral formulation of DMF for the treatment of psoriasis (Procedure No. EMEA/H/C/002157/0000 and see reference2). DMF has furthermore proven efficacious for the treatment of relapsing-remitting multiple sclerosis (MS)3. The clinical response to DMF is slow, with efficacy only appearing after several weeks to months of high-dose administration1. Several mechanisms of action (MOA) have been proposed. DMF reacts, e.g., rapidly with glutathione (GSH)4 and orally administered DMF is released into the bloodstream, absorbed by the cells, and conjugated to GSH5. A proposed MOA for DMF treatment of MS and psoriasis therefore builds on GSH depletion increasing hemoxygenase-1 expression, impairing STAT1 (signal transducer and activator of transcription 1) phosphorylation, and hereby inhibiting Th1 and Th17 (T helper cell 1 and 17) differentiation6,7. However, GSH levels return to normal within 24 h of DMF administration7. DMF furthermore reduces neutrophil recruitment in inflammation8 and reduces microglial and astrocytic inflammation9. DMF inhibition of Th1 and Th17 differentiation has also been demonstrated through suppression of the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), p38 MAPK (mitogen-activated protein kinase) and ERK1/2 (extracelluar signal-regulated kinase 1 and 2) signalling pathways8,10. Along this line, DMF was identified as a NF-κB inhibitor11, and more specifically targeting the C-terminal kinase domain (CTKD) of the ribosomal S6 kinases (RSKs)12 and mitogen- and stress-activated kinases (MSKs)13. The closely related RSKs and MSKs belong to the Ras/Raf/ MAPK signalling pathway and encompass four human RSK isoforms (RSK1–4) and two MSK isoforms (MSK1 and 2)14. RSK1, 2, and 3 are ubiquitously expressed in human tissues and RSKs are generally recognized as regulators of proliferative a processes15. MSK1 and 2 are expressed in most cells but at particularly high levels in cells of the immune system and the central nervous system. MSK1 and 2 display a key role in the regulation of IL-10 (interleukin 10) expression by innate immune cells16. RSKs and MSKs are composed of a N-terminal kinase domain (NTKD), a linker region and a C-terminal kinase domain (CTKD), and both kinase domains have adenosine triphosphate (ATP) binding sites (Fig. 1b). The RSKs are activated by phosphorylation of the activation loop in the CTKD by ERK1/217, and MSKs similarly by ERK1/2 or p38 MAPK. The linker region is phosphorylated by the activated CTKD, and hereby activates the NTKD for phosphorylation of downstream targets18. We find that DMF inhibits RSK/MSK kinases primarily by covalent binding to a conserved cysteine residue placed at an allosteric site, and that this interaction is likely to represent an important component of the mechanism of action of DMF as a clinical drug. Results DMF inhibition and modification of RSK2CTKD. The efficacy of DMF and monomethyl fumarate (MMF) as inhibitors against purified RSK2CTKD and mutants hereof was evaluated in a wellestablished, time-resolved fluorescence resonance energy transfer (FRET) based kinase activity assay (see Methods). We found that DMF leads to full inhibition of RSK2 CTKD, but at moderate efficacy with an IC50, app. of 225 μM, when incubated with RSK2CTKD prior to ERK2 activation, whereas MMF displayed no inhibition of RSK2CTKD (Fig. 1c). Considering the high-dose administration of DMF, the observed effect remains however relevant. Compared with MMF, the double bond in the α,βunsaturated ester of DMF is an excellent (and twofold symmetric) Michael acceptor (Fig. 1a), which led us to consider if the inhibitory effect on RSK/MSK was due to covalent modifications of cysteine residues resulting in a dimethyl 2-S-succinate adduct4. Indeed, a covalent modification was identified by the reaction of [2,3-14C]-labelled DMF with the purified CTKD of RSK2 O * O O * O b C436 C439 C560 ATP pocket T577 C579 C599 αL Activation loop c d 1 2 3 [2,3- C] DMF RSK2 + + + + + + Iodoacetamide GSH pH 8 – – + – – + 14 Inhibition (%) 100 ERK docking 50 RSK2 0 10 100 1000 Inhibitor (μM) 10,000 Fig. 1 DMF inhibits RSK2. a DMF with Michael acceptor reactive carbons marked with red asterisks. b Schematic representation of RSK2CTKD and position of cysteine residues (...truncated)