The anti-inflammatory drug BAY 11-7082 suppresses the MyD88-dependent signalling network by targeting the ubiquitin system

Sam Strickson,Lorna Plater,Axel Knebel,Philip Cohen,Christoph H Emmerich,David G Campbell,Natalia Shpiro,Maria Stella Ritorto

doi:10.1042/bj20121651

Abstract

The compound BAY 11-7082 inhibits IκBα [inhibitor of NF-κB (nuclear factor κB)α] phosphorylation in cells and has been used to implicate the canonical IKKs (IκB kinases) and NF-κB in >350 publications. In the present study we report that BAY 11-7082 does not inhibit the IKKs, but suppresses their activation in LPS (lipopolysaccharide)-stimulated RAW macrophages and IL (interleukin)-1-stimulated IL-1R (IL-1 receptor) HEK (human embryonic kidney)-293 cells. BAY 11-7082 exerts these effects by inactivating the E2-conjugating enzymes Ubc (ubiquitin conjugating) 13 and UbcH7 and the E3 ligase LUBAC (linear ubiquitin assembly complex), thereby preventing the formation of Lys63-linked and linear polyubiquitin chains. BAY 11-7082 prevents ubiquitin conjugation to Ubc13 and UbcH7 by forming a covalent adduct with their reactive cysteine residues via Michael addition at the C3 atom of BAY 11-7082, followed by the release of 4-methylbenzene-sulfinic acid. BAY 11-7082 stimulated Lys48-linked polyubiquitin chain formation in cells and protected HIF1α (hypoxia-inducible factor 1α) from proteasomal degradation, suggesting that it inhibits the proteasome. The results of the present study indicate that the anti-inflammatory effects of BAY 11-7082, its ability to induce B-cell lymphoma and leukaemic T-cell death and to prevent the recruitment of proteins to sites of DNA damage are exerted via inhibition of components of the ubiquitin system and not by inhibiting NF-κB.

Highlights

myeloid differentiation factor 88 (MyD88) is an adaptor protein that plays an essential role in the signalling networks that are activated by pathogen-associated molecular pattern (PAMP), as well as by IL-1, IL-18 and IL-33 [1]
We examined whether compounds reported to inhibit the activity or activation of the canonical IκB kinase (IKK) complex inhibited the proliferation of HBL-1 lymphoma cells expressing the MyD88[L265P] mutation
Since neither the IKKβ inhibitor BI605906 nor the transforming growth factor β-activated kinase 1 (TAK1) inhibitors that prevent the MyD88-dependent activation of the IKK complex in fibroblasts or macrophages had this effect [22,26], it seemed that BAY 11-7082 must be inducing HBL-1 cell death by another mechanism and prompted us to explore which proteins BAY 11-7082 and BAY 11-7085 might be targeting

Summary

Introduction

MyD88 (myeloid differentiation factor 88) is an adaptor protein that plays an essential role in the signalling networks that are activated by PAMPs (pathogen-associated molecular patterns), as well as by IL (interleukin)-1, IL-18 and IL-33 [1]. BAY 11-7082 did not inhibit IRAK4 or IRAK1 in vitro (Supplementary Table S1), which are the most ‘upstream’ protein kinases in the MyD88 signalling network, and nor did it prevent the autophosphorylation of IRAK4 induced by LPS in RAW macrophages (Figure 3A) or IL-1 in IL-1R cells (Figure 3B).

Results

Conclusion