Fig. 2: Idronoxil inhibits formation of TBK1 signalling complexes.

a Interferon [IFN]-β luciferase expression in HEK293T cells expressing the indicated constructs after O/N incubation (n = 3 independent experiments). b Surface plasmon resonance [SPR] analysis of idronoxil (IDX) binding to recombinant human TBK1 (representative of n = 3 independent experiments). c qPCR amplification signal of kinase concentration after KinomeScan^TM assays, for the indicated IDX concentrations (averaged from n = 2 technical replicates). IKKe is IKKε. d In silico modelling of IDX binding (bottom—green) and STING C-terminal tail (top—yellow, PDB: 6nt9)¹⁹ to TBK1 dimer (the blue and red residues refer to the two different TBK1 units in the structure). Immunoblot of Sting^KO immortalised bone marrow-derived macrophages [iBMDMs] expressing mCitrine-STING, after 1 h stimulation with 50 μg/ml DMXAA (STING) and increasing doses of IDX (e), or 5 μM IDX or 100 nM MRT67307 [MRT] (f). e 1 representative blot of three independent experiments shown. f One representative blot of two independent experiments shown. Pink: pull-down of mCitrine-STING with anti-GFP antibody. Black: whole cell lysates. e, f Phosphorylated proteins are indicated with [P]. Molecular weight markers are shown in kDa. g IP-10 protein levels in wild-type (IC₅₀ = 1.6 μM), Tbk1^KO (IC₅₀ = 0.75 μM), Ikbkε^KO [Ikkε^KO](IC₅₀ = 2 μM) and wild-type [WT] iBMDM cells after O/N stimulation with 50 μg/ml DMXAA (STING) and increasing doses of IDX (n = 3 independent experiments). h HEK-TLR3 cells expressing NF-κB and IFN-β luciferase reporters were treated 6 h with 0.5 μg/ml poly(I:C) [pIC] (TLR3) in the presence of 5 μM IDX or 400 nM MRT (n = 3 independent experiments). a, g, h Data are mean ± s.e.m. a–c, g Non-linear regression analyses are shown. h Two-way ANOVA with uncorrected Fisher’s LSD (with single pooled variance) multiple comparisons are shown. Exact p values are shown for all comparisons. Source data and detailed statistical analyses are provided as a Source Data file.