Aberrant activation of innate immune pathways is associated with a variety of diseases. Progress in understanding the molecular mechanisms of innate immune pathways has led to the promise of targeted therapeutic approaches, but the development of drugs that act specifically on molecules of interest remains challenging. Here we report the discovery and characterization of highly potent and selective small-molecule antagonists of the stimulator of interferon genes (STING) protein, which is a central signalling component of the intracellular DNA sensing pathway1,2. Mechanistically, the identified compounds covalently target the predicted transmembrane cysteine residue 91 and thereby block the activation-induced palmitoylation of STING. Using these inhibitors, we show that the palmitoylation of STING is essential for its assembly into multimeric complexes at the Golgi apparatus and, in turn, for the recruitment of downstream signalling factors. The identified compounds and their derivatives reduce STING-mediated inflammatory cytokine production in both human and mouse cells. Furthermore, we show that these small-molecule antagonists attenuate pathological features of autoinflammatory disease in mice. In summary, our work uncovers a mechanism by which STING can be inhibited pharmacologically and demonstrates the potential of therapies that target STING for the treatment of autoinflammatory disease.
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We thank N. Jordan, E. Simeoni and L. Muhandes for technical assistance and advice. We acknowledge the staff of the BSF-ACCESS screening platform at the EPFL for support, especially M. Chambon and J. Bortoli, and the staff of the ISIC Mass Spectrometry platform at the EPFL, especially N. Gasilova. We thank the following core facilities for their support: BIOp, CPG and HCF. A.A. received grants from the SNF (BSSGI0-155984, 31003A_159836), the Gebert Rüf Foundation (GRS-059_14) and the Else Kröner-Fresenius Stiftung (2014_A250). R.B. received funding from an AGS Research Award and the German Research council (DFG - BE 5877/2-1).
Nature thanks Z. Chen, T. Taguchi, H. Wu and the other anonymous reviewer(s) for their contribution to the peer review of this work.
A.A. is a consultant to IFM Therapeutics, LLC. A.A., S.M.H., L.R. and the EPFL have filed provisional patent applications related to STING inhibitors. All other authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Screening workflow. HEK mC-STING, HEK293T cells expressing mCherry–STING. b, Left, summary of the primary screen. Mean of normalized values for IFNβ luciferase activity in cells treated with compound versus cells treated with DMSO. Right, validation of selected candidates from the primary screen. Normalized values for IFNβ luciferase activity (light blue) induced by coexpression of cyclic di-GMP synthase (cdG-Syn) and STING are shown, in comparison to activity triggered by RIG-I (green). c, d, HEK293T cells expressing mCherry–STING were transfected with plasmids that encoded either cdG-Syn or RIG-I, as well as an IFNβ luciferase reporter, and then treated with C-178 or C-176 (1.25 μM–0.01 μM), after which IFNβ luciferase activity (c) or cell viability (using the CellTiter-Blue assay) (d) were measured. e, f, Chemical structures of derivatives of C-176 (e) and their effect (at a concentration of 0.5 μM) on IFNβ luciferase reporter activity triggered by mmSTING or RIG-I (f). Mean ± s.e.m. of n = 3 experiments (c, d, f).
a, b, mRNA expression levels of Tnf in BMDMs activated with cGAMP, cyclic di-GMP (cdG), dsDNA, CMA or LPS for 5 h, after pretreatment for 1 h with C-178 (0.5 μM) or DMSO (n = 3 biological replicates). c, Heat map of RNA sequencing analysis of BMDMs treated with DMSO or CMA, in the presence or absence of C-178. The top 50 upregulated genes in cells treated with DMSO versus cells treated with CMA, in the absence of C-178, are shown for all conditions. d, Scatter plot of the dimensions PC1 versus PC2. e, Levels of expression of IFNB1 mRNA in THP-1 and WI-38 cells pretreated with C-178 (0.25 μM) or DMSO and stimulated with cGAMP for 3 h (n = 2 biological replicates). f, HEK293T cells were transfected with the construct chimaeric 1 (C1: hsSTING (amino acids 1–138)–mmSTING (amino acids 138–378)) or the construct chimaeric 2 (C2: mmSTING (amino acids 1–137)–hsSTING (amino acids 139–379)), together with cdG-Syn and IFNβ luciferase reporter, and treated with C-178 (0.5 μM) (n = 3 biological replicates). Data are mean or mean ± s.e.m. P values were determined by two-tailed t-test.
a, BMDMs were treated with C-178 (0.25 μM) for 1 h, washed or left untreated and after 1 h were stimulated with cGAMP for 3 h. Levels of Ifnb1 mRNA were assessed by RT–qPCR. Data are mean ± s.e.m. (n = 3 biological replicates). b–d, HEK293T cells expressing indicated Flag–mmSTING constructs were exposed to C-178 or C-176 (1 μM), lysed and Flag–mmSTING was immunoprecipitated. The eluted protein was analysed by intact mass spectrometry (LC-MS). b, Deconvoluted electrospray ionization mass spectrum (left), and expected and measured mass shifts after covalent binding of C-176 and C-178 (right) are shown. c, Deconvoluted electrospray ionization mass spectrum for Flag–mmSTING(C91S). d, Fragmentation map of Flag–mmSTING–C-176 analysed by top-down analysis, using LC-HCD-MS/MS (an additional 8 residues are due to N-terminal Flag). Data that were obtained with three different NCE values are combined to create a fragmentation map with assigned b- and y-fragments. Achieved sequence coverage is 15% with 20 p.p.m. mass accuracy for fragment assignment. One representative of n = 2 independent experiments is shown (b, c). For electrospray ionization spectra see Supplementary Information.
a, Structures of C-176-AL and C-176-AZ. b, HEK293T cells transfected with Flag–mmSTING and an IFNβ luciferase reporter were treated with C-176-AL, and luciferase activity was then assessed. Data are mean of n = 2. c, Labelling events of wild-type HEK293T cells and HEK293T with Flag–mmSTING incubated with C-176-AL (0.25 μM). d, Distinct labelling of mmSTING and hsSTING by C-176-AL (0.25 μM). e, Concentration-dependent competitor blockage of C-176-AL (0.25 μM) against C-178 and C-176-09 in HEK293T cells that express Flag–mmSTING. f, Labelling events of HEK293T cells that express Flag–mmSTING (wild-type mmSTING or mmSTING(C91S)) when exposed to iodoacetamide azide or C-176-AZ (both at 0.25 μM). g, HEK293T cells expressing Flag–mmSTING, GSTO1–Flag, MAP27K–Flag, MGMT–Flag or GSTP1–Flag were treated with C-176-AZ (0.25 μM). For in-gel fluorescence imaging, TAMRA azide (c–e) or SiR alkyne (f, g) was used. Immunoblots against Flag or β-actin are shown and data are representative of n = 3 independent experiments (c–g). h, Proposed reaction mechanism. For gel source data, see Supplementary Fig. 1.
a, Single-channel images of Flag and GM130 stainings of mouse embryonic fibroblasts that express Flag–mmSTING (control corresponding to images shown in Fig. 3), treated with CMA or DMSO. Scale bar, 20 μm. b, Protein quantification of p-TBK1, Flag–mmSTING and β-actin by immunoblot of mouse embryonic fibroblasts from a. c, [3H]-palmitate labelling of HEK293T cells that express the indicated Flag–mmSTING constructs, and which were treated with C-178, C176 (1 μM) or DMSO. d, e, [3H]-palmitate labelling of immunoprecipitated endogenous calnexin or transferrin receptor from HEK293T cells treated as indicated (compounds at 1 μM). f, HEK293T cells that express Flag–mmSTING were treated with C-178 (1 μM) or 2-bromopalmitate (2-BP) (50 μM) and stimulated with CMA for 1.5 h. Analysis of indicated proteins was performed by native PAGE or SDS–PAGE. g, Crosslinked lysates (DSS, 1 mM) of HEK293T cells that express Flag–mmSTING treated with C-178 (1 μM) and stimulated with CMA (2 h) were analysed by SDS–PAGE and immunoblotted for STING and p-TBK1. One representative of n = 3 (a–c, e–g) or n = 2 (d) independent experiments. For gel source data, see Supplementary Fig. 1.
a, Labelling of endogenous STING immunoprecipitaed from splenocytes of mice treated with C-176-AL, visualized by in-gel fluorescence. One representative of n = 2. b, Plasma concentration profiles of C-176 after a single-dose intraperitoneal injection into wild-type mice (n = 2 mice per condition). Data are mean of technical replicates. c, Serum levels of type I IFNs and IL-6 from wild-type mice pretreated with C-176 or vehicle 4 h after injection with CMA (n = 3 mice per condition). d, Body weight of wild-type mice during two weeks of daily DMSO and C-176 injection. e–g, Mice from d were euthanized and blood samples were collected for measuring plasma levels of TNFα and IL-6 (e), blood cell counts (f), and liver and kidney parameters (g). Data are mean ± s.d. of n = 10 mice per condition (e–g). P values were calculated by one-way ANOVA (c) or two-tailed t-test (e–g). ns, not significant. For gel source data, see Supplementary Fig. 1. For source data, see Supplementary Table 1. Source Data
a, Wild-type (n = 2) or Trex1−/− (n = 7) mouse embryonic fibroblasts were treated with DMSO or C-178 (2 μM) overnight. mRNA levels of Isg15 and Cxcl10 were measured by RT–qPCR. b, c, Control mice (wild type or Trex1+/− (each n = 3)) were treated with vehicle, and Trex1−/− mice were treated with C-176 (n = 5 mice) or vehicle (n = 4 mice) for 11 days. Serum type I IFN levels (b) were measured and histological analysis of the heart (c) was performed. Scale bars, 50 μm. d, Histological analysis of distinct organs from wild-type (all n = 4) or Trex1−/− mice treated for 3 months. Smooth muscle, n = 3 (C-176) or 5 (vehicle); tongue and stomach, n = 3 (C-176) or 6 (vehicle). Representative histological images are shown. Data are mean ± s.e.m. P values were calculated using two-tailed t-test (a) or one-way ANOVA (b–d). For source data, see Supplementary Table 1. Source Data
a, Structure of C-170 and C-171. b, IFNβ luciferase reporter measurements from HEK293T cells transfected with indicated constructs (C-170 and C-171, 0.02–2 μM) (n = 3). c, d, THP-1 cells were pretreated with C-170 (0.5 μM) and stimulated with cGAMP or triphosphate RNA. IFNB1 and TNF mRNA levels were assessed by RT–qPCR (n = 5) (c), and p-TBK1 was determined by immunoblot (d). e, [3H]-palmitate labelling of HEK293T cells that express Flag–hsSTING, treated with C-170 (1 μM) or DMSO. f, HEK293T cells that express Flag–hsSTING (wild-type hsSTING or hsSTING(C91S)) were treated with C-170 (1 μM), lysed and Flag–hsSTING was analysed by intact mass measurement (LC-MS). Deconvoluted electrospray ionization mass spectrum showing intact mass indicated hsSTING constructs and treatments, are shown. Data are mean ± s.e.m. P values were calculated using two-tailed t-test. NS, not significant. One representative of n = 3 (d) and n = 2 (e–f) independent experiments is shown. For gel source data, see Supplementary Fig. 1. For electrospray ionization spectra see Supplementary Information.
a, THP-1 cells that had been pretreated with H-151 or DMSO were stimulated with cGAMP or triphosphate RNA, or left unstimulated. IP-10 production was quantified by enzyme-linked immunosorbent assay after overnight incubation (n = 3). b, Levels of TNF mRNA assessed by RT–qPCR in THP-1 cells that had been pretreated with H-151 and stimulated with cGAMP or triphosphate RNA (n = 4). c, [3H]-palmitate labelling of immunoprecipitated endogenous calnexin or transferrin receptor from HEK293T cells that had been treated with H-151 (1 μM). d, Structure of H-151-AL. e, Competition assay of H-151-AL with H-151 in HEK293T cells that express Flag–hsSTING. Flag–hsSTING labelled with H-151-AL was visualized by in-gel fluorescence. f, HEK293T cells or HEK293T cells that express Flag–hsSTING were treated with H-151-AL, lysed and clicked to a SiR azide. Whole-cell lysates were analysed by in-gel fluorescence and by immunoblot. g, HEK293T cells that express Flag–hsSTING, GSTO1–Flag, MAP27K–Flag, MGMT–Flag or GSTP1–Flag were exposed to H-151-AL, clicked to TAMRA azide and visualized by in-gel fluorescence or immunoblot. h, Deconvoluted electrospray ionization mass spectrum showing intact mass of Flag–hsSTING(C91S) with or without H-151. i, Flag–hsSTING was purified from HEK293T cells that had been pretreated with H-151 (1 μM) and analysed by top-down analysis using LC-HCD-MS/MS (with additional 8 residues due to N-terminal Flag). Data that were obtained with three different NCE values are combined to create fragmentation map with assigned b- and y-fragments. Achieved sequence coverage is 23% with 20 p.p.m. mass accuracy for fragment assignment. Data are shown as mean ± s.e.m. P values were calculated by two-tailed t-test. NS, not significant. One of n = 2 experiments is shown (c, e–h). For gel source data, see Supplementary Fig. 1. For electrospray ionization spectra, see Supplementary Information.
a, HEK293T cells were transfected with plasmids encoding mmSTING in combination with cdG-Syn or were transfected with a plasmid for TBK1, and an IFNβ luciferase reporter, and then treated with H-151 (concentration 0.02–2 μM). Reporter activity was measured after overnight incubation. Data are mean ± s.e.m. (n = 3; nonlinear regression analysis). Experiments were performed together with those that generated the data displayed in Fig. 5b. b, BMDMs were pretreated with H-151 (0.5 μM) and levels of Ifnb1 mRNA expression induced by cGAMP were assessed by RT–qPCR. Data are mean ± s.e.m. (n = 3); P value was calculated by two-tailed t-test. c, Mean plasma concentration profiles of H-151 following a single-dose intraperitoneal injection into wild-type mice (n = 3 mice per time point). d, Schematic, and in vivo detection of H-176-AL binding to mmSTING. Visualization by in-gel fluorescence was performed using SiR azide. One representative of n = 2. For gel source data, see Supplementary Fig. 1. For source data, see Supplementary Table 1. Source Data
In the absence of inhibition, ligand binding triggers the translocation of STING to the Golgi1,25, where palmitoylation occurs at cytoplasmic proximal cysteine residues (Cys88 and Cys91)26. In turn, this post-translational modification facilitates the multimerization of STING to create a platform—possibly at the lipid raft domain at the trans-Golgi network26—for the recruitment of TBK1, and thereby enables the initiation of downstream signalling. Through covalent interaction with Cys91, the compounds we describe here block the palmitoylation of STING and retain the protein in a signalling-incompetent state.
This file contains the complete NMR spectra data
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This file contains a Supplementary Note, which provides information detailing the palmitoylation of STING
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Haag, S.M., Gulen, M.F., Reymond, L. et al. Targeting STING with covalent small-molecule inhibitors. Nature 559, 269–273 (2018). https://doi.org/10.1038/s41586-018-0287-8
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