Targeting STING oligomerization with small-molecule inhibitors

Abstract

Stimulator of interferon genes (STING) is an essential adaptor protein required for the inflammatory response to cytosolic DNA. dsDNA activates cGAS to generate cGAMP, which binds and activates STING triggering a conformational change, oligomerization, and the IRF3- and NFκB-dependent transcription of type I Interferons (IFNs) and inflammatory cytokines, as well as the activation of autophagy. Aberrant activation of STING is now linked to a growing number of both rare as well as common chronic inflammatory diseases. Here, we identify and characterize a potent small-molecule inhibitor of STING. This compound, BB-Cl-amidine inhibits STING signaling and production of type I IFNs, IFN-stimulated genes (ISGs) and NFκB-dependent cytokines, but not other pattern recognition receptors. In vivo, BB-Cl-amidine alleviated pathology resulting from accrual of cytosolic DNA in Trex-1 mutant mice. Mechanistically BB-Cl-amidine inhibited STING oligomerization through modification of Cys¹⁴⁸. Collectively, our work uncovers an approach to inhibit STING activation and highlights the potential of this strategy for the treatment of STING-driven inflammatory diseases.

Keywords: Trex-1; cytokines; protein arginine deiminases; small-molecule inhibitor; stimulator of interferon genes.

Fig. 1.

BB-Cl-amidine inhibits STING-dependent signaling. (A) Structure of BB-Cl-amidine. (B and C) ELISA analysis of TNF-α and IFN-β in conditioned medium from BMDMs pretreated with vehicle control (DMSO) or BB-Cl-amidine (1 μM) for 1 h followed by treatment with the indicated ligands for 24 h. (D) ELISA analysis of IFN-β in conditioned medium from BMDMs pretreated with vehicle control (DMSO) or BB-Cl-amidine (1 μM) for 1 h followed by infection with HSV1 (MOI 10) or Sendai virus 20 (20 Units) for 24 h. (E) qPCR analysis of Ifnβ expression in BMDMs pretreated with the indicated concentrations of BB-Cl-amidine followed by treatment with diABZI-4 for 2 h. (F) ELISA analysis of IFNβ from BMDMs pretreated with the indicated concentrations of BB-Cl-amidine followed by treatment with diABZI-4 (500 nM) for 24 h. (G) Kinetic cell death analysis of primary BMDMs treated with the indicated concentrations of BB-Cl and stained with Sytox Orange and Hoechst. Cells were imaged using the Cytation5 microscope. One read per hour for 12 h. (H) Immunoblot analysis of phosphorylated STING, IRF3, TBK1, STAT1, P65, and LC3 conversion in whole-cell lysates from BMDMs pretreated with the indicated concentrations of BB-Cl-amidine for 1 h followed by treatment with diABZI-4 for 1 h. (I) qPCR analysis of Ifnβ in human primary monocytes pretreated with BB-Cl-amidine (1 μM) followed by treatment with 500 nM diABZI-4 for 2 h. (J and K) Serum analysis of IFN-β and TNF-α in mice administered BB-Cl-amidine (10 mg/kg) for 1 h followed by diABZI (0.5 mg/kg) for 5 h. B, C, and G representative data. D–F, and H pooled data from three independent experiments. I and J vehicle (n = 4), diABZI-4 (n = 6), diABZI-4 + BB-Cl-amidine (n = 5). *P < 0.05; **P < 0.001. Error bars show mean ± SEM.

Fig. 2.

Transcriptome analysis of BB-Cl-amidine-induced STING inhibition. (A) Heat-map analysis of top 46 expression changes in genes calculated from log (FPKM+1) values from RNA sequencing analysis on RNA extracted from BMDMs pretreated with vehicle control (DMSO) or BB-Cl-amidine (1 μM) followed by treatment with diABZI-4 for 2 or 6 h. (B and C) Log₂ fold change in vehicle control (DMSO) vs. diABZI-4-treated cells (Left) and BB-Cl-amidine-treated cells vs. diABZI-4-treated cells (Right) for 2 h (B) or 6 h (C). Data are the average of two replicates sequenced from each of the indicated samples.

Fig. 3.

BB-Cl-amidine inhibits STING signaling independent of PADs. (A) qPCR analysis of Ifnβ expression in WT and Padi4^−/− BMDMs treated with diABZI-4 for 2 h. (B) Immunoblot analysis of phosphorylated IRF3, STING, and PAD4 in whole-cell lysates from WT and Padi4^−/− BMDMs treated with diABZI-4 for the indicated times. (C and D) qPCR analysis of Ifnβ (C) and Cxcl0 (D) expression in BMDMs pretreated with 1 μM BB-Cl-amidine for 1 h followed by treatment with diABZI-4 for 2 h. (E) Immunoblot analysis of phosphorylated IRF3, PAD4, and β-actin in whole-cell lysates from WT and Padi4^−/− BMDMs pretreated with 1 μM BB-Cl-amidine for 1 h followed by treatment with diABZI-4 for 1 h. (F and G) qPCR analysis of Ifnβ (F) and Cxcl0 (G) expression in WT, Padi2^−/− and Padi2/4^−/− BMDMs treated with diABZI-4 for 2 h. ***P < 0.0001. Error bars show mean ± SEM.

Fig. 4.

BB-Cl-amidine directly targets STING. (A and B) Structure of BB-Cl-Yne and BB-F-Yne alkyne probes. (C) ELISA analysis of IFN-β in conditioned medium from BMDMs pretreated with vehicle control (DMSO), BB-Cl-amidine (1 μM), BB-Cl-Yne (1 μM), or BB-F-Yne (1 μM) for 1 h followed by treatment with 500 nM diABZI-4 for 24 h. (D) Log₁₀ fold change enrichment of proteins from copper-clicked lysates from cells treated with BB-Cl-Yne (1 μM) for 1 h. (E) Representative mass spectrometry spectra of STING identified from streptavidin pull downs of clicked lysates from cells treated with BB-Cl-Yne or cells cotreated with BB-Cl-amidine and BB-Cl-Yne. (F) Peptide coverage analysis of peptides identified in streptavidin bead pull downs from clicked lysate cells treated with BB-Cl-Yne or cells cotreated with BB-Cl-amidine and BB-Cl-Yne. (G) Immunoblot of STING and strep-IR-dye in streptavidin bead pull downs and input from clicked lysates of cells treated with or without BB-Cl-Yne and BB-Cl-amidine. (C) pooled data from two independent experiments performed in triplicate. (D and E) performed in triplicate. (G) representative data of two independent experiments.

Fig. 5.

BB-Cl-amidine alleviates STING-dependent experimental AGS. (A–C) Survival analysis (A), spleen weight (B) and heart:body weight ratio (C) of Trex^D18N/D18N mice administered a control diet or a BB-Cl-amidine-embedded diet. (D) Representative H&E staining of tissue sections from hearts of WT and Trex1^D18N/D18N mice administered a control diet or BB-Cl-amidine embedded diet. (E) Pathology scoring of heart sections from (D). (F) Trichrome staining of tissue sections from hearts of WT and Trex1^D18N/D18N mice administered a control diet or BB-Cl-amidine embedded diet. (A) vehicle (n = 10) BB-Cl-amidine (n = 17), data representative of pooled mice from two independent experiments. (B and C) WT vehicle (n = 5), WT BB-Cl-amidine (n = 5), Trex1^D18N/D18 vehicle (n = 6), Trex1^D18N/D18N BB-Cl-amidine (n = 9). (D) representative images. (E) WT vehicle (n = 3), WT BB-Cl-amidine (n = 3), Trex1^D18N/D18N vehicle (n = 4), Trex1^D18N/D18N BB-Cl-amidine (n = 6). *P < 0.05, two-way ANOVA. Error bars show mean ± SEM.

Fig. 6.

BB-Cl-amidine impairs STING oligomerization via the modification of Cys¹⁴⁸. (A) Representative mass spectrometry spectra of STING modified by BB-Cl-amidine identified in tryptic digests from recombinant STING (10 μg) incubated with BB-Cl-amidine (10 μM) for 1 h at 37 °C. (B) Immunoblot analysis of STING in native and reduced fractions from lysates of HEK293T cells expressing WT murine STING or a murine STING-C147S mutant and treated with diABZI-4 for 15 min. (C) Immunoblot analysis of STING in native and reduced fractions of lysates from BMDMs pretreated with the indicated concentrations of BB-Cl-amidine followed by treatment with diABZI-4. (D) Immunoblot analysis of STING in native and reduced fractions of lysates from WT and Padi4^−/− BMDMs treated with 500 nM diABZI for the indicated times.