human and mouse STING

Pretreated with STING-IN-2 (C-170; 0.5 μM), THP-1 cells were then stimulated using cGAMP. TNF and IFNB1 mRNA levels are decreased, as well as p-TBK1 levels, by STING-IN-2 (C-170) therapy [1].

C-170 could attenuate pathological features of autoinflammatory disease in mice [1]. First we verified that the compounds target STING by using an in vivo click-chemistry approach and also assessed the pharmacokinetic profile of C-176 on single-dose intraperitoneal injection (Extended Data Fig. 6a, b). We next evaluated whether C-176 can suppress the induction of type I IFNs triggered by the administration of CMA. Of note, pretreatment with C-176 markedly reduced the CMA-mediated induction of serum levels of type I IFNs and IL-6. (Fig. 4a and Extended Data Fig. 6c). Thus, C-176 is effective in mice and—as expected for a covalent inhibitor—the short serum half-life does not limit its in vivo inhibitory capacity. To assess the potential of C-176 to antagonize STING in a model of autoinflammatory disease, we investigated its efficacy in Trex1−/− mice. Trex1−/− mice show signs of severe multi-organ inflammation caused by the persistent activation of the cyclic GMP–AMP synthase–STING pathway and recapitulate certain pathogenic features of Aicardi–Goutières syndrome in humans. Having verified that C-178 suppresses interferon-stimulated genes in cells from Trex1−/− mice (Extended Data Fig. 7a), we performed a two-week in vivo efficacy study with C-176. Notably, treatment of Trex1−/− mice with C-176 resulted in a significant reduction in serum levels of type I IFNs and in a strong suppression of inflammatory parameters in the heart (Extended Data Fig. 7b, c). Wild-type mice on a two-week treatment with C-176 showed no evident signs of overt toxicity (Extended Data Fig. 6d–g). We next conducted a three-month trial with C-176 in Trex1−/− mice, which demonstrated marked amelioration of various signs of systemic inflammation (Fig. 4b, c and Extended Data Fig. 7e). Thus, C-176 attenuates STING-associated autoinflammatory disease in mice.

Competition assay[1]
HEK293T cells expressing Flag–STING were incubated with the indicated compounds and after 1 h, C-176-AL was added for 1 h. Cells were collected in PBS and analysed by in-gel analysis of C-176-AL-mediated labelling of STING (see ‘ Gel-based analysis of compound binding to STING’).

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Gel-based analysis of compound binding to STING[1]
HEK293T cells expressing Flag–STING were incubated with C-176-AL, C-175-AZ, iodoacetamide azide or H-151-AL in serum-free medium, collected in PBS and lysed by repetitive freezing and thawing. Forty-three microlitres of lysed cells was treated with a freshly prepared ‘click reagent’ mixture containing tris(benzyltriazolylmethyl)amine (TBTA) (3 μl per sample, 3 mM in 1:4 DMSO:t-ButOH), tetramethylrhodamine (TAMRA) azide), SiR azide or SiR alkyne (2 μl per sample, 1.25 mM in DMSO), and freshly prepared CuSO4 (1 μl per sample) and tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) (1 μl per sample) and incubated at room temperature for 30 min. The reaction was quenched by addition of reducing sample buffer. In-gel fluorescence was visualized using Fusion FX and analysed by Fusion capt advance acquisition software.

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Crosslinking with disuccinimidyl suberate[1]
HEK293T cells expressing Flag–mmSTING were incubated with or without C-176 (1 μM) for 1 h and treated with DMSO or CMA (250 μg ml−1) for 2 h. Crosslinking was performed in PBS with 1 mM disuccinimidyl suberate (DSS) freshly prepared in DMSO at room temperature for 1 h.

Cell-based IFNβ promoter–reporter luciferase measurements[1]
HEK293T cells were seeded in a 96-well plate and transfected using GeneJuice (Millipore) with a IFN-β promoter–reporter plasmid (pIFNβ–GLuc) in combination with indicated expression constructs. After 16 h, gaussia luciferase activity was measured in the supernatants using coelenterazine as substrate.[1]

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High-throughput chemical compound screen[1]
HEK293T cells that expressed mouse STING with an N-terminal mCherry tag30 were transfected using GeneJuice with a construct encoding for cyclic di-GMP synthase in conjunction with an IFN-β firefly luciferase reporter plasmid. Three hours later, transfected cells were seeded in 384-well plates coated with library compounds. For each compound, a 40-nl volume was selected to obtain a concentration of 10 μM in the assay plates. The amount of DMSO in each well was normalized to 0.1%. After overnight treatment, cells were lysed in lysis buffer (25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 10% glycerol, 1% Triton X-100) for 20 min followed by the addition of firefly luciferase substrate. Reporter activity was measured using a Tecan Infinite plate reader. The screen was performed on ~20,000 compounds from a chemically diverse compound collection available at the BSF core facility at EPFL. For the identification of hsSTING-specific compounds, HEK293T cells expressing a human STING construct were transfected with a construct encoding mouse cyclic GMP–AMP synthase in conjunction with an IFN-β firefly luciferase reporter plasmid. Further analysis was performed as described above. The screen was performed on ~30,000 compounds from a chemically diverse compound collection available at the BSF core facility at EPFL.[1]

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Stimulation of cells[1]
BMDMs (1 × 106 cells ml−1) were pretreated with DMSO, C-178 (0.5 μM unless otherwise indicated) or H-151 (0.5 μM) for 1 h, followed by stimulation with either CMA (250 μg ml−1), dsDNA (90-mer, 1.33 μg ml−1), cyclic di-GMP or cGAMP (1.5 μg ml−1). Triphosphate RNA (166 ng ml−1) or LPS (1 μg ml−1) were used as controls. THP-1 cells (1 × 106 cells ml−1) were differentiated with PMA (100 ng ml−1) for at least 3 h and treated with C-170 (0.5 μM) or H-151 (0.5 μM or as indicated) for 2 h, followed by stimulation with cyclic GAMP (375 ng ml−1) or triphosphate RNA (133 ng ml−1) for 2–3 h (for mRNA expression analysis and p-TBK1 immunoblot) or overnight (for IP-10 production). Alternatively, WI-38 cells (0.15 × 106 cells ml−1) and THP-1 (1 × 106 cells ml−1) cells were pretreated with C-178 (0.5 μM) and stimulated with cGAMP (1.5 μg ml−1) for 2–3 h. To transfect dsDNA, triphosphate RNA and cyclic dinucleotides, Lipofectamine 2000 was used. The sequence of the sense strand of the 90-mer DNA is as follows: 5′-TACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACATACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACA-3′.

C57BL/6J mice (stock number 000664) were purchased from Jackson Laboratories. TREX1-deficient mice were a gift from T. Lindahl31 and were backcrossed for >10 generations to C57BL/6NJ. Mice were maintained under specific-pathogen-free (SPF) conditions at EPFL. For the pharmacokinetic studies, wild-type mice were injected intraperitoneally with 750 nmol C-176 per mouse in 200 μl corn oil. Blood was collected at 30 min, 2 h and 4 h and serum C-176 levels were measured by mass spectrometry (liquid chromatography–high-resolution mass spectrometry). To assess the in vivo inhibitory effect of C-176, wild-type mice (8–12 weeks of age) were injected either with vehicle or C-176. After 1 h or 4 h, CMA was administered at a concentration of 224 mg kg−1. Four hours later, mice were euthanized and the serum was collected to measure CMA-induced cytokine levels. To assess the in vivo inhibitory effect of H-151, wild-type mice were injected intraperitoneally with 750 nmol H-151 per mouse in 200 μl 10% Tween-80 in PBS. After 1 h CMA (112 mg kg−1) was administered, and after 4 h mice were euthanized and the serum was collected. The efficacy study in Trex1−/− mice was conducted as follows: mice (2–5 weeks of age) were injected with 7.5 μl of C-176 or DMSO dissolved in 85 μl corn oil twice per day for 11 consecutive days. Mice were euthanized by anaesthetization in a CO2 chamber followed by cervical dislocation. For toxicology studies, 8-week-old mice were injected daily with 562.5 nmol of C-176 for 2 weeks. At day 14, blood samples were collected in lithium-heparin-coated tubes, and plasma was isolated after centrifugation at 4 °C and then stored at −80 °C. Plasma parameters were measured using DimensionXpand Plus. For the peripheral blood cell profile, 100 μl of blood was collected in EDTA-K-coated tubes. Complete blood counts were analysed with an ADVIA120 haematology system. For the detection of luciferase activity, Trex1−/−Ifnb1Δβ-luc/Δβ-luc reporter mice (aged 4–7 weeks) were injected intraperitoneally daily for 7 days with 750 nmol H-151 or DMSO in 200 μl PBS 0.1% Tween-80. For in vivo imaging, mice were anaesthetized with isofluran and injected intravenously with 15 mg kg−1 XenoLight D-luciferin in isotonic sodium chloride. Photon flux was quantified two minutes after injection on an In-vivo Xtreme II imaging device with binning set to 8 × 8 pixels and an integration time of 3 min. Animal experiments were approved either by the Service de la Consommation et des Affaires Vétérinaires of the canton of Vaud or by the Landesdirektion Dresden (Germany) and were performed in accordance with the respective legal regulations.

[1]. Targeting STING with covalent small-molecule inhibitors. Nature. 2018 Jul;559(7713):269-273.

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.[1]

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Western blot analysis Cells were lysed in 2× Laemmli buffer, immobilized protein on beads in sample reducing buffer followed by denaturing at 95 °C for 5 min. Cell lysates were separated by SDS–PAGE and transferred onto PVDF membranes. Blots were incubated with anti-STING (D2P2F), phospho-TBK1 (D52C2), TBK1 (D1B4), anti-transferrin receptor anti-calnexin or anti-Flag M2. As secondary antibodies, anti-rabbit-IgG-HRP or anti-mouse-IgG-HRP (1:2000) were used. Anti-β-actin was used as control. ECL signal was recorded on the ChemiDoc XRS Biorad Imager and data were analysed with Image Laboratory.[1]

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Quantitative RT–qPCR Total RNA was isolated using the RNAeasy Mini Kit (Qiagen) and cDNA was synthesized using the RevertAid First Strand cDNA Synthesis kit. Quantitative RT–qPCR was performed in duplicates using Maxima SYBR Green Master Mix on a QuantStudio 5 machine. GAPDH was used as an endogenous normalization control to obtained relative expression data. Primer sequences are as follows: mmGapdh forward, 5′-GTCATCCCAGAGCTGAACG-3′; mmGapdh reverse, 5′-TCATACTTGGCAGGTTTCTCC-3′; mmIfnb1 forward, 5′-CTCCAGCTCCAAGAAAGGAC-3′; mmIfnb1 reverse, 5′-TGGCAAAGGCAGTGTAAC TC-3′; mmTnf forward, 5′-TATGGCCCAGACCCTCACA-3′, mmTnf reverse, 5′-GGAGTAGACAAGGTACAACCCATC-3′, mmIsg15 forward, 5′-AAGAAGCAGATTGCCCAGAA-3′; mmIsg15 reverse, 5′-TCTGCGTCAGAAAGACCTCA-3′; mmCxcl10 forward, 5′-AAGTGCTGCCGTCATTTTCT-3′; mmCxcl10 reverse, 5′-GTGGCAATGATCTCAACACG-3′; hsGAPDH forward, 5′-GAGTCAACG GATTTGGTCGT-3′; hsGAPDH reverse, 5′- GACAAGCTTCCCGTTCTCAG-3′; hsIFNB1 forward, 5′-CAGCATCTGCTGGTTGAAGA-3′; hsIFNB1 reverse, 5′-CATTACCTGAAGGCCAAGGA-3′; hsTNF forward, 5′-CCCGAGT GACAAGCCTGTAG–3′; hsTNF reverse, 5′- TGAGGTACAGGCCCTCTGAT-3′.[1]

C15H16N2O4EXACTMASS

288.299

288.11

C, 62.49; H, 5.59; N, 9.72; O, 22.20

346691-38-1

2059265

Light yellow to yellow solid powder

1.3±0.1 g/cm3

359.3±37.0 °C at 760 mmHg

171.1±26.5 °C

0.0±0.8 mmHg at 25°C

1.602

4.01

1

4

5

21

361

0

CCCCC1=CC=C(NC(C2=CC=C([N+]([O-])=O)O2)=O)C=C1

QMVOHFICEFYHMK-UHFFFAOYSA-N

InChI=1S/C15H16N2O4/c1-2-3-4-11-5-7-12(8-6-11)16-15(18)13-9-10-14(21-13)17(19)20/h5-10H,2-4H2,1H3,(H,16,18)

N-(4-Butylphenyl)-5-nitrofuran-2-carboxamide

C170; C 170; STING inhibitor C-170; N-(4-butylphenyl)-5-nitrofuran-2-carboxamide; STING-IN-2; N-(4-butylphenyl)-5-nitro-2-furancarboxamide; N-(4-butylphenyl)-5-nitro-2-furamide; C-170; CBMicro_018692; STING inhibitor C-170

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~433.58 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)