STING/stimulator of interferon genes

C-176 significantly inhibits IFNβ reporter activity mediated by STING, but not by RIG-I or TBK1. Type I IFN and IL-6 serum level induction mediated by CMA is significantly inhibited by pretreatment with C-176[1].

Without causing considerable toxicity, C-176 (750/375 nmol C-176 per mouse in 200 μL corn oil) greatly lowers the CMA-mediated activation of type I IFNs and IL-6 blood levels[1]. With no obvious symptoms of overt toxicity in Trex1−/− mice, C-176 significantly lowers serum levels of type I IFNs and strongly suppresses inflammatory markers in the heart[1]. In Trex1−/− mice, C-176 significantly reduces a number of indicators of systemic inflammation [

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.

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Metabolic labelling with [3H]-palmitate[1]
Indicated Flag–STING constructs were expressed in HEK293T cells. For metabolic labelling, cells were starved for 1 h in Glasgow minimal essential medium buffered with 10 mM Hepes, pH 7.4 with C-178 or C-176 (1 μM). Cells were then incubated for 2 h in IM with 200 μCi ml−1 [3H]-palmitic acid (9,10-3H(N)) (American Radiolabelled Chemicals) in presence of C-178 or C-176, and with or without stimulation with CMA (250 μg ml−1). For immunoprecipitation, cells were washed three times in PBS, lysed for 30 min at 4 °C in the following buffer (0.5% Nonidet P-40, 500 mM Tris pH 7.4, 20 mM EDTA, 10 mM NaF, 2 mM benzamidin and protease inhibitor cocktail)) and centrifuged for 3 min at 5000 r.p.m. Supernatants were incubated overnight at 4 °C with the appropriate antibodies (anti-Flag, anti-transferrin receptor and anti-calnexin) and G sepharose beads. For radiolabelling experiments, after immunoprecipitation washed beads were incubated for 5 min at 90 °C in reducing sample buffer before 4–20%-gradient SDS–PAGE. Following SDS–PAGE, gels were incubated in a fixative solution (25% isopropanol, 65% H2O, 10% acetic acid) and incubated for 30 min with signal enhancer Amplify NAMP100. The radiolabelled products were revealed using autoradiography and quantified using the Typhoon Imager.

Immunoprecipitation[1]
Flag–STING expression was induced in HEK293T cells overnight by doxycycline. Cells were incubated with or without C-178 or C-176 (1 μM) for 1 h and treated with DMSO or CMA (250 μg ml−1) for 2 h. Cells were washed in PBS and lysed in lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerin, 1 mM MgCl, 1 mM CaCl, 1% Brij-58 and protease inhibitor cocktail) for 30 min. Flag–STING was immunoprecipitated using anti-Flag M2 affinity gel agarose gel for 2 h at 4 °C. After stringent washing in lysis buffer and PBS, the supernatant was completely removed and the resin was boiled in sample buffer before SDS–PAGE was performed. For immunoprecipitation of endogenous STING, splenocytes were lysed in the above-mentioned lysis buffer and incubated with anti-STING (RD System AF6516) and G sepharose beads overnight. Beads were washed in PBS and gel-based analysis of C-176-AL binding to STING was performed.

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Intact mass measurements for mmSTING[1]
Expression of Flag–mmSTING or Flag–mmSTING(C91S) expression was induced in HEK293T cells overnight by doxycycline. Cells were treated with or without C-178 or C-176 (1 μM) for 30 min and lysed in lysis buffer (20 mM Hepes, 150 mM NaCl, 10% glycerin and 1% DDM). Flag–STING was immunoprecipitated using anti-Flag M2 affinity gel agarose gel for 2 h at 4 °C. Precipitated proteins were eluted using the Flag peptide according to the manufacturer’s instructions. Protein mass spectrometry was performed on a Shimadzu MS2020 connected to a Nexerra UHPLC system, equipped with a Waters ACQUITY UPLC BEH C4 1.7-μm, 2.1 × 50-mm column. Buffer A was 0.05% formic acid in water, and buffer B was 0.05% formic acid in acetonitrile. The analytical gradient was from 10% to 90% buffer B within 6.0 min with 0.75 ml min−1 flow. Mass spectra were collected from 300–2,000 Da and the spectra were deconvoluted using the software MagTran.

Animal/Disease Models: WT type mice.
Doses: 750/375 nmol C-176 per mouse in 200 μL corn oil (~1.34/0.67 mg/mL).
Route of Administration: Intraperitoneally, once.
Experimental Results: Dramatically decreased Serum levels of type I IFNs and IL-6.

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Mice and in vivo studies[1]
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.

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This study was conducted in two separate stages. A total of 198 rats (300–350 g, 9–11 weeks, male) (Stage 1, 90 rats; Stage 2, 108 rats) were used for the experiments (as shown in Figure 1). In total, 7 rats were found dead over the course of experimentation (within 24 h after modeling), and 6 were excluded based on behavioral exclusion criteria (at 30 day after modeling). Animals were monitored for health and killed via cervical dislocation with secondary decapitation.[2]
Stage 1[2]
To investigate the role of STING, the selective STING antagonist C-176 or STING agonist ADU-S100 was administered via intraperitoneal injection to investigate STING signaling following TBI in the first stage. The rats were randomly divided into four groups (n = 18 rats/group): sham + vehicle1 group, TBI + vehicle1 group, TBI + ADU-S100 group, TBI + C-176 group. Rats were randomized using a web-based random group generator (www.pubmed.de/tools/zufallsgenerator). Data acquisition and analyses were blinded to the experimenter. Corn oil (cat. No. ST1177; Beyotime) as vehicle1 was used to dissolve ADU-S100 and C-176. Vehicle1 (corn oil) were administered via intraperitoneal injection at the same time points after TBI or sham modeling. Cerebral tissues for western blot assay were collected (n = 6 rats/group) 24 h after the injection. Neurobehavioral tests, including open field test, force swimming test, and novel object recognition test, were performed 30 days after the TBI induced by a weight-drop model (n = 12 rats/group). In addition, cerebral tissues for Nissl and immunofluorescence staining (n = 6 rats/group) and for ELISA (n = 6 rats/group) were also collected (Figure 1a).

Stage 2[2]
In the second stage, a selective activator of NLRP3 (nigericin) was administered via intracerebroventricular injection to elucidate NLRP3 as a downstream factor in TBI. The rats were divided into five groups (n = 18 rats/group): TBI + ADU-S100 + MCC950 group, TBI + ADU-S100 + VX765 group, TBI + C-176 + nigericin group, TBI +C-176+ vehicle2 group, and TBI + ADU-S100 + vehicle2 group. 10% ethyl alcohol and 90% corn oil as vehicle2 was used to dissolve nigericin, MCC950 and VX-765, and administered via intracerebroventricular injection. Tissues for western blot assay were collected 24 h after the TBI (n = 6 rats/group). After that, behavioral tests (n = 12 rats/group), Nissl, and immunofluorescence staining (n = 6 rats/group) and ELISA (n = 6 rats/group) were performed 30 days post-modeling (Figure 1b). The remaining rats were killed via cervical dislocation.

Drug administration Nigericin, MCC950 or VX765 was administered through intracerebroventricular injection 30 mins before modeling using a needle attached to a microsyringe (5 μl). The injection was made using the following coordinates from bregma: 0.3 mm posterior, 1.0 mm lateral, and 2.5 mm ventral. Nigericin (2 μg/2 μl), MCC950 (2 μg/2 μl) or VX765 (2 μg/2 μl) was infused at a rate of 0.667 μl/min using a microinfusion pump (TJ-4A/SL0107-1A, LongerPump). Nigericin, MCC950 or VX765 was dissolved in 10% ethyl alcohol and 90% corn oil. The needle was held in the brain for an additional 10 min before being slowly removed. Finally, the burr hole was sealed using bone wax. The STING agonist ADU-S100 (0.5 mg/kg) and antagonist C-176(10 mg/kg) were administered via intraperitoneal injection immediately after the TBI. Drug doses used in this study were determined in a preliminary experiment based on previously published studies (Lee et al., 2021; Peng et al., 2020; Tsuchiya et al., 2019).

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

[2]. STING mediates neuroinflammatory response by activating NLRP3-related pyroptosis in severe traumatic brain injury. J Neurochem. 2022 Sep;162(5):444-462.

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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Long-term neurological deficits after severe traumatic brain injury (TBI), including cognitive dysfunction and emotional impairments, can significantly impair rehabilitation. Glial activation induced by inflammatory response is involved in the neurological deficits post-TBI. This study aimed to investigate the role of the stimulator of interferon genes (STING)-nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) signaling in a rodent model of severe TBI. Severe TBI models were established using weight-drop plus blood loss reinfusion model. Selective STING agonist ADU-S100 or antagonist C-176 was given as a single dose after modeling. Further, NLRP3 inhibitor MCC950 or activator nigericin, or caspase-1 inhibitor VX765, was given as an intracerebroventricular injection 30 min before modeling. After that, a novel object recognition test, open field test, force swimming test, western blot, and immunofluorescence assays were used to assess behavioral and pathological changes in severe TBI. Administration of C-176 alleviated TBI-induced cognitive dysfunction and emotional impairments, neuronal loss, and inflammatory activation of glia cells. However, the administration of STING agonist ADU-S100 exacerbated TBI-induced behavioral and pathological changes. In addition, STING activation exacerbated pyroptosis-associated neuroinflammation via promoting glial activation, as evidenced by increased cleaved caspase-1 and GSDMD N-terminal expression. In contrast, the administration of C-176 showed anti-pyroptotic effects. The neuroprotective effects of C-176 were partially reversed by the NLRP3 activator, nigericin. Collectively, glial STING is responsible for neuroinflammation post-TBI. However, pharmacologic inhibition of STING led to a remarkable improvement of neuroinflammation partly through suppressing NLRP3 signaling. The STING-NLRP3 signaling is a potential therapeutic target in TBI-induced neurological dysfunction.[2]

C11H7IN2O4

358.09

357.945

C, 36.90; H, 1.97; I, 35.44; N, 7.82; O, 17.87

314054-00-7

1103958

Light yellow to yellow solid powder

1.9±0.1 g/cm3

361.2±37.0 °C at 760 mmHg

172.3±26.5 °C

0.0±0.8 mmHg at 25°C

1.714

3.38

1

4

2

18

326

0

IC1C=CC(=CC=1)N([H])C(C1=CC=C([N+](=O)[O-])O1)=O

JBIKQXOZLBLMKI-UHFFFAOYSA-N

InChI=1S/C11H7IN2O4/c12-7-1-3-8(4-2-7)13-11(15)9-5-6-10(18-9)14(16)17/h1-6H,(H,13,15)

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

2934.99.03.00

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)

Solubility in Formulation 1: 1.67 mg/mL (4.66 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.25 mg/mL (0.70 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 2.5 mg/mL clear EtOH stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 0.25 mg/mL (0.70 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 2.5 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 4: 0.25 mg/mL (0.70 mM) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 2.5 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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Solubility in Formulation 5: 10 mg/mL (27.93 mM) in Cremophor EL (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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