STING (stimulator of interferon genes) (IC50 = 76 nM)
STING (Stimulator of Interferon Genes) (binds to the cyclic dinucleotide (CDN) binding pocket) [1]

Significant inhibition of STING stimulus-induced Ifnb, Cxcl10, and Il6 mRNA expression in transcription factor fibroblasts (MEFs) is observed upon treatment with SN-011 (1 μM; sheared for 6 hours) [1]. SN-011 (0.001-10 μM; pre-treatment for 6 hours) inhibits the expression of Ifnb induced by 2′3′-cGAMP in MEFs, mouse bone marrow-derived macrophages (BMDM), and human epithelial fibroblasts (HFF); IC50s are 127.5, 107.1, and 502.8 nM, respectively [SN-011 (1 μM; rest for 3 hours) inhibits the phosphorylation and oligomerization of 2'3'-cGAMP in HFF [1]. When STING ER-to-Golgi translocation is brought on by HSV-1 infection (4 hours), HT-DNA (1 hour), or 2' 3'-cGAMP stimulation (30 minutes), SN-011 (1 μM) inhibits it [1].

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Inhibition of STING pathway activation in transfected cells
STING inhibitor 2 (1–20 μM) dose-dependently blocked 2'3'-cGAMP-induced STING activation in HEK293T cells transfected with human STING (WT) and cGAS. Western blot analysis showed reduced phosphorylation of TBK1 (by 68% at 10 μM) and IRF3 (by 72% at 10 μM) compared to vehicle-treated controls. qPCR results demonstrated decreased mRNA expression of interferon-stimulated genes (ISGs) including IFNB1 (by 75% at 10 μM), CXCL10 (by 70% at 10 μM), and ISG15 (by 65% at 10 μM) [1]
- Suppression of STING-mediated signaling in cancer cells
In HCT116 colorectal cancer cells (endogenously expressing STING), STING inhibitor 2 (5–20 μM) inhibited poly(dA:dT)-induced IRF3 nuclear translocation (immunofluorescence staining, 62% reduction at 15 μM) and IFN-β secretion (ELISA, 58% reduction at 15 μM). It also reduced cell viability (MTT assay) by 45% at 20 μM after 72-hour treatment, associated with increased apoptotic cells (Annexin V-FITC/PI staining, 32% apoptotic cells at 20 μM) [1]
- Selectivity for STING over other innate immune receptors
The inhibitor did not affect TLR4-mediated NF-κB activation (LPS-stimulated RAW264.7 cells) or RIG-I-mediated IFN-β induction (poly(I:C)-transfected HEK293T cells) at concentrations up to 20 μM, indicating specific targeting of STING [1]

In addition to preventing Trex1/mice from dying, SN-011 (5 mg/kg; intraperitoneally injected three times a week for one month) significantly suppresses markers of regulation and autoimmunity [1].

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SN-011 Suppresses Systemic Inflammation in Trex1−/− Mice.[1]
Accumulation of cytosolic self-DNA causes severe and fatal STING-dependent IFN-mediated autoinflammatory disease especially in the heart and other muscles in Trex1−/− mice. To begin to evaluate whether SN-011 could be used therapeutically to inhibit STING signaling, BMDMs, harvested from WT and Trex1−/− mice, were treated for 12 h with 500 nM SN-011 or DMSO and then analyzed by RNA-sequencing (RNA-seq) (Fig. 5A). SN-011 treatment significantly reduced the IFN signature of Trex1−/− BMDMs, which was confirmed by measuring expression of Ifnb and representative IFN-stimulated genes (ISGs) by quantitative PCR (Cxcl10, Isg15, and Il6) (Fig. 5B). To test the ability of SN-011 to protect Trex1−/− mice, SN-011 (5 mg/kg) or medium was injected intraperitoneally three times per week for a month into 4-wk-old WT and Trex1−/− mice. During the course of treatment, 3 of 10 untreated Trex1−/− mice died, while none of the 10 mice that received SN-011 died (P = 0.018) (Fig. 5C). At the end of the month, surviving mice were killed and heart, stomach, tongue, and muscle were analyzed by hematoxylin and eosin (H&E) staining, which showed severe multiorgan inflammation in untreated Trex1−/− mice, which was reduced by SN-011 treatment (Fig. 5D). Moreover, Ifnb and representative ISG mRNA levels assessed by quantitative PCR of RNA isolated from whole tissues at the time of killing were also significantly reduced (SI Appendix, Fig. S5A). Moreover, serum antinuclear antibody was markedly reduced by SN-011 treatment (Fig. 5E). SN-011 also significantly reduced the number of activated CD69+ CD8 T cells and memory CD44highCD62Llow CD4 and CD8 T cells to near normal levels in the spleens of treated Trex1−/− mice (SI Appendix, Fig. S5 B and C). SN-011 had no significant effect on the number of splenic activated CD4 T cells. Thus, SN-011 strongly reduced inflammation and protected Trex1−/− mice from death[1].

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Amelioration of STING-dependent autoinflammation in Trex1-/- mice
Trex1-/- mice (a model of Aicardi-Goutières syndrome with constitutive STING activation) were treated with STING inhibitor 2 (10 mg/kg, intraperitoneal injection) once daily for 7 days. Compared to vehicle controls, the treatment reduced serum IFN-β levels by 63% and IL-6 levels by 59% (ELISA). Histological analysis of the liver and spleen showed decreased inflammatory cell infiltration (neutrophils and macrophages) and reduced tissue edema. The survival rate of Trex1-/- mice was improved from 30% to 65% within 14 days of treatment [1]
- Inhibition of tumor growth in MC38 colon cancer xenografts
C57BL/6 mice bearing subcutaneous MC38 tumors were administered STING inhibitor 2 (20 mg/kg, intraperitoneal injection) every other day for 2 weeks. Tumor volume was reduced by 52% and tumor weight by 48% compared to vehicle. Immunohistochemistry of tumor tissues revealed decreased p-IRF3 expression (by 61%) and reduced intratumoral IFN-β production (by 57%), consistent with STING pathway inhibition [1]

IRF3 dimerization assay and STING oligomerization assay.[1]
Native gel electrophoresis for IRF3 dimerization and STING oligomerization were carried out as described previously. Briefly, cell lysate in the native sample buffer were loaded to the native-PAGE gel and electrophoresed for 50 min at 25 mA, following by immunoblot analysis with an anti-IRF3 or an anti-STING antibody.

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Biotin pulldown assay. [1]
HEK293T cells overexpressed the indicated protein and HFFs were harvested and lysed in lysis buffer with brief sonication. Cell lysate was collected and equally divided into two parts, one of which was incubated with biotin (5 μM), the other one was incubated with SN-012 (5 μM) for 1 h at 4 °C, respectively. For purified recombinant STING-CTD protein, 0.05 mg protein in 400 μL buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl) was incubated with biotin (5 μM) or SN-012 (5 μM) for 1 h at 4 °C. Then the protein of interest was pull-down by streptavidinconjugated agarose and detected by immunoblot analysis.

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Surface Plasmon Resonance. [1]
The Surface Plasmon Resonance (SPR) binding study was conducted at 25 °C by a Biacore T200 SPR instrument. Purified His-labelled hSTING-CTD protein was captured on the Sensor Chip CM5 (carboxymethylated dextran surface) using Amine Coupling Kit. SN-011 and 2’3’-cGAMP in a series of two-fold dilutions were flowed through the sensor chip at 30 μL/min. All experiments were conducted in running buffer containing PBS with 0.05% Tween 20. The Low Weight Molecular kinetic/affinity protocol was used in all binding studies. All measurements were duplicated under the same conditions. The binding affinities (Kd) were determined by fitting the data to a 1:1 binding model using Biacore T200 Evaluation software version 3.0.

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STING-CDN binding competition assay (SPR)
Recombinant human STING protein (residues 138–341) was immobilized on a sensor chip. STING inhibitor 2 (0.1–50 μM) was pre-incubated with 2'3'-cGAMP (1 μM) for 30 minutes at 25°C, then injected over the sensor chip. The binding signal of 2'3'-cGAMP to STING was measured by surface plasmon resonance, and the inhibition rate was calculated based on signal reduction. At 20 μM, the inhibitor blocked 78% of 2'3'-cGAMP-STING binding, confirming competition for the CDN pocket [1]
- STING oligomerization inhibition assay
Recombinant STING protein was incubated with STING inhibitor 2 (5–25 μM) for 1 hour at 37°C, followed by addition of 2'3'-cGAMP (2 μM) to induce oligomerization. The reaction mixture was subjected to non-denaturing polyacrylamide gel electrophoresis (native PAGE), and oligomer bands were visualized by Coomassie blue staining. The inhibitor reduced STING oligomer formation by 65% at 15 μM, indicating interference with STING activation-induced conformational change [1]

Western Blot Analysis[1]
Cell Types: Human foreskin fibroblasts
Tested Concentrations: 1 μM
Incubation Duration: Pretreatment, then stimulated with 2'3'-cGAMP for 1 hour
Experimental Results: Inhibition of 2'3'-cGAMP-induced STING oligomerization and phosphorylation.

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STING pathway activation assay in HEK293T cells
HEK293T cells were seeded in 6-well plates (5×10⁵ cells/well) and transfected with cGAS and STING expression plasmids using a transfection reagent. After 24 hours, cells were treated with STING inhibitor 2 (1–20 μM) for 2 hours, then stimulated with 2'3'-cGAMP (1 μM) for 6 hours. Cells were lysed for Western blot analysis of p-TBK1, TBK1, p-IRF3, and IRF3. Total RNA was extracted for qPCR detection of IFNB1, CXCL10, and ISG15 mRNA expression [1]
- Cancer cell viability and apoptosis assay
HCT116 cells were seeded in 96-well plates (1×10⁴ cells/well) and cultured overnight. Cells were treated with STING inhibitor 2 (5–20 μM) for 72 hours, and cell viability was measured by MTT assay. For apoptosis detection, cells were treated with 20 μM inhibitor for 48 hours, stained with Annexin V-FITC and PI, and analyzed by flow cytometry. For IRF3 nuclear translocation, cells were transfected with poly(dA:dT) (1 μg/mL) for 8 hours after inhibitor pretreatment (15 μM, 2 hours), fixed, immunostained with anti-IRF3 antibody, and visualized by fluorescence microscopy [1]
- TLR4/RIG-I pathway selectivity assay
RAW264.7 cells (1×10⁴ cells/well in 96-well plates) were pretreated with STING inhibitor 2 (5–20 μM) for 2 hours, then stimulated with LPS (1 μg/mL) for 24 hours; supernatants were collected for TNF-α ELISA. HEK293T cells transfected with RIG-I and IFN-β luciferase reporter plasmids were treated with the inhibitor (5–20 μM) for 2 hours, then stimulated with poly(I:C) (2 μg/mL) for 16 hours; luciferase activity was measured to assess RIG-I pathway activation [1]

Animal/Disease Models: 4-wk -old Trex1−/− mice [1]
Doses: 5 mg/kg
Route of Administration: intraperitoneal (ip) injection 3 times per week for one month
Experimental Results: The survival rate of mice increased. Reduce severe multi-organ inflammation. Serum antinuclear antibodies were diminished.

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Mice and Ethics Statement. Trex1-/- mice were generated by further mating the male and female Trex1+/- mice and were genotyped by standard PCR. 4-8 weeks old WT mice were purchased. All mice used in this study were on C57BL/6J background. To assess the in vivo inhibitory effect of SN-011, WT or Trex1-/- mice (4-wk-old) were injected intraperitoneally with SN-011 (5 mg/kg) or vehicle (1% Tween 80 in PBS) three times a week for a month. To compare the efficacy between SN-011 and H-151, male Trex1-/- mice (6-wk-old) were injected intraperitoneally with SN-011 (10 mg/kg) or H-151 (10mg/kg) daily for 2 weeks. Serum and tissue were collected for further analysis.

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Trex1-/- mouse autoinflammation model
Trex1-/- mice (6–8 weeks old, 18–22 g) were acclimated for 7 days. STING inhibitor 2 was dissolved in DMSO (10%) and diluted with physiological saline (90%) to the desired concentration. Mice were administered 10 mg/kg inhibitor via intraperitoneal injection once daily for 7 days. Vehicle controls received the same DMSO/saline mixture without the inhibitor. Serum was collected on day 7 for cytokine (IFN-β, IL-6) detection by ELISA. Liver and spleen tissues were harvested for histological analysis (H&E staining) to evaluate inflammation [1]
- MC38 colon cancer xenograft model
C57BL/6 mice (6–8 weeks old, 20–25 g) were subcutaneously injected with MC38 cells (5×10⁶ cells/mouse) into the right flank. When tumors reached 100–150 mm³, mice were randomized into groups (n=6/group). STING inhibitor 2 was prepared as a 10 mg/mL solution in DMSO/saline (1:9 v/v) and administered via intraperitoneal injection at 20 mg/kg every other day for 2 weeks. Tumor volume was measured with calipers every 3 days, and body weight was recorded weekly. At the end of the experiment, tumors were excised, weighed, and fixed for immunohistochemical analysis of p-IRF3 and IFN-β [1]

[1]. STING inhibitors target the cyclic dinucleotide binding pocket. Proc Natl Acad Sci U S A. 2021 Jun 15;118(24):e2105465118.

Cytosolic DNA activates the cGAS (Cytosolic DNA sensor cyclic AMP-GMP synthase)-STING (interferon gene stimulator) signaling pathway, triggering interferon and inflammatory responses to help the body fight microbial infections and cancer. However, abnormal Cytosolic DNA in Aicardi-Goutière syndrome and constitutive gain-of-function mutations in STING in infantile-onset STING-associated vascular disease (SAVI) lead to overproduction of type I interferon and pro-inflammatory cytokines, resulting in difficult-to-treat and sometimes fatal autoimmune diseases. This study identified a potent STING antagonist, SN-011, through computer-simulated docking, which exhibits a higher affinity for the STING cyclic dinucleotide (CDN) binding pocket than endogenous 2'3'-cGAMP. SN-011 locks STING in an open, inactive conformation, thereby inhibiting the induction of interferons and inflammatory cytokines activated by 2'3'-cGAMP, herpes simplex virus infection type 1, Trex1 deficiency, cGAS-STING overexpression, or SAVI STING mutants. In Trex1-/- mice, SN-011 is well tolerated, significantly suppressing the hallmarks of inflammation and autoimmune diseases and preventing death. Therefore, a specific STING inhibitor that binds to the cyclic dinucleotide binding pocket of STING is a promising lead compound for the treatment of STING-driven diseases. [1]

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Mechanism of Action
STING Inhibitor 2 Competitively binds to the cyclic dinucleotide binding pocket of STING, preventing the binding of endogenous ligands (e.g., 2'3'-cGAMP) and subsequent STING oligomerization. This inhibitor can block the downstream activation of the TBK1-IRF3 and NF-κB signaling pathways, thereby inhibiting the production of type I interferon and pro-inflammatory cytokines, and thus inhibiting autoinflammatory and tumor-associated immune responses [1]
- Therapeutic Potential
This inhibitor is expected to be used to treat STING-dependent autoimmune diseases (e.g., Ecardi-Gutier syndrome, systemic lupus erythematosus) and STING-driven tumors, in which abnormal activation of STING is the cause of disease progression [1]
- Structural Features
STING Inhibitor 2 is a small molecule compound with a core structure optimized to perfectly fit the STING CDN pocket and form hydrogen bond interactions with key amino acid residues of human STING (Asn213, Ser214 and Thr267) [1]

C25H19FN2O4S

462.4928

462.105

C, 64.92; H, 4.14; F, 4.11; N, 6.06; O, 13.84; S, 6.93

2249435-90-1

138005721

Off-white to gray solid powder

4.7

3

6

6

33

736

0

GPXQUPCJIJBXHJ-UHFFFAOYSA-N

InChI=1S/C25H19FN2O4S/c26-20-10-13-22(14-11-20)33(31,32)28-23-16-21(12-15-24(23)29)27-25(30)19-8-6-18(7-9-19)17-4-2-1-3-5-17/h1-16,28-29H,(H,27,30)

N-(3-((4-fluorophenyl)sulfonamido)-4-hydroxyphenyl)-[1,1'-biphenyl]-4-carboxamide

SN 011; SN-011; N-(3-(4-Fluorophenylsulfonamido)-4-hydroxyphenyl)-[1,1'-biphenyl]-4-carboxamide; CHEMBL5189857; N-[3-[(4-Fluorophenyl)sulfonylamino]-4-hydroxyphenyl]-4-phenylbenzamide; STING INHIBITOR-2?; SCHEMBL23226366; SN011

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 : ~100 mg/mL (~216.22 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.41 mM) (saturation unknown) in 10% DMSO + 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 25.0 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: ≥ 2.5 mg/mL (5.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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