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STING inhbitor 2 ( SN-011; SN011) is a novel and potent inhibitor of STING with potential anti-inflammatory activity. It can block the activation of the Sting signaling pathway.
| Targets |
STING (stimulator of interferon genes) (IC50 = 76 nM)
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|---|---|
| ln Vitro |
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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| ln Vivo |
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].
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]. |
| Enzyme Assay |
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. 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. 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. |
| Cell Assay |
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. |
| Animal Protocol |
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. 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. |
| References | |
| Additional Infomation |
Cytosolic DNA activates cGAS (cytosolic DNA sensor cyclic AMP-GMP synthase)-STING (stimulator of interferon genes) signaling, which triggers interferon and inflammatory responses that help defend against microbial infection and cancer. However, aberrant cytosolic self-DNA in Aicardi-Goutière's syndrome and constituently active gain-of-function mutations in STING in STING-associated vasculopathy with onset in infancy (SAVI) patients lead to excessive type I interferons and proinflammatory cytokines, which cause difficult-to-treat and sometimes fatal autoimmune disease. Here, in silico docking identified a potent STING antagonist SN-011 that binds with higher affinity to the cyclic dinucleotide (CDN)-binding pocket of STING than endogenous 2'3'-cGAMP. SN-011 locks STING in an open inactive conformation, which inhibits interferon and inflammatory cytokine induction activated by 2'3'-cGAMP, herpes simplex virus type 1 infection, Trex1 deficiency, overexpression of cGAS-STING, or SAVI STING mutants. In Trex1-/- mice, SN-011 was well tolerated, strongly inhibited hallmarks of inflammation and autoimmunity disease, and prevented death. Thus, a specific STING inhibitor that binds to the STING CDN-binding pocket is a promising lead compound for STING-driven disease.[1]
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| Molecular Formula |
C25H19FN2O4S
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|---|---|
| Molecular Weight |
462.4928
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| Exact Mass |
462.105
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| Elemental Analysis |
C, 64.92; H, 4.14; F, 4.11; N, 6.06; O, 13.84; S, 6.93
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| CAS # |
2249435-90-1
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| PubChem CID |
138005721
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| Appearance |
Off-white to gray solid powder
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| LogP |
4.7
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
33
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| Complexity |
736
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
GPXQUPCJIJBXHJ-UHFFFAOYSA-N
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| InChi Code |
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)
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| Chemical Name |
N-(3-((4-fluorophenyl)sulfonamido)-4-hydroxyphenyl)-[1,1'-biphenyl]-4-carboxamide
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| Synonyms |
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
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~216.22 mM)
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|---|---|
| Solubility (In Vivo) |
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. 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1622 mL | 10.8110 mL | 21.6221 mL | |
| 5 mM | 0.4324 mL | 2.1622 mL | 4.3244 mL | |
| 10 mM | 0.2162 mL | 1.0811 mL | 2.1622 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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