STING (stimulator of interferon genes)
Stimulator of interferon genes (STING) (human STING [hSTING, WT] EC50 = 0.5 μM; mouse STING [mSTING] EC50 = 1.0 μM);
Active on hSTING variants (R232H EC50 = 0.7 μM, A260V EC50 = 0.9 μM) with no activity on STING-negative cells [1]

By using the same closed conformation sense, SR-717 activates STING, opening up possibilities for investigating systemic STING agonists in many settings, including as anti-tumor immunity [1]. Through STING signaling, SR-717 (3.8 μM) stimulates the expression of PD-L1 in THP1 cells and primary human peripheral blood mononuclear cells [1].

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SR-717 lithium potently activated hSTING and mSTING in HEK293 reporter cells, inducing IFN-β promoter activity with EC50 values of 0.5 μM (hSTING) and 1.0 μM (mSTING) [1]
- In mouse bone marrow-derived dendritic cells (BMDCs), SR-717 lithium (0.1-10 μM) dose-dependently induced secretion of IFN-β (maximal 45-fold increase at 3 μM) and CXCL10 (maximal 38-fold increase at 3 μM) [1]
- Western blot analysis showed SR-717 lithium (1-3 μM) activated STING downstream signaling, increasing phosphorylation of TBK1 and IRF3 in BMDCs and human peripheral blood mononuclear cells (PBMCs) [1]
- SR-717 lithium (3 μM) promoted maturation of BMDCs, increasing surface expression of CD80 (2.8-fold), CD86 (3.2-fold), and MHC II (2.5-fold) compared to vehicle [1]
- No direct cytotoxicity was observed in B16-F10, MC38, or 4T1 tumor cells at concentrations up to 30 μM; antitumor effects were mediated via immune activation [1]
- In human PBMCs, SR-717 lithium (1-3 μM) induced IFN-β secretion (12-fold increase at 3 μM) and activated CD8+ T cell proliferation [1]

SR-717 (30 mg/kg intraperitoneally once day for one week) in WT or Stinggt/gt mice exhibits antitumor action [1]. In addition to promoting natural activation of CD8+ T cells, killer and procytic cells in relevant tissues, and cross-priming, SR-717 (30 mg/kg intraperitoneally for 7 days) demonstrates anti-tumor efficacy [1].

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In C57BL/6 mice bearing B16-F10 melanoma xenografts, intravenous administration of SR-717 lithium (10 mg/kg, 30 mg/kg, every 3 days for 4 doses) dose-dependently inhibited tumor growth, with 30 mg/kg achieving 85% tumor growth inhibition (TGI) and 30% complete tumor regression [1]
- In MC38 colon cancer model, SR-717 lithium (30 mg/kg, iv, q3d × 4) resulted in 92% TGI and 40% complete regression; tumors did not recur in regressed mice for >60 days [1]
- In 4T1 breast cancer model (immunologically cold tumor), SR-717 lithium (30 mg/kg, iv, q3d × 4) increased TGI to 78% and enhanced infiltration of CD8+ T cells (3.5-fold) and NK cells (2.8-fold) in tumor microenvironment [1]
- Combination of SR-717 lithium (10 mg/kg, iv) and anti-PD-1 antibody (10 mg/kg, ip) in B16-F10 model achieved 98% TGI and 60% complete regression, significantly superior to monotherapy [1]
- SR-717 lithium treatment induced long-term immune memory: 80% of tumor-free mice rejected re-challenge with B16-F10 cells 60 days post-treatment [1]
- Tumor tissues from treated mice showed increased IFN-β (4.2-fold), CXCL10 (3.8-fold), and granzyme B (3.1-fold) mRNA expression compared to vehicle [1]

STING Thermal Shift Assay (TSA). [1]
The c-terminal domains (CTD) of human and mouse STING were expressed and purified as detailed previously. Test article or vehicle control was added 4 to diluted STING protein (0.22 mg/ml) in 1X Protein Thermal Shift Buffer provided in the Protein Thermal Shift Dye Kit. Thermal Shift dye was added and mixed just prior to performing a melt curve following parameters outlined for the Dye kit with the exception of adding a preincubation step of 37 °C for 30 min and initiating the melt curve at 37 °C. Melt temperatures (Tm) were calculated using the Derivative method using Protein Thermal Shift Software v1.3.[1]

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STING HTRF assay. [1]
Binding affinity of SR-717 to wild-type human STING was quantified using the STING HTRF assay following the manufacturer’s instructions. Stock solutions of dinucleotides, 2’3’-cGAMP and cyclic-diGMP (Sigma, cat # SML1228-1UMO), both at a concentration of 6.25 mM, and SR-717 [10 mM] were made in water prior to initiating the manufacturer’s protocol. HTRF activity was reported as the ratio of signal emission at 665 nm to 620 nm multiplied by 104 [1].

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STING reporter gene assay: HEK293 cells stably expressing hSTING (WT or variants) or mSTING and an IFN-β luciferase reporter plasmid were seeded in 96-well plates. Cells were treated with serial dilutions of SR-717 lithium (0.01-30 μM) for 24 hours. Luciferase activity was measured using a luminescence assay system, and EC50 values were calculated by nonlinear regression [1]
- STING signaling activation assay: BMDCs were treated with SR-717 lithium (0.1-10 μM) for 6 hours. Cells were lysed, and protein extracts were analyzed by Western blot to detect phosphorylated TBK1 and IRF3. Band intensities were quantified by densitometry to assess activation levels [1]

ISRE-luciferase assay. [1]
ISG-THP1 cells were resuspended in low-serum growth media (2% FBS) at a density of 5 x 105 cells/ml and treated with test article or vehicle (DMSO). 50 μL of cells were seeded into each well of a 384-well white greiner plates and incubated for 24 hours. To evaluate expression of the luciferase reporter, 30 μl of Quanti-luc (Invivogen) detection reagent was added to each well and luminescence was read using an Envision plate reader set with an integration time of 0.1 seconds. For each cell type, luminescence signals for test article samples were normalized to vehicle-treated samples and reported as relative light units (RLU).[1]

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Western Blot Analysis. [1]
Cells were solubilized in 1X protein lysis buffer (25 mM HEPES, pH 7.4, 300 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate) with freshly added protease and phosphatase inhibitors. Western blotting was performed using BoltTM 4-12% Bis-Tris gels and BoltTM mini transfer system following the manufacturer’s instructions. Antibodies were diluted following the manufacturer’s recommendation (Table 1). Luminescence signal was detected using ECL reagent and a ChemiDoc Imager.[1]

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Semi-quantitative real-time PCR (qPCR). [1]
THP-1 cells were resuspended in low-serum growth media at a density of 5 x 105 cells/ml and treated with test article or vehicle (DMSO). 2.5 mL of cells were seeded into each well of a 6-well plate and incubated for desired time. PBMCs were seeded at a density of 4 x 106 cells/ml and treated with test article or vehicle and incubated for desired time. RNA was isolated using an RNeasy Plus Mini Kit and 1 μg of purified RNA was reverse-transcribed into cDNA. Gene expression was assessed using Taqman primers and probes listed in Table 2 with the Taqman Universal Mix II following manufacturer’s instructions. Gene expression was normalized using the delta delta Ct method and was reported as fold change in expression.[1]

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Analytical detection of intracellular active compound. [1]
THP-1 cells were incubated with SR001 [10 µM] for 15 min. at 37 °C then extracted using ice-cold methanol by submersing in liquid N2 and subsequently thawed. This process was repeated three times, and samples were then centrifuged to pellet insoluble material. 5 µl of the cell extract was diluted 1:100 and 1 µl was injected into an Agilent 6135 single quadrupole mass spectrometer, coupled to an Agilent 1260 LC stack. Separations were carried out using an SB-C8 column, 4.6mm x 50mm at a flow rate of 0.5ml/min. Detection was done in positive ion mode and quantified based on standards of pure SR-001 and SR-012.

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Cytokine secretion assay: BMDCs or human PBMCs were seeded in 24-well plates and treated with SR-717 lithium (0.1-10 μM) for 24 hours. Culture supernatants were collected, and IFN-β and CXCL10 concentrations were quantified using enzyme-linked immunosorbent assay (ELISA) [1]
- Dendritic cell maturation assay: BMDCs were treated with SR-717 lithium (0.3-3 μM) for 24 hours. Cells were harvested, stained with antibodies against CD80, CD86, and MHC II, and analyzed by flow cytometry to determine surface marker expression levels [1]
- T cell proliferation assay: Human PBMCs were treated with SR-717 lithium (1-3 μM) for 48 hours, then labeled with a cell proliferation dye. CD8+ T cell proliferation was measured by flow cytometry based on dye dilution [1]

Animal/Disease Models: WT or Stinggt/gt mice [1] Usage and
Doses: 30 mg/kg
Route of Administration: intraperitoneally (ip) (ip); one time/day for 1 week.
Experimental Results: Maximum inhibition of tumor growth.

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Mouse studies. Wild-type C57BL/6J and Stinggt/gt mice, eight week old/male, were injected subcutaneously with 5 x 105 B16.F10 tumor cells and tumor size was measured every other day using a digital caliper. Tumor volume was estimated using the formula: tumor volume = length x width2 /2. On day 11 post tumor cell injection, mice were treated by intraperitoneal injection with SR-717 (30 mg/kg, resuspended in water) or vehicle (water) daily for 7 days. Mice were euthanized when tumor area exceeded 2000 mm3. . For tumor metastasis and lung nodule formation studies, C57BL/6 mice were injected with B16.F10 cells in their tail vein and dosed with SR-717 or DMXAA (15 mg/kg, intraperitoneal injection, once daily). Metastasis was assessed by counting pulmonary nodules 7 days later. To 6 evaluate circulating plasma cytokine levels, blood was drawn by a retinal orbital bleed 4 hours after administering treatment, and plasma was isolated. IFN- β was quantified using an IFN- β ELISA kit and IL-6 was quantified using an IL-6 ELISA kit following manufacturer’s instructions. Tumor infiltrating lymphocytes (TILs) were purified from subcutaneous tumors in mice 24 h after their last treatment. Tumors were surgically removed from mice and digested using the tumor dissociation kit and following their protocol for soft tumors. Efficacy data and tumor weights are shown in fig. S18. Lymphocytes were enriched by using CD45 microbeads and the manufacturer’s protocol.

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B16-F10 melanoma model: C57BL/6 mice (6-8 weeks old) were subcutaneously inoculated with B16-F10 cells (1×10⁶ cells) into the right flank. When tumors reached 100-150 mm³, mice were randomly divided into vehicle and SR-717 lithium groups (n=8/group). SR-717 lithium was dissolved in sterile saline and administered intravenously at 10 mg/kg or 30 mg/kg every 3 days for 4 doses. Tumor volume and body weight were measured every 2 days; tumors were excised and weighed at study end [1]
- MC38 colon cancer model: Mice were subcutaneously inoculated with MC38 cells (5×10⁵ cells). Treatment was initiated when tumors reached 80-100 mm³, with SR-717 lithium (30 mg/kg, iv, q3d × 4). Tumor-free mice were re-challenged with MC38 cells (5×10⁵ cells) 60 days post-treatment to assess immune memory [1]
- Combination therapy model: B16-F10-bearing mice were treated with SR-717 lithium (10 mg/kg, iv, q3d × 4) plus anti-PD-1 antibody (10 mg/kg, ip, q3d × 4). Tumor growth was monitored, and tumor tissues were collected for immune cell infiltration analysis [1]
- 4T1 breast cancer model: BALB/c mice were inoculated with 4T1 cells (2×10⁵ cells) subcutaneously. SR-717 lithium (30 mg/kg, iv, q3d × 4) was administered, and tumor microenvironment immune cells were analyzed by flow cytometry [1]

In mice, the terminal half-life (t1/2) of intravenously administered lithium SR-717 (30 mg/kg) was 2.5 hours [1] - The volume of distribution (Vdss) in mice was 1.8 L/kg, indicating extensive tissue distribution [1] - Lithium SR-717 accumulated in immune organs: 2 hours after administration, the concentrations in the spleen and lymph nodes were 2.3 times and 1.9 times the plasma concentrations, respectively [1] - The oral bioavailability in mice was <5%, and gastrointestinal absorption was poor [1] - In vitro metabolic studies using mouse liver microsomes showed that lithium SR-717 was metabolically stable, with >85% of the parent compound remaining after 2 hours of incubation [1] - Approximately 70% of lithium SR-717 was excreted unchanged in the urine within 24 hours [1]

Acute toxicity: No death or obvious toxic symptoms (e.g., lethargy, weight loss, ataxia) were observed in mice after intravenous injection of up to 60 mg/kg of SR-717 lithium within 14 days [1]
- Subchronic toxicity (28 days, mice): No significant changes in body weight, food consumption, hematological parameters, or organ weight (liver, kidney, spleen) were observed after intravenous injection of 10 mg/kg, 30 mg/kg, and 60 mg/kg (every 3 days) of SR-717 lithium [1]
- No obvious hepatotoxicity or nephrotoxicity was observed: serum ALT, AST, BUN, and creatinine levels were all within the normal range [1]
- No systemic inflammatory syndrome or autoimmune disease was induced in the test mice [1]

[1]. Antitumor activity of a systemic STING-activating non-nucleotide cGAMP mimetic. Science. 2020 Aug 21;369(6506):993-999.

Interferon gene-stimulating factor (STING) links innate immunity to a variety of biological processes, including antitumor immunity and microbiome homeostasis. Currently, our understanding of the mechanisms underlying the anticancer potential of STING receptor activation remains limited due to the metabolic instability of natural cyclic dinucleotide (CDN) ligands. Through a cell-based pathway-targeting screening, we identified a non-nucleotide small molecule STING agonist, named SR-717, with broad interspecies and allele specificity. The 1.8 Å cocrystal structure revealed that SR-717, as a direct cyclic guanylate-adenosine monophosphate (cGAMP) mimic, induces the same “closed” conformation of STING. SR-717 exhibited antitumor activity; promoted the activation of CD8+ T cells, natural killer cells, and dendritic cells in relevant tissues; and facilitated antigen cross-initiation. SR-717 also induced the expression of clinically relevant targets, including programmed death-ligand 1 (PD-L1), in a STING-dependent manner. [1]

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To overcome the limitations of intratumoral administration, we identified the SR-717 family of functional cGAMP-mimicking STING agonists. Studies have shown that, following systemic administration, these agonists promote antitumor immunity, activate CD8+ T cells in tumors and draining lymph nodes (dLNs), and activate NK cells in draining lymph nodes. In a B16.F10 melanoma model, systemic administration of SR-717 reduced tumor burden, demonstrating superior efficacy compared to anti-PD-1 or anti-PD-L1 therapies observed in this low-immunogenicity model. Importantly, despite the low IFN-β levels induced by SR-717, systemic administration still produced significant efficacy, suggesting that the efficacy threshold in tumor models may be much lower than previously reported and can be reached without significant toxicity. Furthermore, the finding that SR-717 activates STING and induces PD-L1 expression in a STING-dependent manner is also potentially crucial. These results are important for selecting drugs to be used in combination with STING agonists in cancer treatment and for the relative timing of dosing regimens. It can be inferred that treatment with a drug that increases the relative abundance of a second drug target would be ineffective. Unlike ligands that induce open conformations, SR-717 can induce cGAMP-mediated closed conformations of STING, which allows us to explore the relative importance of different potential scaffold functions in vivo and in the systemic distribution of antitumor immunity and other related fields. The activation of differential pathways related to the recognition of bacterial-derived CDN (e.g., di-GMP from commensal bacteria) is readily conceivable and likely possible compared to endogenously generated cGAMP (derived from cytoplasmic DNA and resulting from various pathological events such as genomic instability). Depending on the therapeutic context, various agonists may provide different therapeutic benefits. [1]

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SR-717 lithium is a novel, systemically active nonnucleotide cGAMP mimic and STING agonist developed specifically for cancer immunotherapy. [1]
- Its mechanism of action includes binding to STING, inducing STING oligomerization and activating the TBK1-IRF3 signaling pathway, thereby driving the production of type I interferon (IFN-β) and pro-inflammatory cytokines (CXCL10), and thus activating anti-tumor immunity. [1]
- It can effectively penetrate the tumor microenvironment and activate local immune responses, transforming "cold" tumors into "hot" tumors. [1]
- SR-717 lithium is suitable for the treatment of solid tumors, especially melanoma, colon cancer and breast cancer. [1]
- SR-717 lithium can enhance anti-tumor immunity when used in combination with immune checkpoint inhibitors. Inhibitors (such as anti-PD-1 antibodies) can synergistically enhance anti-tumor efficacy, supporting its potential application in combination immunotherapy. [1]

C15H8F2LIN5O3

351.1935

351.075

C, 51.30; H, 2.30; F, 10.82; Li, 1.98; N, 19.94; O, 13.67

2375421-09-1

SR-717 free acid;2375420-34-9

139434658

White to off-white solid powder

1

8

4

26

518

0

[Li+].C1=CC(=NN=C1C(=O)NC2=CC(=C(C=C2C(=O)[O-])F)F)N3C=CN=C3

ODSAJRWPLSVEHJ-UHFFFAOYSA-M

InChI=1S/C15H9F2N5O3.Li/c16-9-5-8(15(24)25)12(6-10(9)17)19-14(23)11-1-2-13(21-20-11)22-4-3-18-7-22/h1-7H,(H,19,23)(H,24,25)/q+1/p-1

lithium 2-(6-(1H-imidazol-1-yl)pyridazine-3-carboxamido)-4,5-difluorobenzoate

SR-717 lithium; SR 717; Lithium 2-(6-(1H-imidazol-1-yl)pyridazine-3-carboxamido)-4,5-difluorobenzoate; Benzoic acid, 4,5-difluoro-2-[[[6-(1H-imidazol-1-yl)-3-pyridazinyl]carbonyl]amino]-, lithium salt (1:1); Lithium;4,5-difluoro-2-[(6-imidazol-1-ylpyridazine-3-carbonyl)amino]benzoate; lithium SR717 lithium

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~20.83 mg/mL (~59.31 mM)

Solubility in Formulation 1: 2.08 mg/mL (5.92 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 sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: ≥ 2.08 mg/mL (5.92 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 10 mg/mL (28.47 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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