STING/stimulator of interferon genes

By causing the same closed conformation, SR-717 activates STING and opens up possibilities for investigating systemic STING agonists in many contexts, such as anti-tumor immunity [1]. In THP1 cells and primary human peripheral blood mononuclear cells, SR-717 (3.8 μM) stimulates PD-L1 expression in a STING-dependent manner [1].

SR-717 (30 mg/kg intraperitoneally once day for 1 week) demonstrated anticancer efficacy in WT or Stinggt/gt mice [1]. SR-717 (30 mg/kg intraperitoneally for 7 days) demonstrates anti-tumor efficacy; enhances activation of CD8+ T, natural killer cells, and dendritic cells in relevant organs; and promotes antigen cross-priming [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].

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.

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

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Mouse studies. [1]
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.

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

Stimulator of interferon genes (STING) links innate immunity to biological processes ranging from antitumor immunity to microbiome homeostasis. Mechanistic understanding of the anticancer potential for STING receptor activation is currently limited by metabolic instability of the natural cyclic dinucleotide (CDN) ligands. From a pathway-targeted cell-based screen, we identified a non-nucleotide, small-molecule STING agonist, termed SR-717, that demonstrates broad interspecies and interallelic specificity. A 1.8-angstrom cocrystal structure revealed that SR-717 functions as a direct cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) mimetic that induces the same "closed" conformation of STING. SR-717 displayed antitumor activity; promoted the activation of CD8+ T, natural killer, and dendritic cells in relevant tissues; and facilitated antigen cross-priming. SR-717 also induced the expression of clinically relevant targets, including programmed cell death 1 ligand 1 (PD-L1), in a STING-dependent manner.[1]

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To address the limitations of intratumoral delivery, we have identified the SR-717 chemical series of functional cGAMP mimetic STING agonists, which, after systemic administration, were demonstrated to promote antitumor immunity and activate CD8+ T cells within tumors and the dLN, as well as activate NK cells within the dLN. The systemic administration of SR-717 reduced tumor burden in the B16.F10 melanoma model with a level of efficacy that was observed to be superior than what is observed for anti–PD-1 or anti–PD-L1 therapy in this particular poorly immunogenic model. Importantly, systemic administration of SR-717 produced substantial efficacy despite inducing modest levels of IFN-β, suggesting that the threshold for efficacy in tumor models may be far lower than previously reported and can be achieved without considerable toxicity. It is also of potential critical importance that STING activation by SR-717 was found to induce the expression of PD-L1 in a STING-dependent fashion. These results have important implications for the choice of agent to be combined with a STING agonist, as well as the relative timing of a dosing regimen, in the context of cancer treatment. Presumably, it would be unproductive to treat with an agent that increases the relative abundance of the target of the second agent. The ability of SR-717 to induce the cGAMP-induced closed STING conformation, in contrast to open conformation–inducing ligands, enables exploration of the relative importance of different potential scaffolding functions in vivo and in the context of systemic distribution in settings of antitumor immunity and beyond. Differential pathway activation associated with the recognition of bacterial-derived CDNs [e.g., di-GMP derived from commensal bacteria] as compared with endogenously produced cGAMP, derived from cytosolic DNA as a result of diverse pathological events (e.g., genomic instability), is readily conceivable and most likely probable. Each class of agonist may provide differential therapeutic benefits depending on the setting.[1]

C15H9F2N5O3

345.260469198227

345.067345

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

2375420-34-9

SR-717;2375421-09-1

139434659

White to off-white solid powder

1.4

2

8

4

25

512

0

FC1C(=CC(C(=O)O)=C(C=1)NC(C1=CC=C(N=N1)N1C=NC=C1)=O)F

WEBVQIJGIZVRGA-UHFFFAOYSA-N

InChI=1S/C15H9F2N5O3/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)

4,5-difluoro-2-[(6-imidazol-1-ylpyridazine-3-carbonyl)amino]benzoic acid

SR-717 (free acid); CHEMBL4867353; 4,5-difluoro-2-[(6-imidazol-1-ylpyridazine-3-carbonyl)amino]benzoic acid; 2-(6-(1H-Imidazol-1-yl)pyridazine-3-carboxamido)-4,5-difluorobenzoic acid; 4,5-difluoro-2-{[6-(1H-imidazol-1-yl)pyridazine-3-carbonyl]amino}benzoic acid; 4,5-difluoro-2-[6-(1H-imidazol-1-yl)pyridazine-3-amido]benzoic acid;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)