STING/stimulator of interferon genes

ADU-S100 ammonium salts have several characteristics that improve stability and lipophilicity in comparison to endogenous and pathogen-derived cyclic dinucleotides (CDNs), thereby significantly enhancing STING signals [1]. In THP-1 human monocytes, ADU-S100 produced more type I IFN than CDA. Disulfide mixed-linked cyclic dinucleotide (CDN) derivatives, on the other hand, effectively activated all five hSTING alleles, including the refractory hSTINGREF and hSTINGQ alleles (ML RR-CDA, ML RR-S2 CDG, and ML RR-S2 cGAMP). In comparison to endogenous ML cGAMP and the TLR3 agonist Poly I:C, ADU-S100 induced the highest expression of IFN-β and the proinflammatory cytokines TNF-α, IL-6, and MCP-1 on a molar equivalent basis. In mouse bone marrow macrophages (BMM), ADU-S100 was also found to induce STING aggregation and induce TBK1 and IRF3 phosphorylation. When compared to ML cGAMP, ADU-S100 induces noticeably higher levels of IFN-α [1].

ADU-S100 outperformed endogenous ML cGAMP in its anti-tumor control capacity. In B16 tumor-bearing mice, a dose response study of ADU-S100 compounds was carried out to ascertain the best anti-tumor dose level that would maximize tumor antigen-specific CD8+ T cell responses and increase long-term survival to 50%[1].

Luciferase Assay[1]
104 HEK293T cells were seeded in 96-well plates and transiently transfected with human IFN-β firefly reporter plasmid(Fitzgerald et al., 2003) and TK-Renilla luciferase reporter for normalization. The following day, cells were stimulated with 10 μM of eachADU-S100 or 100 μg/ml DMXAA using digitonin permeabilization (50 mM HEPES, 100 mM KCL, 3 mM MgCl2, 0.1 mM DTT, 85 mM Sucrose, 0.2% BSA, 1 mM ATP, 0.1 mM GTP, 10 ug/ml digitonin) to ensure uniform uptake. After 20 min, stimulation mixtures were removed and normal media was added. After a total of 6 hours, cell lysates were prepared and reporter gene activity measured using the Dual Luciferase Assay System on a Spectramax M3 luminometer.

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Differential Scanning Fluorimetry[1]
Thermal shift assays were performed as (Cavlar et al., 2013). Assays were conducted with STING ligand binding domain at 1 mg/ml with or without various ADU-S100 at 1 mM in 20mM Tris-HCL, 150 mM NaCl, pH 7.5 and 1:500 dilution of SYPRO Orange Dye. The fluorescence as a function of temperature was recorded in a CFX 96 real time PCR machine reading on the HEX channel EX 450–490 EM 560–580 nm. The temperature gradient was from 15–80°C ramping 0.5°C per 15 seconds. Curves were fit to a Boltzmann sigmoidal to establish the midpoint of thermal unfolding (Tm).

BM-DCs from WT or STING−/− mice were stimulated with 25 μg/ml DMXAA or 100 ng/ml LPS for 4 hours. Total RNA was isolated using the RNeasy® kit and incubated with Deoxyribonuclease I, Amplification Grade. cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit and expression of cytokines was measured by real-time qRT-PCR using specific primers/probes for mouse INF-β, TNF-α, IL-6 and IL12p40, and pan-specific primers were to quantify expression of the IFN-α family. Primer sequences are listed in Table 1 in Supplementary Materials. PCR reactions were performed in the 7300 Real Time PCR system. The results are expressed as 2−ΔCt using 18s as endogenous control.

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WT BMM were stimulated with ADU-S100 at 5 μM in HBSS with the addition of Effectene transfection reagent (per kit protocol). Human PBMCs were stimulated as indicated. Stimulated cells were and assessed by real-time qRT-PCR for gene expression of IFN-β1, MCP-1, TNF-α and IL-6 using the PrimePCR RNA purification and cDNA analysis system, and run on the CFX96 gene cycler. Relative normalized expression was determined by comparing induced target gene expression to unstimulated controls, using the reference genes Gapdh and Ywhaz (mouse) and GusB and Pgk1 (human), genes confirmed to have a coefficient variable (CV) below 0.5 and M value below 1, and thus did not vary with different treatment conditions.

10~6 of B16-SIY tumor cells, 5 × 10~4 B16.F10 tumor cells, 10~5 4T-1 and CT26, or 106 other tumor cells were injected s.c. in 100 μl DPBS or HBSS on the right flank of mice. Following tumor implantation, mice were randomized into treatment groups. When tumors were 100–200 mm3 in volume (5–7 mm wide), either one single or three doses of DMXAA resuspended in 7.5% of NaHCO3, or CDNs formulated in HBSS or vehicle control, were injected IT. Measurements of tumors were performed twice per week using calipers, and the tumor volume was calculated with the formula: V= (length × width2)/2. In some experiments, tumor-free survivors were rechallenged with tumor cells on the opposite flank several weeks after the injection of the primary tumor. Naïve mice were used as controls. For the contralateral experiments, mice were implanted on both flanks and only one tumor was treated. For the B16 melanoma lung metastasis experiments, mice were implanted on the flank with 5 × 104 cells B16.F10 on day 0, and then injected intravenously with 1 × 105 cells on day 7. Lungs were harvested on day 28. Administration of compounds, measurements of tumors and counting of lung tumors were performed in a blinded fashion.[1]

[1]. Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity. Cell Rep. 2015 May 19;11(7):1018-30.

ADU-S100 (MIW815) is a synthetic cyclic dinucleotide (CDN) agonist (activator) that activates the interferon gene-stimulating factor (STING) receptor. The STING receptor is a key receptor for activating the innate (endogenous) immune system. ADU-S100 (MIW815) activates all known human and mouse STING receptors and effectively induces the expression of cytokines and chemokines, thereby generating a potent and durable antigen-specific T cell-mediated immune response against cancer cells. MIW815, a STING-activating cyclic dinucleotide agonist listed in the DrugBank database, is a synthetic cyclic dinucleotide (CDN) and an agonist of the interferon gene-stimulating factor protein (STING; transmembrane protein 173; TMEM173), possessing potential immunomodulatory and antitumor activity. After intratumoral injection, the STING agonist MIW815 binds to STING and activates the STING-mediated signaling pathway. This activates the immune response by activating certain immune cells, including dendritic cells (DCs), which in turn induces the expression of cytokines and chemokines, ultimately leading to an antigen-specific T cell-mediated immune response against cancer cells. STING is a transmembrane protein that can activate immune cells in the tumor microenvironment and plays a key role in the activation of the innate immune system.

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Tumor-initiated spontaneous T cell initiation depends on IFN-β produced by tumor-resident dendritic cells. Based on recent observations that IFN-β expression depends on the activation of the host STING pathway, we hypothesize that direct activation of the STING pathway by intratumoral (IT) injection of a specific agonist will produce an effective antitumor therapeutic effect. After a proof-of-concept study using the mouse STING agonist DMXAA showed significant therapeutic effects, we synthesized cyclic dinucleotide (CDN) derivatives that can activate all human STING alleles and mouse STING. Intrathecal injection of STING agonists significantly inhibited established tumors in mice and produced a strong systemic immune response that could clear distant metastases and provide durable immune memory. Synthetic CDN has great potential for translational application as a cancer therapeutic agent. [1]

C₂₀H₃₀N₁₂O₁₀P₂S₂

724.60

724.112

1638750-96-5

ADU-S100 disodium salt;1638750-95-4;ADU-S100 enantiomer ammonium salt;ADU-S100;1638241-89-0

123131801

White to off-white solid powder

6

20

2

46

1180

7

C1[C@@H]2[C@H]([C@H]([C@@H](O2)N3C=NC4=C(N=CN=C43)N)O)OP(=S)(OC[C@@H]5C([C@H]([C@@H](O5)N6C=NC7=C(N=CN=C76)N)OP(=O)(O1)[S-])O)[O-].[NH4+].[NH4+]

VZYXAGGQHVUTHM-LJFXOJISSA-N

InChI=1S/C20H24N10O10P2S2.2H3N/c21-15-9-17(25-3-23-15)29(5-27-9)19-12(32)13-8(38-19)2-36-42(34,44)40-14-11(31)7(1-35-41(33,43)39-13)37-20(14)30-6-28-10-16(22)24-4-26-18(10)30;;/h3-8,11-14,19-20,31-32H,1-2H2,(H,33,43)(H,34,44)(H2,21,23,25)(H2,22,24,26);2*1H3/t7-,8-,11?,12-,13-,14-,19-,20-,41?,42?;;/m1../s1

diazanium;(1R,6R,8R,9R,10S,15R,17R)-8,17-bis(6-aminopurin-9-yl)-12-oxido-3-oxo-12-sulfanylidene-3-sulfido-2,4,7,11,13,16-hexaoxa-3λ5,12λ5-diphosphatricyclo[13.2.1.06,10]octadecane-9,18-diol

ADU-S100; ML RR-S2 CDA; MIW-815; ML RR-S2 CDA ammonium salt; ADU-S100 ammonium salt; 1638750-96-5; diazanium;(1R,6R,8R,9R,10S,15R,17R)-8,17-bis(6-aminopurin-9-yl)-3,12-dioxido-3,12-bis(sulfanylidene)-2,4,7,11,13,16-hexaoxa-3lambda5,12lambda5-diphosphatricyclo[13.2.1.06,10]octadecane-9,18-diol; MIW815 ammonium salt; MIW815 (ammonium salt); ML RR-S2 CDA (ammonium salt; MIW815 ammonium salt

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)

H2O : ~100 mg/mL (~138.01 mM)
DMSO : ~15 mg/mL (~20.70 mM)
MEthanol : ~5 mg/mL (~6.90 mM)

Solubility in Formulation 1: ≥ 50 mg/mL (69.00 mM) (saturation unknown) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution.

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