Endogenous Metabolite; second messenger; STING/stimulator of interferon genes
cGAMP binds to and activates the endoplasmic reticulum protein STING (Stimulator of Interferon Genes), leading to the activation of the transcription factor IRF3 and subsequent induction of type I interferons, particularly interferon-β (IFN-β).[1]

The development of mouse tumor cells is stimulated by cGAMP disodium [2]. In vitro, human and mouse dendritic cells are directly activated by cGAMP disodium [2]. Patient fibroblasts exhibited enhanced transcription of IFNB1 but not of genes producing tumor factor (TNF), interleukin 6 (IL6), or interleukin 1 (IL1) when treated with cGAMP disodium [3]. The endoplasmic reticulum (ER) resident protein STING is activated by cGAMP disodium, which results in the induction of an antiviral state and type I segregin walking [4].

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Chemical synthesis of cGAMP was performed, and the synthesized molecule was identical to the endogenous cGAMP purified from L929 cells as confirmed by mass spectrometry.[1]
cGAMP induced IFNβ RNA and protein expression in L929 mouse fibrosarcoma cells after introduction into the cells via digitonin permeabilization. The induction was dose-dependent, with robust IFNβ RNA induction observed at concentrations as low as 10 nM.[1]
cGAMP was significantly more potent than cyclic-di-GMP (c-di-GMP) in inducing IFNβ protein in L929 cells, as measured by ELISA.[1]
cGAMP production was triggered in mammalian cells (L929, THP1, MEF, BMDM) upon transfection with various DNA sequences (e.g., ISD, poly[dA:dT], HT-DNA) or infection with DNA viruses (HSV-1ΔICP34.5, Vaccinia virus), but not upon RNA virus (VSV) infection or RNA transfection.[1]
cGAMP activity was heat-resistant, resistant to Benzonase (DNA/RNA digesting enzyme) and proteinase K treatment, and its production in cytosolic extracts required the presence of both ATP and GTP.[1]
Activation of IRF3 by cGAMP was dependent on STING, as demonstrated using L929 cells with STING knockdown (L929-shSTING) and HEK293T cells ectopically expressing STING.[1]
Direct binding of cGAMP to the recombinant STING protein (residues 139-379) was demonstrated using a UV-induced crosslinking assay with radiolabeled [³²P]-cGAMP. Binding was competed by unlabeled (cold) cGAMP, c-di-GMP, and c-di-AMP, but not by ATP or GTP.[1]

The splenocytes of immunized mice are stimulated to produce suggestive cytokines by cGAMP disodium (5 μg)—a nasal mucosal adjuvant [2].

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The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. In this study, researchers show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity – a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines[2].

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When used as a mucosal adjuvant via intranasal co-administration with the model antigen ovalbumin (OVA) in C57BL/6 mice, cGAMP significantly enhanced antigen-specific adaptive immune responses. It promoted higher OVA-specific total IgG, IgG1, IgG2c, and mucosal IgA antibody titers in sera and nasal lavage compared to OVA alone or other adjuvants like c-di-AMP and CTB.[2]
Spleen cells from mice immunized with OVA plus cGAMP exhibited significantly enhanced antigen-specific proliferation capacity upon re-stimulation with OVA in vitro, as measured by ³H-thymidine incorporation.[2]
Upon OVA re-stimulation, spleen cells from cGAMP-adjuvanted mice produced significantly higher numbers of IFN-γ and IL-2 secreting cells (indicative of a Th1 response) and IL-4 secreting cells (indicative of a Th2 response) in ELISPOT assays, compared to OVA alone. Notably, the induction of IL-17 secreting cells (Th17 response) was comparatively lower than that induced by c-di-AMP, indicating a distinct cytokine profile favoring a balanced Th1/Th2 response with moderated Th17 activity.[2]

Cytosolic DNA induces type I interferons and other cytokines that are important for antimicrobial defense but can also result in autoimmunity. This DNA signaling pathway requires the adaptor protein STING and the transcription factor IRF3, but the mechanism of DNA sensing is unclear. We found that mammalian cytosolic extracts synthesized cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP) in vitro from adenosine triphosphate and guanosine triphosphate in the presence of DNA but not RNA. DNA transfection or DNA virus infection of mammalian cells also triggered cGAMP production. cGAMP bound to STING, leading to the activation of IRF3 and induction of interferon-β. Thus, cGAMP functions as an endogenous second messenger in metazoans and triggers interferon production in response to cytosolic DNA[1].

In vitro stimulation of primary cells[2]
The culture medium of primary cells was supplemented with 5 µg/ml (murine cells) or 60 µg/ml (human cells) of c-di-AMP or cGAMP or left without additive. Cells were incubated for 24 h at 37°C.

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Scrape loading[3]
HEK STING cells were seeded at a density of 2.5 × 105 cells ml−1 in 96-well plates. After 16 h cGAMP(2′-5′) was added to the medium to a final concentration of 50 μg ml−1. Monolayers of cells were manually wounded by six scratches per well using an 18G needle. Images were acquired after 4–8 h.

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To test the generation of a STING activator, L929-shSTING cells were transfected with DNA. Cytoplasmic extracts from these cells were mixed with THP1 or Raw264.7 cells that had been permeabilized with perfringolysin O (PFO). The PFO treatment allowed cytoplasmic exchange while retaining organelles. Activation of IRF3 in the permeabilized cells was assessed by monitoring its phosphorylation and dimerization using native gel electrophoresis.[1]
To detect cGAMP activity in vitro, L929-shSTING cytosolic extracts (S100) were incubated with DNA (e.g., HT-DNA) in the presence of ATP. The reaction mixture was heated to 95°C to denature proteins, and the supernatant was then incubated with PFO-permeabilized Raw264.7 or THP1 cells. IRF3 dimerization in the permeabilized cells served as a readout for cGAMP activity.[1]
For functional assays, cGAMP (chemically synthesized or derived from cells) was introduced into cells via digitonin permeabilization. After incubation for indicated times (e.g., 8 hours), IFNβ mRNA levels were measured by quantitative RT-PCR, and IFNβ protein secretion was measured by ELISA. IRF3 activation was analyzed by native polyacrylamide gel electrophoresis to detect dimer formation.[1]
To assess cGAMP production during viral infection, L929 or THP1 cells were infected with HSV-1ΔICP34.5 (DNA virus) or Vaccinia virus. Cell extracts were prepared, and an aliquot was heated to 95°C. The heat-resistant supernatant was then tested for its ability to induce IRF3 dimerization in PFO-permeabilized Raw264.7 cells. Another aliquot of the extract was fractionated by reverse-phase HPLC, and cGAMP levels in fractions were quantified using nano-liquid chromatography-mass spectrometry with selective reaction monitoring (SRM).[1]

Animal/Disease Models: Female C57BL/6 (H-2b) mice 6-8 weeks old [2]
Doses: 5 µg
Route of Administration: Nostril mucosal adjuvant
Experimental Results: Compared with serum from OVA-immunized mice, using cGAMP adjuvant Ovalbumin (OVA)-specific IgA and total IgG as well as IgG1 and IgG2c titers were higher in the serum of OVA-immunized mice.

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Mouse immunization experiments[2]
Five animals per group were immunized i. n. on days 0, 14 and 28. Animals were anesthetized with Isoflurane and treated 10 µl per nostril with 15 µg OVA alone or co-administered with 5 µg per dose of c-di-AMP, cGAMP or cholera toxin B subunit in Ampuwa or with Ampuwa alone in the control group (mock immunization). On day 42 after immunization animals were sacrificed and samples were collected.

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Female C57BL/6 mice (6-8 weeks old) were immunized intranasally under isoflurane anesthesia on days 0, 14, and 28. Each mouse received 10 µL per nostril of a solution containing 15 µg of OVA alone, or co-administered with 5 µg per dose of cGAMP (dissolved in water), or with control adjuvants (c-di-AMP or cholera toxin B subunit). The control group received water alone (mock). Mice were sacrificed on day 42 post-immunization, and samples (blood, spleen, cervical lymph nodes, nasal lavage) were collected for analysis.[2]

The study points out that molecules produced by mammals themselves, such as cGAMP, are expected to have very low toxicity. [2]

[1]. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science. 2013 Feb 15;339(6121):826-30.

[2]. Cyclic GMP-AMP displays mucosal adjuvant activity in mice. PLoS One. 2014 Oct 8;9(10):e110150.

[3]. Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP. Nature. 2013 Nov 28;503(7477):530-4.

The innate immune defenses of multicellular organisms against microbial pathogens require intercellular cooperation. Communication between immune cells is typically attributed to soluble protein factors secreted by pathogen-sensing cells. Cytokines, such as type I interferon (IFN), alert uninfected cells to potential pathogen attack. Furthermore, they synergize with chemokines to guide specialized immune cells in controlling and clearing microbial infections. Multiple receptors and signaling pathways link pathogen sensing to cytokine induction, and cytoplasmic recognition of nucleic acids appears crucial for the activation of type I interferon (a key regulator of antiviral immunity). Cytoplasmic DNA is sensed by the circular GMP-AMP (cGAMP) synthase (cGAS) receptor, which catalyzes the synthesis of the second messenger cGAMP (2'-5'). This molecule, in turn, activates the endoplasmic reticulum (ER)-resident receptor STING, thereby inducing an antiviral state and promoting the secretion of type I interferon. We found that in mouse and human cells, cGAMP (2'-5') synthesized by cGAS is transferred from the producing cell to neighboring cells via gap junctions and promotes STING activation in neighboring cells, thereby exerting antiviral immune effects independently of the type I interferon signaling pathway. Consistent with the limited cargo specificity of connexins (proteins that assemble gap junction channels), most of the connexins tested confer this bystander immunity, suggesting that this local immune synergy has broad physiological significance. In summary, these observations suggest that cGAS-triggered cGAMP (2'-5') transfer is a novel host strategy that can rapidly deliver antiviral immunity via transcription-independent horizontal transfer. [3] cGAMP (circular GMP-AMP) is the first endogenous cyclic dinucleotide second messenger discovered in metazoans. It is synthesized in the cytosol of mammalian cells by an unknown enzyme (later identified as cGAS) whose synthesis is in response to the presence of cytoplasmic DNA, which serves as a danger signal. cGAMP then binds directly to the adaptor protein STING, triggering a signaling cascade that leads to activation of TBK1 kinase, phosphorylation and dimerization of transcription factor IRF3, and subsequent induction of type I interferon and other cytokines. This pathway is crucial for the innate immune defense against DNA viruses and intracellular bacteria and may also be associated with autoimmune diseases. This study shows that cGAMP has potent interferon-inducing activity and therefore has potential applications in immunotherapy or as a vaccine adjuvant. [1]

C20H25N10NAO13P2

698.408995389938

718.063

2407516-83-8

cGAMP diammonium;cGAMP;849214-04-6

137120249

White to off-white solid powder

Endogenous Metabolite

5

19

2

47

1290

8

O[C@@H]1[C@]2([H])OP(OC[C@@]3([H])O[C@@H](N4C=NC5=C(N=CN=C45)N)[C@H](O)[C@]3([H])OP(O)(=O)OC[C@@]2([H])O[C@H]1N1C=NC2C(N=C(N)NC1=2)=O)(O)=O.[NaH]

MFPHQIYDBUVTRL-DQNSRKNCSA-L

InChI=1S/C20H24N10O13P2.2Na/c21-14-8-15(24-3-23-14)29(4-25-8)18-10(31)12-6(40-18)1-38-45(36,37)43-13-7(2-39-44(34,35)42-12)41-19(11(13)32)30-5-26-9-16(30)27-20(22)28-17(9)33;;/h3-7,10-13,18-19,31-32H,1-2H2,(H,34,35)(H,36,37)(H2,21,23,24)(H3,22,27,28,33);;/q;2*+1/p-2/t6-,7-,10-,11-,12-,13-,18-,19-;;/m1../s1

disodium;2-amino-9-[(1S,6R,8R,9R,10S,15R,17R,18R)-17-(6-aminopurin-9-yl)-9,18-dihydroxy-3,12-dioxido-3,12-dioxo-2,4,7,11,13,16-hexaoxa-3λ5,12λ5-diphosphatricyclo[13.3.0.06,10]octadecan-8-yl]-1H-purin-6-one

Cyclic GMP-AMP; G14522; Cyclic GMP-AMP disodium;3',3'-cGAMP disodium

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 : ~180 mg/mL (~250.57 mM)

Solubility in Formulation 1: 100 mg/mL (139.20 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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