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| Targets |
Endogenous Metabolite; second messenger; STING/stimulator of interferon genes
cGAMP free acid targets the stimulator of interferon genes (STING) pathway in eukaryotic cells. As a cyclic dinucleotide, cGAMP is a potent agonist of STING, activating the innate immune response. In bacteria, cGAMP functions as a second messenger regulating various physiological processes including survival, adhesion, colonization, biofilm formation, and virulence. |
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| ln Vitro |
Mouse splenocytes' capacity for antigen-specific proliferation is enhanced by cGAMP [2]. In vitro, cGAMP directly stimulates human and mouse dendritic cells [2]. Patient fibroblasts exhibit enhanced IFNB1 transcription with cGAMP stimulation, but not transcription of genes encoding TNF, IL6, or interleukin 1 (IL1) [3]. Type I IFN is secreted and an antiviral state is induced when cGAMP activates the receptor STING, which is located in the endoplasmic reticulum (ER) [4].
In vitro, cGAMP free acid is a cyclic dinucleotide that activates the STING pathway and induces type I interferon production. It is a naturally occurring molecule that works as a bacterial second messenger. cGAMP regulates bacterial survival, adhesion, colonization, biofilm formation, and virulence in prokaryotes. These properties make cGAMP a valuable tool for studying bacterial signaling and innate immunity. |
| ln Vivo |
cGAMP promotes the production of antigen-specific cytokines in spleen cells of immunized mice [2].
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]. In vivo, cGAMP free acid functions as a second messenger in both prokaryotes and eukaryotes. In eukaryotic cells, it activates the STING pathway, triggering type I interferon responses. In bacteria, it regulates various physiological processes. However, detailed in vivo pharmacological data are limited, as cGAMP is a naturally occurring molecule rather than a therapeutic agent. |
| Enzyme Assay |
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].
The in vitro receptor binding assay for cGAMP involves measuring its binding to STING. The compound is incubated with recombinant STING protein in the presence of a labeled cGAMP ligand. Binding affinity is determined using surface plasmon resonance, fluorescence polarization, or isothermal titration calorimetry. STING activation can be assessed by measuring downstream signaling events such as TBK1 and IRF3 phosphorylation. |
| Cell Assay |
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. Scrape loading[4] 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. The in vitro cell-based assay for cGAMP involves culturing cells that express STING (e.g., THP-1 macrophages, dendritic cells) and treating them with the compound to assess activation of the STING pathway. Cells are treated with cGAMP at various concentrations, and type I interferon (IFN-β) production is measured by ELISA or qPCR. STING signaling is assessed by measuring phosphorylation of TBK1 and IRF3 by Western blot. Cell viability is assessed using MTT or CellTiter-Glo assays. |
| Animal Protocol |
Animal/Disease Models: 6-8 weeks old female C57BL/6 (H-2b) mice [2]
Doses: 5 µg Route of Administration: Nostril mucosa Adjuvant Experimental Results: Ovalbumin (OVA) specific IgA and total IgG and IgG1 and IgG1 titers were higher compared to IgG2c in sera from OVA-immunized mice with cGAMP adjuvant. In vivo animal studies for cGAMP involve its use as a STING agonist to stimulate antitumor immunity or as a tool to study innate immune responses. Mice are administered cGAMP via various routes (intravenous, intratumoral, intraperitoneal). Immune activation is assessed by measuring cytokine levels, immune cell activation markers, and antitumor responses. Standard protocols for STING agonist evaluation are employed. |
| ADME/Pharmacokinetics |
cGAMP free acid has a molecular weight of 674.41 and a molecular formula of C20H24N10O13P2. As a highly polar cyclic dinucleotide, it has limited oral bioavailability and is typically administered by injection. The compound appears as a white to yellow solid powder and has a purity of ≥98%. Detailed PK parameters are available from preclinical studies of cGAMP and its analogs.
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| Toxicity/Toxicokinetics |
The toxicity profile of cGAMP has been evaluated in the context of STING agonist research. As a potent activator of the innate immune system, its primary safety concerns relate to excessive inflammation and cytokine storm. Standard toxicology assessments include acute and repeated-dose toxicity studies in rodents. The compound is for research use only and is not intended for human therapeutic use.
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| References | |
| Additional Infomation |
c-GMP-AMP is a cyclic purine dinucleotide formed by the cyclization of AMP and GMP units via a 3',5' linker. It is an adenosine monophosphate, a cyclic purine dinucleotide, and a guanylate monophosphate. It is the conjugate acid of c-GMP-AMP(2-).
cGAMP free acid is a research compound and has not been approved for clinical use. It is a naturally occurring cyclic dinucleotide that works as a bacterial second messenger and regulates bacterial survival, adhesion, colonization, biofilm formation, and virulence in prokaryotes. In eukaryotic cells, cGAMP activates the STING pathway and induces type I interferon production. It is a valuable tool for studying bacterial signaling, innate immunity, and STING-mediated antitumor immunity. |
| Molecular Formula |
C20H24N10O13P2
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|---|---|
| Molecular Weight |
674.417
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| Exact Mass |
674.1
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| Elemental Analysis |
C, 35.62; H, 3.59; N, 20.77; O, 30.84; P, 9.19
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| CAS # |
849214-04-6
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| Related CAS # |
cGAMP disodium;2407516-83-8;cGAMP diammonium; 849214-04-6 (free acid)
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| PubChem CID |
135471108
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| Appearance |
White to yellow solid powder
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| Density |
2.6±0.1 g/cm3
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| Index of Refraction |
2.071
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| Source |
Endogenous Metabolite
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| LogP |
-5.9
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| Hydrogen Bond Donor Count |
7
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| Hydrogen Bond Acceptor Count |
19
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
45
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| Complexity |
1300
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| Defined Atom Stereocenter Count |
8
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| SMILES |
O[C@@H]1[C@@H]2OP(OC[C@H]3O[C@@H](N4C=NC5C(=NC=NC4=5)N)[C@H](O)[C@@H]3OP(OC[C@H]2O[C@H]1N1C=NC2C(N=C(NC1=2)N)=O)(O)=O)(O)=O
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| InChi Key |
RFCBNSCSPXMEBK-INFSMZHSSA-N
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| InChi Code |
InChI=1S/C20H24N10O13P2/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)/t6-,7-,10-,11-,12-,13-,18-,19-/m1/s1
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| Chemical Name |
2-amino-9-((2R,3R,3aS,7aR,9R,10R,10aS,14aR)-9-(6-amino-9H-purin-9-yl)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9]tetraoxa[2,8]diphosphacyclododecin-2-yl)-3,9-dihydro-6H-purin-6-one
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| Synonyms |
3′3′-cGAMP; Cyclic AMP-GMP; c-GMP-AMP; Cgamp; 849214-04-6; c-GMP-AMP; 3',5'-cyclic GMP-AMP; CHEMBL4449584; 3',3'-cGAMP; 2-amino-9-[(1S,6R,8R,9R,10S,15R,17R,18R)-17-(6-aminopurin-9-yl)-3,9,12,18-tetrahydroxy-3,12-dioxo-2,4,7,11,13,16-hexaoxa-3lambda5,12lambda5-diphosphatricyclo[13.3.0.06,10]octadecan-8-yl]-1H-purin-6-one; 3'3'-Cyclic guanosine monophosphate-adenosine monophosphate; cyclic GMP-AMP; cGAMP;
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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) |
H2O : ~180 mg/mL (~266.90 mM)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.4828 mL | 7.4138 mL | 14.8276 mL | |
| 5 mM | 0.2966 mL | 1.4828 mL | 2.9655 mL | |
| 10 mM | 0.1483 mL | 0.7414 mL | 1.4828 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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