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]

cGAMP 二钠刺激小鼠肿瘤细胞的发育[2]。在体外，人类和小鼠树突状细胞直接被 cGAMP 二钠激活 [2]。当用 cGAMP 二钠治疗时，患者成纤维细胞表现出 IFNB1 的转录增强，但产生肿瘤因子 (TNF)、白细胞介素 6 (IL6) 或白细胞介素 1 (IL1) 的基因的转录没有增强 [3]。内质网 (ER) 驻留蛋白 STING 被 cGAMP 二钠激活，从而诱导抗病毒状态和 I 型分离蛋白行走 [4]。

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进行了cGAMP的化学合成，并通过质谱分析证实合成的分子与从 L929 细胞中纯化的内源性 cGAMP 一致。[1]
通过洋地黄皂苷透化法将cGAMP导入 L929 小鼠纤维肉瘤细胞后，可诱导 IFNβ RNA 和蛋白的表达。该诱导作用是剂量依赖性的，低至 10 nM 的浓度即可观察到显著的 IFNβ RNA 诱导。[1]
通过 ELISA 检测，在诱导 L929 细胞产生 IFNβ 蛋白方面，cGAMP的效力显著高于环二鸟苷酸 (c-di-GMP)。[1]
转染各种 DNA 序列（如 ISD、poly[dA:dT]、HT-DNA）或感染 DNA 病毒（HSV-1ΔICP34.5、痘苗病毒）后，哺乳动物细胞（L929、THP1、MEF、BMDM）中会触发cGAMP的产生，但 RNA 病毒（VSV）感染或 RNA 转染则不会。[1]
cGAMP的活性具有耐热性，对 Benzonase（DNA/RNA 消化酶）和蛋白酶 K 处理有抗性，其在胞质提取物中的生成需要 ATP 和 GTP 的同时存在。[1]
cGAMP对 IRF3 的激活依赖于 STING，这通过使用 STING 敲低的 L929 细胞 (L929-shSTING) 和异位表达 STING 的 HEK293T 细胞得以证明。[1]
使用放射性标记的[³²P]-cGAMP进行紫外诱导交联实验，证明了cGAMP与重组 STING 蛋白（第139-379位氨基酸）的直接结合。未标记的（冷的）cGAMP、c-di-GMP 和 c-di-AMP 可竞争性抑制该结合，但 ATP 或 GTP 不能。[1]

免疫小鼠的脾细胞被鼻粘膜佐剂 cGAMP 二钠 (5 μg) 刺激产生提示性细胞因子 [2]。

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最近发现的哺乳动物酶环GMP-AMP合酶在被病原体来源的细胞质双链DNA激活后产生环GMP-cAMP（cGAMP）。该产品可以刺激STING依赖性干扰素I型信号传导。在这里，我们探讨了cGAMP作为小鼠粘膜佐剂的疗效。在这项研究中，研究人员表明，cGAMP可以增强对模型抗原卵清蛋白的适应性免疫反应。它促进免疫小鼠的抗原特异性IgG和平衡的Th1/Th2淋巴细胞反应。cGAMP诱导的免疫反应的一个特征是白细胞介素-17的诱导略有减少，这是Th17活性的标志，这是其他环核苷酸佐剂没有观察到的一个明显特征。我们进一步表征了小鼠骨髓来源的树突状细胞和人树突状细胞在体外的先天免疫刺激活性。观察到的结果表明，cGAMP可作为人类疫苗的候选粘膜佐剂[2]。

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在 C57BL/6 小鼠中，通过鼻内与模型抗原卵清蛋白 (OVA) 共同给药作为黏膜佐剂使用时，cGAMP 能显著增强抗原特异性适应性免疫反应。与单独使用 OVA 或其他佐剂（如 c-di-AMP 和 CTB）相比，它能促进血清和鼻灌洗液中更高的 OVA 特异性总 IgG、IgG1、IgG2c 和黏膜 IgA 抗体滴度。[2]
用 OVA 加 cGAMP 免疫的小鼠，其脾细胞在体外用 OVA 再刺激后，显示出显著增强的抗原特异性增殖能力，通过 ³H-胸腺嘧啶核苷掺入法测定。[2]
OVA 再刺激后，通过 ELISPOT 实验检测，与单独使用 OVA 相比，使用 cGAMP 作为佐剂免疫的小鼠，其脾细胞能产生显著更多数量的分泌 IFN-γ 和 IL-2 的细胞（表明 Th1 反应）以及分泌 IL-4 的细胞（表明 Th2 反应）。值得注意的是，诱导分泌 IL-17 的细胞（Th17 反应）的水平相对于 c-di-AMP 诱导的水平较低，这表明其细胞因子谱具有独特性，有利于平衡的 Th1/Th2 反应，同时 Th17 活性受到适度调节。[2]

胞浆DNA诱导I型干扰素和其他细胞因子，这些细胞因子对抗菌防御很重要，但也可能导致自身免疫。这种DNA信号通路需要衔接蛋白STING和转录因子IRF3，但DNA传感的机制尚不清楚。我们发现，哺乳动物细胞质提取物在体外由三磷酸腺苷和鸟苷三磷酸在DNA而非RNA的存在下合成环鸟苷一磷酸腺苷一磷酸（环GMP-AMP或cGAMP）。哺乳动物细胞的DNA转染或DNA病毒感染也会触发cGAMP的产生。cGAMP与STING结合，导致IRF3的激活和干扰素-β的诱导。因此，cGAMP在后生动物中起着内源性第二信使的作用，并触发干扰素的产生以响应细胞质DNA[1]。

原代细胞的体外刺激[2]
原代细胞的培养基中补充了5µg/ml（小鼠细胞）或60µg/ml（人细胞）的c-di-AMP或cGAMP，或者不添加任何添加剂。细胞在37°C下孵育24小时。

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Scrape loading[[3]
HEK STING细胞以2.5×105个细胞ml-1的密度接种在96孔板中。16小时后，向培养基中加入cGAMP（2′-5′）至终浓度为50μg ml−1。使用18G针手动损伤单层细胞，每孔6处划痕。4-8小时后采集图像。

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为检测 STING 激活剂的生成，用 DNA 转染 L929-shSTING 细胞。将这些细胞的胞质提取物与经产气荚膜溶素 O (PFO) 透化的 THP1 或 Raw264.7 细胞混合。PFO 处理允许细胞质交换，同时保留细胞器。通过天然凝胶电泳监测 IRF3 的磷酸化和二聚化，来评估透化细胞中 IRF3 的激活。[1]
为体外检测 cGAMP 活性，将 L929-shSTING 胞质提取物 (S100) 与 DNA（如 HT-DNA）在 ATP 存在下孵育。将反应混合物加热至 95°C 以使蛋白质变性，然后将上清液与 PFO 透化的 Raw264.7 或 THP1 细胞孵育。透化细胞中 IRF3 的二聚化作为 cGAMP 活性的读数。[1]
对于功能实验，通过洋地黄皂苷透化法将cGAMP（化学合成或来源于细胞）导入细胞。在指定时间（例如 8 小时）孵育后，通过定量 RT-PCR 测量 IFNβ mRNA 水平，并通过 ELISA 测量 IFNβ 蛋白分泌。通过天然聚丙烯酰胺凝胶电泳分析 IRF3 的二聚体形成以检测其激活。[1]
为评估病毒感染期间 cGAMP 的产生，用 HSV-1ΔICP34.5（DNA 病毒）或痘苗病毒感染 L929 或 THP1 细胞。制备细胞提取物，并将等分试样加热至 95°C。然后将耐热上清液测试其在 PFO 透化的 Raw264.7 细胞中诱导 IRF3 二聚化的能力。提取物的另一等分试样通过反相 HPLC 进行分离，并使用纳升级液相色谱-质谱联用与选择性反应监测 (SRM) 对馏分中的 cGAMP 水平进行定量。[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 states that molecules such as cGAMP that are produced by the mammalian organism itself are expected to be of 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 defence of multicellular organisms against microbial pathogens requires cellular collaboration. Information exchange allowing immune cells to collaborate is generally attributed to soluble protein factors secreted by pathogen-sensing cells. Cytokines, such as type I interferons (IFNs), serve to alert non-infected cells to the possibility of pathogen challenge. Moreover, in conjunction with chemokines they can instruct specialized immune cells to contain and eradicate microbial infection. Several receptors and signalling pathways exist that couple pathogen sensing to the induction of cytokines, whereas cytosolic recognition of nucleic acids seems to be exquisitely important for the activation of type I IFNs, master regulators of antiviral immunity. Cytosolic DNA is sensed by the receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), which catalyses 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 the secretion of type I IFNs. Here we find in murine and human cells that cGAS-synthesized cGAMP(2'-5') is transferred from producing cells to neighbouring cells through gap junctions, where it promotes STING activation and thus antiviral immunity independently of type I IFN signalling. In line with the limited cargo specificity of connexins, the proteins that assemble gap junction channels, most connexins tested were able to confer this bystander immunity, thus indicating a broad physiological relevance of this local immune collaboration. Collectively, these observations identify cGAS-triggered cGAMP(2'-5') transfer as a novel host strategy that serves to rapidly convey antiviral immunity in a transcription-independent, horizontal manner.[3]

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cGAMP (cyclic GMP-AMP) is identified as the first endogenous cyclic dinucleotide second messenger in metazoans. It is synthesized in the cytosol of mammalian cells by an unknown enzyme (later identified as cGAS) in response to the presence of cytosolic DNA, which acts as a danger signal. cGAMP then binds directly to the adaptor protein STING, triggering a signaling cascade that leads to the activation of TBK1 kinase, phosphorylation and dimerization of the transcription factor IRF3, and subsequent induction of type I interferons and other cytokines. This pathway is crucial for innate immune defense against DNA viruses and intracellular bacteria, and may also contribute to autoimmune conditions. The study suggests cGAMP has potential applications in immunotherapy or as a vaccine adjuvant due to its potent interferon-inducing activity.[1]

C20H25N10NAO13P2

698.408995389938

718.063

2407516-83-8

cGAMP diammonium;cGAMP;849214-04-6

137120249

White to off-white solid powder

Endogenous Metabolite

331

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

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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)

配方 1 中的溶解度: 100 mg/mL (139.20 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。