2',3'-cGAMP is a cyclic A-and G-containing dinucleotide second messenger, which is formed upon cellular recognition of foreign cytosolic DNA as part of the innate immune response. The molecule binds to the adaptor protein STING, which induces an immune response characterized by the production of type I interferons and cytokines. The development of STINGbinding molecules with both agonistic as well as antagonistic properties is currently of tremendous interest to induce or enhance antitumor or antiviral immunity on the one hand, or to treat autoimmune diseases on the other hand. To escape the host innate immune recognition, some viruses encode poxin endonucleases that cleave 2',3'-cGAMP. Here we report that dideoxy-2',3'-cGAMP (1) and analogs thereof, which lack the secondary ribose-OH groups, form a group of poxinstable STING agonists. Despite their reduced affinity to STING, particularly the compound constructed from two A nucleosides, dideoxy-2',3'-cAAMP (2), features an unusually high antitumor response in mice.

Tet3 is the main α-ketoglutarate (αKG)-dependent dioxygenase in neurons that converts 5-methyl-dC into 5-hydroxymethyl-dC and further on to 5-formyl- and 5-carboxy-dC. Neurons possess high levels of 5-hydroxymethyl-dC that further increase during neural activity to establish transcriptional plasticity required for learning and memory functions. How αKG, which is mainly generated in mitochondria as an intermediate of the tricarboxylic acid cycle, is made available in the nucleus has remained an unresolved question in the connection between metabolism and epigenetics. We show that in neurons the mitochondrial enzyme glutamate dehydrogenase, which converts glutamate into αKG in an NAD+-dependent manner, is redirected to the nucleus by the αKG-consumer protein Tet3, suggesting on-site production of αKG. Further, glutamate dehydrogenase has a stimulatory effect on Tet3 demethylation activity in neurons, and neuronal activation increases the levels of αKG. Overall, the glutamate dehydrogenase-Tet3 interaction might have a role in epigenetic changes during neural plasticity.

The cGAS‐STING pathway is known for its role in sensing cytosolic DNA introduced by a viral infection, bacterial invasion or tumorigenesis. Free DNA is recognized by the cyclic GMP‐AMP synthase (cGAS) catalyzing the production of 2’,3’‐cyclic guanosine monophosphate‐adenosine monophosphate (2’,3’‐cGAMP) in mammals. This cyclic dinucleotide acts as a second messenger, activating the stimulator of interferon genes (STING) that finally triggers the transcription of interferon genes and inflammatory cytokines. Due to the therapeutic potential of this pathway, both the production and the detection of cGAMP via fluorescent moieties for assay development is of great importance. Here, we introduce the paralleled synthetic access to the intrinsically fluorescent, cyclic dinucleotides 2’3’‐cthGAMP and 3’3’‐cthGAMP based on phosphoramidite and phosphate chemistry, adaptable for large scale synthesis. We examine their binding properties to murine and human STING and confirm biological activity including interferon induction by 2’3’‐cthGAMP in THP‐1 monocytes. Two‐photon imaging revealed successful cellular uptake of 2’3’‐cthGAMP in THP‐1 cells.

2',3'-cGAMP ist ein zyklischer A-und Ghaltiger Dinukleotid-Botenstoff, der bei der zellulären Erkennung fremder zytosolischer DNA als Teil der angeborenen Immunantwort gebildet wird. Das Molekül bindet an das Adapterprotein STING, welches eine Immunantwort auslöst, die durch die Produktion von Typ-I-Interferonen und Zytokinen gekennzeichnet ist. Die Entwicklung von STING-bindenden Molekülen mit sowohl agonistischen als auch antagonistischen Eigenschaften ist derzeit von großem Interesse, um einerseits eine antitumorale oder antivirale Immunantwort zu induzieren bzw. zu verstärken und andererseits Autoimmunerkrankungen zu behandeln. Um der Erkennung durch das angeborene Wirtsimmunsystem zu entgehen, kodieren einige Viren Poxin-Endonukleasen, die in der Lage sind 2',3'-cGAMP zu spalten. Hier berichten wir, dass Didesoxy-2',3'-cGAMP (1) und dessen Analoga eine Gruppe von Poxin-stabilen STING-Agonisten bilden. Trotz ihrer geringeren Affinität zu STING weist insbesondere die aus zwei A-Nukleosiden aufgebaute Verbindung Didesoxy-2',3'-cAAMP (2) eine ungewöhnlich hohe Antitumorreaktion bei Mäusen auf.