STING agonism turns human T cells into interferon-producing cells but impedes their functionality.

The cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) axis is the predominant DNA sensing system in cells of the innate immune system. However, human T cells also express high levels of STING, while its role and physiological trigger remain largely unknown. Here, we show that the cGAS-STING pathway is indeed functional in human primary T cells. In the presence of a TCR-engaging signal, both cGAS and STING activation switches T cells into type I interferon-producing cells. However, T cell function is severely compromised following STING activation, as evidenced by increased cell death, decreased proliferation, and impaired metabolism. Interestingly, these different phenotypes bifurcate at the level of STING. While antiviral immunity and cell death require the transcription factor interferon regulatory factor 3 (IRF3), decreased proliferation is mediated by STING independently of IRF3. In summary, we demonstrate that human T cells possess a functional cGAS-STING signaling pathway that can contribute to antiviral immunity. However, regardless of its potential antiviral role, the activation of the cGAS-STING pathway negatively affects T cell function at multiple levels. Taken together, these results could help inform the future development of cGAS-STING-targeted immunotherapies.

Novel Poxin Stable cGAMP-Derivatives Are Remarkable STING Agonists.

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 STING-binding 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 poxin-stable 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.

Chemical Synthesis of the Fluorescent, Cyclic Dinucleotides cth GAMP.

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'-cth GAMP and 3'3'-cth GAMP 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'-cth GAMP in THP-1 monocytes. Two-photon imaging revealed successful cellular uptake of 2'3'-cth GAMP in THP-1 cells.

A Click-Chemistry Linked 2'3'-cGAMP Analogue.

2'3'-cGAMP is an uncanonical cyclic dinucleotide where one A and one G base are connected via a 3'-5' and a unique 2'-5' linkage. The molecule is produced by the cyclase cGAS in response to cytosolic DNA binding. cGAMP activates STING and hence one of the most powerful pathways of innate immunity. cGAMP analogues with uncharged linkages that feature better cellular penetrability are currently highly desired. Here, the synthesis of a cGAMP analogue with one amide and one triazole linkage is reported. The molecule is best prepared via a first CuI -catalyzed click reaction, which establishes the triazole, while the cyclization is achieved by macrolactamization.

Investigation of the Deprotonative Generation and Borylation of Diamine-Ligated α-Lithiated Carbamates and Benzoates by in Situ IR spectroscopy.

Diamine-mediated α-deprotonation of O-alkyl carbamates or benzoates with alkyllithium reagents, trapping of the carbanion with organoboron compounds, and 1,2-metalate rearrangement of the resulting boronate complex are the primary steps by which organoboron compounds can be stereoselectively homologated. Although the final step can be easily monitored by 11B NMR spectroscopy, the first two steps, which are typically carried out at cryogenic temperatures, are less well understood owing to the requirement for specialized analytical techniques. Investigation of these steps by in situ IR spectroscopy has provided invaluable data for optimizing the homologation reactions of organoboron compounds. Although the deprotonation of benzoates in noncoordinating solvents is faster than that in ethereal solvents, the deprotonation of carbamates shows the opposite trend, a difference that has its origin in the propensity of carbamates to form inactive parasitic complexes with the diamine-ligated alkyllithium reagent. Borylation of bulky diamine-ligated lithiated species in toluene is extremely slow, owing to the requirement for initial complexation of the oxygen atoms of the diol ligand on boron with the lithium ion prior to boron-lithium exchange. However, ethereal solvent, or very small amounts of THF, facilitate precomplexation through initial displacement of the bulky diamines coordinated to the lithium ion. Comparison of the carbonyl stretching frequencies of boronates derived from pinacol boronic esters with those derived from trialkylboranes suggests that the displaced lithium ion is residing on the pinacol oxygen atoms and the benzoate/carbamate carbonyl group, respectively, explaining, at least in part, the faster 1,2-metalate rearrangements of boronates derived from the trialkylboranes.