The cGAS–STING signalling pathway has emerged as a key mediator of inflammation in the settings of infection, cellular stress and tissue damage. Underlying this broad involvement of the cGAS–STING pathway is its capacity to sense and regulate the cellular response towards microbial and host-derived DNAs, which serve as ubiquitous danger-associated molecules. Insights into the structural and molecular biology of the cGAS–STING pathway have enabled the development of selective small-molecule inhibitors with the potential to target the cGAS–STING axis in a number of inflammatory diseases in humans. Here, we outline the principal elements of the cGAS–STING signalling cascade and discuss the general mechanisms underlying the association of cGAS–STING activity with various autoinflammatory, autoimmune and degenerative diseases. Finally, we outline the chemical nature of recently developed cGAS and STING antagonists and summarize their potential clinical applications.

Aberrant activation of innate immune pathways is associated with a variety of diseases. Progress in understanding the molecular mechanisms of innate immune pathways has led to the promise of targeted therapeutic approaches, but the development of drugs that act specifically on molecules of interest remains challenging. Here we report the discovery and characterization of highly potent and selective small-molecule antagonists of the stimulator of interferon genes (STING) protein, which is a central signalling component of the intracellular DNA sensing pathway. Mechanistically, the identified compounds covalently target the predicted transmembrane cysteine residue 91 and thereby block the activation-induced palmitoylation of STING. Using these inhibitors, we show that the palmitoylation of STING is essential for its assembly into multimeric complexes at the Golgi apparatus and, in turn, for the recruitment of downstream signalling factors. The identified compounds and their derivatives reduce STING-mediated inflammatory cytokine production in both human and mouse cells. Furthermore, we show that these small-molecule antagonists attenuate pathological features of autoinflammatory disease in mice. In summary, our work uncovers a mechanism by which STING can be inhibited pharmacologically and demonstrates the potential of therapies that target STING for the treatment of autoinflammatory disease.

The advances in subunit vaccines development have intensified the search for potent adjuvants, particularly adjuvants inducing cellmediated immune responses. Identification of the C-type lectin Mincle as one of the receptors underlying the remarkable immunogenicity of the mycobacterial cell wall, via recognition of trehalose-6,6′-dimycolate (TDM), has opened avenues for the rational design of such molecules. Using a combination of chemical synthesis, biological evaluation, molecular dynamics simulations, and protein mutagenesis, we gained insight into the molecular bases of glycolipid recognition by Mincle. Unexpectedly, the fine structure of the fatty acids was found to play a key role in the binding of a glycolipid to the carbohydrate recognition domain of the lectin. Glucose and mannose esterified at O-6 by a synthetic α-ramified 32-carbon fatty acid showed agonist activity similar to that of TDM, despite their much simpler structure. Moreover, they were seen to stimulate proinflammatory cytokine production in primary human and murine cells in a Mincle-dependent fashion. Finally, they were found to induce strong Th1 and Th17 immune responses in vivo in immunization experiments in mice and conferred protection in a murine model of Mycobacterium tuberculosis infection. Here we describe the rational development of new molecules with powerful adjuvant properties. mycobacteria | glycolipid | innate immunity

histones in situ, which were partially lost upon aclarubicin treatment (Extended Data Fig. 1c, d). Thus, histones appear to dynamically engage cGAS in the nucleus.Consistent with prior work [13], functional analysis of cGAS in vitro enzymatic activity revealed that mononucleosomes (hereafter nucleosomes) inhibited DNA-induced cGAMP synthesis (Extended Data Fig. 1e). Likewise, compact chromatin fibres (12-mer nucleosome arrays) suppressed cGAS activity (Extended Data Fig. 1e). H2A-H2B dimers also had an inhibitory effect, but neither H2A or H2B monomers nor H3 or H4 monomers, respectively (Extended Data Fig. 1f, g). Thus, H2A-H2B dimers on their own can suppress cGAS (Extended Data Fig. 1h), albeit with weaker overall potency compared to fullassembled nucleosomes with additional features of nucleosomes in chromatin being necessary to exert maximal inhibition. Overall structure of the cGAS-NCP complexTo determine how cGAS interacts with nucleosomes, we pursued structural studies. A 1.5:1 molar mixture of human cGAS (residues 155 to 522) with a 147 bp 601 DNA nucleosome core particle (NCP) resulted in heterogenous particle distributions (Extended Data Fig. 2ad). To select for and stabilize more homogenous cGAS-NCP complexes, we combined gradient centrifugation with chemical crosslinking (GraFix) [15]. Both WT cGAS and cGAS K394E, a mutant impaired in dsDNA-mediated cGAS dimerisation [16], were used for structure determination. For the cGAS K394E mutant, we obtained a 4.1 Å reconstruction revealing two NCPs organized in a NCP 1 -cGAS 1 -cGAS 2 -NCP 2 sandwich arrangement with an expected molecular weight around 560 kDa, consistent with the most prominent peak fraction in multi-angle light scattering (MALS) (Fig. 1a, b, Extended Data Fig. 3, Supplementary Video 1, 2, and Extended Data Table 1a). The two individual nucleosomes are held together by two cGAS protomers. While the first cGAS protomer and its corresponding NCP (designated cGAS 1 and NCP 1 ) are well-resolved, the second nucleosome/cGAS pair (NCP 2 and cGAS 2 ) is less ordered (Extended Data Fig. 3e). In the dimeric NCP 1 -cGAS 1 -cGAS 2 -NCP 2 arrangement, each cGAS protomer interacts with the histone octamer of one NCP through histones H2A and H2B and the nucleosomal DNA (e.g. cGAS 1 and NCP 1 ), while contacting the second nucleosome (e.g. cGAS 1 and NCP 2 ) primarily through interactions with the nucleosomal DNA (Fig. 1a, b). In the WT cGAS structure, we observed a similar overall structural arrangement, with the NCP 1 -cGAS 1 -cGAS 2 -NCP 2 complex at 5.1Å and the focused NCP 1 -cGAS 1 structure at 4.7Å resolution (Extended Data

The appearance of DNA in the cytosol is perceived as a danger signal that stimulates potent immune responses through cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS). How cells regulate the activity of cGAS toward self-DNA and guard against potentially damaging autoinflammatory responses is a fundamental biological question. Here, we identify barrier-to-autointegration factor 1 (BAF) as a natural opponent of cGAS activity on genomic self-DNA. We show that BAF dynamically outcompetes cGAS for DNA binding, hence prohibiting the formation of DNA-cGAS complexes that are essential for enzymatic activity. Upon acute loss of nuclear membrane integrity, BAF is necessary to restrict cGAS activity on exposed DNA. Our observations reveal a safeguard mechanism, distinct from physical separation, by which cells protect themselves against aberrant immune responses toward genomic DNA.

Dectin-2 is a C-type lectin involved in the recognition of several pathogens such as Aspergillus fumigatus, Candida albicans, Schistosoma mansonii, and Mycobacterium tuberculosis that triggers Th17 immune responses. Identifying pathogen ligands and understanding the molecular basis of their recognition is one of the current challenges. Purified M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) was shown to induce signaling via Dectin-2, an activity that requires the (α1 → 2)-linked mannosides forming the caps. Here, using isogenic M. tuberculosis mutant strains, we demonstrate that ManLAM is a bona fide and actually the sole ligand mediating bacilli recognition by Dectin-2, although M. tuberculosis produces a variety of cell envelope mannoconjugates, such as phosphatidyl-myo-inositol hexamannosides, lipomannan or manno(lipo)proteins, that bear (α1 → 2)-linked mannosides. In addition, we found that Dectin-2 can recognize lipoglycans from other bacterial species, such as Saccharotrix aerocolonigenes or the human opportunistic pathogen Tsukamurella paurometabola, suggesting that lipoglycans are prototypical Dectin-2 ligands. Finally, from a structure/function relationship perspective, we show, using lipoglycan variants and synthetic mannodendrimers, that dimannoside caps and multivalent interaction are required for ligand binding to and signaling via Dectin-2. Better understanding of the molecular basis of ligand recognition by Dectin-2 will pave the way for the rational design of potent adjuvants targeting this receptor.

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