The cGAS-STING pathway as a therapeutic target in inflammatory diseases

Abstract

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.

Fig. 1. Overview of the cGAS–STING signalling pathway.

A schematic detailing double-stranded DNA (dsDNA)-induced activation of cytosolic cyclic GMP–AMP synthase (cGAS), which can occur through pathogen infection or cellular stress. On binding dsDNA, cGAS dimers assemble on dsDNA resulting in enzymatic activation of cGAS and synthesis of 2′3′ cyclic GMP–AMP (cGAMP). cGAMP binds to stimulator of interferon genes (STING) dimers localized at the endoplasmic reticulum (ER) membrane, which leads to profound conformational changes that trigger STING oligomerization, liberation from anchoring factors (such as STIM1), interaction with trafficking factors (for example, SEC24/23, STEEP, not depicted in figure) and, finally, incorporation into coatomer protein complex II (COPII) vesicles. On passing through the ER–Golgi intermediate compartment (ERGIC) and Golgi, STING recruits TANK-binding kinase 1 (TBK1), promoting TBK1 autophosphorylation, STING phosphorylation at Ser366 and recruitment of interferon regulatory factor 3 (IRF3). The phosphorylation of IRF3 by TBK1 enables IRF3 dimerization and translocation to the nucleus to induce gene expression of type I interferons, interferon-stimulated genes (ISGs), and several other inflammatory mediators, pro-apoptotic genes and chemokines. Activation of STING also leads to NF-κB activation and the formation of LC3⁺ vesicles (autophagosomes) by a non-canonical mechanism of autophagy. In the end, both STING within autophagosomes and STING from the Golgi traffic to the lysosome, where STING degradation occurs. Steady-state STING translocation through the secretory pathway in the absence of robust cGAMP stimulation is counteracted by continuous retrograde transport to the ER, a step that is mediated by COPI vesicles and facilitated by an interaction of STING with SURF4.

Fig. 2. cGAS–STING effector mechanisms and intercellular cGAMP transmission.

a | Stimulator of interferon genes (STING) mediates inflammatory and antiviral cellular programmes by engaging the transcription factors interferon regulatory factor 3 (IRF3) and canonical NF-κB (RELA–p50). IRF3 activation is strictly controlled by TANK-binding kinase 1 (TBK1), which binds to the C-terminal tail (CTT) of STING through a conserved PLPLRT/SD binding motif to then phosphorylate Ser366 within the CTT (phospho-site). In certain cells, activation of NF-κB is less dependent on the CTT and TBK1. In addition, non-canonical NF-κB (RELB–p52) is activated downstream of STING and counteracts IRF3 and canonical NF-κB signalling. STING impacts cell survival and cellular proliferation in diverse ways and is associated with the induction of apoptosis, necroptosis, pyroptosis and cellular senescence. Both necroptosis and senescence can be triggered by STING-induced cytokines. Apoptosis induction can proceed through the upregulation of pro-apoptotic genes (PUMA, NOXA) or, alternatively, directly engage BAX and BAK at the mitochondrial membrane. STING-mediated lysosomal damage has been shown to activate NLRP3 inflammasome-dependent pyroptosis. b | Intercellular 2′3′ cyclic GMP–AMP (cGAMP) transfer can be mediated by connexin proteins that form gap junctions between neighbouring cells, which can be homotypic or heterotypic. Additionally, various cell types use the voltage-dependent anion channel (VDAC) composed of LRCC8 subunits to import and export cGAMP. In the extracellular space, cGAMP is subject to degradation by the enzyme ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1). Tumour cells have also been shown to export cGAMP whereas myeloid cells use SLC19A1 or P2XR7 to import cGAMP. Connexin channels also mediate heterotypic cellular cGAMP transfer. APC, antigen-presenting cell; cGAS, cyclic GMP–AMP synthase; IFN, interferon; ISG, interferon-stimulated gene.

Fig. 3. Mechanisms underlying cGAS–STING activity in sterile inflammatory conditions.

Corpses of dying or apoptotic cells can become engulfed by phagocytic cells and are targeted to lysosomes (where DNase II is active) via LC3-associated phagocytosis (LAP). The efficient disposal of extracellular DNA through this route restricts cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) activity. Within mitochondria, transcription factor A mitochondrial (TFAM) plays a crucial role in stabilizing mitochondrial (mt) DNA through the formation of nucleoids, which is important to counteract mtDNA stress and aberrant cGAS activation. Exogenous stress signals, including chemotherapy or infection, may also cause mitochondrial dysfunction — in the most severe cases leading to apoptosis — which can promote loss of mtDNA compartmentalization. mtDNA either herniates through the inner mitochondrial membrane to then be released through BAX/BAK macropores or can be released via voltage-dependent anion channel (VDAC) pores in the outer mitochondrial membrane. Alternatively, mitochondrial permeability transition pore (MPTP) may present an alternative route for inner mitochondrial membrane traversal. Mitophagy of stressed mitochondria, which is executed by Parkin and PINK1, limits mtDNA-mediated stimulation of cGAS. Nuclear DNA may become accessible to cytosolic cGAS through the following mechanisms: first, the derepression of retroelements, second, DNA damage or replication stress may lead to direct nuclear DNA leakage, third, in senescence, chromatin herniations can bud off the main nucleus leading to accumulation of cytoplasmic chromatin fragments (CCFs) or, alternatively, in the context of genomic instability nuclear DNA may be encapsulated in micronuclei after mitosis. Both of these latter two events can promote cGAS activity on rupture of the aberrant micro-organelle membrane. All these instances of nuclear-derived cytosolic DNA recognition are efficiently antagonized by the exonuclease TREX1. Within the main nucleus, both nucleosomes and architectural chromatin proteins, including histone H1 and barrier-to-autointegration factor (BAF) act to limit aberrant cGAS activity. ER, endoplasmic reticulum.

Fig. 4. Mechanisms of action of inhibitors targeting cGAS and STING.

a | Inhibitory molecules targeting the catalytic pocket of cyclic GMP–AMP synthase (cGAS) are shown on the left, and those reported to impede DNA binding are shown on the right. b | Inhibitory compounds affecting stimulator of interferon genes (STING) can either occupy the ligand-binding domain (LBD) or covalently bind to C91 of both human and mouse STING. In the 2′3′ cyclic GMP–AMP (cGAMP)-bound state, STING undergoes conformational rearrangements leading to its oligomerization. The inset highlights the position of C88 and C91, residues of STING that are both subject to palmitoylation. Structures shown: cGAS catalytic domain (CD-cGAS) apo form (Homo sapiens PDB: 4O68); CD-cGAS DNA-bound (H. sapiens PDB: 6CT9); STING full-length apo form (H. sapiens PDB: 6NT5); STING full-length cGAMP-bound (H. sapiens PDB: 6NT5, modelled with Gallus gallus PDB: 6NT7 (front view) or G. gallus PDB: 6NT8 (tetramer side-view).