Protein-protein interactions in cGAS-STING pathway: a medicinal chemistry perspective

ABSTRACT

Protein-protein interactions (PPIs) play pivotal roles in biological processes and are closely linked with human diseases. Research on small molecule inhibitors targeting PPIs provides valuable insights and guidance for novel drug development. The cGAS-STING pathway plays a crucial role in regulating human innate immunity and is implicated in various pathological conditions. Therefore, modulators of the cGAS-STING pathway have garnered extensive attention. Given that this pathway involves multiple PPIs, modulating PPIs associated with the cGAS-STING pathway has emerged as a promising strategy for modulating this pathway. In this review, we summarize an overview of recent advancements in medicinal chemistry insights into cGAS-STING PPI-based modulators and propose alternative strategies for further drug discovery based on the cGAS-STING pathway.

Plain language summary

Article highlights.

• Role of cGAS-STING pathway in innate immunity.

• The disordered of cGAS-STING pathway can cause cancer or autoimmune disease.

• The cGAS-STING pathway is contributed to resisting infection.

• The protein-protein interactions of cGAS-STING pathway.

• Using DNA as a scaffold, cGAS proteins form polymers to further form phase separation.

• STING oligomers provide a platform for downstream protein interactions.

• PPIs-related modulators of cGAS-STING pathway.

• Modulators targeting protein-protein interactions in the cGAS-STING pathway are highlighted and their advantages in disease therapy are discussed.

• These summarized strategies may provide inspiration for the development of cGAS-STING modulators.

• cGAS-STING pathway plays crucial roles in the initiating immune system.

• Several structurally diverse cGAS-STING modulators have been used as the anticancer and anti-autoimmune diseases agents.

• The function of cGAS-STING pathway is highly dependent on protein-protein interactions. In this review, we summarize an overview of recent advancements in medicinal chemistry insights into cGAS-STING PPI-based modulators and propose alternative strategies for further drug discovery based on the cGAS-STING pathway.

1. Introduction

In recent years, the cGAS-STING pathway has emerged as a prominent subject of investigation in the field of immunity. This pathway plays a pivotal role in defending against pathogenic microorganisms and regulating innate immunity. In 2008, three independent research groups reported the discovery of STING (Stimulator of Interferon Genes) [1], MITA (Mediator of IRF3 Activation) [2] and ERIS (Endoplasmic Reticulum IFN Stimulator) [3]. Subsequently, these proteins were collectively referred to as STING. In 2013, Chen's research group demonstrated that 2′,3′-cyclic GMP-AMP (2′,3′-cGAMP) can directly activate STING as a second messenger and identified cyclic GMP-AMP synthase (cGAS) as a crucial upstream synthase for 2′,3′-cGAMP production [4]. Shang et al., in 2019, elucidated the full-length structure of STING and provided insights into its molecular activation mechanism [5]. In the same year, Li's group and Gwack's group reported on the mechanism of STING autoinhibition by C-terminal tail (CTT) and the retention of STING by ER-protein stromal-interaction molecule 1 (STIM1), respectively [6,7]. In 2023, Shang's group revealed the head-to-head self-inhibition mode of STING [8]. Several agonists of this pathway, such as IMSA-101, E7766 and GSK37417, are currently undergoing clinical investigation for treatment of tumors. In 2023, VENT-03, an inhibitor targeting cGAS catalytic domain, commenced phase I clinical trials for treatment of systemic lupus erythematosus (SLE). In recent years, there has been an increasing number of co-crystal structures illuminating protein interactions within the cGAS-STING pathway, leading to a more comprehensive understanding of this immune regulatory cascade centered around cGAS-STING.

1.1. Mechanisms of the cGAS-STING pathway

Self-damage and pathogen infection lead to the presence of pathogenic self-DNA and foreign-DNA, respectively [9]. Pathogenic DNA is recognized by pattern recognition receptors such as pathogen-associated molecular patterns and damage-associated molecular patterns. This recognition mechanism is critical for detecting pathogens and triggering innate immune responses [10]. Pathogenic DNA delivered into the cytoplasm is recognized by cGAS [11,12]. Using double-stranded DNA (dsDNA) as a scaffold, cGAS can dimerize and oligomerize to achieve phase separation, enabling efficient production of 2′,3′-cGAMP from ATP and GTP [11,13]. After recognizing 2′,3′-cGAMP, STING undergoes extensive conformational changes that lead to its activation. Activated STING is translocated from the ER to the Golgi apparatus [13]. It then recruits TANK-binding kinase 1 (TBK1) [14,15] and interferon regulatory factor 3 (IRF3) [16]. During this process, STING undergoes oligomerization and modifications, such as phosphorylation, ubiquitination and palmitoylation [17,18]. Oligomerized STING recruits TBK1 at its C-terminal tail and promotes the autophosphorylation of TBK1 [14,15,18]. Phosphorylated TBK1 (pTBK1) phosphorylates STING [18,19], allowing STING to recruit IRF3. And then pTBK1 phosphorylates IRF3 resulting in that IRF3 dimer enter the nucleus to induce the production of type I interferon B (IFNβ) and other proinflammatory factors [17,20]. The adaptor protein complex 1 (AP-1) controls the termination of STING-dependent immune activation [21]. STING is transported by clathrin-coated transport vesicles (CCVs) for endosomal degradation. It is worth mentioning that nuclear factor kappa B (NF-κB) pathway, another crucial pathway of innate immunity, is closely related to cGAS-STING pathway. STING activates canonical IκB kinase dependent NF-κB signal pathway and noncanonical NF-κB-inducing kinase dependent NF-κB signal pathway, resulting in expression of pro-inflammatory cytokines. Zhang et al. found that NF-κB can alter microtubule cytoskeletal transport network, which in turn alter STING trafficking and degradation to enhance STING-mediated immune responses [22]. In addition, STING is contributed to autophagy induced by cytosolic pathogenic DNA. STING-dependent TBK1 activation has been reported to lead to ubiquitination of bacterial phagosomes [23]. This autophagy favors the clearance of pathogens. cGAMP functions not only in individual cells, but also in peripheral cells through intercellular transport. In this process, ENPP1, ENPP3 and SMPDL3A degrade cGAMP, which limits the intercellular effect of cGAMP [24–26]. In the whole pathway, there are extensive protein-protein interaction among its key proteins (Figure 1). The immune signal in this pathway relies on PPIs to complete the transduction [27], hence it is worth that study small molecule modulators based on PPIs of cGAS-STING pathway. Given the molecular pathways of cGAS-STING pathway leading to diseases and physiological function changes, targeting key PPIs with small molecules could be a therapeutic strategy for related diseases. In this review, we will focus on the protein-protein interactions in the cGAS-STING pathway to summarize and analyze whether they can be used as targets for the development of protein-protein interaction modulators.

Figure 1.

A brief activation process of cGAS-STING-TBK1 signaling pathway.

1.2. cGAS-STING pathway related diseases

The cGAS-STING pathway plays an important role in recognizing abnormal dsDNA and activating innate immunity in humans [12,16,17,19]. The disorder of cGAS-STING pathway results in the initiation and progression of various diseases, including inflammatory diseases, autoimmune diseases, neurodegenerative diseases, senescence, metabolic diseases, cancer and pathogen infections. Correlative studies of these diseases have found that intervening in the STING pathway can be preventative and therapeutic for related diseases (Table 1). When this pathway is appropriately activated, innate immunity can effectively exhibit antiviral [28], antibacterial [29] and anti-cancer [30] effects. In the process of infecting the human, the reproduction of viruses and bacteria lead to the necrosis of human cells, which results in endogenous dsDNA and pathogen genome exposure. Extracellular self-derived dsDNA and pathogen dsDNA activate the intracellular cGAS-STING pathway to produce anti-infective effects. In tumor cells, chromosomal instability is thought to activate STING, which in turn produces interferons and pro-inflammatory factors that inhibit tumor growth. However, Li et al. at the Sloan-Kettering Cancer Center found that tumor chromosomal instability activates the cGAS-STING pathway and rebuilds the tumor microenvironment [31]. With the support of a well-established immune system, chromosomally instability tumor cells activate STING leading to produce endoplasmic reticulum stress and promote the metastasis of tumor cells. The overactivated cGAS-STING pathway can result in various autoimmune diseases and inflammation, including Aicardi-Goutières syndrome (AGS) [32], STING-associated vasculopathy of infancy (SAVI) [33] and SLE [34]. Organelle damage (mitochondrial damage and endoplasmic reticulum stress), dsDNA metabolism disorders and cell necrosis release a large amount of endogenous dsDNA to activate the cGAS-STING pathway, which is the main cause of autoimmune diseases caused by the cGAS-STING pathway [35]. In addition, some genetic mutations can lead to overactivation of the STING pathway, such as SAVI-related mutations (V147L, N154S, V155M, V155R, R284S), which lead to STING self-activation. Mutations in the COPα gene lead to defective COP I transport, resulting in abnormal activation of STING. The occurrence and progression of these diseases are based on the disordered immune signaling of this pathway.

Table 1.

Examples of cGAS-STING pathway related disease.

STING modulators	Type of disease	Specific disease	Function	Ref.
cGAS-STING agonists	Cancer	Cancer	Mediate the immune system to suppress cancer	[25]
	Infection	Bacterial and viral infections	Mediate the immune system to suppress infection	[23,24]
cGAS-STING inhibitors	Autoimmune and inflammatory disorders	Aicardi-goutières syndrome (AGS)	Loss of function of three prime repair exonuclease 1 (Trex1) and DNase I lead to failure of DNA hydrolysis. Abnormal activation of cGAS and STING	[26,29]
		STING-associated vasculopathy of infancy (SAVI)	Gain of function of STING mutations (V147L, N154S, V155M, V155R, R284S)	[30–32]
		Systemic lupus erythematosus (SLE)	Loss of function of Trex1 and DNase I lead to failure of DNA hydrolysis and abnormal activation of cGAS and STING	[33,34]
		COPA Syndrome	Single-gene autoimmune disorder, transport defect of COP I leads to abnormal activation of STING on Golgi	[35]
		Familial chilblain lupus	Loss of function in Trex1. Gain of function of STING mutation G166E	[31]
cGAS-STING inhibitors	Senescence and ageing	Senescence and ageing	Gain of function of cGAS-STING pathway related pro-inflammatory cytokine	[36,37]
	Neurodegenerative diseases	Parkinson's disease	Loss of function in STING is able to alleviate PD	[38,45]
		Amyotrophic lateral sclerosis	Abnormal increase of 2′3′-cGAMP level	[39,46]
		Huntington's disease	Abnormal activation of cGAS and STING lead to the increase of type I IFN	[40,47]
	Metabolic diseases	Non-alcoholic fatty liver disease	Loss of function in STING is able to alleviate NAFLD	[41,48]
		Alcoholic liver disease	Abnormal activity of IRF3	[42,49]
		Acute pancreatitis	DAMP released by dead acinar cells directly activate cGAS	[43,50]
	Cancer	Cancer metastasis	Endoplasmic reticulum stress due to cGAS-STING	[44]

2. cGAS-related PPIs & modulators

cGAS is an important DNA-sensing receptor that detects DNA damage or viral DNA in the cytoplasm. As a DNA-binding protein, cGAS is the initiator of the STING-mediated immune response [12]. Under inactive conditions, cGAS is present in the cytoplasm as a monomer. Human cGAS (hcGAS) monomer is a 522-amino-acid protein, including a less-conserved N-terminal domain (1–160 aa) and a highly-conserved NTase and Mab 21 domain (161–522 aa). NTase and Mab21 domain include three DNA binding sites (A, B and C) and Zinc ribbon (390–404 aa) (Figure 2A) [51–53].

Figure 2.

Regulatory strategies targeting cGAS interactions. (A) Schematic of hcGAS domain organization. Amino acidsequences of each binding site are listed in Table 2, along with the corresponding detailed amino acids. (B) Hyperphosphorylation of cGAS prevents its activity. (C) DNA-binding site A (cyan), site B (orange), site C (green), and dimerization interface (red) of cGAS (PDB ID: 5N6I). (D) Compounds targeting the cGAS interaction include DNA intercalators (quinacrine and X6), DNA competitive inhibitor (suramin), dimer interface inhibitors (Z918 and CU-76), and a G3BP1-cGAS inhibitor (Epigallocatechin gallate). (E) The structure-activity relationship of Z918 and CU-76. (F) Proteins that interact with cGAS. (G) The structure of 2:2 cGAS-NCP complex (PDB ID: 7CCR) includes the cGAS monomer (green), the NCP protein (cyan), and the NCP DNA (orange).

2.1. DNA-bound cGAS leads to the interaction of homologous dimers

When DNA binds to site A, cGAS forms a 1:1 complex with dsDNA [11,51]. On the basis of two molecules of 1:1 cGAS-DNA, DNA binding site B of cGAS interacts with dsDNA to form a 2:2 complex [11,51]. In cGAS dimers formed with DNA as a scaffold, there are interactions between cGAS and DNA, as well as interactions between cGAS.

The N-terminus of cGAS has been confirmed to effectively enhance the catalytic activity of cGAS [54]. Studies have shown that the N-terminus of cGAS is rich in a high proportion of positively charged amino acid residues, such as R (10.6% in hcGAS, 17.8% in mice cGAS, mcGAS) and K (6.2% in hcGAS, 4.8% in mcGAS). These residues can form strong interactions with negatively charged DNA [54]. Hyperphosphorylation of cGAS inhibits its own activity. Specifically, there are eight phosphorylation sites in the N terminus (Ser13, Ser37, Ser64, Thr69, Thr91, Ser116, Ser129 and Ser143 in hcGAS) and one in the catalytic domain (Ser305 in hcGAS) [55]. During mitosis, hyperphosphorylation of the N-terminus may disrupt its positively charged surface, resulting in weaker binding of cGAS to DNA (Figure 2B). Deletion of N-terminus (1–160 aa) reduces the activity of cGAS. However, deletion of C-terminal (161–522 aa) causes cGAS to lose its ability to activate the IRF3 pathway [52]. Many studies have revealed that mutations in site A and B, specifically in the positive residues from DNA binding regions, can significantly block the activity of cGAS (Table 2). cGAS dimers form positively charged grooves. In the groove, the electrostatic charge on the protein surface of cGAS will complement the charge of DNA, resulting in a closer cGAS-DNA combination.

Table 2.

Interactions of cGAS-NCP and cGAS oligomer.

Complex	Key residues in cGAS	Interface
cGAS-NCP complex a	R222	E61, E64 (H2A)
	R222	K240, R241 (cGAS)
	R241	E61, D90, E92 (H2A)
	K315	R71 (H2A)
	G316	R71 (H2A)
	K323	P50 (H2B)
	R341	R71 (H2A); D51 (H2B)
	R342	K75, T76 (H2A)
	R300, K301, R302	DNA phosphate backbone (NCP)
cGAS oligomer	K151, K152 R158, K160, R161, K162, S165, K173, R180, K372, K395, K399a	cGAS-DNA (site A)
	R222, K240, R244, K315, K323, K335, R341, R342a	cGAS-DNA (site B)
	K275, K285, K279, K282, R300, K301, G303, K427, K428, K432b	cGAS-DNA (site C)
	K335, K382, E386a	cGAS-cGAS (dimer)

^aAmino acid sequence in mouse cGAS.

^bAmino acid sequence in human cGAS.

Notably, the key residues K347, K394 and E398 in hcGAS (K335, K382 and E386 in mcGAS) are highly conserved in both hcGAS and mcGAS. These mediate the formation of the dimerization interface [56,57]. These residues are critical for the formation of cGAS dimers (Table 2). Mutations of these residues obstruct the formation of cGAS dimers. In the crystal structure of cGAS dimers, these three residues form a “hook-like” conformation (Figure 2C). Because these three amino acids are highly conserved, developing inhibitors of cGAS that target the formation of the cGAS dimer interface will effectively circumvent species and mutant variations.

2.2. Oligomerization & phase separation of cGAS

DNA length affects cGAS oligomerization and phase separation in the cytoplasm. Long dsDNA is more readily recognized by hcGAS than short dsDNA [58]. cGAS can recognize fewer than 20 bp of dsDNA in vitro, but 20 bp DNA is unable to activate cGAS in cells [59]. 45 bp DNA is a “length” threshold for activating intracellular cGAS [59]. Short DNA is able to form a 2:2 complex with cGAS, but it is unable to form an oligomer with cGAS [60]. In the process of cGAS oligomerization, long DNA binds to cGAS sites A and B, forming a “head-to-head”, “tail-to-tail” ladder-like cGAS oligomer [59]. DNA binding site C combines with DNA to form a more complex “networked-ladder” cluster of cGAS, leading to the phase separation of cGAS-DNA [53,61]. In the ladder-like oligomers formed with long DNA as a scaffold, there are interactions between cGAS and DNA, as well as interactions between cGAS molecules themselves. In addition to the interactions in cGAS dimer described earlier, there are “head-to-head”, “tail-to-tail” cGAS-cGAS interactions. But mutant studies have showed that direct “head-to-head”, “tail-to-tail” interactions are not required for oligomerization [59]. In a ladder-like oligomer, the interactions at the dimerization interface of cGAS and the interactions between cGAS and DNA are essential.

2.3. cGAS-NCP interaction leads to inactive conformation of cGAS

cGAS is generally considered as a cytosolic DNA innate immune receptor. But nuclear DNA damage induces cGAS translocation to the nucleus [62]. cGAS accumulates in the nucleus and chromosomes during mitosis. Meanwhile, 2′,3′-cGAMP is able to triggers IFN-independent DNA damage response signaling by suppressing homology-directed repair [63,64]. A large number of nucleosomes (NCP) keep cGAS inactive, preventing cGAS from recognizing self-DNA [65,66]. cGAS can also be recruited to DNA damage sites in combination with polyadenylate diphosphate ribosyltransferase-1 (PARP1) to block the formation of the PARP1-timeless complex and inhibit homologous recombination DNA repair, thereby promoting tumorigenesis [62]. However, low levels of cGAS-dependent IRF3 phosphorylation do not lead to inflammation during mitotic arrest [67,68]. It is capable of inducing apoptosis independently of transcription when there is mitotic aberration [67,68]. cGAS and IRF3 in cancer cells make mouse xenograft tumors responsive to the antimitotic agent paclitaxel [68]. Whereas cGAS knockdown decreases the response of cancer cells to paclitaxel [68]. Whether inhibitors of the cGAS-NCP interaction can be used to treat solid tumors in combination with antimitotic agents is worth exploring.

In the cGAS-NCP complex, DNA binding site B interacts with NCP leading a 1:1 cGAS-NCP complex. On the basis of the two molecule 1:1 cGAS-NCP complex, the DNA binding site C interacts with NCP to form a 2:2 cGAS-NCP complex [69]. The DNA binding site A does not directly interact with NCP. And dsDNA does not make contact with site A due to steric hindrance in cGAS-NCP complex [69]. Positively charged residues in cGAS have extensive hydrogen bond interactions with the acidic patch of NCP. Key residues at site B (R222, R241, K315, G316, K323, R341, R342) interact with the H2A-H2B histone of NCP, while key residues at site C (R300, K301, R302) interact with the DNA of NCP [70].

2.4. Other proteins that interact with cGAS

GTPase activating protein-(SH3 domain)-binding protein 1 (G3BP1) can enhance the binding of cGAS to DNA [71]. The interaction between cGAS and G3BP1 was analyzed by Co-immunoprecipitation. G3BP1 promotes the formation of cGAS-DNA complexes and facilitates cGAS in forming a primary aggregated state in its resting state. Knockout of G3BP1 significantly inhibits the production of type I interferon and the synthesis of 2′,3′-cGAMP induced by dsDNA. In vitro 2′,3′-cGAMP synthesis assay and DNA binding assay further showed that G3BP1 enhances the DNA recognition of cGAS and promotes its catalytic activity.

Poly(rC)-binding protein 1 (PCBP1) is an RNA-binding protein involved in the metabolism and regulation of RNA in cells. Recent studies have shown that PCBP1 is also involved in regulating the binding of cGAS to DNA, thereby affecting the innate immune response [72]. The KH1 domain of PCBP1 (1–80 aa) interacts with the C-terminal domain of cGAS (161–522 aa), and this interaction facilitates the binding of cGAS to DNA. PCBP1 positively regulates cGAS-mediated innate immune responses in a viral-dependent manner. PCBP1 can directly bind to DNA and mediate cGAS-dsDNA interaction, thereby regulating the host immune response.

Polyglutamine-binding protein 1 (PQBP1) mediates innate responses to human immunodeficiency virus 1 (HIV-1) and other lentiviruses by interacting with cGAS. Immunoprecipitation experiments revealed that the PQBP1 WW domain is required to interact with cGAS [73]. Encoded DNA-dependent protein kinase (DNA-PK) phosphorylates cGAS and inhibits its enzymatic activity [74]. Missense mutations in the catalytic subunit of PRKDC (DNA-PKcs) are associated with autoimmune diseases. Cells from mice lacking DNA-PKcs or from patients carrying PRKDC missense mutations exhibit an inflammatory gene expression signature.

2.5. Inhibitor targeting the cGAS-related PPIs

Interfering with the binding of cGAS-dsDNA will fundamentally inhibit the dimerization, oligomerization and phase separation of cGAS (Figure 2D). The antimalarial drug quinacrine (THP1 cells IC₅₀ = 3.7 μM) and its derivatives X6 (THP1 cells IC₅₀ = 14.0 μM) are DNA intercalators [75]. Quinacrine indirectly inhibits the interaction between cGAS and dsDNA by disrupting the conformation of dsDNA. In addition, the anti-Trypanosoma drug suramin can act as a competitive inhibitor to disrupt the interaction between cGAS and dsDNA [76]. Unfortunately, the inhibitory activity of suramin was not disclosed.

Rosaura et al. identified a potential druggable pocket centered on K335 and K382, which also includes R341, K372 and K395 in cGAS [77]. Compound Z918 was identified as a hit in a virtual screen, showing an IC₅₀ of 100 μM in a KinaseGlo cGAS in vitro assay. Compound CU-76 (In vitro ATP consumption assay IC₅₀ = 0.27 ± 0.06 μM) was discovered through structural optimization. In vitro IC₅₀ derived from a concentration-dependent enzymatic activity curve to measure ATP consumption from cGAS-mediated 2′,3′-cGAMP syntheses. Although the authors were not able to obtain a crystal structure of the CU-76-GAS complex, molecular docking studies revealed that CU-76 interacts with Glu386, Lys382 and His378 through hydrogen bonds. And they confirm that it does not disrupt the cGAS-dsDNA interaction in a high-throughput fluorescence polarization assay. In a series of optimizations (Figure 2E), the ester bond of Z918 is optimized, resulting in methyl ester substituted compound 1 (In vitro ATP consumption assay IC₅₀ = 1.9 μM). The amine group on the 1,3,5-triazine is replaced by a dimethyl substituted amine group, resulting in complete loss of activity (compound 2). Subsequently, the hydrophobic benzene ring is optimized to enhance the hydrophobic interaction, resulting in CU-76 (In vitro ATP consumption assay IC₅₀ = 0.27 μM). Epigallocatechin gallate, a natural small molecule found in green tea, can act as an inhibitor of G3BP1 and interfere with the binding of G3BP1 to cGAS. This interference prevents cGAS from recognizing abnormal DNA [71]. The author did not disclose the inhibitory activity of Epigallocatechin gallate.

3. Activation of STING & PPIs-related agonists

STING protein is a sensing and connector protein initially anchored to the ER, which is a transmembrane protein consisting of 379 amino acids [78]. STING protein appears in the form of a dimer on the ER membrane. STING monomers include N-terminal domain (1–18 aa), transmembrane domain (19–134 aa, TMD), connector (135–156 aa), ligand binding domain (157–343 aa, LBD) and C-terminal tail (344–379 aa, CTT). TMD is composed of four transmembrane helices. The connector includes loops (135–140 aa, 150–156 aa) and helix (141–149 aa) (Figure 3A). The connector between the two monomers in the dimer is in a cross formation.

Figure 3.

Activation strategies targeting STING. (A) Schematic of h-STING domain organization. (B) STING is activated by agonists targeting LBD. (C) The artificial LiSmore device replaces the STING platform. (D) Compounds that can synergistically activate STING lead to STING oligomerization, including C53 and NVS-STG2 (PDB ID: 7SII).

3.1. STING homodimer

In STING dimer, there is a close interaction between the two monomers. In LBD interface, key residues form a hydrophobic interface between STING monomers. W161A/M271A mutations may expose hydrophobic interfaces, leading to higher order oligomers [78]. SAVI mutation G166E in LBD significantly enhances the stability of STING dimer [5,6].

The CTT is disordered. Despite conducting relevant molecular dynamics simulations and mutational studies, the conformation of the CTT could not be confirmed [79]. The CTT is required for STING to activate downstream signals. But under inactive circumstances, CTT somehow autoinhibits STING [80]. SAVI mutant of STING, R284S, has been reported to be located at the tetramer interface [5,6]. Co-immunoprecipitation experiments showed that wild-type STING without the CTT can bind to CTT fragments, whereas R284S STING without the CTT cannot [6]. The CTT probably enables STING autoinhibition by interfering with the STING oligomerization interface. Exploring the interaction between CTT and STING oligomerization interface may lay the foundation for development of novel STING inhibitors or molecular glue-like compounds that induce STING polymerization.

STING can recognize 2′,3′-cGAMP synthesized by cGAS, as well as cyclic di-guanylate, cyclic di-adenylate and 3′,3′-cGAMP from bacteria and viruses. After recognizing 2′,3′-cGAMP, generally, STING LBD undergoes a 180° rotation relative to the TMD to form a non-crossover conformation. This rotation allows STING to expose the oligomerization interface and release the CTT [5]. Scientists have developed a series of inhibitors targeting the binding site of cyclic dinucleotides (CDNs) to compete with them, thereby inhibiting the STING pathway. A series of agonists have been developed that target the CDNs binding site, effectively replacing CDNs to activate the STING pathway (Figure 3B). However, given that the key steps for STING function are oligomerization and CTT release, regulating the interaction of STING oligomerization may be more straightforward and clearer.

3.2. Oligomerization & palmitoylation of STING

The STING dimer undergoes oligomerization and palmitoylation upon binding 2′,3′-cGAMP and undergoing conformational changes. STING oligomerization is based on CTT release and disruption of the “head-to-head” autoinhibition pattern [81]. When CTT is released, the oligomerization interface is exposed leading to STING oligomerization in ER, at which point the oligomeric interaction of STING may be weak [82]. In the STING tetramer, there are interactions between STING dimers. The LBDα2-LBDα3 loop (273–280 aa) undergoes a conformational change, transitioning from an outward extension to an inward convergence. This change allows for the interaction between the two dimers [5]. The conformational change of the LBD is based on the conformational reorganization of the connector. Studies have revealed that SAVI mutations V147L, N154S, V155M and V155R at the connector can induce conformational changes in LBD [5,6]. Palmitoylation at the C88/91 of TMD, which occurs in the Golgi, may also induce a conformational transition of TMD that is more favorable for the stabilization of STING oligomerization [83]. SAVI mutations C206Y, R281Q, R284S and R284G at the oligomerization interface have more direct effects on STING oligomerization [5,6]. These SAVI mutants induce STING oligomerization in the absence of 2′,3′-cGAMP. The SAVI mutant of R284S relies on C148 for oligomerization. C148 at the connector forms disulfide bonds between STING dimers, which enhances the stability of STING oligomers [6]. In addition, sulfated glycosaminoglycans synthesized in the Golgi can also bind to STING in the form of a hinge to mediate STING oligomerization [84]. The oligomerization of STING forms a protein-protein interaction platform, which is a crucial mechanism for the functioning of this immune pathway.

3.3. Oligomerization-related agonists

The CTT-bonded oligomer, which mimics the STING oligomerization platform, is a method for activating STING downstream (Figure 3C). In recent years, the small molecule synergistic induction of STING oligomerization has emerged as another important method for activating STING. Apart from STING agonists such as 2′,3′-cGAMP that bind to the CDN binding pocket, some STING agonists can also bind to the TMD to directly induce STING oligomerization (Figure 3D). Lu et al. reported a class of compounds (C53) that bind to the transmembrane domain of STING and can promote strong oligomerization of STING with 2′,3′-cGAMP [85]. However, neither C53 nor 2′,3′-cGAMP alone induced strong oligomerization. C53 binds to the TMD of STING, causing the transmembrane helices to separate from each other. This separation promotes the oligomerization of STING. Unfortunately, the activity of C53 was not disclosed. Liu et al. found that human STING functions as a proton channel [86]. Activated STING on liposomes transports protons, resulting in a pH increase in the Golgi. Proton leakage from organelles leads to the lipidation of light chain 3B (LC3B) and the activation of the inflammasome. Aberrant LC3B lipidation or inflammasome activation may result in excessive activation of the inflammatory response, leading to an inflammatory storm. C53 binding site is located at the channel interface. C53 blocks STING-induced proton flux in the Golgi and liposomes. C53 inhibits STING-induced LC3B lipidation and inflammasome activation. The interferon-induction function of STING can be separated from LC3B lipidation and inflammasome activation. Therefore, combining C53 with other STING agonists may alleviate the inflammatory storm side effect associated with STING agonists in clinical research. Stabilizing the oligomer conformation of STING is a potential direction for the development of STING agonists (Figure 3D).

Li et al. reported that the STING molecular glue-like compound (NVS-STG2, THP1-Dual ISRE-Luc assay AC₅₀ = 5.2 μM) can induce high-order oligomerization of hSTING [87]. NVS-STGs selectively activated cells containing hSTING TMD. NVS-STG2, C53 and 2′,3′-cGAMP can synergistically activate STING. The cryo-EM structure shows that the two NVS-STG2 molecules are arranged side by side through the central phenyl group. Each NVS-STG2 interacts with TM2, TM3 and TM4 of a STING dimer, while also interacting with TM2 and TM4 of the adjacent STING dimer. Among the hSTING mutations, R94A, L134A, R95A and L98A mutations reduce the activity of NVS-STG2 to varying degrees. However, L136A enhances the activity of NVS-STG2 (Figure 3D).

Recently, Dou et al. introduced a light-sensitive optogenetic device called LiSmore (light-inducible SMOC-like repeats) into dendritic cells to activate STING downstream signals [88]. Under the ultra-photosensitive CRY2 clustering system, the LiSmore device allows for the rapid and efficient polymerization of oligomers under biocompatible blue light stimulation. Linking CTT to CRY2 can replace the STING oligomerization platform to achieve light-regulated activation of STING downstream pathways (Figure 3C).

4. Proteins interacting with STING

4.1. STING recruits TBK1, IRF3 & AP-1

STING oligomerization provides a platform for recruiting and phosphorylating TBK1 and IRF3. Oligomerized STING is arranged side by side, and the CTT is released. The CTT has the TBK1 binding motif PLPLRT/SD [14], the IRF3 binding motif pLxIS (p, hydrophilic residue; x, any residue; S, phosphorylation site) [89], and the AP-1 binding motif EXXXLI (X, any residue) [21]. TBK1 undergoes trans-autophosphorylation upon binding to the STING CTT. pTBK1 phosphorylates the adjacent STING CTT at S366, whereas the CTT bound to TBK1 is not phosphorylated [90]. The phosphorylated STING (pSTING) CTT recruits IRF3. TBK1 phosphorylates IRF3, at which point both TBK1 and IRF3 bind to the STING CTT [91]. Phosphorylated IRF3 dissociates from STING, forming a dimer that translocates to the nucleus [92]. AP-1 is recruited by pSTING CTT and controls the trafficking of STING into the endolysosomal system for STING degradation [21]. The development of PPI inhibitors based on STING CTT is an important strategy for regulating the STING pathway. Among the interaction modes of STING with other proteins, the modes of STING interacting with TBK1, IRF3 and AP-1 are deeply studied, and inhibitors can be developed to inhibit or activate STING pathway by targeting these three interactions.

4.2. The interaction of STING-TBK1

TBK1, a serine/threonine kinase belonging to the inhibitor of κB kinases (IKKs) kinase family, is required for type I IFN responses by interacting with STING. TBK1 has 745 amino acids, including the N-terminal kinase domain (KD, 1–308 aa), the ubiquitin-like domain (ULD, 309–384 aa), the scaffold dimerization domain (SDD, 407–657 aa) and the C-terminal domain (CTD, 657–745 aa) [93].

TBK1 phosphorylation is significantly reduced in the STING mutations Q273A, A277Q and Q273A/A277Q which are located at STING tetramer interface [90]. The prerequisite for TBK1 to interact with STING is STING oligomerization, and TBK1 directly interacts with the CTT of STING [14]. During the binding of STING and TBK1, the N-terminus of TBK1 dimer attaches to the top of the LBD of STING. The TBK1 binding motif (369–377 aa, PLPLRT/SD) inserts into the groove formed by the TBK1 dimer in a chain-like conformation [14]. This groove is composed of the KD subunit of one TBK1 monomer and the SDD subunit of another TBK1 monomer. Blocking the interaction between TBK1 and STING would inhibit the downstream activities, such as the phosphorylation of STING, TBK1 and IRF3 [94]. As shown in the x-ray crystal structures of the protein, R27, N578 and Q581 form hydrogen bonds to the backbone of STING CTT (Figure 4A). Pull-down assays showed that mutations P371Q, L374A and R375A in the STING CTT region, as well as mutations Y577A, N578A and Q581A in TBK1, disrupt the interaction between STING and TBK1 [14].

Figure 4.

The binding model of STING-TBK1 complex, STING-IRF3 complex, and STING-AP-1 complex. (A) The binding model of STING-TBK1 (PDB ID: 6O8C), including TBK1 monomer (cyan or green), STING CTT (red and white), and TBK1 binding motif PLPLRT/SD (red). (B) The binding model of STING-IRF3 (PDB ID: 5JEJ), including IRF3 monomer (cyan or green), STING CTT (red and white), and IRF3 binding motif pLxIS (red). (C) The binding model of STING-AP-1 (PDB ID: 7R4H), including AP-1γ (orange), AP-1σ (green), other AP-1 domains (cyan), and AP-1 binding motif EXXXLI (red).

The kinase domain of TBK1 is also polyubiquitinated by K63-linked chains [95]. After this, some adaptor proteins bind to the CTD of TBK1. These adaptor proteins include TRAF family member-associated NF-κB activator, NF-κB activating kinase associated protein 1, similar to NAP1 TBK1 adaptor and optineurin [96]. K63-linked or linear polyubiquitin binds to UBAN domain in the adaptor proteins triggering oligomerization of TBK1 and resulting in trans-autophosphorylation of TBK1 [95].

4.3. The interaction of pSTING-IRF3

IRF3 is a transcription factor that has the ability to regulate interferonand inflammatory factors [97]. The structure of IRF3 consists of two functional domains: an N-terminal DNA-binding domain and a C-terminal regulatory domain [98]. In its inactive state, IRF3 is monomeric, and its DNA-binding domain interacts with its C-terminal regulatory domain, thereby blocking the transcriptional activity of IRF3 [99]. When cells are subjected to viral infection or other stimuli, the C-terminal regulatory domain of IRF3 is phosphorylated, leading to its dimerization and transcriptional activity. This promotes the expression of interferons and inflammatory factors [99].

IRF3 is recruited to the STING pLxIS motif through electrostatic interactions, hydrophobic interactions and hydrogen bonding [100]. Crystal structures show that pS366 of STING interacts with positively charged residues of IRF3 (R285, H288, H290 and K313) through electrostatic interactions (Figure 4B) [89]. pS366, I365 and L363 make contact with the backbone of IRF3 by forming hydrogen bonds. Subsequently, phosphorylation of S386 and S396 of IRF3 occurs by TBK1, which then leads to the release of the C terminus from the DNA-binding domain. The phosphorylated C-terminus interacts with another IRF3, causing IRF3 to dissociate from STING and form a dimer [101]. R211 and R285 interact with pS386 and pS396, respectively, in the IRF3 dimer. IRF3 dimers then translocate into the nucleus, where the DNA-binding domain DNA-binding domain interacts with DNA to initiate transcription of IFN. A class of phenylmethimazole molecules has been reported to block the homodimerization and nuclear translocation of IRF3 [102].

4.4. Interaction of AP-1 with STING ends STING signaling

AP-1 controls the termination of STING-dependent immune signaling [21]. AP-1 is an adaptor protein complex that plays a role in protein transport from the trans-Golgi network to the endolysosomal system. It is composed of β1, γ, μ1 and δ1 subunits. AP-1 plays a role in inhibiting the activation of STING by regulating the entry of STING into CCVs and subsequently into endolysosomes. CTT interacts with AP-1, and STING^LI (L364A/I365A) cannot bind to AP-1. The LI motif of STING CTT mediates the direct interaction in the STING-AP-1 complex (Figure 4C). The specific recognition of STING by AP-1 depends on STING pS366, and the S366A mutant impairs the interaction between STING and AP-1.

T he STING-AP-1 complex exhibits the details of the PPI interface [21]. The EXXXLI motif recruits AP-1 through electrostatic and hydrophobic interactions. L65, F67, H85, V88 and V98 of the δ subunit form a hydrophobic cavity that engages with L364 and I365 of STING through hydrophobic interactions. R15 of γ subunit forms electrostatic interactions with the carboxyl group of E360. The phosphate group of pS366 can interact with K60 and R61 of δ subunit through hydrogen bonds. AP-1 initiates the degradation of STING through autophagy.

4.5. Other proteins that interact with STING

STIM1 is an intracellular calcium sensor protein that is primarily expressed in the endoplasmic reticulum membrane. It plays a crucial role in reducing intracellular calcium concentration [103,104]. Recent studies reveal that STIM1 negatively regulates IFN expression by retaining STING in the ER [105]. Co-immunoprecipitation showed that the N-terminal transmembrane segment (1–249 aa) of STIM1 interacted with STING TMD. Co-expression of full-length STIM1 or STIM1 (1–249 aa) with STING suppressed the autoimmune disease caused by the STING mutant. In addition, the loss of STIM1 significantly enhanced IFN expression following viral infection. Hemophagocytic lymphohistiocytosis-associated protein UNC13D is a negative regulator of STING signaling [106]. UNC13D can interact with STING to inhibit STING oligomerization. Cellular knockdown and deletion of UNC13D promoted IFN-β production induced by DNA viruses, but not RNA viruses. Song et al. generated a series of STING truncates and verified that UNC13D interacts with the TM2-TM3 linker and CTD of STING through co-immunoprecipitation.

Inhibitor of nuclear factor kappa B kinase subunit epsilon (IKKϵ) and TBK1 are homologous protein kinases that play an important role in the immune response [107]. There is also an important interaction between IKKϵ and STING. TBK1 is dispensable for STING-mediated interferon production but redundant for STING-mediated activation of the NF-κB pathway [107]. Neither TBK1^KO nor IKKϵ^KO affected STING-mediated NF-κB pathway activation, whereas TBK1^KO/IKKϵ^KO clearly blocked STING-mediated NF-κB activation. This interaction has significant biological implications in viral infection and tumor immunity.

PKR-like endoplasmic reticulum kinase (PERK) is a signal transduction protein anchored to the endoplasmic reticulum. It plays a role in the context of endoplasmic reticulum stress and abnormal protein folding [108]. Zhang et al. found that the intracellular domain of PERK interacts with STING CTT through a co-immunoprecipitation assay. PERK is activated by autophosphorylation after binding to STING, which is dependent on STING but independent of endoplasmic reticulum stress. Activated PERK phosphorylates eukaryotic initiation factor 2α, leading to an inflammatory- and survival-preferred translation program. This mechanism is temporally prior to STING-TBK1-IRF3 signaling, leading to significant inhibition of mRNA translation but specific promotion of the expression of inflammation- and survive-related proteins.

In addition, STING undergoes post-translational modifications, such as phosphorylation, ubiquitination and palmitoylation, to exert its function. Serine/threonine UNC-51-like kinase 1 is involved in phosphorylating STING. Mg²⁺/Mn²⁺-dependent protein phosphatase 1A is involved in the dephosphorylation of STING [109]. Many ubiquitin ligases can ubiquitinate STING, but their regulation of STING is inconsistent. Ubiquitination, mediated by the ubiquitin ligase RNF5 and tripartite motif 30α, acts as a negative regulatory mechanism for STING, mediating STING degradation [110]. The ubiquitinating enzymes TRIM56, TRIM10, AMFR and INSIG1 serve as positive modulators of STING signaling [111]. The palmitoyl transferase DHHC is involved in the palmitoylation of C88/91, which is particularly crucial for this signaling pathway [112].

5. Small-molecules inhibitors targeting STING oligomerization & palmitoylation

Referring to the binding pocket of endogenous ligands, the researchers developed STING inhibitors targeting CDN binding pockets (Figure 5A). In recent years, a series of STING inhibitors that covalently target STING oligomerization have emerged. STING palmitoylation at the Golgi promotes the oligomerization of STING, which is required for the type I IFN response. Interfering with the interaction of STING oligomeric interfaces is a key focus of STING modulators. Covalent targeting of C148 and C88/91 is an important strategy for interfering with STING oligomerization. Currently, small molecule inhibitors targeting the STING oligomerization interface (C292, C148 and C88/91) that have been developed are covalent inhibitors (Figure 5B).

Figure 5.

Inhibition strategies targeting STING. (A) STING is inhibited by inhibitor targeting LBD. (B) Compounds targetingC88, C91, C148, and C292 of STING result in blocking oligomerization (PDB ID: 6NT5). (C) The structure-activity relationship of BB-Cl-Amidine and LB244. (D) The structure-activity relationship of H151 and C-178.

5.1. BB-Cl-Amidine derivatives

Fiachra Humphries et al. developed a class of STING covalent inhibitors (BB-Cl-Amidine derivatives) targeting C148 [113]. Researchers evaluated the effect of a pan-protein Arginine Deiminases inhibitor on STING signaling. By substituting covalent warheads and conducting mass spectrometry analysis, researchers have discovered that BB-Cl-Amidine (THP1 EC₅₀ = 2.0 μM) selectively binds to STING C148 through covalent interactions. qPCR and immunoblot analysis in PAD knockout cells showed that BB-Cl-Amidine covalently modifies STING in a PAD-independent pathway, thereby impairing STING oligomerization and downstream signaling. Researchers also used BB-F-Yne as a negative control, which is a more selective PAD active probe that does not inhibit STING signaling. This was done to verify that PAD inhibition does not affect STING signaling. Trex1^D18N/D18N mutant mice develop severe myocarditis. BB-Cl-Amidine can effectively reduce myocardial fibrosis. On the basis of BB-Cl-Amidine, researchers substituted the covalent warhead and subsequently developed LB244 (THP1 EC₅₀ = 0.8 ± 0.1 μM) with a nitrofuran covalent warhead [114]. In the optimization of the biphenyl group, potent compounds also generally contain highly hydrophobic groups and hydrophilic substitutions on these moieties were not tolerated (AFM83B, no activity). Substitutions around the benzimidazole lead to maintained activity (AFM140). The length of the linker between covalent and non-covalent parts has little effect on the activity of the compounds (Figure 5C). The covalent warhead is the key group to the biological activity of compounds (LB070, THP1 EC₅₀ >40 μM). Validation of the binding site revealed that LB244 acted at C292, but not C148, and adversely affected STING oligomerization.

5.2. H151 & Nitrofuran derivatives

Ablasser et al. discovered nitrofuran compounds C-176 (IFN-Luc activity = 0.4, inhibition ratio vs DMSO) and C-178 (IFN-Luc activity = 0.1) through cell-based chemical screening [115]. C-178 and C-176 significantly inhibit the STING pathway. C-178 was confirmed to covalently bind to C91 in the STING protein through STING amino acid mutation assay, mass spectrometry and molecular probe assay. C-178 interferes with STING palmitoylation and STING aggregation. The nitrofuran fragment undergoes a nucleophilic addition to the nucleophilic side chain of C91, followed by intramolecular rearrangement to form a covalent bond. The more water-soluble compound C-176 was selected for evaluation of its efficacy. C-176 significantly ameliorates systemic inflammation in Trex1^-/- mice. Compounds C-176 and C-178 exhibit strong activity against mSTING but demonstrate weak activity against hSTING. As a result, the researcher conducted additional structural investigations. In this series of structural optimization, the N-methylation modification of the amide linkage chain leads to inactive compound 3 (IFN-Luc activity = 1.2). Both the removal of nitrofuran (compound 4, IFN-Luc activity = 1.3) and the replacement of the furan ring (compound 5, IFN-Luc activity = 1.3). The activity of halogen group substituted compounds (compound 6, IFN-Luc activity = 0) was partially improved. The activity was maintained for compounds with extended alkyl side chains (compound 7, IFN-Luc activity = 0.4). In addition, the activity of cyano-substituted compounds decreased significantly (compound 8, IFN-Luc activity = 0.7). Subsequently, H151 (IFN-Luc activity = 0.4) was identified from counter screens and validation screens (Figure 5D). The irreversible modification of STING by H151 was confirmed by LC-MS/MS analysis to be C91-dependent. H151 inhibits the type I IFN response, reduces TBK1 phosphorylation and interferes with hSTING palmitoylation.

5.3. Nitro-fatty acid derivatives

Previously, nitro-fatty acid compounds (NO₂-FAs) have been identified as lipids with anti-inflammatory activity. Hansen et al. identified that nitro-conjugated linoleic acid (NO₂-cLA) and nitro-oleic acid (9-NO₂-OA and 10-NO₂-OA) form covalent bond with STING through Michael covalent addition with alkenes [116]. In subsequent mechanistic studies, the researchers found that NO₂-FAs depend on Cys88/91 and N-terminal His16 to disrupt STING palmitoylation. Cys88/91 is non-selectively covalently modified, and His16 is nitro-alkylated by NO₂-FAs. The researchers did not report activity data. However, 10-NO₂-OA has entered multiple Phase II clinical trials (NCT04125745, NCT03449524, NCT04053543 and NCT03422510) in focal segmental glomerulosclerosis and hypertension pulmonary. Unfortunately, the author did not disclose the inhibitory activity of this series of compounds.

5.4. Acrylamide derivatives

Vinogradova et al. identified more than 3000 covalently liganded cysteines on different proteins in primary human T cells [117]. Some electrophilic compounds can influence the biological effects of T cells by covalently targeting cysteine residues. The researchers obtained BPK-25 (ISRE-Luc activity = 3.2 μM) with acrylamide structures by screening a library of electrophilic small molecules. Protein spectrum analysis and molecular docking showed that BPK-25 forms a covalent bond with C91 to prevent STING palmitoylation.

5.5. Briarane-type diterpenoids

The diterpenoid natural product excavatolide B (ExcB) with an epoxy-lactone structure was extracted from the coral Briareum stechei. Briaexcavatolide L is an analog of ExcB where the epoxide ring has been opened, resulting in a loss of anti-inflammatory activity [118]. Researchers speculate that ExcB exerts its anti-inflammatory effect by covalently binding to STING through epoxy-lactone. An ExcB analogue with terminal alkynes (EXCB-16C-AOyne) was designed and synthesized for bioorthogonal conjugation, enabling fluorescence visualization and protein enrichment. Using the probe ExcB-16C-AOyne, the researchers determined that this compound targets STING. Intact mass analysis showed that the epoxy-lactone warhead of ExcB covalently binds to the palmitoylation site C91 of STING. Unfortunately, the author did not disclose the inhibitory activity of this series of compounds.

6. Conclusion

In recent years, several cGAS inhibitors have been developed for the treatment of autoimmune diseases (Table 3). Among these compounds, X6 has progressed to preclinical experiments for the treatment of SLE. VENT-03 commenced phase I clinical trials for the treatment of SLE. It is a strategy for developing cGAS inhibitors that targeting the progression of cGAS phase separation or the interactions between cGAS and proteins. For example, interrupting the interaction between cGAS dimers can effectively block the cGAS phase separation and reduce the production efficiency of 2′,3′-cGAMP. It may be necessary to develop inhibitors of DNA-PKcs or NCP in order to indirectly activate cGAS, in combination with mitogenic antagonists, to achieve a synergistic anticancer immunity. Although the amino acid mutations, based on the ladder-like higher-order oligomerization model of cGAS, show that there are no specific interactions at the oligomerization interface. The development of molecular glue-like compounds based on higher-order oligomerization cocrystal structures may also be worth trying to enhance cGAS activity.

Table 3.

The summary of cGAS-STING modulators which intervening PPIs.

Action effect	Compound name	Target	Condition or disease	Identifier (Phase)
cGAS inhibitor	X6 (Antimalarial drugs)	DNA intercalators	Systemic Lupus Erythematosus	Preclinical
	CU-76	The interface of cGAS dimer	Autoimmune Diseases	Preclinical
	Suramin (Immunomodulator)	DNA competitive inhibitor	/	/
	Epigallocatechin gallatc	G3BP1-cGAS inhibitor	/	/
STING agonist	NVS-STG2	Between the TMD of the two STING dimers	Tumor	Preclinical
	C53	The TMD of STING dimer	/	/
STING inhibitor	BB-Cl-Amidine	Covalent bound at C148	/	/
	LB244	Covalent bound at C292	Autoimmune Diseases	Preclinical
	BPK25	Covalent bound at C91	/	/
	C-178		Inflammatory disease	Preclinical
	H151		Psoriasis	Preclinical
	ExcB		/	/
	NO2-cLA	Covalent bound at C88/C91	Pulmonary arterial hypertension	Phase II clinical trials (terminated)

The development of LiSmore introduces a new concept for artificially constructing a “STING-like” platform. NVS-STG2 offers a novel method for promoting the stabilization of STING oligomerization. Although scientists attempt to mitigate the excessive immune response caused by STING agonists in clinical settings using various techniques, there is still no optimal solution. Blocking the interaction between STING and AP-1 could be a strategy for activating STING pathway, which can increase the STING level in cells. The inhibitors which target the STING-AP-1 interaction to indirectly activate STING may be possible to avoid inflammatory factor storm side effects. The development of STING inhibitors also faces some challenges. In diseases associated with gain-of-function mutations in STING, such as SAVI, inhibitors that target STING CDN binding site have not outstanding effects. Inhibitors, which targeting oligomerization, STING-TBK1 interaction and STING-IRF3 interaction could be exhibit improved efficacy, should be developed. In addition, the bypass activation of STING also contributes to inflammatory diseases. But the mode of interaction of STING with IKKϵ and PERK, respectively, has not been clearly studied.

With a more comprehensive understanding of this pathway in disease, the application of modulators has more potential [119]. The hyperactivation of the cGAS-STING pathway is thought to induce inflammatory diseases [120]. The cGAS-STING pathway, however, plays a dual role in the occurrence and development of tumors [31]. cGAS-STING inhibitors and agonists may be used independently in the same cancer patient at different stages of disease progression. The development of the disease is complex. It is needed that developing more cGAS-STING inhibitors or agonists with novel mechanisms. cGAS-STING modulators, based on protein-protein interactions, can accurately regulate downstream signals in terms of type and intensity, thereby meeting this demand.

7. Future perspective

It is important to note from cGAS-STING pathway that regulations of the cGAS phase separation process, STING oligomer interactions and STING-downstream interactions are important methods for developing modulators. We are working on an exploration of the discovery and development of cGAS-STING modulators. The structure of protein-protein cocrystal structures or a series of biological experiments to verify protein-protein interactions can suggest the key amino acids in PPIs. These key amino acids will lead us to explore potential sites of pharmacological intervention. One of the most common research methods is to design peptides containing key amino acids and further optimize the peptide molecule or redesign the backbone to discover regulators with good activity and druggability. Among the cGAS-STING pathways, cGAS-NCP, STING-TBK1, STING-IRF3 and STING-AP-1 are the promising PPIs for the development of peptide molecules and the discovery of small molecule regulators. For example, STING CTT is a disordered, long-chain peptide bound in a groove formed by TBK1 dimer. We can design peptide molecules based on CTT to block STING-TBK1 interactions. Due to its large molecular weight, peptide molecules can better bind to the STING-TBK1 interaction interface, thereby efficiently blocking the STING-TBK1 interaction. However, it is relatively difficult to develop small molecules to efficiently block STING-TBK1 interactions. The interaction interface between cGAS-NCP and STING-IRF3 is relatively flat and STING-AP-1 has CTT binding pocket but most of the area is exposed to the solvation. Peptides have an advantage in blocking the interaction between cGAS-NCP, STING-IRF3 and STING-AP-1. At present, there is a lack of molecules with clear mechanisms for these interactions. In the cGAS-STING pathway, only the cGAS inhibitor VENT-03 commenced the Phase I clinical trial. STING inhibitors are mostly tool molecules, and their druggability and safety are not good enough. At present, the research of cGAS-STING requires more molecules with novel mechanisms and better druggability. Finally, with the evolving field of cGAS-STING molecular pathways and newer strategies being introduced, more modulators with novel mechanism can be obtained in the near future.

Funding Statement

This work was supported by Projects 82273786 and 82073766 of the National Natural Science Foundation of China; the original exploration project (2632022YC05) and the Project Program of State Key Laboratory of Natural Medicines (No. SKLNMKF202311) of China Pharmaceutical University; the Outstanding Youth Foundation of Jiangsu Province of China (CN) (Grant BK20240199).

Financial disclosure

This work was supported by Projects 82273786 and 82073766 of the National Natural Science Foundation of China; the original exploration project (2632022YC05) and the Project Program of State Key Laboratory of Natural Medicines (No. SKLNMKF202311) of China Pharmaceutical University; the Outstanding Youth Foundation of Jiangsu Province of China (CN) (Grant BK20240199). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Data availability statement

No data was used for the research described in the article.