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Bioactive modulators targeting STING adaptor in cGAS-STING pathway
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Bioactive modulators targeting STING adaptor in cGAS-STING pathway Q2
Xi Feng, Dongyu Liu, Zhiyu Li and Jinlei Bian Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-pathway triggers innate immune responses by recognizing cytosolic DNA. Recent studies revealed that STING adaptor associates with various diseases, and several modulators targeting STING have been identified including three agonists that have entered clinical trials for treating cancer over the past 2 years. In particular, the efficacy of STING agonists and/or antagonists suggests adaptor STING as a potential therapeutic target for diverse diseases. Herein, we summarize the latest advances in understanding STING functioning and provide an overview of recent STING modulator discoveries, including structural details and the potential therapeutic applications of these modulators.
Introduction Innate immunity is the first-line natural barrier of the host for the elimination of pathogenic infections that can be triggered by pattern recognition receptors (PRRs), such as cytosolic DNA sensors [1]. Among them, the cGAS-STING pathway has a key role in detecting cytosolic DNA to activate downstream intracellular signal cascades and inducing type I interferons (IFNs) to trigger innate immune responses for antimicrobial effects, as well as initiating autophagy [2,3]. Increasing evidence has revealed that the cGAS-STING pathway is involved in modulating anticancer immunity and contributing to autoimmune, autoinflammatory disorders [2,4]. In particular, the transmembrane protein STING in the cGAS-STING pathway has received increased attention from both industry and academia in recent years. Hence, in this review, we highlight the latest understanding of STING functioning as well as the discovery and structural features of recently disclosed modulators targeting STING adaptor, including agonists and antagonists.
(cGAMP in eukaryotic cells), a second messenger, after engaging with DNA [5]. The synthesized cGAMP in turn binds to the endoplasmic reticulum (ER)-membrane adaptor STING (also known as MITA, ERIS, MPYS, or TMEM173) as an endogenous ligand and induces the activation of STING [6]. Some bacterial cyclic dinucleotides (CDNs) are able to bind and activate STING directly [7]. After stimulation by CDN, the ER-locating STING then traffics to perinuclear compartments, including Golgi apparatus, endosomes, and autophagy-related compartments [8]. During this process, STING recruits TANK-binding kinase 1 (TBK1) at its C-terminal tail and promotes the autophosphorylation of TBK1, which in turn phosphorylates the transcription factor IRF3 [5,9]. In addition, the transcription factor nuclear factor (NF)-kB is also activated in response to STING cascades [6]. As a result, the activated transcription factors enter the nucleus and function together to induce the production of type I IFNs, such as IFNb, and inflammatory cytokines, including tumor necrosis factor-a (TNF-a) and interleukin 6 (IL-6), which ultimately boost the fast-acting and robust innate immune system as well as the resulting adaptive immunity (Fig. 1) [5].
cGAS-STING pathway The initial sensing of aberrant cytosolic double-stranded (ds)DNA is accomplished by cGAS. It catalyzes the synthesis of cyclic GMP-AMP
Corresponding authors: Li, Z. ([email protected]), Bian, J. ([email protected]) 1359-6446/ã 2019 Elsevier Ltd. All rights reserved. https://doi.org/10.1016/j.drudis.2019.11.007
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Diseases involving STING Among the aforementioned cytosolic DNA sensing pathways, the ER-locating adaptor STING is considered to be the converging point for enhancing or suppressing immune responses [10].
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FIGURE 1
The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is activated by sensing cytosolic DNA. Cytosolic DNA (either from foreign DNA or self-DNA) is recognized by cGAS and leads to the generation of cGAMP. cGAMP directly binds to the endoplasmic reticulum (ER) adaptor STING, which subsequently traffics to the Golgi. After being palmitoylated at Golgi, STING recruits and activates TANK-binding kinase 1 (TBK1), which further recruits interferon regulatory factor 3 (IRF3) for phosphorylation and dimerization. The phosphorylated IRF3 dimer then enters the nucleus and functions with nuclear factor (NF)kB to induce the expression of type I interferons (IFNs) and other cytokines. For additional definitions, please see the main text.
Aberrant activation of STING is correlated with various diseases, and efforts have been made to regulate this pivotal pathway as a pharmaceutical approach. On the one hand, excessive signaling through STING is implicated in chronic inflammation [11,12]. Loss-of-function mutations in Trex1 can lead to the dysfunction of its encoding enzyme, which acts as a 30 -50 DNA exonuclease (TREX1) [13]. This loss of enzyme activity could contribute to the abnormal accumulation of cytosolic DNA, leading to the continuous signaling of the cGAS-STING pathway, and resulting in autoinflammatory disorders, including Aicardi–Goutie`res syndrome (AGS) and systemic lupus erythematosus (SLE) [14,15]. Similarly, gain-of-function mutations in the STING-encoding gene TMEM173 are linked to the hyperactivation of STING, which can result in systemic inflammation, known as STING-associated vasculopathy with onset in infancy (SAVI) [15,16]. In patients with SAVI, several mutants of STING have been identified, including V147L, N154S, and V155M [15,16]. These STING variants are considered to be sensitive and could be activated in a ligandindependent manner [8]. In addition, the cGAS-STING pathway has been shown to enhance tumor development by activating indoleamine 2,3-dioxygenase (IDO, an immune checkpoint) pathway in a murine Lewis lung carcinoma (LLC) model [17]. 2
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On the other hand, initiating the host cGAS-STING pathway for antitumor immunity is a promising approach in cancer immunotherapy. The cGAS-STING pathway is an important tumor-surveillance mechanism in host antitumor immunity [4]. In tumor cells, DNA leakage in the cytoplasm during mitosis is detected by cGAS, leading to the secretion of type I IFNs [18]. Type I IFN signals are responsible for stimulating the cross-presentation of tumor antigens and priming tumor-specific CD8+ effector T cells [18]. In addition, dendritic cells (DCs) can express type I IFNs by taking up tumor-derived DNA [4,19]. Activation of the cGAS-STING pathway is also associated with promoting cellular senescence by mediating the production of type I IFNs and senescence-associated secretory phenotype (SASP) [2,20]. Of note, it has been observed that the cGAS-STING pathway is irregularly inhibited in various cancer types, including melanoma and colorectal carcinoma [21,22]. Increasing evidence indicates that specific activation of the STING pathway might suppress tumor progression by initiating intrinsic immunity, further causing the tumor to become ‘hot’ by restoring T cell infiltration into the tumor microenvironment (TME), which is considered to be beneficial for immune checkpoint blockage therapy [23]. Given that disorder of the cGAS-STING pathway is associated with various diseases, modulating this fundamental pathway
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STING activation STING activation can be divided into an ER-locating stage and a post-ER stage based on our current understanding (Fig. 1). Human STING (hSTING) protein functions as a homodimer, comprising a luminal N-terminal domain (aa 1–154) that anchors ER with four transmembrane helices, and a cytosolic C-terminal domain (CTD, aa 140–379) containing the ligand-binding pocket (Fig. 2a,b) [25]. The crystal structure of an apo-STING CTD dimer showed that the ligand-binding domain (LBD) exhibits an inactive ‘open’ conformation and, correspondingly, the cGAMP-bound STING dimer showed an active ‘closed’ state, which refers to a conformational change driven by the binding of the ligand (Fig. 2c) [26,27]. Recently, Shang et al. revealed that cGAMP could induce a 180 clockwise rotation of the STING dimer CTD relative to the transmembrane domain (TMD), which gradually led to the formation of a b-sheet that caps the binding pocket and the resulting closure [27]. This conformational change is indispensable for STING activation, but the exact mechanism remains unclear. However, this ‘closed’ conformation could contribute to the following ER-toGolgi translocation and the oligomerization of STING [27]. Furthermore, investigations of the gain-of-function mutations of Sting in SAVI showed that variants including V147 L, N154S, and V155 M usually locate at the connector region. Thus, mutated residues in the helix loop might contribute to, or drive, the conformational change because of their larger side chains (Fig. 2b) [27]. After ligand binding, STING translocates from ER to the Golgi. This movement is necessary because blocking ER-to-Golgi traffic with brefeldin A (BFA) or expression of Shigella effector IpaJ abolished forward signaling events, including phosphorylation of IRF3 and induction of IFNb [8,28]. The observation of some SAVI STING variants localizing to the perinuclear compartments without stimulation by ligand is consistent with the implication that the conformational changes in STING contribute to the postER state [8]. After translocation, a post-translational modification of STING at Golgi occurs at the TMD, including palmitoylation at Cys88/91 (Fig. 2b) [29]. It was suggested that the Golgi-located protein palmitoyltransferase DHHCs catalyze this modification. Palmitoylation at cystine was shown to be important for STING activation because either the generation of a STING mutant (C88/ 91S) or using palmitoylation inhibitors abolished downstream gene expression [12,29]. It was proposed that palmitoylation promotes STING activation by facilitating the oligomerization of STING at Golgi membrane, further contributing to the recruitment of TBK1 and IRF3 [9,29]. This modification might be independent of CDN binding and has been considered as a target for inhibiting STING activation [29–31]. The STING dimer could then
bind to the TBK1 dimer via its C-terminal tail and activate TBK1 for further phosphorylation of STING and IRF3 in a ligand-dependent manner [9,32]. The major phosphorylation site of STING is Ser366, located at the C-terminal tail. It was suggested that this phosphorylation is accomplished by an another TBK1 dimer from a neighboring STING-TBK1 complex [9,33]. The phosphorylated tails of STING might further engage and deliver IRF3 to TBK1 for further phosphorylation and signaling [9]. Furthermore, the phosphorylation of Ser172 at TBK1, which is necessary for its activation, was also considered to be achieved by another TBK1 molecule because this residue could not reach any kinase active sites of either monomer [9]. This interaction of STING with TBK1 might in turn reflect the importance of clustering of STING at Golgi.
Bioactive modulators targeting STING adaptor Agonists Given the key role of STING in initiating innate immunity, delivering STING agonists to boost immune signaling in TME offers a potential cancer immunotherapy approach. In recent years, several STING agonists have been identified and three candidates are in Phase I/II clinical trials. In general, most reported STING agonists function by directly connecting with LBD by mimicking the native ligand to trigger STING for the following reactions. This is a straightforward and reliable approach because CDNs have been known as small-molecule cell-signaling mediators for decades [34]. In 2016, Novartis and Aduro BioTech released their synthetic CDN ADU-S100 (ML RR-S2 CDA, Fig. 3a) as a first-in-class STING agonist. This has entered Phase I clinical trials for the treatment of solid tumors or lymphomas as monotherapy or in combination with immune checkpoint inhibitors (ICIs) [35,36] (Clinicaltrials. gov ID: NCT03937141, NCT03172936, and NCT02675439). CDN ADU-S100 is a dithio derivative of natural CDN 20 -30 cAMP and is reported to display improved chemical and/or metabolic stability and higher potency in activating STING compared with cGAMP [35]. In May 2019, a Phase II clinical trial of ADU-S100 was scheduled for the treatment of PD-L1 positive recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). However, the annular structure of CDNs with two nucleotides might be not favored by some medicinal chemists for developing potential drugs. Indeed, the physicochemical properties of CDNs, including high polarity and poor membrane permeability, could limit their efficacy in vivo. In addition, metabolic stability, including enzymatic degradation by phosphodiesterases (e.g., ENPP1) or nucleases as well as the synthetic issue of CDN derivatives for structure-activity relationship (SAR) screening should also be of concern [37]. Interestingly, it was reported that the stereochemical configuration of the two phosphates in ADU-S100 is crucial because the [Rp, Rp] diastereomers (e.g., ADU-S100) showed improved potency in resisting digestion and inducing type I IFNs in human THP1 cells compared with the corresponding [Rp, Sp] diastereomers (Fig. 3a) [35]. In addition, the 20 -30 linkage of CDN enhanced the binding affinity to hSTING, but this observation was not consistent with the outcome of synthetic cAIMP analogs in a luciferase assay [35,38]. The dithio modification of phosphate could increase enzymatic stability while maintaining its high affinity for hSTING [35]. Moreover, the mono or dual fluoro-substitution in the 20 -ribose sugar of 30 -30 cAIMP analogs improved the enzymatic stability, as well as the ability to induce type I IFNs, with EC50 of www.drugdiscoverytoday.com
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could be a potential and powerful tool against diseases ranging from inflammation to cancer. However, despite a few molecules characterized as cGAS enzyme inhibitors, it is difficult to develop drugs targeting cGAS [24]. Indeed, growing attention has been paid to another compelling target, STING adaptor, because several agonist and antagonists and/or inhibitors with different mechanisms of action have been developed and reported to be efficient in vivo. Recent studies also revealed the unique and complex signal process involving STING adaptor. Thus, it would be useful to further understand this side for future drug development.
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FIGURE 2
Structures of stimulator of interferon genes (STING) dimer and the binding of agonists. (a) STING is a transmembrane protein. (b) Cryo-electron microscopy (EM) structure of full-length human STING [Protein Data Bank (PDB) ID: 6NT5], V147/N154/V155 variants are marked in red and Cys88/91 are marked in green. (c) Cocrystal structure and a close-up of cGAMP in complex with human (h)STINGH232 (PDB ID: 4LOH). cGAMP is shown in green, and key residues and hydrogen bonds are shown as sticks and black dashes, respectively. (d) Co-crystal structure of dimethyloxoxanthenyl acetic acid (DMXAA) with hSTINGS162A/Q230I/Q266I (PDB ID: 4QXR). DMXAA is shown in green and mutated residues are marked in dark yellow and pink. (e) Co-crystal structure of di-aminobenzimidazole (diABZI) (compound 3) with hSTING (PDB ID: 6DXL). diABZI is shown in green. For additional definitions, please see the main text.
0.3–1.1 mM in an IRF-luciferase assay, whereas cGAMP exhibited an EC50 of 7.2 mM [38]. Generally, the instability of CDNs limited its application and has been regarded as a challenge and goal when optimizing CDNs. Interestingly, several ENPP1 phosphodiesterase 4
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inhibitors have been designed and developed to activate STING indirectly for improved antitumor efficacy [39,40]. The co-crystal structure of cGAMP with hSTING revealed that the cGAMP is positioned in a U-shaped cavity of the STING dimer,
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FIGURE 3
Structures of stimulator of interferon genes (STING) agonists and antagonists. (a) Cyclic dinucleotide (CDN) agonists: endogenous ligand cGAMP and clinical candidate ADU-S100. (b) Flavonoid and xanthone-derived agonists: flavone acetic acid (FAA) and dimethyloxoxanthenyl acetic acid (DMXAA) are specific mouse (m)STING agonists and a-mangostin shows higher affinity to human (h)STING than to mSTING. (c) Di-aminobenzimidazole (diABZI) agonists. (d) STING antagonists acting by targeting palmitoylation. The reaction sites targeting Cys88/91 are marked with red dots. (e) STING antagonists acting by occupying the ligand-binding pocket. For additional definitions, please see the main text.
with the cyclic sugar-phosphate backbone at the bottom and the purine rings orienting upward in a parallel alignment (Fig. 2c) [25]. The purine bases could be further anchored by the caps of the binding pocket formed during the conformation change of the
STING dimer [25]. Recently, Endo et al. reported that a CDN, E7766, a macrocycle-bridged derivative of ADU-S100, showed good potency in activating hSTING and improved metabolic stability [41]. The alkyl linker between six amino groups of two www.drugdiscoverytoday.com
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adenines immobilized the molecular conformation, which could be beneficial in terms of its affinity and stability. Another two clinical candidates for a STING agonist, MK-1454 from Merck and BMS-986301 from Bristol-Myers Squibb (both structures undisclosed), entered Phase I escalation trials in February 2017 and May 2019, respectively, for the treatment of advanced solid tumors as monotherapy and combination therapy with ICIs (Clinicaltrials.gov ID: NCT03010176 for MK-1454 and NCT03956680 for BMS-986301 [36]). Several non-CDN STING agonists have also been identified recently. The xanthone derivative dimethyloxoxanthenyl acetic acid (DMXAA, Fig. 3b) was the first agent developed to target the cGAS-STING pathway and showed potent antitumor efficacy in a mouse model [35]. DMXAA was originally recognized as a vascular disrupting agent, but failed a Phase III clinical trial targeting nonsmall cell lung cancer (NSCLC) [34]. However, Conlon et al. revealed that DMXAA was a selective mouse STING (mSTING) agonist with poor affinity to hSTING for type I IFN induction in human cells [42]. Similarly, the binding mode of DMXAA with either mSTING or mutated hSTING comprises two DMXAA molecules aligned in parallel in STING LBD, whereas the carboxylic acid moieties point to the lid (Fig. 2d) [25,43]. Critical residues change in hSTING could render STING responsive to DMXAA, whereas limited C7 modification on DMXAA failed to significantly improve the interaction to hSTING [43,44]. In addition, a flavonoid derivative, flavone acetic acid (FAA, which failed a Phase II trial as an anticancer agent) and 10-carboxymethyl-9-acridanone (CMA) were identified as mSTING agonists with weak activities to induce type I IFN responses in human cells (Fig. 3b) [45,46]. Zhang et al. reported a dietary xanthone, a-mangostin, with allyl and hydroxyl groups substituted in the xanthone scaffold, that showed advanced affinity to hSTING but still displayed a weaker potency (five to tenfold) for inducing type I IFN compared with cGAMP in reporter assays (Fig. 3b) [47]. Ramanjulu et al., from GlaxoSmithKline, reported a series of STING agonists (ABZIs) through a hSTING CTD-based highthroughput screening (HTS) method (Fig. 3c) [48]. The initial hit, aminobenzimidazole (ABZI). showed moderate affinity to hSTING with an IC50 of 14 mM from inhibition of the isotopelabeled cGAMP binding to STING [48]. A co-crystal structure of ABZI in complex with hSTING CTD was built to illustrate the binding mode, which revealed a conformation of two close ABZI molecules located at the STING LBD in mirror symmetry [48]. Specifically, the formamide was anchored by hydrogen bonds to Ser241, located at the lid region of STING CTD, and the substituted pyrazole ring sat at the bottom of binding pocket, connecting to Ser162 via an important hydrogen bond [48]. Meanwhile, the hydroxyphenethyl moiety at the N1 position of each of the two Q3 benzimidazole rings posed to the other molecule without further interactions with the binding pocket [48]. Subsequently, the authors designed the dimeric compound, diABZI, using a linking strategy between the two ABZI subunits at N1 benzimidazole with a short alkyl linker, obtaining a 1000-fold shift in binding affinity to hSTING with an IC50 of 20 nM (Fig. 3c) [48]. Two optimized diABZIs were further demonstrated to be 18–400-fold more potent compared with cGAMP for IFNb induction in human peripheral blood mononuclear cells, with EC50s of 3.1 and 0.13 mM, respectively [48]. Interestingly, diABZI could bind to 6
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and activate STING in the ‘open’ conformation, according to its crystal structure, which is similar to the complex of the lead ABZI (Fig. 2e) [49]. Although the linking strategy used in optimizing lead ABZI shows promise for drug design, the dimeric compounds also showed undesirable properties in terms of their druggability. More recently, Zhang et al. reported a 1,3-benzodioxole derivate, BNBC, as a specific human cGAS-STING pathway agonist through a reporter-based HTS method [49]. The authors found that BNBC induced INFb expression in a weak manner compared with cGAMP, and promoted the perinuclear translocation of a truncated STING lacking its C-terminal region, which suggests that BNBC does not target the STING CTD. Indeed, the ER-locating calcium sensor stromal interaction molecule 1 (STIM1) was recently identified as an ER-retention factor of STING that could regulate the anchoring and release of STING on or from the ER [50].
Antagonists Except for synthetic agonists of STING adaptor, additional compounds that could inhibit STING signaling have been identified in recent years. Several have been characterized as cGAS inhibitors, including X-6, PF-06928215, RU-521, and suramin, as well as a few STING antagonists [51–54]. Haag et al. recently reported a series of small-molecule inhibitors targeting STING signaling through a cell-based chemical screen method (Fig. 3d) [30]. The authors identified nitrofuran derivative C-178 as an irreversible inhibitor of mSTING; the species-specific inhibition was accomplished through a covalent bond between C-178 and the TMD-closing residue Cys91, which is associated with palmitoylation of STING together with Cys88 [30]. The 5-nitrofuran moiety was crucial for the selective recognition of mSTING because change of either 5nitro or the furan ring completely abolished the inhibitory activity of each compound [30]. In addition, the compound with a methyl group substituted at the central amide moiety of C-178 lost its ability to inhibit mSTING [30]. This selectively formed covalent bond between C-178 with Cys91 of STING can be achieved by nucleophilic addition of the thiol group to the 4position of the furan ring, and a modified mSTING was subsequently generated [30]. This modification of STING interfered with the palmitoylation of STING at the post-ER state before the engagement of TBK1 and led to the arrest of further signaling [30]. Furthermore, the authors identified an indole derivative, H151, as an hSTING antagonist through further screening; this interacted with hSTING via an irreversible addition to Cys91 and exhibited efficacy in the following in vivo evaluation in a Trex1/ mouse model (Fig. 3d) [30]. Hansen et al. also identified several nitro-fatty acids (NO2-FAs and CXA-10) as potent STING antagonists by inhibiting palmitoylation, and these have entered Phase II trials as an oral peroxisome proliferator-activated receptor-g (PPARg) agonist (Fig. 3d) [31]. The authors found that CXA-10 could post-translationally modify STING at both palmitoylation target sites of Cys88/91 as well as the N-terminal residue His16 through a Michael addition, and inhibited palmitoylation and suppressed the phosphorylation of TBK1 [31]. CXA-10 was then further tested in an in vitro evaluation and exhibited potency by inhibiting type I IFN expression in SAVI patient-derived fibroblast cells [31]. Together, these two independent studies revealed that palmitoylation of STING is a potential target for STING inhibition and that STING antagonists
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Concluding remarks Targeting STING adaptor has a focus in recent years because of its pivotal role in regulating the innate immune response. Several STING modulators have been identified with different chemical scaffolds and mechanisms of action for the treatment of cancer, autoimmune, and autoinflammatory disease. Among them, three STING agonist candidates have entered clinical trials over the past 2 years and one STING antagonist is being prepared for a clinical trial in 2020 [24]. However, the scaffolds reported so far have been relatively limited and current clinical candidates have exhibited some deficiency in terms of their druggability. Although the activation of the STING pathway is complex and not well studied, there are various opportunities for the development of new modulators during STING activation, including ligand binding, translocating, palmitoylation, and others. In addition, it is difficult to test STING modulators in murine models because of the difference between hSTING and mSTING. Nonetheless, discovering novel STING modulators could be an important advance in the development of treatments for various diseases, including cancer. Q4
Acknowledgments We gratefully acknowledge financial support from the National Natural Science Foundation of China (no. 81703347, 21672260, and 21372260), the National Natural Science Foundation of Jiangsu Province of China (no. BK20170743 and BK20171393), and the ‘Double First-class’ University Project (CPU2018GY07), the State Key Laboratory of Drug Research (SIMM1903KF-03).
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18 Fuertes, M.B. et al. (2013) Type I interferon response and innate immune sensing of cancer. Trends Immunol. 34, 67–73 19 Woo, S.R. et al. (2014) STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors. Immunity 41, 830–842 20 Yang, H. et al. (2017) cGAS is essential for cellular senescence. Proc. Natl. Acad. Sci. U. S. A. 114, E4612–E4620 21 Xia, T. et al. (2016) Recurrent loss of STING signaling in melanoma correlates with susceptibility to viral oncolysis. Cancer Res. 76, 6747–6759 22 Xia, T. et al. (2016) Deregulation of STING signaling in colorectal carcinoma constrains DNA damage responses and correlates with tumorigenesis. Cell Rep. 14, 282–297 23 Duwell, P. et al. (2019) Innate immune stimulation in cancer therapy. Hematol. Oncol. Clin. N. Am. 33, 215–231 24 Sheridan, C. (2019) Drug developers switch gears to inhibit STING. Nat. Biotechnol. 37, 199–201 25 Gao, P. et al. (2013) Structure-function analysis of STING activation by cG(2’,5’)pA (3’,5’)p and targeting by antiviral DMXAA. Cell 154, 748–762 26 Diner, E.J. et al. (2013) The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING. Cell Rep. 3, 1355– 1361 27 Shang, G. et al. (2019) Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP. Nature 567, 389–393 28 Saitoh, T. et al. (2009) Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response. Proc. Natl. Acad. Sci. U. S. A. 106, 20842– 20846 29 Mukai, K. et al. (2016) Activation of STING requires palmitoylation at the Golgi. Nat. Commun. 7, 11932 30 Haag, S.M. et al. (2018) Targeting STING with covalent small-molecule inhibitors. Nature 559, 269–273 31 Hansen, A.L. et al. (2018) Nitro-fatty acids are formed in response to virus infection and are potent inhibitors of STING palmitoylation and signaling. Proc. Natl. Acad. Sci. U. S. A. 115, E7768–E7775 32 Zhao, B. et al. (2019) A conserved PLPLRT/SD motif of STING mediates the recruitment and activation of TBK1. Nature 569, 718–722
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showed efficacy in the treatment of autoinflammatory and autoimmune disorders [31]. Wang et al. reported a natural cyclopeptide, astin C, from Aster tataricus as an antagonist of hSTING (Fig. 3e) [54]. The authors showed that astin C exhibited good potency for reducing infb mRNA expression under ISD stimulation in murine and human fibroblasts, with IC50s of 3.4 and 10.8 mM, respectively [54]. Apart from the covalent inhibitors, astin C could specifically bind to STING CTD with a Kd of 2.3 mM compared with cGAMP in a microscale thermophoresis assay [54]. Moreover, the authors suggested that astin C could occupy the binding pocket via in silico docking with an interaction between Ser162, Tyr163, and Arg238. This was supported by the finding that astin C decreased the binding affinity of cGAMP to STING by approximately tenfold in an isothermal titration calorimetry assay [54]. Interestingly, an astin C analog, M11, which was chosen as a negative control because of its inefficiency, shares the same basic scaffold as astin C expect for one amino acid change from dichloridated proline to pyrrole-2-carboxylated threonine (Fig. 3e) [54]. This single exchange resulting in shifted activity requires further investigation in terms of its SAR. Meanwhile, Siu et al. identified a series of carboxylate-containing derivatives as weak antagonists of hSTING (Fig. 3e) [55]. The authors found that compound 18 bound to STING in a 2:1 binding stoichiometry and stabilized the ‘open’ conformation. According to the co-crystal structure, this binding was stabilized by a hydrogen bond interaction between the carboxyl acid moieties and Thr263 [Protein Data Bank (PDB) ID: 6MXE] [55].
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33 Liu, S. et al. (2015) Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation. Science 347 aaa2630 34 Berger, G. et al. (2019) Pharmacological modulation of the STING pathway for cancer immunotherapy. Trends. Mol. Med. 25, 412–427 35 Corrales, L. et al. (2015) Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity. Cell Rep. 11, 1018–1030 36 https://clinicaltrials.gov [Accessed 14 November 2019]. 37 Li, L. et al. (2014) Hydrolysis of 2’3’-cGAMP by ENPP1 and design of nonhydrolyzable analogs. Nat. Chem. Biol. 10, 1043–1048 38 Lioux, T. et al. (2016) Design, synthesis, and biological evaluation of novel cyclic adenosine-inosine monophosphate (cAIMP) analogs that activate stimulator of interferon genes (STING). J. Med. Chem. 59, 10253–10267 39 Weston, A. et al. (2019) Preclinical studies of SR-8314, a highly selective ENPP1 inhibitor and an activator of STING pathway. Cancer Res. 79 (13 Suppl.), 3077 40 Baird, J. et al. (2018) MV-626, a potent and selective inhibitor of ENPP1 enhances STING activation and augments T-cell mediated anti-tumor activity in vivo. Proc. 33rd Annu. Meet. Soc. Immunother. Cancer 2018, P410 41 Endo, A. et al. (2019) Discovery of E7766: a representative of a novel class of macrocycle-bridged STING agonists (MBSAs) with superior potency and pangenotypic activity. Cancer Res. 79 (13 Suppl.), 4456 42 Conlon, J. et al. (2013) Mouse, but not human STING, binds and signals in response to the vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid. J. Immunol. 190, 5216–5225 43 Gao, P. et al. (2014) Binding-pocket and lid-region substitutions render human STING sensitive to the species-specific drug DMXAA. Cell Rep. 8, 1668–1676
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44 Hwang, J. et al. (2019) Design, synthesis, and biological evaluation of C7functionalized DMXAA derivatives as potential human-STING agonists. Org. Biomol. Chem. 17, 1869–1874 45 Kerr, D.J. and Kaye, S.B. (1989) Flavone acetic acid–preclinical and clinical activity. Eur. J. Cancer Clin. Oncol. 25, 1271–1272 46 Cavlar, T. et al. (2013) Species-specific detection of the antiviral small-molecule compound CMA by STING. EMBO J. 32, 1440–1450 47 Zhang, Y. et al. (2018) Identification of alpha-mangostin as an agonist of human STING. ChemMedChem 13, 2057–2064 48 Ramanjulu, J.M. et al. (2018) Design of amidobenzimidazole STING receptor agonists with systemic activity. Nature 564, 439–443 49 Zhang, X. et al. (2019) Discovery and mechanistic study of a novel humanstimulator-of-interferon-genes agonist. ACS Infect. Dis. 5, 1139–1149 50 Srikanth, S. et al. (2019) The Ca21 sensor STIM1 regulates the type I interferon response by retaining the signaling adaptor STING at the endoplasmic reticulum. Nat. Immunol. 20, 152–162 51 An, J. et al. (2018) Inhibition of cyclic GMP-AMP synthase using a novel antimalarial drug derivative in trex1-deficient mice. Arthritis Rheumatol. 70, 1807–1819 52 Hall, J. et al. (2017) Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay. PLoS One 12, e0184843 53 Vincent, J. et al. (2017) Small molecule inhibition of cGAS reduces interferon expression in primary macrophages from autoimmune mice. Nat. Commun. 8, 750 54 Wang, C. et al. (2018) The cyclopeptide Astin C specifically inhibits the innate immune CDN sensor STING. Cell Rep. 25, 3405–3421 55 Siu, T. et al. (2019) Discovery of a novel cGAMP competitive ligand of the inactive form of STING. ACS Med. Chem. Lett. 10, 92–97
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Bioactive modulators targeting STING adaptor in cGAS-STING pathway Q2
Xi Feng, Dongyu Liu, Zhiyu Li and Jinlei Bian Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-pathway triggers innate immune responses by recognizing cytosolic DNA. Recent studies revealed that STING adaptor associates with various diseases, and several modulators targeting STING have been identified including three agonists that have entered clinical trials for treating cancer over the past 2 years. In particular, the efficacy of STING agonists and/or antagonists suggests adaptor STING as a potential therapeutic target for diverse diseases. Herein, we summarize the latest advances in understanding STING functioning and provide an overview of recent STING modulator discoveries, including structural details and the potential therapeutic applications of these modulators.
Introduction Innate immunity is the first-line natural barrier of the host for the elimination of pathogenic infections that can be triggered by pattern recognition receptors (PRRs), such as cytosolic DNA sensors [1]. Among them, the cGAS-STING pathway has a key role in detecting cytosolic DNA to activate downstream intracellular signal cascades and inducing type I interferons (IFNs) to trigger innate immune responses for antimicrobial effects, as well as initiating autophagy [2,3]. Increasing evidence has revealed that the cGAS-STING pathway is involved in modulating anticancer immunity and contributing to autoimmune, autoinflammatory disorders [2,4]. In particular, the transmembrane protein STING in the cGAS-STING pathway has received increased attention from both industry and academia in recent years. Hence, in this review, we highlight the latest understanding of STING functioning as well as the discovery and structural features of recently disclosed modulators targeting STING adaptor, including agonists and antagonists.
(cGAMP in eukaryotic cells), a second messenger, after engaging with DNA [5]. The synthesized cGAMP in turn binds to the endoplasmic reticulum (ER)-membrane adaptor STING (also known as MITA, ERIS, MPYS, or TMEM173) as an endogenous ligand and induces the activation of STING [6]. Some bacterial cyclic dinucleotides (CDNs) are able to bind and activate STING directly [7]. After stimulation by CDN, the ER-locating STING then traffics to perinuclear compartments, including Golgi apparatus, endosomes, and autophagy-related compartments [8]. During this process, STING recruits TANK-binding kinase 1 (TBK1) at its C-terminal tail and promotes the autophosphorylation of TBK1, which in turn phosphorylates the transcription factor IRF3 [5,9]. In addition, the transcription factor nuclear factor (NF)-kB is also activated in response to STING cascades [6]. As a result, the activated transcription factors enter the nucleus and function together to induce the production of type I IFNs, such as IFNb, and inflammatory cytokines, including tumor necrosis factor-a (TNF-a) and interleukin 6 (IL-6), which ultimately boost the fast-acting and robust innate immune system as well as the resulting adaptive immunity (Fig. 1) [5].
cGAS-STING pathway The initial sensing of aberrant cytosolic double-stranded (ds)DNA is accomplished by cGAS. It catalyzes the synthesis of cyclic GMP-AMP
Corresponding authors: Li, Z. ([email protected]), Bian, J. ([email protected]) 1359-6446/ã 2019 Elsevier Ltd. All rights reserved. https://doi.org/10.1016/j.drudis.2019.11.007
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Diseases involving STING Among the aforementioned cytosolic DNA sensing pathways, the ER-locating adaptor STING is considered to be the converging point for enhancing or suppressing immune responses [10].
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FIGURE 1
The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is activated by sensing cytosolic DNA. Cytosolic DNA (either from foreign DNA or self-DNA) is recognized by cGAS and leads to the generation of cGAMP. cGAMP directly binds to the endoplasmic reticulum (ER) adaptor STING, which subsequently traffics to the Golgi. After being palmitoylated at Golgi, STING recruits and activates TANK-binding kinase 1 (TBK1), which further recruits interferon regulatory factor 3 (IRF3) for phosphorylation and dimerization. The phosphorylated IRF3 dimer then enters the nucleus and functions with nuclear factor (NF)kB to induce the expression of type I interferons (IFNs) and other cytokines. For additional definitions, please see the main text.
Aberrant activation of STING is correlated with various diseases, and efforts have been made to regulate this pivotal pathway as a pharmaceutical approach. On the one hand, excessive signaling through STING is implicated in chronic inflammation [11,12]. Loss-of-function mutations in Trex1 can lead to the dysfunction of its encoding enzyme, which acts as a 30 -50 DNA exonuclease (TREX1) [13]. This loss of enzyme activity could contribute to the abnormal accumulation of cytosolic DNA, leading to the continuous signaling of the cGAS-STING pathway, and resulting in autoinflammatory disorders, including Aicardi–Goutie`res syndrome (AGS) and systemic lupus erythematosus (SLE) [14,15]. Similarly, gain-of-function mutations in the STING-encoding gene TMEM173 are linked to the hyperactivation of STING, which can result in systemic inflammation, known as STING-associated vasculopathy with onset in infancy (SAVI) [15,16]. In patients with SAVI, several mutants of STING have been identified, including V147L, N154S, and V155M [15,16]. These STING variants are considered to be sensitive and could be activated in a ligandindependent manner [8]. In addition, the cGAS-STING pathway has been shown to enhance tumor development by activating indoleamine 2,3-dioxygenase (IDO, an immune checkpoint) pathway in a murine Lewis lung carcinoma (LLC) model [17]. 2
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On the other hand, initiating the host cGAS-STING pathway for antitumor immunity is a promising approach in cancer immunotherapy. The cGAS-STING pathway is an important tumor-surveillance mechanism in host antitumor immunity [4]. In tumor cells, DNA leakage in the cytoplasm during mitosis is detected by cGAS, leading to the secretion of type I IFNs [18]. Type I IFN signals are responsible for stimulating the cross-presentation of tumor antigens and priming tumor-specific CD8+ effector T cells [18]. In addition, dendritic cells (DCs) can express type I IFNs by taking up tumor-derived DNA [4,19]. Activation of the cGAS-STING pathway is also associated with promoting cellular senescence by mediating the production of type I IFNs and senescence-associated secretory phenotype (SASP) [2,20]. Of note, it has been observed that the cGAS-STING pathway is irregularly inhibited in various cancer types, including melanoma and colorectal carcinoma [21,22]. Increasing evidence indicates that specific activation of the STING pathway might suppress tumor progression by initiating intrinsic immunity, further causing the tumor to become ‘hot’ by restoring T cell infiltration into the tumor microenvironment (TME), which is considered to be beneficial for immune checkpoint blockage therapy [23]. Given that disorder of the cGAS-STING pathway is associated with various diseases, modulating this fundamental pathway
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STING activation STING activation can be divided into an ER-locating stage and a post-ER stage based on our current understanding (Fig. 1). Human STING (hSTING) protein functions as a homodimer, comprising a luminal N-terminal domain (aa 1–154) that anchors ER with four transmembrane helices, and a cytosolic C-terminal domain (CTD, aa 140–379) containing the ligand-binding pocket (Fig. 2a,b) [25]. The crystal structure of an apo-STING CTD dimer showed that the ligand-binding domain (LBD) exhibits an inactive ‘open’ conformation and, correspondingly, the cGAMP-bound STING dimer showed an active ‘closed’ state, which refers to a conformational change driven by the binding of the ligand (Fig. 2c) [26,27]. Recently, Shang et al. revealed that cGAMP could induce a 180 clockwise rotation of the STING dimer CTD relative to the transmembrane domain (TMD), which gradually led to the formation of a b-sheet that caps the binding pocket and the resulting closure [27]. This conformational change is indispensable for STING activation, but the exact mechanism remains unclear. However, this ‘closed’ conformation could contribute to the following ER-toGolgi translocation and the oligomerization of STING [27]. Furthermore, investigations of the gain-of-function mutations of Sting in SAVI showed that variants including V147 L, N154S, and V155 M usually locate at the connector region. Thus, mutated residues in the helix loop might contribute to, or drive, the conformational change because of their larger side chains (Fig. 2b) [27]. After ligand binding, STING translocates from ER to the Golgi. This movement is necessary because blocking ER-to-Golgi traffic with brefeldin A (BFA) or expression of Shigella effector IpaJ abolished forward signaling events, including phosphorylation of IRF3 and induction of IFNb [8,28]. The observation of some SAVI STING variants localizing to the perinuclear compartments without stimulation by ligand is consistent with the implication that the conformational changes in STING contribute to the postER state [8]. After translocation, a post-translational modification of STING at Golgi occurs at the TMD, including palmitoylation at Cys88/91 (Fig. 2b) [29]. It was suggested that the Golgi-located protein palmitoyltransferase DHHCs catalyze this modification. Palmitoylation at cystine was shown to be important for STING activation because either the generation of a STING mutant (C88/ 91S) or using palmitoylation inhibitors abolished downstream gene expression [12,29]. It was proposed that palmitoylation promotes STING activation by facilitating the oligomerization of STING at Golgi membrane, further contributing to the recruitment of TBK1 and IRF3 [9,29]. This modification might be independent of CDN binding and has been considered as a target for inhibiting STING activation [29–31]. The STING dimer could then
bind to the TBK1 dimer via its C-terminal tail and activate TBK1 for further phosphorylation of STING and IRF3 in a ligand-dependent manner [9,32]. The major phosphorylation site of STING is Ser366, located at the C-terminal tail. It was suggested that this phosphorylation is accomplished by an another TBK1 dimer from a neighboring STING-TBK1 complex [9,33]. The phosphorylated tails of STING might further engage and deliver IRF3 to TBK1 for further phosphorylation and signaling [9]. Furthermore, the phosphorylation of Ser172 at TBK1, which is necessary for its activation, was also considered to be achieved by another TBK1 molecule because this residue could not reach any kinase active sites of either monomer [9]. This interaction of STING with TBK1 might in turn reflect the importance of clustering of STING at Golgi.
Bioactive modulators targeting STING adaptor Agonists Given the key role of STING in initiating innate immunity, delivering STING agonists to boost immune signaling in TME offers a potential cancer immunotherapy approach. In recent years, several STING agonists have been identified and three candidates are in Phase I/II clinical trials. In general, most reported STING agonists function by directly connecting with LBD by mimicking the native ligand to trigger STING for the following reactions. This is a straightforward and reliable approach because CDNs have been known as small-molecule cell-signaling mediators for decades [34]. In 2016, Novartis and Aduro BioTech released their synthetic CDN ADU-S100 (ML RR-S2 CDA, Fig. 3a) as a first-in-class STING agonist. This has entered Phase I clinical trials for the treatment of solid tumors or lymphomas as monotherapy or in combination with immune checkpoint inhibitors (ICIs) [35,36] (Clinicaltrials. gov ID: NCT03937141, NCT03172936, and NCT02675439). CDN ADU-S100 is a dithio derivative of natural CDN 20 -30 cAMP and is reported to display improved chemical and/or metabolic stability and higher potency in activating STING compared with cGAMP [35]. In May 2019, a Phase II clinical trial of ADU-S100 was scheduled for the treatment of PD-L1 positive recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). However, the annular structure of CDNs with two nucleotides might be not favored by some medicinal chemists for developing potential drugs. Indeed, the physicochemical properties of CDNs, including high polarity and poor membrane permeability, could limit their efficacy in vivo. In addition, metabolic stability, including enzymatic degradation by phosphodiesterases (e.g., ENPP1) or nucleases as well as the synthetic issue of CDN derivatives for structure-activity relationship (SAR) screening should also be of concern [37]. Interestingly, it was reported that the stereochemical configuration of the two phosphates in ADU-S100 is crucial because the [Rp, Rp] diastereomers (e.g., ADU-S100) showed improved potency in resisting digestion and inducing type I IFNs in human THP1 cells compared with the corresponding [Rp, Sp] diastereomers (Fig. 3a) [35]. In addition, the 20 -30 linkage of CDN enhanced the binding affinity to hSTING, but this observation was not consistent with the outcome of synthetic cAIMP analogs in a luciferase assay [35,38]. The dithio modification of phosphate could increase enzymatic stability while maintaining its high affinity for hSTING [35]. Moreover, the mono or dual fluoro-substitution in the 20 -ribose sugar of 30 -30 cAIMP analogs improved the enzymatic stability, as well as the ability to induce type I IFNs, with EC50 of www.drugdiscoverytoday.com
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could be a potential and powerful tool against diseases ranging from inflammation to cancer. However, despite a few molecules characterized as cGAS enzyme inhibitors, it is difficult to develop drugs targeting cGAS [24]. Indeed, growing attention has been paid to another compelling target, STING adaptor, because several agonist and antagonists and/or inhibitors with different mechanisms of action have been developed and reported to be efficient in vivo. Recent studies also revealed the unique and complex signal process involving STING adaptor. Thus, it would be useful to further understand this side for future drug development.
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FIGURE 2
Structures of stimulator of interferon genes (STING) dimer and the binding of agonists. (a) STING is a transmembrane protein. (b) Cryo-electron microscopy (EM) structure of full-length human STING [Protein Data Bank (PDB) ID: 6NT5], V147/N154/V155 variants are marked in red and Cys88/91 are marked in green. (c) Cocrystal structure and a close-up of cGAMP in complex with human (h)STINGH232 (PDB ID: 4LOH). cGAMP is shown in green, and key residues and hydrogen bonds are shown as sticks and black dashes, respectively. (d) Co-crystal structure of dimethyloxoxanthenyl acetic acid (DMXAA) with hSTINGS162A/Q230I/Q266I (PDB ID: 4QXR). DMXAA is shown in green and mutated residues are marked in dark yellow and pink. (e) Co-crystal structure of di-aminobenzimidazole (diABZI) (compound 3) with hSTING (PDB ID: 6DXL). diABZI is shown in green. For additional definitions, please see the main text.
0.3–1.1 mM in an IRF-luciferase assay, whereas cGAMP exhibited an EC50 of 7.2 mM [38]. Generally, the instability of CDNs limited its application and has been regarded as a challenge and goal when optimizing CDNs. Interestingly, several ENPP1 phosphodiesterase 4
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inhibitors have been designed and developed to activate STING indirectly for improved antitumor efficacy [39,40]. The co-crystal structure of cGAMP with hSTING revealed that the cGAMP is positioned in a U-shaped cavity of the STING dimer,
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FIGURE 3
Structures of stimulator of interferon genes (STING) agonists and antagonists. (a) Cyclic dinucleotide (CDN) agonists: endogenous ligand cGAMP and clinical candidate ADU-S100. (b) Flavonoid and xanthone-derived agonists: flavone acetic acid (FAA) and dimethyloxoxanthenyl acetic acid (DMXAA) are specific mouse (m)STING agonists and a-mangostin shows higher affinity to human (h)STING than to mSTING. (c) Di-aminobenzimidazole (diABZI) agonists. (d) STING antagonists acting by targeting palmitoylation. The reaction sites targeting Cys88/91 are marked with red dots. (e) STING antagonists acting by occupying the ligand-binding pocket. For additional definitions, please see the main text.
with the cyclic sugar-phosphate backbone at the bottom and the purine rings orienting upward in a parallel alignment (Fig. 2c) [25]. The purine bases could be further anchored by the caps of the binding pocket formed during the conformation change of the
STING dimer [25]. Recently, Endo et al. reported that a CDN, E7766, a macrocycle-bridged derivative of ADU-S100, showed good potency in activating hSTING and improved metabolic stability [41]. The alkyl linker between six amino groups of two www.drugdiscoverytoday.com
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adenines immobilized the molecular conformation, which could be beneficial in terms of its affinity and stability. Another two clinical candidates for a STING agonist, MK-1454 from Merck and BMS-986301 from Bristol-Myers Squibb (both structures undisclosed), entered Phase I escalation trials in February 2017 and May 2019, respectively, for the treatment of advanced solid tumors as monotherapy and combination therapy with ICIs (Clinicaltrials.gov ID: NCT03010176 for MK-1454 and NCT03956680 for BMS-986301 [36]). Several non-CDN STING agonists have also been identified recently. The xanthone derivative dimethyloxoxanthenyl acetic acid (DMXAA, Fig. 3b) was the first agent developed to target the cGAS-STING pathway and showed potent antitumor efficacy in a mouse model [35]. DMXAA was originally recognized as a vascular disrupting agent, but failed a Phase III clinical trial targeting nonsmall cell lung cancer (NSCLC) [34]. However, Conlon et al. revealed that DMXAA was a selective mouse STING (mSTING) agonist with poor affinity to hSTING for type I IFN induction in human cells [42]. Similarly, the binding mode of DMXAA with either mSTING or mutated hSTING comprises two DMXAA molecules aligned in parallel in STING LBD, whereas the carboxylic acid moieties point to the lid (Fig. 2d) [25,43]. Critical residues change in hSTING could render STING responsive to DMXAA, whereas limited C7 modification on DMXAA failed to significantly improve the interaction to hSTING [43,44]. In addition, a flavonoid derivative, flavone acetic acid (FAA, which failed a Phase II trial as an anticancer agent) and 10-carboxymethyl-9-acridanone (CMA) were identified as mSTING agonists with weak activities to induce type I IFN responses in human cells (Fig. 3b) [45,46]. Zhang et al. reported a dietary xanthone, a-mangostin, with allyl and hydroxyl groups substituted in the xanthone scaffold, that showed advanced affinity to hSTING but still displayed a weaker potency (five to tenfold) for inducing type I IFN compared with cGAMP in reporter assays (Fig. 3b) [47]. Ramanjulu et al., from GlaxoSmithKline, reported a series of STING agonists (ABZIs) through a hSTING CTD-based highthroughput screening (HTS) method (Fig. 3c) [48]. The initial hit, aminobenzimidazole (ABZI). showed moderate affinity to hSTING with an IC50 of 14 mM from inhibition of the isotopelabeled cGAMP binding to STING [48]. A co-crystal structure of ABZI in complex with hSTING CTD was built to illustrate the binding mode, which revealed a conformation of two close ABZI molecules located at the STING LBD in mirror symmetry [48]. Specifically, the formamide was anchored by hydrogen bonds to Ser241, located at the lid region of STING CTD, and the substituted pyrazole ring sat at the bottom of binding pocket, connecting to Ser162 via an important hydrogen bond [48]. Meanwhile, the hydroxyphenethyl moiety at the N1 position of each of the two Q3 benzimidazole rings posed to the other molecule without further interactions with the binding pocket [48]. Subsequently, the authors designed the dimeric compound, diABZI, using a linking strategy between the two ABZI subunits at N1 benzimidazole with a short alkyl linker, obtaining a 1000-fold shift in binding affinity to hSTING with an IC50 of 20 nM (Fig. 3c) [48]. Two optimized diABZIs were further demonstrated to be 18–400-fold more potent compared with cGAMP for IFNb induction in human peripheral blood mononuclear cells, with EC50s of 3.1 and 0.13 mM, respectively [48]. Interestingly, diABZI could bind to 6
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and activate STING in the ‘open’ conformation, according to its crystal structure, which is similar to the complex of the lead ABZI (Fig. 2e) [49]. Although the linking strategy used in optimizing lead ABZI shows promise for drug design, the dimeric compounds also showed undesirable properties in terms of their druggability. More recently, Zhang et al. reported a 1,3-benzodioxole derivate, BNBC, as a specific human cGAS-STING pathway agonist through a reporter-based HTS method [49]. The authors found that BNBC induced INFb expression in a weak manner compared with cGAMP, and promoted the perinuclear translocation of a truncated STING lacking its C-terminal region, which suggests that BNBC does not target the STING CTD. Indeed, the ER-locating calcium sensor stromal interaction molecule 1 (STIM1) was recently identified as an ER-retention factor of STING that could regulate the anchoring and release of STING on or from the ER [50].
Antagonists Except for synthetic agonists of STING adaptor, additional compounds that could inhibit STING signaling have been identified in recent years. Several have been characterized as cGAS inhibitors, including X-6, PF-06928215, RU-521, and suramin, as well as a few STING antagonists [51–54]. Haag et al. recently reported a series of small-molecule inhibitors targeting STING signaling through a cell-based chemical screen method (Fig. 3d) [30]. The authors identified nitrofuran derivative C-178 as an irreversible inhibitor of mSTING; the species-specific inhibition was accomplished through a covalent bond between C-178 and the TMD-closing residue Cys91, which is associated with palmitoylation of STING together with Cys88 [30]. The 5-nitrofuran moiety was crucial for the selective recognition of mSTING because change of either 5nitro or the furan ring completely abolished the inhibitory activity of each compound [30]. In addition, the compound with a methyl group substituted at the central amide moiety of C-178 lost its ability to inhibit mSTING [30]. This selectively formed covalent bond between C-178 with Cys91 of STING can be achieved by nucleophilic addition of the thiol group to the 4position of the furan ring, and a modified mSTING was subsequently generated [30]. This modification of STING interfered with the palmitoylation of STING at the post-ER state before the engagement of TBK1 and led to the arrest of further signaling [30]. Furthermore, the authors identified an indole derivative, H151, as an hSTING antagonist through further screening; this interacted with hSTING via an irreversible addition to Cys91 and exhibited efficacy in the following in vivo evaluation in a Trex1/ mouse model (Fig. 3d) [30]. Hansen et al. also identified several nitro-fatty acids (NO2-FAs and CXA-10) as potent STING antagonists by inhibiting palmitoylation, and these have entered Phase II trials as an oral peroxisome proliferator-activated receptor-g (PPARg) agonist (Fig. 3d) [31]. The authors found that CXA-10 could post-translationally modify STING at both palmitoylation target sites of Cys88/91 as well as the N-terminal residue His16 through a Michael addition, and inhibited palmitoylation and suppressed the phosphorylation of TBK1 [31]. CXA-10 was then further tested in an in vitro evaluation and exhibited potency by inhibiting type I IFN expression in SAVI patient-derived fibroblast cells [31]. Together, these two independent studies revealed that palmitoylation of STING is a potential target for STING inhibition and that STING antagonists
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Concluding remarks Targeting STING adaptor has a focus in recent years because of its pivotal role in regulating the innate immune response. Several STING modulators have been identified with different chemical scaffolds and mechanisms of action for the treatment of cancer, autoimmune, and autoinflammatory disease. Among them, three STING agonist candidates have entered clinical trials over the past 2 years and one STING antagonist is being prepared for a clinical trial in 2020 [24]. However, the scaffolds reported so far have been relatively limited and current clinical candidates have exhibited some deficiency in terms of their druggability. Although the activation of the STING pathway is complex and not well studied, there are various opportunities for the development of new modulators during STING activation, including ligand binding, translocating, palmitoylation, and others. In addition, it is difficult to test STING modulators in murine models because of the difference between hSTING and mSTING. Nonetheless, discovering novel STING modulators could be an important advance in the development of treatments for various diseases, including cancer. Q4
Acknowledgments We gratefully acknowledge financial support from the National Natural Science Foundation of China (no. 81703347, 21672260, and 21372260), the National Natural Science Foundation of Jiangsu Province of China (no. BK20170743 and BK20171393), and the ‘Double First-class’ University Project (CPU2018GY07), the State Key Laboratory of Drug Research (SIMM1903KF-03).
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showed efficacy in the treatment of autoinflammatory and autoimmune disorders [31]. Wang et al. reported a natural cyclopeptide, astin C, from Aster tataricus as an antagonist of hSTING (Fig. 3e) [54]. The authors showed that astin C exhibited good potency for reducing infb mRNA expression under ISD stimulation in murine and human fibroblasts, with IC50s of 3.4 and 10.8 mM, respectively [54]. Apart from the covalent inhibitors, astin C could specifically bind to STING CTD with a Kd of 2.3 mM compared with cGAMP in a microscale thermophoresis assay [54]. Moreover, the authors suggested that astin C could occupy the binding pocket via in silico docking with an interaction between Ser162, Tyr163, and Arg238. This was supported by the finding that astin C decreased the binding affinity of cGAMP to STING by approximately tenfold in an isothermal titration calorimetry assay [54]. Interestingly, an astin C analog, M11, which was chosen as a negative control because of its inefficiency, shares the same basic scaffold as astin C expect for one amino acid change from dichloridated proline to pyrrole-2-carboxylated threonine (Fig. 3e) [54]. This single exchange resulting in shifted activity requires further investigation in terms of its SAR. Meanwhile, Siu et al. identified a series of carboxylate-containing derivatives as weak antagonists of hSTING (Fig. 3e) [55]. The authors found that compound 18 bound to STING in a 2:1 binding stoichiometry and stabilized the ‘open’ conformation. According to the co-crystal structure, this binding was stabilized by a hydrogen bond interaction between the carboxyl acid moieties and Thr263 [Protein Data Bank (PDB) ID: 6MXE] [55].
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