Human Stimulator Of Interferon Genes Research Articles

Unraveling the Binding Mode of Cyclic Adenosine-Inosine Monophosphate (cAIMP) to STING through Molecular Dynamics Simulations.

The stimulator of interferon genes (STING) plays a significant role in immune defense and protection against tumor proliferation. Many cyclic dinucleotide (CDN) analogues have been reported to regulate its activity, but the dynamic process involved when the ligands activate STING remains unclear. In this work, all-atom molecular dynamics simulations were performed to explore the binding mode between human STING (hSTING) and four cyclic adenosine-inosine monophosphate analogs (cAIMPs), as well as 2',3'-cGMP-AMP (2',3'-cGAMP). The results indicate that these cAIMPs adopt a U-shaped configuration within the binding pocket, forming extensive non-covalent interaction networks with hSTING. These interactions play a significant role in augmenting the binding, particularly in interactions with Tyr167, Arg238, Thr263, and Thr267. Additionally, the presence of hydrophobic interactions between the ligand and the receptor further contributes to the overall stability of the binding. In this work, the conformational changes in hSTING upon binding these cAIMPs were also studied and a significant tendency for hSTING to shift from open to closed state was observed after binding some of the cAIMP ligands.

Abstract 2057: Monitoring cGAMP Biochemically and in cell using homogenous HTS formatted bioluminescent assay

Abstract The STimulator of INterferon Genes (STING) is a transmembrane protein located at the endoplasmic reticulum, which serves as a molecular hub in response to cytoplasmic cyclic dinucleotide (CDN) second messengers such as cyclic GMP-AMP (cGAMP). Upon its generation via cyclic GMP-AMP synthase (cGAS) by a wide array of pathogens that provide extracellular microbial DNA and by intracellular (nuclear and mitochondrial) DNA, it binds directly to the STING protein resulting in the activation of the STING pathway. This activation by cytosolic DNA initiates a cascade of events, cGAS-STING-TBK1-IRF3 signaling, that induces type 1 IFN response, and eventually the signaling is terminated by degradation of pathway components and clearance of stimulatory DNA. Given its significant role as an adaptor for DNA sensors to pathogens and its involvement in immune sensing, tumor growth control, and control of autoinflammatory and autoimmune disorders, the STING pathway has become a very important drug target. Credible evidence suggests that the cGAS–cGAMP–STING pathway makes fundamental contributions to at least three major cancer therapies: radiation therapy, chemotherapy, and immunotherapy, and it thus represents a promising drug target. Therefore, there is a need to identify lead compounds that effectively modulate human STING for further drug development. Towards this goal, we have developed a bioluminescent assay to monitor the concentration of cGAMP in intracellular as well as extracellular compartments. The assay relies on the new concept of Protein Complementation Assay (PCA) using nanoluciferase (nanoluc) complementation technology where a small peptide (SmBiT) derived from nanoluc linked to cGAMP through a linker (cGAMP tracer) can be recognized by an anti cGAMP antibody that is linked to a nanoluc complementary fragment (LgBiT) resulting in bioluminescence upon the addition of nanoluc substrate. The presence of free cGAMP that is generated in the reaction competes with cGAMP tracer and decreases bioluminescence. We have optimized the assay for higher sensitivity down to less than 10 nM cGAMP and less than one ng of cGAS and thus, and it is suitable for testing compounds that modulate cGAS activity. We also improved cell lysis condition to increase recovery of cGAMP that is generated intracellularly using several cGAS activators and correlating its concentration with cytokine release. We envision using this assay to monitor cGAMP presence in sera and spinal fluids of patients who have ALS and Alzheimer diseases. The assay is homogenous, with minimal or no false hits using LOPAC screening and HTS formatted and can be completed in less than one hour. Thus, this assay provides valuable contribution for investigating modulators of the STING pathway and facilitates the search for novel therapeutics. Citation Format: Said A. Goueli, kevin Hsiao, Nate Murray, dareen Mikheil, Matt Larsen, Hui Wang. Monitoring cGAMP Biochemically and in cell using homogenous HTS formatted bioluminescent assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2057.

Abstract 6735: A systemically administered non-nucleotide STING agonist in albumin nanoformulation with potent antitumor activity and low toxicity

Abstract Background: Stimulator of interferon genes (STING), a dimeric transmembrane adapter protein, plays a pivotal role in regulating tumor immune microenvironment and has been explored as a therapeutic strategy against tumors. Most STING agonists were tested in clinical trials using intratumoral injection with limited systemic efficacy. Several systemically administered STING agonists have progressed to clinical trials, with their efficacy yet to be determined, while their systemic toxicity may limit their application. Our study aimed to develop a novel systemically administrated non-nucleotide STING agonist formulated within albumin nanoparticles, demonstrating potent antitumor activity and low systemic toxicity. Methods & Results: We synthesized a novel non-nucleotide small molecule STING dimer ZSA-51D. The acid form of ZSA-51D exhibited a high binding affinity (EC 50: 1.3 nM) to human STING. ZSA-51D demonstrated potent STING activation in THP1-BlueTM ISG cells, with an EC50 of 5.1 nM. To enable systemic delivery, ZSA-51D was encapsulated in albumin nanoparticles (nano ZSA-51D) with a particle size of 115 nm. Surprisingly, nano ZSA-51D exhibited a tenfold increase in STING activation (EC50 of 0.44 nM) compared to free ZSA-51D. Furthermore, nano ZSA-51D demonstrated sevenfold greater activation of bone marrow-derived dendritic cells (EC 50 of 3.5 nM), and eightfold enhanced repolarization of bone marrow-derived macrophages (EC 50 of 4.2 nM) from M2 to M1 compared to free ZSA-51D. The in-vivo anticancer efficacy was evaluated on MC-38 and KPC 6620 xenograft in C57BL/6 mice using intravenous administration of ZSA-51D (1 mg/kg) or nano ZSA-51D, in combination with PD-1 antibody (α-PD1, 100 μg) for 5 dose every 3 days. The results revealed that nano ZSA-51D exhibited superior anticancer effects compared to free ZSA-51D. Remarkedly, nano ZSA-51D, in combination with α-PD1, completely eradicated cancer in the MC-38 xenograft model. These cured mice completely rejected the rechallenge with MC38 cells 120 days after the initial tumor inoculation, indicating potent and long-term anticancer immune memory for anticancer activity. In addition, we performed the toxicity evaluation of nano ZSA-51D and free ZSA-51D at the same dose regime as the efficacy study. Nano ZSA-51D shows no hematological and liver toxicity. However, free ZSA-51D has a sever local inflammation after injections, whereas nano ZSA-51D has no local toxicity. Conclusion: In this study, we developed a novel systemically administered non-nucleotide sting agonist formulated within albumin nanoparticles. Nano ZSA-51, in combination with α-PD1, demonstrated superior anticancer efficacy and low toxicity, holding the potential for future clinical trials. The significantly enhanced STING activation and in vivo efficacy of nano ZSA-51D warrant further investigation into its underlying mechanisms. Citation Format: Jinsong Tao, Hongyi Zhao, Zhongwei Liu, Hanning Wen, Chengyi Li, Qiuxia Li, Miao He, Bo Wen, Wei Gao, Duxin Sun. A systemically administered non-nucleotide STING agonist in albumin nanoformulation with potent antitumor activity and low toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6735.

Drosophila melanogaster Sting mediates Coxiella burnetii infection by reducing accumulation of reactive oxygen species.

The Gram-negative bacterium Coxiella burnetii is the causative agent of query fever in humans and coxiellosis in livestock. C. burnetii infects a variety of cell types, tissues, and animal species including mammals and arthropods, but there is much left to be understood about the molecular mechanisms at play during infection in distinct species. Human stimulator of interferon genes (STING) induces an innate immune response through the induction of type I interferons (IFNs), and IFN promotes or suppresses C. burnetii replication, depending on tissue type. Drosophila melanogaster contains a functional STING ortholog (Sting) which activates NF-κB signaling and autophagy. Here, we sought to address the role of D. melanogaster Sting during C. burnetii infection to uncover how Sting regulates C. burnetii infection in flies. We show that Sting-null flies exhibit higher mortality and reduced induction of antimicrobial peptides following C. burnetii infection compared to control flies. Additionally, Sting-null flies induce lower levels of oxidative stress genes during infection, but the provision of N-acetyl-cysteine (NAC) in food rescues Sting-null host survival. Lastly, we find that reactive oxygen species levels during C. burnetii infection are higher in Drosophila S2 cells knocked down for Sting compared to control cells. Our results show that at the host level, NAC provides protection against C. burnetii infection in the absence of Sting, thus establishing a role for Sting in protection against oxidative stress during C. burnetii infection.

Antitumor Effect of Platinum-Modified STING Agonist MSA-2.

The stimulator of interferon genes (STING)-activated innate immune pathway is strong and durable for tumor immunotherapy. MSA-2 is an available non-nucleotide human STING agonist that promotes the tumor immunotherapy of STING activation. However, strategies for remolding and improving the immunotherapy effects of MSA-2 are of value for clinical applications. Here, we synthesized the platinum salt-modified MSA-2 (MSA-2-Pt) due to platinum salt being a classic chemotherapeutic drug. We found that MSA-2-Pt could achieve double-effect antitumor immunotherapy, including inducing cell death by platinum and activating the STING pathway by MSA-2. In the colon carcinoma MC38 model (sensitive to immune checkpoint immunotherapy tumor) and melanoma B16F10 model (poorly immunogenic and highly aggressive tumor), the MSA-2-Pt had a good antitumor effect, which was a little better than MSA-2 with intratumor injections. The results present a promising strategy for STING activation in tumor immunotherapy and broadening platinum-based drugs.

Activation of human STING by a molecular glue-like compound

Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models.

Human STING is a proton channel.

Proton leakage from organelles is a common signal for noncanonical light chain 3B (LC3B) lipidation and inflammasome activation, processes induced upon stimulator of interferon genes (STING) activation. On the basis of structural analysis, we hypothesized that human STING is a proton channel. Indeed, we found that STING activation induced a pH increase in the Golgi and that STING reconstituted in liposomes enabled transmembrane proton transport. Compound 53 (C53), a STING agonist that binds the putative channel interface, blocked STING-induced proton flux in the Golgi and in liposomes. STING-induced LC3B lipidation and inflammasome activation were also inhibited by C53, suggesting that STING's channel activity is critical for these two processes. Thus, STING's interferon-induction function can be decoupled from its roles in LC3B lipidation and inflammasome activation.

Arabinose- and xylose-modified analogs of 2′,3′-cGAMP act as STING agonists

Stimulator of interferon genes (STING) agonists are promising candidates for vaccine adjuvants and antitumor immune stimulants. The most potent natural agonist of STING, 2',3'-cyclic GMP-AMP (2',3'-cGAMP), is subject to nuclease-mediated inherent metabolic instability, thereby placing limits on its clinical efficacy. Here, we report on a new class of chemically synthesized sugar-modified analogs of 2',3'-cGAMP containingarabinose and xylose sugar derivatives that bind mouse and human STING alleles with high affinity. The co-crystal structures demonstrate that such analogs act as 2',3'-cGAMP mimetics that induce the "closed" conformation of human STING. These analogs show significant resistance to hydrolysis mediated by ENPP1 and increased stability in human serum, while retaining similar potency as 2',3'-cGAMP at inducing IFN-β secretion from human THP1 cells. The arabinose- and xylose-modified 2',3'-cGAMP analogs open a new strategy for overcoming the inherent nuclease-mediated vulnerability of natural ribose cyclic nucleotides, with the additional benefit of high translational potential as cancer therapeutics and vaccine adjuvants.

Abstract 2765: Detection and quantification of cellular GAMP using homogenous HTS formatted bioluminescent assay

Abstract The STimulator of INterferon Genes (STING) is a transmembrane protein located at the endoplasmic reticulum, which serves as a molecular hub in response to cytoplasmic cyclic dinucleotide (CDN) second messengers such as cyclic GMP-AMP (cGAMP). Upon stimulation of cyclic GMP-AMP synthase (cGAS) by a wide array of pathogens that provide extracellular microbial DNA and by intracellular (nuclear and mitochondrial) DNA, the second messenger cGAMP is generated, which then binds directly to the STING protein resulting in the activation of the STING pathway. This activation by cytosolic DNA initiates a cascade of events, cGAS-STING-TBK1-IRF3 signaling, that induces type 1 IFN response, and eventually the signaling is terminated by degradation of pathway components and clearance of stimulatory DNA. Given the significant roles of STING as an adaptor for DNA sensors to pathogens and its involvement in immune sensing, tumor growth control, and control of autoinflammatory and autoimmune disorders, the STING pathway has become a very important drug target. cGAS is well-positioned to act as a DNA sensor that triggers innate immune responses and initiates host immune responses against pathogens and cancer, but inappropriate activation of STING signaling causes severe and often fatal autoimmune or autoinflammatory diseases. Credible evidence suggests that the cGAS-cGAMP-STING pathway makes fundamental contributions to at least three major cancer therapies: radiation therapy, chemotherapy, and immunotherapy, and it thus represents a promising drug target. Therefore, there is a need to identify lead compounds that effectively modulate human STING for further drug development. Towards this goal, we have developed a bioluminescent assay to monitor the concentration of cGAMP in intracellular as well as extracellular compartments. The assay relies on the new concept of nanoluciferase (nanoluc) complementation where a small peptide (SmBiT) linked to cGAMP through a linker (cGAMP tracer) can be recognized by an anti cGAMP antibody that is linked to a nanoluc complementary fragment (LgBiT) resulting in bioluminescence upon the addition of nanoluc substrate. The presence of free cGAMP that is generated in the reaction competes with cGAMP tracer and decreases bioluminescence. The assay is sensitive and can detect less than 10 nM cGAMP and less than one ng of cGAS and thus, it is suitable for testing compounds that modulate cGAS activity. We will show data that correlate the activation of cGAS using less than 10 ng of dsDNA stimulation, and the activation of IFN response element reporter assays to detect cytokine release upon cGAS-STING activation. The assay is homogenous, HTS formatted and can be completed in less than one hour. Thus, this assay provides valuable contribution for investigating modulators of the STING pathway and facilitates the search for novel therapeutics. Citation Format: Said A. Goueli, Kevin Hsiao, Hui Wang, Matt Larsen. Detection and quantification of cellular GAMP using homogenous HTS formatted bioluminescent assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2765.

Acid-Ionizable Iron Nanoadjuvant Augments STING Activation for Personalized Vaccination Immunotherapy of Cancer.

The critical challenge for cancer vaccine-induced T-cell immunity is the sustained activation of antigen cross-presentation in antigen-presenting cells (APCs) with innate immune stimulation. In this study, it is first discovered that the clinically used magnetic contrast agents, iron oxide nanoparticles (IONPs), markedly augment the type-I interferon (IFN-I) production profile of the stimulator of interferon genes (STING) agonist MSA-2 and achieve a 16-fold dosage-sparing effect in the human STING haplotype. Acid-ionizable copolymers are coassembled with IONPs and MSA-2 into iron nanoadjuvants to concentrate STING activation in the draining lymph nodes. The top candidate iron nanoadjuvant (PEIM) efficiently delivers the model antigen ovalbumin (OVA) to CD169+ APCs and facilitates antigen cross-presentation to elicit a 55-fold greater frequency of antigen-specific CD8+ cytotoxic T-lymphocyte response than soluble antigen. PEIM@OVA nanovaccine immunization induces potent and durable antitumor immunity to prevent tumor lung metastasis and eliminate established tumors. Moreover, PEIM nanoadjuvant is applicable to deliver autologous tumor antigen and synergizes with immune checkpoint blockade therapy for prevention of postoperative tumor recurrence and distant metastasis in B16-OVA melanoma and MC38 colorectal tumor models. The acid-ionizable iron nanoadjuvant offers a generalizable and readily translatable strategy to augment STING cascade activation and antigen cross-presentation for personalized cancer vaccination immunotherapy.

Structure-activity relationship study of amidobenzimidazole derivatives as stimulator of interferon genes (STING) agonists

Stimulator of interferon genes (STING) is a crucial adaptor protein that can regulate the innate immune response by inducing the secretion of type Ι interferons and other cytokines after recognizing endogenous or exogenous DNA. Due to the key role of STING in the innate immune system, the activation of STING pathway is expected to be an efficacious immunotherapeutic tactic to treat cancer. In this study, we performed a structure-activity relationship study of amidobenzimidazole monomer, led to a series of ABZI STING agonist derivatives with potent STING-activating effects. Among them, compound 72, as a representative compound, markedly activated the STING-TBK1-IRF3 signaling pathway and significantly increased the mRNA and protein levels of IFN-β, CXCL10 and IL-6 in both WT THP-1 cells and human peripheral blood mononuclear cells (hPBMCs). In addition, it was confirmed that compound 72 was highly selective for human STING, specifically targeting human STING signaling and showing no activation of m-STING.

Design, Synthesis, and Biochemical and Biological Evaluation of Novel 7-Deazapurine Cyclic Dinucleotide Analogues as STING Receptor Agonists.

Cyclic dinucleotides (CDNs) are second messengers that activate stimulator of interferon genes (STING). The cGAS-STING pathway plays a promising role in cancer immunotherapy. Here, we describe the synthesis of CDNs containing 7-substituted 7-deazapurine moiety. We used mouse cyclic GMP-AMP synthase and bacterial dinucleotide synthases for the enzymatic synthesis of CDNs. Alternatively, 7-(het)aryl 7-deazapurine CDNs were prepared by Suzuki-Miyaura cross-couplings. New CDNs were tested in biochemical and cell-based assays for their affinity to human STING. Eight CDNs showed better activity than 2'3'-cGAMP, the natural ligand of STING. The effect on cytokine and chemokine induction was also evaluated. The best activities were observed for CDNs bearing large aromatic substituents that point above the CDN molecule. We solved four X-ray structures of complexes of new CDNs with human STING. We observed π-π stacking interactions between the aromatic substituents and Tyr240 that are involved in the stabilization of CDN-STING complexes.

Lanthanide-Nucleotide Coordination Nanoparticles for STING Activation.

Activation of the stimulator of interferon genes (STING) is essential for blocking viral infections and eliciting antitumor immune responses. Local injection of synthetic STING agonists, such as 2'3'-cGAMP [cGAMP = cyclic 5'-guanosine monophosphate (cGMP)-adenosine monophosphate (AMP)], is a promising approach to enhance antiviral functions and cancer immunotherapy. However, the application of such agonists has been hindered by complicated synthetic procedures, high doses, and unsatisfactory systemic immune responses. Herein, we report the design and synthesis of a series of 2'3'-cGAMP surrogates in nanoparticle formulations formed by reactions of AMP, GMP, and coordinating lanthanides. These nanoparticles can stimulate the type-I interferon (IFN) response in both mouse macrophages and human monocytes. We further demonstrate that the use of europium-based nanoparticles as STING-targeted adjuvants significantly promotes the maturation of mouse bone-marrow-derived dendritic cells and major histocompatibility complex class I antigen presentation. Dynamic molecular docking analysis revealed that these nanoparticles bind with high affinity to mouse STING and human STING. Compared with soluble ovalbumin (OVA), subcutaneously immunized europium-based nanovaccines exhibit significantly increased production of primary and secondary anti-OVA antibodies (∼180-fold) in serum, as well as IL-5 (∼28-fold), IFN-γ (∼27-fold), and IFN-α/β (∼4-fold) in splenocytes ex vivo. Compared with the 2'3'-cGAMP/OVA formulation, subcutaneous administration of nanovaccines significantly inhibits B16F10-OVA tumor growth and prolongs the survival of tumor-bearing mice in both therapeutic and protective models. Given the rich supramolecular chemistry with lanthanides, this work will enable a readily accessible platform for potent humoral and cellular immunity while opening new avenues for cost-effective, highly efficient therapeutic delivery of STING agonists.

Cryo-EM structure of an active bacterial TIR–STING filament complex

Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes1–4. Activation of STING requires its assembly into an oligomeric filament structure through binding of a cyclic dinucleotide4–13, but the molecular basis of STING filament assembly and extension remains unknown. Here we use cryogenic electron microscopy to determine the structure of the active Toll/interleukin-1 receptor (TIR)–STING filament complex from a Sphingobacterium faecium cyclic-oligonucleotide-based antiphage signalling system (CBASS) defence operon. Bacterial TIR–STING filament formation is driven by STING interfaces that become exposed on high-affinity recognition of the cognate cyclic dinucleotide signal c-di-GMP. Repeating dimeric STING units stack laterally head-to-head through surface interfaces, which are also essential for human STING tetramer formation and downstream immune signalling in mammals5. The active bacterial TIR–STING structure reveals further cross-filament contacts that brace the assembly and coordinate packing of the associated TIR NADase effector domains at the base of the filament to drive NAD+ hydrolysis. STING interface and cross-filament contacts are essential for cell growth arrest in vivo and reveal a stepwise mechanism of activation whereby STING filament assembly is required for subsequent effector activation. Our results define the structural basis of STING filament formation in prokaryotic antiviral signalling.

Abstract 1996: Intravenous administration of the small molecule STING agonist CRD5500 elicits potent anti-tumor immune responses in cold tumors

Abstract Background: Innate immune signaling mediated through STING (Stimulator of Interferon Genes) generates a Type I interferon (Type 1 IFN) signal that increases T cell infiltration into cold or non-inflamed tumors leading to significant regression1. CRD5500 is a small molecule STING agonist that has demonstrated excellent systemic tolerability in primate GLP toxicology studies and is scheduled for IND filing with the US FDA in the first half of 2022. The compound binds to STING at a unique location that is outside the dimeric cleft that is the natural ligand binding site2. While this first-in class agent cross-reacts with cynomolgus monkey STING and activates all five major variants of human STING with similar potency, it does not cross-react with rodent STING. Here we demonstrate the potent anti-tumor activity of CRD5500 administered intravenously in a range of immunologically cold murine tumors implanted in human STING knock-in mice. Methods: The functional potency of STING across species was evaluated in STING over-expressing HEK293T cells and PBMCs. Human STING KI mice (Genoway) were used to monitor anti-tumor effects of CRD5500 dosed IV or IT against the following tumors: B16F10 (melanoma), PANO2 (pancreatic), C1498 (acute myeloid leukemia) and LL2 (lung). Intravenous pharmacokinetic and pharmacodynamic evaluation was carried out in cynomolgus monkeys. Results: CRD5500’s activated the different variants of human STING with EC50 values that ranged from 10-70 nM in an pIRF3 reporter assay. Treatment of hSTING transfected cells, human tumor cell lines or hPBMC with CRD5500 led to dose dependent activation of STING. Further, CRD5500 treatment of human DCs and PBMCs led to the induction of inflammatory cytokines such as IFNβ and TNFα. Intravenous administration of CRD5500 in human STING knock-in mice caused tumor regression in several cold tumor models such as B16F10, PANO2, LL2, C1498. Further, CRD5500 demonstrated the abscopal effect in B16F10 tumors when administered via intratumoral route. The compound was well tolerated at high doses in the primate GLP study when administered intravenously and caused dose and exposure dependent increases in CXCL10. Conclusion: The ability of CRD5500 to generate impressive anti-tumor immune responses in cold tumors as a single agent coupled with its excellent IV safety profile in non-human primates make it well suited for FIH investigations planned for mid-2022 under a US FDA IND.

Abstract 667: Inhibition of protein-protein interaction between STING and TRIM29 is a new approach to enhance anti-tumor immune response

Abstract Introduction: STimulator of INterferon Genes (STING) has been reported to be essential for antitumor immunity by inducing type I IFN expression, resulting in activation of both innate and adaptive immunity. Thus, pharmacological activation of STING pathway is actively being tested in many clinical studies. However, the clinical efficacy was not that clear, probably due to the limited expression of STING protein, which is tightly regulated by TRIM29 (E3 ligase)-mediated protein degradation pathway. Here, we present a novel small-molecule drug candidate with strong antitumor response in murine syngeneic model by upregulating cellular levels of STING through the inhibition of protein-protein interaction. Methods: The inhibitors of STING-TRIM29 interaction were identified using luciferase-complementation high-throughput screening system. Target protein of the inhibitor was identified in cellular thermostability shift assay. Their immunological activity was tested in Raw264.7 and A431 cells as well as CT26 murine syngeneic model. Results: The lead compound, SB24011, bound to STING and upregulated cellular levels of STING by inhibiting ubiquitin-mediated degradation. Together with STING agonist, SB24011 synergistically enhanced the inflammatory cytokine expression in cell lines as well as in CT26 syngeneic animal model. Intratumoral injection of SB24011 showed strong abscopal activity as single agent as well as synergistic antitumor responses with anti-PD1 antibody in CT26 syngeneic animal model. Conclusions: We identified novel small-molecule drug candidate, SB24011, which prevents the degradation of both murine and human STING proteins, resulting in enhanced immune responses together with STING agonist, cGAMP. SB24011 showed strong abscopal antitumor activity as single agent and synergistic antitumor activity as combination agent with other IO agents in CT26 syngeneic models. Citation Format: Seung Bum Park, Wansang Cho, Won Sol Chan, young il choi, Sungoh Ahn, Dong-Sup Lee. Inhibition of protein-protein interaction between STING and TRIM29 is a new approach to enhance anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 667.

Abstract 1402: Monitoring the concentration of cGAMP using homogenous bioluminescent HTS-formatted assay

Abstract Genomic instability engages the cGAS (cytosolic DNA sensor cyclic AMP-GMP synthase)-STING (stimulator of interferon genes) signaling (cGAS-cGAMP-STI NG) pathway on both sides of cancer either by being instrumental for antitumor immunity and blocking malignancy or by promoting inflammation-driven carcinogenesis and metastasis. The cGAS-STING axis also plays an essential role in initiating host immune defense against microbial invasion and STING-deficient mice are more susceptible to some viral infections. Credible evidence suggests that the cGAS-cGAMP-STI NG pathway makes fundamental contributions to at least three major cancer therapies, radiation therapy, chemotherapy, immunotherapy, and thus represents a promising pharmaceutical target. cGAS is well-positioned to act as a DNA sensor that triggers innate immune responses through production of the second messenger cyclic GMP-AMP (cGAMP) upon binding to DNA and initiating host immune responses against pathogens and cancer. However, inappropriate activation of STING signaling causes severe and often fatal autoimmune or autoinflammatory diseases. Hence, STING is an attractive drug target for the treatment of STING-driven autoimmune and inflammatory disorders. Therefore, there is a need to identify lead compounds that effectively inhibit human STING for further drug development. An attempt to search for such molecules have been described recently with the report on a STING-specific inhibitor that showed high efficiency, specificity, and safety, paving the way for therapeutically manipulating STING-mediated clinical diseases. Towards this goal, we have developed a bioluminescent assay to monitor the concentration of cGAMP in intracellular as well as extracellular compartments. The assay relies on the use of ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) to release AMP which can be converted to ATP via a coupled enzyme reaction. The ATP generated can be quantified using luciferase/luciferin detection reagents. The assay is homogenous, HTS formatted and can be completed in less than one hour. The assay is sensitive and can detect as low as 10 nM cGAMP. We will show data on the use of agents that stimulate GAS activity such as dsDNA, and the use of reporter assays to detect cytokine release upon cGAS-STING activation. Citation Format: Said A. Goueli, kevin Hsiao. Monitoring the concentration of cGAMP using homogenous bioluminescent HTS-formatted assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1402.

Discovery of novel Thieno[2,3-d]imidazole derivatives as agonists of human STING for antitumor immunotherapy using systemic administration

The activation of stimulator of interferon genes (STING) signaling pathways plays an important role in the innate immune response. Although several STING agonists have been developed recently, the majority of clinical CDN STING agonists are administered by intratumoral (IT) injection. Therefore, there remains a need to develop diverse non-CDN small-molecule STING agonists with systemic administration. Herein, by using a scaffold hopping strategy, we designed a series of thieno [2,3-d]imidazole derivatives as novel STING agonists. Further structure-activity relationship study and optimization led to the discovery of compound 45 as a highly potent human STING agonist with an EC50 value of 1.2 nM. Compound 45 was found to bind to multiple human STING isoforms and accordingly activated the downstream TBK1/IRF3 and NF-κB signaling pathways in the reporter cells bearing with different STING isoforms. The activation on STING signaling pathway was abolished in the STING knock-out cells, indicating that it is a specific STING agonist. Compound 45 significantly inhibited the tumor growth in allograft 4T1 and CT26 tumor models by systemic administration, and more significantly, 45 was able to induce tumor regression in CT26 tumor model without inducing weight loss, suggesting that compound 45 is a highly promising candidate worthy for further development.

LL-37 transports immunoreactive cGAMP to activate STING signaling and enhance interferon-mediated host antiviral immunity.

Cyclic 2',3'-GMP-AMP (cGAMP) binds to and activates stimulator of interferon genes (STING), which then induces interferons to drive immune responses against tumors and pathogens. Exogenous cGAMP produced by infected and malignant cells and synthetic cGAMP used in immunotherapy must traverse the cell membrane to activate STING in target cells. However, as an anionic hydrophilic molecule, cGAMP is not inherently membrane permeable. Here, we show that LL-37, a human host defense peptide, can function as a transporter of cGAMP. LL-37 specifically binds cGAMP and efficiently delivers cGAMP into target cells. cGAMP transferred by LL-37 activates robust interferon responses and host antiviral immunity in a STING-dependent manner. Furthermore, we report that LL-37 inducers vitamin D3 and sodium butyrate promote host immunity by enhancing endogenous LL-37 expression and its mediated cGAMP immune response. Collectively, our data uncover an essential role of LL-37 in innate immune activation and suggest new strategies for immunotherapy.

Activation of STING by targeting a pocket in the transmembrane domain.

Stimulator of interferon genes (STING) is an adaptor protein in innate immunity against DNA viruses or bacteria1-5. STING-mediated immunity could be exploited in the development of vaccines or cancer immunotherapies. STING is a transmembrane dimeric protein that is located in the endoplasmic reticulum or in the Golgi apparatus. STING is activated by the binding of its cytoplasmic ligand-binding domain to cyclic dinucleotides that are produced by the DNA sensor cyclic GMP-AMP (cGAMP) synthase or by invading bacteria1,6,7. Cyclic dinucleotides induce a conformational change in the STING ligand-binding domain, which leads to a high-order oligomerization of STING that is essential for triggering the downstream signalling pathways8,9. However, the cGAMP-induced STING oligomers tend to dissociate in solution and have not been resolved to high resolution, which limits our understanding of the activation mechanism. Here we show that a small-molecule agonist, compound 53 (C53)10, promotes the oligomerization and activation of human STING through a mechanism orthogonal to that of cGAMP. We determined a cryo-electron microscopy structure of STING bound to both C53 and cGAMP, revealing a stable oligomer that is formed by side-by-side packing and has a curled overall shape. Notably, C53 binds to a cryptic pocket in the STING transmembrane domain, between the two subunits of the STING dimer. This binding triggers outward shifts of transmembrane helices in the dimer, and induces inter-dimer interactions between these helices to mediate the formation of the high-order oligomer. Our functional analyses show that cGAMP and C53 together induce stronger activation of STINGthan either ligand alone.