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.

2',4'-LNA-Functionalized 5'-S-Phosphorothioester CDNs as STING Agonists.

Cyclic dinucleotides (CDNs) have garnered popularity over the last decade as immunotherapeutic agents, which activate the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway to trigger an immune response. Many analogs of 2'3'-cGAMP, c-di-GMP, and c-di-AMP have been developed and shown as effective cancer vaccines and immunomodulators for the induction of both the adaptive and innate immune systems. Unfortunately, the effectiveness of these CDNs is limited by their chemical and enzymatic instability. We recently introduced 5'-endo-phosphorothoiate 2'3'-cGAMP analogs as potent STING agonist with improved resistance to cleavage by clinically relevant phosphodiesterases. We herein report the synthesis of locked nucleic acid-functionalized (LNA) endo-S-CDNs and evaluate their ability to activate STING in THP1 monocytes. Interestingly, some of our synthesized LNA 3'3'-endo-S-CDNs can moderately activate hSTING REF haplotype (R232H), which exhibit diminished response to both 2'3'-cGAMP and ADU-S100. Also, we show that one of our most potent endo-S-CDNs has remarkable chemical (oxidants I2 and H2O2) and phosphodiesterase stability.

Combination of MHI148 Targeted Photodynamic Therapy and STING Activation Inhibits Tumor Metastasis and Recurrence.

Metastasis and recurrence are notable contributors to mortality associated with breast cancer. Although immunotherapy has shown promise in mitigating these risks after conventional treatments, its effectiveness remains constrained by significant challenges, such as impaired antigen presentation by dendritic cells (DCs) and inadequate T cell infiltration into tumor tissues. To address these limitations, we developed a multifunctional nanoparticle platform, termed GM@P, which consisted of a hydrophobic shell encapsulating the photosensitizer MHI148 and a hydrophilic core containing the STING agonist 2'3'-cGAMP. This design elicited robust type I interferon responses to activate antitumor immunity. The GM@P nanoparticles loaded with MHI148 specifically targeted breast cancer cells. Upon exposure to 808 nm laser irradiation, the MHI148-loaded nanoparticles produced toxic reactive oxygen species (ROS) to eradicate tumor cells through photodynamic therapy (PDT). Notably, PDT stimulated immunogenic cell death (ICD) to foster the potency of antitumor immune responses. Furthermore, the superior photoacoustic imaging (PAI) capabilities of MHI148 enabled the simultaneous visualization of diagnostic and therapeutic procedures. Collectively, our findings uncovered that the combination of PDT and STING activation facilitated a more conducive immune microenvironment, characterized by enhanced DC maturation, infiltration of CD8+ T cells, and proinflammatory cytokine release. This strategy stimulated local immune responses to augment systemic antitumor effects, offering a promising approach to suppress tumor growth, inhibit metastasis, and prevent recurrence.

Fluorinated cGAMP analogs, which act as STING agonists and are not cleavable by poxins: Structural basis of their function.

The cGAS-STING pathway is a crucial part of innate immunity; it serves to detect DNA in the cytoplasm and to defend against certain cancers, viruses, and bacteria. We designed and synthesized fluorinated carbocyclic cGAMP analogs, MD1203 and MD1202D (MDs), to enhance their stability and their affinity for STING. These compounds demonstrated exceptional activity against STING. Despite their distinct chemical modifications relative to the canonical cyclic dinucleotides (CDNs), crystallographic analysis revealed a binding mode with STING that was consistent with the canonical CDNs. Importantly, MDs were resistant to cleavage by viral poxin nucleases and MDs-bound poxin adopted an unliganded-like conformation. Moreover, MDs complexed with poxin showed a conformation distinct from cGAMP bound to poxin, closely resembling their conformation when bound to STING. In conclusion, the development of MD1203 and MD1202D showcases their potential as potent STING activators with remarkable stability against poxin-mediated degradation-a crucial characteristic for future development of antivirals.

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.

A photoaffinity labeling strategy identified EF1A1 as a binding protein of cyclic dinucleotide 2'3'-cGAMP.

2'3'-cyclic GMP-AMP (2'3'-cGAMP), generated by cyclic GMP-AMP synthase (cGAS) under activation by cytosolic DNA, has a vital role in innate immune response via its receptor protein stimulator of interferon genes (STING) to fight viral infections and tumors. In order to have a complete understanding of biological functions of 2'3'-cGAMP, it is important to find out whether 2'3'-cGAMP has other unrevealed binding proteins present in mammalian cells and executes unknown functions. Here we report the 2'3'-cGAMP-based photoaffinity probes that capture and isolate 2'3'-cGAMP-binding proteins. These probes enable the identification of some potential 2'3'-cGAMP-binding proteins from HeLa cells. EF1A1, an essential protein regulating protein synthesis, is further validated to associate with 2'3'-cGAMP in vitro and in cells to impede protein synthesis. Thus, our studies provide a powerful approach to enable identification of the 2'3'-cGAMP interactome, discover unknown functions of 2'3'-cGAMP, and understand its physiological/pathological roles in tumor immunity and immune-related diseases.

2',3'-Cyclic GMP-AMP Dinucleotides for STING-Mediated Immune Modulation: Principles, Immunotherapeutic Potential, and Synthesis.

The cGAS-STING pathway discovered ten years ago is an important component of the innate immune system. Activation of cGAS-STING triggers downstream signalling, such as TBK1-IRF3, NF-κB and autophagy, which in turn leads to antipathogen responses, durable antitumour immunity or autoimmune diseases. 2',3'-Cyclic GMP-AMP dinucleotides (2',3'-cGAMP), the key second messengers produced by cGAS, play a pivotal role in cGAS-STING signalling by binding and activating STING. Thus, 2',3'-cGAMP has immunotherapeutic potential, which in turn has stimulated research on the design and synthesis of 2',3'-cGAMP analogues for clinical applications over the past ten years. This review presents the discovery, metabolism, and function of 2',3'-cGAMP in the cGAS-STING innate immune signalling axis. The enzymatic and chemical syntheses of 2',3'-cGAMP analogues as STING-targeting therapeutics are also summarized.

Structural basis of cyclic oligoadenylate degradation by ancillary Type III CRISPR-Cas ring nucleases.

Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. These enzymes contain a CRISPR-associated Rossman-fold (CARF) domain, which binds and cleaves the cA molecule. Here, we present the structures of the standalone ring nuclease from Sulfolobus islandicus (Sis) 0811 in its apo and post-catalytic states. This enzyme is composed by a N-terminal CARF and a C-terminal wHTH domain. Sis0811 presents a phosphodiester hydrolysis metal-independent mechanism, which cleaves cA4 rings to generate linear adenylate species, thus reducing the levels of the second messenger and switching off the cell antiviral state. The structural and biochemical analysis revealed the coupling of a cork-screw conformational change with the positioning of key catalytic residues to proceed with cA4 phosphodiester hydrolysis in a non-concerted manner.

Au naturale: use of biologically derived cyclic di-nucleotides for cancer immunotherapy.

Cyclic di-nucleotides (CDNs) are widespread second messenger signalling molecules that regulate fundamental biological processes across the tree of life. These molecules are also potent modulators of the immune system, inducing a Type I interferon response upon binding to the eukaryotic receptor STING. Such a response in tumours induces potent immune anti-cancer responses and thus CDNs are being developed as a novel cancer immunotherapy. In this review, I will highlight the use, challenges and advantages of using naturally occurring CDNs to treat cancer.

Cyclic CMP and cyclic UMP mediate bacterial immunity against phages.

The cyclic pyrimidines 3',5'-cyclic cytidine monophosphate (cCMP) and 3',5'-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria.

Design and synthesis of gene-directed caged cyclic nucleotides exhibiting cell type selectivity.

We designed a new caging group that can be photoactivated only in the presence of a non-endogenous enzyme when exposed to 405 nm light. Because cells or tissues can be genetically tagged by an exogenously expressed enzyme, this novel method can serve as a strategy for adding targeting abilities to photocaged compounds.

Identification of Novel Carbocyclic Pyrimidine Cyclic Dinucleotide STING Agonists for Antitumor Immunotherapy Using Systemic Intravenous Route.

Stimulator of Interferon Genes (STING) plays an important role in innate immunity by inducing type I interferon production upon infection with intracellular pathogens. STING activation can promote increased T-cell activation and inflammation in the tumor microenvironment, resulting in antitumor immunity. Natural and synthetic cyclic dinucleotides (CDNs) are known to activate STING, and several synthetic CDN molecules are being investigated in the clinic using an intratumoral administration route. Here, we describe the identification of STING agonist 15a, a cyclic dinucleotide structurally diversified from natural ligands with optimized properties for systemic intravenous (iv) administration. Our studies have shown that STING activation by 15a leads to an acute innate immune response as measured by cytokine secretion and adaptive immune response via activation of CD8+ cytotoxic T-cells, which ultimately provides robust antitumor efficacy.

Crystal structure and functional implication of a bacterial cyclic AMP-AMP-GMP synthetase.

Mammalian cyclic GMP-AMP synthase (cGAS) and its homologue dinucleotide cyclase in Vibrio cholerae (VcDncV) produce cyclic dinucleotides (CDNs) that participate in the defense against viral infection. Recently, scores of new cGAS/DncV-like nucleotidyltransferases (CD-NTases) were discovered, which produce various CDNs and cyclic trinucleotides (CTNs) as second messengers. Here, we present the crystal structures of EcCdnD, a CD-NTase from Enterobacter cloacae that produces cyclic AMP-AMP-GMP, in its apo-form and in complex with ATP, ADP and AMPcPP, an ATP analogue. Despite the similar overall architecture, the protein shows significant structural variations from other CD-NTases. Adjacent to the donor substrate, another nucleotide is bound to the acceptor binding site by a non-productive mode. Isothermal titration calorimetry results also suggest the presence of two ATP binding sites. GTP alone does not bind to EcCdnD, which however binds to pppApG, a possible intermediate. The enzyme is active on ATP or a mixture of ATP and GTP, and the best metal cofactor is Mg2+. The conserved residues Asp69 and Asp71 are essential for catalysis, as indicated by the loss of activity in the mutants. Based on structural analysis and comparison with VcDncV and RNA polymerase, a tentative catalytic pathway for the CTN-producing EcCdnD is proposed.

Extracting meaningful circuit-based calcium dynamics in astrocytes and neurons from adult mouse brain slices using single-photon GCaMP imaging.

Confocal, multiphoton, or other advanced microscopy techniques produce high-quality datasets of calcium activity in live tissue. However, researchers without access to such expensive equipment can still produce meaningful observations from single-photon datasets. Here, we describe a protocol to extract meaningful features of both somatic neuronal and membranous astrocytic calcium dynamics obtained from charge-coupled device (CCD)-based camera setups, typical of electrophysiology rigs and highly relevant for investigating neuronal and astrocytic involvement in brain circuitry. For complete details on the use and execution of this protocol, please refer to Asrican et al. (2020).

Catalytic asymmetric and stereodivergent oligonucleotide synthesis.

We report the catalytic stereocontrolled synthesis of dinucleotides. We have demonstrated, for the first time to our knowledge, that chiral phosphoric acid (CPA) catalysts control the formation of stereogenic phosphorous centers during phosphoramidite transfer. Unprecedented levels of diastereodivergence have also been demonstrated, enabling access to either phosphite diastereomer. Two different CPA scaffolds have proven to be essential for achieving stereodivergence: peptide-embedded phosphothreonine-derived CPAs, which reinforce and amplify the inherent substrate preference, and C2-symmetric BINOL-derived CPAs, which completely overturn this stereochemical preference. The presently reported catalytic method does not require stoichiometric activators or chiral auxiliaries and enables asymmetric catalysis with readily available phosphoramidites. The method was applied to the stereocontrolled synthesis of diastereomeric dinucleotides as well as cyclic dinucleotides, which are of broad interest in immuno-oncology as agonists of the stimulator of interferon genes (STING) pathway.

Design, Synthesis and Biological Evaluation of (2',5' and 3'5'-Linked) cGAMP Analogs that Activate Stimulator of Interferon Genes (STING).

Stimulator of interferon genes (STING) is an endoplasmic reticulum adaptor transmembrane protein that plays a pivotal role in innate immune system. STING agonists, such as endogenous cyclic dinucleotide (CDN) cyclic GMP-AMP (cGAMP), have been used in diverse clinical research for immunogenic tumor clearance, antiviral treatments and vaccine adjuvants. CDNs containing noncanonical mixed 3'-5' and 2'-5' phosphodiester linkages show higher potency in the activation of the STING pathway. In this study, a series of 2'3'-CDNs were designed and synthesized through a modified one-pot strategy. We then established a surface plasmon resonance (SPR)-based binding assay to quantify the binding affinities of synthesized CDNs for human STING, which requested a minuscule amount of sample without any pretreatment. Using this assay, we identified compound 8d (KD = 0.038 µM), a novel CDN that showed higher binding affinity with hSTING than cGAMP (KD = 0.543 µM). Cellular assays confirmed that 8d could trigger the expression of type I IFNs and other proinflammatory cytokines more robust than cGAMP. 8d also exhibited more resistant than cGAMP to enzymatic cleavage in vitro, indicating the successful improvement in drug availability. These findings provide guidelines for the design and structural optimization of CDNs as STING agonists.

Tumour initiation, store-operated calcium entry (SOCE) and apoptosis: cyclic nucleotide dependence.

Chemical instigators and modulators of tumourigenesis influence cell signal transduction pathways. Cyclic nucleotides and steroid hormones may contribute to the process of carcinogenesis or provide protection via apoptotic mechanisms. Although several pharmacologic classes of compounds influence cyclic nucleotide levels markedly, less is known about the class effects of promoters and blockers of tumourigenesis and apoptosis. This molecular modeling study uses cyclic nucleotide templates to investigate relative molecular similarity within compounds modulating tumourigenesis and apoptosis. Findings, in respect of superimposition and molecular fit of the investigated compounds, are related to their individual effects on cyclic nucleotide pharmacology. Modulators of tumourigenesis and estrogen receptor sub-type ligands relate to cyclic nucleotide structure. Estradiol and GPER ligands provide a similar pattern of fit to adenine nucleotide. Chemically diverse modulators of apoptosis, including K+ channel ligands, fit to different components of cyclic nucleotide structure. Compounds modulating Ca2+ entry and IP3 receptors relate structurally to the nucleotide dioxaphosphinin moiety. Relative molecular similarity within the structures of apoptosis and tumourigenesis modulators identifies a unifying property within chemically disparate compounds. The ubiquitous generation of oxidative stress and ROS in cells by apoptosis modulating compounds may relate to the disruption of cyclic nucleotide regulated homeostasis mechanisms.

Antitumor activity of a systemic STING-activating non-nucleotide cGAMP mimetic.

Stimulator of interferon genes (STING) links innate immunity to biological processes ranging from antitumor immunity to microbiome homeostasis. Mechanistic understanding of the anticancer potential for STING receptor activation is currently limited by metabolic instability of the natural cyclic dinucleotide (CDN) ligands. From a pathway-targeted cell-based screen, we identified a non-nucleotide, small-molecule STING agonist, termed SR-717, that demonstrates broad interspecies and interallelic specificity. A 1.8-angstrom cocrystal structure revealed that SR-717 functions as a direct cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) mimetic that induces the same "closed" conformation of STING. SR-717 displayed antitumor activity; promoted the activation of CD8+ T, natural killer, and dendritic cells in relevant tissues; and facilitated antigen cross-priming. SR-717 also induced the expression of clinically relevant targets, including programmed cell death 1 ligand 1 (PD-L1), in a STING-dependent manner.

Structure-Aided Development of Small-Molecule Inhibitors of ENPP1, the Extracellular Phosphodiesterase of the Immunotransmitter cGAMP.

Cancer cells initiate an innate immune response by synthesizing and exporting the small-molecule immunotransmitter cGAMP, which activates the anti-cancer Stimulator of Interferon Genes (STING) pathway in the host. An extracellular enzyme, ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), hydrolyzes cGAMP and negatively regulates this anti-cancer immune response. Small-molecule ENPP1 inhibitors are much needed as tools to study the basic biology of extracellular cGAMP and as investigational cancer immunotherapy drugs. Here, we surveyed structure-activity relationships around a series of cell-impermeable and thus extracellular-targeting phosphonate inhibitors of ENPP1. In addition, we solved the crystal structure of an exemplary phosphonate inhibitor to elucidate the interactions that drive potency. This study yielded several best-in-class inhibitors with Ki < 2 nM and excellent physicochemical and pharmacokinetic properties. Finally, we demonstrate that an ENPP1 inhibitor delays tumor growth in a breast cancer mouse model. Together, we have developed ENPP1 inhibitors that are excellent tool compounds and potential therapeutics.

HD-[HD-GYP] Phosphodiesterases: Activities and Evolutionary Diversification within the HD-GYP Family.

Cyclic dinucleotides are signaling molecules that modulate many processes, including immune response and virulence factor production. Their cellular levels in bacteria are fine-tuned by metal-dependent phosphodiesterases, namely, the EAL and HD-GYP proteins, with HD-GYPs belonging to the larger HD domain superfamily. In this study, we first focus on the catalytic properties and the range of metal ions and substrates of the HD-[HD-GYP] subfamily, consisting of two HD domains. We identified SO3491 as a homologue of VCA0681 and the second example of an HD-[HD-GYP]. Both proteins hydrolyze c-di-GMP and 3'3'c-GAMP and coordinate various metal ions, but only Fe and to a lesser extent Co support hydrolysis. The proteins are active only in the diferrous form and not in the one-electron more oxidized FeIIFeIII state. Although the C-terminal HD-GYP domain is essential for activity, the role of the N-terminal HD domain remains unknown. We show that the N-terminal site is important for protein stability, influences the individual apparent kcat and KM (but not kcat/KM), and cannot bind c-di-GMP, thus precluding its involvement in cyclic dinucleotide sensing. We proceeded to perform phylogenetic analyses to examine the distribution and functional relationships of the HD-[HD-GYP]s to the rest of the HD-GYPs. The phylogeny provides a correlation map that draws a link between the evolutionary and functional diversification of HD-GYPs, serving as a template for predicting the chemical nature of the metallocofactor, level of activity, and reaction outcome.