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A strategy for creating potent and pan-genotypic stimulator of interferon genes (STING) agonists is described. Locking a bioactive U-shaped conformation of cyclic dinucleotides by introducing a transannular macrocyclic bridge between the nucleic acid bases leads to a topologically novel macrocycle-bridged STING agonist (MBSA). In addition to substantially enhanced potency, the newly designed MBSAs, exemplified by clinical candidate E7766, exhibit broad pan-genotypic activity in all major human STING variants. E7766 is shown to have potent antitumor activity with long lasting immune memory response in a mouse liver metastatic tumor model. Two complementary stereoselective synthetic routes to E7766 are also described.

Enzymatic shortcut: Cyclic dinucleotides, which are of great interest to study immunology and immune oncology, can be synthesized in a one-step biotransformation significantly shortening the chemical synthesis route. The enzyme displays a surprisingly large substrate scope. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that catalyzes the synthesis of the cyclic GMP-AMP dinucleotide 2���3���-cGAMP. 2���3���-cGAMP functions as inducer for the production of type I interferons. Derivatives of this important second messenger are highly valuable for pharmaceutical applications. However, the production of these analogues requires complex, multistep syntheses. Herein, human cGAS is shown to react with a series of unnatural nucleotides, thus leading to novel cyclic dinucleotides. Most substrate derivatives with modifications at the nucleobase, ribose, and the ��-thio phosphate were accepted. These results demonstrate the catalytic promiscuity of human cGAS and its utility for the biocatalytic synthesis of cyclic dinucleotide derivatives., ChemBioChem;Vol. 21. 2020, Issue 22, pp 3225-3228

Bacterial cyclic dinucleotides (CDNs) play important roles in regulating biofilm formation, motility and virulence. In eukaryotic cells, theses bacterial CDNs are recognized as pathogen-associated molecular patterns (PAMPs) and trigger an innate immune response. We report the photophysical analyses of a novel group of enzymatically synthesized emissive CDN analogues comprised of two families of isomorphic ribonucleotides. The highly favorable photophysical features of the CDN analogues, when compared to their non-emissive natural counterparts, are used to monitor in real time the dinucleotide cyclase-mediated synthesis and phosphodiesterase (PDE)-mediated hydrolysis of homodimeric and mixed CDNs, providing effective means to probe the activities of two classes of bacterial enzymes and insight into their biomolecular recognition and catalytic features.

Cyclic dinucleotides (CDNs) trigger the innate immune response in eukaryotic cells through the STING (stimulator of interferon genes) signalling pathway. To decipher this complex cellular process, a better correlation between structure and downstream function is required. Here we report the design and immunostimulatory effect of a novel group of c-di-GMP analogues. By employing an “atomic mutagenesis” strategy, changing one atom at a time, a class of gradually modified CDNs was prepared. These c-di-GMP analogues induce type-I interferon (IFN) production, with some being more potent than c-di-GMP, their native archetype. To the best of our knowledge, this study demonstrates for the first time that CDN analogues bearing modified nucleobases can tune the innate immune response in eukaryotic cells.

Cyclic dinucleotides (CDNs) have been recognized as second messengers in both prokaryotes and eukaryotes. Until now, four CDNs were identified in the natural organism. In mammalian cells, CDNs could activate stimulator of interferon genes (STING), leading to the induction of type I interferon pro-inflammatory cytokines. It indicated that CDNs could be used for the treatment of tumor and infectious diseases in immune-therapy. Here, we will review the latest progress of the CDNs analogs and their bioactivities. What’s more, the preparation of CDNs through biosynthetic and chemical synthetic methods was summarized as well. It was hoped that this review would provide a comprehensive understanding of CDNs based STING agonists on the chemical perspective for readers who are interested in this area.

Cyclic dinucleotides have emerged as important secondary messengers and cell signaling molecules that regulate several cell responses. A guanine-deficit G-quadruplex structure formation by a sequence containing (4n – 1) guanines, n denoting the number of G-tetrad layers, was previously reported. Here, a (4n – 1) G-quadruplex structure is shown to be capable of binding guanine-containing dinucleotides in micromolar affinity. The guanine base of the dinucleotides interacts with a vacant G-triad, forming four additional Hoogsteen hydrogen bonds to complete a G-tetrad. Solution structures of two complexes, both comprised of a (4n – 1) G-quadruplex structure, one bound to a linear dinucleotide (d(AG)) and the other to a cyclic dinucleotide (cGAMP), are solved using NMR spectroscopy. The latter suggests sufficiently strong interaction between the guanine base of the dinucleotide and the vacant G-triad, which acts as an anchor point of binding. The binding interfaces from the two solution structures provide useful information for specific ligand design. The results also infer that other guanine-containing metabolites of a similar size have the capability of binding G-quadruplexes, potentially affecting the expression of the metabolites and functionality of the bound G-quadruplexes. NRF (Natl Research Foundation, S’pore) Accepted version

Stimulator of interferon genes (STING) binds cyclic dinucleotides (CDNs), which induce a large conformational change of the protein. The structural basis of activation of STING by CDNs is rather well understood. Unliganded STING forms an open dimer that undergoes a large conformational change (∼10 Å) to a closed conformation upon the binding of a CDN molecule. This event activates downstream effectors of STING and subsequently leads to activation of the type 1 interferon response. However, a previously solved structure of STING with 3′,3′-c-di-GMP shows Mg atoms mediating the interaction of STING with this CDN. Here, it is shown that no Mg atoms are needed for this interaction; in fact, magnesium can in some cases obstruct the binding of a CDN to STING.

The human stimulator of interferon genes protein (hSTING) can bind cyclic dinucleotides (CDNs) to activate the production of type I interferons and inflammatory cytokines. These CDNs can be either bacterial secondary messengers, 3'3'-CDNs, or endogenous 2'3'-cGAMP. cGAMP, with a unique 2'-5' bond, is the most potent activator of hSTING among all CDNs. However, current understanding of the molecular principles underlying the unique ability of 2'3'-cGAMP to potently activate hSTINGs other than 3'3'-CDNs remains incomplete. In this work, molecular dynamics simulations were used to provide an atomistic picture of the binding of 2'3'-cGAMP and one 3'3'-CDN (c-di-GMP) to hSTING. The results suggest that hSTING binds more strongly to 2'3'-cGAMP than to c-di-GMP, which prefers to bind with a more open and flexible state of hSTING. Finally, a potential "dock-lock-anchor" mechanism is proposed for the activation of hSTING upon the binding of a potent ligand. It is believed that deep insights into understanding the binding of hSTING with 3'3'-CDNs and the endogenous 2'3'-cGAMP would help to establish the principles underlying powerful 2'3'-cGAMP signaling and the nature of hSTING activation, as well as related drug design.

Cyclic dinucleotides (CDNs) could activate stimulator of interferon genes (STING) protein to produce type I interferon and other pro-inflammation cytokines in mammalian cells. To explore new types of potentially efficient STING activators targeting all five major hSTING variants (WT, R232H, HAQ, AQ and R293Q), we here reported the synthesis of a total of 19 inosine-containing CDNs based on the combinations of hypoxanthine with four natural bases (A, G, C and U) and three phosphodiester linkage backbones (3'-3', 2'-3', 2'-2'). The IFN-β induction results showed that all of the 2'-3' and 2'-2' CDNs linked by inosine and purine nucleosides favored the stacking interaction with Y167 and R238 residues of hSTING protein, and several CDNs constructed by hypoxanthine and pyrimidine like c[I(2',5')U(2',5')] could also activate all five hSTING variants. The molecular dynamic simulation and the isothermal titration calorimetric (ITC) assay further demonstrated the potential of cAIMP isomers with 2'-5' phosphate to form the hydrogen binding with R232 and R238 residues of hSTING in an entropically driven manner compared to cGAMP isomers. It would be promising to exploit novel inosine-mixed CDNs as activators of hSTING variants in immune therapy.

Bacteria utilize nucleotide-based second messengers to regulate a myriad of physiological processes. Cyclic dinucleotides have emerged as central regulators of bacterial physiology, controlling processes ranging from cell wall homeostasis to virulence production, and so far over thousands of manuscripts have provided biological insights into c-di-NMP signaling. The development of small molecule inhibitors of c-di-NMP signaling has significantly lagged behind. Recent developments in assays that allow for high-throughput screening of inhibitors suggest that the time is right for a concerted effort to identify inhibitors of these fascinating second messengers. Herein, we review c-di-NMP signaling and small molecules that have been developed to inhibit cyclic dinucleotide-related enzymes.

Bacterial and host cyclic dinucleotides (cdNs) mediate cytosolic immune responses through the STING signaling pathway, though evidence suggests alternative pathways exist. We used cdN-conjugated beads to biochemically isolate host receptors for bacterial cdNs, and identified the oxidoreductase RECON. High-affinity cdN binding inhibited RECON enzyme activity by simultaneously blocking the substrate and co-substrate sites, as revealed by structural analyses. During bacterial infection of macrophages, RECON antagonized STING activation by acting as a molecular sink for cdNs. Bacterial infection of hepatocytes, which do not express STING, revealed that RECON negatively regulates NF-κB activation. Loss of RECON activity, via genetic ablation or inhibition by cdNs, resulted in increased NF-κB activation and reduced bacterial survival, suggesting that cdN inhibition of RECON promotes a proinflammatory, antibacterial state that is distinct from the anti-viral state associated with STING activation. Thus, RECON functions as a cytosolic pattern recognition receptor specific for bacterial cdNs, shaping inflammatory gene activation via its effects on STING and NF-κB.

The regulation of multiple bacterial phenotypes was found to depend on different cyclic dinucleotides (CDNs) that constitute intracellular signaling second messenger systems. Most notably, c-di-GMP, along with proteins related to its synthesis, sensing, and degradation, was identified as playing a central role in the switching from biofilm to planktonic modes of growth. Recently, this research topic has been under expansion, with the discoveries of new CDNs, novel classes of CDN receptors, and the numerous functions regulated by these molecules. In this review, we comprehensively describe the three main bacterial enzymes involved in the synthesis of c-di-GMP, c-di-AMP, and cGAMP focusing on description of their three-dimensional structures and their structural similarities with other protein families, as well as the essential residues for catalysis. The diversity of CDN receptors is described in detail along with the residues important for the interaction with the ligand. Interestingly, genomic data strongly suggest that there is a tendency for bacterial cells to use both c-di-AMP and c-di-GMP signaling networks simultaneously, raising the question of whether there is crosstalk between different signaling systems. In summary, the large amount of sequence and structural data available allows a broad view of the complexity and the importance of these CDNs in the regulation of different bacterial behaviors. Nevertheless, how cells coordinate the different CDN signaling networks to ensure adaptation to changing environmental conditions is still open for much further exploration.

Cytosolic detection of pathogen-derived nucleic acids is critical for the initiation of innate immune defense against diverse bacterial, viral and eukaryotic pathogens. Conversely, inappropriate responses to cytosolic nucleic acids can produce severe autoimmune pathology. The host protein STING has been identified as a central signaling molecule in the innate immune response to cytosolic nucleic acids. STING seems to be especially critical for responses to cytosolic DNA and the unique bacterial nucleic acids called 'cyclic dinucleotides'. Here we discuss advances in the understanding of STING and highlight the many unresolved issues in the field.

Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3', 3'-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.

Cyclic dinucleotides (CDN) such as c-di-GMP and c-di-AMP are important multi-functional compounds that regulate a diverse range of processes in bacteria. The diversity of the functions is enabled by the two distinct conformations, that is, open and close conformations. We herein report the concise synthesis of triazole-linked analogues (c-di-GM P TL , c-di-AM P TL ) through click dimerization of nucleoside analogues. The implementation of a cyclophane system in the central macrocycle furnished the structure with the conformational restriction to maintain the structure similar to the open conformer of natural congeners.

© 2015 Taylor and Francis Group, LLC. Cyclic di-GMP (c-di-GMP) is a second messenger that is important in regulating bacterial physiology and behavior, including motility and virulence. Many questions remain about the role and regulation of this signaling molecule, but current methods of detection are limited by either modest sensitivity or requirements for extensive sample purification. We have taken advantage of a natural, high affinity receptor of c-di-GMP, the Vc2 riboswitch aptamer, to develop a sensitive and rapid electrophoretic mobility shift assay (EMSA) for c-di-GMP quantitation that required minimal engineering of the RNA.

Cyclic dinucleotides (cdNs) are novel second messengers that are synthesized directly from two “molecular units of energy” in the cell, namely ATP and GTP. In prokaryotes, all three combinations of cdNs have been discovered: cyclic di-AMP, cyclic AMP-GMP, and cyclic di-GMP. Their effects on bacterial host physiology have been shown to be important and diverse, impacting processes ranging from biofilm formation to motility to sporulation. Many of their prokaryotic effectors are protein-based; however, cyclic dinucleotides have also been shown to exert genetic expression control at the RNA level by binding to mRNA regulatory elements called riboswitches. While cyclic dinucleotide signaling in prokaryotes has been appreciated for over 25 years, it was not until the last few years that cyclic dinucleotide signaling has been shown to exist and have a profound impact in mammals. cGAMP (a non-canonically linked cyclic AMP-GMP) has been discovered endogenously within humans and found to be a critical second messenger for signaling pathogen invasion to the host. cGAS, a cytosolic mammalian cyclic dinucleotide synthase, produces cGAMP upon binding pathogen DNA. Subsequently, cGAMP binds to the ER-resident transmembrane protein STING, which triggers a downstream cascade of interferon, cytokine, and chemokine production. Therefore from prokaryotes to humans, cyclic dinucleotide signaling spans billions of years in evolution, and today has led to great interest in its therapeutic targeting.This work details the discovery of cyclic AMP-GMP and its cognate riboswitch within the bacteria Geobacter sulfurreducens. Interestingly, cyclic AMP-GMP signaling through its riboswitch plays a previously unappreciated role in exoelectrogenesis within Geobacter. The structural elucidation and signaling role of cyclic AMP-GMP (cGAMP) in humans is subsequently described, showing that humans utilize a novel regioisomer of cyclic AMP-GMP containing mixed 2′-5′ and 3′-5′ phosphodiester bonds. The ancient origin of human cGAMP signaling is illuminated by the discovery of a functional cGAS-cdN-STING pathway in Nematostella vectensis, an anemone species >500 million years diverged from humans. The structure of anemone STING is highly conserved compared to human STING, yet anemone cGAS appears to produce only a 3′-5′ linked cdN. Finally, the signaling space of the cGAS-cdN-STING pathway is examined with a focus on its potential functions in lower organisms. Taken holistically, this work details the signaling mechanisms of cyclic dinucleotides in multiple organisms, while profiling the chemical evolution of a unique second messenger.

Cyclic dinucleotides such as cyclic dimeric guanosine monophosphate (c-di-GMP,) cyclic dimeric adenosine monophosphate (c-di-AMP) and cyclic guanosine-adenosine monophosphate (cGAMP) are found in both prokaryotes and eukaryotes and are important in cell signaling processes. The work discussed herein aims at understanding the aggregative properties of these cyclic dinucleotides for interest in the biological implications of aggregation and for the purpose of various applications of these higher order structures. The applications include simple detection methods for these molecules as well as a unique G-quadruplex peroxidase that can be used for catalysis. To that end, a new fluorescent cyclic dinucleotide which incorporated the fluorescent purine analog, 2-aminopurine, was synthesized. This analog is capable of forming intercalated dimers with natural cyclic dinucleotides containing guanine which allows for a simple turn-off fluorescent method for these second messengers. Additionally, ligands containing four guanine bases were utilized to bind c-di-GMP and leading to a fluorescent turn-on fluorescent detection method. The c-di-GMP octamer was also studied for its ability to enhance the peroxidation ability of the iron-containing cofactor hemin. Its proficient ability to enhance catalysis suggests that DNA loops found in other G-quadruplex forming molecules are not necessary for catalytic enhancement of hemin.

Cyclic dinucleotides are synthesized using a ring-closing metathesis protocol and incorporated into oligonucleotides. A stabilization of a three-way junction is observed by an oligodeoxynucleotide containing a central 2'-C to 3'-phosphate connection.

Second messengers are intracellular signaling molecules that enable cells to respond to environmental changes by relaying signals received at the cell surface to enact downstream physiological effects. A recently expanded class of second messengers in bacteria are the cyclic dinucleotides, which include cyclic di-GMP (cdiG), cyclic di-AMP (cdiA), and 3',3' cyclic AMP-GMP (cAG). In bacteria, these messengers regulate processes ranging from chemotaxis and sporulation to antibiotic resistance and virulence. Recent studies have found that bacterial cyclic dinucleotides, as well as a mammalian 2',3' cAG homolog, trigger the innate immune response upon detection in the mammalian cell cytosol. However, many components and roles of cyclic dinucleotide signaling remain unidentified or poorly understood. A clear understanding of cyclic dinucleotide signal transduction would further the pursuit of these signaling pathways as therapeutic targets and the effects of using cyclic dinucleotides as small molecule adjuvants. Herein, novel strategies for cyclic dinucleotide detection are reported that use that natural ability of riboswitch RNAs to discriminate between these nucleotide-based messengers. First, a microfluidic electrophoretic mobility shift assay (µMSA) is presented that enables highly sensitive and rapid detection of cdiG in vitro. This method takes advantage of the ligand-induced conformational change of a natural cdiG GEMM-I class riboswitch aptamer, termed Vc2. This same aptamer was then converted to a fluorescent biosensor for live-cell imaging by fusing it to the Spinach aptamer, an RNA mimic of GFP. This Vc2-Spinach RNA-based biosensor senses low levels of endogenous cdiG in Escherichia coli and responds to increased levels of cdiG upon coexpression of a heterologous cdiG synthase. A similar Spinach-based strategy was used to develop a cdiA RNA-based biosensor through fusion to riboswitches from the recently reported ydaO class. This cdiA sensor is the first tool for live cell imaging of cdiA and provided the first demonstration of the Spinach system in Listeria monocytogenes, an important gram-positive intracellular pathogen. Finally, as no known biosensor existed for 3',3' cAG, rational engineering of a cdiG aptamer led to the discovery of a subclass of GEMM-I riboswitches in Geobacter species that is naturally specific for 3',3' cAG. Further analysis revealed that this novel riboswitch subclass, which represents the first known effector for this messenger, is important in regulating the process of extracellular electron transfer in Geobacter and these riboswitches were used to develop the first biosensor specific for 3',3' cAG. It is envisioned that the use of these cyclic dinucleotide-specific sensors can be used to further identify unknown components in the signal transduction of these important regulatory molecules.

We report synthesis of each of the ten cyclic 2′-deoxyribodinucleotides by a solution-phase H-phosphonate method. The cyclic dimers have been characterized by 31P NMR, MS, UV, and enzymatic degradation.

Deciphering the molecular basis of HCN channel regulation by cGMP leads to the serendipitous discovery of cyclic dinucleotides as potent inhibitors of If current in the heart.

An integrated set of reactions and conditions that allow an eight-step one-flask synthesis of the protected derivatives of c-di-GMP and the [R(p),R(p)] and [R(p),S(p)] thiophosphate analogues is reported. Deprotection is also carried out as a one-flask procedure, with the final products isolated by crystallization from the reaction mixture. Chromatography is only used for separation of the thiophosphate diastereomers.

Four different nucleosides with olefinic 2′-modifications were prepared; 2′- C -methylene, 2′- C -(propen-1-yl), 2′- C -allyl and 2′- O -allyl uridines, respectively. These were incorporated into dinucleotides with allyl phosphate or vinyl phosphonate linkages. Hence, six different dinucleotides were studied as substrates for RCM reactions, and from four of these, cyclic dinucleotides with connections between 2′-C and phosphorus of 3–6 atoms were obtained.

The novel dinucleotide 5′-phosphate, [(L,D)-pIsodApdC], discovered in our laboratory, is a strong inhibitor of HIV-1 integrase for both the 3′-processing and the strand transfer steps. The rationale used in this molecular design was that residues immediately upstream of the dinucleotide cleavage site in the 3′-processing step might provide critical recognition/binding sites on integrase. The rationale for the second type of inhibitors was based on the elimination products (linear and cyclic dinucleotides) of 3′-processing. However, while the linear dinucleotide 5′-phosphate (pdGpdT) was active, its cyclic counterpart was inactive against both wild-type and mutant HIV integrase.

Cyclic dinucleotides with saturated connections between a nucleobase and the phosphate are synthesised using a tandem ring-closing metathesis (RCM) and hydrogenation protocol and found to be significantly stabilised towards ammonia.

A defined chemical synthesis of the cyclic dinucleotides, ApAp, ApUp, and UpUp has been devised, based on modern phosphotriester methods. These cyclic dinucleotides have been shown to inhibit RNA polymerase. The 1H NMR spectra of the protected dimers show a preference for the 2E form, while the spectra of the unprotected dimers show the 3E form to be favored. The circular dichroism spectra show a negative long wavelength transition; however, ApUp does not show the short wavelength maximum present in the spectra of APAP and UpUp.

Ever since their initial discovery few years ago, cyclic dinucleotides (cDNs), their biosynthesis, and their biological function have been in focus of intense research efforts. In this issue, Kato et al. (2015) present strong evidence that the key enzyme in cDN biosynthesis, DncV, is poised on a tipping point such that, given a nudge, the enzyme, can link the nucleotides into a distinct cyclic loop, leading to a specific innate immune response.