Immunostimulatory silica nanoparticle boosts innate immunity in brain tumors.

The high mortality associated with glioblastoma multiforme (GBM) is attributed to its invasive nature, hypoxic core, resistant cell subpopulations and a highly immunosuppressive tumor microenvironment (TME). To support adaptive immune function and establish a more robust antitumor immune response, we boosted the local innate immune compartment of GBM using an immunostimulatory mesoporous silica nanoparticle, termed immuno-MSN. The immuno-MSN was specifically designed for systemic and proficient delivery of a potent innate immune agonist to dysfunctional antigen-presenting cells (APCs) in the brain TME. The cargo of the immuno-MSN was cyclic diguanylate monophosphate (cdGMP), a Stimulator of Interferon Gene (STING) agonist. Studies showed the immuno-MSN promoted the uptake of STING agonist by APCs in vitro and the subsequent release of the pro-inflammatory cytokine interferon ß, 6-fold greater than free agonist. In an orthotopic GBM mouse model, systemically administered immuno-MSN particles were taken up by APCs in the near-perivascular regions of the brain tumor with striking efficiency. The immuno-MSNs facilitated the recruitment of dendritic cells and macrophages to the TME while sparing healthy brain tissue and peripheral organs, resulting in elevated circulating CD8+ T cell activity (2.5-fold) and delayed GBM tumor growth. We show that an engineered immunostimulatory nanoparticle can support pro-inflammatory innate immune function in GBM and subsequently augment current immunotherapeutic interventions and improve their therapeutic outcome.

An Improved Strategy for the Chemical Synthesis of 3',5'-Cyclic Diguanylic Acid.

The physiological functions of c-di-GMP and its involvement in many key processes led to its recognition as a major and ubiquitous bacterial second messenger. Aside from being a bacterial signaling molecule, c-di-GMP is also an immunostimulatory molecule capable of inducing innate and adaptive immune responses through maturation of immune mammalian cells. Given the broad biological functions of c-di-GMP and its potential applications as a nucleic-acid-based drug, the chemical synthesis of c-di-GMP has drawn considerable interest. An improved phosphoramidite approach to the synthesis of c-di-GMP is reported herein. The synthetic approach is based on the use of a 5'-O-formyl protecting group, which can be rapidly and chemoselectively cleaved from a key dinucleotide phosphoramidite intermediate to enable a cyclocondensation reaction leading to a fully protected c-di-GMP product in a yield ∼80%. The native c-di-GMP is isolated, after complete deprotection, in an overall yield of 36% based on the commercial ribonucleoside used as starting material. © 2019 by John Wiley & Sons, Inc.

Targeting STING with cyclic di-GMP greatly augmented immune responses of glycopeptide cancer vaccines.

Cyclic di-GMP (CDG) was applied to MUC1 glycopeptide-based cancer vaccines with physical mixing and built-in (at 2'-OH of CDG) strategies for activating the STING pathway. CDG in both strategies behaved as a potent immunostimulant and contributed to high titers of IgG antibodies and the expression of multiple cytokines.

Synthesis of All Possible Canonical (3'-5'-Linked) Cyclic Dinucleotides and Evaluation of Riboswitch Interactions and Immune-Stimulatory Effects.

The cyclic dinucleotides (CDNs) c-di-GMP, c-di-AMP, and c-AMP-GMP are widely utilized as second messengers in bacteria, where they signal lifestyle changes such as motility and biofilm formation, cell wall and membrane homeostasis, virulence, and exo-electrogenesis. For all known bacterial CDNs, specific riboswitches have been identified that alter gene expression in response to the second messengers. In addition, bacterial CDNs trigger potent immune responses, making them attractive as adjuvants in immune therapies. Besides the three naturally occurring CDNs, seven further CDNs containing canonical 3'-5'-linkages are possible by combining the four natural ribonucleotides. Herein, we have synthesized all ten possible combinations of 3'-5'-linked CDNs. The binding affinity of novel CDNs and GEMM riboswitch variants was assessed utilizing a spinach aptamer fluorescence assay and in-line probing assays. The immune-stimulatory effect of CDNs was evaluated by induction of type I interferons (IFNs), and a novel CDN c-AMP-CMP was identified as a new immune-stimulatory agent.

Synthesis of [32P]-c-di-GMP for Diguanylate Cyclase and Phosphodiesterase Activity Determinations.

Diguanylate cyclases that synthesize and phosphodiesterases that hydrolyze the second messenger cyclic-di-GMP (c-di-GMP) are at the center of bacterial signaling pathways that control behaviors relevant to all aspects of microbial physiology and pathogenesis (Romling et al., Microbiol Mol Biol Rev 77(1):1-52, 2013). Bioinformatics tools can easily predict the presence of the diguanylate cyclase GGDEF domain, or the EAL and HD-GYP domains associated with phosphodiesterase activity. However, experimental confirmation of enzymatic activity is still necessary, as many proteins contain degenerate domains that lack catalytic activity but nonetheless function as c-di-GMP receptors.

Synthesis and Characterization of 8-Nitroguanosine 3',5'-Cyclic Monophosphorothioate Rp-Isomer as a Potent Inhibitor of Protein Kinase G1α.

Guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinases (PKG) are kinases regulating diverse physiological functions including vascular smooth muscle relaxation, neuronal synaptic plasticity, and platelet activities. Certain PKG inhibitors, such as Rp-diastereomers of derivatives of guanosine 3',5'-cyclic monophosphorothioate (Rp-cGMPS), have been designed and used to study PKG-regulated cell signaling. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is an endogenous cGMP derivative formed as a result of excess production of reactive oxygen species and nitric oxide. 8-Nitro-cGMP causes persistent activation of PKG1α through covalent attachment of cGMP moieties to cysteine residues of the enzyme (i.e., the process called protein S-guanylation). In this study, we synthesized a nitrated analogue of Rp-cGMPS, 8-nitroguanosine 3',5'-cyclic monophosphorothioate Rp-isomer (Rp-8-nitro-cGMPS), and investigated its effects on PKG1α activity. We synthesized Rp-8-nitro-cGMPS by reacting Rp-8-bromoguanosine 3',5'-cyclic monophosphorothioate (Rp-8-bromo-cGMPS) with sodium nitrite. Rp-8-Nitro-cGMPS reacted with the thiol compounds cysteine and glutathione to form Rp-8-thioalkoxy-cGMPS adducts to a similar extent as did 8-nitro-cGMP. As an important finding, a protein S-guanylation-like modification was clearly observed, by using Western blotting, in the reaction between recombinant PKG1α and Rp-8-nitro-cGMPS. Rp-8-Nitro-cGMPS inhibited PKG1α activity with an inhibitory constant of 22 µM in a competitive manner. An organ bath assay with mouse aorta demonstrated that Rp-8-nitro-cGMPS inhibited vascular relaxation induced by acetylcholine or 8-bromo-cGMP more than Rp-8-bromo-cGMPS did. These findings suggest that Rp-8-nitro-cGMPS inhibits PKG through induction of an S-guanylation-like modification by attaching the Rp-cGMPS moiety to the enzyme. Additional study is warranted to explore the potential application of Rp-8-nitro-cGMPS to biochemical and therapeutic research involving PKG1α activation.

Synthesis of Triazole-Linked Analogues of c-di-GMP and Their Interactions with Diguanylate Cyclase.

Cyclic di-GMP (c-di-GMP) is a widespread second messenger that plays a key role in bacterial biofilm formation. The compound's ability to assume multiple conformations allows it to interact with a diverse set of target macromolecules. Here, we analyzed the binding mode of c-di-GMP to the allosteric inhibitory site (I-site) of diguanylate cyclases (DGCs) and compared it to the conformation adopted in the catalytic site of the EAL phosphodiesterases (PDEs). An array of novel molecules has been designed and synthesized by simplifying the native c-di-GMP structure and replacing the charged phosphodiester backbone with an isosteric nonhydrolyzable 1,2,3-triazole moiety. We developed the first neutral small molecule able to selectively target DGCs discriminating between the I-site of DGCs and the active site of PDEs; this molecule represents a novel tool for mechanistic studies, particularly on those proteins bearing both DGC and PDE modules, and for future optimization studies to target DGCs in vivo.

Enzymatic synthesis of 2'-ara and 2'-deoxy analogues of c-di-GMP.

The substrate specificity of recombinant full-length diguanylate cyclase (DGC) of Thermotoga maritima with mutant allosteric site was investigated. It has been originally shown that the enzyme could use GTP closest analogues - 2'-deoxyguanosine-5'-triphosphate (dGTP) and 9-ß-D-arabinofuranosyl-guanine-5'-triphosphate (araGTP) as the substrates. The first demonstrations of an enzymatic synthesis of bis-(3'-5')-cyclic dimeric deoxyguanosine monophosphate (c-di-dGMP) and the previously unknown bis-(3'-5')-cyclic dimeric araguanosine monophosphate (c-di-araGMP) using DGC of T. maritima in the form of inclusion bodies have been provided.

Synthesis of c-di-GMP analogs with thiourea, urea, carbodiimide, and guanidinium linkages.

The first syntheses of neutral thiourea, urea, and carbodiimide analogs, along with two guanidinium analogs, of the bacterial signaling molecule cyclic diguanosine monophosphate (c-di-GMP) are reported. The key intermediate, obtained in nine steps, is a 3'-amino-5'-azido-3',5'-dideoxy derivative. The 5'-azide serves as a masked amine from which the amine is obtained by Staudinger reduction, while the 3'-amine is converted to an isothiocyanate that, while stable to chromatography, and Staudinger conditions, nevertheless reacts well with the 5'-amine.

Potent suppression of c-di-GMP synthesis via I-site allosteric inhibition of diguanylate cyclases with 2'-F-c-di-GMP.

Cyclic-di-GMP (c-di-GMP) is a central regulator of bacterial behavior. Various studies have implicated c-di-GMP in biofilm formation and virulence factor production in multitudes of bacteria. Hence it is expected that the disruption of c-di-GMP signaling could provide an effective means to disrupt biofilm and/or virulence factor formation in several bacteria of clinical relevance. C-di-GMP achieves the regulation of bacterial phenotype via binding to several effector molecules including transcription factors, enzymes and riboswitches. Crystal structure analyses of c-di-GMP effector molecules, in complex with the ligand, reveal that various classes of c-di-GMP receptors recognize this dinucleotide using different sets of recognition elements. Therefore, it is plausible that different analogues of c-di-GMP could be used to selectively modulate a specific class of c-di-GMP binding receptors, and hence modulate the bacterial phenotype. Thus far only a detailed study of the differential binding of c-di-GMP analogues to riboswitches, but not proteins, has been reported. In this report, we prepared various 2'-modified analogues of c-di-GMP and studied both polymorphisms of these analogues using DOSY NMR and the binding to several effector proteins, such as PilZ-containing proteins, diguanylate cyclases (DGC) containing I-sites, and phoshphodiesterases (PDE). 2'-Modification of c-di-GMP did not adversely affect the propensity to form higher aggregates, such as octameric forms, in the presence of potassium salts. Interestingly, we find that the selective binding to different classes of c-di-GMP binding proteins could be achieved with the 2'-modified analogues and that 2'-F analogue of c-di-GMP binds to the I-site of DGCs better (four times) than the native dinucleotide, c-di-GMP, whereas c-di-GMP binds to PDEs better (10 times) than 2'-F-c-di-GMP. 2'-F-c-di-GMP potently inhibits c-di-GMP synthesis by DGCs and hence raises the potential that cell permeable analogues of 2'-F-c-di-GMP could be used to disrupt c-di-GMP signaling in bacteria.

Structural and energetic factors controlling the enantioselectivity of dinucleotide formation under prebiotic conditions.

Recently, it has been reported that the montmorillonite-catalyzed oligomerization of activated nucleotides exhibits remarkable enantioselectivity. In the current paper we investigate the structures and intrinsic energies of homochiral and heterochiral cyclic dinucleotides by means of accurate quantum chemical calculations in gas-phase and in bulk water. The steric effect of the clay is represented with geometrical constraints. Our computations reveal that the heterochiral dimer geometries are systematically less stable than their homochiral counterparts due to steric clashes inside the sugar-phosphate ring geometry. Thus we suggest that the homochiral selectivity observed in the cyclic dinucleotide formation in confined spaces may arise from the energetic destabilization of the heterochiral ring geometries as compared to their homochiral analogues. In the present paper we provide the first model of the 3D structure of d,l cyclic dinucleotides, which until now has eluded experimental observation.

Synthesis of biotinylated c-di-gmp and c-di-amp using click conjugation.

The biotinylated c-di-GMP and c-di-AMP conjugates 10a/b were synthesized by a straightforward set of procedures from standard, commercially available phosphoramidites. Their availability should allow isolation and characterization of new protein and RNA receptors for these key bacterial signaling molecules.

Endo-S-c-di-GMP analogues-polymorphism and binding studies with class I riboswitch.

C-di-GMP, a cyclic guanine dinucleotide, has been shown to regulate biofilm formation as well as virulence gene expression in a variety of bacteria. Analogues of c-di-GMP have the potential to be used as chemical probes to study c-di-GMP signaling and could even become drug leads for the development of anti-biofilm compounds. Herein we report the synthesis and biophysical studies of a series of c-di-GMP analogues, which have both phosphate and sugar moieties simultaneously modified (called endo-S-c-di-GMP analogues). We used computational methods to predict the relative orientation of the guanine nucleobases in c-di-GMP and analogues. DOSY NMR of the endo-S-c-di-GMP series showed that the polymorphism of c-di-GMP can be tuned with conservative modifications to the phosphate and sugar moieties (conformational steering). Binding studies with Vc2 RNA (a class I c-di-GMP riboswitch) revealed that conservative modifications to the phosphate and 2'-positions of c-di-GMP dramatically affected binding to class I riboswitch.

The bacterial second messenger c-di-GMP: probing interactions with protein and RNA binding partners using cyclic dinucleotide analogs.

The ability of bacteria to adapt to a changing environment is essential for their survival. One mechanism used to facilitate behavioral adaptations is the second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). c-di-GMP is widespread throughout the bacterial domain and plays a vital role in regulating the transition between the motile planktonic lifestyle and the sessile biofilm forming state. This second messenger also controls the virulence response of pathogenic organisms and is thought to be connected to quorum sensing, the process by which bacteria communicate with each other. The intracellular concentration of c-di-GMP is tightly regulated by the opposing enzymatic activities of diguanlyate cyclases and phosphodiesterases, which synthesize and degrade the second messenger, respectively. The change in the intracellular concentration of c-di-GMP is directly sensed by downstream targets of the second messenger, both protein and RNA, which induce the appropriate phenotypic response. This review will summarize our current state of knowledge of c-di-GMP signaling in bacteria with a focus on protein and RNA binding partners of the second messenger. Efforts towards the synthesis of c-di-GMP and its analogs are discussed as well as studies aimed at targeting these macromolecular effectors with chemically synthesized cyclic dinucleotide analogs.

Nitric oxide promotes recycling of 8-nitro-cGMP, a cytoprotective mediator, into intact cGMP in cells.

8-Nitro-cGMP is an endogenous nucleotide discovered under inflammation conditions as an important mediator of nitric oxide (NO) signaling. Besides cGMP-like behaviour, 8-nitro-cGMP exerts unique cytoprotective effects against oxidative stress. Although the formation of 8-nitro-cGMP from 8-nitro-GTP has previously been proposed, the mechanism by which excess or unused 8-nitro-cGMP is removed from cells remains unknown. In this study, we report a nitric oxide-dependent cellular conversion of 8-nitro-cGMP to intact cGMP in RAW 264.7 macrophage cells. In our experiments, we synthesized isotopically labeled 8-nitro-cGMP as a tool for metabolites analysis and identified 8-amino-cGMP as an initial metabolite of 8-nitro-cGMP using a LC-MS/MS technique. We also proved that endogenous 8-nitro-cGMP can be converted into 8-amino-cGMP by immunocytochemical staining with an antibody that specifically recognizes 8-amino-cGMP. Moreover, we showed that isotopically labeled 8-amino-cGMP is metabolized into cGMP under inflammation conditions. We propose that nitrosylation of 8-amino-cGMP occurs by NO formation under stress conditions and gives putative 8-diazonium-cGMP, which subsequently decomposes into cGMP. To the best of our knowledge, this study is the first to report reductive deamination of aminoguanine nucleotide at the C-8 position. The findings of this study collectively indicate that NO plays a crucial role not only in the production of 8-nitro-cGMP but also in its elimination under oxidative stress or inflammation.

Synthesis and characterization of a fluorescent analogue of cyclic di-GMP.

Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger, has emerged as a key controller of several biological processes. Numbers of reports that deal with the mechanistic aspects of this second messenger have appeared in the literature. However, the lack of a reporter tag attached to the c-di-GMP at times limits the understanding of further details. In this study, we have chemically coupled N-methylisatoic anhydride (MANT) with c-di-GMP, giving rise to Mant-(c-di-GMP) or MANT-CDG. We have characterized the chemical and physical properties and spectral behavior of MANT-CDG. The fluorescence of MANT-CDG is sensitive to changes in the microenvironment, which helped us study its interaction with three different c-di-GMP binding proteins (a diguanylate cyclase, a phosphodiesterase, and a PilZ domain-containing protein). In addition, we have shown here that MANT-CDG can inhibit diguanylate cyclase activity; however, it is hydrolyzed by c-di-GMP specific phosphodiesterase. Taken together, our data suggest that MANT-CDG behaves like native c-di-GMP, and this study raises the possibility that MANT-CDG will be a valuable research tool for the in vitro characterization of c-di-GMP signaling factors.

One-flask synthesis of cyclic diguanosine monophosphate (c-di-GMP).

The bacterial signaling molecule, cyclic diguanosine monophosphate (c-di-GMP), plays a key role in controlling biofilm formation and pathogenic virulence, among many other functions. It has widespread consequences for human health, and current research is actively exploring its molecular mechanisms. The convenient one-flask, gram-scale synthesis of c-di-GMP described here will facilitate these efforts.

The overlapping host responses to bacterial cyclic dinucleotides.

Macrophages respond to infection with Legionella pneumophila by the induction of inflammatory mediators, including type I Interferons (IFN-Is). To explore whether the bacterial second messenger cyclic 3'-5' diguanylate (c-diGMP) activates some of these mediators, macrophages were infected with L. pneumophila strains in which the levels of bacterial c-diGMP had been altered. Intriguingly, there was a positive correlation between c-diGMP levels and IFN-I expression. Subsequent studies with synthetic derivatives of c-diGMP, and newly described cyclic 3'-5' diadenylate (c-diAMP), determined that these molecules activate overlapping inflammatory responses in human and murine macrophages. Moreover, UV crosslinking studies determined that both dinucleotides physically associate with a shared set of host proteins. Fractionation of macrophage extracts on a biotin-c-diGMP affinity matrix led to the identification of a set of candidate host binding proteins. These studies suggest that mammalian macrophages can sense and mount a specific inflammatory response to bacterial dinucleotides.

Synthesis and characterization of C8 analogs of c-di-GMP.

We have synthesized five analogs of c-di-GMP with different substituents at the guanine C8 position, to study their effects on the metal-dependent polymorphism we had previously demonstrated for the parent compound. Of these, only the K(+) salt of c-di-Br-GMP, 2, forms higher order complexes, predominantly two different syn octamolecular ones. Its Na(+) salt, as well as both the K(+) and Na(+) salts of c-di-thio-GMP, 3, c-di-methylthio-GMP, 4, c-di-phenyl-GMP, 5, and c-di-acetylphenyl-GMP, 6, all form primarily a syn bimolecular structure.

Differential analogue binding by two classes of c-di-GMP riboswitches.

The ability of bacteria to adapt to a changing environment is essential for their survival. One mechanism bacteria have evolved to sense environmental cues and translate these signals into phenotypic changes uses the second messenger signaling molecule, cyclic diguanosine monophosphate (c-di-GMP). In addition to several classes of protein receptors, two classes of c-di-GMP-binding riboswitches (class I and class II) have been identified as downstream targets of the second messenger in this signaling pathway. The crystal structures of both riboswitch classes bound to c-di-GMP were previously reported. Here, we further investigate the mechanisms that RNA has evolved for recognition and binding of this second messenger. Using a series of c-di-GMP analogues, we probed the interactions made in the RNA-ligand complex for both classes of riboswitches to identify the most critical elements of c-di-GMP for binding. We found that the structural features of c-di-GMP required for binding differ between these two effectors and that the class II riboswitch is much less discriminatory in ligand binding than the class I riboswitch. These data suggest an explanation for the predicted preferential use of the class I motif over the class II motif in the c-di-GMP signaling pathway.