Acidity constants and protonation sites of cyclic dinucleotides determined by capillary electrophoresis, quantum chemical calculations, and NMR spectroscopy.

Cyclic dinucleotides (CDNs) are important second messengers in bacteria and eukaryotes. Detailed characterization of their physicochemical properties is a prerequisite for understanding their biological functions. Herein, we examine acid-base and electromigration properties of selected CDNs employing capillary electrophoresis (CE), density functional theory (DFT), and nuclear magnetic resonance (NMR) spectroscopy to provide benchmark pKa values, as well as to unambiguously determine the protonation sites. Acidity constants (pKa ) of the NH+ moieties of adenine and guanine bases and actual and limiting ionic mobilities of CDNs were determined by nonlinear regression analysis of the pH dependence of their effective electrophoretic mobilities measured by CE in aqueous background electrolytes in a wide pH range (0.98-11.48), at constant temperature (25°C), and constant ionic strength (25 mM). The thermodynamic pKa values were found to be in the range 3.31-4.56 for adenine and 2.28-3.61 for guanine bases, whereas the pKa of enol group of guanine base was in the range 10.21-10.40. Except for systematic shifts of ∼2 pKa , the pKa values calculated by the DFT-D3//COSMO-RS composite protocol that included large-scale conformational sampling and "cross-morphing" were in a relatively good agreement with the pKa s determined by CE and predict N1 atom of adenine and N7 atom of guanine as the protonation sites. The protonation of the N1 atom of adenine and N7 atom of guanine in acidic background electrolytes (BGEs) and the dissociation of the enol group of guanine in alkaline BGEs was confirmed also by NMR spectroscopy.

Vinylphosphonate-based cyclic dinucleotides enhance STING-mediated cancer immunotherapy.

Cyclic dinucleotides (CDNs) trigger the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, which plays a key role in cytosolic DNA sensing and thus in immunomodulation against infections, cell damage and cancer. However, cancer immunotherapy trials with CDNs have shown immune activation, but not complete tumor regression. Nevertheless, we designed a novel class of CDNs containing vinylphosphonate based on a STING-affinity screening assay. In vitro, acyloxymethyl phosphate/phosphonate prodrugs of these vinylphosphonate CDNs were up to 1000-fold more potent than the clinical candidate ADU-S100. In vivo, the lead prodrug induced tumor-specific T cell priming and facilitated tumor regression in the 4T1 syngeneic mouse model of breast cancer. Moreover, we solved the crystal structure of this ligand bound to the STING protein. Therefore, our findings not only validate the therapeutic potential of vinylphosphonate CDNs but also open up opportunities for drug development in cancer immunotherapy bridging innate and adaptive immunity.

Silver Ion High-Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectrometry: A Tool for Analyzing Cuticular Hydrocarbons.

Aliphatic hydrocarbons (HCs) are usually analyzed by gas chromatography (GC) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. However, analyzing long-chain HCs by GC is difficult because of their low volatility and the risk of decomposition at high temperatures. MALDI cannot distinguish between isomeric HCs. An alternative approach based on silver ion high-performance liquid chromatography (Ag-HPLC) is shown here. The separation of HC standards and cuticular HCs was accomplished using two ChromSpher Lipids columns connected in series. A gradient elution of the analytes was optimized using mobile phases prepared from hexane (or isooctane) and acetonitrile, 2-propanol, or toluene. HCs were detected by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Good separation of the analytes according to the number of double bonds, cis/trans geometry, and position of double bonds was achieved. The retention times increased with the number of double bonds, and trans isomers eluted ahead of cis isomers. The mobile phase significantly affected the mass spectra of HCs. Depending on the mobile phase composition, deprotonated molecules, molecular ions, protonated molecules, and various solvent-related adducts of HCs were observed. The optimized Ag-HPLC/APCI-MS was applied for characterizing cuticular HCs from a flesh fly, Neobellieria bullata, and cockroach, Periplaneta americana. The method made it possible to detect a significantly higher number of HCs than previously reported for GC or MALDI-MS. Unsaturated HCs were frequently detected as isomers differing by double-bond position(s). Minor HCs with trans double bonds were found beside the prevailing cis isomers. Ag-HPLC/APCI-MS has great potential to become a new tool in chemical ecology for studying cuticular HCs.

Modulation of the antagonistic properties of an insulin mimetic peptide by disulfide bridge modifications.

Insulin is a peptide responsible for regulating the metabolic homeostasis of the organism; it elicits its effects through binding to the transmembrane insulin receptor (IR). Insulin mimetics with agonistic or antagonistic effects toward the receptor are an exciting field of research and could find applications in treating diabetes or malignant diseases. We prepared five variants of a previously reported 20-amino acid insulin-mimicking peptide. These peptides differ from each other by the structure of the covalent bridge connecting positions 11 and 18. In addition to the peptide with a disulfide bridge, a derivative with a dicarba bridge and three derivatives with a 1,2,3-triazole differing from each other by the presence of sulfur or oxygen in their staples were prepared. The strongest binding to IR was exhibited by the peptide with a disulfide bridge. All other derivatives only weakly bound to IR, and a relationship between increasing bridge length and lower binding affinity can be inferred. Despite their nanomolar affinities, none of the prepared peptide mimetics was able to activate the insulin receptor even at high concentrations, but all mimetics were able to inhibit insulin-induced receptor activation. However, the receptor remained approximately 30% active even at the highest concentration of the agents; thus, the agents behave as partial antagonists. An interesting observation is that these mimetic peptides do not antagonize insulin action in proportion to their binding affinities. The compounds characterized in this study show that it is possible to modulate the functional properties of insulin receptor peptide ligands using disulfide mimetics.

Electrochemistry of Tetrathiafulvalene Ligands Assembled on the Surface of Gold Nanoparticles.

The synthesis of a tetrathiafulvalene (TTF) derivative, S-[4-({4-[(2,2'-bi-1,3-dithiol-4-ylmethoxy)methyl] phenyl}ethynyl)phenyl] ethanethioate, suitable for the modification of gold nanoparticles (AuNPs), is described in this article. The TTF ligand was self-assembled on the AuNP surface through ligand exchange, starting from dodecanethiol-stabilized AuNPs. The resulting modified AuNPs were characterized by TEM, UV-Vis spectroscopy, and electrochemistry. The most suitable electrochemical method was the phase-sensitive AC voltammetry at very low frequencies of the sine-wave perturbation. The results indicate a diminishing electronic communication between the two equivalent redox centers of TTF and also intermolecular donor-acceptor interactions manifested by an additional oxidation wave upon attachment of the ligand to AuNPs.

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.

Push-pull amino-nitro helicenes: synthesis, (chir)optical properties and self-assembly into thin films.

A series of regioisomeric push-pull amino-nitro [6]helicenes and a related [7]helicene derivative were prepared and their racemates resolved into enantiomers. Compared to the parent helicenes, they exhibit red-shifted UV-Vis spectra, pronounced dipole moments, altered chiroptical properties such as remarkable optical rotatory power, and can form nanocrystalline Langmuir-Blodgett films.

Discovery of isonucleotidic CDNs as potent STING agonists with immunomodulatory potential.

Stimulator of interferon genes (STING) is an adaptor protein of the cGAS-STING signaling pathway involved in the sensing of cytosolic DNA. It functions as a receptor for cyclic dinucleotides (CDNs) and, upon their binding, mediates cytokine expression and host immunity. Besides naturally occurring CDNs, various synthetic CDNs, such as ADU-S100, have been reported to effectively activate STING and are being evaluated in clinical trials for the treatment of cancer. Here, we describe the preparation of a unique new class of STING agonists: isonucleotidic cyclic dinucleotides and the synthesis of their prodrugs. The presented CDNs stimulate STING with comparable efficiency to ADU-S100, whereas their prodrugs demonstrate activity up to four orders of magnitude better due to the improved cellular uptake. The compounds are very potent inducers of inflammatory cytokines by peripheral blood mononuclear cells (PBMCs). We also report the X-ray crystal structure of the lead inhibitor bound to the wild-type (WT) STING.

Functional stapled fragments of human preptin of minimised length.

Preptin is a 34-amino-acid-long peptide derived from the E-domain of a precursor of insulin-like growth factor 2 (pro-IGF2) with bone-anabolic and insulin secretion amplifying properties. Here, we describe the synthesis, structures, and biological activities of six shortened analogues of human preptin. Eight- and nine-amino-acid-long peptide amides corresponding to the C-terminal part of human preptin were stabilised by two types of staples to induce a higher proportion of helicity in their secondary structure. We monitored the secondary structure of the stapled peptides using circular dichroism. The biological effect of the structural changes was determined afterwards by the ability of peptides to stimulate the release of intracellular calcium ions. We confirmed the previous observation that the stabilisation of the disordered conformation of human preptin has a deleterious effect on biological potency. However, surprisingly, one of our preptin analogues, a nonapeptide stabilised by olefin metathesis between positions 3 and 7 of the amino acid chain, had a similar ability to stimulate calcium ions' release to the full-length human preptin. Our findings could open up new ways to design new preptin analogues, which may have potential as drugs for the treatment of diabetes and osteoporosis.

Stereo-Defined Acyclic Nucleoside Phosphonates are Selective and Potent Inhibitors of Parasite 6-Oxopurine Phosphoribosyltransferases.

Pathogens such as Plasmodium and Trypanosoma spp. are unable to synthesize purine nucleobases. They rely on the salvage of these purines and their nucleosides from the host cell to synthesize the purine nucleotides required for DNA/RNA production. The key enzymes in this pathway are purine phosphoribosyltransferases (PRTs). Here, we synthesized 16 novel acyclic nucleoside phosphonates, 12 with a chiral center at C-2', and eight bearing a second chiral center at C-6'. Of these, bisphosphonate (S,S)-48 is the most potent inhibitor of the Plasmodium falciparum and P. vivax 6-oxopurine PRTs and the most potent inhibitor of two Trypanosoma brucei (Tbr) 6-oxopurine PRTs yet discovered, with Ki values as low as 2 nM. Crystal structures of (S,S)-48 in complex with human and Tbr 6-oxopurine PRTs show that the inhibitor binds to the enzymes in different conformations, providing an explanation for its potency and selectivity (i.e., 35-fold in favor of the parasite enzymes).

5'-Phosphonate modified oligoadenylates as potent activators of human RNase L.

The oligoadenylate synthetase-ribonuclease L pathway is a major player in the interferon-induced antiviral defense mechanism of cells. Upon sensing viral dsRNA, 5'-phosphorylated 2',5'-oligoadenylates are synthesized, and subsequently activate latent RNase L. To determine the influence of 5'-phosphate end on the activation of human RNase L, four sets of 5'-phosphonate modified oligoadenylates were prepared on solid-phase. The ability of these 5'-modified oligoadenylates bearing shortened, isosteric and prolonged phosphonate linkages to activate RNase L was explored. We found that isosteric linkages and linkages prolonged by one atom were in general well tolerated by the enzyme with the EC50 values comparable to that of the natural activator. In contrast, linkages shortened by one atom or prolonged by two atoms exhibited decrease in the activity.

The Ad-MD method to calculate NMR shift including effects due to conformational dynamics: The 31 P NMR shift in DNA.

A method for averaging of NMR parameters by molecular dynamics (MD) has been derived from the method of statistical averaging in MD snapshots, benchmarked and applied to structurally dynamic interpretation of the 31 P NMR shift (δ31P ) in DNA phosphates. The method employs adiabatic dependence of an NMR parameter on selected geometric parameter(s) that is weighted by MD-calculated probability distribution(s) for the geometric parameter(s) (Ad-MD method). The usage of Ad-MD for polymers is computationally convenient when one pre-calculated structural dependence of an NMR parameter is employed for all chemically equivalent units differing only in dynamic behavior. The Ad-MD method is benchmarked against the statistical averaging method for δ31P in the model phosphates featuring distinctively different structures and dynamic behavior. The applicability of Ad-MD is illustrated by calculating 31 P NMR spectra in the Dickerson-Drew DNA dodecamer. δ31P was calculated with the B3LYP/IGLO-III/PCM(water) and the probability distributions for the torsion angles adjacent to the phosphorus atoms in the DNA phosphates were calculated using the OL15 force field.

Spirostanol Saponins from Flowers of Allium Porrum and Related Compounds Indicating Cytotoxic Activity and Affecting Nitric Oxide Production Inhibitory Effect in Peritoneal Macrophages.

Saponins, a diverse group of natural compounds, offer an interesting pool of derivatives with biomedical application. In this study, three structurally related spirostanol saponins were isolated and identified from the leek flowers of Allium porrum L. (garden leek). Two of them were identical with the already known leek plant constituents: aginoside (1) and 6-deoxyaginoside (2). The third one was identified as new component of A. porrum; however, it was found identical with yayoisaponin A (3) obtained earlier from a mutant of elephant garlic Allium ampeloprasun L. It is a derivative of the aginoside (1) with additional glucose in its glycosidic chain, identified by MS and NMR analysis as (2α, 3ß, 6ß, 25R)-2,6-dihydroxyspirostan-3-yl ß-D-glucopyranosyl-(1 → 3)-ß-D-glucopranosyl-(1 → 2)-[ß-D-xylopyranosyl-(1 → 3)]-ß-D-glucopyranosyl]-(1 → 4)-ß-D-galactopyranoside, previously reported also under the name alliporin. The leek native saponins were tested together with other known and structurally related saponins (tomatonin and digitonin) and with their related aglycones (agigenin and diosgenin) for in vitro cytotoxicity and for effects on NO production in mouse peritoneal cells. The highest inhibitory effects were exhibited by 6-deoxyaginoside. The obtained toxicity data, however, closely correlated with the suppression of NO production. Therefore, an unambiguous linking of obtained bioactivities of saponins with their expected immunobiological properties remained uncertain.

Enzymatic Synthesis of 3'-5', 3'-5' Cyclic Dinucleotides, Their Binding Properties to the Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations.

The 3'-5', 3'-5' cyclic dinucleotides (3'3'CDNs) are bacterial second messengers that can also bind to the stimulator of interferon genes (STING) adaptor protein in vertebrates and activate the host innate immunity. Here, we profiled the substrate specificity of four bacterial dinucleotide synthases from Vibrio cholerae (DncV), Bacillus thuringiensis (btDisA), Escherichia coli (dgcZ), and Thermotoga maritima (tDGC) using a library of 33 nucleoside-5'-triphosphate analogues and then employed these enzymes to synthesize 24 3'3'CDNs. The STING affinity of CDNs was evaluated in cell-based and biochemical assays, and their ability to induce cytokines was determined by employing human peripheral blood mononuclear cells. Interestingly, the prepared heterodimeric 3'3'CDNs bound to the STING much better than their homodimeric counterparts and showed similar or better potency than bacterial 3'3'CDNs. We also rationalized the experimental findings by in-depth STING-CDN structure-activity correlations by dissecting computed interaction free energies into a set of well-defined and intuitive terms. To this aim, we employed state-of-the-art methods of computational chemistry, such as quantum mechanics/molecular mechanics (QM/MM) calculations, and complemented the computed results with the {STING:3'3'c-di-ara-AMP} X-ray crystallographic structure. QM/MM identified three outliers (mostly homodimers) for which we have no clear explanation of their impaired binding with respect to their heterodimeric counterparts, whereas the R2 = 0.7 correlation between the computed ΔG'int_rel and experimental ΔTm's for the remaining ligands has been very encouraging.

Cytotoxicity of Amino-BODIPY Modulated via Conjugation with 2-Phenyl-3-Hydroxy-4(1H)-Quinolinones.

The combination of cytotoxic amino-BODIPY dye and 2-phenyl-3-hydroxy-4(1H)-quinolinone (3-HQ) derivatives into one molecule gave rise to selective activity against lymphoblastic or myeloid leukemia and the simultaneous disappearance of the cytotoxicity against normal cells. Both species' conjugation can be realized via a disulfide linker cleavable in the presence of glutathione characteristic for cancer cells. The cleavage liberating the free amino-BODIPY dye and 3-HQ derivative can be monitored by ratiometric fluorescence or by the OFF-ON effect of the amino-BODIPY dye. A similar cytotoxic activity is observed when the amino-BODIPY dye and 3-HQ derivative are connected through a non-cleavable maleimide linker. The work reports the synthesis of several conjugates, the study of their cleavage inside cells, and cytotoxic screening.

Synthesis of phosphonate derivatives of 2'-deoxy-2'-fluorotetradialdose d-nucleosides and tetradialdose d-nucleosides.

Analogs of nucleosides and nucleotides represent a promising pool of potential therapeutics. This work describes a new synthetic route leading to 2'-deoxy-2'-fluorotetradialdose D-nucleoside phosphonates. Moreover, a new universal synthetic route leading to tetradialdose d-nucleosides bearing purine nucleobases is also described. All new compounds were tested as triphosphate analogs for inhibitory potency against a variety of viral polymerases. The fluorinated nucleosides were transformed to phosphoramidate prodrugs and evaluated in cell cultures against various viruses including influenza and SARS-CoV-2.

Conformational energies and equilibria of cyclic dinucleotides in vacuo and in solution: computational chemistry vs. NMR experiments.

Performance of computational methods in modelling cyclic dinucleotides - an important and challenging class of compounds - has been evaluated by two different benchmarks: (1) gas-phase conformational energies and (2) qualitative agreement with NMR observations of the orientation of the χ-dihedral angle in solvent. In gas-phase benchmarks, where CCSD(T) and DLPNO-CCSD(T) methods have been used as the reference, most of the (dispersion corrected) density functional approximations are accurate enough to justify prioritizing computational cost and compatibility with other modelling options as the criterion of choice. NMR experiments of 3'3'-c-di-AMP, 3'3'-c-GAMP, and 3'3'-c-di-GMP show the overall prevalence of the anti-conformation of purine bases, but some population of syn-conformations is observed for guanines. Implicit solvation models combined with quantum-chemical methods struggle to reproduce this behaviour, probably due to a lack of dynamics and explicitly modelled solvent, leading to structures that are too compact. Molecular dynamics simulations overrepresent the syn-conformation of guanine due to the overestimation of an intramolecular hydrogen bond. Our combination of experimental and computational benchmarks provides "error bars" for modelling cyclic dinucleotides in solvent, where such information is generally difficult to obtain, and should help gauge the interpretability of studies dealing with binding of cyclic dinucleotides to important pharmaceutical targets. At the same time, the presented analysis calls for improvement in both implicit solvation models and force-field parameters.

Multipodal insulin mimetics built on adamantane or proline scaffolds.

Multi-orthogonal molecular scaffolds can be applied as core structures of bioactive compounds. Here, we prepared four tri-orthogonal scaffolds based on adamantane or proline skeletons. The scaffolds were used for the solid-phase synthesis of model insulin mimetics bearing two different peptides on the scaffolds. We found that adamantane-derived compounds bind to the insulin receptor more effectively (Kd value of 0.5 µM) than proline-derived compounds (Kd values of 15-38 µM) bearing the same peptides. Molecular dynamics simulations suggest that spacers between peptides and central scaffolds can provide greater flexibility that can contribute to increased binding affinity. Molecular modeling showed possible binding modes of mimetics to the insulin receptor. Our data show that the structure of the central scaffold and flexibility of attached peptides in this type of compound are important and that different scaffolds should be considered when designing peptide hormone mimetics.

Acid-Stable Ester Linkers for the Solid-Phase Synthesis of Immobilized Peptides.

A series of N-terminally Fmoc-protected linkers of the general formula Fmoc-X-CO-O-Y-COOH have been prepared, where X is -NH-CH2 -CH2 - or -p-(aminomethyl)phenyl- and Y is -(CH2 )n - (n is 1 or 4) or -p-(methyl)phenyl-. These linkers can easily be covalently attached via their C-terminal carboxyl group to a resin bearing a free amino group. After cleavage of the N-terminal Fmoc group, the linkers can be extended by standard solid-phase peptide synthesis techniques. These ester linkers are acid-stable and resistant to the base-mediated diketopiperazine formation that often occurs during the synthesis of ester-bound peptides; they are stable at neutral pH in aqueous buffers for days but can be effectively cleaved with 0.1 m NaOH or aq. ammonia within minutes or hours, respectively. These properties make these ester handles well suited for use as linkers for the solid-phase peptide synthesis of immobilized peptides when the stable on-resin immobilization of the peptides and the testing of their biological properties in aqueous buffers at neutral pH are necessary.