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.

Oesophageal safety in voltage-guided atrial fibrillation ablation using ablation index or contact force only: a prospective comparison.

AIMS: Left atrial ablation using radiofrequency (RF) is associated with endoscopically detected thermal oesophageal lesions (EDELs). The aim of this study was to compare EDEL occurrence after conventional contact force-guided (CFG) RF ablation vs. an ablation index-guided (AIG) approach in clinical routine of voltage-guided ablation (VGA). Predictors of EDEL were also assessed. METHODS AND RESULTS: This study compared CFG (n = 100) with AIG (n = 100) in consecutive atrial fibrillation ablation procedures, in which both pulmonary vein isolation and VGA were performed. In the AIG group, AI targets were ≥500 anteriorly and ≥350-400 posteriorly. Upper endoscopy was performed after ablation.The CFG and AIG groups had comparable baseline characteristics. The EDEL occurred in 6 and 5% (P = 0.86) in the CFG and AIG groups, respectively. Category 2 lesions occurred in 4 and 2% (P = 0.68), respectively. All EDEL healed under proton pump inhibitor therapy. The AI > 520 was the only predictor of EDEL [odds ratio (OR) 3.84; P = 0.039]. The more extensive Category 2 lesions were predicted by: AI max > 520 during posterior ablation (OR 7.05; P = 0.042), application of posterior or roof lines (OR 5.19; P = 0.039), existence of cardiomyopathy (OR 4.93; P = 0.047), and CHA2DS2-VASc score (OR 1.71; P = 0.044). The only Category 2 lesion with AI max < 520 (467) occurred in a patient with low body mass index. CONCLUSIONS: Both methods were comparable with respect to clinical complications and EDEL. In consideration of previous reconnection data and our study results regarding oesophageal safety, optimal AI target range might be between 400 and 450.

Synthesis and Antitrypanosomal Activity of 6-Substituted 7-Methyl-7-deazapurine Nucleosides.

Human African Trypanosomiasis caused by Trypanosoma brucei species is one of the most damaging neglected tropical diseases. While the number of newly diagnosed cases per year is record low, there is still high interest in the development of new antitrypanosomal agents in case of resistance to currently used drugs and their combinations, and to replace drugs with serious side effects. We report a series of 7-methyl-7-deazapurine (5-methyl-pyrrolo[2,3-d]pyrimidine) ribonucleosides bearing alkyl, methylsulfanyl, methylamino, or diverse alkoxy groups at position 6 that was prepared through glycosylation of 6-chloro-7-methyl-7-deazapurine followed by nucleophilic substitutions or cross-coupling reactions at position 6 and deprotection. Most of the title nucleosides displayed significant activity against Trypanosoma brucei brucei and T. b. gambiense at submicromolar or nanomolar concentrations and low cytotoxicity and thus represent promising candidates for further development.

Antiviral Activity of 7-Substituted 7-Deazapurine Ribonucleosides, Monophosphate Prodrugs, and Triphoshates against Emerging RNA Viruses.

A series of 7-deazaadenine ribonucleosides bearing alkyl, alkenyl, alkynyl, aryl, or hetaryl groups at position 7 as well as their 5'-O-triphosphates and two types of monophosphate prodrugs (phosphoramidates and S-acylthioethanol esters) were prepared and tested for antiviral activity against selected RNA viruses (Dengue, Zika, tick-borne encephalitis, West Nile, and SARS-CoV-2). The modified triphosphates inhibited the viral RNA-dependent RNA polymerases at micromolar concentrations through the incorporation of the modified nucleotide and stopping a further extension of the RNA chain. 7-Deazaadenosine nucleosides bearing ethynyl or small hetaryl groups at position 7 showed (sub)micromolar antiviral activities but significant cytotoxicity, whereas the nucleosides bearing bulkier heterocycles were still active but less toxic. Unexpectedly, the monophosphate prodrugs were similarly or less active than the corresponding nucleosides in the in vitro antiviral assays, although the bis(S-acylthioethanol) prodrug 14h was transported to the Huh7 cells and efficiently released the nucleoside monophosphate.

Synthesis and Cytotoxic and Antiviral Activity Profiling of All-Four Isomeric Series of Pyrido-Fused 7-Deazapurine Ribonucleosides.

All four isomeric series of novel 4-substituted pyrido-fused 7-deazapurine ribonucleosides possessing the pyridine nitrogen atom at different positions were designed and synthesized. The total synthesis of each isomeric fused heterocycle through multistep heterocyclization was followed by glycosylation and derivatization at position 4 by cross-coupling reactions or nucleophilic substitutions. All compounds were tested for cytostatic and antiviral activity. The most active were pyrido[4',3':4,5]pyrimidine nucleosides bearing MeO, NH2 , MeS, or CH3 groups at position 4, which showed submicromolar cytotoxic effects and good selectivity for cancer cells. The mechanism involved activation by phosphorylation and incorporation to DNA where the presence of the modified ribonucleosides causes double-strand breaks and apoptosis.

Enzymatic synthesis of base-modified RNA by T7 RNA polymerase. A systematic study and comparison of 5-substituted pyrimidine and 7-substituted 7-deazapurine nucleoside triphosphates as substrates.

We synthesized a small library of eighteen 5-substituted pyrimidine or 7-substituted 7-deazapurine nucleoside triphosphates bearing methyl, ethynyl, phenyl, benzofuryl or dibenzofuryl groups through cross-coupling reactions of nucleosides followed by triphosphorylation or through direct cross-coupling reactions of halogenated nucleoside triphosphates. We systematically studied the influence of the modification on the efficiency of T7 RNA polymerase catalyzed synthesis of modified RNA and found that modified ATP, UTP and CTP analogues bearing smaller modifications were good substrates and building blocks for the RNA synthesis even in difficult sequences incorporating multiple modified nucleotides. Bulky dibenzofuryl derivatives of ATP and GTP were not substrates for the RNA polymerase. In the case of modified GTP analogues, a modified procedure using a special promoter and GMP as initiator needed to be used to obtain efficient RNA synthesis. The T7 RNA polymerase synthesis of modified RNA can be very efficiently used for synthesis of modified RNA but the method has constraints in the sequence of the first three nucleotides of the transcript, which must contain a non-modified G in the +1 position.

Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases.

DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2'-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. colienzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription.

Ultrasonography analysis of gallbladder motility in patients with functional dyspepsia.

BACKGROUND: Gallbladder motility has been studied in patients with functional gastrointestinal disorders, such as functional dyspepsia, irritable bowel syndrome and biliary disorders without gallstones and results of these observations are often inconclusive and conflicting. METHODS: The investigation was performed on 180 therapy-naïve newly diagnosed patients with functional dyspepsia (97 females and 83 males), aged 20-79 in which we have investigate ultrasonographically parameters of gallbladder motility. RESULTS: Bonferroni post hoc correction stressed that fasting gallbladder volume and ejection fraction were significantly distorted in individuals with postprandial distress syndrome, although, the residual gallbladder volume was significantly lower in patients with epigastric pain syndrome comparing with other examinees. Ejection fraction of the gallbladder negatively correlated with body mass index. CONCLUSION: The impaired contractibility of the gall bladder in patients with functional dyspepsia, based on the results of this study, is illustrated by the changes in the ejection fraction, which was more pronounced in patients with the postprandial distress syndrome.

Celiac disease diagnosed after uncomplicated pregnancy in a patient with history of bulimia nervosa.

INTRODUCTION: The association between celiac disease and eating disorders has been very rarely reported. This is the first report on celiac disease associated with bulimia in this part of Europe. CASE REPORT: An adult female patient with history of bulimia and one uncomplicated pregnancy was admitted to the Gastroenterology Department, due to long lasting dyspeptic symptoms, constipation, major weight loss and fatigue. After positive serological screening, the diagnosis of celiac disease was confirmed with histopathology examination of duodenal biopsy specimen. CONCLUSION: Complicated interactions between celiac disease and bulimia can make them difficult to diagnose and treat. It is important to consider the presence of celiac disease in patients with bulimia and gastrointestinal symptoms.

Dilated cardiomyopathy associated with celiac disease: case report and literature review.

INTRODUCTION: Celiac disease is an inflammatory condition of the small intestinal mucosa induced by gluten consumption in genetically susceptible individuals, leading to a spectrum of gastrointestinal presentation. A number of autoimmune and other disorders are highly associated with celiac disease. Cardiomyopathy associated with celiac disease has been rarely reported in the literature. CASE OUTLINE: We present a case of a 27-year-old male with one month history of diarrhea, weight loss, fatigue, dyspeptic symptoms, peripheral edema, and cardiac palpitations. After positive serological screening with immunoglobulin A anti-tissue transglutaminase antibody test, the diagnosis of celiac disease was confirmed with histopathology examination of duodenal biopsy specimen. Echocardiographic findings were consistent with acute myocarditis. After common causes of myocarditis had been excluded, probable celiac disease-associated autoimmune myocarditis was diagnosed. The patient was recommended to undergo a strict life-long gluten-free diet. IgA anti-transglutaminase antibodies, and anti-gliadin antibodies, were both significantly elevated during the 6-, 12- and 18-month follow-up. Low compliance to gluten-free diet in our patient led to progressive worsening of the left ventricular ejective fraction and other serious cardiac complications which warranted invasive cardiac interventions. CONCLUSION: Dilated cardiomyopathy associated with celiac disease is a serious condition which requires multidisciplinary approach involving gastroenterologist and cardiologist. Compliance with gluten-free diet is mandatory if patients are to avoid progression of cardiomyopathy. Screening of patients with idiopathic dilated cardiomyopathy for celiac disease is advisable.