Stable Oxidative Cytosine Modifications Accumulate in Cardiac Mesenchymal Cells From Type2 Diabetes Patients: Rescue by α-Ketoglutarate and TET-TDG Functional Reactivation.

RATIONALE: Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes. OBJECTIVE: To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs. METHODS AND RESULTS: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors. Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high-fat diet (HFD), injected with streptozotocin, or both in combination (streptozotocin/HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in diabetic CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised TDG (thymine DNA glycosylase) and TET1 (ten-eleven translocation protein 1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5-formylcytosine. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization, and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5-formylcytosine accumulation, thus partially mimicking the diabetic epigenetic landscape in cells of nondiabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG dehydrogenase, increased the αKG level in diabetic CMSCs and in the heart of HFD and streptozotocin mice eliciting, in HFD, DNA demethylation, glucose uptake, and insulin response. CONCLUSIONS: Restoring the epimetabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.

Design and synthesis of N-benzoyl amino acid derivatives as DNA methylation inhibitors.

The inhibition of human DNA Methyl Transferases (DNMT) is a novel promising approach to address the epigenetic dysregulation of gene expression in different diseases. Inspired by the validated virtual screening hit NSC137546, a series of N-benzoyl amino acid analogues was synthesized and obtained compounds were assessed for their ability to inhibit DNMT-dependent DNA methylation in vitro. The biological screening allowed the definition of a set of preliminary structure-activity relationships and the identification of compounds promising for further development. Among the synthesized compounds, L-glutamic acid derivatives 22, 23, and 24 showed the highest ability to prevent DNA methylation in a total cell lysate. Compound 22 inhibited DNMT1 and DNMT3A activity in a concentration-dependent manner in the micromolar range. In addition, compound 22 proved to be stable in human serum and it was thus selected as a starting point for further biological studies.

Design, Synthesis, and Evaluation of Acrylamide Derivatives as Direct NLRP3 Inflammasome Inhibitors.

NLRP3 inflammasome plays a key role in the intracellular activation of caspase-1, processing of pro-inflammatory interleukin-1ß (IL-1ß), and pyroptotic cell death cascade. The overactivation of NLRP3 is implicated in the pathogenesis of autoinflammatory diseases, known as cryopyrin-associated periodic syndromes (CAPS), and in the progression of several diseases, such as atherosclerosis, type-2 diabetes, gout, and Alzheimer's disease. In this study, the synthesis of acrylamide derivatives and their pharmaco-toxicological evaluation as potential inhibitors of NLRP3-dependent events was undertaken. Five hits were identified and evaluated for their efficiency in inhibiting IL-1ß release from different macrophage subtypes, including CAPS mutant macrophages. The most attractive hits were tested for their ability to inhibit NLRP3 ATPase activity on human recombinant NLRP3. This screening allowed the identification of 14, 2-(2-chlorobenzyl)-N-(4-sulfamoylphenethyl)acrylamide, which was able to concentration-dependently inhibit NLRP3 ATPase with an IC50 value of 74 µm. The putative binding pose of 14 in the ATPase domain of NLRP3 was also proposed.

In vitro pharmacological evaluation of multitarget agents for thromboxane prostanoid receptor antagonism and COX-2 inhibition.

PURPOSE: Patients with high cardiovascular risk due to ageing and/or comorbidity (diabetes, atherosclerosis) that require effective management of chronic pain may take advantage from new non-steroidal anti-inflammatory drugs (NSAIDs) that at clinical dosages may integrate the anti-inflammatory activity and reduced gastrointestinal side effects of selective cyclooxygenase-2 (COX-2) inhibitor (coxib) with a cardioprotective component involving antagonism of thromboxane A2 prostanoid (TP) receptor. METHODS: New compounds were obtained modulating the structure of the most potent coxib, lumiracoxib, to obtain novel multitarget NSAIDs endowed with balanced coxib and TP receptor antagonist properties. Antagonist activity at TP receptor (pA2) was evaluated for all compounds in human platelets and in an heterologous expression system by measuring prevention of aggregation and Gq-dependent production of intracellular inositol phosphate induced by the stable thromboxane A2 (TXA2) agonist U46619. COX-1 and COX-2 inhibitory activities were assessed in human washed platelets and lympho-monocytes suspension, respectively. COX selectivity was determined from dose-response curves by calculating a ratio (COX-2/COX-1) of IC50 values. RESULTS: The tetrazole derivative 18 and the trifluoromethan sulfonamido-isoster 20 were the more active antagonists at TP receptor, preventing human platelet aggregation and intracellular signalling, with pA2 values statistically higher from that of lumiracoxib. Comparative data regarding COX-2/COX-1 selectivity showed that while compounds 18 and 7 were rather potent and selective COX-2 inhibitor, compound 20 was somehow less potent and selective for COX-2. CONCLUSION: These results indicate that compounds 18 and 20 are two novel combined TP receptor antagonists and COX-2 inhibitors characterized by a fairly balanced COX-2 inhibitor activity and TP receptor antagonism and that they may represent a first optimization of the original structure to improve their multitarget activity.

Simultaneous ultrasound-assisted water extraction and ß-cyclodextrin encapsulation of polyphenols from Mangifera indica stem bark in counteracting TNFα-induced endothelial dysfunction.

This study proposes an alternative technique to prevent heat degradation induced by classic procedures of bioactive compound extraction, comparing classical maceration/decoction in hot water of polyphenols from Mango (Mangifera indica L.) (MI) with ultrasound-assisted extraction (UAE) in a water solution of ß-cyclodextrin (ß-CD) at room temperature and testing their biological activity on TNFα-induced endothelial dysfunction. Both extracts counteracted TNFα effects on EAhy926 cells, down-modulating interleukin-6, interleukin-8, cyclooxygenase-2 and intracellular adhesion molecule-1, while increasing endothelial nitric oxide synthase levels. ß-CD extract showed higher efficacy in improving endothelial function. These effects were abolished after pre-treatment with the oestrogen receptor inhibitor ICI1182,780. Moreover, the ß-CD extract induced Akt activation and completely abolished the TNFα-induced p38MAPK phosphorylation. UAE and ß-CD encapsulation provide an efficient extraction protocol that increases polyphenol bioavailability. Polyphenols from MI play a protective role on endothelial cells and may be further considered as oestrogen-like molecules with vascular protective properties.

Electrophilic warhead-based design of compounds preventing NLRP3 inflammasome-dependent pyroptosis.

Pyroptosis is a caspase-1-dependent pro-inflammatory form of programmed cell death implicated in the pathogenesis of autoinflammatory diseases as well as in disorders characterized by excessive cell death and inflammation. Activation of NLRP3 inflammasome is a key event in the pyroptotic cascade. In this study, we describe the synthesis and chemical tuning of α,ß-unsaturated electrophilic warheads toward the development of antipyroptotic compounds. Their pharmacological evaluation and structure-activity relationships are also described. Compound 9 was selected as a model of this series, and it proved to be a reactive Michael acceptor, irreversibly trapping thiol nucleophiles, which prevented both ATP- and nigericin-triggered pyroptosis of human THP-1 cells in a time- and concentration-dependent manner. Moreover, 9 and other structurally related compounds, inhibited caspase-1 and NLRP3 ATPase activities. Our findings can contribute to the development of covalent, multitarget antipyroptotic compounds targeting molecular components of the NLRP3 inflammasome regulatory pathway.

Enabling technologies for the rapid dechlorination of polychloroarenes and PCBs.

Dielectric heating and acoustic cavitation (ultrasound or high-performance disperser) may all dramatically enhance conversion rates and yields in heterogeneous metal-assisted organic reactions even when low reagent excesses are used. These so called "enabling technologies" bring with them process intensification, safer protocols, cost reduction and energy savings. We herein describe a series of rapid polychlorinated aromatic and PCBs dechlorinations (15min) carried out in a moderate excess of metallic sodium and using non-conventional techniques. We compared the results with those obtained for reactions carried out under conventional heating and with those performed with less reactive metals such as magnesium and zinc. In this comparison, high-intensity ultrasound stands out as the technique of choice.

Solvent-free chemoselective oxidation of thioethers and thiophenes by mechanical milling.

Organosulphur compounds can be easily and selectively oxidized to sulfones using a small excess of Oxone(®) (1.6 eq.) under solventless mechanical milling conditions. This green procedure has been efficiently applied to a series of model compounds and to the desulphurization of medium/high sulphur content paraffins (up to 3000 mg kg(-1)).

Ultrasound-enhanced one-pot synthesis of 3-(Het)arylmethyl-4-hydroxycoumarins in water.

3-(Aryl)methyl-4-hydroxycoumarins were produced in good to excellent yields by reaction between 4-hydroxycoumarin and (hetero)aromatic aldehydes in the presence of Hantzsch 1,4-dihydropyridine (HEH) which works as an hydride donor (i.e., in a sequential Knoevenagel-reductive Michael addition). The sonochemical-assisted procedure (method B) provides an improved and accelerated conversion when compared to conventional silent reactions (method A). Experiments carried out according to method B showed that the reaction could be more efficiently run in the absence of organic solvents, at 30-40°C in open vessel, without the need of an excess HEH and with simplified work-up and separation procedures.