Pharmacological inhibition of NLRP3 inflammasome activation may offer a new option in the treatment of inflammatory bowel disease. In this work, we report the design, synthesis, and biological screening of a series of acrylate derivatives as NLRP3 inhibitors. The in vitro determination of reactivity, cytotoxicity, NLRP3 ATPase inhibition, and antipyroptotic properties allowed the selection of 11 (INF39), a nontoxic, irreversible NLRP3 inhibitor able to decrease interleukin-1β release from macrophages. Bioluminescence resonance energy transfer experiments proved that this compound was able to directly interfere with NLRP3 activation in cells. In vivo studies confirmed the ability of the selected lead to alleviate the effects of colitis induced by 2,4-dinitrobenzenesulfonic acid in rats after oral administration.

1 The e ect of L-glutamate (Glu) on human lymphocyte function was studied by measuring anti-CD 3 monoclonal antibody (mAb) or phytohaemagglutinin (PHA)-induced intracellular Ca 2+ ([Ca 2+ ] i ) rise (Fura-2 method), and cell proliferation (MTT assay). 2 Glu (0.001 ± 100 mM) did not modify basal lymphocyte [Ca 2+ ] i , but signi®cantly potentiated the e ects of anti-CD 3 mAb or PHA. Maximal [Ca 2+ ] i rises over resting cells were: 165+8 and 247+10 nM at 3.0610 72 mg ml 71 anti-CD 3 mAb; 201+4 and 266+9 nM at 5.0610 72 mg ml 71 PHA, in the absence or presence of 1 mM Glu, respectively. 3 The Glu e ect showed a bell-shape concentration-dependent relationship, with a maximum (+90+3% for anti-CD 3 mAb and +57+2% for PHA over Glu-untreated cells) at 1 mM. 4 Non-NMDA receptor agonists (1 mM) showed a greater e cacy (+76+2% for (S)-AMPA; +78+4% for KA), if compared to NMDA (+46+2%), or Glu itself. 5 Ionotropic Glu receptor antagonists completely inhibited the e ects of the corresponding speci®c receptor agonists (1 mM). The IC 50 values calculated were: 0.9 mM for D-AP5; 0.6 mM for (+)-MK801; 0.3 mM for NBQX. Both NBQX and KYNA were able to abolish Glu e ect. The IC 50s calculated were: 3.4 mM for NBQX; 0.4 mM for KYNA. 6 Glu (0.1 ± 1 mM) did not change the resting cell proliferation, whereas Glu (1 mM) signi®cant inhibited (727+4%) PHA (1.0610 72 mg ml 71 )-induced lymphocyte proliferation at 72 h.

No abstract

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,β-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.

Background and purpose: Phenolic compounds exert cytoprotective effects; our purpose was to investigate whether the isosteric polyphenolic compounds clovamide and rosmarinic acid are neuroprotective. Experimental approach: Three in vitro models of neuronal death were selected: (i) differentiated SH-SY5Y human neuroblastoma cells exposed to tert-butylhydroperoxide (t-BOOH), for oxidative stress; (ii) differentiated SK-N-BE(2) human neuroblastoma cells treated with L-glutamate, for excitotoxicity; and (iii) differentiated SH-SY5Y human neuroblastoma cells exposed to oxygen-glucose deprivation/reoxygenation, for ischaemia-reperfusion. Cell death was evaluated by lactate dehydrogenase measurements in the cell media, while the mechanisms underlying the effects by measuring: (i) t-BOOH-induced glutathione depletion and increase in lipoperoxidation; and (ii) L-glutamate-induced intracellular Ca 2+ overload (fura-2 method) and inducible gene expression (c-fos, c-jun), by reverse transcriptase-PCR. The ability of compounds to modulate nuclear factor-kB and peroxisome proliferator-activated receptor-g activation was evaluated by Western blot in SH-SY5Y cells not exposed to harmful stimuli. Key results: Both clovamide and rosmarinic acid (10-100 mmol·L -1 ) significantly protected neurons against insults with similar potencies and efficacies. The EC50 values were in the low micromolar range (0.9-3.7 mmol·L -1 ), while the maximal effects ranged from 40% to -60% protection from cell death over untreated control at 100 mmol·L -1. These effects are mediated by the prevention of oxidative stress, intracellular Ca 2+ overload and c-fos expression. In addition, rosmarinic acids inhibited nuclear factor-kB translocation and increased peroxisome proliferator-activated receptor-g expression in SH-SY5Y cells not exposed to harmful stimuli. Conclusion and implications: Clovamide and rosmarinic acid are neuroprotective compounds of potential use at the nutritional/pharmaceutical interface.

No abstract

Background and purpose: Nutrient overload leads to obesity and insulin resistance. Pioglitazone, a selective peroxisome proliferator-activated receptor (PPAR)g agonist, is currently used to manage insulin resistance, but the specific molecular mechanisms activated by PPARg are not yet fully understood. Recent studies suggest the involvement of suppressor of cytokine signalling (SOCS)-3 in the pathogenesis of insulin resistance. This study aimed to investigate the hepatic signalling pathway activated by PPARg activation in a non-genetic insulin-resistant animal model. Experimental approach: Male Wistar rats were maintained on a high-cholesterol fructose (HCF) diet for 15 weeks. Pioglitazone (3 mg·kg -1 ) was administered orally for the last 4 weeks of this diet. At the end of the treatment, serum was collected for biochemical analysis. Levels of PPARg, SOCS-3, pro-inflammatory markers, insulin receptor substrate-2 and Akt/glycogen synthase kinase-3b phosphorylation were assesed in rat liver. Key results: Rats fed the HCF diet exhibited hyperlipidemia, hyperinsulinemia, impaired glucose tolerance and insulin resistance. Pioglitazone administration evoked a significant improvement in lipid metabolism and insulin responsiveness. This was accompanied by reduced hepatic expression of SOCS-3, interleukin-6, tumour necrosis factor-a and markers of neutrophil infiltration. Diet-induced PPARg expression was unaffected by the pioglitazone treatment. Conclusion and implications:Chronic pioglitazone administration reduced hepatic inflammatory responses in rats fed a HCF diet. These effects were associated with changes in hepatic expression of SOCS-3, which may be a crucial link between the reduced local inflammation and the improved insulin signalling.

Our preliminary data suggest that the CYP2D6 polymorphism influences both donepezil metabolism and therapeutic outcome and that a knowledge of a patient's CYP2D6 genotype together with donepezil concentration measurements might be useful in the context of improving the clinical efficacy of donepezil therapy.