Effects of Menadione, Hydrogen Peroxide, and Quercetin on Apoptosis and Delayed Luminescence of Human Leukemia Jurkat T-Cells

Menadione (MD) is an effective cytotoxic drug able to produce intracellularly large amounts of superoxide anion. Quercetin (QC), a widely distributed bioflavonoid, can exert both antioxidant and pro-oxidant effects and is known to specifically inhibit cell proliferation and induce apoptosis in different cancer cell types. We have investigated the relation between delayed luminescence (DL) induced by UV-laser excitation and the effects of MD, hydrogen peroxide, and QC on apoptosis and cell cycle in human leukemia Jurkat T-cells. Treatments with 500 μM H₂O₂ and 250 μM MD for 20 min produced 66.0 ± 4.9 and 46.4 ± 8.6% apoptotic cell fractions, respectively. Long-term (24 h) pre-exposure to 5 μM, but not 0.5 μM QC enhanced apoptosis induced by MD, whereas short-term (1 h) pre-incubation with 10 μM QC offered 50% protection against H₂O₂-induced apoptosis, but potentiated apoptosis induced by MD. Since physiological levels of QC in the blood are normally less than 10 μM, these data can provide relevant information regarding the benefits of flavonoid-combined treatments of leukemia. All the three drugs exerted significant effects on DL. Our data are consistent with (1) the involvement of Complex I of the mitochondrial respiratory chain as an important source of delayed light emission on the 10 μs–10 ms scale, (2) the ability of superoxide anions to quench DL on the 100 μs–10 ms scale, probably via inhibition of reverse electron transfer at the Fe/S centers in Complex I, and (3) the relative insensitivity of DL to intracellular OH^? and H₂O₂ levels.     

The synthesis and SAR of novel diarylsulfone 11β-HSD1 inhibitors

In this communication, human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitory activities of a novel series of diarylsulfones are described. Optimization of this series resulted in several highly potent 11β-HSD1 inhibitors with excellent pharmacokinetic (PK) properties. Compound (S)-25 showed excellent efficacy in a non-human primate ex vivo pharmacodynamic model.     

Contrasting Properties of α7-Selective Orthosteric and Allosteric Agonists Examined on Native Nicotinic Acetylcholine Receptors

Subtype-selective ligands are important tools for the pharmacological characterisation of neurotransmitter receptors. This is particularly the case for nicotinic acetylcholine receptors (nAChRs), given the heterogeneity of their subunit composition. In addition to agonists and antagonists that interact with the extracellular orthosteric nAChR binding site, a series of nAChR allosteric modulators have been identified that interact with a distinct transmembrane site. Here we report studies conducted with three pharmacologically distinct nicotinic ligands, an orthosteric agonist (compound B), a positive allosteric modulator (TQS) and an allosteric agonist (4BP-TQS). The primary focus of the work described in this study is to examine the suitability of these compounds for the characterisation of native neuronal receptors (both rat and human). However, initial experiments were conducted on recombinant nAChRs demonstrating the selectivity of these three compounds for α7 nAChRs. In patch-clamp recordings on rat primary hippocampal neurons we found that all these compounds displayed pharmacological properties that mimicked closely those observed on recombinant α7 nAChRs. However, it was not possible to detect functional responses with compound B, an orthosteric agonist, using a fluorescent intracellular calcium assay on either rat hippocampal neurons or with human induced pluripotent stem cell-derived neurons (iCell neurons). This is, presumably, due to the rapid desensitisation of α7 nAChR that is induced by orthosteric agonists. In contrast, clear agonist-evoked responses were observed in fluorescence-based assays with the non-desensitising allosteric agonist 4BP-TQS and also when compound B was co-applied with the non-desensitising positive allosteric modulator TQS. In summary, we have demonstrated the suitability of subtype-selective orthosteric and allosteric ligands for the pharmacological identification and characterisation of native nAChRs and the usefulness of ligands that minimise receptor desensitisation for the characterisation of α7 nAChRs in fluorescence-based assays.     

Discovery and optimization of benzenesulfonanilide derivatives as a novel class of 11β-HSD1 inhibitors

A novel series of benzenesulfonanilide derivatives of 11β-HSD1 inhibitors were identified via modification of the sulfonamide core of the arylsulfonylpiperazine lead structures. The synthesis, in vitro biological evaluation, and structure-activity relationship of these compounds are presented. Optimization of this series rapidly resulted in the discovery of compounds (S)-10 and (S)-23 (11β-HSD1 SPA IC(50)=1.8 and 1.4 nM, respectively).     

Altered stress stimulation of inward rectifier potassium channels in Andersen-Tawil syndrome

Inward rectifier potassium channels of the Kir2 subfamily are important determinants of the electrical activity of brain and muscle cells. Genetic mutations in Kir2.1 associate with Andersen-Tawil syndrome (ATS), a familial disorder leading to stress-triggered periodic paralysis and ventricular arrhythmia. To identify the molecular mechanisms of this stress trigger, we analyze Kir channel function and localization electrophysiologically and by time-resolved confocal microscopy. Furthermore, we employ a mathematical model of muscular membrane potential. We identify a novel corticoid signaling pathway that, when activated by glucocorticoids, leads to enrichment of Kir2 channels in the plasma membranes of mammalian cell lines and isolated cardiac and skeletal muscle cells. We further demonstrate that activation of this pathway can either partly restore (40% of cases) or further impair (20% of cases) the function of mutant ATS channels, depending on the particular Kir2.1 mutation. This means that glucocorticoid treatment might either alleviate or deteriorate symptoms of ATS depending on the patient's individual Kir2.1 genotype. Thus, our findings provide a possible explanation for the contradictory effects of glucocorticoid treatment on symptoms in patients with ATS and may open new pathways for the design of personalized medicines in ATS therapy.     

Optimization of novel di-substituted cyclohexylbenzamide derivatives as potent 11β-HSD1 inhibitors

Novel 4,4-disubstituted cyclohexylbenzamide inhibitors of 11β-HSD1 were optimized to account for liabilities relating to in vitro pharmacokinetics, cytotoxicity and protein-related shifts in potency. A representative compound showing favorable in vivo pharmacokinetics was found to be an efficacious inhibitor of 11β-HSD1 in a rat pharmacodynamic model (ED₅₀ = 10 mg/kg).     

FRET detection of lymphocyte function–associated antigen-1 conformational extension

Lymphocyte function–associated antigen 1 (LFA-1, CD11a/CD18, αLβ2-integrin) and its ligands are essential for adhesion between T-cells and antigen-presenting cells, formation of the immunological synapse, and other immune cell interactions. LFA-1 function is regulated through conformational changes that include the modulation of ligand binding affinity and molecular extension. However, the relationship between molecular conformation and function is unclear. Here fluorescence resonance energy transfer (FRET) with new LFA-1–specific fluorescent probes showed that triggering of the pathway used for T-cell activation induced rapid unquenching of the FRET signal consistent with extension of the molecule. Analysis of the FRET quenching at rest revealed an unexpected result that can be interpreted as a previously unknown LFA-1 conformation.     

Exposure to silver nanoparticles induces oxidative stress and memory deficits in laboratory rats

Currently most of the applications of silver nanoparticles are in antibacterial/antifungal agents in medicine and biotechnology, textile engineering, water treatment and silver-based consumer products. However, the effects of silver nanoparticles on human body, especially on the central nervous system, are still unclear. To study the mechanisms underlying the effects of silverpoly(amidehydroxyurethane) coated silver nanoparticles on brain functions, we subjected male Wistar rats to chronic treatments with silver-29 nm (5 μg/kg and 10 μg/kg) and silver-23 nm (5 μg/kg and 10 μg/kg) nanoparticles for 7 days. We evaluated the effects of nanoparticles size and structure on rat memory function. Memory processes were studied by means of two cognitive tasks (Y-maze and radial arm-maze). Exposure to silver nanoparticles significantly decreased spontaneous alternation in the Y-maze task and working memory functions in the radial arm-maze task, suggesting that nanoparticles have effects on short-term memory. We found no effects on long-term memory, which we assessed by reference memory trials in the radial arm-maze task. We found that memory deficits were significantly correlated with oxidative stress generation only in the Y-maze task. Our findings suggest that silver nanoparticles may induce an impairment of memory functions by increasing oxidative stress in the brain. The use of silver nanoparticles for medical purposes therefore requires careful consideration, particularlyif it involves exposure of the human brain.     

Discovery and initial SAR of arylsulfonylpiperazine inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1)

High-throughput screening of a small-molecule compound library resulted in the identification of a series of arylsulfonylpiperazines that are potent and selective inhibitors of human 11beta-Hydroxysteroid Dehydrogenase Type 1 (11beta-HSD1). Optimization of the initial lead resulted in the discovery of compound (R)-45 (11beta-HSD1 IC(50)=3nM).