Redox active or thiol reactive? Optimization of rapid screens to identify less evident nuisance compounds

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action.     

Modulation of infection-induced inflammation and locomotive deficit and longevity in senescence-accelerated mice-prone (SAMP8) model by the oligomerized polyphenol Oligonol.

Oligonol is produced from the oligomerization of polyphenols (typically proanthocyanidin from a variety of fruits such as lychees, grapes, apples, persimmons, etc.) and contains catechin-type monomers and oligomers of proanthocyanidins. The ability of Oligonol to affect infection-dependent eye inflammation, locomotion and longevity in senescence-accelerated prone mice (SAMP8) (a model of senescence acceleration and geriatric disorders with increased oxidative stress and neuronal deficit) was investigated. Oligonol (60mg/kg) significantly modulated the extent of inflammation scores in the eye of SAMP8 mice. Examination of the mice indicated infection with mouse hepatitis virus and pinworm (Syphacia obvelata) in both males and females and with the intestinal protozoa (trichomonad) in males. A comparison of the two groups (using log-rank test) and the difference in the mean life span between groups (using Student's t-test) indicated significant differences in survival (p=0.043) and the mean life span (p=0.033) in male SAMP8 mice. Oligonol increased the mean life span and this was statistically significant. In the open-field locomotive test, the 7-week-old SAMP8 mice crossed more than 40 partitioned lines in 1min. At 48-week-old control untreated male SAMP8 crossed 2 lines. The Oligonol-treated 48-week-old male SAMP8 mice crossed 17 lines however. The improved locomotive activity was statistically significant even after 36weeks in the Oligonol-treated male SAMP8 but this was not the case throughout the time course of the study in the Oligonol-treated female SAMP8. Thus Oligonol treatment to SAMP8 mice modulated the severity of infection-dependent inflammation, prolonged life-span and significantly improved locomotive activity indicating potential benefit to aging-associated diseases such as Alzheimer's or Parkinson's diseases. This presents potential for further research to define infection-dependent inflammation associated with degenerative conditions and the molecular mechanism of dietary antioxidant protection.     

Glutathione effects on vitamin D metabolism in control and streptozotocin diabetic rat

The circulating concentration of 1,25-dihydroxycholecalciferol [1,25-(OH)₂D₃] is a physiologic index of enzymatic activity of the renal 1-hydroxylase of 25-hydroxychole-calciferol (25-OH-D₃). Hydroxylation of 25-OH-D₃ and circulating 1,25-(OH)₂D₃ are decreased in the streptozotocin diabetic rat. We previously found that activity of another redox enzyme system, cytosolic superoxide dismutase, also decreased in streptozotocin diabetes, can be restored by treatment with glutathione. In the present experiment we tested the effect of glutathione treatment on vitamin D metabolism in control and diabetic rats. Enteral glutathione increased circulating 1,25-(OH)₂D₃ and decreased 25-OH-D₃ in both control and diabetic animals. These results suggest that exogenous glutathione increases 25-OH-D₃ 1-hydroxylation both under basal conditions in the normal animal and in diabetes-induced depression.     

Contribution of redox-active iron and copper to oxidative damage in Alzheimer disease

Metal-catalyzed hydroxyl radicals are potent mediators of cellular injury, affecting every category of macromolecule, and are central to the oxidative injury hypothesis of Alzheimer disease (AD) pathogenesis. Studies on redox-competent copper and iron indicate that redox activity in AD resides exclusively within the neuronal cytosol and that chelation with deferoxamine, DTPA, or, more recently, iodochlorhydroxyquin, removes this activity. We have also found that while proteins that accumulate in AD possess metal-binding sites, metal-associated cellular redox activity is primarily dependent on metals associated with nucleic acid, specifically cytoplasmic RNA. These findings indicate aberrations in iron homeostasis that, we suspect, arise primarily from heme, since heme oxygenase-1, an enzyme that catalyzes the conversion of heme to iron and biliverdin, is increased in AD, and mitochondria, since mitochondria turnover, mitochondrial DNA, and cytochrome C oxidative activity are all increased in AD. These findings, as well as studies demonstrating a reduction in microtubule density in AD neurons, suggest that mitochondrial dysfunction, acting in concert with cytoskeletal pathology, serves to increase redox-active heavy metals and initiates a cascade of abnormal events culminating in AD pathology.     

马来酸二乙酯对半乳糖胺/脂多糖致小鼠急性肝损伤发生中核转录因子表达的影响

目的　耗竭肝脏还原型谷胱甘肽 (GSH)导致氧化还原失平衡后 ,是否可通过改变库普弗细胞核转录因子表达而影响肝损伤发生。方法　给小鼠注射马来酸二乙酯 (DEM) ,耗竭 GSH。结果　马来酸二乙酯在一定程度上 ,抑制了半乳糖胺 /脂多糖 (Galn/ L PS)诱导胞质 IκBα的降解和核 NF- κBp6 5的表达。结论　 DEM可能是通过抑制 IκBα降解 ,影响 NF-κBp6 5易位入核 ,从而抑制肿瘤坏死因子 (TNF) -α释放 ,减轻肝损伤     

In Vitro and In Vivo Evaluations of Dihydroquinoline- and Dihydroisoquinoline-based Targetor Moieties for Brain-specific Chemical Delivery Systems

Brain-targeted delivery of various drugs can be successfully achieved by chemical delivery systems (CDS) that contain a 1,4-dihydropyridine-based redox targetor moiety and undergo a sequential metabolism. However, the susceptibility of this moiety toward hydration in acidic media may limit the shelf-life of such compounds in aqueous formulation. Here, a systematic investigation of the chemical stability toward oxidation and hydration of ester and amide derivatives of 3-substituted 1,4-dihydropyridine, 1,4-dihydroquinoline, and 4-substituted 1,2-dihydroisoquinoline is reported, together with the in vitro stability and in vivo (rat) distribution of isoquinoline-based testosterone and hydrocortisone chemical delivery systems, which were selected as having the most suitable acid-resistant targetor moieties.     

The sensitivity of biomarkers for genotoxicity and acute cytotoxicity in nasal and buccal cells of welders

Welders are inhalatively exposed to fumes which contain genotoxic carcinogens and it was found in epidemiological studies that they have increased cancer rates which may be causally related to DNA damage. In order to assess their health risks and to find out which chemicals cause the adverse effects, bioassays can be performed which enable the detection of genetic damage. The aim of the present study was a comparative investigation with exfoliated buccal and nasal cells in regard to induction of chromosomal alterations and acute cytotoxicity in welders and unexposed controls (n = 22 per group). To elucidate the factors which account for genotoxic and cytotoxic effects, additional biochemical parameters were monitored reflecting the redox status as well as concentrations of different metals and 1-hydroxypyrene (1-OHP) in body fluids. We found in the nasal cells significant induction of alterations which are indicative for DNA damage, i.e. of micronuclei (MNi) and nuclear buds, while elevated rates of nuclear anomalies reflecting cytotoxic effects (condensed chromatin, karyorrhexis, karyolylsis) were detected in cells from both organs. The levels of certain metals (Cr, Cu, Mn, Mo, Ni), but not markers of oxidative damage were significantly higher in the body fluids of the welders. Multivariate Poisson regression analyses indicate that exposure to Mo (15% MNi increase by one standard deviation increase of Mo in serum), Ni (9% increase) and Mn (14% increase) are positively associated with the induction of MNi in nasal cells while Ni was associated with cytotoxic effects in both types of cells (12 and 16% increase). Taken together, our findings indicate that epithelial cells from the respiratory tract are suitable for the detection of DNA-damaging and cytotoxic effects in welders and can be used to assess health risks associated with genomic instability.     

Effects of Hyperkinetic,a β Subunit of Shaker Voltage-Dependent K+ Channels,on the Oxidation State of Presynaptic Nerve Terminals

The Drosophila Hyperkinetic (Hk) gene encodes a β subunit of Shaker (Sh) K⁺ channels and shows high sequence homology to aldoketoreductase. Hk mutations are known to modify the voltage dependence and kinetics of Sh currents, which are also influenced by the oxidative state of the N-terminus region of the Sh channel, as demonstrated in heterologous expression experiments in frog oocytes. However, an in vivo role of Hk in cellular reduction/oxidation (redox) has not been demonstrated. By using a fluorescent indicator of reactive oxygen species (ROS), dihydrorhodamine-123 (DHR), we show that the presynaptic nerve terminal of larval motor axons is metabolically active, with more rapid accumulation of ROS in comparison with muscle cells. In Hk terminals, DHR fluorescence was greatly enhanced, indicating increased ROS levels. This observation implicates a role of the Hk β subunit in redox regulation in presynaptic terminals. This phenomenon was paralleled by the expected effects of the mutations affecting glutathione S-transferase S1 as well as applying H₂O₂ to wild-type synaptic terminals. Thus, our results also establish DHR as a useful tool for detecting ROS levels in the Drosophila neuromuscular junction.     

The redox state of the alarmin HMGB1 is a pivotal factor in neuroinflammatory and microglial priming: A role for the NLRP3 inflammasome

The alarmin high mobility group box-1 (HMGB1) has been implicated as a key factor mediating neuroinflammatory processes. Recent findings suggest that the redox state of HMGB1 is a critical molecular feature of HMGB1 such that the reduced form (fr-HMGB1) is chemotactic, while the disulfide form (ds-HMGB1) is pro-inflammatory. The present study examined the neuroinflammatory effects of these molecular forms as well as the ability of these forms to prime the neuroinflammatory and microglial response to an immune challenge. To examine the neuroinflammatory effects of these molecular forms in vivo, animals were administered intra-cisterna magna (ICM) a single dose of fr-HMGB1 (10 μg), ds-HMGB1 (10 μg) or vehicle and basal pro-inflammatory effects were measured 2 and 24 h post-injection in hippocampus. Results of this initial experiment demonstrated that ds-HMGB1 increased hippocampal pro-inflammatory mediators at 2 h (NF-κBIα mRNA, NLRP3 mRNA and IL-1β protein) and 24 h (NF-κBIα mRNA, TNFα mRNA, and NLRP3 protein) after injection. fr-HMGB1 had no effect on these mediators. These neuroinflammatory effects of ds-HMGB1 suggested that ds-HMGB1 may function to prime the neuroinflammatory response to a subsequent immune challenge. To assess the neuroinflammatory priming effects of these molecular forms, animals were administered ICM a single dose of fr-HMGB1 (10 μg), ds-HMGB1 (10 μg) or vehicle and 24 h after injection, animals were challenged with LPS (10 μg/kg IP) or vehicle. Neuroinflammatory mediators and the sickness response (3, 8 and 24 h after injection) were measured 2 h after immune challenge. We found that ds-HMGB1 potentiated the neuroinflammatory (NF-κBIα mRNA, TNFα mRNA, IL-1β mRNA, IL-6 mRNA, NLRP3 mRNA and IL-1β protein) and sickness response (reduced social exploration) to LPS challenge. fr-HMGB1 failed to potentiate the neuroinflammatory response to LPS. To examine whether these molecular forms of HMGB1 directly induce neuroinflammatory effects in isolated microglia, whole brain microglia were isolated and treated with fr-HMGB1 (0, 1, 10, 100, or 1000 ng/ml) or ds-HMGB1 (0, 1, 10, 100, or 1000 ng/ml) for 4 h and pro-inflammatory mediators measured. To assess the effects of these molecular forms on microglia priming, whole brain microglia were pre-exposed to these forms of HMGB1 (0, 1, 10, 100, or 1000 ng/ml) and subsequently challenged with LPS (10 ng/ml). We found that ds-HMGB1 increased expression of NF-κBIα mRNA and NLRP3 mRNA in isolated microglia, and potentiated the microglial pro-inflammatory response (TNFα mRNA, IL-1β mRNA and IL-1β protein) to LPS. fr-HMGB1 failed to potentiate the microglial pro-inflammatory response to LPS. Consistent with prior reports, the present findings demonstrate that the disulfide form of HMGB1 not only potentiates the neuroinflammatory response to a subsequent immune challenge in vivo, but also potentiates the sickness response to that challenge. Moreover, the present findings demonstrate for the first time that ds-HMGB1 directly potentiates the microglia pro-inflammatory response to an immune challenge, a finding that parallels the effects of ds-HMGB1 in vivo. In addition, ds-HMGB1 induced expression of NLRP3 and NF-κBIα in vivo and in vitro suggesting that the NLRP3 inflammasome may play role in the priming effects of ds-HMGB1. Taken together, the present results suggest that the redox state of HMGB1 is a critical determinant of the priming properties of HMGB1 such that the disulfide form of HMGB1 induces a primed immunophenotype in the CNS, which may result in an exacerbated neuroinflammatory response upon exposure to a subsequent pro-inflammatory stimulus.