Glutathione Peroxidase-like Antioxidant Activity of 1,3-Benzoselenazoles: Synthesis and In Silico Molecular Docking Studies as Pancreatic Lipase Inhibitors.

The synthesis of 1,3-benzoselenazoles was achieved by the reaction of corresponding bis[3-amino-N-(p-tolyl)benzamide-2-yl] diselenide, bis[3-amino-N-(4-methoxyphenyl)benzamide-2-yl] diselenide, and bis[3-amino-N-(4-(dimethylamino)phenyl) benzamide-2-yl] diselenide with aryl aldehydes. The 1,3-benzoselenazoles continued to exist as planar molecules due to the presence of secondary Se···O interactions as revealed by the single-crystal X-ray analysis. The presence of secondary Se···O interactions in 1,3-benzoselenazoles was confirmed using natural bond orbital (NBO) and atoms in molecules (AIM) calculations. Nucleus-independent chemical shift (NICS) values suggested the presence of aromatic character in a five-membered benzoselenazole heterocyclic ring. The glutathione peroxidase (GPx)-like antioxidant activity of all 1,3-benzoselenazoles was assessed using a thiophenol assay, exhibiting greater antioxidant activity than Ph2Se2 used as a reference. The most active catalyst carrying a strong electron-donating group (-NMe2) at the ortho-position to the benzoselenazole ring was further investigated at different concentrations of thiophenol, H2O2, and 1,3-benzoselenazoles as catalyst for determining their catalytic parameters. Moreover, the potential applications of all 1,3-benzoselenazoles against pancreatic lipase (PL) have been identified using in silico interactions between the active sites of the 1LPB protein as evaluated using a molecular docking study.

Highly Electrophilic Intermediates in the Bypass Mechanism of Glutathione Peroxidase: Synthesis, Reactivity, and Structures of Selenocysteine-Derived Cyclic Selenenyl Amides.

Selenocysteine (Sec)-derived cyclic selenenyl amides, formed by the intramolecular cyclization of Sec selenenic acids (Sec-SeOHs), have been postulated to function as protective forms in the bypass mechanism of glutathione peroxidase (GPx). However, their chemical properties have not been experimentally elucidated in proteins or small-molecule systems. Recently, we reported the first nuclear magnetic resonance observation of Sec-SeOHs and their cyclization to the corresponding cyclic selenenyl amides by using selenopeptide model systems incorporated in a molecular cradle. Herein, we elucidate the structures and reactivities of Sec-derived cyclic selenenyl amides. The crystal structures and reactions toward a cysteine thiol or a 1,3-diketone-type chemical probe indicated the highly electrophilic character of cyclic selenenyl amides. This suggests that they can serve not only as protective forms to suppress the inactivation of Sec-SeOHs in GPx but also as highly electrophilic intermediates in the reactions of selenoproteins.

Glutathione peroxidase 3 is a novel clinical diagnostic biomarker and potential therapeutic target for neutrophils in rheumatoid arthritis.

BACKGROUND: Neutrophils have a critical role in the pathogenesis of rheumatoid arthritis (RA) with immune system dysfunction. However, the molecular mechanisms of this process mediated by neutrophils still remain elusive. The purpose of the present study is to identify hub genes in neutrophils for diagnosis and treatment of RA utilizing publicly available datasets. METHODS: Gene expression profiles were downloaded from the Gene Expression Omnibus, and batch-corrected and normalized expression data were obtained using the ComBat package. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were used to conduct significantly functional analysis and crucial pathways. The resulting co-expression genes modules and hub genes were generated based on the weighted gene co-expression network analysis and visualization by Cytoscape. Flow cytometry was conducted to detect reactive oxygen species (ROS) levels in neutrophils. RESULTS: Neutrophils underwent transcriptional changes in synovial fluid (SF) of RA patients, different from peripheral blood of healthy controls or patients with RA. Especially, glycolysis, HIF-1 signaling, NADH metabolism, and oxidative stress were affected. These hub genes were strongly linked with classical glycolysis-related genes (ENO1, GAPDH, and PKM) responsible for ROS production. The antioxidant enzyme glutathione peroxidase 3 (GPX3), a ROS scavenger, was first identified as a hub gene in RA neutrophils. Neutrophils from patients with autoinflammatory and autoimmune diseases had markedly enhanced ROS levels, most notably in RA SF. CONCLUSION: This research recognized hub genes and explored the characteristics of neutrophils in RA. Our findings suggest that the novel hub gene GPX3 is involved in the neutrophil-driven oxidative stress-mediated pathogenesis of RA. It has the potency to be a target for neutrophil-directed RA therapy.

Synthesis, Reactions, and Antioxidant Properties of Bis(3-amino-1-hydroxybenzyl)diselenide.

Bis(3-amino-1-hydroxybenzyl)diselenide containing two ortho groups was synthesized from 7-nitro-3H-2,1-benzoxaselenole and in situ generated sodium benzene tellurolate (PhTeNa). One-pot synthesis of 1,3-benzoselenazoles was achieved from bis(3-amino-1-hydroxybenzyl)diselenide and aryl aldehydes using acetic acid as a catalyst. The X-ray crystal structure of chloro-substituted benzoselenazole revealed a planar structure with T-shaped geometry around the Se atom. Both natural bond orbital and atoms in molecules calculations confirmed the presence of secondary Se···H interactions in bis(3-amino-1-hydroxybenzyl)diselenide and Se···O interactions in benzoselenazoles, respectively. The glutathione peroxidase (GPx)-like antioxidant activities of all compounds were evaluated using a thiophenol assay. Bis(3-amino-1-hydroxybenzyl)diselenide and benzoselenazoles showed better GPx-like activity compared to that of the diphenyl diselenide and ebselen, used as references, respectively. Based on 77Se{1H} NMR spectroscopy, a catalytic cycle for bis(3-amino-1-hydroxybenzyl)diselenide using thiophenol and hydrogen peroxide was proposed involving selenol, selenosulfide, and selenenic acid as intermediates. The potency of all GPx mimics was confirmed by their in vitro antibacterial properties against the biofilm formation of Bacillus subtilis and Pseudomonas aeruginosa. Additionally, molecular docking studies were used to evaluate the in silico interactions between the active sites of the TsaA and LasR-based proteins found in Bacillus subtilis and Pseudomonas aeruginosa.

Analysis and Characterization of Glutathione Peroxidases in an Environmental Microbiome and Isolated Bacterial Microorganisms.

Glutathione peroxidases (Gpx) are a group of antioxidant enzymes that protect cells or tissues against damage from reactive oxygen species (ROS). The Gpx proteins identified in mammals exhibit high catalytic activity toward glutathione (GSH). In contrast, a variety of non-mammalian Gpx proteins from diverse organisms, including fungi, plants, insects, and rodent parasites, show specificity for thioredoxin (TRX) rather than GSH and are designated as TRX-dependent peroxiredoxins. However, the study of the properties of Gpx in the environmental microbiome or isolated bacteria is limited. In this study, we analyzed the Gpx sequences, identified the characteristics of sequences and structures, and found that the environmental microbiome Gpx proteins should be classified as TRX-dependent, Gpx-like peroxiredoxins. This classification is based on the following three items of evidence: i) the conservation of the peroxidatic Cys residue; ii) the existence and conservation of the resolving Cys residue that forms the disulfide bond with the peroxidatic cysteine; and iii) the absence of dimeric and tetrameric interface domains. The conservation/divergence pattern of all known bacterial Gpx-like proteins in public databases shows that they share common characteristics with that from the environmental microbiome and are also TRX-dependent. Moreover, phylogenetic analysis shows that the bacterial Gpx-like proteins exhibit a star-like radiating phylogenetic structure forming a highly diverse genetic pool of TRX-dependent, Gpx-like peroxidases.

The role of glutathione peroxidase-1 in health and disease.

Glutathione peroxidase 1 (GPx1) is an important cellular antioxidant enzyme that is found in the cytoplasm and mitochondria of mammalian cells. Like most selenoenzymes, it has a single redox-sensitive selenocysteine amino acid that is important for the enzymatic reduction of hydrogen peroxide and soluble lipid hydroperoxides. Glutathione provides the source of reducing equivalents for its function. As an antioxidant enzyme, GPx1 modulates the balance between necessary and harmful levels of reactive oxygen species. In this review, we discuss how selenium availability and modifiers of selenocysteine incorporation alter GPx1 expression to promote disease states. We review the role of GPx1 in cardiovascular and metabolic health, provide examples of how GPx1 modulates stroke and provides neuroprotection, and consider how GPx1 may contribute to cancer risk. Overall, GPx1 is protective against the development and progression of many chronic diseases; however, there are some situations in which increased expression of GPx1 may promote cellular dysfunction and disease owing to its removal of essential reactive oxygen species.

Modeling of selenocysteine-derived reactive intermediates utilizing a nano-sized molecular cavity as a protective cradle.

In the biological functions of selenoproteins, various highly reactive species formed by oxidative modification of selenocysteine residues have been postulated to play crucial roles. Representative examples of such species are selenocysteine selenenic acids (Sec-SeOHs) and selenocysteine selenenyl iodides (Sec-SeIs), which have been widely recognized as important intermediates in the catalytic cycle of glutathione peroxidase (GPx) and iodothyronine deiodinase, respectively. However, examples of even spectroscopic observation of Sec-SeOHs and Sec-SeIs in either protein or small-molecule model systems remain elusive so far, most likely due to their notorious instability. For the synthesis of small-molecule model compounds of these reactive species, it is essential to suppress their very facile bimolecular decomposition such as self-condensation and disproportionation. Here we outline a novel method for the synthesis of stable small-molecule model compounds of the selenocysteine-derived reactive species, in which a nano-sized molecular cavity is used as a protective cradle to accommodate the reactive selenocysteine unit. Stabilization by the molecular cradle led to the successful synthesis of Sec-SeOHs, which are stable in solution at low temperatures, and a Sec-SeI, which can be isolated as crystals. The catalytic cycle of GPx was investigated using the NMR-observable Sec-SeOH models, and all the chemical processes proposed for the catalytic cycle of GPx, including the bypass process from Sec-SeOH to the corresponding cyclic selenenyl amide, were experimentally confirmed. Detailed protocols for the syntheses of selenopeptide derivatives bearing the molecular cradle and for the spectroscopic monitoring of their reactions are provided.

Selenium Dihalides Click Chemistry: Highly Efficient Stereoselective Addition to Alkynes and Evaluation of Glutathione Peroxidase-Like Activity of Bis(E-2-halovinyl) Selenides.

Highly efficient stereoselective syntheses of novel bis(E-2-chlorovinyl) selenides and bis(E-2-bromovinyl) selenides in quantitative yields by reactions of selenium dichloride and dibromide with alkynes were developed. The reactions proceeded at room temperature as anti-addition giving products exclusively with (E)-stereochemistry. The glutathione peroxidase-like activity of the obtained products was estimated and compounds with high activity were found. The influence of substituents in the products on their glutathione peroxidase-like activity was discussed.

PEGylation and antioxidant effects of a human glutathione peroxidase 1 mutant.

Human glutathione peroxidase1 (hGPx1) is a good antioxidant and potential drug, but the limited availability and poor stability of hGPx1 have affected its development and application. To solve this problem, we prepared a hGPx1 mutant (GPx1M) with high activity in an Escherichia coli BL21(DE3)cys auxotrophic strain using a single protein production (SPP) system. In this study, the GPx1M was conjugated with methoxypolyethylene glycol-succinimidyl succinate (SS-mPEG, Mw = 5 kDa) chains to enhance its stability. SS-mPEG-GPx1M and GPx1M exhibited similar enzymatic activity and stability toward pH and temperature change, and in a few cases, SS-mPEG-GPx1M was discovered to widen the range of pH stability and increase the temperature stability. Lys 38 was confirmed as PEGylated site by liquid-mass spectrometry. H9c2 cardiomyoblast cells and Sprague-Dawley (SD) rats were used to evaluate the effects of GPx1M and SS-mPEG-GPx1M on preventing or alleviating adriamycin (ADR)-mediated cardiotoxicity, respectively. The results indicated that GPx1M and SS-mPEG-GPx1M had good antioxidant effects in vitro and in vivo, and the effect of SS-mPEG-GPx1M is more prominent than GPx1M in vivo. Thus, PEGylation might be a promising method for the application of GPx1M as an important antioxidant and potential drug.

Hydroperoxide-Reducing Enzymes in the Regulation of Free-Radical Processes.

The review presents current concepts of the molecular mechanisms of oxidative stress development and describes main stages of the free-radical reactions in oxidative stress. Endogenous and exogenous factors of the oxidative stress development, including dysfunction of cell oxidoreductase systems, as well as the effects of various external physicochemical factors, are discussed. The review also describes the main components of the antioxidant defense system and stages of its evolution, with a special focus on peroxiredoxins, glutathione peroxidases, and glutathione S-transferases, which share some phylogenetic, structural, and catalytic properties. The substrate specificity, as well as the similarities and differences in the catalytic mechanisms of these enzymes, are discussed in detail. The role of peroxiredoxins, glutathione peroxidases, and glutathione S-transferases in the regulation of hydroperoxide-mediated intracellular and intercellular signaling and interactions of these enzymes with receptors and non-receptor proteins are described. An important contribution of hydroperoxide-reducing enzymes to the antioxidant protection and regulation of such cell processes as growth, differentiation, and apoptosis is demonstrated.

Modeling the Catalytic Cycle of Glutathione Peroxidase by Nuclear Magnetic Resonance Spectroscopic Analysis of Selenocysteine Selenenic Acids.

Although selenocysteine selenenic acids (Sec-SeOHs) have been recognized as key intermediates in the catalytic cycle of glutathione peroxidase (GPx), examples of the direct observation of Sec-SeOH in either protein or small-molecule systems have remained elusive so far, mostly due to their instability. Here, we report the first direct spectroscopic (1H and 77Se NMR) evidence for the formation of Sec-SeOH in small-molecule selenocysteine and selenopeptide model systems with a cradle-type protective group. The catalytic cycle of GPx was investigated using NMR-observable Sec-SeOH models. All the hitherto proposed chemical processes, i.e., not only those of the canonical catalytic cycle but also those involved in the bypass mechanism, including the intramolecular cyclization of Sec-SeOH to the corresponding five-membered ring selenenyl amide, were examined in a stepwise manner.

Catalytically potent and selective clusterzymes for modulation of neuroinflammation through single-atom substitutions.

Emerging artificial enzymes with reprogrammed and augmented catalytic activity and substrate selectivity have long been pursued with sustained efforts. The majority of current candidates have rather poor catalytic activity compared with natural molecules. To tackle this limitation, we design artificial enzymes based on a structurally well-defined Au25 cluster, namely clusterzymes, which are endowed with intrinsic high catalytic activity and selectivity driven by single-atom substitutions with modulated bond lengths. Au24Cu1 and Au24Cd1 clusterzymes exhibit 137 and 160 times higher antioxidant capacities than natural trolox, respectively. Meanwhile, the clusterzymes demonstrate preferential enzyme-mimicking catalytic activities, with Au25, Au24Cu1 and Au24Cd1 displaying compelling selectivity in glutathione peroxidase-like (GPx-like), catalase-like (CAT-like) and superoxide dismutase-like (SOD-like) activities, respectively. Au24Cu1 decreases peroxide in injured brain via catalytic reactions, while Au24Cd1 preferentially uses superoxide and nitrogenous signal molecules as substrates, and significantly decreases inflammation factors, indicative of an important role in mitigating neuroinflammation.

Structural and functional characterization of the glutathione peroxidase-like thioredoxin peroxidase from the fungus Trichoderma reesei.

Glutathione peroxidases (GPx) are a family of enzymes with the ability to reduce organic and inorganic hydroperoxides to the corresponding alcohols using glutathione or thioredoxin as an electron donor. Here, we report the functional and structural characterization of a GPx identified in Trichoderma reesei (TrGPx). TrGPx was recombinantly expressed in a bacterial host and purified using affinity. Using a thioredoxin coupled assay, TrGPx exhibited activity of 28 U and 12.5 U in the presence of the substrates H2O2 and t-BOOH, respectively, and no activity was observed when glutathione was used. These results indicated that TrGPx is a thioredoxin peroxidase and hydrolyses H2O2 better than t-BOOH. TrGPx kinetic parameters using a pyrogallol assay resulted at Kmapp = 11.7 mM, Vmaxapp = 10.9 IU/µg TrGPx, kcat = 19 s-1 and a catalytic efficiency of 1.6 mM-1 s-1 to H2O2 as substrate. Besides that, TrGPx demonstrated an optimum pH ranging from 9.0-12.0 and a half-life of 36 min at 80 °C. TrGPx 3D-structure was obtained in a reduced state and non-catalytic conformation. The overall fold is similar to the other phospholipid-hydroperoxide glutathione peroxidases. These data contribute to understand the antioxidant mechanism in fungi and provide information for using antioxidant enzymes in biotechnological applications.

Proton Transfer and SN 2 Reactions as Steps of Fast Selenol and Thiol Oxidation in Proteins: A Model Molecular Study Based on GPx.

The so-called peroxidatic cysteines and selenocysteines in proteins reduce hydroperoxides through a dual attack to the peroxide bond in a two-step mechanism. First, a proton dislocation from the thiol/selenol to a close residue of the enzymatic pocket occurs. Then, a nucleophilic attack of the anionic cysteine/selenocysteine to one O atom takes place, while the proton is shuttled back to the second O atom, promoting the formation of a water molecule. In this computational study, we use a molecular model of GPx to demonstrate that the enzymatic environment significantly lowers the barrier of the latter SN 2 step. Particularly, in our Se-based model the energy barriers for the two steps are 29.82 and 2.83 kcal mol-1 , both higher than the corresponding barriers computed in the enzymatic cluster, i. e., 21.60 and null, respectively. Our results, obtained at SMD-B3LYP-D3(BJ)/6-311+G(d,p), cc-pVTZ//B3LYP-D3(BJ)/6-311G(d,p), cc-pVTZ level of theory, show that the mechanistic details can be well reproduced using an oversimplified model, but the energetics is definitively more favorable in the GPx active site. In addition, we pinpoint the role of the chalcogen in the peroxide reduction process, rooting the advantages of the presence of selenium in its acidic and nucleophilic properties.

Design, microwave synthesis, and molecular docking studies of catalpol crotonates as potential neuroprotective agent of diabetic encephalopathy.

Catalpol has gained increasing attention for its potential contributions in controlling glycolipid metabolism and diabetic complications, which makes used as a very promising scaffold for seeking new anti-diabetic drug candidates. Acylation derivatives of catalpol crotonate (CCs) were designed as drug ligands of glutathione peroxidase (GSH-Px) based on molecular docking (MD) using Surfex-Docking method. Catalpol hexacrotonate (CC-6) was synthesized using microwave assisted method and characterized by FT-IR, NMR, HPLC and HRMS. The MD results indicate that with the increasing of esterification degree of hydroxyl, the C log P of CCs increased significantly, and the calculated total scores (Total_score) of CCs are all higher than that of catalpol. It shows that CCs maybe served as potential lead compounds for neuroprotective agents. It was found that the maximum Total_score of isomers in one group CCs is often not that the molecule with minimum energy. MD calculations show that there are five hydrogen bonds formed between CC-6 and the surrounding amino acid residues. Molecular dynamics simulation results show that the binding of CC-6 with GSH-Px is stable. CC-6 was screened for SH-SY5Y cells viability by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay, the result indicates CC-6 can effectively reverse SZT induced cells apoptosis with dose-dependent manner, which can indirectly show that CC-6 is a potential neuroprotective agent.

Serum and milk concentrations of oxidant and anti-oxidant markers in dairy cows affected with bloody milk.

This study was conducted to determine the serum and milk levels of thiobarbturic acid-reac- tive substances (TBARS), nitric oxide (NO), superoxide dismutase (SOD), glutathione peroxi- dase (GSH-Px), vitamin E and selenium, IL-4 and IL-6 in lactating dairy cows affected with bloody milk using commercially available ELISA kits. Milk and whole blood samples were collected from 60 cows affected with bloody milk and 20 apparently healthy cows for control. In the serum, levels of GSH-Px and SOD were significantly (p˂0.05) higher in healthy cows compared to cows affected with bloody milk while the levels of TBARS and NO were significantly (p˂0.05) higher in affected cows. In the milk, levels of SOD, TBARS and NO were significantly (p˂0.05) higher in affected cows. In the serum, levels of vitamin E were significantly (p˂0.05) lower in affected cows compared to healthy cows, while no significant changes were observed in the levels of this vitamin in the milk between healthy and affected cows. In the serum, levels of selenium were significantly (p˂0.05) lower in affected cows while in milk, selenium levels were significantly (p˂0.05) higher in affected cows compared to healthy ones. Levels of IL-4 were significantly (p˂0.05) lower in the serum and milk of affected cows compared to healthy cows while levels of IL-6 were significantly (p˂0.05) higher in both serum and milk of affected cows. Results of this study suggest a possible role of oxidative stress in the pathogenesis of bloody milk in dairy cows.

Phytochemical Analysis and Antioxidant and Anti-Inflammatory Capacity of the Extracts of Fruits of the Sechium Hybrid.

In addition to their own antioxidants, human cells feed on external antioxidants, such as the phenolic compounds of fruits and vegetables, which work together to keep oxidative stress in check. Sechium edule, an edible species of chayote, has phenolic compounds with antioxidant activity and antineoplastic activity. A Sechium hybrid shows one thousand times greater antineoplastic activity than edible species, but its antioxidant and anti-inflammatory activities and the content of phenolic compounds are unknown. The aim of this study was to determine the antioxidant and anti-inflammatory capacity of the extract of fruits of the Sechium hybrid in vitro and in vivo. Phytochemical analysis using HPLC showed that the extract of the Sechium hybrid has at least 16 phenolic compounds; galangin, naringenin, phloretin and chlorogenic acid are the most abundant. In an in vitro assay, this extract inhibited 2,2-diphenyl-L-picrylhydrazyl (DPPH) activity and protected the dimyristoylphosphatidylethanolamine (DMPE) phospholipid model cell membrane from oxidation mediated by hypochlorous acid (HClO). In vivo, it was identified that the most abundant metabolites in the extract enter the bloodstream of the treated mice. On the other hand, the extract reduces the levels of tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and interleukin-6 (IL-6) but increases interleukin-10 (IL-10) and glutathione peroxidase levels. Our findings indicate that intake of the fruits of the Sechium hybrid leads to antioxidant and anti-inflammatory effects in a mouse model. Therefore, these results support the possibility of exploring the clinical effect of this hybrid in humans.

The glutathione peroxidase family of Theobroma cacao: Involvement in the oxidative stress during witches' broom disease.

The glutathione peroxidases (GPXs) are enzymes which are part of the cell antioxidant system inhibiting the ROS-induced damages of membranes and proteins. In cacao (Theobroma cacao L.) genome, five GPX genes were identified. Cysteine insertion codons (UGU) were found in TcPHGPX, TcGPX2, TcGPX4, TcGPX6 and tryptophan insertion codon (UGG) in TcGPX8. Multiple alignments revealed conserved domains between TcGPXs and other plants and human GPXs. Homology modeling was performed using the Populus trichocarpa GPX5 structure as template, and the molecular modeling showed that TcGPXs have affinity with selenometionine in their active site. In silico analysis of the TcGPXs promoter region revealed the presence of conserved cis-elements related to biotic stresses and hormone responsiveness. The expression analysis of TcGPXs in cacao plantlet meristems infected by M. perniciosa showed that TcGPXs are most expressed in susceptible variety than in resistant one, mainly in disease stages in which oxidative stress and programmed cell death occurred. This data, associated with phylogenetic and location analysis suggested that TcGPXs may play a role in protecting cells from oxidative stress as a try of disease progression reduction. To our knowledge, this is the first study of the overall GPX family from T. cacao.

Synthesis and in vitro anti-bladder cancer activity evaluation of quinazolinyl-arylurea derivatives.

Based on the structural modification of molecular-targeted agent sorafenib, a series of quinazolinyl-arylurea derivatives were synthesized and evaluated for their anti-proliferative activities against six human cancer cell lines. Compared with other cell lines tested, T24 was more sensitive to most compounds. Compound 7j exhibited the best profile with lower IC50 value and favorable selectivity. In this study, we focused on 7j-induced death forms of T24 cells and tried to elucidate the reason for its potent proliferative inhibitory activity. Compound 7j treatment could trigger three different cell death forms including apoptosis, ferroptosis, and autophagy; which form would occur depended on the concentrations and incubation time of 7j: (1) Lower concentrations within the initial 8 h of 7j treatment led to apoptosis-dependent death. (2) Ferroptosis and autophagy occurred in the case of higher concentrations combining with extended incubation time through effectively regulating the Sxc-/GPx4/ROS and PI3K/Akt/mTOR/ULK1 pathways, respectively. (3) The above death forms were closely associated with intracellular ROS generation and decreased mitochondrial membrane potential induced by 7j. In molecular docking and structure-activity relationship analyses, 7j could bind well to the active site of the corresponding receptor glutathione peroxidase 4 (GPx4). Compound 7j could be a promising lead for molecular-targeted anti-bladder cancer agents' discovery.

In vivo and in vitro studies on inactivation of selenium containing protein- glutathione peroxidase 3 in mice nephrocytes caused by lead.

Glutathione peroxidases (Gpxs) play vital roles in elimination of hydroperoxide and other reactive oxygen species through catalyzing reduced glutathione to protect from oxidative stress caused by heavy metals such as lead. Among the family of Gpxs, Gpx3 is the only extracellular enzyme synthesized in the kidney and actively secreted into the plasma. This study investigated mechanisms of lead-induced GPx3 inactivation both at the animal and molecular levels. Six-week-old mice were randomly divided into 4 groups, and exposed to different lead concentrations (0, 1, 2 and 4 g/L) in their drinking water for 4 weeks. Contents of GPx3 in blood serum were tested by enzyme-linked immunosorbent assay (ELISA) and the mRNA levels of Gpx3 in mice nephrocytes were determined by quantitative real-time PCR (qPCR), both of which showed significantly inhibited at higher lead concentrations accompanied by the decreased Gpx3 activities and the elevated levels of malondialdehyde (MDA) in nephrocytes, which indicated that lead could induce strongly oxidative stress through affecting Gpx3 function. So we further investigated molecular mechanisms of GPx3 inactivation caused by lead with multiple spectroscopic techniques, isothermal titration calorimetry (ITC) and molecular docking studies in vitro. Results showed that lead statically quenched GPx3 fluorescence by tightly binding to the structural domain of GPx3 in a 3:1 ratio with high binding affinity (K = 3.1(±0.087) × 107 mol-1). Further investigation of the conformation of GPx3 by UV-visible spectroscopy and circular dichroism (CD) spectroscopy indicated that lead changed the secondary structure of GPx3 by loosening the GPx3 skeleton and decreasing the hydrophobicity around tryptophan residues. This work proved in vivo and in vitro experiments that lead could induce oxidative stress in mice nephrocytes by interacting with GPx3.