Exploration of the underlying mechanism of Astragaloside III in attenuating immunosuppression via network pharmacology and vitro/vivo pharmacological validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus mongholicus Bunge (AM, recorded in http://www.worldfloraonline.org, 2023-08-03) is a kind of medicine food homology plant with a long medicinal history in China. Astragaloside III (AS-III) has immunomodulatory effects and is one of the most active components in AM. However, its underlying mechanism of action is still not fully explained. AIM OF THE STUDY: The research was designed to discuss the protective effects of AS-III on immunosuppression and to elucidate its prospective mechanism. MATERIALS AND METHODS: Molecular docking methods and network pharmacology analysis were used to comprehensively investigate potential targets and relative pathways for AS-III and immunosuppression. In order to study and verify the pharmacological activity and mechanism of AS-III in alleviating immunosuppression, immunosuppression mouse model induced by cyclophosphamide (CTX) in vivo and macrophage RAW264.7 cell model induced by hypoxia/lipopolysaccharide (LPS) in vitro were used. RESULTS: A total of 105 common targets were obtained from the AS-III-related and immunosuppression-related target networks. The results of network pharmacology and molecular docking demonstrate that AS-III may treat immunosuppression through by regulating glucose metabolism-related pathways such as regulation of lipolysis in adipocytes, carbohydrate digestion and absorption, cGMP-PKG signaling pathway, central carbon metabolism in cancer together with HIF-1 pathway. The results of molecular docking showed that AS-III has good binding relationship with LDHA, AKT1 and HIF1A. In CTX-induced immunosuppressive mouse model, AS-III had a significant protective effect on the reduction of body weight, immune organ index and hematological indices. It can also protect immune organs from damage. In addition, AS-III could significantly improve the expression of key proteins involved in energy metabolism and serum inflammatory factors. To further validate the animal results, an initial inflammatory/immune response model of macrophage RAW264.7 cells was constructed through hypoxia and LPS. AS-III improved the immune function of macrophages, reduced the release of NO, TNF-α, IL-1ß, PDHK-1, LDH, lactate, HK, PK and GLUT-1, and restored the decrease of ATP caused by hypoxia. Besides, AS-III was also demonstrated that it could inhibit the increase of HIF-1α, PDHK-1 and LDH by adding inhibitors and agonists. CONCLUSIONS: In this study, the main targets of AS-III for immunosuppressive therapy were initially analyzed. AS-III was systematically confirmed to attenuates immunosuppressive state through the HIF-1α/PDHK-1 pathway. These findings offer an experimental foundation for the use of AS-III as a potential candidate for the treatment of immunosuppression.

Systems pharmacology reveals the mechanism of Astragaloside IV in improving immune activity on cyclophosphamide-induced immunosuppressed mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Myelosuppression, also known as bone marrow suppression (BMS), is a pathological phenomenon of the decrease in the production of blood cells and further lead to immune homeostasis disorder. Astragalus mongholicus Bunge (AM, checked with The World Flora Online, http://www.worldfloraonline.org, updated on January 30, 2023) is a traditional Chinese medicine with efficacy of tonifying Qi and strengthening body immunity in thousands of years of clinical practice in China. Astragaloside IV (AS-IV) is a major active ingredient of AM, which plays an important role in regulating immune system through different ways. AIM OF THE STUDY: This study was aimed to investigate the protective effect and mechanism of AS-IV on macrophages in vitro and cyclophosphamide (CTX)-induced immunosuppressive mice in vivo, and to provide experimental basis for the prevention and treatment of AS-IV in myelosuppression. MATERIALS AND METHODS: Based on network pharmacology and molecular docking technology, the core targets and signaling pathways of saponins of AM against myelosuppression were screened. And then, the immunoregulatory effect of AS-IV on RAW264.7 cells was investigated by cellular immune activity and cellular secretion analysis in vitro. In this way, the effects of AS-IV on the main potential targets of HIF-1α/NF-κB signaling pathway were analyzed by qRT-PCR and Western blot methods. Furthermore, comprehensive analysis of the effects of AS-IV against CTX-induced mice were conducted on the basis of immune organs indices analysis, histopathological analysis, hematological analysis, natural killer cell activity analysis and spleen lymphocyte transformation activity analysis. In order to further verify the relationship between active ingredients and action targets, drug inhibitor experiments were finally conducted. RESULTS: AS-IV, as a potential anti-myelosuppressive compound, was screened by systematic pharmacological methods to act on target genes including HIF1A and RELA together with the HIF-1α/NF-κB signaling pathway. Further studies by molecular docking technology showed that AS-IV had good binding activity with HIF1A, RELA, TNF, IL6, IL1B and other core targets. Besides, cellular and animal experiments validation results showed that AS-IV could enhance the migration and phagocytosis of RAW264.7 cells, and protect the immune organs such as spleen and thymus together with bone tissues from damage. By this means, immune cell function including spleen natural killer cell and lymphocyte transformation activity were also enhanced. In addition, white blood cells, red blood cells, hemoglobin, platelets and bone marrow cells were also significantly improved in the suppressed bone marrow microenvironment (BMM). In kinetic experiments, the secretion of cytokines such as TNF-α, IL-6 and IL-1ß were increased, and IL-10, TGF-ß1 were decreased. The key regulatory proteins such as HIF-1α, NF-κB, PHD3 in HIF-1α/NF-κB signaling pathway were also regulated in the results of upregulated expression of HIF-1α, p-NF-κB p65 and PHD3 at the protein or mRNA level. Finally, the inhibition experiment results suggested that AS-IV could significantly improve protein response in immunity and inflammation such as HIF-1α, NF-κB and PHD3. CONCLUSION: AS-IV could significantly relieve CTX-induced immunosuppressive and might improve the immune activity of macrophages by activating HIF-1α/NF-κB signaling pathway, and provide a reliable basis for the clinical application of AS-IV as a potentially valuable regulator of BMM.

Saikosaponin B1 and Saikosaponin D inhibit tumor growth in medulloblastoma allograft mice via inhibiting the Hedgehog signaling pathway.

Medulloblastoma (MB), accounting for nearly 10% of all childhood brain tumors, are implicated with aberrant activation of the Hedgehog (Hh) signaling pathway. Saikosaponin B1 (SSB1) and Saikosaponin D (SSD), two bioactive constituents of Radix Bupleuri, are reported to have many biological activities including anticancer activities. In our work, we evaluated the inhibition of SSB1 and SSD on MB tumor growth in allograft mice and explored the underlying mechanisms. The associated biological activity was investigated in Shh Light II cells, an Hh-responsive fibroblast cell line, using the Dual-Glo® Luciferase Assay System. First, SSB1 (IC50, 241.8 nM) and SSD (IC50, 168.7 nM) inhibited GLI-luciferase activity in Shh Light II cells stimulated with ShhN CM, as well as Gli1 and Ptch1 mRNA expression. In addition, both compounds suppressed the Hh signaling activity provoked by smoothened agonist (SAG) or excessive Smoothened (SMO) expression. Meanwhile, SSB1 and SSD did not inhibit glioma-associated oncogene homolog (GLI) luciferase activity activated by abnormal expression of downstream molecules, suppressor of fuse (SUFU) knockdown or GLI2 overexpression. Consequently, SSB1 (30 mg/kg, ip) and SSD (10 mg/kg, ip) displayed excellent in vivo inhibitory activity in MB allografts, and the tumor growth inhibition ratios were approximately 50% and 70%, respectively. Our findings, thus, identify SSB1 and SSD significantly inhibit tumor growth in MB models by inhibiting the Hedgehog pathway through targeting SMO.

Biomimetic Cascade Polymer Nanoreactors for Starvation and Photodynamic Cancer Therapy.

The selective disruption of nutritional supplements and the metabolic routes of cancer cells offer a promising opportunity for more efficient cancer therapeutics. Herein, a biomimetic cascade polymer nanoreactor (GOx/CAT-NC) was fabricated by encapsulating glucose oxidase (GOx) and catalase (CAT) in a porphyrin polymer nanocapsule for combined starvation and photodynamic anticancer therapy. Internalized by cancer cells, the GOx/CAT-NCs facilitate microenvironmental oxidation by catalyzing endogenous H2O2 to form O2, thereby accelerating intracellular glucose catabolism and enhancing cytotoxic singlet oxygen (1O2) production with infrared irradiation. The GOx/CAT-NCs have demonstrated synergistic advantages in long-term starvation therapy and powerful photodynamic therapy (PDT) in cancer treatment, which inhibits tumor cells at more than twice the rate of starvation therapy alone. The biomimetic polymer nanoreactor will further contribute to the advancement of complementary modes of spatiotemporal control of cancer therapy.

Laser soliton microcombs heterogeneously integrated on silicon.

Silicon photonics enables wafer-scale integration of optical functionalities on chip. Silicon-based laser frequency combs can provide integrated sources of mutually coherent laser lines for terabit-per-second transceivers, parallel coherent light detection and ranging, or photonics-assisted signal processing. We report heterogeneously integrated laser soliton microcombs combining both indium phospide/silicon (InP/Si) semiconductor lasers and ultralow-loss silicon nitride (Si3N4) microresonators on a monolithic silicon substrate. Thousands of devices can be produced from a single wafer by using complementary metal-oxide-semiconductor-compatible techniques. With on-chip electrical control of the laser-microresonator relative optical phase, these devices can output single-soliton microcombs with a 100-gigahertz repetition rate. Furthermore, we observe laser frequency noise reduction due to self-injection locking of the InP/Si laser to the Si3N4 microresonator. Our approach provides a route for large-volume, low-cost manufacturing of narrow-linewidth, chip-based frequency combs for next-generation high-capacity transceivers, data centers, space and mobile platforms.

Cascade catalytic nanoplatform constructed by laterally-functionalized pillar[5]arenes for antibacterial chemodynamic therapy.

Chemodynamic therapy (CDT) is an emerging approach to overcome bacterial infections that can efficiently convert hydrogen peroxide (H2O2) to generate highly toxic hydroxyl radicals (˙OH). How to develop safe and effective CDT-based strategies is in high demand but challenging. Herein, a cascade catalytic nanoplatform (GOx-NCs/Fe3O4) was designed by absorbing glucose oxidase (GOx) onto the surface of covalent-assembled polymer capsules (NCs) encapsulating Fe3O4 nanoparticles. With the presence of glucose, GOx could effectively catalyze it to produce H2O2 and result in a decrease in pH value, both of which would assist the subsequent Fenton reaction. Encapsulated Fe3O4 nanoparticles would subsequently trigger H2O2 to produce ˙OH, which could make antibacterial CDT come true. More importantly, the polymer capsules exhibited little to no cytotoxicity towards mammalian cells, which might provide more opportunities and potential to apply in other fields.

Systematic exploration of Astragalus membranaceus and Panax ginseng as immune regulators: Insights from the comparative biological and computational analysis.

BACKGROUND: Immune system plays a decisive role for defending various pathogenic microorganisms. Astragalus membranaceus (AM) and Panax ginseng (PG) are two tonic herbs used in traditional Chinese medicine (TCM) as immune booster and help to control diseases with their healthy synergistic effect on immune system. PURPOSE: This study was aimed to investigate the promote effect and molecular mechanisms of AM and PG on immune system as booster and to control the target diseases using animal and computational systematic study. METHODS: Computational models including absorption, distribution, metabolism, and elimination (ADME) with weighted ensemble similarity (WES) algorithm-based models and ClueGo network analysis were used to find the potential bioactive compounds targets and pathways, which were responsible for immune regulation. Viscera index analysis, proliferation activity of splenic lymphocytes and cytotoxic activity of NK cells assays were performed to validate the effect of AM and PG on immune system of long-term administrated mice. Metabonomic study of mice plasma was conducted to investigate effect of AM and PG on the endogenous metabolic perturbations, together with correlation analysis. RESULTS: AM and PG simultaneously showed the ability to strengthen the immune system function including enhancement of spleen and thymus index, proliferation of splenic lymphocytes and cytotoxic activity of NK cells. Besides, the different molecular mechanisms of AM and PG on immune regulation were also investigated by analyzing the potential bioactive compounds, enzymes actions and pathways. Quercetin, formononetin and kaempferol were the main immune-related compounds in AM, while ginsenoside Ra1, ginsenoside Rh1 and kaempferol in PG. About 10 target proteins were found close to immune regulation, including acetylcholinesterase (ACHE, common target in AM and PG), sphingosine kinase 1(SPHK1), cytidine deaminase (CDA), and Choline O-acetyltransferase (CHAT). Glycerophospholipid metabolism was regulated in both AM and PG groups. Pyrimidine metabolism and sphingolipid metabolism were considered as the special pathway in AM groups. Energy metabolism and glycerolipid metabolism were the special pathways in PG groups. CONCLUSION: A novel comprehensive molecular mechanism analysis method was established and applied to clarify the scientific connotation of AM and PG as immune regulation, with similar herbal tonic effect provided in clinical practice of TCM, which can provide a new line of research for drug development (immune booster) using AM and PG.

Saikosaponin B2 attenuates kidney fibrosis via inhibiting the Hedgehog Pathway.

BACKGROUND: Renal interstitial fibrosis is a common pathway through which chronic kidney disease progresses to end-stage renal disease. There are currently no effective drugs available to treat kidney fibrosis, so traditional medicine is likely to be a candidate. The therapeutic potential of saikosaponin B2 (SSB2), a biologically active ingredient of Radix Bupleuri, on renal fibrosis has not been reported. METHODS: A unilateral ureteral obstruction (UUO) model was conducted to induce renal interstitial fibrosis in mice. SSB2's effect was valuated by histological staining and exploring the changes in expression of relative proteins and mRNAs. A conditional medium containing sonic hedgehog variant protein stimulating normal rat kidney interstitial fibroblast cells (NRK-49F) was used in an in vitro model to determine the possible mechanism. The molecular target of SSB2 was verified using several mutation plasmids. RESULTS: SSB2 administration reduced kidney injury and alleviated interstitial fibrosis by decreasing excessive accumulation of extracellular matrix components in UUO mice. It could also reduce the expression of α-SMA, fibronectin and Gli1, a crucial molecule of the hedgehog (Hh) signaling pathway both in vivo and in vitro. In NIH-3T3 cells simulated by conditional medium containing sonic hedgehog variant protein, SSB2 showed the ability to decrease the expression of Gli1 and Ptch1 mRNA. Using a dual-luciferase reporter assay, SSB2 suppressed the Gli-luciferase reporter activity in NIH-3T3 cells, and the IC50 was 0.49 µM, but had no effect on the TNF-α/NF-κB and Wnt/ß-catenin signaling pathways, indicating the inhibition selectivity on the Hh signaling pathway. Furthermore, SSB2 failed to inhibit the Hh pathway activity evoked by ectopic expression of Gli2ΔN and Smo D473H, suggesting that SSB2 might potentially act on smoothened receptors. CONCLUSION: SSB2 could attenuate renal fibrosis and decrease fibroblast activation by inhibiting the Hh signaling pathway.

Diselenium-containing ultrathin polymer nanocapsules for highly efficient targeted drug delivery and combined anticancer effect.

The combination of selenium and pillararenes to prepare selenium-containing pillararene-based biomaterials is of great significance for the development of biomedicine. Herein, using a covalent self-assembly strategy, we successfully developed new diselenium-containing ultrathin polymer nanocapsules based on lateral cross-linked pillararenes. The new system exhibited a very potent anticancer effect; additionally, the incorporation of the cleavable redox diselenium bond into the polymer nanocapsules provided a smart nanocarrier for drug delivery. Moreover, the polymer nanocapsules were developed for anticancer drug targeting delivery by loading an anticancer drug and introducing the tumor-penetrating peptide RGD through the host-guest interaction strategy. The targeting DOX-loaded diselenium-containing polymer nanocapsules exhibited enhanced stability, self-anticancer effect, targeted delivery and controlled drug release, resulting in effective combined inhibition of tumor progression.

Exploitation of apple pomace towards extraction of triterpenic acids, antioxidant potential, cytotoxic effects, and inhibition of clinically important enzymes.

Apple pomace (AP) utilised for analysis of triterpenic acids (TTAs) using HPLC-MS/MS. The methanol, ethanol and ethyl acetate extracts showed high phenolic content with significant antioxidant activity compared to chloroform and n-hexane. AP TTAs; ursolic acid, betulinic acid and maslinic acid showed potent antioxidant and enzyme inhibitory effects. The IC50 values were 13.2-30.8 µg/mL (tyrosinase), 19.6-42.5 µg/mL (xanthine oxidase) and 16.6-38.6 µg/mL (urease) for AP extracts and 8.4-25.8 µg/mL (tyrosinase), 12.6-30.2 µg/mL (xanthine oxidase) and 10.1-28.6 µg/mL (urease) for TTAs, compared to the positive controls; kojic acid (10.4 ±â€¯0.06 µg/mL), allopurinol (9.6 ±â€¯0.04 µg/mL) and thiourea (8.9 ±â€¯0.02 µg/mL) towards respective enzymes. UA showed a competitive type of inhibition for tyrosinase, while BA showed a noncompetitive type of inhibition towards xanthine oxidase. In addition, the AP extracts and TTAs exerted significant cytotoxic effects towards the proliferation of cancer cell lines. AP methanol extract (IC50 of 38.5 ±â€¯4.1, 47.1 ±â€¯3.5, 70.6 ±â€¯2.3, and 50.5 ±â€¯3.9 µg/mL) and ursolic acid (IC50 of 6.5 ±â€¯0.7, 15.5 ±â€¯1.4, 20.8 ±â€¯1.3, and 5.6 ±â€¯0.8 µg/mL) showed prominent anticancer activity on Hela, Skov-3, Caski, and NCL cancer cell lines, respectively. Thus, this study shows that the AP & TTAs could be utilized for functional food development and as a potent antioxidant, anticancer, skin whitening, and anti-urolithic agents.

A systematic study of the dissolution and relative bioavailability of four ginsenosides in the form of ultrafine granular powder, common powder and traditional pieces of Panax quinquefolius L, in vitro and in beagles.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax quinquefolius L (PQ), also known as American ginseng, has been used as a medicinal herb for thousands of years in the Far East, which was wildly used actively in healing the cardiovascular, endocrine and immune systems, in supporting chemoprevention of cancer. MATERIALS AND METHODS: An integrated, rapid, sensitive and reliable UHPLC-ESI-QQQ MS/MS method was validated and successfully applied in a pharmacokinetics study in which four representative ginsenosides were measured in beagle plasma following oral administration of Panax quinquefolius L (PQ) in the form of ultrafine granular powder, standard powder and an extract. RESULTS: Two paired ions ([M+Na](+) in the positive MS process, and two characteristic ions [Q3](+) in the positive MS/MS process) of the target compounds were optimized and selected for improved qualitative and quantitative analysis of ginsenosides in beagle plasma. The relative bioavailability of the target ginsenosides in these three formulations was measured by the pharmacokinetic parameters, including Cmax, Tmax, AUC0-∞ and so on. The ultrafine granular powder had the highest bioavailability, as well as the greatest extent of and fastest dissolution in vitro. CONCLUSION: Our results show that improved formulations of PQ could facilitate the dissolution and promote absorption of the important compounds it contains.

Selenium-containing organic nanoparticles as silent precursors for ultra-sensitive thiol-responsive transmembrane anion transport.

An anion transporter with a selenoxide group was able to form nanoparticles in water, whose activity was fully turned off due to the aggregation effect. The formed nanoparticles have a uniform size and can be readily dispersed in water at high concentrations. Turn-on of the nanoparticles by reducing molecules is proposed to be a combined process, including the reduction of selenoxide to selenide, disassembly of the nanoparticles and location of the transporter to the lipid membrane. Accordingly, a special acceleration phase can be observed in the turn-on kinetic curves. Since turn-on of the nanoparticles is quantitatively related to the amount of reductant, the nanoparticles can be activated in a step-by-step manner. Due to the sensibility of this system to thiols, cysteine can be detected at low nanomolar concentrations. This ultra-sensitive thiol-responsive transmembrane anion transport system is quite promising in biological applications.

Reversible Ca(2+) switch of an engineered allosteric antioxidant selenoenzyme.

A Ca(2+) -responsive artificial selenoenzyme was constructed by computational design and engineering of recoverin with the active center of glutathione peroxidase (GPx). By combining the recognition capacity for the glutathione (GSH) substrate and the steric orientation of the catalytic selenium moiety, the engineered selenium-containing recoverin exhibits high GPx activity for the catalyzed reduction of H2 O2 by glutathione (GSH). Moreover, the engineered selenoenzyme can be switched on/off by Ca(2+) -induced allosterism of the protein recoverin. This artificial selenoenzyme also displays excellent antioxidant ability when it was evaluated using a mitochondrial oxidative damage model, showing great potential for controlled catalysis in biomedical applications.

A dual enzyme microgel with high antioxidant ability based on engineered seleno-ferritin and artificial superoxide dismutase.

An antioxidant microgel with both glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities is reported. Using computational design and genetic engineering methods, the main catalytic components of GPx are fabricated onto the surface of ferritin. The resulting seleno-ferritin (Se-Fn) monomers can self-assemble into nanocomposites that exhibit remarkable GPx activity due to the well organized multi-GPx catalytic centers. Subsequently, a porphyrin derivative is synthesized as an SOD mimic, and is employed to construct a synergistic dual enzyme system by crosslinking Se-Fn nanocomposites into a microgel. Significantly, this dual enzyme microgel is demonstrated to display better antioxidant ability than single GPx or SOD mimics in protecting cells from oxidative damage.

A novel selenium and copper-containing peptide with both superoxide dismutase and glutathione peroxidase activities.

Superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS. In order to imitate the synergism of these enzymes, we designed and synthesized a novel 32-mer peptide (32P) on the basis of the previous 15-mer peptide with GPX activity and a 17-mer peptide with SOD activity. Upon the selenation and chelation of copper, the 32-mer peptide is converted to a new Se- and Cu-containing 32-mer peptide (Se-Cu-32P) and displays both SOD and GPX activities and its kinetics was studied. Moreover, the novel peptide was demonstrated to be able to better protect vero cells from the injury induced by xanthine oxidase (XOD)/xanthine/Fe2+ damage system than its parents. Thus, this bifunctional enzyme imitated the synergism of SOD and GPX and could be a better candidate of therapeutic medicine.

Engineered selenium-containing glutaredoxin displays strong glutathione peroxidase activity rivaling natural enzyme.

Insertion of selenocysteine (Sec) into protein scaffolds provides an opportunity for designing enzymes with improved and unusual catalytic properties. The use of a common thioredoxin fold with a high affinity for glutathione in glutaredoxin (Grx) and glutathione peroxidase (GPx) suggests a possibility of engineering Grx into GPx and vice versa. Here, we engineered a Grx domain of mouse thioredoxin/glutathione reductase (TGR) into a selenium-containing enzyme by substituting the active site cysteine (Cys) with selenocysteine (Sec) in a Cys auxotrophic system. The resulting selenoenzyme displayed an unusually high GPx catalytic activity rivaling that of several native GPxs. The engineered seleno-Grx was characterized by mass spectrometry and kinetic analyses. It showed a typical ping-pong kinetic mechanism, and its catalytic properties were similar to those of naturally occurring GPxs. For example, its second rate constant (k(cat)/K(mH2O2)) was as high as 1.55x10(7) M(-1) min(-1). It appears that glutathione-dependent Grx, GPx and glutathione transferase (GST) evolved from a common thioredoxin-like ancestor to accommodate related glutathione-dependent functions and can be interconverted by targeted Sec insertion.

A novel selenium-containing glutathione transferase zeta1-1, the activity of which surpasses the level of some native glutathione peroxidases.

Glutathione peroxidase (GPX) is a critical antioxidant selenoenzyme in organisms that protects cells against oxidative damage by catalyzing the reduction of hydroperoxides by glutathione (GSH). Thus, some GPX mimics have been generated because of their potential therapeutic value. The generation of a semisynthetic selenoenzyme with peroxidase activity, which matches the catalytic efficiencies of naturally evolved GPX, has been a great challenge. Previously, we semisynthesized a GPX mimetic with high catalytic efficiency using a rat theta class glutathione transferase (rGST T2-2) as a scaffold, in which the highly specific GSH-binding site is adjacent to an active site serine residue that can be chemically modified to selenocysteine (Sec). In this study, we have taken advantage of a new scaffold, hGSTZ1-1, in which there are two serine residues in the active site, to achieve both high thiol selectivity and highly catalytic efficiency. The GPX activity of Se-hGSTZ1-1 is about 1.5 times that of rabbit liver GPX, indicating that the selenium content at the active site plays an important role in enhancement of catalytic performance. Kinetic studies revealed that the catalytic mechanism of Se-hGSTZ1-1 belong in a ping-pong mechanism similar to that of the natural GPX.

Functional mimicry of the active site of glutathione peroxidase by glutathione imprinted selenium-containing protein.

For imitating the active site of antioxidant selenoenzyme glutathione peroxidase (GPx), an artificial enzyme selenosubtilisin was employed as a scaffold for reconstructing substrate glutathione (GSH) specific binding sites by a bioimprinting strategy. GSH was first covalently linked to selenosubtilisin to form a covalent complex GSH-selenosubtilisin through a Se-S bond, then the GSH molecule was used as a template to cast a complementary binding site for substrate GSH recognition. The bioimprinting procedure consists of unfolding the conformation of selenosubtilisin and fixing the new conformation of the complex GSH-selenosubtilisin. Thus a new specificity for naturally occurring GPx substrate GSH was obtained. This bioimprinting procedure facilitates the catalytic selenium moiety of the imprinted selenosubtilisin to match the reactive thiol group of GSH in the GSH binding site, which contributes to acceleration of the intramolecular catalysis. These imprinted selenium-containing proteins exhibited remarkable rate enhancement for the reduction of H2O2 by GSH. The average GPx activity was found to be 462 U/micromol, and it was approximately 100 times that for unimprinted selenosubtilisin. Compared with ebselen, a well-known GPx mimic, an activity enhancement of 500-fold was observed. Detailed steady-state kinetic studies demonstrated that the novel selenoenzyme followed a ping-pong mechanism similar to the naturally occurring GPx.

A novel dicyclodextrinyl diselenide compound with glutathione peroxidase activity.

A 6A,6A'-dicyclohexylamine-6B,6B'-diselenide-bis-beta-cyclodextrin (6-CySeCD) was designed and synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX). In this novel GPX model, beta-cyclodextrin provided a hydrophobic environment for substrate binding within its cavity, and a cyclohexylamine group was incorporated into cyclodextrin in proximity to the catalytic selenium in order to increase the stability of the nucleophilic intermediate selenolate. 6-CySeCD exhibits better GPX activity than 6,6'-diselenide-bis-cyclodextrin (6-SeCD) and 2-phenyl-1,2-benzoisoselenazol-3(2H)-one (Ebselen) in the reduction of H(2)O(2), tert-butyl hydroperoxide and cumenyl hydroperoxide by glutathione, respectively. A ping-pong mechanism was observed in steady-state kinetic studies on 6-CySeCD-catalyzed reactions. The enzymatic properties showed that there are two major factors for improving the catalytic efficiency of GPX mimics. First, the substrate-binding site should match the size and shape of the substrate and second, incorporation of an imido-group increases the stability of selenolate in the catalytic cycle. More efficient antioxidant ability compared with 6-SeCD and Ebselen was also seen in the ferrous sulfate/ascorbate-induced mitochondria damage system, and this implies its prospective therapeutic application.