A Redox Modulatory SOD Mimetic Nanozyme Prevents the Formation of Cytotoxic Peroxynitrite and Improves Nitric Oxide Bioavailability in Human Endothelial Cells.

The endothelium-derived signalling molecule nitric oxide (NO) in addition to controlling multifarious servo-regulatory functions, suppresses key processes in vascular lesion formation and prevents atherogenesis and other vascular abnormalities. The conversion of NO into cytotoxic and powerful oxidant peroxynitrite (ONOO- ) in a superoxide (O2 .- )-rich environment has emerged as a major reason for reduced NO levels in vascular walls, leading to endothelial dysfunction and cardiovascular complications. So, designing superoxide dismutase (SOD) mimetics that can selectively catalyze the dismutation of O2 .- in the presence of NO, considering their rapid reaction is challenging and is of therapeutic relevance. Herein, the authors report that SOD mimetic cerium vanadate (CeVO4 ) nanozymes effectively regulate the bioavailability of both NO and O2 .- , the two vital constitutive molecules of vascular endothelium, even in the absence of cellular SOD enzyme. The nanozymes optimally modulate the O2 .- level in endothelial cells under oxidative stress conditions and improve endogenously generated NO levels by preventing the formation of ONOO- . Furthermore, nanoparticles exhibit size- and morphology-dependent uptake into the cells and internalize via the clathrin-mediated endocytosis pathway. Intravenous administration of CeVO4 nanoparticles in mice caused no definite organ toxicity and unaltered haematological and biochemical parameters, indicating their biosafety and potential use in biological applications.

Antioxidant and Prooxidant Nanozymes: From Cellular Redox Regulation to Next-Generation Therapeutics.

Nanozymes, nanomaterials with enzyme-mimicking activity, have attracted tremendous interest in recent years owing to their ability to replace natural enzymes in various biomedical applications, such as biosensing, therapeutics, drug delivery, and bioimaging. In particular, the nanozymes capable of regulating the cellular redox status by mimicking the antioxidant enzymes in mammalian cells are of great therapeutic significance in oxidative-stress-mediated disorders. As the distinction of physiological oxidative stress (oxidative eustress) and pathological oxidative stress (oxidative distress) occurs at a fine borderline, it is a great challenge to design nanozymes that can differentially sense the two extremes in cells, tissues and organs and mediate appropriate redox chemical reactions. In this Review, we summarize the advances in the development of redox-active nanozymes and their biomedical applications. We primarily highlight the therapeutic significance of the antioxidant and prooxidant nanozymes in various disease model systems, such as cancer, neurodegeneration, and cardiovascular diseases. The future perspectives of this emerging area of research and the challenges associated with the biomedical applications of nanozymes are described.

Platelet activation and ferroptosis mediated NETosis drives heme induced pulmonary thrombosis.

Cell-free heme (CFH) is a product of hemoglobin, myoglobin and hemoprotein degradation, which is a hallmark of pathologies associated with extensive hemolysis and tissue damage. CHF and iron collectively induce cytokine storm, lung injury, respiratory distress and infection susceptibility in the lungs suggesting their key role in the progression of lung disease pathology. We have previously demonstrated that heme-mediated reactive oxygen species (ROS) induces platelet activation and ferroptosis. However, interaction of ferroptotic platelets and neutrophils, the mechanism of action and associated complications remain unclear. In this study, we demonstrate that heme-induced P-selectin expression and Phosphatidylserine (PS) externalization in platelets via ASK-1-inflammasome axis increases platelet-neutrophil aggregates in circulation, resulting in Neutrophil extracellular traps (NET) formation in vitro and in vivo. Further, heme-induced platelet activation in mice increased platelet-neutrophil aggregates and accumulation of NETs in the lungs causing pulmonary damage. Thus, connecting CFH-mediated platelet activation to NETosis and pulmonary thrombosis. As lung infections induce acute respiratory stress, thrombosis and NETosis, we propose that heme -mediated platelet activation and ferroptosis might be crucial in such clinical manifestations. Further, considering the ability of redox modulators and ferroptosis inhibitors like FS-1, Lpx-1 and DFO to inhibit heme-induced ferroptotic platelets-mediated NETosis and pulmonary thrombosis. They could be potential adjuvant therapy to regulate respiratory distress-associated clinical complications.

A Simple Substitution on Thyroid Hormones Remarkably Alters the Regioselectivity of Deiodination by a Deiodinase Mimic.

The regioselective deiodinations of L-thyroxine (T4) play key roles in the thyroid hormone homeostasis. These reactions are catalyzed by three isoforms of the selenoenzymes, iodothyronine deiodinases (Dio1, Dio2 and Dio3), which are highly homologous in nature. Dio1 mediates 5'- or 5-deiodinations of T4 to produce T3 and rT3, respectively. In contrast, Dio2 and Dio3 are selective to 5'- or 5-deiodination to produce T3 and rT3, respectively. Understanding of the regioselectivity of deiodination at the molecular level is important as abnormal levels of thyroid hormone have been implicated in various clinical conditions, such as hypoxia, myocardial infarction, neuronal ischemia and cancer. In this paper, we report that the electronic properties of the iodine atoms in thyroxine (T4) can be modulated through a simple substitution in the 4'-phenolic moiety. This leads to the change in the regioselectivity of deiodination by different small molecule mimics of Dio enzymes. By using this chemical approach, we also show that the substitution of a strong electron withdrawing group facilitates the removal of all four iodine atoms in the T4 derivative. Theoretical investigations on the hydrogen bonded adducts of T4 with imidazole indicate that the charge on the iodine atoms depend on the nature of hydrogen bond between the -OH group of T4 and the imidazole moiety. While the imidazole can act as either hydrogen bond acceptor (HBA) or hydrogen bond donor (HBD), the protonated imidazole acts exclusively as HBD in T4-imidazole complex. These studies support the earlier observations that the histidine residue at the active sites of the deiodinases play an important role not only in the substrate binding, but also in altering the regioselectivity of the deiodination reactions.

A GPx-mimetic copper vanadate nanozyme mediates the release of nitric oxide from S-nitrosothiols.

Although reactive oxygen and nitrogen species (ROS/RNS), such as hydrogen peroxide (H2O2), nitric oxide (NO), hydroxyl radicals (OH˙), superoxide (O2-) etc., play crucial roles in redox biology and cellular signaling, higher concentrations of these species lead to oxidative and nitrosative stress, which are associated with various pathophysiological conditions like neurodegeneration, cardiovascular diseases and cancer. There is growing evidence that functional impairment of the endothelium is one of the first recognizable signs of the development of atherosclerotic cardiovascular disease. A decreased bioavailability of NO and increased generation of ROS are the two major molecular changes associated with endothelial dysfunction. Therefore, it is a viable strategy to increase the bioavailability of NO while reducing the amount of ROS to prevent the progression of cardiovascular diseases. In this paper, we discuss for the first time that copper vanadate (CuV2O6) can not only release NO from S-nitrosothiols but can also control the ROS levels by functionally mimicking the antioxidant enzyme glutathione peroxidase (GPx) at physiological pH. We used several imaging techniques and spectroscopic measurements to understand the catalysis on the surface of the material during the reactions. The denitrosylation, as well as GPx-like activity, by CuV2O6 can be carried out multiple times without affecting the catalytic activity.

Targeting the DIO3 enzyme using first-in-class inhibitors effectively suppresses tumor growth: a new paradigm in ovarian cancer treatment.

The enzyme iodothyronine deiodinase type 3 (DIO3) contributes to cancer proliferation by inactivating the tumor-suppressive actions of thyroid hormone (T3). We recently established DIO3 involvement in the progression of high-grade serous ovarian cancer (HGSOC). Here we provide a link between high DIO3 expression and lower survival in patients, similar to common disease markers such as Ki67, PAX8, CA-125, and CCNE1. These observations suggest that DIO3 is a logical target for inhibition. Using a DIO3 mimic, we developed original DIO3 inhibitors that contain a core of dibromomaleic anhydride (DBRMD) as scaffold. Two compounds, PBENZ-DBRMD and ITYR-DBRMD, demonstrated attenuated cell counts, induction in apoptosis, and a reduction in cell proliferation in DIO3-positive HGSOC cells (OVCAR3 and KURAMOCHI), but not in DIO3-negative normal ovary cells (CHOK1) and OVCAR3 depleted for DIO3 or its substrate, T3. Potent tumor inhibition with a high safety profile was further established in HGSOC xenograft model, with no effect in DIO3-depleted tumors. The antitumor effects are mediated by downregulation in an array of pro-cancerous proteins, the majority of which known to be repressed by T3. To conclude, using small molecules that specifically target the DIO3 enzyme we present a new treatment paradigm for ovarian cancer and potentially other DIO3-dependent malignancies.

Antioxidant nanozyme counteracts HIV-1 by modulating intracellular redox potential.

Reactive oxygen species (ROS) regulates the replication of human immunodeficiency virus (HIV-1) during infection. However, the application of this knowledge to develop therapeutic strategies remained unsuccessful due to the harmful consequences of manipulating cellular antioxidant systems. Here, we show that vanadium pentoxide (V2 O5 ) nanosheets functionally mimic natural glutathione peroxidase activity to mitigate ROS associated with HIV-1 infection without adversely affecting cellular physiology. Using genetic reporters of glutathione redox potential and hydrogen peroxide, we showed that V2 O5 nanosheets catalyze ROS neutralization in HIV-1-infected cells and uniformly block viral reactivation and replication. Mechanistically, V2 O5 nanosheets suppressed HIV-1 by affecting the expression of pathways coordinating redox balance, virus transactivation (e.g., NF-κB), inflammation, and apoptosis. Importantly, a combination of V2 O5 nanosheets with a pharmacological inhibitor of NF-κB (BAY11-7082) abrogated reactivation of HIV-1. Lastly, V2 O5 nanosheets inhibit viral reactivation upon prostratin stimulation of latently infected CD4+ T cells from HIV-infected patients receiving suppressive antiretroviral therapy. Our data successfully revealed the usefulness of V2 O5 nanosheets against HIV and suggested nanozymes as future platforms to develop interventions against infectious diseases.

A Cerium Vanadate Nanozyme with Specific Superoxide Dismutase Activity Regulates Mitochondrial Function and ATP Synthesis in Neuronal Cells.

Nanoparticles that functionally mimic the activity of metal-containing enzymes (metallo-nanozymes) are of therapeutic importance for treating various diseases. However, it is still not clear whether such nanozymes can completely substitute the function of natural enzymes in living cells. In this work, we show for the first time that a cerium vanadate (CeVO4 ) nanozyme can substitute the function of superoxide dismutase 1 and 2 (SOD1 and SOD2) in the neuronal cells even when the natural enzyme is down-regulated by specific gene silencing. The nanozyme prevents the mitochondrial damage in SOD1- and SOD2-depleted cells by regulating the superoxide levels and restores the physiological levels of the anti-apoptotic Bcl-2 family proteins. Furthermore, the nanozyme effectively prevents the mitochondrial depolarization, leading to a significant improvement in the cellular levels of ATP under oxidative stress.

Modulation of Redox Signaling and Thiol Homeostasis in Red Blood Cells by Peroxiredoxin Mimetics.

Red blood cell death or erythrocyte apoptosis (eryptosis) is generally mediated by oxidative stress, energy depletion, heavy metals exposure, or xenobiotics. As erythrocytes are a major target for oxidative stress due to their primary function as O2-carrying cells, they possess an efficient antioxidant defense system consisting of glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), and peroxiredoxin 2 (Prx2). The oxidative stress-mediated activation of the Ca2+-permeable cation channel results in Ca2+ entry into the cells and subsequent cell death. Herein, we describe for the first time that selenium compounds having intramolecular diselenide or selenenyl sulfide moieties can prevent the oxidative stress-induced eryptosis by exhibiting an unusual Prx2-like redox activity under conditions when the cellular Prx2 and CAT enzymes are inhibited.

Nanoceria-Based Phospholipase-Mimetic Cell Membrane Disruptive Antibiofilm Agents.

Continuous mounting of antibiotic resistance due to the narrow range of mechanisms targeted poses tremendous threat to global health. Highly resistant pathogenic bacteria dwelling in the biofilm mode on the surface of medical devices has increased the susceptibility of chronic as well as healthcare-associated infections. Lantipeptides have shown promising membrane disruption of Gram-positive bacteria, leading to programmed cell death, but they are impermeable and hence ineffective to the outer cell membrane of Gram-negative bacteria. Herein, we report for the first time that a polymer-coated nanoceria (PAA-Cnp) having phospholipase-mimetic activity can target the cell membrane of both Gram-negative and Gram-positive bacteria. The nanozyme shows promising membrane disruption-based bactericidal activity against a broad spectrum of pathogenic as well as biofilm-encased bacteria. The unprecedented nanozyme-based strategy described in this paper is useful in preventing biofilm formation on medical devices such as urinary catheters.

Highly Stable Pyrimidine Based Luminescent Copper Nanoclusters with Superoxide Dismutase Mimetic and Nitric Oxide Releasing Activity.

Copper nanoclusters (CuNCs) are emerging as an interesting class of materials for various biomedical applications. In this work, we have designed highly stable nucleobase-capped luminescent CuNCs and studied the effect of substituents on the cluster composition and photophysical properties. The NCs exhibit exceptional stability in ambient atmosphere and show significant variation in the emission properties with a change in position of substituents on the ligand, thiouracil. This study represents the first example of a nanocluster that functionally mimics the activity of a major antioxidant enzyme, superoxide dismutase (SOD). In addition to their enzyme-mimetic activity, the CuNCs evince controlled release of nitric oxide (NO), a key gaseous molecule of endothelial system from S-nitrosothiol, S-nitrosoglutathione (GSNO). Further, to a greater significance, these luminescent CuNCs are readily taken up by the mammalian cells and exhibit low toxicity. The superoxide dismutase and NO releasing activity of the fluorescent, biocompatible copper nanoclusters suggest their potential application in both therapeutics and bioimaging.

Halogen Bonding in Biomimetic Deiodination of Thyroid Hormones and their Metabolites and Dehalogenation of Halogenated Nucleosides.

Thyroid hormones (THs) are key players in the endocrine system and play pivotal roles in carbohydrate and fat metabolism, protein synthesis, overall growth, and brain development. The thyroid gland predominantly produces thyroxine or 3,5,3',5'-tetraiodothyronine (T4) as a prohormone; three isoforms of a mammalian selenoenzyme-iodothyronine deiodinase (DIO1, DIO2 and DIO3)-catalyze the regioselective deiodination of T4 to produce biologically active and inactive metabolites. Whereas DIO1 catalyzes both 5- and 5'-deiodination of T4, DIO2 and DIO3 selectively mediate 5- and 5'-deiodination, respectively. In this review we discuss the regioselective deiodination of THs in the presence of organochalcogen compounds. Naphthalene-based compounds containing sulfur and/or selenium at the peri positions mediate regioselective 5-deiodination of THs, detailed mechanistic studies having revealed that the heterolytic cleavage of the C-I bond is facilitated by the formation of cooperative Se/S⋅⋅⋅I halogen bonds and Se/S⋅⋅⋅Se chalcogen bonds. We also discuss the biomimetic deiodination of several TH metabolites, including sulfated THs, iodothyronamines, and iodotyrosines. A brief discussion on the dehalogenation of halogenated nucleosides and nucleobases in the presence of organochalcogen compounds is also included.

Modeling Thioredoxin Reductase-Like Activity with Cyclic Selenenyl Sulfides: Participation of an NH⋠⋠⋠Se Hydrogen Bond through Stabilization of the Mixed Se-S Intermediate.

At the redox-active center of thioredoxin reductase (TrxR), a selenenyl sulfide (Se-S) bond is formed between Cys497 and Sec498, which is activated into the thiolselenolate state ([SH,Se- ]) by reacting with a nearby dithiol motif ([SHCys59 ,SHCys64 ]) present in the other subunit. This process is achieved through two reversible steps: an attack of a cysteinyl thiol of Cys59 at the Se atom of the Se-S bond and a subsequent attack of a remaining thiol at the S atom of the generated mixed Se-S intermediate. However, it is not clear how the kinetically unfavorable second step progresses smoothly in the catalytic cycle. A model study that used synthetic selenenyl sulfides, which mimic the active site structure of human TrxR comprising Cys497, Sec498, and His472, suggested that His472 can play a key role by forming a hydrogen bond with the Se atom of the mixed Se-S intermediate to facilitate the second step. In addition, the selenenyl sulfides exhibited a defensive ability against H2 O2 -induced oxidative stress in cultured cells, which suggests the possibility for medicinal applications to control the redox balance in cells.

Directing Traffic: Halogen-Bond-Mediated Membrane Transport.

The plasma membrane regulates the transport of molecules into the cell. Small hydrophobic molecules can diffuse directly across the lipid bilayer. However, larger molecules require specific transporters for their entry into the cell. Regulating the cellular entry of small molecules and proteins is a challenging task. The introduction of halogen, particularly iodine, to small molecules and proteins is emerging to be a promising strategy to improve the cellular uptake. Recent studies reveal that a simple substitution of hydrogen atom with iodine not only increases the cellular uptake, but also regulates the membrane transport. The strong halogen-bond-forming ability of iodine atoms plays a crucial role in the transport and the introduction of iodine may provide an efficient strategy for studying membrane activity and cellular functions and improving the delivery of therapeutic agents. This Concept article does not provide a comprehensive picture of membrane transport but highlights halogen-substitution as a novel strategy for understanding and regulating the cell-membrane traffic.

Crystal-facet-dependent denitrosylation: modulation of NO release from S-nitrosothiols by Cu2O polymorphs.

Nitric oxide (NO), a gaseous small molecule generated by the nitric oxide synthase (NOS) enzymes, plays key roles in signal transduction. The thiol groups present in many proteins and small molecules undergo nitrosylation to form the corresponding S-nitrosothiols. The release of NO from S-nitrosothiols is a key strategy to maintain the NO levels in biological systems. However, the controlled release of NO from the nitrosylated compounds at physiological pH remains a challenge. In this paper, we describe the synthesis and NO releasing ability of Cu2O nanomaterials and provide the first experimental evidence that the nanocrystals having different crystal facets within the same crystal system exhibit different activities toward S-nitrosothiols. We used various imaging techniques and time-dependent spectroscopic measurements to understand the nature of catalytically active species involved in the surface reactions. The denitrosylation reactions by Cu2O can be carried out multiple times without affecting the catalytic activity.

CeVO4 Nanozymes Catalyze the Reduction of Dioxygen to Water without Releasing Partially Reduced Oxygen Species.

In this study, we report a remarkably active CeVO4 nanozyme that functionally mimics cytochrome c oxidase (CcO), the terminal enzyme in the respiratory electron transport chain, by catalyzing a four-electron reduction of dioxygen to water. The nanozyme catalyzes the reaction by using cytochrome c (Cyt c), the biological electron donor for CcO, at physiologically relevant pH. The CcO activity of the CeVO4 nanozymes depends on the relative ratio of surface Ce3+ /Ce4+ ions, the presence of V5+ and the surface-Cyt c interactions. The complete reduction of oxygen to water takes place without release of any partially reduced oxygen species (PROS) such as superoxide, peroxide and hydroxyl radicals.

Probing the Formation of a Seleninic Acid in Living Cells by the Fluorescence Switching of a Glutathione Peroxidase Mimetic.

Glutathione peroxidase (GPx) is a selenoenzyme that protects cells against oxidative damage. Although the formation of a seleninic acid (-SeO2 H) by this enzyme during oxidative stress has been proposed, a selenic acid has not been identified in cells. Herein, we report that the formation of a seleninic acid can be monitored in living cells by using a redox-active ebselen analogue with a naphthalimide fluorophore. The probe reacts with H2 O2 to generate the highly fluorescent seleninic acid. The electron withdrawing nature of the -SeO2 H moiety and strong Se⋅⋅⋅O interactions, which prevent the photoinduced electron transfer, are responsible for the fluorescence.

Modelling the Inhibition of Selenoproteins by Small Molecules Using Cysteine and Selenocysteine Derivatives.

Small molecule-based electrophilic compounds such as 1-chloro-2,4-dinitrobenzene (CDNB) and 1-chloro-4-nitrobenzene (CNB) are currently being used as inhibitors of cysteine- and selenocysteine-containing proteins. CDNB has been used extensively to determine the activity of glutathione S-transferase and to deplete glutathione (GSH) in mammalian cells. Also, CDNB has been shown to irreversibly inhibit thioredoxin reductase (TrxR), a selenoenzyme that catalyses the reduction of thioredoxin (Trx). Mammalian TrxR has a C-terminal active site motif, Gly-Cys-Sec-Gly, and both the cysteine and selenocysteine residues could be the targets of the electrophilic reagents. In this paper we report on the stability of a series of cysteine and selenocysteine derivatives that can be considered as models for the selenoenzyme-inhibitor complexes. We show that these derivatives react with H2 O2 to generate the corresponding selenoxides, which undergo spontaneous elimination to produce dehydroalanine. In contrast, the cysteine derivatives are stable towards such elimination reactions. We also demonstrate, for the first time, that the arylselenium species eliminated from the selenocysteine derivatives exhibit significant redox activity by catalysing the reduction of H2 O2 in the presence of GSH (GPx (glutathione peroxidase)-like activity), which suggests that such redox modulatory activity of selenium compounds may have a significant effect on the cellular redox state during the inhibition of selenoproteins.