Boronate-Based Oxidant-Responsive Derivatives of Acetaminophen as Proinhibitors of Myeloperoxidase.

Myeloperoxidase (MPO) is an important component of the human innate immune system and the main source of a strong oxidizing and chlorinating species, hypochlorous acid (HOCl). Inadvertent, misplaced, or excessive generation of HOCl by MPO is associated with multiple human inflammatory diseases. Therefore, there is a considerable interest in the development of MPO inhibitors. Here, we report the synthesis and characterization of a boronobenzyl derivative of acetaminophen (AMBB), which can function as a proinhibitor of MPO and release acetaminophen, the inhibitor of chlorination cycle of MPO, in the presence of inflammatory oxidants, i.e., hydrogen peroxide, hypochlorous acid, or peroxynitrite. We demonstrate that the AMBB proinhibitor undergoes conversion to acetaminophen by all three oxidants, with the involvement of the primary phenolic product intermediate, with relatively long half-life at pH 7.4. The determined rate constants of the reaction of the AMBB proinhibitor with hydrogen peroxide, hypochlorous acid, or peroxynitrite are equal to 1.67, 1.6 × 104, and 1.0 × 106 M-1 s-1, respectively. AMBB showed lower MPO inhibitory activity (IC50 > 0.3 mM) than acetaminophen (IC50 = 0.14 mM) toward MPO-dependent HOCl generation. Finally, based on the determined reaction kinetics and the observed inhibitory effects of two plasma components, uric acid and albumin, on the extent of AMBB oxidation by ONOO- and HOCl, we conclude that ONOO- is the most likely potential activator of AMBB in human plasma.

Bio-Approach for Obtaining Enantiomerically Pure Clopidogrel with the Use of Ionic Liquids.

Clopidogrel is a chiral compound widely used as an antiplatelet medication that lowers the risk of blood clots, strokes, and heart attacks. The main aim of the study presented herein was to obtain (S)-clopidogrel, which is commercially available in treatments, via the kinetic resolution of racemic clopidogrel carboxylic acid with the use of lipase from Candida rugosa and a two-phase reaction medium containing an ionic liquid. For this purpose, the enantioselective biotransformation of clopidogrel carboxylic acid and chiral chromatographic separation with the use of a UPLC-MS/MS system were optimized. The best kinetic resolution parameters were obtained by using a catalytic system containing lipase from Candida rugosa OF as a biocatalyst, cyclohexane and [EMIM][BF4] as a two-phase reaction medium, and methanol as an acyl acceptor. The enantiomeric excess of the product was eep = 94.21% ± 1.07 and the conversion was c = 49.60% ± 0.57%, whereas the enantioselectivity was E = 113.40 ± 1.29. The performed study proved the possibility of obtaining (S)-clopidogrel with the use of lipase as a biocatalyst and a two-phase reaction medium containing an ionic liquid, which is in parallel with green chemistry methodology and does not require environmentally harmful conditions.

Degradation of Chemical Components of Thermally Modified Robinia pseudoacacia L. Wood and Its Effect on the Change in Mechanical Properties.

Currently, emphasis is placed on using environmentally friendly materials both from a structural point of view and the application of protective means. For this reason, it is advisable to deal with the thermal modification of wood, which does not require the application of protective substances, to increase its service life. The main reason for the thermal modification of black locust is that although black locust grows abundantly in our country, it has no industrial use. It is mainly used outdoors, where thermal modification could increase its resistance. This article deals with the thermal modification of black locust wood (Robinia pseudoacacia L.) and the impact of this modification on the chemical components of the wood with an overlap in the change in mechanical properties compared to untreated wood. Static (LOP, MOR, and MOE) and dynamic (IBS) bending properties were evaluated as representative mechanical properties. At the same time, the impact of thermal modification on the representation of chemical components of wood (cellulose, lignin, hemicellulose) was also tested. As a result of the heat treatment, the mechanical properties gradually decreased as the temperature increased. The highest decrease in mechanical values found at 210 °C was 43.7% for LOP and 45.1% for MOR. Thermal modification caused a decrease in the content of wood polysaccharides (the decrease in hemicelluloses content was 33.2% and the drop in cellulose was about 29.9% in samples treated at 210 °C), but the relative amount of lignin in the wood subjected to increased temperature was higher due to autocondensation, and mainly because of polysaccharide loss. Based on the correlations between chemical and mechanical changes caused by thermal modification, it is possible to observe the effects of reducing the proportions of chemical components and changes in their characteristic properties (DP, TCI) on the reduction in mechanical properties. The results of this research serve to better understand the behavior of black locust wood during thermal modification, which can primarily be used to increase its application use.

Kinetic Study on the Reactivity of Azanone (HNO) toward Cyclic C-Nucleophiles.

Azanone (HNO) is an elusive electrophilic reactive nitrogen species of growing pharmacological and biological significance. Here, we present a comparative kinetic study of HNO reactivity toward selected cyclic C-nucleophiles under aqueous conditions at pH 7.4. We applied the competition kinetics method, which is based on the use of a fluorescein-derived boronate probe FlBA and two parallel HNO reactions: with the studied scavenger or with O2 (k = 1.8 × 104 M-1s-1). We determined the second-order rate constants of HNO reactions with 13 structurally diverse C-nucleophiles (k = 33-20,000 M-1s-1). The results show that the reactivity of HNO toward C-nucleophiles depends strongly on the structure of the scavenger. The data are supported with quantum mechanical calculations. A comprehensive discussion of the HNO reaction with C-nucleophiles is provided.

Testing for Ketoprofen Binding to HSA Coated Magnetic Nanoparticles under Normal Conditions and after Oxidative Stress.

Binding and transport of ligands is one of the most important functions of human blood serum proteins. Human serum albumin is found in plasma at the highest concentration. Because of this, it is important to study protein-drug interactions for this albumin. Since there is no single model describing this interaction, it is necessary to measure it for each active substance. Drug binding should also be studied in conditions that simulate pathological conditions of the body, i.e., after oxidative stress. Due to this, it is expected that the methods for testing these interactions need to be easy and fast. In this study, albumin immobilized on magnetic nanoparticles was successfully applied in the study of protein-drug binding. Ketoprofen was selected as a model drug and interactions were tested under normal conditions and artificially induced oxidative stress. The quality of obtained results for immobilized protein was confirmed with those for free albumin and literature data. It was shown that the type of magnetic core coverage does not affect the quality of the obtained results. In summary, a new, fast, effective, and universal method for testing protein-drug interactions was proposed, which can be performed in most laboratories.

Decomposition of Piloty's acid derivatives - Toward the understanding of factors controlling HNO release.

The recent interest in the clinical applications of Piloty's acid derivatives as HNO donors for the treatment of cardiovascular system dysfunction has led us to the examination of factors controlling HNO release from selected ortho-substituted N-hydroxysulfonamides. Here we present the kinetic and quantum mechanical studies on the mechanism of HNO release from selected ortho-substituted N-hydroxysulfonamides and in vivo examination of the antiaggregatory properties of N-hydroxy-(2-bromobenzene)sulfonamide complex with sodium salt of ß-cyclodextrin sulfobutyl ethers-ethyl ethers as compared with Angeli's salt.

Mitochondria-Targeted Triphenylphosphonium-Based Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications.

Mitochondria are recognized as one of the most important targets for new drug design in cancer, cardiovascular, and neurological diseases. Currently, the most effective way to deliver drugs specifically to mitochondria is by covalent linking a lipophilic cation such as an alkyltriphenylphosphonium moiety to a pharmacophore of interest. Other delocalized lipophilic cations, such as rhodamine, natural and synthetic mitochondria-targeting peptides, and nanoparticle vehicles, have also been used for mitochondrial delivery of small molecules. Depending on the approach used, and the cell and mitochondrial membrane potentials, more than 1000-fold higher mitochondrial concentration can be achieved. Mitochondrial targeting has been developed to study mitochondrial physiology and dysfunction and the interaction between mitochondria and other subcellular organelles and for treatment of a variety of diseases such as neurodegeneration and cancer. In this Review, we discuss efforts to target small-molecule compounds to mitochondria for probing mitochondria function, as diagnostic tools and potential therapeutics. We describe the physicochemical basis for mitochondrial accumulation of lipophilic cations, synthetic chemistry strategies to target compounds to mitochondria, mitochondrial probes, and sensors, and examples of mitochondrial targeting of bioactive compounds. Finally, we review published attempts to apply mitochondria-targeted agents for the treatment of cancer and neurodegenerative diseases.

Triazene salts: Design, synthesis, ctDNA interaction, lipophilicity determination, DFT calculation, and antiproliferative activity against human cancer cell lines.

Synthesis, characterization and investigation of antiproliferative activity of nine triazene salts against human cancer cells lines (MV-4-11, MCF-7, JURKAT, HT-29, Hep-G2, HeLa, Du-145 and DAUDI), and normal human mammary epithelial cell line (MCF7-10A) is presented. The structures of novel compounds were determined using 1H and 13C NMR, and GC-APCI-MS analyses. Among the derivatives, compound 2c, 2d, 2e and 2f has very strong activity against biphenotypic B myelomonocytic leukemia MV4-11, with IC50 values from 5.42 to 7.69 µg/ml. The cytotoxic activity of compounds 2c-2f against normal human mammary gland epithelial cells MCF-10A is 6-11 times lower than against cancer cell lines. Our results also show that compounds 2c and 2f have very strong activity against DAUDI and HT-29 with IC50 4.91 µg/ml and 5.59 µg/ml, respectively. Their lipophilicity was determined using reversed-phase ultra-performance liquid chromatography and correlated with antiproliferative activity. Our UV-Vis spectroscopic results indicate also that triazene salts tends to interact with negatively charged DNA phosphate chain. To support the experiment, theoretical calculations of the 1H NMR shifts were carried out within the Density Functional Theory.

Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation.

Using high throughput screening-compatible assays for superoxide and hydrogen peroxide, we identified potential inhibitors of the NADPH oxidase (Nox2) isoform from a small library of bioactive compounds. By using multiple probes (hydroethidine, hydropropidine, Amplex Red, and coumarin boronate) with well defined redox chemistry that form highly diagnostic marker products upon reaction with superoxide (O2 (̇̄)), hydrogen peroxide (H2O2), and peroxynitrite (ONOO(-)), the number of false positives was greatly decreased. Selected hits for Nox2 were further screened for their ability to inhibit ONOO(-)formation in activated macrophages. A new diagnostic marker product for ONOO(-)is reported. We conclude that the newly developed high throughput screening/reactive oxygen species assays could also be used to identify potential inhibitors of ONOO(-)formed from Nox2-derived O2 (̇̄)and nitric oxide synthase-derived nitric oxide.

A kinetic study on the reactivity of azanone (HNO) toward its selected scavengers: Insight into its chemistry and detection.

Recently, azanone (HNO), which is the protonated one-electron reduction product of ·NO, has gained considerable attention due to its unique pharmacological effects. Although there has been much progress in understanding HNO biology and chemistry, it remains the most elusive reactive nitrogen species. Herein, we applied the competition kinetics method, based on two parallel HNO reactions with the different scavengers and molecular oxygen (kO2 = (1.8 ± 0.3) × 104 M-1 s-1), to determine the rate constants for the reactions of HNO with its selected co-reactants. The rate constants for the reactions of HNO with nitrite (k = (5.0 ± 0.9) × 103 M-1s-1), hydroxylamine (k = (2.1 ± 0.4) × 104 M-1s-1), sulfite (k = (1.2 ± 0.2) × 106 M-1 s-1), thiosulfate (k = (2.2 ± 0.7) × 104 M-1 s-1), benzenesulfinate (k = (4.4 ± 0.9) × 104 M-1 s-1), 2-bromobenzenesulfinate (k = (5.0 ± 1.2) × 104 M-1 s-1), nitrosoglutathione (k = (2.4 ± 0.7) × 104 M-1s-1), nitrosobenzene (k > 1.5 × 105 M-1 s-1), 2-nitroso-1-naphthol (k = (1.0 ± 0.2) × 106 M-1 s-1), triphenylphosphine (k > 7.3 × 106 M-1 s-1), triphenylphosphine-3,3',3″-trisulfonate (k = (3.0 ± 0.5) × 106 M-1 s-1), tris-carboxyethylphosphine (k = (1.2 ± 0.3) × 107 M-1 s-1), a triphenylphosphine-based P-CM fluorogenic probe (k > 1.2 × 107 M-1 s-1), the TEMPO-9-AC fluorogenic probe (k = (9 ± 2) × 104 M-1 s-1) and 4-acetamido-TEMPO (k = (8 ± 2) × 104 M-1s-1) are reported. The implications of these HNO reactions are also discussed. The data presented in this paper are a valuable contribution to the incompletely understood reactivity of HNO.

Characterization of Fluorescein-Based Monoboronate Probe and Its Application to the Detection of Peroxynitrite in Endothelial Cells Treated with Doxorubicin.

Boronate probes have emerged recently as a versatile tool for the detection of reactive oxygen and nitrogen species. Here, we present the characterization of a fluorescein-based monoboronate probe, a 4-(pinacol boronate)benzyl derivative of fluorescein methyl ester (FBBE), that proved to be useful to detect peroxynitrite in cell culture experiments. The reactivity of FBBE toward peroxynitrite as well hypochlorite, hydrogen peroxide, and tyrosyl hydroperoxide was determined. Second-order rate constants of the reactions of FBBE with peroxynitrite, HOCl, and H2O2 at pH 7.4 were equal to (2.8 ± 0.2) × 10(5) M(-1) s(-1), (8.6 ± 0.5) × 10(3) M(-1) s(-1), and (0.96 ± 0.03) M(-1) s(-1), respectively. The presence of glutathione completely blocked the oxidation of the probe by HOCl and significantly inhibited its oxidation by H2O2 and tyrosyl hydroperoxide but not by peroxynitrite. The oxidative conversion of the probe was also studied in the systems generating singlet oxygen, superoxide radical anion, and nitric oxide in the presence and absence of glutathione. Spectroscopic characterization of FBBE and its oxidation product has been also performed. The differences in the reactivity pattern were supported by DFT quantum mechanical calculations. Finally, the FBBE probe was used to study the oxidative stress in endothelial cells (Ea.hy926) incubated with doxorubicin, a quinone anthracycline antibiotic. In endothelial cells pretreated with doxorubicin, FBBE was oxidized, and this effect was reversed by PEG-SOD and L-NAME but not by catalase.

Nitroxyl (HNO) reacts with molecular oxygen and forms peroxynitrite at physiological pH. Biological Implications.

Nitroxyl (HNO), the protonated one-electron reduction product of NO, remains an enigmatic reactive nitrogen species. Its chemical reactivity and biological activity are still not completely understood. HNO donors show biological effects different from NO donors. Although HNO reactivity with molecular oxygen is described in the literature, the product of this reaction has not yet been unambiguously identified. Here we report that the decomposition of HNO donors under aerobic conditions in aqueous solutions at physiological pH leads to the formation of peroxynitrite (ONOO(-)) as a major intermediate. We have specifically detected and quantified ONOO(-) with the aid of boronate probes, e.g. coumarin-7-boronic acid or 4-boronobenzyl derivative of fluorescein methyl ester. In addition to the major phenolic products, peroxynitrite-specific minor products of oxidation of boronate probes were detected under these conditions. Using the competition kinetics method and a set of HNO scavengers, the value of the second order rate constant of the HNO reaction with oxygen (k = 1.8 × 10(4) m(-1) s(-1)) was determined. The rate constant (k = 2 × 10(4) m(-1) s(-1)) was also determined using kinetic simulations. The kinetic parameters of the reactions of HNO with selected thiols, including cysteine, dithiothreitol, N-acetylcysteine, captopril, bovine and human serum albumins, and hydrogen sulfide, are reported. Biological and cardiovascular implications of nitroxyl reactions are discussed.

Application of vibrational spectroscopy supported by theoretical calculations in identification of amorphous and crystalline forms of cefuroxime axetil.

FT-IR and Raman scattering spectra of cefuroxime axetil were proposed for identification studies of its crystalline and amorphous forms. An analysis of experimental spectra was supported by quantum-chemical calculations performed with the use of B3LYP functional and 6-31G(d,p) as a basis set. The geometric structure of a cefuroxime axetil molecule, HOMO and LUMO orbitals, and molecular electrostatic potential were also determined by using DFT (density functional theory). The benefits of applying FT-IR and Raman scattering spectroscopy for characterization of drug subjected to degradation were discussed.

Kinetic resolution of profens by enantioselective esterification catalyzed by Candida antarctica and Candida rugosa lipases.

Profens (2-arylpropionic acids) are known as one of the major nonsteroidal antiinflammatory drugs (NSAIDs) used in the treatment of inflammation associated with tissue injury. The inflammatory activity of profens is mainly due to their (S)-enantiomer, whereas they are commercially available not only as pure enantiomers, but as racemates as well. There are several methods widely used in order to obtain enantiomerically pure compounds, however, the kinetic resolution with the application of lipases as biocatalysts may have an added advantage in the production of optically pure active pharmaceutical ingredients, such as milder reaction conditions, reduced energy requirements, and production costs. The aim of this study was to compare the results described in the literature in the case of the influence of reaction medium, alcohol moiety, and reaction temperature on the catalytic activity of lipases from Candida antarctica and Candida rugosa.

Evaluation of a novel pyridinium cation-linked styryl-based boronate probe for the detection of selected inflammation-related oxidants.

Reactive oxygen and nitrogen species (RONS) are a range of chemical individuals produced by living cells that contribute to the proper functioning of organisms. Cells under oxidative and nitrative stress show excessive production of RONS (including hydrogen peroxide, H2O2, hypochlorous acid, HOCl, and peroxynitrite, ONOO-) which may result in a damage proteins, lipids, and genetic material. Thus, the development of probes for in vivo detection of such oxidants is an active area of research, focusing on molecular redox sensors, including boronate-caged fluorophores. Here, we report a boronate-based styryl probe with a cationic pyridinium moiety (BANEP+) for the fluorescent detection of selected biological oxidants in vitro and in vivo. We compare the chemical reactivity of the BANEP+ probe toward H2O2, HOCl, and ONOO- and examine the influence of the major intracellular non-enzymatic antioxidant molecule, glutathione (GSH). We demonstrate that, at the physiologically relevant GSH concentration, the BANEP+ probe is efficiently oxidized by peroxynitrite, forming its phenolic derivative HNEP+. GSH does not affect the fluorescence properties of the BANEP+ and HNEP+ dyes. Finally, we report the identification of a novel type of molecular marker, with the boronate moiety replaced by the iodine atom, formed from the probe in the presence of HOCl and iodide anion. We conclude that the reported chemical reactivity and structural features of the BANEP+ probe may be a basis for the development of new red fluorescent probes for in vitro and in vivo detection of ONOO-.

Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses.

Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants.

Reaction between peroxynitrite and triphenylphosphonium-substituted arylboronic acid isomers: identification of diagnostic marker products and biological implications.

Aromatic boronic acids react rapidly with peroxynitrite (ONOO(-)) to yield phenols as major products. This reaction was used to monitor ONOO(-) formation in cellular systems. Previously, we proposed that the reaction between ONOO(-) and arylboronates (PhB(OH)2) yields a phenolic product (major pathway) and a radical pair PhB(OH)2O(•-)···(•)NO2 (minor pathway). [Sikora, A. et al. (2011) Chem. Res. Toxicol. 24, 687-697]. In this study, we investigated the influence of a bulky triphenylphosphonium (TPP) group on the reaction between ONOO(-) and mitochondria-targeted arylboronate isomers (o-, m-, and p-MitoPhB(OH)2). Results from the electron paramagnetic resonance (EPR) spin-trapping experiments unequivocally showed the presence of a phenyl radical intermediate from meta and para isomers, and not from the ortho isomer. The yield of o-MitoPhNO2 formed from the reaction between o-MitoPhB(OH)2 and ONOO(-) was not diminished by phenyl radical scavengers, suggesting a rapid fragmentation of the o-MitoPhB(OH)2O(•-) radical anion with subsequent reaction of the resulting phenyl radical with (•)NO2 in the solvent cage. The DFT quantum mechanical calculations showed that the energy barrier for the dissociation of the o-MitoPhB(OH)2O(•-) radical anion is significantly lower than that of m-MitoPhB(OH)2O(•-) and p-MitoPhB(OH)2O(•-) radical anions. The nitrated product, o-MitoPhNO2, is not formed by the nitrogen dioxide radical generated by myeloperoxidase in the presence of the nitrite anion and hydrogen peroxide, indicating that this specific nitrated product may be used as a diagnostic marker product for ONOO(-). Incubation of o-MitoPhB(OH)2 with RAW 264.7 macrophages activated to produce ONOO(-) yielded the corresponding phenol o-MitoPhOH as well as the diagnostic nitrated product, o-MitoPhNO2. We conclude that the ortho isomer probe reported here is most suitable for specific detection of ONOO(-) in biological systems.

Wood Surface Finishing with Transparent Lacquers Intended for Indoor Use, and the Colour Resistance of These Surfaces during Accelerated Aging.

This work evaluates the effects of accelerated aging on the discolouration of surface-treated spruce wood and oak wood coated with solvent-based polyurethane lacquers, and surface-treated spruce wood coated with water-based transparent coating systems. All concerned coating materials were intended for indoor use. It was also explored how the colour stability of spruce wood and oak wood surfaces treated with solvent-based polyurethane lacquers was affected by wood surface layer modifications with pigment or stain mordants applied before these lacquers. Another issue studied was how the lignin stabilizer admixed into the primer and pigments admixed into the top coating layers affected the stability of water-based coating systems on spruce. The experimental results showed that the accelerated aging process with a simulation of indoor conditions induced significant discolouration of wood surfaces coated with solvent-based polyurethane lacquers and water-based coating systems. There were also confirmed significant impacts of all the studied factors (wood species, lacquer/coating system type, lacquer modification, wood pre-treatment with pigment and stain mordants). The spruce wood surfaces coated with solvent-based polyurethane lacquers were less stable (ΔE = 10-19, dependent on the lacquer type) than the oak surfaces treated in the same ways (ΔE = 4-11). There were also confirmed significant impacts of the particular surface treatment on the colour stability as well as significant impacts of wood surface pre-treatment with pigment and stain mordants (ΔE = 4-17-for spruce wood, and ΔE = 5.5-13-for oak wood). In the case of water-based lacquers, the ΔE values ranged between 3 and 11 (according to the coating system type). The results show that an appropriate UV absorbent combined with an appropriate lignin stabilizer and pigment mordant may enable attaining the required colour stability for a given surface treatment applied on a given wood species.

Effect of a Nanocellulose Addition on the Mechanical Properties of Paper.

Nowadays, the emphasis is on increasing the durability of all products. For this reason, it is also advisable to look into extending the durability of paper products. The main reason for using flax pulp is that flax and cotton pulp are widely used for the production of banknotes due to their higher strength. This paper deals with flax pulp with the addition of nanocellulose, which should further enhance the mechanical properties of the pulp. The tensile strength, breaking length, and tensile energy absorption index were evaluated as the key mechanical properties. At the same time, the effect of the addition of nanocellulose, whether it was added to the pulp mass or applied to the later produced paper as a spray or coating, was tested in comparison to paper without the addition of nanocellulose. The best mechanical properties, i.e., tensile strength, were achieved for the highest addition of 5% of nanocellulose into the pulp, at 24.3 Nm∙g-1, and for the coating application, at 28.7 Nm∙g-1, compared to the flax pulp without the addition, where the tensile strength was 20.5 Nm∙g-1. The results of this research are used for the assessment of nanocellulose as a natural compatible additive to enhance the strength properties of cellulose-based materials.

VEO-IBD NOX1 variant highlights a structural region essential for NOX/DUOX catalytic activity.

Inflammatory bowel diseases (IBD) are chronic intestinal disorders that result from an inappropriate inflammatory response to the microbiota in genetically susceptible individuals, often triggered by environmental stressors. Part of this response is the persistent inflammation and tissue injury associated with deficiency or excess of reactive oxygen species (ROS). The NADPH oxidase NOX1 is highly expressed in the intestinal epithelium, and inactivating NOX1 missense mutations are considered a risk factor for developing very early onset IBD. Albeit NOX1 has been linked to wound healing and host defence, many questions remain about its role in intestinal homeostasis and acute inflammatory conditions. Here, we used in vivo imaging in combination with inhibitor studies and germ-free conditions to conclusively identify NOX1 as essential superoxide generator for microbiota-dependent peroxynitrite production in homeostasis and during early endotoxemia. NOX1 loss-of-function variants cannot support peroxynitrite production, suggesting that the gut barrier is persistently weakened in these patients. One of the loss-of-function NOX1 variants, NOX1 p. Asn122His, features replacement of an asparagine residue located in a highly conserved HxxxHxxN motif. Modelling the NOX1-p22phox complex revealed near the distal heme an internal pocket restricted by His119 and Asn122 that is part of the oxygen reduction site. Functional studies in several human NADPH oxidases show that substitution of asparagine with amino acids with larger side chains is not tolerated, while smaller side chains can support catalytic activity. Thus, we identified a previously unrecognized structural feature required for the electron transfer mechanism in human NADPH oxidases.