The role of S-nitrosoglutathione reductase (GSNOR) in human disease and therapy

S-nitrosoglutathione reductase (GSNOR), or ADH5, is an enzyme in the alcohol dehydrogenase (ADH) family. It is unique when compared to other ADH enzymes in that primary short-chain alcohols are not its principle substrate. GSNOR metabolizes S-nitrosoglutathione (GSNO), S-hydroxymethylglutathione (the spontaneous adduct of formaldehyde and glutathione), and some alcohols. GSNOR modulates reactive nitric oxide (?NO) availability in the cell by catalyzing the breakdown of GSNO, and indirectly regulates S-nitrosothiols (RSNOs) through GSNO-mediated protein S-nitrosation. The dysregulation of GSNOR can significantly alter cellular homeostasis, leading to disease. GSNOR plays an important regulatory role in smooth muscle relaxation, immune function, inflammation, neuronal development and cancer progression, among many other processes. In recent years, the therapeutic inhibition of GSNOR has been investigated to treat asthma, cystic fibrosis and interstitial lung disease (ILD). The direct action of ?NO on cellular pathways, as well as the important regulatory role of protein S-nitrosation, is closely tied to GSNOR regulation and defines this enzyme as an important therapeutic target.     

ADH IB Expression,but Not ADH III,Is Decreased in Human Lung Cancer

Endogenous S-nitrosothiols, including S-nitrosoglutathione (GSNO), mediate nitric oxide (NO)-based signaling, inflammatory responses, and smooth muscle function. Reduced GSNO levels have been implicated in several respiratory diseases, and inhibition of GSNO reductase, (GSNOR) the primary enzyme that metabolizes GSNO, represents a novel approach to treating inflammatory lung diseases. Recently, an association between decreased GSNOR expression and human lung cancer risk was proposed in part based on immunohistochemical staining using a polyclonal GSNOR antibody. GSNOR is an isozyme of the alcohol dehydrogenase (ADH) family, and we demonstrate that the antibody used in those studies cross reacts substantially with other ADH proteins and may not be an appropriate reagent. We evaluated human lung cancer tissue arrays using monoclonal antibodies highly specific for human GSNOR with minimal cross reactivity to other ADH proteins. We verified the presence of GSNOR in ≥85% of specimens examined, and extensive analysis of these samples demonstrated no difference in GSNOR protein expression between cancerous and normal lung tissues. Additionally, GSNOR and other ADH mRNA levels were evaluated quantitatively in lung cancer cDNA arrays by qPCR. Consistent with our immunohistochemical findings, GSNOR mRNA levels were not changed in lung cancer tissues, however the expression levels of other ADH genes were decreased. ADH IB mRNA levels were reduced (>10-fold) in 65% of the lung cancer cDNA specimens. We conclude that the previously reported results showed an incorrect association of GSNOR and human lung cancer risk, and a decrease in ADH IB, rather than GSNOR, correlates with human lung cancer.     

Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings

Nitric oxide (NO) and related molecules such as peroxynitrite, S-nitrosoglutathione (GSNO), and nitrotyrosine, among others, are involved in physiological processes as well in the mechanisms of response to stress conditions. In sunflower seedlings exposed to five different adverse environmental conditions (low temperature, mechanical wounding, high light intensity, continuous light, and continuous darkness), key components of the metabolism of reactive nitrogen species (RNS) and reactive oxygen species (ROS), including the enzyme activities L-arginine-dependent nitric oxide synthase (NOS), S-nitrosogluthathione reductase (GSNOR), nitrate reductase (NR), catalase, and superoxide dismutase, the content of lipid hydroperoxide, hydrogen peroxide, S-nitrosothiols (SNOs), the cellular level of NO, GSNO, and GSNOR, and protein tyrosine nitration [nitrotyrosine (NO(2)-Tyr)] were analysed. Among the stress conditions studied, mechanical wounding was the only one that caused a down-regulation of NOS and GSNOR activities, which in turn provoked an accumulation of SNOs. The analyses of the cellular content of NO, GSNO, GSNOR, and NO(2)-Tyr by confocal laser scanning microscopy confirmed these biochemical data. Therefore, it is proposed that mechanical wounding triggers the accumulation of SNOs, specifically GSNO, due to a down-regulation of GSNOR activity, while NO(2)-Tyr increases. Consequently a process of nitrosative stress is induced in sunflower seedlings and SNOs constitute a new wound signal in plants.     

Structural and functional characterization of a plant S-nitrosoglutathione reductase from Solanum lycopersicum

S-nitrosoglutathione reductase (GSNOR), also known as S-(hydroxymethyl)glutathione (HMGSH) dehydrogenase, belongs to the large alcohol dehydrogenase superfamily, namely to the class III ADHs. GSNOR catalyses the oxidation of HMGSH to S-formylglutathione using a catalytic zinc and NAD⁺ as a coenzyme. The enzyme also catalyses the NADH-dependent reduction of S-nitrosoglutathione (GSNO). In plants, GSNO has been suggested to serve as a nitric oxide (NO) reservoir locally or possibly as NO donor in distant cells and tissues. NO and NO-related molecules such as S-nitrosothiols (S-NOs) play a central role in the regulation of normal plant physiological processes and host defence. The enzyme thus participates in the cellular homeostasis of S-NOs and in the metabolism of reactive nitrogen species. Although GSNOR has recently been characterized from several organisms, this study represents the first detailed biochemical and structural characterization of a plant GSNOR, that from tomato (Solanum lycopersicum). SlGSNOR gene expression is higher in roots and stems compared to leaves of young plants. It is highly expressed in the pistil and stamens and in fruits during ripening. The enzyme is a dimer and preferentially catalyses reduction of GSNO while glutathione and S-methylglutathione behave as non-competitive inhibitors. Using NAD⁺, the enzyme oxidizes HMGSH and other alcohols such as cinnamylalcohol, geraniol and ω-hydroxyfatty acids. The crystal structures of the apoenzyme, of the enzyme in complex with NAD⁺ and in complex with NADH, solved up to 1.9 Å resolution, represent the first structures of a plant GSNOR. They confirm that the binding of the coenzyme is associated with the active site zinc movement and changes in its coordination. In comparison to the well characterized human GSNOR, plant GSNORs exhibit a difference in the composition of the anion-binding pocket, which negatively influences the affinity for the carboxyl group of ω-hydroxyfatty acids.     

S-Nitrosoglutathione is a component of wound- and salicylic acid-induced systemic responses in Arabidopsis thaliana

S-Nitrosoglutathione (GSNO) is a bioactive, stable, and mobile reservoir of nitric oxide (NO), and an important player in defence responses to herbivory and pathogen attack in plants. It has been demonstrated previously that GSNO reductase (GSNOR) is the main enzyme responsible for the in vivo control of intracellular levels of GSNO. In this study, the role of S-nitrosothiols, in particular of GSNO, in systemic defence responses in Arabidopsis thaliana was investigated further. It was shown that GSNO levels increased rapidly and uniformly in injured Arabidopsis leaves, whereas in systemic leaves GSNO was first detected in vascular tissues and later spread over the parenchyma, suggesting that GSNO is involved in the transmission of the wound mobile signal through the vascular tissue. Moreover, GSNO accumulation was required to activate the jasmonic acid (JA)-dependent wound responses, whereas the alternative JA-independent wound-signalling pathway did not involve GSNO. Furthermore, extending previous work on the role of GSNOR in pathogenesis, it was shown that GSNO acts synergistically with salicylic acid in systemic acquired resistance activation. In conclusion, GSNOR appears to be a key regulator of systemic defence responses, in both wounding and pathogenesis.     

Function of S-nitrosoglutathione reductase (GSNOR) in plant development and under biotic/abiotic stress

During the last decade, it was established that the class III alcohol dehydrogenase (ADH3) enzyme, also known as glutathione-dependent formaldehyde dehydrogenase (FALDH; EC 1.2.1.1), catalyzes the NADH-dependent reduction of S-nitrosoglutathione (GSNO) and therefore was also designated as GSNO reductase. This finding has opened new aspects in the metabolism of nitric oxide (NO) and NO-derived molecules where GSNO is a key component. In this article, current knowledge of the involvement and potential function of this enzyme during plant development and under biotic/abiotic stress is briefly reviewed.Key words: nitric oxide, nitrosative stress, S-nitrosoglutathione reductase     

Structure-activity relationship of pyrrole based S-nitrosoglutathione reductase inhibitors: carboxamide modification

The enzyme S-nitrosoglutathione reductase (GSNOR) is a member of the alcohol dehydrogenase family (ADH) that regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). GSNO and SNOs are implicated in the pathogenesis of many diseases including those in respiratory, gastrointestinal, and cardiovascular systems. The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious GSNOR inhibitor which is currently in clinical development for acute asthma. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogs of N6022 focusing on carboxamide modifications on the pendant N-phenyl moiety. We have identified potent and novel GSNOR inhibitors that demonstrate efficacy in an ovalbumin (OVA) induced asthma model in mice.     

Discovery of 5-(2-chloro-4′-(1H-imidazol-1-yl)-[1,1′-biphenyl]-4-yl)-1H-tetrazole as potent and orally efficacious S-nitrosoglutathione reductase (GSNOR) inhibitors for the potential treatment of COPD

Endogenous nitrosothiols (SNOs) including S-nitrosoglutathione (GSNO) serve as reservoir for bioavailable nitric oxide (NO) and mediate NO-based signaling, inflammatory status and smooth muscle function in the lung. GSNOR inhibition increases pulmonary GSNO and induces bronchodilation while reducing inflammation in lung diseases. In this letter, design, synthesis and structure–activity relationships (SAR) of novel imidazole-biaryl-tetrazole based GSNOR inhibitors are described. Many potent inhibitors (30, 39, 41, 42, 44, 45 and 58) were identified with low nanomolar activity (IC₅₀s: <15?nM) along with adequate metabolic stability. Lead compounds 30 and 58 exhibited good exposure and oral bioavailability in mouse pharmacokinetic (PK) study. Compound 30 was selected for further profiling and revealed comparable mouse and rat GSNOR potency, high selectivity against alcohol dehydrogenase (ADH) and carbonyl reductase (CBR1) family of enzymes, low efflux ratio and permeability in PAMPA, a high permeability in CALU-3 assay, significantly low hERG activity and minimal off-target activity. Further, an in vivo efficacy of compound 30 is disclosed in cigarette smoke (CS) induced mouse model for COPD.     

Reduction of S-nitrosoglutathione by alcohol dehydrogenase 3 is facilitated by substrate alcohols via direct cofactor recycling and leads to GSH-controlled formation of glutathione transferase inhibitors

GSNO (S-nitrosoglutathione) is emerging as a key regulator in NO signalling as it is in equilibrium with S-nitrosated proteins. Accordingly, it is of great interest to investigate GSNO metabolism in terms of competitive pathways and redox state. The present study explored ADH3 (alcohol dehydrogenase 3) in its dual function as GSNOR (GSNO reductase) and glutathione-dependent formaldehyde dehydrogenase. The glutathione adduct of formaldehyde, HMGSH (S-hydroxymethylglutathione), was oxidized with a k(cat)/K(m) value approx. 10 times the k(cat)/K(m) value of GSNO reduction, as determined by fluorescence spectroscopy. HMGSH oxidation in vitro was greatly accelerated in the presence of GSNO, which was concurrently reduced under cofactor recycling. Hence, considering the high cytosolic NAD(+)/NADH ratio, formaldehyde probably triggers ADH3-mediated GSNO reduction by enzyme-bound cofactor recycling and might result in a decrease in cellular S-NO (S-nitrosothiol) content in vivo. Formaldehyde exposure affected S-NO content in cultured cells with a trend towards decreased levels at concentrations of 1-5 mM, in agreement with the proposed mechanism. Product formation after GSNO reduction to the intermediate semimercaptal responded to GSH/GSNO ratios; ratios up to 2-fold allowed the spontaneous rearrangement to glutathione sulfinamide, whereas 5-fold excess of GSH favoured the interception of the intermediate to form glutathione disulfide. The sulfinamide and its hydrolysis product, glutathione sulfinic acid, inhibited GST (glutathione transferase) activity. Taken together, the findings of the present study provide indirect evidence for formaldehyde as a physiological trigger of GSNO depletion and show that GSNO reduction can result in the formation of GST inhibitors, which, however, is prevented under normal cellular redox conditions.     

Immunolocalization of S-nitrosoglutathione, S-nitrosoglutathione reductase and tyrosine nitration in pea leaf organelles

S-Nitrosoglutathione (GSNO) is a nitrosothiol which plays a major role in the metabolism of NO in higher plants mediating signaling processes. Protein tyrosine nitration (NO₂–Tyr) is a post-translational modification which contributes to protein regulation. The subcellular localization of GSNO, S-nitrosoglutathione reductase (GSNOR), an enzyme which catalyzes its decomposition and protein tyrosine nitration was studied in pea (Pisum sativum L.) leaf plants with the aid of the electron microscopy immunogold-labeling technique. Our findings show that GSNO, GSNOR and nitrated proteins are present in the different subcellular compartments of leaf cells which include chloroplasts, cytosol, mitochondria, and peroxisomes. Given that pea peroxisomes are one of the cell compartments where nitric oxide (NO) has been thoroughly studied, our results provide additional insights into the metabolism of NO in this organelle where NO and GSNO could function as signal molecules in cross talk between the different cell compartments.     

Detection and quantification of S-nitrosoglutathione (GSNO) in pepper (Capsicum annuum L.) plant organs by LC-ES/MS

Glutathione (GSH) is one of the major, soluble, low molecular weight antioxidants, as well as the major non-protein thiol in plant cells. However, the relevance of this molecule could be even greater considering that it can react with nitric oxide (NO) to generate S-nitrosoglutathione (GSNO) which is considered to function as a mobile reservoir of NO bioactivity in plants. Although this NO-derived molecule has an increased physiological and phytopathological relevance in plants cells, its identification and quantification in plant tissues have not be reported so far. Using liquid chromatography-electrospray/mass spectrometry (LC-ES/MS), a method was set up to detect and quantify simultaneously GSNO as well reduced and oxidized glutathione (GSH and GSSG, respectively) in different pepper plant organs including roots, stems and leaves, and in Arabidopsis leaves. The analysis of NO and GSNO reductase (GSNOR) activity in these pepper organs showed that the content of GSNO was directly related to the content of NO in each organ and oppositely related to the GSNOR activity. This approach opens up new analytical possibilities to understand the relevance of GSNO in plant cells under physiological and stress conditions.     

Mechanism of inhibition for N6022, a first-in-class drug targeting S-nitrosoglutathione reductase

N6022 is a novel, first-in-class drug with potent inhibitory activity against S-nitrosoglutathione reductase (GSNOR), an enzyme important in the metabolism of S-nitrosoglutathione (GSNO) and in the maintenance of nitric oxide (NO) homeostasis. Inhibition of GSNOR by N6022 and related compounds has shown safety and efficacy in animal models of asthma, chronic obstructive pulmonary disease, and inflammatory bowel disease [Sun, X., et al. (2011) ACS Med. Chem. Lett. 2, 402-406]. N6022 is currently in early phase clinical studies in humans. We show here that N6022 is a tight-binding, specific, and fully reversible inhibitor of GSNOR with an IC(50) of 8 nM and a K(i) of 2.5 nM. We accounted for the fact that the NAD(+)- and NADH-dependent oxidation and reduction reactions, catalyzed by GSNOR are bisubstrate in nature in our calculations. N6022 binds in the GSNO substrate binding pocket like a competitive inhibitor, although in kinetic assays it behaves with a mixed uncompetitive mode of inhibition (MOI) toward the GSNO substrate and a mixed competitive MOI toward the formaldehyde adduct, S-hydroxymethylglutathione (HMGSH). N6022 is uncompetitive with cofactors NAD(+) and NADH. The potency, specificity, and MOI of related GSNOR inhibitor compounds are also reported.     

Discovery of potent and novel S-nitrosoglutathione reductase inhibitors devoid of cytochrome P450 activities

The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious S-nitrosoglutathione reductase (GSNOR) inhibitor and is currently undergoing clinical development for the treatment of acute asthma. GSNOR is a member of the alcohol dehydrogenase family (ADH) and regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). Reduced levels of GSNO, as well as other nitrosothiols (SNOs), have been implicated in the pathogenesis of many diseases including those of the respiratory, cardiovascular, and gastrointestinal systems. Preservation of endogenous SNOs through GSNOR inhibition presents a novel therapeutic approach with broad applicability. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogues of N6022 focusing on removal of cytochrome P450 inhibition activities. We identified potent and novel GSNOR inhibitors having reduced CYP inhibition activities and demonstrated efficacy in a mouse ovalbumin (OVA) model of asthma.     

Structure-activity relationships of pyrrole based S-nitrosoglutathione reductase inhibitors: pyrrole regioisomers and propionic acid replacement

S-Nitrosoglutathione reductase (GSNOR) is a member of the alcohol dehydrogenase family (ADH) that regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). GSNO and SNOs are implicated in the pathogenesis of many diseases including those in respiratory, cardiovascular, and gastrointestinal systems. The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious GSNOR inhibitor which is currently undergoing clinical development. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogues of N6022 focusing on scaffold modification and propionic acid replacement. We identified equally potent and novel GSNOR inhibitors having pyrrole regioisomers as scaffolds using a structure based approach.     

Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase

S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by S-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function.